C H A P T E R

5
Prolactin
Nadine Binart

INTRODUCTION pituitary tumors in whom galactorrhea and amenorrhea

were the cardinal clinical features did not have acrome-
Prolactin (PRL) is mainly produced by pituitary lacto- galic features and patients who were known to have
trophs and is tonically inhibited by the hypothalamus by isolated, congenital GH deficiency were able to undergo
the neurotransmitter dopamine. The discovery of multi- postpartum lactation. Finally, in 1970, Frantz and
ple extrapituitary sites of PRL secretion also increases the Kleinberg developed a sensitive in vitro bioassay which
range of known functions of this hormone. Its primary involved staining milk produced by cultured, lactating
function is to enable breast milk production, although mouse mammary tissue in response to PRL that was
PRL receptors (PRLRs) are found in many other tissues. capable of measuring PRL levels as low as 5 ng/mL. In
The major isoform, 23-kDa PRL, acts via a membrane this assay they added excess antibody to GH to neutral-
receptor, the prolactin receptor (PRLR), a member of ize any potential lactogenic effects it had and, for the
the hematopoietic cytokine superfamily, and for which first time, were able to demonstrate measurable PRL
the mechanism of activation has been elucidated. High levels in women with puerperal and nonpuerperal
levels of PRL in humans may interfere with reproduc- galactorrhea but not in most normal men and women.
tive function mainly by actions at the hypothalamus. Shortly thereafter, an RIA for human PRL was devel-
oped which could finally measure PRL levels in the
sera of normal individuals, permitting the entire amino
HISTORICAL OVERVIEW acid sequencing of human PRL [2] and determination
of its cDNA sequence.
In the late 1920s, it was found that pituitary extracts
induce milk secretion. Riddle and coworkers found
that this substance, which they named prolactin (PRL), CELL OF ORIGIN
could be differentiated from the known growth- and
gonad-stimulating substances [1]. In these experi- PRL is made by the pituitary lactotrophs (also known
ments, they showed that PRL stimulated milk produc- as mammotrophs). In the normal human pituitary, the
tion by guinea pig mammary glands and a milk-like lactotrophs comprise about 15 25% of the total number
substance from the crop sacs of pigeons and doves, of cells, are similar in number in both sexes, and do not
giving rise to the pigeon crop sac bioassay for PRL. change significantly with age. During pregnancy and
Over the ensuing years, PRL was characterized, subsequent lactation, however, lactotroph hyperplasia
sequenced and specific radioimmunoassays (RIAs) may be observed, presumably as a result of lactotroph
developed for PRL from a number of species. Because proliferation, transdifferentiation of somatotrophs, and/
of the high lactogenic activity of even very highly or expansion from a stem cell population [3]. The hyper-
purified preparations of human growth hormone (GH), plastic process involutes within several months after
however, it was impossible to separate human PRL delivery, although breastfeeding retards this process.
from GH using the relatively crude pigeon crop assay. This stimulatory effect of pregnancy on the lactotrophs
However, several human disease states provided strong also holds true for prolactinomas, which may be subject
evidence that these two hormones were separate. For to significant pregnancy-induced tumor enlargement
example, it was observed that most patients with (see chapter: Prolactinoma).

The Pituitary. DOI: http://dx.doi.org/10.1016/B978-0-12-804169-7.00005-2 129 © 2017 Elsevier Inc. All rights reserved.
130 5. PROLACTIN

Lactotroph Ontogeny synthesize α-subunits. The final differentiation of

somatomammotrophs occurs at least in part in rela-
Continuous hypothalamic pituitary interaction tionship to estrogen stimulation.
takes place during embryologic development. During
the formation of Rathke’s pouch, the primordium of
the anterior pituitary, the ectodermal primordial cells
of the anterior and intermediate lobes of the pituitary PROLACTIN GENE
make contact with the neuroectoderm of the floor of
the diencephalon and experimental studies have PRL belongs to the somatotrophin/PRL family, a
shown inductive interactions between these tissues large family of proteins that includes GH, placental
that are necessary for their subsequent interdependent lactogens (PLs), PRL-like and PRL-related proteins, pro-
development. Elegant studies using a series of targeted liferins, and proliferin-related proteins. The human PRL
mutations have shown that there are a number of tran- gene is located on chromosome 6p22.2-p21.3 and con-
scription factors (Six-3, Hesx1, Lhx3, Lhx4, Sox2, Sox 3, sists of five coding exons, one noncoding exon, and four
Pitx2, Otx2, Bmp2, Bmp4, and Gli2) that are sequen- introns. It is believed that PRL, GH, and PL arose from
tially expressed in the developing hypothalamus and duplication of a common ancestral gene B400 million
pituitary that lead to the final determination of the five years ago. The entire PRL locus in both humans and rats
mature pituitary cell types and the functional integra- spans a region of B10 kb. The human PRL cDNA is 914
tion of the hypothalamic pituitary system [4]. nucleotides long and contains a 681-nucleotide open
Mutations in most of the genes for these factors have reading frame encoding mRNA for a prohormone
been shown in humans to result in disordered devel- (preprolactin) of 227 amino acids. During PRL proces-
opment of hypothalamic, pituitary, and other brain sing, the 28-amino-acid signal peptide is proteolytically
structures with varying degrees of hypopituitarism. cleaved, resulting in a mature 199-amino-acid PRL poly-
The POU homeodomain transcription factor Pou1f1 peptide with a molecular weight of 23 kDa. Genes for
(also called Pit-1) gene becomes activated relatively the related hormones GH and PL (or chorionic somato-
late in development and is necessary for the activation mammotrophin) are clustered on chromosome 17.
of the PRL, GH, growth hormone-releasing hormone Much of the knowledge regarding regulation of PRL
(GHRH) receptor, and TSHβ genes as well as being nec- gene expression has been derived from studies utiliz-
essary for the differentiation and proliferation of these ing the rat pituitary PRL promoter. Less information is
cell lineages. A point mutation in the POU homeodo- available on regulation of hPRL and mouse PRL.
main of Pou1f1 has been found to be the cause of the Although attempts have been made to investigate
GH, PRL, and TSH deficiencies found in the Snell hPRL regulation by transfecting the hPRL promoter
dwarf mouse, with absence of somatotroph, lactotroph, into rat GH3 cells, it is uncertain how reliably this type
and thyrotroph cells. Similar mutations in Pou1f1 have of experimental system recapitulates hPRL promoter
now been found to cause a similar deficiency of GH, activity, since transformed rat and human lactotrophs
PRL, and TSH in humans [4]. A second paired-like may not share the same repertoire of endogenous tran-
homeodomain factor, known as Prophet of Pit-1 scriptional and/or epigenetic regulators. The rprl gene
(Prop-1) has also been found to be necessary for the is controlled by a proximal promoter and a distal
expression of Pit-1 and mutations of the Prop-1 gene enhancer, located 2433/ 220 bp and 1800/ 21500 bp,
cause the dwarfism in mice known as the Ames mouse respectively, relative to the pituitary transcriptional
(defects of somatotrophs, lactotrophs, and thyrotrophs) start site. In rats, 3 kb of 5’-flanking region is sufficient
and similar mutations in human cause variable defi- to direct lactotroph-specific transgene expression and a
ciencies of GH, PRL, TSH, LH, and FSH [4]. synergistic interaction between the distal enhancer and
Many lactotrophs arise from cells that at least at proximal promoter region is required for high levels of
some point expressed the GH gene. However, at least expression. Sequences flanking the enhancer restrict
in mice, it appears that many, if not most, lactotrophs prl expression to pituitary lactotrophs.
may derive from an earlier precursor [5]. Subsequent Organization of the hPRL gene is more complex,
lactotroph proliferation occurs once estrogen recep- and its transcription is regulated by two main inde-
tors (ERs) appear. Estrogen stimulates PRL gene tran- pendent promoter regions (Fig. 5.1). The proximal
scription (see below) only if Pou1f1 is bound to the region directs pituitary-specific expression, while a
PRL promoter. Stimulation by Prop-1 is necessary for more upstream promoter region, designated the
the subsequent development of all noncorticotroph “superdistal PRL promoter,” is responsible for direct-
cells. Pou1f1 is then necessary for the development ing extrapituitary expression [6]. Although most of
and proliferation of thyrotrophs and somatomammo- the circulating PRL in serum is produced by pituitary
trophs, but the separation of these two cell lines lactotrophs, PRL is also expressed in several extrapi-
occurs before the appearance of the ability to tuitary tissues, including uterine decidualized

I. HYPOTHALAMIC PITUITARY FUNCTION

 PROLACTIN GENE 131

FIGURE 5.1 Schematic diagram of the human PRL 5’ gene regulatory region, gene, and mRNA transcript. The PRL gene consists of five
exons, designated by the numbers (1 5). The region from 25800 base pairs (bp) to 0 indicates the region known to regulate pituitary PRL syn-
thesis whereas the region from 28789 to 25800 is referred to as the “superdistal” or “extrapituitary” promoter region. Thirteen Pit-1 binding
sites (blue) are present in the regulatory region. A single degenerate estrogen response element (ERE) (red) has been identified at 21189 bp.
The extrapituitary PRL mRNA is B150 bp longer than the pituitary transcript, and has a different 5’ UTR, but transcription from either pro-
moter produces identical protein-coding sequences.

endometrium, breast, brain, ovary, prostate, endothe- sequence, occur within a long terminal repeat-like
lial cells, lymphocytes, skin, adipose tissue, and transposable element (TE) of the medium reiteration
cochlea [6]. PRL expression at these sites is cell type- frequency (MER) family. The LTR sequence, named
specific and Pit-1-independent. In human extrapitui- MER39, appears to have been inserted B30 million
tary tissues, PRL mRNA transcription is driven by the years ago before the divergence of monkeys from
alternative “superdistal PRL promoter” located 5.8 kb higher apes. In addition, an older TE, MER20,
upstream of the pituitary transcription start site, result- provides an additional 198 bp of the 5’ flanking
ing in transcription of an extra exon, designated exon region of exon 1a. TE-derived sequences typically
1a. This alternative exon 1a of hPRL gene is noncoding, regulate nearby human genes, and in most cases, the
and transcription from either promoter produces LTR acts as an alternative promoter, but does not
mRNAs with differing 5’ untranslated regions (UTRs) alter the coding sequence. Some of the transcription
but identical protein-coding sequences. Although the factor binding sites that mediate extrapituitary PRL
purpose of the alternative promoter is not known for expression are located within these TE sites. Thus,
certain, it appears to confer tissue-specific expression, the evolutionary acquisition of these two TEs may
and it is likely that the variant 5’ UTRs may influence underlie the ability of mammals to express PRL in
the stability or translational efficiency of the disparate extrapituitary tissues.
PRL transcripts [7].
Characterization and analysis of the far upstream
elements dictating extrapituitary hPRL expression
are incomplete but evolving. In silico analysis of the
Pit-1
extrapituitary hPRL promoter has revealed that the Transcriptional regulators that control the develop-
5’ proximal region of the extrapituitary hPRL pro- ment of anterior pituitary lactotrophs also control PRL
moter, as well as exon 1a and a portion of intronic synthesis during adult life. Prominent among these is

I. HYPOTHALAMIC PITUITARY FUNCTION

132 5. PROLACTIN

the Pit-1 protein, a POU homeodomain transcription to adopt specific configurations; these subsequently
factor that dictates the terminal differentiation of pitui- control the recruitment of various coregulators to fine-
tary somatotrophs, lactotrophs, and thyrotrophs and tune PRL gene transcription [11].
regulates expression of their respective genes, GH, A Pit-1 splice isoform known as Pit-1β contains a
PRL, and TSHβ. Multiple signaling pathways ulti- 26-amino-acid motif inserted at amino acid 48, in the
mately converge upon Pit-1, and as such, Pit-1 serves middle of the Pit-1 transcription activation domain,
as the main transcriptional regulator of PRL gene resulting from an alternative splicing event that uses
expression. Pit-1 integrates information from a wide an in-frame acceptor 78 nucleotides upstream of
range of signaling pathways (triiodothyronine, estra- exon 2. In contrast to the activation of anterior pitui-
diol, glucocorticoids, protein kinase A (PKA), protein tary hormone promoters by Pit-1, Pit-1β represses GH,
kinase C (PKC), and Ras) in a cell-specific manner by PRL, and TSHβ promoters in a pituitary cell-specific
functionally interacting with numerous nuclear hor- manner [12]. The Pit-1β repression is potent as it is
mone receptors and coregulators (including itself, ER, capable of inhibiting the oncogenic Ras response of the
TR, GR, cJun, Oct1, GATA2, P-Lim, Ptx-1, Ets), and rprl promoter. Mechanistically, this Pit-1β motif-
recruiting them to Pit-1 regulated promoters. dependent transcriptional repression results from the
Through its interactions with specific DNA ele- active recruitment of corepressors as well as interfer-
ments in target gene promoters, Pit-1 recruits coregu- ence with efficient recruitment of CBP [13,14].
latory proteins that alter histone acetylation and Studies investigating Pit-1 regulation of PRL and
modify the chromatin structure, providing either a pituitary gene expression have determined that several
permissive or repressive environment for transcrip- mechanisms exist that enable Pit-1 to respond to multi-
tion [8]. Precise homeostatic control is achieved ple diverse stimuli. For example, Pit-1 largely binds to
through a network of interactions between Pit-1 and DNA elements as a dimer, but under the influence of
several different classes of transcription factors, certain signaling events, will bind as a monomer.
including the nuclear receptors, other homeodomain Crystal structure analysis reveals that Pit-1 homodi-
proteins, Ets family proteins, and basic region-leucine merizes in a head-to-tail fashion to achieve optimal
zipper (B-Zip) transcription factors. DNA binding on an idealized palindromic sequence.
Transcriptional activation of the PRL gene requires The activity of Pit-1 homodimers on dimeric sites in
the assembly of specific coactivator complexes at Pit-1 the rprl promoter is determined by the balance
composite DNA binding sites. There are eight Pit-1 between corepressor (NCoR/SMRT, mSin3A/B) and
binding sites (four within the proximal promoter, four coactivator (histone acetylases, CBP, P300/CBP) com-
within the enhancer) in the regulatory region of the rat plexes at the transcriptional start site. In fact, homodi-
prl gene, whereas there are 13 known Pit-1 binding merization of Pit-1 appears to be important to
sites (three within the proximal promoter, eight within maximize CBP recruitment in response to insulin and
the distal enhancer, two within the superdistal region) other growth factor stimulation. In contrast, Ras or ER-
in the human PRL promoter [9]. CBP/p300 is required α activation of the rprl promoter at a monomeric Pit-1/
for Pit-1 activation of PRL promoter in response to Ets composite binding site is independent of CBP and
PKA activation. instead relies upon a p160 SRC coactivator complex.
Pit-1 contains two well-defined functional motifs The recruitment of these specific transcriptional com-
that affect its activity: the transcriptional activation plexes is dependent upon structural features that are
domain and DNA binding domain (DBD). The amino- revealed when Pit-1 binds to DNA sites as a monomer
terminal TAD contains a regulatory domain and a or as a dimer. In addition, differences in the spacing
basal and Ras-responsive region. This latter region between DNA contact points for the POUS and POUHD
contains a basal activation region and an overlapping, domains alter the structure of the linker region, such
dual-function, Ras-responsive and inhibitory segment. that the 4-bp spacing on a Pit-1 site in the prl promoter
Distinct coactivators mediate basal and Ras-activated results in an activating Pit-1 conformation, whereas a
Pit-1 TAD activity, with CBP/p300 being a key effector 6-bp spacing in the gh promoter coverts Pit-1 to a
of Pit-1’s basal TAD, and steroid receptor coactivator-1 repressor in pituitary lactotrophs [15]. Finally, phos-
(SRC-1) a mediator of Ras responsiveness [10]. DNA phorylation of Pit-1 by PKA, PKC, and cyclin-
binding of Pit-1 is achieved through a carboxy- dependent kinases modifies its conformation on DNA
terminal DBD composed of two motifs, referred to as recognition elements and thus alters its DNA binding
the POU-specific (POUS) and POU-homeo (POUHD) affinity. Phosphorylation specifically inhibits binding
domains, both of which are necessary for high-affinity of Pit-1 to monomeric DNA sites (i.e., those used by
DNA binding. The combination of this bipartite DBD Ras and estradiol) and consequently decreases its tran-
and specific DNA binding sites generates a complex scriptional activity in response to these signaling
“code” which enables various subdomains within Pit-1 events, whereas phosphorylation has no effect on

I. HYPOTHALAMIC PITUITARY FUNCTION

 PROLACTIN GENE 133
basal, dimeric Pit-1 binding [16]. Thus, a plethora of expression also requires an intact mitogen-activated pro-
mechanisms, including monomeric versus dimeric Pit- tein kinase (MAPK) signaling transduction pathway as
1 binding, relative spacing of the Pit-1 POUS and interfering with MAPK activation ablates the ability of
POUHD binding sites, and the phosphorylation status estrogen to induce PRL expression [19].
of Pit-1 altogether generate a unique structural inter- A degenerate yet functional ERE sequence also
face for the binding of distinct coactivator/corepressor exists in the hPRL promoter at 21189 bp relative to the
complexes to regulate the expression of the PRL gene transcription start site. This ERE differs significantly
in pituitary lactotrophs. from the rprl ERE, although it is located within the cor-
responding sequence. ERα and Pit-1 exert synergistic
transcriptional effects on both the rat and human PRL
Estrogen promoters, suggesting that Pit-1 and ER are involved
Estrogen is an essential physiological activator of in the formation of a multiprotein complex at this site.
both PRL gene synthesis and lactotroph proliferation. The hPRL ERE has a relatively low binding affinity for
At present, three identified ERs are expressed in the ERα, and when stimulated by estradiol alone, exhibits
adenohypophysis: two classical nuclear ERs, ER alpha modest transcriptional activity. However, a marked
(α), and ER beta (β), which function as ligand- synergistic transcriptional effect is observed in the
activated nuclear transcription factors, and G protein- presence of estradiol and TNFα and it appears that
coupled estrogen receptor 1 (GPR30, GPER1), a seven- this particular ERE sequence is essential for the TNFα-
transmembrane G-protein-coupled receptor (GPCR) induced, NFκB-mediated activation of the hPRL pro-
that binds with high affinity to estradiol and mediates moter [20]. Thus, the ERE sequence in the hPRL pro-
rapid signaling events. Both ERα and ERβ are moter appears to be a target for at least two signaling
expressed in human and rat lactotrophs. The mouse pathways and, as such, may represent an important
pituitary expressed ERα, but not ERβ. GPR30 expres- converging point for integrating multiple physiological
sion in rodent adenohypophysis has been confirmed endocrine signals in vivo.
by in situ hybridization and immunohistochemistry,
but detailed information regarding specific cell-type
Ets
localization of this receptor, or possible functional roles
in mediating or contributing to estradiol effects in lac- Members of the Ets transcription factors family are
totrophs, are not known. The in vivo function of ERα key regulators controlling PRL gene expression.
in regulating prl expression has been demonstrated in Analyses of the rprl promoter in somatolactotroph cell
ERα2/2 mice. Although the specification of the lacto- lines have identified two critical Ets binding sites
troph lineage occurs normally in these animals, there is (EBS): a composite Ets-1/Pit-1 binding site located at
a 10 20-fold reduction in prl mRNA levels and a 2212 and a more proximal EBS located at 296. The
decrease in the number of lactotrophs [17]. Curiously, composite Ets-1/Pit-1 binding site confers synergy
plasma PRL levels are only slightly reduced, probably between the two proteins and mediates stimulation by
as a result of compensatory mechanisms, such as the Ras/MAPK signaling transduction pathway,
heightened sensitivity to PRL secretagogues. Pituitaries including those initiated by fibroblast growth factor
from ERβ 2/2 mice exhibit normal prl expression, but (FGF) and thyrotrophin-releasing hormone (TRH).
the lack of ERβ expression in wild-type mouse pituitary Although Pit-1 and Ets interact directly, the synergistic
precludes the ability to draw conclusions about the role activation of PRL gene expression does not necessitate
of ERβ in human PRL expression and/or lactotroph this physical interaction, but does require the assembly
function on the basis of this genetic mouse model [18]. of distinct Pit-1 transcriptional activation domains, as
The ER selectively binds to a single estrogen response well as the specific sequence of the composite site. The
element (ERE) located within the distal rPRL enhancer synergy between Pit-1 and Ets-1 can be prevented by
adjacent to the monomeric Pit-1d site at approximately an Ets-2 repressor factor apparently by preventing Pit-
1.5 kb upstream from the transcription initiation site. 1 from binding to the composite site. The proximal
This ERE has been shown to mediate a dramatic syner- EBS centered at 296, the target of several growth fac-
gistic interaction between Pit-1 and ER that results in a tor signaling pathways, is recognized and activated by
60-fold induction of rPRL transcription in response to Ets factors GA-binding protein α and β.
estrogen. Binding of Pit-1 to this distal enhancer site as a
monomer dictates the use of a specific aminoterminal
Other Transcription Factors
transactivation domain that is necessary to synergize
with ERα in a cell-specific manner. Interestingly, this Several additional transcription-regulating proteins
Pit-1 synergy domain is not required for synergistic identified in the pituitary have been implicated as key
events on the GH promoter. Estrogen-induced PRL elements in the regulation of the PRL promoter. These

I. HYPOTHALAMIC PITUITARY FUNCTION

134 5. PROLACTIN

include Pitx factors, thyroid hormone receptor (via involving crosstalk among intracellular signaling path-
activating protein-1 [AP-1] transcription factor), ways of BMP-4, Smad-4, and estradiol. In addition to
CCAAT/enhancer-binding protein (C/EBPα), SMAD4, effects on lactotroph proliferation, BMP-4 and estradiol
and Ikaros. The Pitx family is a class of bicoid synergistically activate transcription of the rprl pro-
homeodomain proteins required for the development moter independently of EREs, but in a manner depen-
of several organs. Among the three members of this dent upon a Smad binding element located between
family, Pitx1 and Pitx2 are expressed in the anterior 22000 and 21500 bp relative to the pituitary tran-
pituitary and in several pituitary cell lines. Both Pitx1 scriptional start site.
and Pitx2 can interact and synergize with Pit-1 in acti- In mammosomatotroph cells that express both GH
vating pituitary-specific promoters. The synergy and PRL, the zinc finger transcription factor Ikaros
between Pitx2 and Pit-1 is achieved by Pit-1 binding to suppresses gh but activates prl gene expression. The
the carboxy-terminal tail of Pitx2, which relieves the mechanism for this differential effect on hormone
autorepression imposed by this region, thereby expression appears to rely on the state of chromatin
increasing DNA binding of Pitx2 to a canonical bicoid accessibility for Pit-1, as mediated in part by Ikaros.
site. Two bicoid sites, B1 and B2, located at 227 and Access of Pit-1 is modulated in a gene-specific manner
2110, respectively, have been identified in the human such that Ikaros selectively deacetylates histone 3 resi-
PRL proximal promoter; the B2 site and two Pit-1 bind- dues on the GH promoter, and therefore restricts the
ing sites are necessary for the synergistic interaction of access of Pit-1 [25]. In contrast, Ikaros acetylates his-
Pitx2 and Pit-1 [21]. Although an intact B2 Pitx binding tone 3 on the proximal prl promoter and thereby facili-
site is necessary for full responsiveness to several sig- tates Pit-1 binding to this region in the same cells.
naling pathways regulating the hPRL promoter, Pitx Thus, Ikaros-mediated histone acetylation and chroma-
factors play a secondary role to factors such as Pit-1 tin remodeling provides one potential mechanism for
and Ets in the regulation of hPRL gene expression, the selective regulation of pituitary GH and PRL gene
since mutation of the B1 and B2 sites has only modest expression in cells that are capable of elaborating both.
inhibitory impact on promoter activity [21].
Triiodothyronine (T3) downregulates transcription
of hPRL gene. The hPRL promoter contains two T3
Signaling Pathways That Converge Upon
responsive regions. The first, located in the proximal
promoter, mediates a strong negative effect, while the
the PRL Promoter
second, located in the distal promoter, mediates a PRL expression is dynamically regulated by neuro-
weak positive effect. The overall effect of the two com- transmitters, hormones, and growth factors through
bined regions is negative. T3 exerts its inhibitory effect activation or inhibition of GPCRs and receptor tyrosine
by binding to thyroid hormone receptor (TR) and kinases that feed into several signal transduction path-
interfering with the AP-1 transactivation mediated by ways (Fig. 5.2). These positive and negative signals
an AP-1-binding site located in the proximal hPRL pro- ultimately converge upon the regulatory promoter
moter. The T3/TR complex has different effects on prl region. The intracellular signal transduction pathway
expression in the rat, where it mediates an overall acti- linking dopamine (DA) to the PRL gene involves inhi-
vation of the rprl promoter [22]. bition of adenylate cyclase and the cAMP/PKA path-
C/EBPα, a member of the bZip family of transcrip- way. PKA-dependent cAMP signals typically activate
tion factors, also synergizes with Pit-1 to stimulate the the transcription factor cAMP response element (CRE)-
rprl promoter and the rgh promoter [23]. The impor- binding protein (CREB), leading to its homodimeriza-
tance of C/EBPα interactions with Pit-1 is underscored tion on DNA complexes. However, the human and rat
by the observation that mutations in Pit-1 that disrupt PRL promoters lack functional cAMP DNA response
physical binding between Pit-1 and C/EBPα lead to elements (CREs), and although a one-half CRE is
combined pituitary hormone deficiency in humans. located in the rPRL promoter, CREB does not bind to
The DNA binding site utilized by the Pit-1-C/EBPα this sequence with high affinity. Therefore alternative
complex in the prl promoter overlaps with the proxi- undefined molecular mechanisms are involved in
mal EBS which is recognized by GABPα/GABPβ [23]. cAMP-dependent PRL gene regulation [26]. Growth
Bone morphogenetic protein-4 (BMP-4), one of the factors and hormones, such as insulin, epidermal
members of the TGF-β superfamily, plays a role in growth factor (EGF), and TRH, utilize PKC-dependent
pituitary development from the initial induction of pathways to directly phosphorylate the coactivator
Rathke’s pouch to cell specification in the anterior lobe CBP, which stimulates its subsequent recruitment to
and differentiation of the lactotroph lineage [24]. BMP- Pit-1, to regulate transcription of the PRL gene. Other
4, which is also overexpressed in different rodent pro- signaling pathways involving phosphoinositide
lactinoma models, exerts proliferative effects on post- 3-kinase (PI3K)-Akt phosphorylation of CREB have
natal lactotrophs through a complex mechanism also been proposed to mediate induction of the PRL

I. HYPOTHALAMIC PITUITARY FUNCTION

 HORMONE BIOSYNTHESIS 135

FIGURE 5.2 Schematic diagram of the membrane receptors and corresponding signal transduction pathways in pituitary lactotrophs that
are involved in PRL gene regulation. The dopamine D2 receptor (D2R) exerts the main inhibitory influence upon PRL synthesis. TRHR,
thyrotrophin-releasing hormone receptor; VIPR, vasoactive inhibitory peptide receptor; EGFR, epidermal growth factor receptor; FGFR, fibro-
blast growth factor receptor; PLC, phospholipase C; AC, adenylate cyclase; cAMP, cyclic adenosine monophosphate; PI3K, phosphoinositide
3-kinase; PKA, protein kinase A; PKC, protein kinase C; AKT, protein kinase B; Creb, cAMP response element binding protein; ER, estrogen
receptor.

promoter by insulin and prolactin-releasing peptide. In signaling pathways that modulate PRL expression and
this latter pathway, CREB probably regulates PRL pro- release [30]. The cytokine TNF-α activates the hPRL
moter activity through interaction with an Ets family promoter through NF-κB signaling in a manner that is
member. Extracellular signal-regulated kinase-1/2 also dependent upon ERα [20,31]. Since most of these
(Erk-1/2) activation serves as a point of convergence observations are based upon data derived from trans-
for PRL gene regulation by numerous stimuli, including formed rodent cell lines that lack normal dopaminergic
vasoactive intestinal peptide (VIP), insulin-like growth control, the relevance of these crosstalk patterns for
factor 1 (IGF-1), pituitary adenylyl cyclase activating human PRL expression and lactotroph function
polypeptide (PACAP), and fibroblast growth factor 2 remains to be confirmed.
(FGF2). These stimuli initially signal through pathways
characterized by differential utilization of monomeric
G proteins. Thus, VIP and IGF-1 stimulate PRL gene HORMONE BIOSYNTHESIS
expression through a Ras/Raf/Erk/Ets cascade,
whereas PACAP signals through Rap1/Braf/Erk, and Prolactin Protein
FGF2 signals through Rac-1/phospholipase C (PLC)/
PKC/Erk to control PRL gene expression [27,28]. Structural Characteristics and Posttranslational
Some of the intra- and extracellular factors that par- Modifications
ticipate in lactotroph proliferation and PRL gene The mature human PRL protein is composed of 199
expression appear to participate in complex crosstalk amino acids [32,33]. A comparison of the sequence
signaling pathways when studied in vitro. For exam- homology at the amino acid level from different species
ple, BMP4 and estradiol exert a synergistic effect on shows that the degree of conservation is highly variable
PRL gene expression in a manner dependent upon among mammalian and nonmammalian species,
Smad1, but independent of EREs [29]. TGFβ, however, reflecting their phylogenetic relationships. Primate PRL
inhibits PRL transcription in a manner that overrides has 97% homology to hPRL, whereas rodent PRL has
both BMP4 and estradiol stimulatory actions. only 61 64% homology. Moreover, rat PRL is capable
Similarly, EGFR and ERα participate in overlapping of activating the human PRL receptor (PRLR), whereas

I. HYPOTHALAMIC PITUITARY FUNCTION

136 5. PROLACTIN

inhibits cell proliferation, promotes differentiation, is

proapoptotic, and antiangiogenic. These biological
properties may result from differential use of postre-
ceptor signaling pathways [35]. Although both phos-
phomimetic S179D and unmodified PRL interact with
the same PRLR, unmodified PRL preferentially acti-
vates the Jak-Stat signaling cascade, whereas S179D
predominantly activates Erk 1/2.
In the rat pituitary, the relative ratio of phosphory-
lated to nonphosphorylated PRL isoforms is altered
during different phases of male reproductive develop-
ment [36], and various stages of the estrous cycle, sug-
gesting that this posttranslational modification may
have functional significance. Whether phosphorylation
modifications have biological relevance and, the extent
to which endogenous phosphorylated forms of hPRL
FIGURE 5.3 Schematic illustration of PRL proteins as they exist in participate in biologically significant PRLR signaling,
the serum as three forms: monomeric 23-kDa PRL ( . 95%), “big has not been conclusively determined.
PRL,” consisting of PRL aggregates, and “big, big PRL,” or macropro- Glycosylated PRL has been identified in the pitui-
lactin, consisting of PRL bound to IgG. The native PRL protein is com-
tary glands of several mammalian and nonmammalian
posed of 199 amino acids. Red hatching indicates the relative locations
of the three disulfide bonds. A single N-glycosylation site has been species at highly variable degrees (1 60%) [36]. hPRL
identified on human PRL at codon 31. Two putative phosphorylation is N-glycosylated on N31. Like other PRL variants, gly-
sites (not depicted) have been proposed at serines 163 and 194. cosylation lowers its biological activity as well as its
receptor binding and metabolic clearance rate, glycosy-
lated PRL may account for rare cases of mild asymp-
mouse PRL cannot. The PRL polypeptide is arranged tomatic unexplained hyperprolactinemia.
in a single chain of amino acids with three highly con-
served intramolecular disulfide bonds between six cys- Proteolysis
teine residues (Fig. 5.3). According to nuclear magnetic Several PRL variants are generated from proteolytic
resonance spectroscopy, PRL folds into four antiparallel cleavage of the 23-kDa protein [37]. The major 16-kDa
α-helices, similar to the tertiary structure of GH and variant is a product of cleavage occurring outside the
other close relatives [34]. cells in the interstitial medium and, in the vicinity of
Posttranslational modifications of the PRL polypep- blood capillaries, which implies that tissue-specific
tide, such as glycosylation, phosphorylation, proteo- mechanisms of regulation exist. This 16-kDa PRL vari-
lytic cleavage, and polymerization influence its ant contains only the N-terminal part of the mature
stability, receptor binding, measurement, and biologi- protein and does not bind the PRLR. New evidence
cal activity. Phosphorylated forms of PRL have been suggests that this variant is produced in several tis-
identified in most species, although it is not known sues. The 16-kDa variant seems to bind to endothelial
whether these forms appear in the plasma in vivo. cells and has inherent antiangiogenic properties,
Mass spectrometry of standard human pituitary which has led to use of the term “vasoinhibin” for this
extracts indicates that B19% of human PRL exists in a protein [38,39].
monophosphorylated form, another 19% in a dipho-
sphorylated form, and B62% is unphosphorylated. In Macroprolactin
the human pituitary, PRL is phosphorylated at serine In addition to monomeric 23-kDa PRL, two other
(ser)194 and ser163; in sera, ser163 is primarily depho- major forms of the protein are present in the circula-
sphorylated. The physiologic role of phosphorylated tion. Referred to as “big PRL,” which has a molecular
PRL may be inferred from investigations using a weight of 48 56 kDa and “big big PRL” (also known
recombinant artificial mutant of PRL that has been as macroprolactin), which has a molecular weight of
developed as a PRLR antagonist. S179D is a mimic of .100 kDa, these complexes of 23-kDa PRL and IgG
monophosphorylated hPRL in which the putative ser- autoantibodies can be detected to varying degrees by
ine phosphorylation site is replaced by an aspartate PRL immunoassays [40]; however these forms of PRL
residue [35]. Phosphorylated PRL has reduced potency have minimal biological activity in vivo and no known
in standard bioassays, and it antagonizes the proproli- pathological functions [40]. Macroprolactinemia is also
ferative action of the predominant unphosphorylated termed analytical hyperprolactinemia, and its presence
form. As compared to unmodified PRL, S179D PRL in the sera of patients can lead to clinical dilemmas

I. HYPOTHALAMIC PITUITARY FUNCTION

 HORMONE SECRETION: BIOCHEMISTRY 137
due to the potential misinterpretations of biochemical cell but due to functional heterogeneity of the cells so
testing (see Clinical testing, below). that some cells synthesize and secrete PRL rapidly
while others secrete more slowly.
Placental, Decidual, and Lymphoblastoid Forms Much of the storage pool of PRL in the pituitary
A variety of PRL-like proteins produced by the pla- appears to exist in a high-molecular-weight, disulfide-
centa have been identified in rodents, in addition to bonded, poorly immunoreactive polymeric form that is
PL [41]. These PRL-like proteins are secreted at differ- converted to a releasable, immunoreactive monomeric
ent times during gestation by the placenta and thus form within the secretory granule when processed for
may have different functions, including alterations in release.
blood vessel formation, hematopoiesis, and lympho-
cyte function. Although the expanded PRL family
ligands are not conserved in the human, the cells they Measurement of Prolactin
target are conserved and undergo fundamental
Assays and Bioassays
pregnancy-dependent adaptations.
PRL levels in maternal blood rise throughout gesta- PRL levels in sera are measured by two-site immu-
tion and are of pituitary origin. However, PRL concen- noradiometric assays and chemiluminometric assays
trations in amniotic fluid are 10 100-fold higher than utilizing the sandwich principle, whereby the PRL
either maternal or fetal blood levels. Utilizing the “extra- molecule reacts with an immobilized capture antibody
pituitary” or “decidual” PRL promoter (as detailed and a labeled detector antibody at two distinct sites.
above), human chorion-decidual tissues synthesize and Following removal of unused reagents with a wash
release a PRL species that is identical to pituitary PRL. step, the signal generated is proportional to the con-
Most recent reports have focused on the expression centration of PRL in the sample. Most immunoassays
and regulation of decidual PRL in humans [6]. The regu- are calibrated against the WHO third international
lation of decidual PRL secretion differs from that of standard for PRL, IS 84/500, consisting of human
pituitary PRL (detailed below). Dopamine, bromocrip- 23-kDa monomeric PRL.
tine, and TRH have no effects on the decidual The standard reference bioassay for PRL and other
production of PRL in vitro. Decidual PRL production is lactogens is the Nb2 cell proliferation assay. In this
increased by progesterone and progesterone plus estro- assay, cultured rat lymphoma cells that are completely
gen, but not estrogen alone. Insulin, through the insulin dependent on lactogenic hormones for growth are
receptor, IGF-1, through the IGF-1 receptor and relaxin, incubated with a biologic sample, and the rate of cell
a third peptide related to insulin and IGF-1 have all been division is quantified to provide a measurement for
reported to stimulate synthesis and release of PRL. the amount of lactogens present. This assay is highly
Although there is some evidence in animal studies sensitive (10 pg/mL) but has the potential theoretical
that it may contribute to the osmoregulation of the amni- disadvantage of inaccuracy due to species differences
otic fluid, fetal lung maturation, and uterine contractil- in PRLR responsiveness. Newer bioassays for human
ity, the function of decidual PRL in the human remains lactogens have been developed to address this issue,
obscure [6]. Expression of decidual PRL is controlled by as well as to analyze structure function studies of
many cytokines, transcription factors, and signaling human lactogen analogues, and to assess for the pres-
peptides that act either via well-defined regulatory ence of macroprolactin (see below). These bioassays
pathways or by binding directly to putative control utilize cell lines that stably express the human PRLR
elements within the superdistal promoter regions [6,42]. alone, as part of a proliferation assay, or in the pres-
ence of a luciferase reporter to measure transcriptional
activity [43,44]. Experience with these assays is limited
HORMONE SECRETION: BIOCHEMISTRY and testing to determine accuracy, validity, and repro-
ducibility in larger series of patients is awaited. In
Studies in the early 1970s demonstrated the exis- addition, a recent ultrasensitive-ELISA assay was
tence of two pools of PRL within the rat lactotroph developed to detect mouse PRL in very small volumes
cell, one turning over rapidly and the other turning of whole blood [45].
over slowly. Newly synthesized PRL is preferentially
released compared to older, stored PRL in response to Clinical Testing
some stimuli and constitutes the rapidly turning over PRL is secreted episodically and some PRL levels
pool. However, other stimuli, such as TRH, result in a obtained during the day may rise above the upper
preferential release of older, stored PRL. These two limit of normal established for a given laboratory.
types of secretion, rapid and slow, occur not so much Thus the finding of minimally elevated levels in blood
due to differences in the type of stimulation for a given requires confirmation in several samples. Several

I. HYPOTHALAMIC PITUITARY FUNCTION

138 5. PROLACTIN

nonhypothalamic pituitary conditions can cause mod-

erate PRL elevations, generally to levels ,250 ng/mL.
A careful history and physical examination, screening
blood chemistries, thyroid function tests, and a
pregnancy test will identify virtually all causes except
for hypothalamic pituitary disease. When there is
no obvious cause of the hyperprolactinemia from rou-
tine screening, radiologic evaluation of the
hypothalamic pituitary area is mandatory to exclude
a mass lesion. This includes patients with even mild
PRL elevations. Magnetic resonance imaging with gad-
olinium enhancement is the preferred study for pitui-
tary imaging (see chapter: Prolactinoma). It must be FIGURE 5.4 Schematic diagram illustrating the measurement
emphasized that it is essential to distinguish between a artifact referred to as the “hook effect.” Under usual circumstances
large nonfunctioning tumor causing modest PRL ele- (left side), PRL is detected in immunoassays by a solid-phase capture
vations (usually ,250 ng/mL) from a PRL-secreting antibody and a labeled detection antibody. When PRL levels are
grossly elevated (right side), the PRL protein saturates both antibo-
macroadenoma (PRL levels usually .. 250 ng/mL), as
dies, preventing sandwich formation and quantitative detection.
the management approaches to these two entities are
different. Most PRL-secreting macroadenomas gener-
ally respond readily to DA agonist therapy with size
reduction, whereas only about 10% of nonsecreting MACROPROLACTIN
pituitary tumors respond in this manner. It is also A major diagnostic conundrum facing laboratories
important to be aware of potential artifacts in PRL and clinicians is the differentiation of patients with
measurement that may lead to misdiagnoses. true hyperprolactinemia from those with macroprolac-
Stimulation and suppression tests using TRH, hypo- tinemia [50]. Based on several clinical series, the esti-
glycemia, chlorpromazine, domperidone, and other mated incidence of macroprolactin accounting for a
medications yield nonspecific results and reveal no significant proportion of hyperprolactinemic sera is
more information than simple measurement of basal approximately 10 20% [48,51]. Current best practice
PRL levels. Thus, consensus has developed that such recommends that serum is subfractionated using poly-
stimulation and suppression tests are not recom- ethylene glycol precipitation to provide increased qual-
mended in the differential diagnosis of hyperprolacti- ity of the measurement of bioactive monomeric PRL
nemia [46]. [52]. With this pretreatment step, the larger molecular
weight forms of PRL are removed by precipitation,
leaving the residual monomeric forms in the superna-
Artifacts
tant [48]. Clinicians should be aware that due to meth-
HOOK EFFECT odological issues, PEG precipitation is not technically
Although current PRL immunoassays are highly feasible on all commercial immunoassays.
sensitive and specific, artifacts due to saturation of the Normative ranges have been published for sera trea-
antibodies at excessively high PRL concentrations, ted with PEG for several of the most widely used
which prevent antibody PRL antibody sandwich for- assay systems [53]. Nevertheless, disagreement persists
mation may nevertheless occur (Fig. 5.4). This phe- regarding the threshold concentration of residual
nomenon, referred to as the “hook effect,” grossly monomeric PRL at which a hyperprolactinemic sample
underestimates the true PRL concentration leading the should be designated as being attributable to macro-
laboratory to report a falsely low value. St.-Jean et al. prolactin. Some investigators assert that in a patient
noted this high-end “hook effect” in 5.6% of 69 with hyperprolactinemia, if the recovery of monomeric
patients who were thought to have clinically nonfunc- PRL left following PEG precipitation is less than 40%
tioning adenomas [47]. Interference from the “hook or 50% of the initial total value, then the hyperprolacti-
effect” generally becomes problematic when PRL nemia is due to macroprolactin [51]. Others suggest
levels exceed 100,000 mU/L [48], but the threshold for that since some macroprolactin is present in normal
this effect varies among immunoassay platforms. serum and/or some monomer is precipitated with
Therefore, in patients with large macroadenomas if PEG, that the “normal” reference range should be
there is suspicion regarding the susceptibility of an recalculated from normal samples after PEG treatment;
assay to this phenomenon, PRL assessments should be therefore, hyperprolactinemia is attributable to macro-
performed in both undiluted and 1:100 diluted serum prolactin only when the level of nonprecipitated PRL
to exclude the “hook effect” [49]. is within the normal range [54]. It is clear that in many

I. HYPOTHALAMIC PITUITARY FUNCTION

 HORMONE SECRETION: BIOCHEMISTRY 139
cases, especially when sera contain high concentrations Physiology
of PRL, even when the amount of nonprecipitated PRL
is less than 40% of the total, the residual PRL level still Metabolic Clearance and Production Rates
exceeds the normal range. of Prolactin
An additional issue that remains unresolved is a reli- Using a labeled PRL method, the metabolic clear-
able estimation of the bioactivity of macroprolactin, as it ance rate has been found to be 46 6 4 and 40 6 6 mL/
is presumed that its presence in serum does not confer min/m2 and the calculated production rates using the
biologic significance since it is unable to pass through labeled PRL method were 200 6 63 and 536 6 218 μg/
the capillary endothelial barrier. The bioactivity of day/m2 in two studies [57]. Studies in patients with
macroprolactin as measured by the classic rat Nb2 cell chronic renal failure have shown the MCR to be
proliferation assay is comparable to the bioactivity of reduced by 33%; increased uptake by the liver has
monomeric PRL. However, when bioactivity is mea- been found in nephrectomized rabbits.
sured using human PRLR constructs as tested in Ba/F-3
or human embryonic kidney-derived 293 (HEK-293) cell
lines, the bioactivity of macroprolactin is reduced Hormone Secretion Patterns
[43,44]. Technical pitfalls could potentially account for PRL is secreted episodically (Fig. 5.5). There is an
the findings in either of these cell-based systems. For innate pulsatility to pituitary PRL secretion with an
example, under the bioassay conditions employed in interpulse interval of about 8 minutes, as determined
the Nb2 cell assay, dissociation of macroprolactin into by studies of media obtained from primate pituitaries
its constituents may free monomeric PRL to react, yield- cultured in vitro. When plasma is sampled from nor-
ing a result suggestive of hormonal activity [55]. On the mal individuals in whom hypothalamic function is
other hand, transfection conditions used to express superimposed upon this innate pulsatility, it becomes
exogenous human PRLR plasmids could alter the struc- apparent that there are 4 14 secretory episodes per
ture or function of the PRL added from the sample. day. Using cluster analysis, 13 14 peaks per day in
In most clinical series investigating suspected or possi- young subjects were found with a peak duration of
ble macroprolactinemia, symptoms are fewer and less 67 76 minutes, a mean peak amplitude of 3 4 ng/mL,
severe in patients whose hyperprolactinemia is and an interpulse interval of 93 95 minutes [58].
attributable to macroprolactin than in those patients Disinhibition caused by hypothalamic tumors causes
who are determined to have true hyperprolactinemia an increase in basal PRL levels due to an increase in
[48,56]. In studies in which patients with suspected pulse amplitude and not pulse frequency.
macroprolactinemia (often in retrospect) are treated There is an increase in the amplitude of the PRL
with DA agonists, galactorrhea, when present, generally secretory pulses that begins about 60 90 minutes after
disappears, but oligo/amenorrhea is variably respon- sleep onset; secretory pulses increase with non-REM
sive [56]. Long-term follow-up studies of patients diag- sleep and fall prior to the next period of REM sleep.
nosed with macroprolactinemia indicate that PRL levels The lowest PRL concentrations are found during REM
show considerable instability (up to fivefold) [56]. sleep and the highest concentrations are found during
In clinical practice, if a patient has typical symp- non-REM sleep. When subjects are kept awake to
toms, such as galactorrhea, amenorrhea, or impotence reverse the sleep waking cycle, PRL levels do not rise
and is found to have mild hyperprolactinemia, the until sleep onset. Thus, the diurnal variation of PRL
usual conditions should be excluded (medications, secretion is not an inherent rhythm but depends on the
hypothyroidism, elevated creatinine, pregnancy) to be occurrence of sleep. Interestingly, the diurnal variation
followed by pituitary MRI, primarily to exclude a large
lesion such as a craniopharyngioma or clinically non-
functioning adenoma. In patients with mild hyperpro-
lactinemia who have equivocal symptoms (such as
headaches or decreased libido) but normal menses and
no galactorrhea, assessment for macroprolactin using
PEG precipitation is reasonable. A decision that hyper-
prolactinemia is due to macroprolactin then would
depend on demonstrating an abnormal amount of PRL
precipitated by PEG, and a residual PRL monomer
level that falls within the normal range. Under these
circumstances, pituitary imaging may not be indicated, FIGURE 5.5 PRL levels throughout the day in a single individual
but would warrant continued clinical and biochemical superimposed upon the range from five normal individuals. Note
assessment of such patients on a periodic basis. the episodic nature of secretion and the nocturnal rise.

I. HYPOTHALAMIC PITUITARY FUNCTION

140 5. PROLACTIN

of PRL with sleep-induced rises persists despite other hypothalamic dopamine and stimulate lactotroph
powerful physiologic influences such as breastfeeding. proliferation [30].
There is an increase in circulating PRL levels of PRL begins to rise in the fetal circulation at 20 24
50 100% within 30 minutes of meals that is due to the weeks, increasing from week 30 to term, when it
amino acids generated from the protein component of reaches levels similar to maternal serum PRL. The PRL
the meals, phenylalanine, tyrosine, and glutamic acid rise is fetal autonomous. Unique to humans, the
being the most potent in this regard. Evidence was decidua produces very large amounts of PRL, which
provided that this stimulatory action of these amino accumulates in the amniotic fluid, attaining peak levels
acids is centrally mediated by showing that large neu- as high as 4000 5000 ng/mL between 16 22 weeks
tral amino acids such as valine inhibit the transport of gestation and reducing to 400 500 ng/mL at term.
phenylalanine across the blood brain barrier and Despite such profound changes in PRL in the fetal
blunt the stimulatory action of this amino acid. compartment, there is little knowledge of its impor-
tance in human fetal physiology. These elevated PRL
Changes in Prolactin With Age levels found at term prepare the breast for lactation.
PRL levels are elevated almost 10-fold in infants fol-
lowing delivery but then gradually decrease so that
Changes in Prolactin Levels With Postpartum
levels achieve normal ranges by 3 months of age. Lactation
These high levels of PRL at birth are probably related
to the stimulatory effect of high maternal estrogen Within the first 4 6 weeks postpartum basal PRL
levels. PRL levels are lowest between the ages of 3 levels remain elevated in lactating women and each
months and 9 years and then rise modestly during suckling episode triggers a rapid release of pituitary
puberty to adult levels. In some studies, there is a PRL resulting in a 3 5-fold increase in serum PRL
gradual fall of basal PRL levels with age but in others levels, peaking about 10 minutes after the end of suck-
no changes with age have been found. In hyperprolac- ling. Following termination of suckling PRL levels
tinemic women, estrogen replacement therapy does gradually fall to reach prenursing levels by about 3
not alter PRL levels. PRL levels are lower by 55% in hours after the beginning of the suckling episode. Over
older men compared to younger men due to both a the next 4 12 weeks, basal PRL levels gradually fall to
decreased basal secretion as well as the amount normal and the PRL increase which occurs with each
secreted with each secretory burst. suckling episode decreases. Eventually there is little
or no rise in PRL with suckling, despite continued
milk production.
Changes in Prolactin Levels during the Menstrual
The decreases in basal and stimulated PRL levels
Cycle
between 3 and 6 months postpartum are largely the
Some, but not all, women have higher levels at mid- result of decreased breastfeeding as formula is intro-
cycle and lower levels in the follicular compared to the duced into the baby’s diet. If intense nursing behavior
luteal phase. In most of these studies, no correlations is maintained, basal PRL levels remain elevated and
were found between PRL and estradiol, progesterone, postpartum amenorrhea persists. Eighty minutes of
LH, and FSH levels. However, some studies have nursing per day with a minimum of six nursing epi-
shown that PRL and LH secretion are often synchro- sodes will usually result in persistent hyperprolactine-
nous in the luteal phase and that very small doses of mia and amenorrhea. However, delayed onset of
gonadotrophin-releasing hormone (GnRH) can cause menses is more associated with high suckling duration
the secretion of both PRL and LH at this time. and frequency than with a specific PRL level. High-
intensity lactation-induced failure to ovulate and men-
Changes in Prolactin Levels During Pregnancy struate has been used as a method of contraception in
The profile of PRL release during human pregnancy some developing countries for many years.
is entirely different from that in rodent lactation [30]. It To support a period of hyperprolactinemia during
involves three independently regulated compartments: lactation, and thereby promote milk production, there
maternal, fetal, and decidual. Maternal serum PRL levels is an apparent loss of sensitivity of the short-loop feed-
start rising at 6 8 weeks gestation. Basal PRL levels back system during late pregnancy and lactation [59].
gradually increase throughout the course of pregnancy This is a remarkable example of adaptive plasticity
to reach 200 300 ng/mL at term. Simultaneously, the within a neuroendocrine control network, allowing a
pituitary gland enlarges due to increases in lactotroph sustained period of high PRL secretion to be main-
size and number. Indirect evidence suggests that tained unused by a regulatory feedback pathway [60].
increased PRL release and lactotroph hyperplasia are Breast stimulation may cause an increase in PRL
driven by estrogens, which presumably suppress levels in some nonbreastfeeding normal women, but

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 NEUROENDOCRINE REGULATION 141
chronic nipple stimulation with nipple rings has been humans, naloxone has generally not been found to be
reported to cause sustained galactorrhea [61]. able to block the PRL response to hypoglycemia.
PRL is also produced directly by human breast Hypoglycemia has been regarded as a form of
glandular and adipose tissue [62,63]. Although proges- stress, and PRL does indeed rise with hypoglycemia.
terone inhibits the glandular PRL production, it has no Acute exercise also results in an acute, transient
effect on the adipocyte PRL production. It is unclear increase in PRL levels. Although chronic, high-level
whether the PRL present in breast milk is of local or exercise often results in menstrual disturbance, it is not
systemic origin. associated with sustained hyperprolactinemia.

Changes in Prolactin Secretion With Stress

PRL is one of the pituitary hormones released by NEUROENDOCRINE REGULATION
stress, along with adrenocorticotrophic hormone and
GH. Stress-induced PRL generally occurs with a dou- The hypothalamus exerts a predominantly inhibi-
bling or tripling of PRL levels and lasts less than 1 tory influence on PRL secretion through one or more
hour. In humans, prolonged critical illness does not PRL inhibitory factors (PIF) that reach the pituitary via
cause a sustained elevation of PRL; rather there is a the hypothalamic pituitary portal vessels (Fig. 5.6).
reduction in the pulsatile secretion with an overall There are PRL-releasing factors (PRF) as well.
lowering of levels. The teleological significance for Disruption of the pituitary stalk leads to a moderate
these stress-induced changes in PRL is not clear. increase in PRL secretion as well as to decreased secre-
The neuroendocrine mediation of the acute stress tion of the other pituitary hormones.
response is probably multifactorial but does not
include a decrease in DA. It was attempted to dissect
out the neurotransmitter regulation of the PRL stress Prolactin-Inhibiting Factors
response in humans by administering various blocking
agents immediately prior to surgery. Blockade of hista- Dopamine
mine H1 receptors using chlorpheniramine, serotonin In the 1950s, it was demonstrated that the luteotro-
receptors using cyproheptadine, and DA receptors phic properties of the pituitary, due to PRL, were
using pimozide had little effect on the peak PRL level increased when pituitary glands were transplanted to
reached during surgery. Blockade of opiate receptors beneath the renal capsule, a site away from the regula-
with high-dose naloxone resulted in a significant tion by the hypothalamus, thus demonstrating the
blunting, but not complete inhibition, of the PRL predominance of the inhibitory component of hypotha-
response. These studies imply that the endogenous lamic regulation of PRL secretion. Later, it was demon-
opiate-like peptidergic pathways may play a role in strated that tuberoinfundibular DA (TIDA) released
the PRL stress response. On the other hand, in into the hypothalamic pituitary portal vessels in the

FIGURE 5.6 Neuroendocrine regulation of PRL secretion. TRH, thyrotrophin-releasing hormone; VIP, vasoactive intestinal peptide.

I. HYPOTHALAMIC PITUITARY FUNCTION

142 5. PROLACTIN

median eminence was the physiologic PIF with direct The axons responsible for the release of DA into the
action on the pituitary [64]. median eminence originate in perikarya in the dor-
A number of experiments firmly established that somedial portion of the arcuate nucleus and inferior
DA is the predominant, physiologic PIF, including portion of the ventromedial nucleus of the hypothala-
the findings that the concentration of DA found in the mus [30]. This pathway is known as the TIDA path-
pituitary stalk plasma (about 6 ng/mL) was sufficient way. The DA that traverses the TIDA pathway binds
to decrease PRL levels in rats and that stimuli which to D2 receptors on the lactotroph cell membrane [64].
result in an acute release of PRL usually also result in As discussed above, activation of this receptor results
an acute decrease in portal vessel DA levels [64]. in (1) an inhibition of adenyl cyclase with lowered
However, in many experiments it was found that the intracellular cAMP levels, (2) inhibition of phosphoino-
PRL increase obtained by simply reducing DA was sitide metabolism, and (3) decreased intracellular cal-
considerably less than the elevation of PRL achieved cium mobilization and inhibition of calcium transport
by simultaneous stimulation by a PRF; similarly, the through calcium channels. It has been proposed that
PRL level achieved with the simultaneous stimulation these different actions of DA may actually be mediated
by a PRF with the reduction in DA is usually greater by multiple similar D2 receptors that are produced by
than that achieved by a PRF alone. It is likely that in alternative RNA splicing [64].
most physiologic circumstances that cause a PRL rise, The inhibitory action of DA on PRL secretion is par-
such as lactation, there is a simultaneous fall in DA tially blocked by estrogen administration. This may be
along with a rise in a PRF, such as VIP, although there largely due to the direct action of estrogen on the ERE
may well be circumstances in which various PRFs may of the PRL gene (see above). However, there may also
stimulate PRL release with no concomitant lowering of be other mechanisms but with considerable interspe-
DA levels or DA may be lowered with no concomitant cies differences. Estradiol is able to block the inhibitory
increase in a PRF. action of DA on PRL release from rat lactotroph cells
Newer work with mice in which the D2 receptor in vitro. In studies in humans, the same dose of
(D2R) or DA transporter (DAT) have been “knocked infused DA results in a greater suppression of PRL
out” has confirmed these earlier studies that during the early follicular phase, when estrogen levels
employed pharmacologic methods or lesioning. Thus, are low, compared to the late follicular or periovula-
D2r2/2 mice develop lactotroph hyperplasia and tory phases, when estrogen levels are higher.
sustained hyperprolactinemia, followed by lactotroph Estrogens result in a decrease in DA receptor abun-
adenomas in aged mice, demonstrating that a chronic dance in rats but the DA receptor population showed
loss of neurohormonal DA inhibition promotes a no sex-related differences in a limited number of
hyperplasia neoplasia sequence in adenohypophysial human pituitaries.
lactotrophs [65]. DA action within the synapse termi-
nates by DA reuptake by the DA-secreting neurons Gonadotrophin-Associated Peptide
via the DA transporter. In contrast to the findings
Whether DA alone can account for all of the PIF
with the D2r2/2, Dat2/2 mice have increased dopami-
activity of the hypothalamus has long been a question.
nergic tone and lactotroph hypoplasia [66]. Although
In 1985 Nikolics et al. reported the PRL-inhibiting abil-
such mice have normal circulating levels of PRL, they
ity of a 56-amino-acid polypeptide that is in the car-
cannot increase these levels with various stimuli and
boxyterminal region of the precursor to GnRH and
are unable to lactate [66].
which they termed GAP [68]. However, the GAP
Although much of the direct work demonstrating
sequences in human and rat have 17 amino acid differ-
DA in hypothalamic pituitary portal vessels and the
ences and subsequent studies showed that GAP has no
effects of DA on PRL release in vitro have been done in
PRL-suppressing activity when tested against human
animals, it is clear that DA is the primary PIF in
prolactinomas cultured in vitro. To date, there is no
humans as well. Infusion of DA causes a rapid sup-
evidence that GAP has physiologic significance in
pression of basal PRL levels that can be reversed by
humans as a PIF.
metoclopramide, a DA receptor blocker. Dopamine also
blocks the PRL increments induced by various stimuli.
Studies with low-dose DA infusions in humans have γ-Aminobutyric Acid
shown that DA blood concentrations similar to those γ-Aminobutyric acid (GABA) has an inhibitory
found in rat and monkey hypothalamic pituitary por- effect on PRL secretion in vivo and in vitro in rats and
tal blood are able to suppress PRL secretion. Blockade high-affinity GABA receptors are present on lacto-
of endogenous DA receptors by a variety of drugs, trophs. A tuberoinfundibular GABAergic system has
including phenothiazines, butyrophenones, metoclopra- been described with perikarya located in the arcuate
mide, and domperidone causes a rise in PRL [67]. nucleus and nerve endings demonstrated in the

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 NEUROENDOCRINE REGULATION 143
median eminence and GABA has been demonstrated PRL levels are not low basally but the PRL response to
to be present in portal blood. TRH is markedly blunted and returns to normal with
Studies of the GABA system in humans have correction of the hyperthyroidism.
yielded conflicting results in studies of widely differ- The above conflicting data from passive immuniza-
ing experimental designs. GABA itself causes a modest tion studies, trh2/2 mice, observation of TSH levels
decrease in PRL levels when given to humans for sev- during lactation, and examination of PRL levels in var-
eral days and activation of the endogenous GABAergic ious thyroid states support a role for TRH as a physio-
system with sodium valproate causes a suppression in logic PRF, albeit not the primary one or even one of
the PRL rise induced by mechanical breast stimulation major importance. Moreover, a TRH stimulation test is
in puerperal women. The physiologic role of GABA often used to diagnose hyperprolactinemia in patients,
remains to be fully elucidated in the human. but its physiological importance as a regulator of PRL
release in humans is unclear.

VIP and Peptide Histidine Methionine (PHM)/PHI

Prolactin-Releasing Factors
VIP stimulates PRL release and is found in neuronal
Thyrotrophin-Releasing Hormone perikarya in the parvocellular region of the paraventri-
TRH binds to type 1 TRH receptors expressed in cular nucleus with axons terminating in the external
both thyrotrophs and lactotrophs. Shortly after its ini- zone of the median eminence. Its effects are selective
tial isolation and characterization, TRH causes a rapid for PRL and additive to TRH in causing PRL release at
release of PRL from rat pituitary cell cultures and in concentrations found in hypothalamic pituitary portal
humans after intravenous injection. Release of PRL is blood. The effects of VIP appear to be mediated by
biphasic, the initial peak being mediated by activation stimulation of adenyl cyclase, although recent evidence
of intracellular phosphoinositide pathways with IP3 suggests that transport of calcium through membrane
generation and mobilization of intracellular calcium calcium channels may also be important. In addition to
causing release of stored hormone; the second, more stimulating PRL release, VIP also stimulates pituitary
sustained phase is mediated influx of extracellular PRL mRNA content and PRL synthesis. In conditions
calcium through calcium channels, which causes of increased PRL synthesis, such as lactation, hypotha-
sustained secretion and synthesis of new hormone. lamic VIP mRNA levels are also increased.
A number of different experimental approaches Intravenously administered VIP has also been shown
have failed to clarify the physiologic role of TRH as a to increase PRL levels in humans at serum levels simi-
PRF. The smallest dose of TRH that releases TSH also lar to those demonstrated in rat portal blood.
releases PRL in humans. Immunoneutralization of A number of experiments have been performed
endogenous TRH with TRH antisera causes a 50% sup- using passive immunoneutralization techniques to
pression of basal PRL levels in rats in some studies but determine the physiologic role of VIP as a PRF. Anti-
not in others. The trh2/2 mice became hypothyroid VIP antisera administered to rats have been shown to
with elevated levels of TSH with reduced biological partially inhibit the PRL responses to suckling and
activity but had normal PRL levels, further casting ether-induced stress.
doubt on the essential role of TRH in PRL regulation. Part of the 20-kDa 170-amino-acid VIP precursor is
If TRH mediates the PRL response to suckling, even another similarly sized peptide known as PHM. PHM
in part, it ought to be accompanied by an increase in and VIP colocalize in the hypothalamus and median
TSH, unless there were a concomitant increase in eminence. PHM given to humans has caused a PRL
somatostatin. Studies in humans failed to show any increment in some experiments but not others.
elevations of TSH with suckling. Very small doses of Further complicating the role of VIP as a PRF is the
TRH given systemically were effective in releasing finding that VIP is actually synthesized by anterior
PRL and TSH in lactating rats and women in those pituitary tissue. Antisera to VIP inhibit basal PRL
studies, however, so it is unlikely that failure to show secretion from dispersed pituitary cells in vitro, sug-
a rise in TSH was due to an increase in somatostatin. gesting a local “autocrine” role for VIP in PRL regula-
In hypothyroidism, TRH synthesis is increased, por- tion within the pituitary.
tal vessel TRH levels are increased, and there is an The physiologic role of VIP as a PRF appears to be
increased number of TRH receptors. In human hypo- warranted by the experimental data. The precise roles of
thyroidism, basal TSH and PRL levels are increased as VIP versus. PHM and hypothalamic VIP versus. pitui-
are their responses to injected TR. Correction of the tary VIP still are not clear. How VIP/PHM interact with
hypothyroidism with thyroid hormone corrects both other PRFs such as TRH are additional areas requiring
the elevated TSH and PRL levels and their responses clarification. The general current consensus is that VIP is
to TRH. Conversely, in hyperthyroidism in humans, not a potent PRL secretagogue in humans [30].

I. HYPOTHALAMIC PITUITARY FUNCTION

144 5. PROLACTIN

Serotonin beyond the scope of this discussion. Approaches to

A considerable number of experiments have demon- determining the roles of the opioid peptides and path-
strated a role for serotonin as a neurotransmitter ways in the regulation of PRL secretion have focused
involved in the release of PRL. Most serotoninergic on using opioid agonists and antagonists in experi-
neuronal perikarya are in the dorsal and median raphe mental animals and humans.
nuclei and their axons project forward to the hypothal- In rats, morphine, Met- and Leu-enkephalin,
amus and other limbic and cortical areas. Lesions of β-endorphin, dynorphin, and leumorphin injected sys-
the dorsal but not the median raphe nuclei decrease temically or intracerebroventricularly have all been
forebrain serotonin levels and basal and stimulated shown to elicit PRL release. Subsequent studies
serum PRL levels. employing specific agonists and antagonists operative
Studies in humans also suggest a role for serotonin on the μ, δ, and κ opioid receptors and antibodies
in PRL secretion. Infusion of the serotonin precursor, directed against several opioid peptides have shown
5-hydroxytryptophan, elicits a prompt increase in PRL that it is the μ receptor that is the predominant one
levels. Nocturnal PRL secretion is inhibited by cypro- involved in PRL release, the κ receptor is involved to a
heptadine. On the other hand, pizotifen, a specific, lesser extent, and the δ receptor is not at all involved.
nonergot serotonin antagonist, had no effect on Opioids stimulate PRL release by inhibiting DA turn-
suckling-induced PRL in postpartum women. over and release by the TIDA pathway. Orphanin FQ
Whether serotonin effects are mediated solely (also called nociceptin), which binds to an opioid-like
through brain pathways or whether it has direct effects orphan receptor, also causes an increase in PRL levels
on the pituitary is controversial. One possibility is that in rats when administered intracerebroventricularly.
serotonin causes a decrease in hypothalamic DA gen- In humans, morphine and morphine analogues
eration. Synaptic junctions between serotoninergic increase PRL release acutely and chronically. However,
nerve terminals and dopaminergic perikarya in the blockade of the μ receptor with naloxone has minimal to
arcuate nucleus have been demonstrated. Furthermore, no effect on PRL levels either basally or with stimulation
intraventricular injections of serotonin decrease portal by hypoglycemia, exercise, sleep, TRH, or physical stress.
vessel DA concentrations. It has also been proposed Overall, it appears that the endogenous opioid pathways
that serotonin acts by increasing VIP and oxytocin via play at most only a minor role in the regulation of PRL
effects at the paraventricular nucleus. secretion, especially in humans.
Serotonin has been found within the anterior pitui-
Growth Hormone-Releasing Hormone
tary serotoninergic nerve terminals and has been dem-
onstrated within the median eminence. High-affinity A number of studies have found GHRH to have
S2 serotonin receptors have been found in the anterior PRL-releasing properties. The initial clue to this effect
pituitary as well as uptake of labeled serotonin into of GHRH was the finding that many of the patients
cells of the pituitary. In direct tests of the effects of with acromegaly due to GHRH-secreting tumors were
serotonin on pituitary PRL release, serotonin has been hyperprolactinemic and PRL levels fell in parallel with
found to increase basal and stimulated PRL secretion GH following excision of the GHRH-secreting tumor.
from pituitaries in vitro and this appears to be medi- Large doses of GHRH have been reported to release
ated by the serotonin subtype 4 receptor [69]. PRL in vivo in normal humans. Chronic therapy with
Thus, although it is possible that serotonin is a GHRH in children with GH neurosecretory dysfunc-
direct secretagogue for PRL, via transport from the tion results in a sustained elevation of PRL levels. In
hypothalamus by the portal vessels or through an rat pituitary cell cultures, GHRH causes PRL and GH
autocrine action within the pituitary, its role in this release but an increase only in gh mRNA and not prl
regard is still uncertain. It may mediate the nocturnal mRNA, indicating no stimulation of PRL synthesis.
surge of PRL and may well participate in the suckling- The similarity of GH and PRL responses to a variety of
induced rise in PRL via the ascending serotoninergic stimuli, such as exercise, stress, hypoglycemia, argi-
pathways from the dorsal raphe nucleus and mediated nine infusion, and sleep and the pathological condi-
by activation of VIP release. Serotonin reuptake inhibi- tions of renal failure and hepatic cirrhosis suggest but
tors are widely prescribed antidepressants but only do not prove that GHRH may serve as a physiologic
rarely have cases been reported of hyperprolactinemia PRF under some circumstances.
due to their use.
Posterior Pituitary, Oxytocin, and Vasopressin
Studies in animals have shown that oxytocin, in
Opioid Peptides doses found in the hypothalamic pituitary portal
A detailed description of the various opioid pep- vessels can stimulate PRL release when added to the
tides, their receptors, and their neuronal pathways is medium of pituitary cell cultures or incubations or

I. HYPOTHALAMIC PITUITARY FUNCTION

 NEUROENDOCRINE REGULATION 145
when given intravenously, but it lowers PRL levels cells and in PRL-secreting adenomas. Angiotensin II
when directly injected into the third ventricle. Studies incubated with rat pituitary cells stimulates release of
in which endogenous oxytocin was eliminated by pas- PRL, an effect blocked by AT1 but not AT2 antagonists.
sive immunization with oxytocin antisera or by oxytocin However, in humans, blockade of ACE with enalapril
antagonists show a reduction and a delay in the suck- results in no change in basal PRL levels, no change in
ling and cervical-stimulation-induced PRL surges in the TRH and metoclopramide 5 induced PRL rises and
some but not all studies [70]. Experimental data also only a minimal decrease in the PRL response to hypo-
support the possibility that the oxytocin-induced PRL glycemia. It is unlikely, therefore, that the endogenous
increase in these experimental paradigms is mediated renin angiotensin system of the hypothalamus and
by a decrease in DA [70]. Very limited studies in pituitary has significant physiologic effects on PRL
humans suggest that oxytocin administered intrave- regulation.
nously has no effect on basal PRL levels and causes
only a minimal increase in TRH-stimulated PRL levels. Other Neuroactive Peptides and Neurotransmitters
It is likely that oxytocin plays at most a minimal role Somatostatin receptors are expressed on human PRL
even in suckling-induced PRL secretion in humans [30]. as well as GH-secreting adenomas. Somatostatin inhibits
Vasopressin also has PRL-releasing properties when adenyl cyclase activity of rat anterior pituitary homoge-
injected intravenously into normal rats and sheep and nates and spontaneous and stimulated PRL release [71].
rats with pituitaries transplanted to the renal capsule Furthermore, administration of somatostatin antiserum
but not sheep with hypothalamic pituitary disconnec- to rats causes a rise in PRL levels, implying a physio-
tion. The neurophysin portions of the precursors to logic inhibitory action of somatostatin basally. In
oxytocin and vasopressin also stimulate PRL secretion humans, however, somatostatin administered exoge-
in rats. There are no studies to date of the effects of nously has no effect on TRH-induced PRL release.
vasopressin on PRL secretion in humans. A number of other peptides (neurotensin, substance
Whether other PRFs in the posterior pituitary exist P, cholecystokin, bombesin, calcitonin, endothelin,
in addition to oxytocin, vasopressin, and their galanin, gastrin, transforming growth factor β) and
respective neurophysins has been a matter of contro- bioamines such as glutamine have been found to have
versy, but isolation of such substances has not been varying effects on PRL levels in rats in different experi-
successful. Overall, it is felt that the posterior pitui- mental paradigms, but very limited studies in humans
tary may play only a minor role in the regulation of have shown no effect.
PRL secretion [30].
HISTAMINE
Gonadotrophin-Releasing Hormone This neurotransmitter plays an uncertain role in
GnRH was initially found to release PRL from rat PRL regulation. Histamine neuronal perikarya are
pituitary cells in vitro. Subsequently, GnRH has been present in the posterior hypothalamic region and
found to cause a release of PRL in anovulatory axons project to almost all of the nuclei of the hypo-
women. Postmenopausal women also have a PRL thalamus. Although some experiments show an effect
response to GnRH that is augmented with estrogen of histamine via hypothalamic mechanisms, it has no
supplementation. There is no PRL release in response effect on PRL release from pituitaries in vitro or in
to GnRH in normal, eugonadal males but such a stalk-sectioned rats. In humans, intravenous H2 but
release does occur with high doses of estrogen pre- not H1 blockers cause a rise in PRL levels but pro-
treatment (given to transsexual men). Analysis of PRL longed oral administration of H2 blockers does not
and LH secretory pulses suggests a high degree of con- result in sustained PRL elevation. The fact that the
cordance in women, thereby arguing for a physiologic administration of high doses of H2 blockers increases
role for GnRH in PRL secretion. A subset of human PRL levels in humans, that histamine cannot cross the
prolactinomas that also contain the glycoprotein α sub- blood brain barrier and that histamine has no effect
unit has been shown to bind GnRH specifically and on pituitaries in vitro suggests that histamine may
with high affinity and to release PRL in response to play a physiologic facilitatory role in PRL secretion
GnRH in vitro. within the median eminence.
The roles of other bioamines in the regulation of
Renin Angiotensin System PRL secretion are even less well established. Central
Angiotensin-converting enzyme (ACE) and angio- adrenergic α2-agonists such as clonidine usually have
tensin II receptors and activity have been identified in no effect on PRL secretion although both increases and
the rat pituitary and median eminence of the hypothal- decreases have been reported, the differences being
amus. In the human pituitary, renin, ACE, and angio- due to the doses used. α-Methyl dopa, another central
tensinogen have been detected in normal lactotroph adrenergic agonist, causes a sustained elevation of PRL

I. HYPOTHALAMIC PITUITARY FUNCTION

146 5. PROLACTIN

but this may be due to inhibition of the synthesis of found of suppression of TSH, LH, or FSH levels with
norepinephrine or DA centrally by inhibiting the short-term administration of human PRL which
enzyme l-aromatic acid decarboxylase, which is resulted in a 2 3-fold elevation of PRL levels.
responsible for conversion of dopa to DA and by act- However, these acute studies do not rule out the possi-
ing as a false neurotransmitter to decrease DA secre- bility that such feedback may occur with more pro-
tion or synthesis by a local feedback inhibitory action. longed states of hyperprolactinemia. Alternatively,
However, monoamine oxidase inhibitors increase PRL such feedback might occur via other mechanisms, such
levels. Such an elevation is unexpected, since these as a decrease in a PRF such as VIP.
drugs should increase levels of synaptic norepineph-
rine and DA.

ACETYLCHOLINE PROLACTIN ACTION

This neurotransmitter inhibits adenyl cyclase and
PRL has a great diversity of actions in many species
cAMP accumulation, lowers intracellular free calcium
of animals from fish and birds to mammals, including
levels, and decreases PRL release from pituitary cell
osmoregulation, growth and developmental effects,
cultures, acting through muscarinic and not nicotinic
metabolic effects, actions on ectodermal and integu-
receptors. Although atropine, a muscarinic receptor
mentary structures, and actions related to reproduction
antagonist blocks acetylcholine inhibition of PRL
[39,77]. However, in humans it has as its primary
in vitro, it had no effect on basal or TRH-induced PRL
physiologic action the preparation of the breast for lac-
release in humans and pirenzepine, another musca-
tation in the postpartum period. A number of effects
rinic receptor blocker actually caused a modest
of increased levels of PRL may be seen on many tis-
decrease of PRL levels in humans in vivo. The wide-
sues. Although the roles of physiologic levels of PRL
spread presence of acetylcholine as a neurotransmitter
in such tissues are quite speculative, considerable clar-
in the CNS and the possibility that pituitary tissue
ification of these roles has been elucidated from stud-
itself may synthesize acetylcholine, making interpreta-
ies in Prl22 [76] and Prlr2/2 [78] mice.
tion of studies testing this system difficult and the true
role of acetylcholine in the regulation of PRL secretion
is uncertain.
Prolactin Receptor
Prolactin Short-Loop Feedback The prolactin receptor (PRLR) belongs to the class I
Considerable evidence in rats suggests that PRL cytokine receptor superfamily, a family of single-pass
feedsback negatively on its own secretion (short-loop transmembrane proteins that transduce signals follow-
feedback or autofeedback) [59]. Most evidence sug- ing phosphorylation by cytoplasmic kinases [79]. The
gests that such feedback occurs via augmentation of human PRLR (hPRLR) gene is located on chromosome
hypothalamic TIDA turnover, including direct mea- 5p14-p13.2 and consists of eight or nine coding exons
surements of DA in portal vessels. Electrophysiological and two noncoding exons, which constitute the 5’
data have demonstrated rapid actions of PRL on the UTR. Six alternative forms of the first exon (hE1N1-
electrical activity of TIDA in mice [72,73] or in rats hE1N5) are expressed in a tissue-specific manner, and
[60,74]. Studies using Prl2/2 mice show that they have are spliced into the noncoding exon 2. Exons 3 10
markedly decreased DA in TIDA neurons, along with encode the full-length activating long form of the
hyperplasia of lactotrophs that do not synthesize PRL receptor. Multiple PRLR transcripts, resulting from
[75,76]. alternative splicing and transcriptional start sites, give
Direct evidence for such PRL short-loop feedback in rise to intermediate and short forms of the receptor
the human has not been demonstrated. In a number of (described below). Transcripts from exon 11 are only
reports, however, it has been suggested that altered present in the short forms of the receptor [80]. The
regulation of gonadotrophin and TSH secretion in entire hPRLR locus spans a region that exceeds 200 kb.
hyperprolactinemic patients may constitute indirect The hPRLR cDNA is 1869 nucleotides long and
evidence of PRL-induced augmented TIDA activity. In encodes a protein of 622 amino acids, 24 of which rep-
hyperprolactinemic patients, decreases in gonadotro- resent the signal peptide.
phin pulse amplitude and frequency are usually The PRLR is ubiquitously expressed. Regulation of
found, being attributed to altered gonadotrophin- PRLR protein levels occurs at both transcriptional and
releasing hormone secretion (see below). Such an alter- post-transcriptional levels. Transcription of hPRLR is
ation of GnRH secretion has been postulated to be controlled by multiple promoters, with each of the
due, in part, to PRL-induced DA increase. In a direct alternative noncoding exons 1 utilizing a separate pro-
test of this hypothesis, however, no evidence was moter. The preferentially utilized, generic promoter

I. HYPOTHALAMIC PITUITARY FUNCTION

 PROLACTIN ACTION 147
1/exon-1 (PIII/hE13) contains functional Sp1 and The functional significance of the various isoforms
C/EBP sites that bind transcription factors Sp1/Sp3 has not been determined conclusively, but some
and C/EBPβ, respectively. Estradiol, operating insights into their function have been gained through
through a nonclassical ERα signaling mechanism, the phenotypic analysis of genetic mouse models in
activates the hPRLR hPIII promoter in breast cancer which a short PRLR isoform is overexpressed on a
cells through ERα-mediated recruitment of Sp1 and long Prlr null background (Prlr2/2;rstg) [87]. These
C/EBPβ, and assembly of a coactivator complex con- mice display premature ovarian follicular development
sisting of p300, SRC-1, and pCAF [81]. PRLR levels followed by massive follicular cell death. On a molecu-
are negatively regulated at a posttranslational level lar level, PRL signaling through this particular short
by proteolytic degradation via receptor ubiquitina- PRLR isoform in ovarian tissue represses transcription
tion, facilitated by the SCF β-Trcp E3 ubiquitin ligase, of several genes, including Foxo3a and Galt, which are
and targeting to the lysosomal complex [82,83]. important for normal follicular development.
Alterations in PRLR degradation may contribute to Moreover, this short PRLR isoform does not activate
transformation of breast cells [84]. Jak/Stat, but instead utilizes a unique intracellular sig-
The PRLR contains an extracellular domain (ECD), naling pathway involving calmodulin-dependent pro-
required for ligand binding, a transmembrane domain, tein kinase (CamK) [88]. The phenotypic alterations
and an intracellular domain (ICD), required for signal demonstrated in Prlr2/2rstg ovaries are reversed by the
transduction (Fig. 5.7). The hPRLR exists as at least re-coexpression of the long PRLR, suggesting that
nine recognized isoforms, which have different signal- the two isoforms inhibit the activity of each other, and
ing properties. Mice and rats also express multiple the proper ratio of their expression is important for the
PRLR isoforms [85]. Within a species, the ECDs of normal physiological development of ovarian follicles.
most PRLR isoforms are identical, whereas the ICDs Whether improper ovarian PRLR isoform expression
are of variable length and composition. The long PRLR occurs in humans, and contributes to ovarian failure is
serves as the canonical sequence, and is the only iso- not known, but remains an important question. The
form that signals properly. The intermediate form short PRLR isoforms also appear to inhibit long PRLR
results from a frameshift, and leads to absence of a function through heterodimerization with long PRLR
portion of the ICD. The ΔS1 isoform lacks exons 4 and isoforms resulting in dominant negative activity, and
5, and has reduced affinity for hormone, but displays by enhancing PRLR degradation [89,90]. Alterations in
effective signal transduction. Short forms of the hPRLR the expression of PRLR isoforms and ratio of short to
known as S1a, S1b, and Δ4-S1b are derived from alter- long PRLR isoforms have been observed in breast
native splicing. These forms have similar binding affin- tumor tissue and cancer cell lines compared with the
ity for PRL as the long form, but cannot transduce normal breast and control mammary cells. Accordingly,
signal and exhibit dominant negative activity when abnormal signaling as a consequence of these changes
coexpressed with the long form. In addition to could theoretically contribute to breast tumor develop-
membrane-anchored receptor, a soluble, freely circulat- ment and/or progression.
ing form of the receptor, PRL receptor binding protein The PRLR has three defining domains, two within
(PRLRBP), is generated by proteolytic cleavage of the the ECD and one in the ICD. The ECD contains two
long hPRLR [86]. signature motifs: an aminoterminal region (S1) and a

FIGURE 5.7 Schematic illustration of eight of the

most common human PRLR isoforms. The PRLR gene
(not depicted) undergoes alternative splicing to yield
several transcripts, and subsequently proteins, of vari-
able length. In general, the extracellular and transmem-
brane domains are nearly identical, whereas the
intracellular domains vary in length and composition.
Motifs, such as the disulfide bonds, WSXWS, box 1, and
box 2 are highly conserved.

I. HYPOTHALAMIC PITUITARY FUNCTION

148 5. PROLACTIN

FIGURE 5.8 Schematic illustration demonstrat-

ing two models for PRLR activation. (A) In the
ligand-dependent dimerization model, one mole-
cule of PRL binds to a PRLR monomer at binding
site 1, which leads to the recruitment of the second
PRLR monomer to bind PRL at site 2.
Dimerization of the two PRLRs leads to phosphor-
ylation of Janus kinase and signal transducer and
activator of transcription 5a (Stat5). (B) In the
ligand-independent dimerization model, the
PRLRs exist as preformed dimers at the cell mem-
brane in the absence of ligand. PRL binding to the
dimeric PRLR receptor induces conformational
changes that subsequently activate the receptor.

membrane-proximal region (S2). Two pairs of disulfide bioluminescence resonance energy transfer (BRET1)
bonds in S1 are highly conserved and critical for ter- analyses support an alternative model in which the
tiary receptor folding of the ligand binding domain. A PRLR dimerizes independently of ligand binding [92].
highly conserved WSXWS motif constitutes the S2 It is now recognized that, like GH receptor (GHR)
motif and may be involved in receptor trafficking. The dimers [93], human PRLR dimers exist constitutively
PRLR ECD, as resolved by X-ray crystallography, con- on the cell surface [89,92]. These dimers are linked by
tains two subdomains, which are related to the type III transmembrane domains. PRLR homodimers cannot
repeats of fibronectin. Each domain is composed of drive signal transmission in the absence of the PRL
seven β-strands folded into two antiparallel β-sheets ligand. Heterodimerization of long and short PRLR
[79]. The third defining motif, which lies in the ICD of isoforms produces inactive complexes. The mechanism
the PRLR, is referred to as “box 1” and consists of an of GHR activation was elucidated by Brooks et al. [94].
eight-amino-acid proline-rich hydrophobic sequence It appears that the same mechanism could apply to all
that directly interacts with intracellular tyrosine class I cytokine receptors, which include PRLR. PRLR,
kinases. Mutations in residues of box 1 completely dis- like GHR and other homologous cytokine receptors,
rupt Jak/Stat PRLR signaling. does not possess intrinsic tyrosine kinase activity but
Increased PRL concentrations promote binding to transmits its signal through associated cytoplasmic
monomeric or dimeric forms of the PRLR and induce proteins such as Janus protein kinase2 (Jak2) [95] The
structural changes in the ECD [79]. The active PRL/ initial event is the binding of a single ligand molecule
PRLR complex has a stoichiometry of one hormone to predimerized receptor monomers on the membrane
bound to two receptors (Fig. 5.8). As such, two ECDs surface, via two extracellular interaction sites. This
of the PRLR interact with two asymmetric ligand bind- triggers a change in the conformation of the receptor
ing sites located opposite to each other within the dimer that enables signal transduction to occur. For
receptor core. The formation of 1:2 complexes is an GHR, Brooks et al. propose a fascinating multistep
essential first step for subsequent signal transduction. scissor-like mechanical model in which movements of
Two different mechanisms of PRL binding to PRLR the receptor dimer ultimately lead to receptor activa-
have been proposed. The conventional view holds tion [94]. These recent findings show that, in the
that binding of PRL to a monomeric first receptor absence of hormone, helices of the transmembrane
induces the sequential recruitment and dimerization domains of the predimerized monomers lie parallel to
of a second receptor [91]. This leads to activating each another. Binding of hormone converts the helices
changes in the ICD and initiates signal transduction. into a crossover state that induces their separation at
Recent data garnered from coimmunoprecipitation and the lower transmembrane boundary, hence inducing

I. HYPOTHALAMIC PITUITARY FUNCTION

 PROLACTIN ACTION 149
cytoplasmic Box 1 separation. This helix state transi- interacts with the SH2 domain of a Stat protein.
tion requires rotation of the receptors, but the key out- Following phosphorylation by activated Jak2, Stat pro-
come is separation of the Box 1 sequences. Indeed, in teins hetero- or homodimerize, translocate to the
the basal state (in the absence of ligand), the Box 1 nucleus, and transactivate γ-interferon activation
domains are attached to the N-terminal domain of sequence (GAS) consensus elements on target genes.
Jak2, which is a pseudo-Jak2 kinase sequence whose The tyrosine phosphatase short heterodimer partner
function is to inhibit the inherent kinase activity of (Shp)-2 promotes PRL stimulated assembly of the Jak-
Jak2. Distancing of the two Box 1 domains allows sepa- PRLR complex, and is a required component for Stat
ration of the pseudokinase inhibitory domain of one 5a activation during pregnancy and lactation. Jak2-Stat
Jak2 molecule, which blocks the kinase domain of the signaling is attenuated through an intracellular
other Jak2, and vice versa, allowing Jak2 activation. negative-feedback system involving the action of sev-
This may be a general mechanism underlying class I eral negative regulators, including (1) suppressor of
cytokine receptor action [96]. cytokine signaling (SOCS) proteins, which inhibit Jak2
kinases; (2) cytokine-inducible SH2-containing (CIS)
proteins, which compete with Stat proteins for docking
sites on the PRLR; and (3) protein tyrosine phospha-
PRLR Signal Transduction tases, PTP1B1 and TC-PTP, which dephosphorylate
Ligand binding to the PRLR results in rapid activa- PRL-activated Stats [77].
tion of Jak2, which is constitutively associated with Although the Jak/Stat pathway mediates most
box 1 of the PRLR. Jak2 activation is the most proximal physiological actions of PRL in mammary develop-
event in the intracellular events that occur after ligand ment and lactation, binding of PRL to its receptor also
binding (Fig. 5.9). Jak2 phosphorylates tyrosine resi- activates several additional intracellular cascades to
dues on the PRLR ICD and autophosphorylates resi- promote specific cellular responses. Effectors of these
dues within itself. Receptor-associated Jak2 also cascades engage in signaling crosstalk, and likely
phosphorylates cytoplasmic signal transducer and acti- operate as a complex network rather than hierar-
vator of transcription proteins. Four Stat family mem- chically depending upon cell type and context.
bers (Stat 1, Stat 3, Stat 5a, Stat 5b) serve as the central Phosphotyrosine residues of the PRLR can serve as
transducer molecules of the signal transduction path- docking sites for adapter proteins (Shc/Grb2/SOS)
ways initiated by PRLR activation, but Stat 5a and, to a connecting the receptor to the Ras/Raf/MAPK cas-
lesser extent, Stat 5b, are especially important for cade [79]. This pathway appears to mediate at least
mammary gland development and lactogenesis [97]. A some of PRL mitogenic effects. Activation of PRLR
phosphorylated tyrosine residue of the activated PRLR also facilitates docking of Src family kinases, which

FIGURE 5.9 Schematic illustration depicting

intracellular signal transduction pathways
downstream of the PRLR. Following activation
of the PRLR, Janus kinase 2 tyrosine kinase
becomes activated through auto- or transpho-
sphorylation. This triggers association with sig-
nal transducer and activator of transcription
protein 5a (Stat5) followed by Stat5 phosphoryla-
tion, dimerization, and nuclear translocation.
Association with adaptor proteins such as Shc
leads to signaling through the mitogen-activated
protein kinase (MAPK) pathway to stimulate
mitogenesis. Association with Src family kinases
triggers phosphoinositide 3-kinase (PI3K) and
Akt (protein kinase B) signaling to affect cell
proliferation and survival. Members of the sup-
pressors of cytokine signaling (SOCS) and
cytokine-inducible inhibitor of signaling (CIS)
proteins decrease the actions of Jak.

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150 5. PROLACTIN

couple to multiple signaling effectors, including phos- coupled with high levels of PRL and hPL promote pro-
phatidylinositol (PI) 3’-kinase/AKT and Erk 1/2, link- liferation of the lobuloalveolar epithelium. During and
ing PRLR activation to cell survival and proliferation after parturition, progesterone, estradiol, and hPL
[98,99]. The requirement of Src as an essential media- levels decline, whereas PRL levels rise. These hor-
tor of PRLR signaling in normal mammary tissue is monal changes, together with the effects of local
underscored by findings in female Src2/2 mice, which growth factors such as RANK-ligand and insulin-like
demonstrate lactation failure and precocious mam- growth factor 2 (IGF-2) induce the lobuloalveolar epi-
mary gland involution [100]. As they become avail- thelium to convert into secretory acini [106,107].
able, detailed characterization of mouse genetic Epithelial PRLRs are required for this process, as
models lacking components of PRLR signaling cas- Prlr2/2 mammary transplants fail to develop lobuloal-
cades should provide information on their physiologic veoli, and cannot produce milk proteins during preg-
roles and relevance. nancy [105]. PRL also acts in concert with insulin and
hydrocortisone to induce differentiation of pluripotent
mammary epithelial cells that produce progeny that
Female Reproductive Tissues subsequently grow into alveolar structures [108].
Members of the PRLR signaling pathway whose func-
PRL Effects on Breast tions are essential for mediating PRL effects on alveo-
PRL plays a dominant role in several aspects of the lar morphogenesis include the transcription factors
breast, including growth and development of the Stat5a, Id2, Socs-2, Gata-3, and Elf5 [101,109 112].
mammary gland (mammogenesis), synthesis of milk Both lactogenesis and galactopoiesis require pitui-
(lactogenesis), and maintenance of milk secretion tary PRL, since hypophysectomy during pregnancy
(galactopoiesis). Development of the mammary gland prevents or stops, respectively, lactation. Prl22 and
is a tightly coordinated process involving several hor- Prlr2/2 mice cannot produce milk as a result of defec-
mones, in addition to PRL, that occurs in defined tive mammogenesis. PRL is also essential to maintain
stages that include embryonic, prepubertal, pubertal, sustained lactation. During lactation, PRL regulates the
and pregnancy [101]. During puberty, the epithelial synthesis of milk proteins, including β-casein, lacto-
cell compartment, which consists of a branched ductal globulin, lactalbumin, and whey acidic protein [113].
system, expands while, during pregnancy, the lobu- PRL also regulates the synthesis of enzymes involved
loalveolar compartment differentiates and develops. in lipid metabolism, including lactose synthetase, lipo-
Mammary gland development during the embryonic protein lipase, and fatty acid synthase [113].
and prepubertal stages occurs independently of the
actions of PRL, and PRL plays only a minor role in the Galactorrhea
pubertal stage. However PRL, together with ovarian Clinically, nonpuerperal galactorrhea has been
steroids, local growth factors, and cytokines, is essen- regarded as being a sign of possible hyperprolactine-
tial for the morphologic changes that occur in the mia. The presence of even minute amounts of milk
mammary gland during pregnancy and lactation [102]. expressible from one or both breasts indicates a diag-
The combination of experimental mouse genetics nosis of galactorrhea. Its persistence for more than 1
and transcriptomic profiling has been useful for con- year after normal delivery and cessation of breastfeed-
firming the roles of genes and identifying PRL- ing or its occurrence in the absence of pregnancy gen-
dependent signaling pathways that control mammary erally is regarded as a definition of inappropriate
development. At the onset of puberty, the ovarian ster- lactation. If the material expressible from the nipple
oids estradiol and progesterone and pituitary GH initi- looks like milk, it probably is milk; if there is any
ate and drive ductal morphogenesis [103]. Mammary uncertainty, examination of the breast secretion by
gland development in Prl2/2 and Prlr2/2 mice is staining of fat globules with Sudan IV is diagnostic.
arrested at the stage of ductal elongation and these The incidence of galactorrhea has been variously
mice completely lack lobuloalveolar units, indicating reported in normal women as ranging from 1 to 45%
that it is at this stage and beyond that PRL exerts of subjects tested. This variability is probably due to
developmental influences [76,78]. PRL indirectly influ- differences in the techniques used to express milk
ences the process of ductal side branching during from the breast and the way in which nonmilky secre-
puberty by promoting ovarian progesterone synthesis tions are classified. The volume of milk expressed does
[104,105]. In contrast to these relatively minor effects, not correlate with PRL levels. However, in individuals
PRL plays a major role in the morphologic and func- with hyperprolactinemia, lowering the blood PRL level
tional changes that occur in the breast during preg- to normal almost always will lead to a marked
nancy. During pregnancy, high concentrations of decrease in or abolition of lactation. Inappropriate lac-
estradiol and progesterone, produced by the placenta, tation may be an important clue to the presence of

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 PROLACTIN ACTION 151
pituitary hypothalamic disease, especially if accompa- mammary epithelia develop mammary carcinomas at
nied by amenorrhea. Clinical experience suggests that a long latency, whereas transient overexpression of
galactorrhea may be present in about 5 10% of nor- PRL in differentiated, lactating mammary tissue leads
mally menstruating women and basal PRL levels are to the development of benign mammary adenomas. By
normal in more than 90% of these women. contrast, genetic ablation of the PRLR delays, but does
not prevent or reduce the incidence of, the develop-
PRL and Breast Cancer ment of SV40 large T-antigen-induced breast carcino-
Apart from hyperprolactinemia, which is the most mas [120]. Taken together, findings from these murine
widely characterized disorder in humans that is models support a modulatory role for PRLR function
related to PRL signaling, the role of PRL and its recep- in mammary neoplasia. In humans, a substantial per-
tor in the initiation and/or progression of human centage of primary human breast carcinomas express
breast cancer remains an active area of debate. A wide PRL and PRLR, but immunostaining for the receptor
range of studies has attempted to address the possible does not correlate well with clinicopathological stage
contribution of PRL to breast cancer through epidemi- or disease-free survival. A heterozygous nonsynon-
ological analyses, cellular and molecular studies, and ymous gain-of-function PRLR variant in the PRLR
transgenic mouse models. Overall, epidemiological (I146L substitution in the ECD) has been identified in a
studies examining the relationship between serum small percentage (B5.6%) of human benign breast
PRL levels and the risk of breast cancer in women tumors [121]. However, this variant has also been
have shown conflicting results. The Nurses’ Health reported as a common polymorphism that occurs in
Study, the largest prospective cohort study reported, B2.4% of European and US populations, which argues
found a significantly (34%) increased risk of breast against a role of this variant in this disorder.
cancer when comparing top to bottom quartiles of In light of the above-listed observations that have
serum PRL in postmenopausal women and a nonsig- suggested a role for PRL or PRLR in promoting mam-
nificant 30% increase in risk comparing top to bottom mary tumor growth, efforts have been initiated to
PRL quartiles in premenopausal women [114] investigate the potential usefulness of pharmacologic
(reviewed in [39]). These findings are similar to results inhibition of the PRLR for the treatment of breast neo-
of previously reported smaller studies that found non- plasms or other conditions associated with hyperpro-
significant increases in breast cancer risk [115]. The lactinemia [122]. Preclinical studies using competitive
clinical relevance of these epidemiological studies is PRLR antagonists have demonstrated proof-of-
limited by observations that serum levels of PRL in the principal PRLR antagonism and inhibition of cell pro-
study participants remained within the normal range liferation, but have been associated with unfavorable
and were not associated with clinical symptoms. In pharmacokinetics and the potential adverse effects of
women with obvious hyperprolactinemia, two studies PRLR inhibition, as observed in Prlr2/2 mice, has not
failed to show an association of this state with the risk been ascertained.
of breast cancer [116,117]. The number of studies
investigating associations between genetic variability PRL Effects on Gonadotrophin Secretion
in the PRL or PRLR genes and the risk of breast cancer The effects of normal circulating PRL levels on
are limited, but favor lack of association. Analysis of gonadotrophin secretion are not known. Prl2/2 and
high-density single-nucleotide polymorphism (SNP) Prlr2/2 female mice are sterile, and both types have
data from the Multiethnic Cohort Study which disordered estrous cycles, but whether the sterility in
included 1600 cases of breast cancer and 1900 controls, these cases is due to cell-autonomous effects in the
did not find a significant association between PRL and gonads, altered gonadotrophin secretion, or to a com-
PRLR haplotypes or individual SNPs in relation to bination of these is not clear from these global knock-
breast cancer risk [118]. out mice (see PRL effects on the ovary and PRL effects
Recent attention has turned to the question as to on the testes, below) [76,78]. Prl2/2 male mice have
whether local production of PRL within breast tissue reduced plasma LH levels; gonadotrophin levels in
plays an autocrine or paracrine role in the etiology or Prlr2/2 male mice are not altered. In normal women
progression of breast cancer [30,119]. Accumulating treated with short-term bromocriptine to lower PRL
data from in vitro and animal studies have suggested levels to about 5 ng/mL, there is no change in the pul-
that PRL and/or actions of the PRLR may be involved satile secretion of LH and FSH but estradiol levels are
in mammary tumorigenesis by promoting cell prolifer- higher during the last 3 days of the follicular cycle,
ation and survival, increasing cell motility, supporting and progesterone levels lower during the luteal phase.
tumor vascularization, and/or contributing to cell Hyperprolactinemia, on the other hand, has a num-
transformation [84]. Transgenic mice that universally ber of effects on various steps in the reproductive axis
overexpress PRL systemically or locally within the (Fig. 5.10). Hyperprolactinemia has been found in most

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152 5. PROLACTIN

FIGURE 5.10 Diagram demonstrating how hyperprolactinemia

induces anovulatory infertility. As PRLRs are not expressed on
GnRH neurons, hyperprolactinemia induces infertility via its
actions on other intermediate cells. Increased serum PRL levels
result in decreased kisspeptin expression in hypothalamic Kiss1
neurons (arcuate [ARC] and anteroventral periventricular [AVPV]
nuclei), mediated by PRLR expressed on these cells. Suppression
of kisspeptin reduces hypothalamic GnRH secretion. This leads to
reduced LH and FSH secretion and loss of ovarian stimulation,
which result in infertility. PRL may also have direct effects on
other GnRH afferent neurons. Moreover, the intermediate partici-
pation of other non-neural factors acting on Kiss1 and GnRH neu-
rons secretions cannot be excluded.

studies to suppress LH pulsatile secretion by decreas- inhibition of kisspeptin-1 in the arcuate nucleus and
ing pulse amplitude and frequency. At the menopausal anteroventral periventricular nucleus.
transition in humans, hyperprolactinemia can prevent
the expected rise in gonadotrophins; normalization of PRL Effects on the Ovary and Fertility
PRL levels with bromocriptine results in a rise in The effects of PRL on ovarian function and fertility
gonadotrophin levels and hot flashes. are complex and to some extent species-specific. PRL
Hyperprolactinemia is a well established cause of is an essential luteotrophic hormone in rodents, but
hypogonadotrophic hypogonadism and anovulatory not in humans. Instead, in humans, pituitary LH sup-
infertility [123], but the mechanism by which PRL inhi- ports luteal development and steroidogenesis during
bits hypothalamic secretion of GnRH-I was unclear. the menstrual cycle, and the embryonic trophoblast
Recently, it was demonstrated that this inhibition sustains the corpus luteum during pregnancy. The
involves metastasis suppressor kisspeptin-1 neurons action of PRL on the ovaries has been fairly well-
that express PRLR. Mice rendered hyperprolactinemic characterized in rats, but the role of PRL in normal
do not ovulate, have low circulating levels of LH and ovarian physiology is not well-defined in humans. A
FSH, and exhibit reduced hypothalamic expression of comparison of PRL profiles across the estrous and
the Kiss1 gene, which encodes kisspeptin-1. menstrual cycles in rodents and humans, respectively,
Intraperitoneal injections of kisspeptin-1 restored both reveals marked differences, supporting the notion that
hypothalamic GnRH-I and gonadotrophin secretion, as the function of PRL during these cycles differs
well as ovarian cyclicity suggesting that kisspeptin-1 between the two species. In humans, serum PRL levels
neurons have a major role in hyperprolactinemic ano- remain relatively stable throughout the menstrual
vulation [124]. These experiments suggest PRL- cycle, except for a slight increase during the luteal
mediated inhibition of GnRH-I occurs, in part, through phase, the functional significance of which is not
decreased secretion of kisspeptin-1. PRL might also known. By contrast, in rats, PRL levels rise in a tripha-
have direct effects on other GnRH-I afferent neurons, sic manner showing a sharp rise just prior to ovula-
and the possibility that other non-neural factors could tion, followed by a plateau and an extended
act on kisspeptin-1 and GnRH-I neuronal secretion termination phase [30]. The PRLR is expressed in
cannot be excluded. Likewise, during lactation, a state human granulosa cells and the human ovary produces
of physiological hyperprolactinemia, a selective loss of its own PRL, but the specific roles of circulating versus
kisspeptin-1 input to GnRH-I neurons has been autocrine-derived PRL on ovarian function are
observed in mice [125] and PRL contributes to unknown. PRL is found in human follicular fluid

I. HYPOTHALAMIC PITUITARY FUNCTION

 PROLACTIN ACTION 153
where it has been shown to stimulate ovarian endothe- antagonizing the stimulatory effects of FSH on aroma-
lial cell proliferation [126]. tase activity and (2) directly inhibiting aromatase syn-
Although the role(s) of normal physiologic levels of thesis itself. In fact, PRL is required at low doses (,20
PRL on ovarian function and female fertility are not ng/mL) for progesterone production by granulosa cell
fully known in humans, it is well established that the cultures, but at higher concentrations (i.e., those that
actions of PRL and the activity of its receptor are correlate with hyperprolacinemia in women), PRL
essential for fertility in female mice. Both Prl2/2 and inhibits progesterone production. These in vitro find-
Prlr2/2 female mice are completely infertile. Female ings are in line with in vivo studies of luteal function
Prl2/2 mice have irregular estrous cycles. Whether in women where treatment with bromocriptine to
this is due to an ovarian or hypothalamic defect is not lower normal PRL levels to hypoprolactinemic levels
known for certain, but obvious histological defects in resulted in lowered progesterone levels and shorter
the ovaries are not observed. Prlr2/2 female mice, on luteal phases.
the other hand, display multiple reproductive abnor- As noted above, during pregnancy, human maternal
malities, including reduced rates of mating, fertiliza- PRL secretion rises gradually beginning at 6 8 weeks
tion, and ovulation, as compared to wild-type mice. gestation until term. In the human fetal circulation,
Prlr-deficient ovaries contain fewer primary follicles PRL rises slightly starting at around 10 weeks, pla-
and those eggs that do become fertilized develop teaus, and then rises again very sharply at 30 weeks
poorly as oocyte development is arrested almost until term. The decidua produces and secretes high
immediately after fertilization. In these mice, the amounts of PRL into the amniotic fluid beginning at 12
corpus luteum regresses, and is unable to support weeks, peaking at around 20 weeks, and then gradu-
implantation and placental development [127]. ally declining until term. The exact function of decid-
Administration of progesterone to Prl2/2 and Prlr2/2 ual or amniotic fluid PRL is not known, although
females rescues implantation and early embryonic several functions of decidual PRL have been postu-
development. Thus, regulation of sustained progester- lated. Putative functions of decidual PRL, such as facil-
one production, permitting the proper expression of itation of trophoblast growth, inhibition of myometrial
progesterone-dependent genes, is the essential function contractility, and regulation of angiogenesis, have been
of PRL that is required for normal implantation in suggested but limited data are available to support
Prl2/2 and Prlr2/2 mice in early pregnancy. The cause these theories [129]. Human PL shows a similar pat-
of late embryonic lethality is not known for certain, tern of rise to that of PRL, only on a larger scale, such
but may be a result of the absence of local decidual that by 30 weeks, PL levels exceed PRL levels by
PRL production, leading to derepression of two genes, 10-fold.
IL-6 and 20α-hydroxysteroid dehydrogenase (20α-
Hsd), whose expression is detrimental for the normal Clinical Effects of Hyperprolactinemia on
progress of pregnancy. The phenotypic differences Menstrual Function
between the Prl2/2 and Prlr2/2 mouse models could Elevated serum PRL levels (hyperprolactinemia) in
reflect compensatory action by numerous murine PRL- women cause oligomenorrhea or amenorrhea. The
like proteins that are capable of activating the PRLR, amenorrhea caused by hyperprolactinemia is typically
whereas Prlr2/2 mice cannot respond to any of the secondary, but primary amenorrhea can occur if the
PRL family members. disorder begins before the usual age of puberty. In
The major mechanism by which PRL performs its patients with primary amenorrhea due to hyperprolac-
luteotrophic action in rodents is through stimulation tinemia, estrogen deficiency and failure to develop
of progesterone production by luteal cells [128]. normal secondary sexual characteristics may be the
Specifically, PRL downregulates the expression of presenting problem. Galactorrhea is variable in this
20α-Hsd, which prevents the catabolism of progester- setting because the breast may not have been exposed
one to an inactive metabolite. This serves to increase to appropriate priming with estrogen and progester-
progesterone secretion from the corpus luteum. one. Patients with primary amenorrhea tend to have
Paradoxically, in some experimental settings, PRL macroadenomas more commonly than those with sec-
can also induce luteolysis. ondary amenorrhea, for uncertain reasons. When
In human granulosa cells, PRL stimulates expres- amenorrhea or oligomenorrhea is associated with
sion of type II 3β-hydroxysteroid dehydrogenase, the galactorrhea, it usually is a manifestation of
enzyme responsible for catalyzing the final step in pro- hyperprolactinemia.
gesterone biosynthesis, and increases IGF-2 secretion. Hyperprolactinemia is found in many women with
Perfusion studies of human ovaries in vitro show that a short luteal phase. It is likely that a short luteal phase
PRL directly suppresses progesterone and estrogen is the first evidence of interference in the normal cycle
secretion. PRL inhibits estrogen formation by (1) by hyperprolactinemia. Infertility also may be a

I. HYPOTHALAMIC PITUITARY FUNCTION

154 5. PROLACTIN

presenting symptom of patients with hyperprolactine- receptor expression, and potentiation of LH-induced
mia and is invariable when gonadotrophin levels are steroidogenesis. In humans, PRLRs are expressed in
suppressed with anovulation. In three series of women germ cells undergoing spermatogenesis in seminifer-
(combined number of 367 cases) studied for infertility, ous tubules, and in Leydig cells, vas deferens, epididy-
one-third were found to have hyperprolactinemia. mis, prostate, and seminal vesicles. Human semen
Most of these women presented with amenorrhea and contains significant quantities of PRL. The functional
galactorrhea as well, but hyperprolactinemia without significance of PRL or PRLR expression at many of
other symptoms was found in five of the 22 hyperpro- these sites is still unclear. However, the presence of
lactinemic women in one series of 113 cases of infertil- PRLR in differentiating germ cells in the testes is sup-
ity. That PRL excess may be important in this type of portive of data that PRL acts as a prosurvival factor for
patient is suggested by the finding that treatment of human spermatozoa by preserving motility, suppres-
similar patients with dopamine agonists restored fertil- sing sperm capacitation, and enhancing vitality by
ity. In some infertile women, transient hyperprolacti- inhibiting entry into the cell death pathway [136].
nemia lasting for 1 2 days during the cycle can be Moreover, reduction of normal PRL levels by adminis-
documented; this subset usually responds to bromo- tration of dopamine agonists in men results in sup-
criptine with increased progesterone during the luteal pression of basal and hCG-stimulated testosterone
phase and improved fertility. levels, implying a physiologic role for PRL in testoster-
Reduced libido and orgasmic dysfunction are found one production in humans.
in most hyperprolactinemic amenorrheic women when A substantial body of data from in vitro and in vivo
such complaints are specifically elicited. Reduction of studies supports mitogenic or prosurvival roles for
PRL levels to normal restores normal libido and sexual PRL in the prostate. PRL and PRL isoforms are
function in most of these women. expressed and functional in normal human prostate
PRL levels have been reported as elevated in epithelia and malignant human prostate tissue [137].
13 50% of women with polycystic ovary syndrome Some studies in human prostate cancer cell lines have
(PCOS) [130]. The relationship between hyperprolacti- shown that autocrine-derived PRL promotes prostate
nemia and PCOS is debated. Recent studies in PCOS cancer cell growth [138], whereas others show that
patients with increased PRL levels have implicated exogenous PRL inhibits apoptosis. In rodents, elevated
other causes of hyperprolactinemia, because hyperpro- levels of PRL are associated with increased prostate
lactinemia is not a clinical manifestation of PCOS growth. Ubiquitous transgenic overexpression of PRL
[131,132]. The presence of stress-related hyperprolacti- in mice leads to prostatic hyperplasia with elevated
nemia and macroprolactinemia may interfere with the serum testosterone levels [139]. Prostate-specific trans-
diagnosis of PCOS and leads to unnecessary and genic overexpression of PRL leads to stromal hyper-
expensive diagnostic and therapeutic approaches [133]. plasia, ductal dilatation, focal epithelial dysplasia, but
without changes in serum androgen levels, indicating
that the abnormal prostate findings are not consequen-
tial to hyperandrogenemia, and that autocrine-derived
Male Reproductive Tissues PRL is at least a contributing factor to these effects. By
While PRL clearly plays an essential reproductive contrast, the prostate glands of Prl2/2- mice are B30%
role in females, the role of PRL in male reproductive smaller than those of wild-type mice, and, as found in
function is less defined. A conclusive determination murine models in the breast, PRLR deficiency reduces
regarding the involvement of PRL in male fertility has the incidence of SV40 T-antigen-induced prostate carci-
not emerged from Prl2/2 and Prlr2/2 mouse models. noma [140]. Thus, these studies, which support pro-
The deletion of the PRLR does not alter fertility para- proliferative and/or antiapoptotic properties for PRL
meters, sperm reserves, plasma gonadotrophin levels, in the prostate, suggest potential (patho)physiological
testosterone levels, or weight or histology of the testes roles for PRL in human prostate development and/or
or epididymides [134]. Deficiency of PRL itself in disease, possibly elaborated at an autocrine level.
Prl2/2 mice is associated with reduced plasma LH However, data on the frequency of prostate hyperpla-
levels, but not with effects on male fertility parameters sia or carcinoma in humans with sustained systemic
or basal plasma testosterone levels [135]. hyperprolactinemia are lacking.
In other rodents and humans, indirect evidence sup-
ports the view that PRL plays a subtle role in testes Clinical Effects of PRL in Males
and/or germ cell function. PRL promotes Leydig cell Chronic hyperprolactinemia in males results in impo-
proliferation and differentiation in prepubertal tence and decreased libido in over 90% of cases [124].
hypophysectomized rats and is involved in the mainte- Other findings of hypogonadism, such as decreased beard
nance of Leydig cell morphology, upregulation of LH growth and strength are less commonly encountered.

I. HYPOTHALAMIC PITUITARY FUNCTION

 PROLACTIN ACTION 155
Galactorrhea in men is reported in 10 20% of cases of may constitute a molecular link between PRL signaling
hyperprolactinemia, and is virtually pathognomonic of a and cell ontogenesis [143]. Upregulation of the func-
prolactinoma. The frequency of hyperprolactinemia tional β 2 cell mass is required to match the physiolog-
among men with complaints of impotence or infertility as ical demands of mother and fetus during pregnancy.
assessed by surveys ranges between 2 and 25% among This increase is dependent on PLs and PRLR that play
various series. However, only 1 5% of men with infertil- a particularly central role in the adaptation of islets to
ity have been found to be hyperprolactinemic. pregnancy, as illustrated by studies in heterozygous
Hyperprolactinemia in men is associated with Prlr1/2 female mice. Pregnant Prlr1/2 female mice
decreased pulsatile secretion of LH and FSH (as noted exhibit impaired glucose clearance, reduced glucose-
above), and low or low-normal testosterone levels. The stimulated insulin release, and lower insulin levels as
testosterone response to stimulation with hCG has a result of impaired islet expansion [144]. In support of
been reported to be both decreased and normal; in the critical role of PRLR for islet expansion during ges-
those with decreased responses there is improvement tation, it has been shown that Stat 5, PI3 kinase,
in the response when PRL levels are lowered with bro- MAPK, and pathways involving the endocrine tumor
mocriptine. If there is sufficient normal pituitary tis- suppressor menin all collaborate to mediate the prolif-
sue, reduction of elevated PRL levels to normal erative effects of PRL and PL on pancreatic islets dur-
usually results in a return of normal testosterone levels ing pregnancy [145]. Furthermore, a strong lactogen-
[141]. Although some studies have suggested that dependent upregulation of serotonin biosynthesis
drug-induced hyperprolactinemia partially inhibits the occurs in a subpopulation of mouse islet β-cells during
enzyme 5-αreductase, resulting in reduced dihydrotes- pregnancy. Since newly formed serotonin is rapidly
tosterone (DHT) levels, hyperprolactinemia in men released, this lactogen-induced β-cell function may
with prolactinomas is not associated with this effect. serve local or endocrine tasks, the nature of which
Hyperprolactinemia has an effect on impotence that is remains to be identified [146]. Moreover, male mouse
independent of testosterone levels: Testosterone ther- islets, when exposed to the environmental conditions
apy of hyperprolactinemic men does not always cor- of pregnancy, undergo similar changes in gene expres-
rect the impotence until PRL levels are brought down sion as do female transplanted islets. These results cor-
to normal. Whether this is due to a decrease in DHT roborate the finding that the key environmental factor
levels has not been verified directly. Elevated PRL driving the phenotypic plasticity of β-cells is a rise in
levels have adverse effects on male germ cell and testes circulating PL that activates PRLR on β-cells. This con-
function. Sperm counts and motility are decreased with clusion is supported by experiments of cultured and
an increase in abnormal form [142]. Histology of the transplanted islets and results obtained from Prlr2/2
testes reveals abnormal seminiferous tubule walls and mice [147].
altered Sertoli cell ultrastructure. Hyperprolactinemia Although studies in humans are limited, in vitro
may have a sustained effect on male reproductive func- experiments confirm that PRL increases β-cell number
tion, as the semen analysis does not always return to and stimulates insulin secretion in cultured human
normal despite therapy that successfully normalizes islets. A protective effect of lactogens has been demon-
testosterone and PRL levels [142]. strated on human β-cell apoptosis. The improvement
of cell survival may involve, at least in part, inhibition
of cell death pathways controlled by the BCL2 gene
family members. These findings are relevant for
Carbohydrate Metabolism and Adiposity improvement of the islet isolation procedure and for
PRL functions as a metabolic regulator in two chief clinical islet transplantation [148].
areas: (1) pancreatic β-cell development and function; With regard to appetite regulation and adiposity,
(2) appetite regulation and adiposity. The phenotypic in vivo studies in rodents and humans support a mod-
assessment of Prlr2/2 mice indicates that signaling est orexigenic effect of PRL. In rats, higher PRL levels
through the PRLR, which serves as a common receptor are associated with greater food intake and body
for both PL and PRL, is important to fully attain nor- weight, while suppression of PRL levels leads to the
mal β-cell mass, insulin content, and insulin secretory opposite effects [149]. Prlr2/2 mice exhibit a subtle
capacity The expansion of β-cell mass during both reduction in parametrial and subcutaneous adipose tis-
embryogenesis and the postnatal period is impaired in sue mass as compared to wild-type littermates,
this mouse model [143]. Prlr2/2 newborns display a although no differences are observed in overall body
30% reduction of β-cell mass, consistent with reduced weight [150]. Prlr2/2 mice are highly resistant to high
proliferation index at the 18.5th day of embryonic fat diet-induced obesity, owing to the emergence of a
development. Reduced pancreatic Igf-II expression in brown adipose-like phenotype in peculiar white fat
both rat and mouse models suggests that this factor depots. This finding is associated with a concomitant

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156 5. PROLACTIN

increase of Prdm16, Pgc1α, AdRβ3, and Foxc2 that con- activity relies on a crosstalk between Jak2 and the PKA
stitutes a molecular switching mechanism. This con- signaling pathway through control of the stability of
verges toward an activation of thermogenic brown the activated CREB protein [157].
capacity with the final increase of UCP1 responsible Plasma dehydroepiandrosterone (DHEA) and DHEA
for heat dissipation and resistance to high-calorie sulfate (DHEAS) levels are mildly elevated in about
weight gain. Then, PRL signaling represents an addi- 50% of women with hyperprolactinemia in some series.
tional determinant of energy homeostasis during phys- In most of these studies, however, the investigators did
iological and pathophysiological conditions [151]. not correlate androgen levels with the presence of hir-
Prl2/2 mice exhibit normal body weight and adiposity sutism or other indices of virilization. The abnormal
[152], suggesting that PLs or other ligands act in con- androgen levels return to normal with correction of the
junction with PRL to influence adiposity. hyperprolactinemia by dopamine agonists.
In humans, patients with hyperprolactinemia have
been described to exhibit altered energy metabolism
and are candidates to obesity. Treatment of these Calcium and Bone Metabolism
patients to normalize their PRL levels is accompanied
PRL may have a physiologic role in calcium and
by a reduction of body weight and an improvement of
bone metabolism, and increases intestinal calcium
glucose tolerance and insulin sensitivity [153].
absorption even in vitamin D-deficient rats and can
In contrast to these results, suppression of PRL
stimulate 1-α-hydroxylation of renal 25-hydroxyvitamin
levels with bromocriptine has no effect on glycemic
D, resulting in increased plasma 1,25 (OH)2D levels.
control in normoprolactinemic subjects with insulin-
In humans, however, plasma 1,25 (OH)2D levels and
dependent diabetes. A new preparation of bromocrip-
intestinal calcium absorption are normal in hyperpro-
tine, which has been approved in the US for the treat-
lactinemic subjects. Prlr2/2 mice have decreased bone
ment of diabetes [154], has been shown to cause a
formation rates and bone mineral density in associa-
modest reduction in plasma glucose and hemoglobin
tion with increased parathyroid hormone levels but
A1c levels. However, the safety of lowering normal
decreased estradiol and progesterone levels so that it
PRL levels to subnormal levels, with respect to fertility
is difficult to delineate the contribution of a lack of
and sexual function, has not been proven.
PRL [158].
Other in vitro studies suggest a possible role for
The initial observation by Klibanski et al. that
PRL in adipogenesis. PRL itself is produced in small
hyperprolactinemic women have decreased bone min-
amounts by human adipocytes, including sources from
eral density [159] was confirmed by others but
the breast, visceral, and subcutaneous adipose tissue.
whether this effect is mediated by estrogen deficiency
The PRLR is expressed in both brown and white adi-
or is a direct effect of the hyperprolactinemia has been
pose tissue [149]. PRL upregulates the mRNA expres-
controversial. Correction of hyperprolactinemia results
sion of its receptor, and two transcription factors
in increased bone mass. Studies of hyperprolactinemic
principally involved in adipocyte differentiation,
women who were not amenorrheic and hypoestro-
CEBP/β and PPARγ, and it also stimulates the conver-
genemic have shown that bone mineral density is nor-
sion of NIH-3T3 fibroblasts into adipocytes. Complex
mal, confirming the initial hypothesis that estrogen
interactions between PRL and several adipokines, such
deficiency mediates bone mineral loss. A similar,
as leptin and adiponectin, have been described.
androgen-dependent loss of bone mineral is reported
However, the data regarding effects of exogenous PRL,
in hyperprolactinemic men, which is reversible with
transgenic overexpression of PRL, and PRL deficiency
reversal of the hypoandrogenic state.
on leptin levels in vivo are inconsistent and preclude
reliable conclusions [155].
Immune System
Hypophysectomized rats evince thymic involution
Adrenal Cortex
and decreased cell-mediated immune function, both of
It has been suggested that PRL acts on the adrenal which are reversed by the administration of ovine PRL
cortex to regulate adrenocortical function even though [160]. Similarly, hypoprolactinemia induced in animals
the molecular link between PRL/Stat5 and steroido- by bromocriptine or anti-PRL antibodies has been
genic genes has not yet been established [156]. Using shown to lead to impaired lymphocyte proliferation
Prlr2/2 mice and both cell lines and adrenocortical pri- and macrophage-activating factor production, again
mary culture, it was shown that PRL has no significant reversed by ovine PRL. PRL has long been proposed
effect on adrenal steroidogenesis in vivo and in vitro. as an immune-stimulating and detrimental factor in
However, Jak2 controls adrenal steroidogenesis. This autoimmune disorders. In the murine model of

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