Prolactin promotes a partial recovery from the atrophy of both male and female gerbil prostates caused by castration.

BACKGROUND: The male and female prostates are controlled by steroid hormones, suffering important morphological and physiological changes after castration. Prolactin is involved in the regulation of the male prostate, having already been identified in the tissue, acting through its receptor PRLR. In the Mongolian gerbil, in addition to the male prostate, the female prostate is also well developed and active in its secretion processes. The aim of the present study was to evaluate the effects of exposure to exogenous prolactin in the prostate of both intact and castrated male and female gerbils in order to establish if prolactin administration can sustain prostate cell activity in conditions of sexual hormone deprivation. METHODS: The morphological analyses were performed by biometric analysis, lesion histological analysis and morphometric-stereological aspects. In addition, immune-cytochemical tests were performed for prolactin and its receptor, as well as for the receptors of androgen and oestrogen and serum prolactin dosage. All data were submitted to ANOVA or Kruskal-Wallis tests for comparison between groups. P < 0.05 was considered to be statistically significant. RESULTS: The results showed a strong influence of prolactin on the morphology of the prostate, with the development of important epithelial alterations, after only 3 days of administration, and an expressive epithelial cell discard process after 30 days of administration. Prolactin acts in synergy with testosterone in males and mainly with oestrogens in females, establishing different steroid hormonal receptor immunoreactivity according to sex. It was also demonstrated that prolactin can assist in the recovery from some atrophic effects caused in the gland after castration, without causing additional tissue damage. CONCLUSIONS: The prolactin and its receptor are involved in the maintenance of the homeostasis of male and female gerbils, and also cause distinct histological alterations after exogenous exposure for 3 and 30 days. The effects of prolactin are related to its joint action on androgens and oestrogens and it can also assist in the recovery from the atrophic effects of castration.

Stimulation of prolactin receptor induces STAT-5 phosphorylation and cellular invasion in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in humans and is characterized with poor outcome. In this study, we investigated components of prolactin (Prl) system in cell models of GBM and in histological tissue sections obtained from GBM patients. Expression of Prolactin receptor (PrlR) was detected at high levels in U251-MG, at low levels in U87-MG and barely detectable in U373 cell lines and in 66% of brain tumor tissues from 32 GBM patients by immunohistochemical technique. In addition, stimulation of U251-MG and U87-MG cells but not U373 with Prl resulted in increased STAT5 phosphorylation and only in U251-MG cells with increased cellular invasion. Furthermore, STAT5 phosphorylation and cellular invasion induced in Prl stimulated cells were significantly reduced by using a Prl receptor antagonist that consists of Prl with four amino acid replacements. We conclude that Prl receptor is expressed at different levels in the majority of GBM tumors and that blocking of PrlR in U251-MG cells significantly reduce cellular invasion.

Synergy in ERK activation by cytokine receptors and tyrosine kinase growth factor receptors.

Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) signal through EGF and PDGF receptors, which are important receptor tyrosine kinases (RTKs). Growth hormone (GH) and prolactin (PRL) are four helical bundle peptide hormones that signal via GHR and PRLR, members of the cytokine receptor superfamily. In this study, we examine crosstalk between signaling pathways emanating from these disparate receptor groups (RTKs and cytokine receptors). We find that GH and EGF specifically synergize for activation of ERK in murine preadipocytes. The locus of this synergy resides at the level of MEK activation, but not above this level (i.e., not at the level of EGFR, SHC, or Raf activation). Furthermore, dephosphorylation of the scaffold protein, KSR, at a critical serine residue is also synergistically promoted by GH and EGF, suggesting that GH sensitizes these cells to EGF-induced ERK activation by augmenting the actions of KSR in facilitating MEK-ERK activation. Similarly specific synergy in ERK activation is also detected in human T47D breast cancer cells by cotreatment with PRL and PDGF. This synergy also resides at the level of MEK activation. Consistent with this synergy, PRL and PDGF also synergized for c-fos-dependent transactivation of a luciferase reporter gene in T47D cells, indicating that events downstream of ERK activation reflect this signaling synergy. Important conceptual and physiological implications of these findings are discussed.

Identification of a gain-of-function mutation of the prolactin receptor in women with benign breast tumors.

There is currently no known genetic disease linked to prolactin (Prl) or its receptor (PrlR) in humans. Given the essential role of this hormonal system in breast physiology, we reasoned that genetic anomalies of Prl/PrlR genes may be related to the occurrence of breast diseases with high proliferative potential. Multiple fibroadenomas (MFA) are benign breast tumors which appear most frequently in young women, including at puberty, when Prl has well-recognized proliferative actions on the breast. In a prospective study involving 74 MFA patients and 170 control subjects, we identified four patients harboring a heterozygous single nucleotide polymorphism in exon 6 of the PrlR gene, encoding Ile(146)-->Leu substitution in its extracellular domain. This sole substitution was sufficient to confer constitutive activity to the receptor variant (PrlR(I146L)), as assessed in three reconstituted cell models (Ba/F3, HEK293 and MCF-7 cells) by Prl-independent (i) PrlR tyrosine phosphorylation, (ii) activation of signal transducer and activator of transcription 5 (STAT5) signaling, (iii) transcriptional activity toward a Prl-responsive reporter gene, and (iv) cell proliferation and protection from cell death. Constitutive activity of PrlR(I146L) in the breast sample from a patient was supported by increased STAT5 signaling. This is a unique description of a functional mutation of the PrlR associated with a human disease. Hallmarks of constitutive activity were all reversed by a specific PrlR antagonist, which opens potential therapeutic approaches for MFA, or any other disease that could be associated with this mutation in future.

Prolactin upregulates its receptors and inhibits lipolysis and leptin release in male rat adipose tissue.

Prolactin (PRL) is recognized as a metabolic regulator during lactation, but little information exists on its actions in male adipose tissue. We examined whether PRL affects the expression of its receptors (PRLR), lipolysis, and adipokine secretion in male rats. Both long and short PRLR isoforms were induced 40-50-fold during differentiation of epididymal preadipocytes, with a 10-fold higher expression of the long isoform. PRL upregulated both isoforms before and after differentiation. PRL suppressed lipolysis in epididymal explants and mature adipocytes in a dose- and time-dependent manner, which was reversed by a Jak2 inhibitor. PRL also inhibited leptin, but not adiponectin, release. We conclude that PRL inhibits lipolysis and leptin release by acting directly on adipocytes via interaction with either of its receptors and activation of a Jak2-dependent signaling pathway(s). This is the first demonstration of substantial effects of PRL on male adipocytes.

Involvement of PI3'-K, mitogen-activated protein kinase and protein kinase B in the up-regulation of the expression of nNOSalpha and nNOSbeta splicing variants induced by PRL-receptor activation in GH3 cells.

It is well known that GH-PRL secreting GH3 cells express constitutive neuronal nitric oxide synthase (nNOS) and produce nitric oxide (NO*). In addition, these cells possess plasma membrane prolactin (PRL) receptors which can be responsible for an autocrine 'short-loop' feedback. The aim of the present study was to investigate whether the activation of PRL receptors modulates the expression of the different spliced forms of nNOS gene, and the transductional mechanisms involved in this action. In GH3 cells, both exon 2-containing nNOSalpha and exon 2-lacking nNOSbeta were time-dependently expressed, whereas the other two isoforms eNOS and iNOS were not. The antibodies directed against the residues 53-68 of the external domain common to both the long and short form of rat PRL receptors, and the selective D2 agonist cabergoline (1 nm) reduced both basal and exogenous PRL-induced expressions of nNOSalpha and nNOSbeta, but to a greater extent for the beta splicing form. In line with these results, oPRL (1 and 10 microm) added to the incubation medium increased to a greater extent the expression of nNOSbeta form than of the nNOSalpha. The receptor and non-receptor protein tyrosine kinase (PTK) inhibitors, genistein (10 microm), the Src-specific tyrosine kinase inhibitor PP2 (100 microm), the MAPK inhibitor PD 098059 (50 nm) and the two PI3'-K inhibitors, wortmannin (300 nm) and LY-294002 (25 microm) prevented both basal and exogenous PRL-induced expression of nNOSalpha and nNOSbeta isoforms. In addition, exogenous PRL induced a phosphorylation of protein kinase B (PKB) (Akt) that was prevented both by the two MAPK inhibitors PD 098059 and U 0126, and by the PI3'-K inhibitors wortmannin and LY-294002. Up-regulation of the expression of the two splicing forms of nNOS elicited by PRL-receptor activation was mirrored by the increased synthesis of NO*. In conclusion, PRL receptor activation up-regulated the expression of both nNOSalpha and nNOSbeta proteins via a PTK, PI3'-K, MAPK and PKB signalling transduction components. This action may represent the molecular mechanism by which PRL exerts the 'short-loop' feedback on its own secretion.

Effects of prolactin on intracellular calcium concentration and cell proliferation in human glioma cells.

Prolactin (PRL) has several physiological effects on peripheral tissues and the brain. This hormone acts via its membrane receptor (PRL-R) to induce cell differentiation or proliferation. Using reverse transcription-polymerase chain reaction (RT-PCR) combined with Southern blot analysis, we detected PRL-R transcripts in a human glioma cell line (U87-MG) and in primary cultured human glioblastoma cells. These transcripts were deleted or not in their extracellular domains. We examined the effects of PRL on intracellular free Ca2+ concentration ([Ca2+](i)) in these cells in order to improve our understanding of the PRL transduction mechanism, which is still poorly documented. [Ca2+](i) was measured by microspectrofluorimetry using indo-1 as the Ca2+ fluorescent probe. Spatiotemporal aspects of PRL-induced Ca2+ signals were investigated using high-speed fluo-3 confocal imaging. We found that physiological concentrations (0.4-4 nM) of PRL-stimulated Ca2+ entry and intracellular Ca2+ mobilization via a tyrosine kinase-dependent mechanism. The two types of Ca2+ responses observed were distinguishable by their kinetics: one showing a slow (type I) and the other a fast (type II) increase in [Ca2+](i). The amplitude of PRL-induced Ca2+ increases may be sufficient to provoke several physiological responses, such as stimulating proliferation. Furthermore, PRL induced a dose-dependent increase in [3H]thymidine incorporation levels and in cellular growth and survival, detected by the MTT method. These data indicate that PRL induced mitogenesis of human glioma cells.

Human prolactin (hPRL) antagonists inhibit hPRL-activated signaling pathways involved in breast cancer cell proliferation.

The involvement of human prolactin (hPRL) in breast cancer has been recently reconsidered based on its autocrine/paracrine proliferative effect described in human mammary tumor epithelial cells. Therefore, there is growing interest in the development of potent hPRL antagonists that may inhibit this effect. We previously designed hPRL analogs displaying antagonistic properties in a human transcriptional bioassay. We now report that the most potent of those analogs, G129R-hPRL, antagonizes all hPRL-induced effects analysed in various breast cancer cell lines, including cell proliferation. The analog per se lacks intrinsic agonistic activity on PRL receptor-activated signaling cascades, cell proliferation and apoptosis, indicating that its mode of action only occurs through competitive inhibition of hPRL. We provide some molecular basis of this antagonistic effect by demonstrating that G129R-hPRL competitively inhibits hPRL-activation of the JAK-STAT and MAPK pathways, two signaling cascades involved in the mitogenic effect of hPRL in mammary epithelial cells. This competitive inhibition persists for at least 48 h, as evidenced by long term analysis of STAT5b activation or of progression through cell cycle. These results are the first demonstration at the molecular level that hPRL antagonists interfering with receptor dimerization disrupt signaling events in breast cancer cells, which prevents hPRL-induced cell proliferation.

Characterization and regulation of prolactin receptors in MA-10 Leydig cells.

The aim of this study is to further characterize the prolactin receptors (PRL-R) previously reported in the murine Leydig tumor MA-10 cell line, as well as to study their homologous and heterologous regulation. Two forms of PRL-R, a high and a low molecular weight form, were revealed by studies of covalent crosslinking of 125I-human GH to cultured MA-10 cells or cell membranes and immunoprecipitation of the solubilized PRL-R complexes with polyclonal anti PRL-R antibody, followed by SDS-PAGE and autoradiography. The long form had a molecular weight of 101 kDa and was predominant when the study was performed in the presence of protease inhibitors. The short form, with a molecular weight of 39 kDa, appeared, at least in part, to be a proteolytic product of the longer form. The same size forms of PRL-R were detected by crosslinking studies in the parental C57BL/6 mouse testicular Leydig cells, indicating the physiological relevance of the MA-10 cell model to the study of Leydig cell PRL-R. Homologous down-regulation of PRL-R was demonstrated in cultured MA-10 cells exposed for 24 h to increasing concentrations of PRL. In contrast, heterologous, 3 5-fold up-regulation of PRL-R was induced by various cAMP-elevating agents, including 8-bromo-cAMP (10(-4) -10(-3) M), dibutyryl cAMP (3 x 10(-3) M) and cholera toxin (1-10 ng/ml), although not by hCG (up to 100 ng/ml). This up-regulatory effect was apparently the result of a change in affinity, since cholera toxin caused a 2.4-fold increase in PRL-R affinity, with no change in the number of binding sites. In summary, these studies provide further evidence that MA-10 Leydig cells can serve as a physiologically relevant model for the study of PRL and PRL-R interactions, both at the functional level, as shown in our previous study, and at the structural and regulatory levels as shown in the current study.

Antagonistic properties of human prolactin analogs that show paradoxical agonistic activity in the Nb2 bioassay.

Based on the assumption that the prolactin receptor (PRLR) is activated by PRL-induced sequential dimerization, potential human PRL (hPRL) antagonists were designed that sterically interfere with binding site 2. We previously reported the unexpected agonistic properties of these hPRL analogs in the rat Nb2 bioassay (Goffin, V., Struman, I., Mainfroid, V., Kinet, S., and Martial, J. A. (1994) J. Biol. Chem. 269, 32598-32606). In order to investigate whether such paradoxical agonistic behavior might result from characteristic features of the Nb2 assay (e.g. species specificity), we transfected in the same cell system the cDNA encoding the PRLR from rat or human species along with reporter genes containing PRL-responsive DNA sequences. We characterized the agonistic, self-antagonistic and/or antagonistic effects of wild type rat PRL, wild type hPRL, and three hPRL analogs, mutated either at binding site 1 or at binding site 2. Our results clearly show that the agonistic/antagonistic properties of PRLs are species-specific. We thus propose different models of receptor activation, depending on the relative affinities of each hormonal binding site, which is directed by species specificity. Finally, this is the first report of hPRL binding site 2 analogs showing antagonistic properties on human and, to a lesser extent, rat receptors.

Cloning and characterization of an ovarian-specific protein that associates with the short form of the prolactin receptor.

Prolactin (PRL) is essential for progesterone biosynthesis and luteal cell hypertrophy of the rat corpus luteum during pregnancy. Both the long and short form of the PRL receptor have been identified in the corpus luteum of pregnant rat. The long form has been shown to transduce PRL signal in other cells, whereas no information is available on the role of the short form, especially in the corpus luteum. In the present study, we have cloned a rat ovarian-specific phosphoprotein, PRAP (PRL Receptor Associated Protein), which has no significant homology to other known proteins. We have demonstrated that this protein is immunoprecipitated by anti-PRL receptor and anti-phosphotyrosine antibodies. To determine whether PRAP associates with either the long or the short form of the PRL receptor, fusion proteins with glutathione S-transferase containing the cytoplasmic domain of the long or short form of the PRL receptor were produced, purified, and incubated with luteal proteins. Our results indicate that PRAP preferentially binds to the short form of the PRL receptor. Thus, the long form and short forms of the PRL receptor may signal through distinct pathways. These data provide evidence for the involvement of a novel protein in PRL signal transduction and suggest that PRAP may contribute to the luteotropic effects of PRL on the corpus luteum during pregnancy.