Estrogen receptor α/prolactin receptor bilateral crosstalk promotes bromocriptine resistance in prolactinomas.

Prolactinomas are the most common type of functional pituitary adenoma. Although bromocriptine is the preferred first line treatment for prolactinoma, resistance frequently occurs, posing a prominent clinical challenge. Both the prolactin receptor (PRLR) and estrogen receptor α (ERα) serve critical roles in the development and progression of prolactinomas, and whether this interaction between PRLR and ERα contributes to bromocriptine resistance remains to be clarified. In the present study, increased levels of ERα and PRLR protein expression were detected in bromocriptine-resistant prolactinomas and MMQ cells. Prolactin (PRL) and estradiol (E2) were found to exert synergistic effects on prolactinoma cell proliferation. Furthermore, PRL induced the phosphorylation of ERα via the JAK2-PI3K/Akt-MEK/ERK pathway, while estrogen promoted PRLR upregulation via pERα. ERα inhibition abolished E2-induced PRLR upregulation and PRL-induced ERα phosphorylation, and fulvestrant, an ERα inhibitor, restored pituitary adenoma cell sensitivity to bromocriptine by activating JNK-MEK/ERK-p38 MAPK signaling and cyclin D1 downregulation. Collectively, these data suggest that the interaction between the estrogen/ERα and PRL/PRLR pathways may contribute to bromocriptine resistance, and therefore, that combination treatment with fulvestrant and bromocriptine (as opposed to either drug alone) may exert potent antitumor effects on bromocriptine-resistant prolactinomas.

Effects of metoclopramide-induced hyperprolactinemia on the prolactin and prolactin receptor expression of murine adrenal.

The aim of this study was to evaluate the effects of metoclopramide-induced hyperprolactinemia on the prolactin (PRL) and prolactin receptor's (PRLR) expression in the adrenal. For this purpose, a total of 12 animals with intact ovaries were allocated to two groups: G1 (saline solution) and G2 (metoclopramide). A total of 30 oophorectomized animals was randomized to five subgroups: G3 (saline solution), G4 (metoclopramide), G5 (metoclopramide + 17ß-estradiol), G6 (metoclopramide + progesterone), and G7 (metoclopramide + 17ß-estradiol + progesterone). Immunohistochemical analyses were evaluated semi-quantitatively. For PRLR, the area fraction of labeled cells (ALC) varied from 1 (0-10%) to 3 (> 50%). Based on the mean of the immunostaining intensity, G2 and G4 showed strong expression; G6 and G7 presented a mild reaction; and G1, G3, and G5 exhibited a weak reaction. Concerning PRL, the ALC varied from 1 (0-10%) to 3 (> 50%), and groups G6 and G7 showed a strong reaction; G2, G4, and G5 showed a mild reaction; and G1 and G3 exhibited a weak reaction. These findings suggest that metoclopramide-induced hyperprolactinemia increases PRL expression in the adrenal glands of mice. Furthermore, progesterone alone or in association with estrogen also increases PRL expression, but to a lesser extent.

Gene expression profiles of ductal versus acinar adenocarcinoma of the prostate.

Ductal adenocarcinoma is an uncommon variant of prostatic adenocarcinoma with a generally more aggressive clinical course than usual acinar adenocarcinoma. However, the molecular distinction between ductal and acinar adenocarcinomas is not well characterized. The aim of this investigation was to evaluate the relatedness of ductal versus acinar prostatic adenocarcinoma by comparative gene expression profiling. Archived, de-identified, snap frozen tumor tissue from 5 ductal adenocarcinomas, 3 mixed ductal-acinar adenocarcinomas, and 11 acinar adenocarcinomas cases were analyzed. All cases of acinar and ductal adenocarcinomas were matched by Gleason grade. RNA from whole tissue sections of the 5 ductal and 11 acinar adenocarcinomas cases were subjected to gene expression profiling on Affymetrix U133Plus2 microarrays. Independently, laser-capture microdissection was also performed on the three mixed ductal-acinar cases and five pure acinar cases to isolate homogeneous populations of ductal and acinar carcinoma cells from the same tumor. Seven of these laser-capture microdissected samples (three ductal and four acinar cell populations) were similarly analyzed on U133Plus2 arrays. Analysis of data from whole sections of ductal and acinar carcinomas identified only 25 gene transcripts whose expression was significantly and at least two-fold different between ductal and acinar adenocarcinomas. A similar analysis of microdissected cell populations identified 10 transcripts, including the prolactin receptor, with more significant differences in expression of 5- to 27-fold between ductal and acinar adenocarcinomas cells. Overexpression of prolactin receptor protein in ductal versus acinar adenocarcinoma was confirmed by immunohistochemistry in an independent set of tumors. We conclude that ductal and acinar adenocarcinomas of the prostate are strikingly similar at the level of gene expression. However, several of the genes identified in this study, including the prolactin receptor, represent targets for further investigations on the molecular basis for histomorphological and clinical behavioral differences between acinar and ductal adenocarcinomas.

The prolactin/growth hormone receptor family.

PRL and GH are hormones with a wide spectrum of actions. Specific receptors are widely distributed in a number of classical target organs, but other tissues that are not known targets also contain measurable binding sites or receptor mRNA. The most likely explanation is that PRL and GH cause effects that have not yet been characterized in certain tissues. Cloning of the cDNAs encoding PRL and GH receptors has led to the discovery that the receptors, like the hormones themselves, form a gene family. Multiple receptor forms have been identified, including a short form, which for PRL is a membrane-bound receptor or for GH is a soluble BP, and a long form, which for both PRL and GH is a membrane-bound receptor. PRL and GH receptors, and the mRNAs encoding them, can be up- and down-regulated. GH induces an up-regulation of both GH and PRL receptors, whereas PRL stimulates an increase of only its own receptor. High concentrations of either hormone induce a homologous down-regulation of receptor expression. An assay has been developed to measure the functional activity of different forms of PRL receptor by cotransfecting a milk protein fusion gene specific to PRL coupled to a reporter-gene along with the cDNA of the PRL receptor. Although the short form represents the major form present in rat mammary gland, only the long form of receptor is able to stimulate milk protein gene transcription. For GH, increased expression of the receptor in some target cells is accompanied by a modest enhancement of the response to GH. No single second messenger mediating the action of either PRL or GH has been identified. Several potential components of the signal transduction pathways have been identified, but as yet none has clearly been shown to be able to mimic the effect of PRL or GH. Because of the wide range of biological actions associated with PRL, and the existence of various forms of PRL receptors, it is doubtful that one unifying mechanism of action will be found for this hormone. No human or animal model of a genetic defect of the PRL receptor has thus far been published. Mutations in the GH receptor gene have been demonstrated in Laron type dwarfism. Different exon deletions or point or nonsense mutations resulting in modifications in the extracellular, GH binding region of the GH receptor have been reported.(ABSTRACT TRUNCATED AT 400 WORDS)

Diminished prolactin from chlordecone treatment in ovariectomized (NZBxNZW)F(1) mice.

In murine models of systemic lupus erythematosus (SLE), administration of either prolactin or estradiol (E2) increases autoimmunity, and there is evidence that elevated prolactin in response to E2 administration may contribute substantially to E2 effects. Hormonal influence on SLE can extend to environmental agents, as demonstrated by the ability of estrogenic organochlorine pesticides such as chlordecone to accelerate the development of lupus in female (NZBxNZW)F(1) mice. In order to evaluate a potential role for prolactin in chlordecone effects on SLE, it was necessary to first determine whether treatment with chlordecone, like E2, results in elevated prolactin levels. Ovariectomized (NZBxNZW)F(1) mice were treated for 5-6 weeks with chlordecone or E2 in doses shown previously to significantly shorten the time to onset of SLE. At the end of the treatment period, serum prolactin levels were increased 10- to 20-fold in E2-treated mice compared to untreated controls, but decreased in an apparent dose-dependent manner in mice treated with chlordecone. Prolactin receptor in purified splenic B and CD4 T cells from treated animals, assessed through measurement of mRNA using quantitative real-time PCR, was increased by E2 treatment but unchanged in response to chlordecone. These observations suggest that the role of prolactin in eliciting autoimmunity in E2-treated animals is absent in the case of chlordecone, and by implication, that chlordecone possesses other actions that can replace the contribution of prolactin to development of SLE.

Prolactin Receptor-Mediated Internalization of Imaging Agents Detects Epithelial Ovarian Cancer with Enhanced Sensitivity and Specificity.

Poor prognosis of ovarian cancer, the deadliest of the gynecologic malignancies, reflects major limitations associated with detection and diagnosis. Current methods lack high sensitivity to detect small tumors and high specificity to distinguish malignant from benign tissue, both impeding diagnosis of early and metastatic cancer stages and leading to costly and invasive surgeries. Tissue microarray analysis revealed that >98% of ovarian cancers express the prolactin receptor (PRLR), forming the basis of a new molecular imaging strategy. We fused human placental lactogen (hPL), a specific and tight binding PRLR ligand, to magnetic resonance imaging (gadolinium) and near-infrared fluorescence imaging agents. Both in tissue culture and in mouse models, these imaging bioconjugates underwent selective internalization into ovarian cancer cells via PRLR-mediated endocytosis. Compared with current clinical MRI techniques, this targeted approach yielded both enhanced signal-to-noise ratio from accumulation of signal via selective internalization and improved specificity conferred by PRLR upregulation in malignant ovarian cancer. These features endow PRLR-targeted imaging with the potential to transform ovarian cancer detection. Cancer Res; 77(7); 1684-96. ©2017 AACR.

Prolactin role in the bovine uterus during adenomyosis.

Adenomyosis is uterine dysfunction defined as the presence of endometrial glands within the myometrium. It is suggested that adenomyosis is estrogen-dependent pathology, and prolactin (PRL) also affects its development. In the uterus of ruminants, PRL stimulates gland proliferation and function. We hypothesized that in the bovine uterus, the expression of PRL and its receptors (PRLRs) during adenomyosis is disturbed and modulated by estradiol (E2). Uterine tissues were collected postmortem from cows; epithelial, stromal, and myometrial cells were isolated; and cultured and treated with E2. Material was divided into 2 groups: control (nonadenomyotic) and uteri with adenomyosis. In adenomyotic uterine tissue, PRL and its long-form receptor protein were increased, as determined by Western blotting. Immunohistostaining showed that during adenomyosis, PRL and its receptors are highly expressed in adenomyotic lesions. In cultured myometrial cells, protein expression of PRL and its receptors was increased during adenomyosis. Estradiol decreased PRLRs protein expression in nonadenomyotic stromal cells and in adenomyotic myometrial cells, and increased PRL secretion by adenomyotic myometrial cells. Moreover, PRL secretion was increased in untreated epithelial and stromal cells during adenomyosis. On the other hand, in stromal cells, PRLRs messenger RNA and protein expression was decreased, as determined by real-time PCR and Western blotting, respectively. Obtained results show that significant changes in PRL and PRLRs expression are observed in uterine tissue and cells during adenomyosis, which were also affected by E2. These data suggest involvement of PRL in adenomyosis development and the link between PRL and E2 actions during the dysfunction in cows.

Prolactin inhibits the apoptosis of chondrocytes induced by serum starvation.

The apoptosis of chondrocytes plays an important role in endochondral bone formation and in cartilage degradation during aging and disease. Prolactin (PRL) is produced in chondrocytes and is known to promote the survival of various cell types. Here we show that articular chondrocytes from rat postpubescent and adult cartilage express the long form of the PRL receptor as revealed by immunohistochemistry of cartilage sections and by RT-PCR and Western blot analyses of the isolated chondrocytes. Furthermore, we demonstrate that PRL inhibits the apoptosis of these same chondrocytes cultured in low-serum. Chondrocyte apoptosis was measured by hypodiploid DNA content determined by flow cytometry and by DNA fragmentation evaluated by the ELISA and the TUNEL methods. The anti-apoptotic effect of PRL was dose-dependent and was prevented by heat inactivation. These data demonstrate that PRL can act as a survival factor for chondrocytes and that it has potential preventive and therapeutic value in arthropathies characterized by cartilage degradation.

Anti-prolactin (PRL) autoantibody-binding sites (epitopes) on PRL molecule in macroprolactinemia.

Macroprolactinemia, in which serum prolactin (PRL) mainly consists of PRL with a molecular mass greater than 100 kDa, has been demonstrated to be associated with hyperprolactinemia. We previously reported that anti-PRL autoantibody is the major cause of macroprolactinemia. In this study, the autoantibody-binding sites (epitopes) on the PRL molecule were examined using deletion mutant PRL. The sera from 159 patients with hyperprolactinemia were screened for macroprolactinemia using the polyethylene glycol method and 18 patients (11%) were diagnosed with macroprolactinemia. The sera from these patients were incubated with glutathione S-transferase-human prolactin (hPRL) fragment fusion proteins immobilized on glutathione sepharose and the amounts of bound immunoglobulin G (IgG) were measured using ELISA. IgG was bound to full-length hPRL1-199 in significantly greater amounts in sera from 14 of 18 patients with macroprolactinemia than in controls. hPRL, but not PRL of other species such as bovine, porcine, rat, or human GH, dose-dependently displaced the binding, suggesting that these patients had hPRL-specific autoantibodies. Deletion of 34 amino acid residues from N-and/or C-terminals significantly reduced the binding and N- or C-terminal fragment alone showed partial but significant binding, suggesting that the major epitopes recognized by anti-PRL autoantibodies are located in both N- and C-terminal residues of the PRL molecule.

Long isoform of prolactin receptor predominates in rat intrahepatic bile ducts and further increases under obstructive cholestasis.

Prolactin participates in the regulation of liver function. However, prolactin receptor (PrlR) expression and its regulation have been described only for hepatocytes. In this study, we investigated the expression and regulation of PrlR isoforms in the other important intrahepatic cellular compartment: the biliary epithelial cells, or cholangiocytes. Our aim was to determine whether prolactin should be considered as a potential regulator of cholangiocyte function under normal and pathological conditions. Cholangiocytes and hepatocytes were differentially isolated from rat liver. PrlR expression was analysed at the mRNA level by isoform-specific semiquantitative PCR, and at the protein level by immunostaining of liver sections. Hormonal regulation of PrlR expression was evaluated by comparing intact rats with gonadectomized, pituitary-grafted or bromocriptine-treated animals. Common bile-duct ligation was used as the experimental model of cholestasis. Our results demonstrate that the expression pattern and regulation of PrlR isoforms is totally different in cholangiocytes compared with hepatocytes: (1) mature rat cholangiocytes express low levels of PrlR, while it is very high in hepatocytes, (2) only the long isoform is detected in cholangiocytes, while the short isoform predominates in hepatocytes and (3) PrlR levels in cholangiocytes are induced by obstructive cholestasis, but not by sex hormones or prolactin, while it is the opposite in hepatocytes. From these data, the actions of prolactin on liver are anticipated to exhibit strong cell-type specificity in both normal and pathological conditions.

Prolactin delays hair regrowth in mice.

Mammalian hair growth is cyclic, with hair-producing follicles alternating between active (anagen) and quiescent (telogen) phases. The timing of hair cycles is advanced in prolactin receptor (PRLR) knockout mice, suggesting that prolactin has a role in regulating follicle cycling. In this study, the relationship between profiles of circulating prolactin and the first post-natal hair growth cycle was examined in female Balb/c mice. Prolactin was found to increase at 3 weeks of age, prior to the onset of anagen 1 week later. Expression of PRLR mRNA in skin increased fourfold during early anagen. This was followed by upregulation of prolactin mRNA, also expressed in the skin. Pharmacological suppression of pituitary prolactin advanced dorsal hair growth by 3.5 days. Normal hair cycling was restored by replacement with exogenous prolactin for 3 days. Increasing the duration of prolactin treatment further retarded entry into anagen. However, prolactin treatments, which began after follicles had entered anagen at 26 days of age, did not alter the subsequent progression of the hair cycle. Skin from PRLR-deficient mice grafted onto endocrine-normal hosts underwent more rapid hair cycling than comparable wild-type grafts, with reduced duration of the telogen phase. These experiments demonstrate that prolactin regulates the timing of hair growth cycles in mice via a direct effect on the skin, rather than solely via the modulation of other endocrine factors.

Mouse mammary tumors induced by medroxyprogesterone acetate: immunohistochemistry and hormonal receptors.

Mammary adenocarcinomas induced by medroxyprogesterone acetate (MPA) in female BALB/c mice were investigated as to their morphology and immunohistochemistry and their content of steroid, prolactin (PRL), and epidermal growth factor (EGF) receptors. Histologically, these tumors were mainly of ductal origin, since hyperplastic alveolar nodules were observed only in 3 cases. No viral particles were encountered in electron microscopic studies. Estrogen and/or progesterone, PRL, and EGF receptors were detected in MPA-induced tumors, as well as in the occasional spontaneous mammary tumors of multiparous females. EGF was detected, by a radioimmunoassay, in the cystic fluid of 12 mammary adenocarcinomas. MPA treatment was found to induce uterine secretory changes, glandular cystic hyperplasia, and eventually deciduomas that stained strongly for desmin and to a lesser degree for vimentin, suggesting a muscular differentiation. Consequently, MPA-induced adenocarcinomas can be considered as ductal tumors that possess estrogen and/or progesterone, PRL, and EGF receptors. Whether MPA induces tumor growth directly via progesterone receptors remains to be investigated.

Prognostic significance of prolactin receptors in human breast cancer.

Overall survival and relapse free survival (RFS) were studied in 547 patients according to the presence of prolactin receptors (PRL-R), either free or total (after 3 M MgCl2 desaturation). All these patients were surgically treated for locoregional disease in the same institution between 1978 and 1984. In actuarial survival studies, RFS was higher in total PRL-R positive patients in the whole population (P less than 0.02). When the population was divided into two groups, according to either the presence or the absence of node metastasis or the presence or absence of estradiol receptor, the higher RFS was restricted to node positive (P less than 0.001) and to estradiol receptor positive patients (P less than 0.01). The Cox analysis on RFS showed that free PRL-R alone was a significant prognostic factor in estradiol receptor positive patients; total PRL-R alone was never significant; when considered together with steroid receptors, free as well as total PRL-Rs were significant prognostic factors in some subgroups of patients.

The Role of Prolactin in Bone Metastasis and Breast Cancer Cell-Mediated Osteoclast Differentiation.

BACKGROUND: Metastasis to the bone is a deleterious aspect of breast cancer and is a preferred site that results in bone loss. Hormones such as prolactin (PRL) have not yet been studied for their role in modulating the secondary tumor bone microenvironment. METHODS: We used quantitative immunohistochemistry with 134 samples of human primary breast cancer and 17 matched primary breast cancers and bone metastases. A Cox proportional hazards regression model was fitted to evaluate the associations between high prolactin receptor (PRLR) expression and time to bone metastasis, adjusting for estrogen receptor status, lymph node status, and chemotherapy status. We assessed osteoclast differentiation, osteoclast size, and measured pit formation in dentine slices. Statistical tests were two-sided. RESULTS: High PRLR expression in the primary breast tumor was associated with a shorter time to metastasis that includes bone (PRLRAQUA Max-per 100 unit hazard ratio = 1.04, 95% confidence interval = 1.00 to 1.07, P = .03). We observed the PRLR in rare samples of bone metastases and matched primary breast cancer. PRL treatment of breast cancer cells induced osteoclast differentiation and bone lysis via secreted factors and was abrogated by a PRLR antagonist (delta1-9-G129R-hPRL). We demonstrated that sonic hedgehog is a PRL-regulated cytokine in breast cancer cells and part of the mechanism that induces osteoclast differentiation. CONCLUSIONS: Our evidence indicates that PRL-PRLR can escalate the impact of breast cancer on bone metastasis and that the presence of the PRLR in the tumor microenvironment of breast cancer bone metastasis has the potential to modulate the microenvironment to induce lytic osteoclast formation.

Proteins associated with somatogenic and lactogenic receptors in microsomal membranes and intact rat hepatocytes.

Lactogenic and somatogenic receptors present in rat liver have been examined by cross-linking with a derivative of human somatotropin (AP-hGH1) followed by SDS-polyacrylamide gel electrophoresis. AP-hGH1, which has a content of 2.2 azidophenacyl groups per molecule, mainly linked to half Cys-182 and half Cys-189, exerted a specificity similar to that of the native hormone (hGH), with an ability of 46% with respect to hGH to compete with the radiolabelled hormone for the binding sites of microsomal preparations. Photolysis of the 125I-labelled derivative bound to the lactogenic receptors present in either microsomal membranes or Triton X-100 solubilized preparations gave rise to a 63 kDa species. In addition, 30% of the covalent complexes formed in microsomal membranes belonged to a species with a molecular mass of 70 kDa. Incubation of viable rat hepatocytes with the radiolabelled derivative at either 0 degrees C for 3 h or 15 degrees C for 1.5 h and subjection to irradiation, yielded covalent complexes of molecular masses estimated at 130, 73, 63, 45 and 35 kDa. Experiments performed in the presence of 1 mM NaCN, gave rise to the previous species in a similar yield as that obtained in the absence of cyanide. The 130 kDa complex is related to the somatogenic binding sites, since it was not visualized in the presence of unlabelled bovine somatotropin, while the 70-73, 63, 45 and 35 kDa bands disappeared when the incubations were performed in the presence of unlabelled ovine prolactin.

Multiple regulation of prolactin receptor gene expression in rat liver.

Sex steroids are major regulators of PRL receptor expression in rat liver. Using a probe encoding the rat PRL receptor we have studied receptor mRNA levels in female rat liver during ontogeny and in response to estrogen treatment. Steady state mRNA levels were determined by Northern blot and densitometric analysis. Messenger RNA levels have been compared to the number of binding sites, which was assessed by Scatchard analysis of [125I]ovine PRL binding in membrane preparations. Our results show that steady state mRNA and binding levels of PRL receptors are both regulated by development and estrogens, but that binding does not exactly parallel mRNA levels. From the developmental stages of prepuberty to adult, receptor numbers increase 8-fold, whereas mRNA levels increase 3-fold. Estrogen treatment stimulates receptor levels 6-fold, but mRNA levels are only increased 3-fold. These results suggest that PRL receptor gene expression in rat liver is regulated at the transcriptional or posttranscriptional level as well as at the translational level.

The protein tyrosine kinase P59fyn is associated with prolactin (PRL) receptor and is activated by PRL stimulation of T-lymphocytes.

The clonal expansion of antigen-stimulated T-lymphocytes during an immune response is mediated by several lymphokines. Strong evidence now exists that the neuroendocrine hormone PRL is necessary, but not sufficient, for T-cell proliferation. Little is known, however, of the signal transduction mechanisms of the PRL receptor (PRLR) within T-cells. We demonstrate here that PRL stimulation of the T-cell line Nb2 induced the concentration- and time-dependent activation of the protein tyrosine kinase p59fyn, but not of four other src family protein tyrosine kinases. Activation of fyn was also observed in Concanavalin-A-primed peripheral blood lymphocytes stimulated with PRL and in Nb2 cells incubated with anti-PRLR antibodies. The activation of fyn by PRL stimulation correlated with Nb2 cell proliferation. Immunoblot analysis of anti-fyn and anti-PRLR immune complexes revealed an association between each PRLR isoform and p59fyn. These studies demonstrate for the first time an association between the PRLR and a src family protein tyrosine kinase affiliated with signal transduction.

Normal rat uterine stromal cells in continuous culture: characterization and progestin regulation of growth.

Stromal cells were isolated from rat uterus by sequential enzymatic digestion and density fractionation on Percoll gradient and subcultured by trypsinization. Two stable subcultures, named UII and UIII, were obtained. UII cells exhibited a spindle-shaped, elongated, fibroblast-like morphology, while UIII cells were rounded and polygonal. Both cell types expressed the intermediate filament vimentin but not cytokeratin, nor desmin, suggesting that both were of stromal origin. In UIII cells, the presence of progesterone and prolactin (PRL) receptors was demonstrated by immunocytochemical and binding studies. Cross-linking and Western blotting showed that PRL receptor in UIII cells corresponded to 3 molecular forms of 54, 42 and 32 kDa. The growth properties of these cells were studied under different conditions of culture. In fetal calf serum (FCS) supplemented medium, proliferation of UIII cells was dependent on serum concentration and was not affected by estradiol and progesterone. In 10% FCS supplemented medium, the doubling time was 41.5 +/- 0.8 h. When cultured in 10% dextran-charcoal-treated FCS, cells were maintained in a viable but quiescent state. Under these conditions, progesterone was able to induce growth of these cells in a dose-dependent manner. A 3-fold increase in DNA content was measurable in 10(-7) M progesterone-treated versus control cultures after 5 days. Reduction of serum concentration from 10% to 2% abolished the effect of progesterone suggesting that this effect requires the presence of serum factor(s). In conclusion, this study showed that uterine stromal cells, in continuous culture, retained progesterone and prolactin receptors and progesterone regulation of growth.

Prolactin receptors are found on heterogeneous subpopulations of rat splenocytes.

An increasingly large body of evidence implicates PRL as an immunoregulatory molecule. While most of the data relate to PRL levels and immunocompetence in vivo, we have shown that PRL is mitogenic for splenocytes from ovariectomized rats and rats in certain other hormonal states. This finding suggests that these lymphocytes express PRL receptors. Here, we wished to determine whether all or only a subset of splenocytes were PRL receptor positive. By using polyclonal as well as monoclonal antibodies to PRL receptor, we determined that as many as 20% of the primary splenocytes expressed PRL receptors. In a culture of Nb2 cells, a PRL receptor-positive lymphoid line, as many as 70% were PRL receptor positive. Dual labeling for lymphoid-specific antigen surface markers and PRL receptor indicated that about one third of the PRL receptor-positive splenocytes were kappa-light chain-positive B-cells, while the others stained with antibodies to T-cell markers, CD4 or CD8. These data confirm that lymphocytes express PRL receptors and show for the first time that PRL receptor-positive lymphocytes are a heterogenous subset of total primary splenocytes. These cells may be the target for PRL-mediated immunoregulation.

Autocrine interaction between prolactin and its receptor occurs intracellularly in the 235-1 mammotroph cell line.

We have previously demonstrated that PRL acts as an autocrine growth factor in the pituitary tumor GH3 cell line. In this study we present evidence that the 235-1 cell line also uses PRL as an autocrine growth factor, and that in this case interaction between PRL and its receptor occurs intracellularly. First, the PRL produced by 235-1 cells was shown to be capable of initiating a proliferative response by placement in the Nb2 bioassay. Second, PRL receptors were demonstrated to be present, but, unusually, primarily in the Golgi complex by light and electron microscope immunocytochemistry. Incubation of the cells in anti-PRL had no effect on 235-1 cell proliferation until interleukin-1 treatment caused the PRL receptors to move to the cell surface, whereupon cell proliferation was inhibited. Specific interference with PRL biosynthesis in noninterleukin-1-treated cells also inhibited cell proliferation. Thus, the essential elements of an autocrine loop are present and can be demonstrated to be functional by antibody inhibition of growth after movement of the receptors to the cell surface and by antisense inhibition of growth with the receptors in their normal intracellular location.