Prolactin Receptor Gene Expression Research Articles

泌乳開始における乳腺プロラクチン受容体遺伝子発現の役割

泌乳開始における乳腺プロラクチン受容体遺伝子発現の役割

Distribution of prolactin receptor immunoreactivity in ovine skin and changes during the wool follicle growth cycle.

Prolactin is believed to mediate seasonal cues entraining seasonal reproductive and hair follicle growth cycles. Prolactin receptor binding activity and prolactin receptor gene expression in mammalian skin have recently been described. In this report, prolactin receptor immunoreactivity is identified in sheep skin using a monoclonal antibody against the rat liver prolactin receptor. Western blotting analysis of microsomal membrane proteins from skin showed major bands corresponding to molecular weights of 87 and 71 kDa and minor bands at 101 and 21 kDa. RNase protection analysis revealed the presence of mRNA species coding for long and short forms of the prolactin receptor. Formalin-fixed sections, exposed to the monoclonal antibody and stained by an immunogold method, revealed prolactin receptor-immunoreactivity in the dermal papilla, germinal matrix, outer root sheath, lower regions of the inner root sheath and connective tissue sheath of wool follicles. Staining was absent from keratinised cell populations. In all samples, the interfollicular epidermis, sebaceous and sweat glands were positively stained. The distribution of prolactin receptor is described in both growing and inactive wool follicles and related to postulated cycle-specific actions of circulating prolactin in the control of seasonal fibre growth.

The different forms of the prolactin receptor in the rat corpus luteum: developmental expression and hormonal regulation in pregnancy.

The corpora lutea of pregnancy in the rat are highly dependent on the action of PRL and PRL-like hormones to hypertrophy and to produce progesterone needed for the maintenance of gestation. Two forms of the PRL receptor (PRL-R), designated as long (PRL-RL) and short (PRL-RS), have been described in rat tissues. To determine whether both forms are present in the corpus luteum during pregnancy and to examine the developmental and hormonal regulation of their expression, total RNA isolated from corpora lutea at different stages of pregnancy and from highly luteinized granulosa cells subjected to different hormonal treatments were analyzed by semiquantitative RT-PCR. Immunoblotting of luteal proteins from early and late pregnancy was also performed to determine if the pattern of PRL-R proteins follows that of PRL-R messenger RNA (mRNA) expression. In addition, the correlation between the well characterized PRL-regulated gene, 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), and PRL-R gene expression was investigated during the time of luteolysis. Both PRL-RL and PRL-RS mRNA and protein were expressed in corpora lutea of pregnancy, with the long form being the most dominant at all stages. Whereas no changes in mRNA level of either PRL-RL or PRL-RS were found until day 20 of gestation, a profound decline in PRL-R mRNA and protein for both receptor types occurred at the end of pregnancy. This drop in PRL-R expression was accompanied by a sharp and abrupt expression of 20alpha-HSD mRNA. Studies performed in vivo and in luteinized cells in culture indicate that PRL can up-regulate the expression of the PRL-RL mRNA, an effect prevented by the tyrosine kinase inhibitor, genistein. PRL-RL mRNA was also selectively increased by cAMP. In summary, the results of this investigation have established that: 1) the corpus luteum of pregnancy expresses both the short and long forms of the PRL-R with the long form being more abundant; 2) the mRNA for both forms of the PRL-R remains at constant levels throughout pregnancy but drops before parturition; 3) the decline in PRL-R mRNA at the end of pregnancy is accompanied by a dramatic rise in 20alpha-HSD; 4) PRL is able to increase the expression of PRL-R mRNA; and that 5) both A kinase and tyrosine kinase mediated pathways appear to participate in the up-regulatory mechanism involved in PRL-R mRNA expression.

Prolactin gene expression and prolactin protein in premenopausal and postmenopausal human ovaries

Objective: To investigate intraovarian prolactin and prolactin-receptor gene expression and to assess local prolactin synthesis with emphasis on possible differences between premenopausal and postmenopausal status. Design: The RNA extracted from human premenopausal and postmenopausal tissues was subjected to reverse transcription and polymerase chain reaction by using prolactin-specific intron- and exon-spanning primers. Prolactin-receptor expression was investigated accordingly. The amplified complementary DNA fragments were analyzed by gel electrophoresis and restriction enzyme mapping. Local prolactin hormone synthesis was verified by a time-resolved immunofluorometric assay based on our monoclonal antibodies. Result(s): Prolactin and prolactin-receptor gene expression was observed in all analyzed human ovaries ( n = 18). Several other human tissue specimens, such as lung and kidney, served as negative control tissues. Significantly elevated concentrations of prolactin were detected in cytosolic extracts of premenopausal ( nf = 6; mean ± SD; 20.6 ± 3.3 ng/g tissue wet weight) versus postmenopausal ( n = 6; 3.6 ± 3.0 ng/g tissue wet weight) ovaries. Conclusion(s): The human ovary not only serves as a target for endocrine prolactin action but also as a site of local prolactin hormone production. In agreement with previous reports on extrapituitary sources of prolactin, we consider prolactin as a hormone as well as an autocrine or paracrine growth or regulatory factor. Significantly increased concentrations of prolactin in premenopausal ovarian tissue verifies its role in human reproduction.

Corticosterone is required for the prolactin receptor gene expression in the late pregnant mouse mammary gland

In order to clarify the prolactin receptor (PRL-R) gene expression at lactogenesis, the levels of the long and short forms of PRL-R mRNA were determined by the competitive RT-PCR in the pregnant, lactating and ovariectomized midpregnant mouse mammary gland. Plasma concentrations of corticosterone and progesterone were determined by RIA. The long form of PRL-R mRNA level was low until 10:00 on day 18, increased 3.3-fold at 22:00 on day 18 of pregnancy and further increased to 4.6-fold at 10:00 on day 0 of lactation. The short form of PRL-R mRNA level remained unchanged during this time period. The corticosterone:progesterone ratio increased 15.5-fold during the last 1.5 days of pregnancy. Corticosterone increased the long form of PRL-R mRNA level when the tissues on day 17 were cultured. On day 12 of pregnancy and following ovariectomy, corticosterone was exceedingly high from 2 h to 8 h and the corticosterone:progesterone ratio changed prior to the increase in the long form of PRL-R mRNA level. We conclude that corticosterone increases the PRL-R gene expression in the mammary gland before the onset of parturition.

Removal of milk by suckling acutely increases the prolactin receptor gene expression in the lactating mouse mammary gland

To determine the effect of suckling on the prolactin receptor (PRL-R) gene expression, we measured the quantity of PRL-R mRNA in the lactating mouse mammary gland. When the pups were separated from their mother on day 5 of lactation, the long form of PRL-R (PRL-R L) mRNA disappeared with a half-life of 12.5 h for the first 9 h and 3.0 h for the following 9–15 h. By supplying pups to mice which had been weaned for 24 h, PRL-R L mRNA increased 2.5-fold during the next 6 h-period. The increase in PRL-R L mRNA was found in the mammary glands from which the pups removed milk. The number of mammary PRL-R protein decreased or increased following weaning or following the removal of milk by suckling, respectively. From these observations, it was concluded that the removal of milk acutely increases the level of PRL-R L mRNA during lactation.

Prolactin receptor gene expression in rat splenocytes and thymocytes during oestrous cycle, pregnancy and lactation

Much evidence suggests that prolactin (PRL) has an immunoregulatory function. Part of this evidence is that the receptors for PRL are present on lymphocytes. Probably the effects of PRL on cells of the immune system depend on the level and specific forms of PRL-R present on the target cells. Therefore, PRL-R expression at both protein and mRNA levels was examined during oestrous cycle, pregnancy and lactation using Western blotting and PCR analysis. Antibody to the long form of PRL-R detected 84 and 42 kDa protein bands in the spleen but only 84 kDa band in the thymus. The expression pattern of these two protein bands was different in the spleen, suggesting that these two isoforms of PRL-R long form are differentially regulated by the hormones of oestrous cycle. In addition, depending on the tissue, the level of mRNA for the short and long forms of PRL-R showed a significant change at different stages of oestrous cycle. Moreover, 42 and 84 kDa PRL-R bands were detected in both spleen and thymus throughout the pregnancy and lactation; however, the expression pattern of 84 kDa protein band was different between tissues. This finding suggests that each tissue exhibits differential response to hormones which affect PRL-R content.

Prolactin receptor gene expression in rat splenocytes and thymocytes during oestrous cycle, pregnancy and lactation

Prolactin receptor gene expression in rat splenocytes and thymocytes during oestrous cycle, pregnancy and lactation

Hepatic prolactin receptor gene expression increases in the sheep fetus before birth and after cortisol infusion.

We have investigated the effect of increasing gestational age and cortisol on prolactin receptor (PRLR) gene expression in the fetal sheep liver during late gestation. RNA was extracted from the liver of sheep fetuses between 90 and 144 days (d) gestation (n = 18) and after intrafetal infusion of either cortisol (2-2.5 mg cortisol i.v./24 h; n = 6) or saline (n = 6) between 109 and 116 d gestation. A ribonuclease protection assay for the mRNAs encoding the long (PRLR1) and short (PRLR2) forms of the PRLR was developed using an antisense RNA probe complementary to ovine PRLR2. There was a significant increase (p < 0.05) in the relative levels of liver PRLR1: GAPDH mRNA and PRLR2: GAPDH mRNA levels in fetal sheep between 90 and 144d gestation (PRLR1 mRNA: 90-95 d 0.6 +/- 0.1, 131-133 d 1.2 +/- 0.2, 141-144 d 3.6 +/- 0.5; PRLR2 mRNA: 90-95 d 0.7 +/- 0.1; 131-133 d 1.4 +/- 0.2, 141-144 d 3.0 +/- 0.4). The relative levels of liver PRLR1 and PRLR2: GAPDH mRNA levels were higher (p < 0.05) after cortisol administration (1.7 +/- 0.3 and 0.9 +/- 0.1 respectively) when compared with the saline infused group (0.7 +/- 0.1 and 0.5 +/- 0.1 respectively). We have demonstrated therefore that there is in increase in the levels of the mRNA encoding PRLR1 and PRLR2 in the fetal sheep liver during late gestation and that physiological increases in fetal cortisol stimulate PRLR1 and PRLR2 expression in the liver of the sheep fetus. These data suggest that fetal PRL may play a role in the growth and maturation of the fetal liver which occurs before birth.

Hepatic Prolactin Receptor Gene Expression Increases in the Sheep Fetus Before Birth and After Cortisol Infusion

Hepatic Prolactin Receptor Gene Expression Increases in the Sheep Fetus Before Birth and After Cortisol Infusion

Expression of Prolactin Receptors in Human Osteosarcoma Cells

The expression of prolactin receptor (PRL-R) mRNA was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) combined with Southern analysis in total RNA extracts from two human osteosarcoma cell lines (MG-63 and Saos-2). The level of PRL-R transcript was significantly enhanced in cells cultured in the presence of 1,25-(OH)2vitaminD3(10−7M) and to a lesser extent in the presence of dexamethasone (10−6M). This first demonstration of PRL-R gene expression in osteoblast-like cells supports the hypothesis of a direct action of prolactin in bone cells, which is further strongly suggested by the stimulatory effect of 1,25-(OH)2vitaminD3and dexamethasone on PRL-R mRNA level in these cells.

Inhibition of Prolactin Receptor Gene Expression by 2,3,7,8-Tetrachlorodibenzo-p-dioxin in MCF-7 Human Breast Cancer Cells

Treatment of MCF-7 human breast cancer cells with 10 nM2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) did not decrease prolactin receptor (PRLR) binding. In contrast, PRLR mRNA levels were significantly decreased within 12 h after treatment with TCDD and persisted for up to 48 h. The effects of TCDD on PRLR mRNA levels were inhibited by the aryl hydrocarbon (Ah) receptor antagonist α-naphthoflavone and were not observed in Ah nonresponsive benzo[a]pyrene-resistant MCF-7 cells. These results suggest that the effects of TCDD were mediated through the Ah receptor. After treatment of MCF-7 cells with 10 nM17β-estradiol (E2), there was a 2.3-fold increase in PRLR mRNA levels, and in cells cotreated with E2 plus TCDD, there was a 72% decrease in E2-induced PRLR mRNA levels. Previous studies have showed that TCDD also effects estrogen receptor (ER) binding and mRNA levels through the aryl hydrocarbon receptor pathway; however, the effects of TCDD on PRLR levels and binding in MCF-7 cells were different from those previously observed for ER.

Differential promoter usage in prolactin receptor gene expression: hepatocyte nuclear factor 4 binds to and activates the promoter preferentially active in the liver.

Characterization of the rat PRL receptor (PRLR) gene has revealed three separate untranslated exon 1 sequences, each associated with a different transcription start site and 5'-flanking sequence. We show by RT-PCR that exon 1A is expressed primarily in liver but is also detectable in ovary and mammary gland. Exon 1B expression is observed exclusively in the ovary, whereas exon 1C is expressed in all three tissues. Transient transfection of luciferase reporter constructs containing parts of the 5'-flanking regions (0.3-1.1 kb) of exon 1A, 1B, and 1C, respectively, showed activity of the 1A promoter in Chinese hamster ovary (CHO) cells, the human hepatoma cell line, HepG2, and the rat hepatoma cell line, H4II, which was 10- to 14-fold increased compared with the activity of the promoter-less luciferase vector. No activity of the 1A promoter was detected in the human mammary cell line, T-47D. Relative to a vector containing the Simian virus 40 (SV40) promoter, the 1A promoter had 20% activity in H4II cells and 1-3% activity in CHO and HepG2 cells. The 1B promoter produced a 6.1-fold increase of luciferase activity in CHO cells (approximately 2% of the SV40 promoter), whereas no significant activity was detected in HepG2, H4II, and T-47D cells. The 1C promoter was strongly active in T-47D cells (approximately 64-fold over control) and moderately active in the other cell lines tested (9- to 13-fold over control). 5'-Deletion analysis of the 1A promoter revealed that a fragment containing -83/ +81 bp, relative to the transcription start site, was sufficient to drive transcription in hepatoma cells, whereas this construct was inactive in CHO cells. Cotransfection of CHO cells with the -83/+81 construct and an expression vector encoding the liver-enriched transcription factor, hepatocyte nuclear factor 4 (HNF4), revealed a dose-dependent transactivation of the proximal 1A promoter with a maximal stimulation of approximately 10-fold. Electrophoretic mobility shift assays showed binding of HNF4 to the sequence -14/+24 of the 1A promoter, and mutational analysis revealed that the sequence GGGCAAAGTCA at position +11/+21 is required for this binding. We conclude that the 1A, 1B, and 1C promoters of the PRLR gene are used in a cell type- dependent way that may play a role in differential hormonal regulation of the gene. In particular, we have shown that HNF4 operates on the proximal 1A promoter and may be responsible, in combination with other factors, for the increased activity of this promoter in adult female liver.

Differential promoter usage in prolactin receptor gene expression: hepatocyte nuclear factor 4 binds to and activates the promoter preferentially active in the liver

Differential promoter usage in prolactin receptor gene expression: hepatocyte nuclear factor 4 binds to and activates the promoter preferentially active in the liver

Developmental aspects of prolactin receptor gene expression in fetal and neonatal mice.

The prolactin receptor (PRL-R), a member of the hematopoietin cytokine receptor superfamily, is widely distributed among mammalian tissues. To understand better the potential sites of action and onset of potential PRL responsiveness, the developmental distribution pattern of PRL-R mRNA expression in fetal and neonatal mice was examined. Fetal mouse tissues were collected at distinct stages from timed pregnancies. Following extraction of total RNA, onset of gene expression was evaluated via reverse transcription-polymerase chain reaction (RT-PCR) and Southern hybridization was employed for verification. Expression of PRL-R mRNA was first observed on day 14 in the liver and cranium and on day 15 in the kidney, lung and thymus gland. Pituitary and adrenal glands were positive for PRL-R at day 18 of gestation through to day 1 of postnatal life. Neither whole fetuses prior to day 14 (days 10-13) of gestation nor skin and bladder tissues from 2-day-old mice generated detectable RT-PCR signals for PRL-R. The presence of PRL-R mRNA in fetal thymus and spleen tissues suggests a possible role for PRL in the development of the immune system. Prolactin may act directly on the pituitary to influence its own secretion and/or that of other pituitary-derived factors, as evidenced by the presence of PRL-R mRNA in the pituitary glands of fetal and 1-day-old mice. These data are the first to show the presence of PRL-R gene expression in various organ systems in fetal mice and suggest that PRL is among several factors necessary to coordinate developmental activities.

Prolactin receptor gene expression in specific hypothalamic nuclei increases with age.

Increasing prolactin levels or increasing responsiveness to prolactin may contribute to reproductive aging by influencing the secretory patterns of hypothalamic GnRH, pituitary gonadotropins, and/or ovarian steroids. Some studies have documented changes in the levels of prolactin in peripheral plasma. The goal of this study was to determine whether prolactin receptor mRNA levels in the brain change with aging, which may lead to increasing responsiveness to prolactin. Young (2-4 months) and middle-aged (9-11 months), demonstrating 3 consecutive estrous cycles, and old (16-19 months) and very old (20-21 months) rats, exhibiting repeated pseudopregnancies, were bilaterally ovariectomized. They were implanted with Silastic capsules containing estradiol-17 beta one week later, and killed 2 days after capsule implantation. Changes in prolactin receptor gene expression were assessed using in situ hybridization. The level of prolactin receptor mRNA in choroid plexus, periventricular area of the preoptic nucleus, and arcuate nucleus increased significantly by the time the animals were old. In the lateral ventromedial nucleus, prolactin receptor gene expression did not change significantly during aging, even in the oldest group of rats. These findings suggest that changes in the prolactin receptor gene may influence the ability of prolactin to exert effects and may allow older animals to be more responsive to prolactin than young rats.

Multiple and Tissue-specific Promoter Control of Gonadal and Non-gonadal Prolactin Receptor Gene Expression

Prolactin receptors (PRLRs) are widely expressed, and multiple mRNA transcripts encoding PRLRs are present in prolactin target tissues. The molecular basis for the control of the PRLR gene expression is currently unknown. Analyses of the 5'-untranslated regions of PRLR mRNAs expressed in gonadal and non-gonadal tissues and their genomic organization revealed three alternative first exons designated as E11, E12, and E13. Each of these exons is alternatively spliced to a common noncoding exon (exon 2, nucleotides -115 to -56) that precedes the third exon containing the translation initiation codon. Alternative utilization of exons E11, E12, and E13, as well as alternative splicing of exon 2, generates multiple 5'-untranslated regions in PRLR transcripts. These alternative first exons (E11, E12, and E13) were found to be utilized in a tissue-specific manner in vivo. E11 is predominantly expressed in the ovary, E12 is specifically expressed in the liver, and E13 is expressed as a predominant form in the Leydig cell and as a minor form in the ovary and liver. Genomic 5'-flanking regions containing the three putative PRLR gene promoters (PI, PII, and PIII) that initiate the transcription of E11, E12, and E13, respectively, were identified. E11 was found to initiate from a single site at -549, E12 from multiple sites at -405, -461, and -506, and E13 from two major sites at -340 and -351. These findings indicate that multiple promoters control transcription of the PRLR gene and provide a molecular basis for the differential regulation of PRLR expression in diverse tissues.

Prolactin receptor gene expression in rat splenocytes and thymocytes from birth to adulthood

In vivo and in vitro studies have indicated that the anterior pituitary hormone prolactin (PRL) is an immunoregulator and functions in the development of the neonatal immune system. In this study, prolactin receptor (PRL-R) expression from birth to adulthood as well as the effect of milk ingestion on the PRL-R expression were examined in splenocytes and thymocytes of neonatal rats. Three approaches were taken to measure PRL-R expression: (i) polymerase chain reaction (RT-PCR); (ii) antibody to PRL-R and Western blotting; (iii) antibody to PRL-R and flow cytometry. RT-PCR analysis revealed the short and long form of PRL-R mRNA in both spleen and thymus at every age tested. However, the long form of PRL-R mRNA was always more abundant than that of the short form. In addition, antipeptide antibody against the long form of PRL-R recognized 84 and 42 kD proteins in the spleen, but only the 84 kD protein in the thymus. A monoclonal antibody U6 recognized 38 and 40 kD proteins in both the spleen and thymus. Although the mRNA level of PRL-R was relatively low at birth and increased with age in both the spleen and thymus, the levels of protein bands detected with both antibodies correlated with development in the spleen; whereas the levels remained steady in the thymus. Therefore, we concluded that the expression of PRL-R at the protein level is developmentally regulated in the spleen but not in the thymus. Finally, milk ingestion in the first seven hours decreased the percentage of cells expressing cell surface PRL-R, suggesting that milk-borne PRL may have a direct effect on lymphocytes.

Ontogenesis of prolactin receptor gene expression in the rat olfactory system: potential roles for lactogenic hormones in olfactory development.

The PRL receptor (PRLR) is expressed at very low levels in the olfactory bulb of the adult rat but is detected in abundance in the olfactory epithelium and olfactory bulb of the fetal rat in late gestation. To explore potential roles for the lactogenic hormones in olfactory differentiation and development, we have used in situ hybridization and immunohistochemistry to examine the ontogeny of PRLR gene expression in the rat olfactory system. At midgestation (embryonic day 12.5), messenger RNAs (mRNAs) encoding the long and short isoforms of the rat PRLR were detected in the medial and lateral nasal processes, the epithelial lining of the olfactory pit, and the neuroepithelium lining the cerebral ventricles in the region of the rhinencephalon. PRLR mRNA was also expressed prominently in the frontonasal mesenchyme and in mesenchymal tissue underlying the developing brain and overlying the pontine flexure in the interpeduncular fossa. The distribution of PRLR immunoreactivity was similar to that of PRLR mRNA, indicating that the PRLR gene is translated to that of PRLR mRNA, indicating that the PRLR gene is translated to lactogenic binding protein in the rat embryo in vivo. With advancing gestation, the PRLR was expressed intensely, although discontinuously, in the olfactory epithelium and was detected in the cartilage primordia of the ethmoid, sphenoid, temporal, and mandibular bones. PRLR expression in the vomeronasal organ was confined to the luminal epithelial surface. PRLR mRNA and immunoreactive protein were first detected in the olfactory bulb on embryonic day 18. PRLR expression was most intense initially in the periventricular neuroepithelium; subsequently, robust staining of the mitral and tufted cell neurons became apparent, accompanied by intense PRLR expression in the sensory neuronal cell bodies of the olfactory epithelium. By postnatal day 5, the PRLR was expressed in abundance in mitral and tufted cells of the olfactory bulb and in neuronal cell bodies of the anterior olfactory nucleus and the piriform cortex. PRLR mRNA was also detected in the mitral cells of the olfactory bulb of the lactating rat, although at levels far lower than those in the fetal or neonatal rat. The expression of the PRLR in the olfactory system of the fetal and neonatal rat implicates novel roles for the lactogenic hormones in olfactory differentiation and development and may provide new mechanisms by which the lactogens may regulate neonatal behavior and maternal-infant interactions.

Ontogenesis of prolactin receptor gene expression in the rat olfactory system: potential roles for lactogenic hormones in olfactory development

Ontogenesis of prolactin receptor gene expression in the rat olfactory system: potential roles for lactogenic hormones in olfactory development