Estrogen induces phosphorylation of prolactin through p21-activated kinase 2 activation in the mouse pituitary gland.

A phosphorylated prolactin is a kind of modified prolactin, which is produced through phosphorylation of native prolactin (PRL) by p21-activated kinase 2 (PAK2) in secretory granules in lactotrophs. Phosphorylated prolactin is involved in the regulation of the estrous cycle and in apoptosis in cancer cells, which seem to have important physiological and pathological roles, respectively. In previous research, it has been reported that estrogen induced the phosphorylation of prolactin in the mouse pituitary gland. However, the relationship between estrogen and PAK2 in the production of phosphorylated PRL has not been clarified yet. In order to examine whether PAK2 is involved in PRL phosphorylation by estrogen, we analyzed PAK2 protein levels in mice and phosphorylated prolactin levels in mouse pituitary cells by western blot analysis. The ratio of phosphorylated PAK2/total PAK2 was increased in estrogen implanted mice, but PAK2 protein and gene expression levels were decreased. In addition, the ratio of phosphorylated prolactin/non-phosphorylated prolactin was decreased in primary pituitary cells with introduced siPAK2. These findings suggest that estrogen could induce the phosphorylation of PRL through PAK2 activation. Therefore, this study contributes to better understanding of the mechanism of phosphorylated PRL production in physiological and pathological conditions associated with estrogen.

Inhibition of epidermal growth factor receptor stimulates prolactin expression in primary culture of the mouse pituitary gland.

The roles of epidermal growth factor (EGF) in the regulation of prolactin (PRL) gene expression in the normal pituitary gland remain poorly understood. In the present study, the effects of EGF and an inhibitor of the EGF receptor, erlotinib, on PRL gene expression were examined both in the pituitary tumour cell line GH3 and in a primary culture of the mouse pituitary gland under similar experimental conditions. The results showed that EGF stimulated PRL expression in GH3 cells, but not in normal cells. Erlotinib was found to counteract EGF in GH3 cells inhibiting the PRL expression enhanced by EGF. By contrast, erlotinib induced an elevation in the PRL mRNA levels in the primary culture of the adult pituitary gland and the initiation of PRL production in the culture of the foetal pituitary gland in which PRL production had not yet occurred. Western blot analyses showed that EGF induced and erlotinib inhibited the activation of extracellular regulated protein kinase equally in GH3 and normal cells. These results suggest that the consequences of EGF receptor activation in normal PRL cells contradict those in adenomatous PRL cells.

16 kDa vasoinhibin binds to integrin alpha5 beta1 on endothelial cells to induce apoptosis.

Many functions of vasoinhibins have been reported, but its receptor has not been clarified yet. Vasoinhibins, 11-18 kDa N-terminal fragments of prolactin, have anti-angiogenic activity and act on endothelial cells to induce apoptosis and to inhibit migration and proliferation, which are opposite to the effects of prolactin. Although vasoinhibins bind to the prolactin receptor, its binding activity is very weak compared to prolactin. Therefore, in this study, we evaluated the binding activity between 16 kDa vasoinhibin and integrin beta1, alpha5 beta1, alpha1 beta1 and alphaV beta3 to identify a specific receptor for vasoinhibins. Moreover, we examined whether 16 kDa vasoinhibin induced apoptosis through integrin beta1 and alpha5 beta1 in endothelial cells. In this study, binding assays and co-immunoprecipitation experiments demonstrated that 16 kDa vasoinhibin could bind strongly to integrin beta1 and alpha5 beta1. Moreover, neutralizing with integrin beta1 and alpha5 beta1 antibody could inhibit 16 kDa vasoinhibin-induced apoptosis in endothelial cells. These findings suggest that vasoinhibins can act on endothelial cells through integrin alpha5 beta1 to induce apoptosis.

Vasoinhibin, an N-terminal Prolactin Fragment, Directly Inhibits Cardiac Angiogenesis in Three-dimensional Heart Culture.

Vasoinhibins (Vi) are fragments of the growth hormone/prolactin (PRL) family and have antiangiogenic functions in many species. It is considered that Vi derived from PRL are involved in the pathogenesis of peripartum cardiomyopathy (PPCM). However, the pathogenic mechanism of PPCM, as well as heart angiogenesis, is not yet clear. Therefore, the aim of the present study is to clarify whether Vi act directly on angiogenesis inhibition in heart blood vessels. Endothelial cell viability was decreased by Vi treatment in a culture experiment. Furthermore, expression of proangiogenic genes, such as vascular endothelial growth factor, endothelial nitric oxide synthase, and VE-cadherin, were decreased. On the other hand, apoptotic factor gene, caspase 3, and inflammatory factor genes, tumor necrosis factor α and interleukin 6, were increased by Vi treatment. In three-dimensional left ventricular wall angiogenesis assay in mice, Vi treatment also inhibited cell migration, neovessel sprouting, and growth toward collagen gel. These data demonstrate that Vi treatment directly suppresses angiogenesis of the heart and support the hypothesis that Vi induce PPCM.

Vasoinhibins, N-terminal mouse prolactin fragments, participate in mammary gland involution.

Vasoinhibins are a family of peptides that act on endothelial cells to suppress angiogenesis and promote apoptosis-mediated vascular regression. Vasoinhibins include the N-terminal fragments from prolactin (PRL), GH, and placental lactogen. One of the vasoinhibins, the N-terminal PRL fragment of 16 kDa, is generated by the lysosomal representative protease cathepsin D (Cath D). Because the normal growth and involution of the mammary gland (MG) are profoundly affected by the expansion and regression of blood vessels and also because PRL stimulates the growth and differentiation of MG, we proposed that intact PRL produced during lactation contributes to MG angiogenesis and increased blood flow, whereas during involution, the N-terminal PRL fragment would have proapoptotic effects on mammary epithelial cells (MECs). Therefore, we investigated the production of the N-terminal PRL fragment and its direct effect on the MG. Mouse PRL (mPRL) was proteolytically cleaved by Cath D between amino acids 148 and 149. N-terminal PRL fragment and Cath D expression increased during MG involution. Furthermore, incubation of MG fragments and MCF7 with recombinant 16 kDa mPRL revealed a proapoptotic effect in MECs. Ectopic mPRL in MECs was cleaved to 16 kDa PRL by Cath D in the MG lysosomal fraction. The majority of PRL derived from pituitary gland was cleaved to 16 kDa PRL in culture medium. Therefore, N-terminal PRL fragment increases during the involution period, has a proapoptotic effect on MECs, and is mainly generated by secreted Cath D in the extracellular space of MG.

Analysis of prolactin gene expression and cleaved prolactin variants in the mouse testis and spermatozoa.

Prolactin (PRL) has long been known to be a hormone responsible for mammary gland development and lactation in females, whereas its role in males is still unclear. Thus, we investigated male mouse (m) PRL protein and mRNA expression in spermatozoa at various differentiation stages in the testes. Quantitative RT-PCR and in situ hybridization detected the expression of PRL not only in Leydig cells but also in germ cells, in particular in spermatogonia. The nucleotide sequence of testis PRL mRNA was the same as that in the pituitary. The mPRL was detected in Leydig cells and in round and elongated spermatids of the testes by immunohistochemistry. Immunoblotting detected 2 forms of mPRL in the testes, one form was 23-kDa PRL, and the other form was smaller than full-length PRL. Based on these results, we focused on N-terminal cleaved PRL to determine its involvement in spermatogenesis. Immunohistochemistry using two sets of antibodies, one that recognized full-length PRL and N-terminal cleaved PRL and another that recognized full-length PRL and C-terminal cleaved PRL, suggested that intact PRL was localized in the nucleus of round spermatids, while N-terminal cleaved PRL variants were localized in the Golgi apparatus of the sperrmatid nuclei of round spermatids, cytoplasms of elongated spermatids and in the spermatozoa tails. These findings suggest that PRL is ectopically expressed in the spermiogenesis and spermatogenesis and that cleaved PRL variants were localized in the Golgi apparatus of spermatids and in spermatozoa tails.

Hormonal regulation of prolactin cell development in the fetal pituitary gland of the mouse.

The developmental process of prolactin (PRL) cells in the fetal pituitary gland was studied in mice. Although PRL cells were hardly detectable in the pituitary gland of intact fetuses, a treatment with 17beta-estradiol (E(2)) in vitro induced a number of PRL cells that varied drastically in number depending on the stage of gestation with a peak at embryonic d 15. This effect was specific to E(2), with epidermal growth factor, insulin, and forskolin failing to induce PRL cells. Although both estrogen receptor (ER)alpha and ERbeta were expressed in the fetal pituitary gland, the results from ER knockout models showed that only ERalpha mediates E(2) action on PRL cells. A few PRL cells were observed in ERalpha-deficient mice as well as in their control littermates, suggesting that estrogen is not required for the phenotype determination of PRL cells. Unexpectedly, the effect of E(2) on the induction of PRL cells in vitro was diminished after embryonic d 15. Present results suggest that the exposure of fetal PRL cells to glucocorticoids (GCs) results in a reduction of sensitivity to E(2). The mechanism underlying the down-regulation of estrogen sensitivity by GCs was found not to be down-regulation of ER levels, induction of annexin 1, a GC-inducible inhibitor of PRL secretion, or a decrease in the number of PRL precursors by apoptosis. The effect of GCs appeared within 2 h and did not require a de novo protein synthesis. GCs are considered to be involved in the mechanisms of silencing pituitary PRL in gestation possibly through a novel mechanism.

Estrogen regulates the serum level of phosphorylated prolactin in mice.

Phosphorylated prolactin (PPRL) is considered to be the most quantitatively important post-translationally modified form of prolactin (PRL) in rodents. We recently detected two different types of PPRL in the mouse pituitary gland; one was phosphorylated at serine and the other was phosphorylated at serine/threonine. Furthermore, we showed that there are obvious differences in the ratios between PPRLs and non-phosphorylated PRL in the pituitary gland based on age and sex and that estrogen influences PRL phosphorylation at serine in female mice. In the present study, we examined whether estradiol (E2) increases serine PPRL in the male pituitary gland in the same manner as in the female pituitary gland and examined whether PPRL is released into serum. We first determined the relative amounts of intrapituitary PPRLs in male mice under different pharmacological conditions that increased PRL secretion. The results indicated that treatment with E2 increases serine PPRL. We then performed two-dimensional electrophoresis and immunoblotting analysis after immunoprecipitation with anti-mouse PRL antibody using male and female sera under different pharmacological conditions that increased PRL secretion. The results of this experiment indicated that there were PRLs phosphorylated at serine and serine/threonine in the female serum but not in the male serum. The levels of PPRLs in sera were greatly increased with the E2 treatment for both male and female sera. Furthermore, we examined the effect of E2 on PPRL synthesis in cultured male pituitary glands. In this experiment, we observed increased serine PPRL synthesis and stronger immunohistochemical staining of PRL cells with E2 treatment. These findings suggested that serine PPRL synthesis and secretion were influenced by estrogen.

The effect of estrogen on phosphorylation of prolactin in the mouse pituitary gland.

Several studies have indicated that prolactin (PRL) assumes oligomeric, proteolytically cleaved, phosphorylated and glycosylated forms. Phosphorylated PRL (PPRL) is considered to be the most important posttranslationally modified form in the rat. In the present study, we examined whether or not PRL is present in the mouse pituitary gland in the phosphorylated form. Mouse pituitary PRL was digested with acid phosphatase, resolved by two-dimensional gel electrophoresis, stained with Coomassie brilliant blue, and then immunoblotted against the anti-PRL, anti-phosphoserine and anti-phosphothreonine antibodies. We also examined whether PRL is phosphorylated by protein kinases and semi-quantified the ratios of PPRL to PRL in the pituitary gland. The results indicated that three types of PRL are present in the pituitary glands of both male and female mice. One was non-phosphorylated (isoform 1), and the other two were immunoreactive to anti-phosphoserine (isoform 2) and/or anti-phosphothreonine (isoform 3) antibodies. The ratio between isoforms 2 and 1 of the 30-day-old female mice was higher than that of the 20-day-old female mice. However, the ratios among the three isoforms in the male pituitary glands did not differ with age. The ratio of PPRL to isoform 1 was obviously reduced after ovariectomy (OVX), and it recovered with estrogen replacement. These results suggest that estrogen influences PRL phosphorylation in female mice.

Expression profiling of mouse placental lactogen II and its correlative genes using a cDNA microarray analysis in the developmental mouse placenta.

The placenta is a highly differentiated organ essential for embryonic growth and development. In order to search for key molecules that are associated with mouse placental lactogen II (mPL-II) gene expression, we applied mouse cDNA microarray analysis to RNAs extracted from placentae on days 10, 12, 14, 16 and 18 of pregnancy. Changes in gene expression were categorized between days 10 and 12, 12 and 14, 14 and 16 and 16 and 18 of pregnancy. After microarray analysis, which had a minimum detectable fold change for differential expression of 2, we selected 10 genes, Apoa2, Apoc2, Ceacam14, Creg1, Fmo1, Igf2, Slc2a1, Spink3, Spi1-1 and Tpbpa, exhibiting a expression pattern similar to the mPL-II gene. Furthermore, we performed real-time PCR analysis and in situ hybridization (ISH) to find correlative expression genes for the mPL-II gene. From these results, we identified a resemblance in gene expression between mPL-II and Igf2 and selected these genes for performance of double-fluorescence immunohistochemical staining. We colocalized these proteins in labyrinthine trophoblast cells. These results strongly suggest that the expression of mPL-II and Igf2 is highly related to placental development in mice. This large-scale identification of genes regulated during placentogenesis assists in further elucidation of the molecular basis of extraembryonic development and function.

Cathepsin gene expression in mouse placenta during the latter half of pregnancy.

Gene expressions and their interaction are complex and have not been definitely clarified in the placenta. To identify interactions of gene products previously not studied, we applied cDNA subtraction analyses to the placenta between days 12 and 16, days 12 and 14, days 14 and 16 of pregnancy. Among subtracted cDNAs cathepsin M, Q and R in PECs were specifically identified on days 14 and 16 pregnancy. All of these gene expressions exhibited a similar pattern to the mPL-II gene expression determined by northern blot and RT-PCR analyses. By means of in situ hybridization, these mRNAs were localized in the basal and labyrinth zones of the placenta on day 16 of pregnancy. Double staining studies of cathepsin Q or cathepsin R mRNA by in situ hybridization followed by immunohistochemical staining of mPL-II in the same section revealed that signals for cathepsin Q and cathepsin R mRNAs were colocalized in mPL-II immunopositive trophoblast cells in the basal and labyrinth zones of the placenta on day 16 of pregnancy. Possible association of cathepsins with mPL-II may play important roles in placental functions during the latter half of pregnancy in mice.

Bisphenol A affects glucose transport in mouse 3T3-F442A adipocytes.

Recently, environmental chemicals have appeared in daily human life, and these chemicals have been incidentally taken in by humans. The serum concentrations of some of these chemicals have been found to be associated with the onset and incidence rate of diabetes mellitus. It has been suggested that one of the environmental chemicals, bisphenol A (BPA), has hormone-like activity. It has also been demonstrated that some hormones affect insulin resistance and fat distribution in the body. To study the effects of these environmental chemicals on glucose metabolism, the effect of BPA on glucose transport in mouse 3T3-F442A adipocytes was investigated. The 3T3-F442A adipocytes were incubated with various concentrations of BPA in a medium. Deoxyglucose uptake assay was performed with and without insulin. Immunoblot analysis was performed with a glucose transporter (GLUT) 4-specific antibody and antiphosphotyrosine antibody. The BPA treatment enhanced basal and insulin-stimulated glucose uptake, and caused an increased amount of GLUT4 protein. Thus, the enhanced glucose uptake resulting from the BPA treatment was at least partially due to the increased amount of GLUT4. Tyrosine phosphorylation of insulin receptor substrate-1 with insulin stimulation was not significantly affected. In conclusion, it was demonstrated that BPA, one of the chemicals that we intake incidentally, affects the glucose transport in adipocytes, and also that the environmental chemicals may be identified as one of the environmental factors that affect diabetes and obesity.

Prolactin gene expression in the mouse nipple.

In this study, we attempted to examine the presence of prolactin (PRL) messenger ribonucleic acid (mRNA) and protein in the mouse nipple and mammary gland in pregnancy and lactation. PRL-like substances were found by immunohistochemistry using an antibody against the mouse PRL (mPRL) in the sebaceous gland cells of the nipple during late pregnancy and lactation, and the cistern of alveoli in mammary glands during lactation. Western blot analysis of proteins extracted from the nipple and the mammary gland showed immunoreactive bands corresponding to molecular weights of approximate 16 kDa and 32 kDa, respectively. The expression of mRNA for mPRL in the nipple and mammary gland during late pregnancy and lactation was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR), Southern blotting, and nucleotide sequence analyses. These results suggest that mPRL mRNA and its translation product are synthesized in the mouse nipple.

A composite hormone response element regulates transcription of the rat GHRH receptor gene.

To further elucidate the molecular mechanisms underlying the transcriptional regulation of the GHRH receptor (GHRH-R) gene, hormonal regulation of the promoter activity of this gene was examined. An approximately 3-kb genomic fragment spanning the promoter region of the gene was sequenced and the transcription start site was determined by RT-PCR and RNase protection assay. A major start site was localized at -105 (relative to the translation initiation codon, ATG), and a pit-1 binding sequence characteristic of pituitary specific genes was found at -155 to -146. Deletion and mutation studies demonstrated this site to be functional. In the presence of dexamethasone, the GHRH-R promoter (from -2935 to -11) directed luciferase expression in MtT-S cells, a somatotropic cell line, but not in the PC12 cells that normally do not express GHRH-R. While T(3), all trans-RA, and 9cis-RA alone weakly enhanced the reporter gene expression, each of these substances was found to act as a synergistic enhancer in the presence of dexamethasone. Additional deletion and mutation analyses demonstrated a functional RA response element at -1090 to -1074. Two functional glucocorticoid response elements and a T(3) response element were found in an 80-bp 5'-flanking sequence of the pit-1 site. Interestingly, it is suggested that the 6-bp half-site AGGACA (from -209 to -204) functions as a 3'-half-site of T(3) response element as well as a 5'-half-site of one of the glucocorticoid response elements.