Prolactin cooperates with loss of p53 to promote claudin-low mammary carcinomas.

TP53 is one of the most commonly mutated genes in cancer. In breast cancer, it is mutated in about 40% of primary clinical tumors and is associated with poor survival. The mammotrophic hormone, prolactin (PRL), and/or its receptor are also expressed in many breast cancers, and accumulating epidemiologic data link PRL to breast cancer development and progression. Like TP53 mutations, evidence for PRL activity is evident across several molecular cancer subtypes, and elevated PRL expression and loss of p53 have been observed in some of the same clinical tumors. In order to examine the interaction of these factors, we used genetically modified mouse models of mammary-specific p53 loss and local overexpression of PRL. We demonstrated that mammary PRL decreased the latency of tumors in the absence of p53, and increased the proportion of triple-negative claudin-low carcinomas, which display similarities to human clinical metaplastic carcinomas. Moreover, PRL/p53(-/-) carcinomas displayed higher rates of proliferation and more aggressive behavior. Transcripts associated with cell cycle progression, invasion and stromal reactivity were differentially expressed in carcinomas that developed in the presence of elevated PRL. PRL/p53(-/-) carcinomas also exhibited selectively altered expression of activating protein-1 components, including higher levels of c-Jun and FosL1, which can drive transcription of many of these genes and the epithelial-mesenchymal transition. The ability of PRL to promote claudin-low carcinomas demonstrates that PRL can influence this subset of triple-negative breast cancers, which may have been obscured by the relative infrequency of this cancer subtype. Our findings suggest novel therapeutic approaches, and provide a preclinical model to develop possible agents.

Janus kinase 2 is required for the initiation but not maintenance of prolactin-induced mammary cancer.

The prolactin receptor (PRLR), its associated Janus kinase 2 (Jak2) and the signal transducer and activator of transcription 5 (Stat5) are essential for normal mammary gland development. Owing to the upregulation of the PRLR and the local synthesis of its ligand in neoplastic cells, it has been proposed that PRL can act as a local growth factor in human breast cancers. This notion is supported by experimental evidence in transgenic mice, which showed that the mammary-specific expression of PRL contributes to carcinogenesis in vivo. To assess the importance of Jak2/Stat5 signaling during mammary cancer initiation and progression, we generated a PRL-induced mammary cancer model that allows the functional ablation of the Jak2 gene in the mammary epithelium before and after neoplastic transformation. Collectively, the results of this study show that the functional ablation of Jak2 protects against the onset of PRL-induced mammary tumorigenesis, suggesting that targeting this kinase is a relevant strategy for mammary cancer prevention. Surprisingly, Jak2 deficiency did not affect the growth and survival of PRL-induced mammary cancer cells in culture and in vivo. Consequently, Jak2 cannot be a sole therapeutic target to treat the established disease. PRL-induced mammary cancers exhibited an upregulation of ErbB2 and other ErbB receptor tyrosine kinases that may supersede the functionality of PRLR signaling through Jak2.

Stat5 activation inhibits prolactin-induced AP-1 activity: distinct prolactin-initiated signals in tumorigenesis dependent on cell context.

The essential role of prolactin (PRL) in normal mammary gland growth and differentiation has implicated this hormone in the development and progression of breast cancer. Although Stat5 is the best-characterized mediator of PRL signals, PRL also activates multiple other signals, whose roles in normal and pathologic processes are not well understood. We have shown that PRL stimulates activating protein-1 (AP-1) activity in breast cancer cells, and can cooperate with estradiol in this pathway. AP-1 modulates many processes critical for carcinogenesis, including cell proliferation, survival, transformation, invasion and angiogenesis, and is elevated in many neoplasms, including breast tumors. Here, we investigated the relationship between PRL signals to AP-1 and Stat5. We found that PRL activation of Stat5a and Stat5b, but not Stat1 or Stat3, reduced PRL signals to AP-1, without altering estradiol-induced AP-1 activity. The truncation mutant, Stat5/Delta53C, but not Stat5Y699F, was an effective inhibitor, consistent with a requirement for Stat5 dimerization and nuclear accumulation, but not its C-terminal transactivation activity. The association of Stat5 with AP-1 proteins suggests that this underlies the inhibition. Predictably, the ability of PRL to activate Stat5 and AP-1 was inversely related in mammary cell lines. Further, reduction of Stat5 protein with siRNA in T47D cells, which contain elevated Stat5, increased PRL-induced AP-1 signals, transcripts for the AP-1 target, matrix metalloproteinase-2 and associated invasive behavior. This study points to the importance of cell context in determining the spectrum of PRL-induced actions, which is critical for understanding the contributions of PRL to breast cancer.

Estrogen receptor-positive mammary tumorigenesis in TGFalpha transgenic mice progresses with progesterone receptor loss.

We characterized the novel NRL-transforming growth factor alpha (NRL-TGFalpha) transgenic mouse model in which growth factor - steroid receptor interactions were explored. The NRL promoter directs transgene expression to mammary ductal and alveolar cells and is nonresponsive to estrogen manipulations in vitro and in vivo. NRL-TGFalpha mice acquire proliferative hyperplasias as well as cystic and solid tumors. Quantitative transcript analysis revealed a progressive decrease in estrogen receptor alpha (ER) and progesterone receptor (PR) mRNA levels with tumorigenesis. However, ER protein was evident in all lesion types and in surrounding stromal cells using immunohistochemistry. PR protein was identified in normal epithelial cells and in very few cells of small epithelial hyperplasias, but never in stromal or tumor cells. Prophylactic ovariectomy significantly delayed tumor development and decreased incidence. Finally, while heterozygous (+/-) p53 mice did not acquire mammary lesions, p53+/- mice carrying the NRL-TGFalpha transgene developed ER negative/PR negative undifferentiated carcinomas. These data demonstrate that unregulated TGFalpha expression in the mammary gland leads to oncogenesis that is dependent on ovarian steroids early in tumorigenesis. Resulting tumors resemble a clinical phenotype of ER+/PR-, and when combined with a heterozygous p53 genotype, ER-/PR-.

Temporospatial expression of placental lactogen and prolactin-related protein-1 genes in the bovine placenta and uterus during pregnancy.

The anatomical location of binucleate cells (BNC) influences protein expression but not steroid synthesis in ruminants. In order to determine if BNC in disparate locations differentially express bovine placental lactogen (bPL) and prolactin-related protein-1 (bPRP-1), we quantitated bPL and bPRP-1 transcripts in placentomal (cotyledonary, caruncular) and interplacentomal (intercotyledonary, intercaruncular) tissues throughout pregnancy in the bovine using real-time reverse transcription PCR (RT-PCR) and in situ hybridization. Levels of both bPL and bPRP-1 transcripts at peri-implantation were significantly higher (P < 0.01) in the fetal membrane than in caruncular and intercaruncular tissues. Thereafter, mRNA for these related proteins demonstrated different spatial as well as temporal patterns of expression. Levels of bPRP-1 transcripts peaked at day 60 of pregnancy. Between day 60 and 100, bPRP-1 transcripts fell by approximately sevenfold (P < 0.01) in cotyledonary and intercotyledonary tissues, and fourfold in caruncular (P < 0.01) tissue. Levels of bPRP-1 transcripts remained low in the cotyledonary, intercotyledonary, and caruncular tissues until peripartum. In contrast, bPL expression in placentomes increased with progression of gestation (P < 0.01), but decreased in interplacentomal tissue around peripartum. To conclude, disparate patterns of bPRP-1 and bPL genes are transcribed in the placentomal and interplacentomal tissues during gestation in the bovine, suggesting that these prolactin-like hormones serve distinct functions and are regulated differently in the uteroplacental unit in this species.

Expression of prolactin-related protein I at the fetomaternal interface during the implantation period in cows.

The bovine placenta secretes multiple molecules during implantation and placentation, many of which are produced by binucleate cells. In this study, production of prolactin-related protein I (PRP-I), a member of the non-classical prolactin-related family, was investigated during the implantation period in cows. Expression of bovine PRP-I (bPRP-I) in the placentome was examined during the preimplantation (days 17-19), implantation (days 20-25) and post-implantation (days 30-60) periods by immunohistochemistry, immunofluorescence and in situ hybridization. During the preimplantation period, both bPRP-I and bovine placental lactogen (bPL) were undetectable in trophoblastic cells. Both bPRP-I mRNA and protein appeared first at day 20 of gestation in trophoblastic binucleate cells and multinuclear cells that might migrate into the endometrium and fuse to epithelium; however, no bPL was detected in binucleate cells at this time. After implantation, on day 30, both bPRP-I and bPL were detected in binucleate cells and were co-expressed in the same cells. These data indicate that bPRP-I may play a role before implantation and that bPRP-I may be an excellent marker for trophoblastic cell differentiation, as well as a candidate for pregnancy diagnosis.

Prolactin receptor heterogeneity: processing and signalling of the long and short isoforms during development.

During development, the fetus is exposed to prolactin activity from the placenta, as well as from the developing fetal pituitary. Distinct prolactin receptor isoforms, having different cytoplasmic domains generated by alternative splicing, are expressed as development proceeds at different levels in different organs. The "long" receptors are able to mediate transduction of all signals examined, in contrast with the "short" isoforms, whose truncated cytoplasmic domains are able to mediate a much smaller repertoire of signals and can act as dominant negatives. Our studies demonstrate that, although these forms share internalization mechanisms, the long form is internalized faster, resulting in more rapid down-regulation of this form. In order to examine the mechanisms by which prolactin may exert trophic effects on its target tissues during development, we have examined the signalling pathways through which prolactin binding to the long receptor regulates the transcription of cyclin D1. Our studies reveal the importance of the JAK/STAT (Janus kinase/signal transduction and activators of transcription) pathway, and the complexity of prolactin signalling to this promoter.

Dysregulation of mammary Stats 1,3 and 5 and PRL receptors by overexpression of TGFalpha.

Mammary TGFalpha overexpression results in delayed involution and eventually mammary cancer in transgenic mice. We hypothesized that STATs and PRL receptors (PRLR), critical regulators of mammary function, are altered in these animals and may contribute to this phenotype. We examined these factors late in the first pregnancy (d.18) and during normal involution (d.4 post-lactation) in WAP-TGFalpha transgenic mice and non-transgenic controls. Long form PRLR mRNA in WAP-TGFalpha glands at both pregnant d.18 and d.4 post-lactation was significantly reduced compared to controls, and PRLR-S3 failed to rise during involution. Total and pTyr STAT 1,3,5a and 5b also were altered. STAT 3 was higher at both times in WAP-TGFalpha glands. STAT 5a and 5b were lower at late pregnancy, but higher post-lactation; however, pTyr(694) STAT 5 was abnormally low at both times. Thus overexpression of TGFalpha has direct or indirect effects on both STATs and PRL responsiveness in vivo, which may reflect mechanisms of TGFalpha-induced mammary epithelial abnormalities.

Structural and functional effects of high prolactin levels on injured endothelial cells: evidence for an endothelial prolactin receptor.

Stress has been linked to health problems such as atherosclerosis and prolonged wound healing, which involve the responses of injured endothelial cells. Though prolactin (PRL) levels become increased during the physiological response to stress, the significance and effects of these increases are largely unknown. Here we examined the effects of elevated, though physiological, concentrations of PRL on the responses of cultured endothelial cells after mechanical injury to cell monolayers. When treated at the time of injury with PRL levels of 62.5-1000 ng/mL, cells at the wound front became abnormal in shape and had reductions in f-actin staining in comparison to controls that were not PRL-treated. High PRL concentrations also inhibited the adhesion of cells to their growth surface in a dose-dependent manner. Using rhodamine-labeled PRL, we observed specific PRL uptake by these cells that suggested the presence of a PRL receptor. Finally, mRNA for the long form of the PRL receptor was detected by RT-PCR. To our knowledge, this is the first report demonstrating that (1) high PRL concentrations alter the actin cytoskeleton and adhesion of injured endothelial cells and (2) endothelial cells express the transcript for the PRL receptor. Thus, we report novel effects of PRL that may be mediated by activation of an endothelial cell PRL receptor.

SmMAK16, the Schistosoma mansoni homologue of MAK16 from yeast, targets protein transport to the nucleolus.

The SmMAK16 gene from Schistosoma mansoni was cloned by chance when an adult worm cDNA library was probed with antiserum to affinity-purified S. mansoni GSH S-transferases. SmMAK16 encodes a hydrophilic protein of 259 amino acids with a molecular mass of 31 kDa. The protein shares 43% sequence identity and 66% similarity to the nuclear protein MAK16 of Saccharomyces cerevisiae that has been implicated both in cell cycle progression and biogenesis of 60S ribosomal subunits. Both proteins display a similar degree of sequence similar to the hypothetical protein CeMAK16 from Caenorhabditis elegans. These proteins share a number of apparent protein motifs, including two nuclear localization signals (NLS), multiple sites for phosphorylation by protein kinase CK2 and four conserved cysteine residues that resemble a zinc binding domain. SmMAK16 mRNA is more highly expressed in adult female worm than males. Recombinant SmMAK16 was phosphorylated by human protein kinase CK2. When chimeric constructs containing SmMAK16 fused the green fluorescent protein (GFP) were transiently transfected into COS-7s cells, the reporter was localized not in nuclei, but exclusively in nucleoli. The yeast and nematode homologues were likewise able to direct nucleolar accumulation of the fluorescent reporter. The high degree of sequence conservation together with the ability to direct nucleolar protein transport supports the hypothesis that MAK16 proteins play a key role in the biogenesis of 60S subunits.

Prolactin receptor and uterine milk protein expression in the ovine endometrium during the estrous cycle and pregnancy.

Lactogenic hormones regulate epithelial proliferation, differentiation, and function in a variety of epitheliomesenchymal organs. During pregnancy, the ovine uterus is a potential site for endocrine and paracrine actions of lactogenic hormones in the form of pituitary prolactin (PRL) and placental lactogen (PL). These studies determined temporal and spatial alterations in PRL receptor (PRL-R) and expression of uterine milk proteins (UTMP), a marker of endometrial secretory activity, in the ovine endometrium during the estrous cycle and pregnancy. Slot-blot hybridization analysis indicated that steady-state levels of endometrial PRL-R mRNA increased during pregnancy. In situ hybridization and immunohistochemical analyses indicated that PRL-R mRNA and protein were exclusively expressed in the endometrial glandular epithelium (GE). No PRL-R mRNA expression was detected in luminal epithelium, stroma, myometrium, or conceptus trophectoderm. Reverse transcription-polymerase chain reaction analyses determined that the endometrial GE expressed both long and short alternative splice forms of the ovine PRL-R gene. Slot-blot hybridization analysis indicated that steady-state levels of intercaruncular endometrial UTMP mRNA increased about 3-fold between Days 20 and 60, increased another 3-fold between Days 60 and 80, and then declined slightly to Day 120. In pregnant ewes, UTMP mRNA expression was restricted to the endometrial GE in the stratum spongiosum (sGE), increased substantially between Days 15 and 17, and, between Days 17 to 50 of gestation, was markedly higher in upper than lower sGE. After Day 50, hyperplasia of the sGE was accompanied by increased UTMP mRNA expression by all sGE. Collectively, results indicate that 1) endometrial sGE is a primary target for actions of lactogenic hormones and 2) UTMP mRNA expression is correlated with PL production by the trophectoderm and state of sGE differentiation during pregnancy. It is proposed that activation of PRL-R signal transduction pathways by PRL and PL plays a major role in endometrial GE remodeling and differentiated function during pregnancy in support of conceptus growth and development.

Differential gene regulation of interleukin-1 ligands and receptors in bovine peripheral blood neutrophils and mononuclear cells in response to E. coli lipopolysaccharide (LPS).

The proinflammatory cytokine, interleukin-1, plays a prominent role in the inflammatory reactions that characterize numerous diseases. In this study, we examined the gene expression for bovine IL-1 ligands and receptors by bovine peripheral blood mononuclear cells (MNCs) and neutrophils (PMNs) in response to E. coli lipopolysaccharide (LPS) in vitro. Gene expression of mRNA for IL-1alpha, IL-1beta, IL-1 receptor antagonist (IL-1ra), type 1 IL-1 receptor, type 2 IL-1 receptor, and IL-1 beta converting enzyme (ICE), were measured by a semi-quantitative RT-PCR technique. LPS had little effect on type 1 IL-1R expression in MNC, whereas, it strongly up-regulated type 1 IL-1R expression in PMNs. Co-incubation of PMNs with LPS and bovine recombinant IL-1beta had little additional effect on type 1 IL-1R expression. Incubation of MNCs with LPS resulted in up-regulation of IL-1beta, IL-1ra, and type 2 IL-1R, no change in IL-1alpha, and a decrease in ICE gene expression. Incubation of PMNs with LPS up-regulated IL-1beta gene expression, whereas, IL-1alpha, IL-1ra, type 2 IL-1R and ICE were unchanged. This study provides evidence for differential regulation of gene products of the bovine IL-1 family by peripheral blood mononuclear cells (MNC) and neutrophils (PMNs) in response to E. coli LPS.

cDNA cloning and gene expression of the type 1 bovine interleukin-1 receptor.

Regulation of interleukin-1 (IL-1) mediated biological responses is complicated by the multiple ligands and receptors of the IL-1 family. Most studies of IL-1 receptors have used human or rodent cells. Here, we report that the coding region of the bovine type 1 interleukin-1 receptor (type 1 IL-1R) cDNA extends 1719 bp in length. Northern analysis of specific bovine cell and tissue RNA demonstrated a 4.5 kb transcript. Overall, the bovine type 1 IL-1R coding region exhibits approximately 81 and 76% similarity with the human type 1 IL-1R at the nucleotide and amino acid level, respectively, and somewhat less similarity with the mouse and rat sequences. Type 1 IL-1R transcripts were confirmed by RT-PCR in several bovine cell types, including peripheral blood mononuclear cells (PBMCs), neutrophils (PMNs), and fibroblast, peritoneal macrophage, and arterial endothelial cell lines. It is expected that molecular clones for the bovine type 1 and 2 IL-1 receptors will provide us with the tools needed to decipher species-and cell-specific regulation of IL-1 action in the bovine.

Regulation of bovine interleukin-1 receptors.

Interleukin-1 is a key player in inflammation and the immune response. The interleukin-1 family consists of three ligands (IL-1 alpha, IL-1 beta, and the IL-1 receptor antagonist) and two receptors (IL-1RI and IL-1RII). Previous studies suggest a dynamic relationship among these receptors and ligands that regulates the magnitude and extent of IL-1 mediated activities. Our laboratory has cloned and sequenced the bovine type I and II interleukin-1 receptors, and has begun to investigate their regulation in bovine leukocytes in vitro and in vivo. IL-1RI and IL-1RII mRNA levels were upregulated in vitro by various mediators, including dexamethasone, rBoIL-4, rBoGM-CSF, and rHuTNF alpha. Conversely, IL-1RI mRNA levels were down-regulated by IFN-gamma. An in vivo study indicated that IL-1RII mRNA levels increased earlier than IL-1RI mRNA levels in dexamethasone-treated cattle. These findings suggest that early upregulation of IL-1RII, which is a decoy receptor, may be part of the anti-inflammatory action of glucocorticoids. Our investigations suggest that anti-inflammatory agents increase expression of the biologically inactive IL-1RII, as compared with the biologically active IL-1RI, in bovine leukocytes.

Growth hormone receptor and regulation of gene expression in fetal lymphoid cells.

The role of growth hormone (GH) in modulating the adult immune response is receiving increased attention; however, its role in the development of immune competence in the fetus has not been defined. In order to begin to address the role of GH in the ontogeny of the immune response. cells from bovine fetal spleen and thymus were examined for GH receptor and responsiveness to GH. Northern analysis and ligand binding studies showed that growth hormone receptor (GHR) was readily detected in early- and mid-gestational fetal thymocytes, but it was less readily detected in thymocytes from older fetuses. In contrast, GHR was easily detected in splenocytes at all fetal ages. Thymocytes and splenocytes from mid-gestational fetuses expressed low levels of cell surface GHR by flow cytofluorometric analysis, and CD4+ and CD8 (single positive) thymocyte subsets were positive. Northern analyses were employed to determine the effects of in vitro GH treatment on expression of several proto-oncogenes, cytokines, and GHR in thymocytes from fetuses at approximately mid-gestation. GH treatment for 30 min down-regulated c-jun and c-fos mRNA approximately 2- and 2.8-fold, respectively. After 6 h treatment, GH increased transcript levels for interleukin (IL)-1alpha, IL-1beta, IL-6, and GM-CSF about 2.5-, 2.2-, 3-, and 2-fold, respectively. GH also down-regulated the expression of its own receptor about 3.2-fold after 8 h of incubation. The presence of GHR in fetal lymphoid cells and its temporal and spatial regulation suggest a potential role for GH in the development and or function of the fetal bovine immune system. Although the mechanism(s) is unclear, our results suggest that GH is intimately involved in lymphocyte function and expression of certain cytokines during a critical period of fetal immune development.

Transcriptional regulation of interleukin-1beta gene by interleukin-1beta itself is mediated in part by Oct-1 in thymic stromal cells.

Interleukin (IL)-1 is involved in many processes, including thymic development. However, control of IL-1 expression in thymic-derived stromal cells (TSC) has not been reported. We found that IL-1beta increased steady-state mRNA levels for IL-1alpha and IL-1beta in TSC-936 and TSC-2C4 cells; stability was not a major determinant of this effect. To study transcriptional regulation of IL-1beta, we functionally characterized 4 kilobase pairs of the 5'-flanking region and first intron of the bovine IL-1beta gene. The -470/+14 fragment was sufficient to confer maximal responsiveness to IL-1beta upon transfection into these cell lines. Progressive 5' deletions identified several IL-1beta-responsive regions, including -308 to -226, which we further characterized. Electrophoretic mobility shift and supershift analyses showed that IL-1beta induced the ability to form multiple protein complexes with -261/-226 and that one of these contained nuclear factor Oct-1. A competitor containing a mutated Oct consensus site failed to compete not only for this complex but others as well, suggesting that this sequence regulates binding of other proteins to this region. Functional analysis confirmed that this element was essential for maximal induction of transcription. These findings document a heretofore undescribed mechanism utilized by TSC for regulation of IL-1beta transcription by IL-1beta itself.

Long and short forms of the ovine prolactin receptor: cDNA cloning and genomic analysis reveal that the two forms arise by different alternative splicing mechanisms in ruminants and in rodents.

Prolactin is a pituitary hormone that binds to specific receptors in numerous tissues. Depending on the size of their cytoplasmic domain, long and short prolactin receptors (l-PR, s-PR) have been described. Up to now, s-PR were found in rodents only. We report here the cloning of full-length coding sequences for short and long ovine prolactin receptors (s-oPR, l-oPR). The only difference between s- and l-oPR coding sequences was, respectively, the presence or absence of a 39 base pair insert at the beginning of the cytoplasmic domain, with two contiguous inframe stop codons at its 3' end. Sequence comparison revealed that the alternative splicing producing s- and l-oPR was different from that of rodents, although the resulting proteins were very similar. PCR experiments on ovine genomic DNA showed that the 39 base pair insert was directly linked to the downstream exon, and separated from the upstream exon by an 800 base pair intron. Thus, the alternative splicing used a single intron with one 5' and two 3' sites. The same organization was found in bovine and caprine genomes, suggesting that this feature is general in ruminants and different from rodents, which use mutually exclusive exons to produce s-PR and l-PR.

Effects of dexamethasone treatment on IL-1 receptor mRNA levels in vivo.

Using semiquantitative reverse transcriptasepolymerase chain reaction and Northern analysis, we observed in vivo up-regulation of interleukin-1 (IL-1) RI and IL-1RII mRNA levels in peripheral blood mononuclear cells (PBMCs) and neutrophils (PMNs) from Holstein cattle injected with dexamethasone (0.04 mg/kg). Baseline levels of IL-1RI mRNA were greater than IL-1RII mRNA levels in PBMCs and PMNs before dexamethasone treatment. This is in contrast with the previously reported predominance of IL-1RII in unstimulated human PMNs. IL-1RII mRNA was strongly induced in both bovine PBMCs and PMNs at 24 h and returned to baseline levels by 72 h, after dexamethasone injection. Conversely, the greatest increase in IL-1RI mRNA in PBMCs and PMNs was not detected until 72 h after dexamethasone injection. These data provide evidence for sequential in vivo up-regulation of first IL-1RII mRNA and later IL-1RI mRNA by dexamethasone that is consistent with the anti-inflammatory activity of glucocorticoids.

Prolactin receptor heterogeneity in bovine fetal and maternal tissues.

Study of diverse PRL actions at a variety of fetal and maternal targets during pregnancy is complicated by receptor heterogeneity and multiple ligands circulating at this time. In the present studies, we have examined PRL receptors at a variety of potential targets by reverse transcription-PCR and Western analysis. Bovine tissues contain two different transcripts for the PRL receptor; the one that encodes a short form includes an additional 39 bases at a position identical to the deviation from the long form found in rodents and sheep. Western analyses of PRL receptors in microsomal fractions from various maternal and fetal tissues revealed considerable size heterogeneity. Collectively, the larger immunoreactive moieties (apparent Mr 100 kDa or greater) and the smaller species (47-55 kDa) correlated well with the relative abundance of the transcripts for the different forms of the receptor and varied considerably among tissues. N-Glycosylation was shown to be the major, but not the only, modification of both receptor forms when transiently transfected into COS-7 and END-6.2 cells. Much of the short form could be reduced to the mobility predicted from the complementary DNA by culture with tunicamycin; this was not true of the long form, suggesting modifications specific for its cytoplasmic domain. Differences in the pattern of immunoreactive species in the COS-7 and END-6.2 cells are consistent with cell-specific modifications. The ability of these receptor forms to mediate a transcriptional response to PRL and its placental relative, placental lactogen, was evaluated with a PRL response element inserted upstream from a thymidine kinase promoter/reporter gene construct transiently transfected into CHO-K1 cells. Both hormones were able to stimulate reporter gene expression through the long form, but not the short form, of the receptor. These studies will facilitate examination of tissue-specific actions of PRL and related hormones during pregnancy.

Regulation of interleukin (IL)-1alpha, IL-1beta, and IL-6 expression by growth hormone and prolactin in bovine thymic stromal cells.

Growth hormone (GH) and prolactin (PRL) have been implicated in T-cell development, but relatively little is known about the mechanism(s) of their actions on the multiple cell types in this complex tissue. Here, we investigated the effects of GH and PRL on the expression of interleukin (IL)-1alpha, IL-1beta and IL-6 in thymic stromal cells (TSC). These cytokine mRNAs were increased by GH, PRL and placental lactogen (PL) in primary cultures prepared from mid-gestational fetuses in a dose-dependent manner. IL-1 receptor antagonist (IL-1ra) abolished the hormone-induced IL-6 expression, suggesting that the induction of IL-6 was secondary to IL-1 activity. To examine the effects of these hormones on an individual cell type and develop a system in which signalling mechanisms can be studied, we generated immortalized cell lines using a strategy of conditional transformation. In the cell line, TSC-936, which displayed vimentin-positive staining and morphological characteristics of mesenchymal cells, both GH and PRL increased levels of steady-state mRNAs for IL-1alpha and IL-1beta. Nuclear run-on analysis revealed that the transcription rate of the IL-1beta gene was significantly increased by GH and PRL at 30 and 60 min, respectively, but that for IL-1alpha was not significantly changed, suggesting the possibility of an alternative mechanism mediating this response. These data suggest that modulation of cytokine gene expression is one mechanism by which GH and PRL facilitate thymic development and T-cell maturation.