Prolactin stimulates ubiquitination, initial internalization, and degradation of its receptor via catalytic activation of Janus kinase 2

Prolactin (PRL) activates its receptor to initiate signal transduction pathways (including activation of Janus kinases, Jak) but also stimulates downregulation of this receptor to limit the magnitude and duration of signaling. Degradation of the long form of PRL receptor (PRLr) depends on its phosphorylation on Ser349 that is required to facilitate PRLr ubiquitination. Signaling events that mediate PRL-induced degradation of PRLr remain to be elucidated. Here, we investigated the role of Jak2 activity in ligand-triggered increase of PRLr phosphorylation on Ser349, PRLr ubiquitination, endocytosis, and degradation. Using Jak2 reconstitution in Jak2-null cells as well as pharmacologic approaches, we found that treatment with PRL (but not with PRLr antagonist) promotes phosphorylation of PRLr on Ser349 and accelerates endocytosis of PRLr. Furthermore, PRL-stimulated PRLr phosphorylation, endocytosis, and degradation in Jak2-null cells reconstituted with wild type but not with catalytically inactive Jak2. We discuss how Jak2-mediated signaling might be transduced into Ser349 phosphorylation of PRLr as well as its ubiquitination and endocytosis.     

Prolactin receptor signal transduction.

Within the immune system, multiple isoforms of the human prolactin receptor (PRLr) serve to mediate the effects of its ligand (PRL). Now numbering four, these isoforms are structurally and functionally distinct, demonstrating significant differences in ligand affinities, kinetics of transduction and the transduction proteins activated. The proximal transduction pathways activated during PRLr-associated signaling include the tyrosine kinases Jak2, Fyn and Tec, the phosphatase SHP-2, the guanine nucleotide exchange factor Vav, and the signaling suppressor SOCS. Differential activation of these pathways may contribute to the pleiotropism of PRL action in tissues of the immune system.     

Modulation of growth factor receptor function by isoform heterodimerization.

Activation of prolactin (PRL)-dependent signaling occurs as the result of ligand-induced dimerization of receptor (PRLr). Although three PRLr isoforms (short, intermediate, and long) have been characterized and are variably coexpressed in PRL-responsive tissues, the functional effects of ligand-induced PRLr isoform heterodimerization have not been examined. To determine whether heterodimeric PRLr complexes were capable of ligand-induced signaling and cellular proliferation, chimeras consisting of the extracellular domain of either the alpha or beta subunit of human granulocyte-macrophage colony-stimulating factor receptor (GM-CSFr) and the intracellular domain of the rat intermediate or short PRLr isoforms (PRLr-I or PRLr-S) were synthesized. Because high affinity binding of GM-CSF is mediated by the extracellular domain of one alpha and beta GM-CSFr pair, use of GM-CSFr/PRLr chimera specifically directed the dimerization of the PRLr intracellular domains within ligand-receptor complexes. Stable transfection of these constructs into the Ba/F3 line was demonstrated by Northern blot and immunoprecipitation analyses. Flow cytometry revealed specific binding of a phycoerythrin-conjugated human GM-CSF to the transfectants, confirming cell surface expression of the chimeric receptors. When tested for their ability to proliferate in response to GM-CSF, only chimeric transfectants expressing GM-CSFr/PRLr-I homodimers demonstrated significant [3H]thymidine incorporation. GM-CSF stimulation of transfectants expressing either GM-CSFr/PRLr-S homodimers or GM-CSFr/PRLr-S+1 heterodimers failed to induce proliferation. Consistent with these data, the GM-CSF-induced activation of two phosphotyrosine kinases, Jak2 and Fyn, was observed only in homodimeric GM-CSFr/PRLr-I transfectants. These results show that the PRLr-S functions as a dominant negative isoform, down-regulating both signaling and proliferation mediated by the receptor complex. Thus, structural motifs necessary for Jak2 and Fyn activation within the carboxy terminus of the PRLr-I, absent in the PRLr-S, are required in each member of the dimeric PRLr complex.     

ACTH受体在人淋巴组织中的表达

目的　通过检测ACTH受体及其mRNA在人淋巴组织中的表达情况 ,寻求神经内分泌系统 免疫系统之间功能双向调节的形态学依据。方法　应用免疫组化SP法及核酸分子原位杂交法检测ACTH受体及其mRNA在人淋巴结、脾脏及肠道淋巴组织中的表达。结果　免疫组化显示ACTH受体在人各种淋巴组织中表达的阳性率为 82 .5 % ,与原位杂交阳性率 70 .0 %之间的差异无显著性。结论　人的免疫活性细胞不但可以表达ACTH受体蛋白 ,还可以合成其mRNA ,ACTH受体为神经内分泌系统 免疫系统网络共有的生物学语言媒介 ,内分泌源性的ACTH与免疫源性的ACTH都可以通过免疫细胞膜上的ACTH受体发挥对免疫系统的调节作用     

Implication of alpha4 phosphoprotein and the rapamycin-sensitive mammalian target-of-rapamycin pathway in prolactin receptor signalling

A prolactin (PRL)-responsive 3'-end cDNA encoding rat alpha4 phosphoprotein was previously isolated from a rat lymphoma cDNA library. Rat alpha4 is a homologue of yeast Tap42 and is a component of the mammalian target-of-rapamycin (mTOR) signalling pathway that stimulates translation initiation and G1 progression in response to nutrients and growth factors. In the present study, the full-length rat alpha4 cDNA was obtained by 5'-RACE and the 1023 bp open reading frame predicted a 340 amino acid protein of 39.1 kDa. The alpha4 mRNA was expressed in quiescent PRL-dependent Nb2 lymphoma cells deprived of PRL for up to 72 h but expression was downregulated within 4 h of PRL treatment. In contrast, PRL-independent Nb2-Sp cells showed constitutive expression of alpha4 that was not affected by PRL. Western analysis of Nb2 cell lysates or of V5-tagged-alpha4 expressed in COS-1 cells detected a single immunoreactive band of approximately 45 kDa. Enzymatic deglycosylation of affinity-purified 45 kDa alpha4 yielded the predicted 39 kDa protein. Phosphorylation of Nb2 alpha4 was induced by PRL or 2-O-tetradecanoyl-phorbol-13-acetate (TPA) and further enhanced by a combination of PRL and TPA. The Nb2 alpha4 associated with the catalytic subunit of protein phosphatase 2A and localized predominantly in Nb2 nuclear fractions with trace amounts in the cytosol. The immunosuppressant drug rapamycin inhibited proliferation of Nb2 cells in response to PRL or interleukin-2, but had no effect on Nb2-Sp cells. Furthermore, transient overexpression of alpha4 in COS-1 cells inhibited PRL stimulation of the immediate-early gene interferon regulatory factor-1 promoter activity. Therefore, PRL downregulation of alpha4 expression and/or PRL-inducible phosphorylation of alpha4 may be necessary for PRL receptor (PRLr) signalling to the interferon regulatory factor-1 promoter in the Nb2 cells and, furthermore, implicates cross-talk between the mTOR and PRLr signalling cascades during Nb2 cell mitogenesis.     

Photoperiod and bromocriptine treatment effects on expression of prolactin receptor mRNA in bovine liver, mammary gland and peripheral blood lymphocytes

Recent evidence suggests that photoperiod influences immune function. Interestingly, photoperiod has profound effects on concentrations of prolactin (PRL), a hormone also known to be involved in fluctuations of the immune system. However, the impact of photoperiod on PRL receptor (PRL-R) expression is poorly understood, particularly in tIssues of the immune system. Two experiments were performed to increase the general understanding of how photoperiod interacts with the immune system. Our first objective was to determine the effects of photoperiod on PRL-R mRNA expression and cellular immune function. Lymphocytes were isolated from blood collected from calves (n=10) and PRL-R mRNA expression of both long and short forms was quantified using real-time PCR. Lymphocytes expressed PRL-R mRNA, suggesting that PRL could act directly on these cells. To determine the relationship between photoperiod and PRL-R mRNA expression in other tIssues, hepatic and mammary biopsies were collected after calves were exposed to long days (LDPP; 16 h light:8 h darkness) or short days (SDPP; 8 h light:16 h darkness). Relative to LDPP, SDPP decreased circulating PRL, but increased expression of both forms of PRL-R mRNA in liver, mammary gland and lymphocytes. Short days also increased lymphocyte proliferation compared with long days. Reversal of photoperiodic treatments reversed the effects on circulating PRL, PRL-R mRNA expression and lymphocyte proliferation. Our second objective was to manipulate PRL concentration in photoperiod-treated animals, using bromocriptine. Concentrations of PRL in LDPP animals injected daily with bromocriptine for 1 week were decreased compared with LDPP controls, to a level similar to SDPP animals. Receptor expression was increased in LDPP+bromocriptine-treated animals relative to LDPP controls, as was lymphocyte proliferation. Overall, our results indicate that photoperiodic effects on PRL-R mRNA expression were inverse to those on circulating PRL, with short days stimulating expression of both forms of PRL-R mRNA. Expression of PRL-R mRNA changed in the same direction as lymphocyte proliferation with regard to photoperiod treatment, suggesting a link between photoperiodic effects on PRL sensitivity and immune function. Thus, PRL signaling may mediate photoperiodic effects on immune function.     

Prolactin as a chemoattractant for human breast carcinoma.

Prolactin (PRL) is recognized as a growth and differentiating hormone in the human breast. These effects are mediated by the PRL receptor (PRLr); when stimulated the PRL-PRLr complex activates several signaling cascades, including those involving the GTP-binding proteins Ras and Rac. The activation of these signaling pathways has been associated with cytoskeletal alterations and increased cellular motility. We hypothesized that such changes could occur in PRL-stimulated human breast cancer cells. To test this hypothesis, complementary studies, including wound closure, time-lapse video microscopy (TLVM), and Boyden chamber assay were performed. These studies revealed that PRL significantly enhanced the migration of the breast cancer cell lines T47D, MCF7, and MDA23 1. Co-stimulation with PRL was noted to potentiate epidermal growth factor (EGF)-induced cell motility. IF microscopy of filamentous actin using rhodamine-conjugated phalloidin revealed a significant and rapid generation of both membrane ruffling and stress fibers in response to PRL, an effect inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. In sum, these data reveal that PRL stimulation modulates the cytoskeleton and induces the motility of human breast cancer cells in vitro, events that have been associated with the progression of mammary carcinoma in vivo. Given the recently delineated autocrine-paracrine role for PRL in human breast cancer, these findings could be of appreciable clinical significance.     

BCG-induced protection in guinea pigs vaccinated and challenged via the respiratory route

Since studies on cellular immune responses have demonstrated the role of the mucosal lymphoid system of the respiratory tract, we have studied responses obtained from the local respiratory route, compared to the systemic intradermal route, of BCG immunization. Guinea pigs vaccinated with different doses of BCG via both routes served to follow lymphoid cell proliferation, hilar lymph node and lung BCG clearance, lung granuloma formation and protection induced after virulent challenge.Results demonstrate that the aerogenic route of vaccination with BCG has no harmful side-effects for the host. In comparison with the intradermal route of vaccination, aerogenic vaccination with 10⁵ BCG cfu induced higher local cellular immune responses and a substantially improved protective effect.     

催乳素对Jurkat细胞CD154表达的影响

经RT-PCR法证实Jurkat细胞株表达催乳素(PRL)受体mRNA。发现在植物血凝素(PHA)和重组人PRL(rhPRL)共刺激下，Jurkat细胞CD154表达量明显增加。推测PRL与其受体结合，增加CD154表达而发挥免疫调节作用。     

Prolactin suppresses glucocorticoid-induced thymocyte apoptosis in vivo

The hypothesis that prolactin (PRL) functions as an immunomodulator was based on studies showing lymphocyte PRL receptors, and its effects on growth, differentiation, and apoptosis in lymphoid cells. However, studies of PRL (PRL-/-) and PRL receptor knockout mice indicated that PRL was not required for immune system development or function under basal conditions. Because PRL maintains survival in glucocorticoid (GC)-treated Nb2-T lymphocytes in vitro, and PRL and GCs are elevated during stress, we investigated whether PRL protected T cells in vivo from GC-induced apoptosis. Adrenalectomized mice [PRL -/-, undetectable PRL; pituitary grafted PRL-/- (PRL-/-Graft), elevated PRL; and PRL+/-, normal PRL] were treated with dexamethasone (DEX) or PBS. Thymocytes and splenocytes were isolated and annexin V labeling of phosphatidylserine, DNA fragmentation, and caspase-3 activation were assessed as indices of apoptosis. Total thymocytes and CD4+ and CD8+ T cells obtained from DEX-treated PRL-/- mice exhibited significantly increased annexin V binding. In contrast, binding was not altered by DEX in PRL-/-Graft thymocytes. In addition, DEX induced classic DNA fragmentation in PRL-/- thymocytes. Elevated serum PRL reduced this effect. Thymocytes from DEX-treated PRL-/- mice exhibited increased caspase-3 activation, which was inhibited in cells from PRL-/-Graft mice. Finally, elevated expression of X-linked inhibitor of apoptosis, XIAP, was observed in thymi from DEX-treated PRL -/-Graft mice. This is the first demonstration that elevated PRL antagonizes apoptosis in thymocytes exposed to GCs in vivo. These observations suggest that, under conditions of increased GCs, such as during stress, elevated PRL functions physiologically to maintain survival and function of T-lymphocytes.     

Gamma/Delta T cell mRNA levels decrease at mucosal sites and increase at lymphoid sites following an oral SIV infection of macaques

The oral and esophageal mucosa have been identified as possible sites of HIV/SIV entry following oral infection. Here, gamma/delta (gammadelta) T cells, a multi-functional T cell subset, were assessed at oral/esophageal mucosa and lymphoid sites at the earliest times (1-14 days) post-oral SIV inoculation utilizing quantitative RT-PCR. During these earliest times post-infection, decreased gammadelta TCR mRNA levels were observed at the oral gingiva and esophageal mucosa, while increased levels were observed within regional lymph nodes (cervical and retropharyngeal). Higher lymph node gammadelta TCR levels were associated with increased mRNA expression of the lymphoid homing chemokine/receptor (CCL21/CCR7) pair in these lymph nodes. In contrast to gammadelta TCR levels, CD4 mRNA expression remained relatively stable through 4 days post-infection, and depletion of CD4 T cells was only evident after 7 or 14 days post-infection. The decrease of gammadelta T cell mRNA from mucosal sites and the corresponding increase at lymphoid sites suggest a rapid redistribution of these immune cells at these earliest times post-SIV infection.     

Modulating the intestinal immune system: the role of lymphotoxin and GALT organs

The gut associated immune system fences off potentially harmful intestinal antigens from the systemic circulation and induces systemic tolerance against luminal antigens. Intestinal immune responses against luminal antigens include IgA secretion and induction of regulatory cells. Unlike few other cytokines, lymphotoxin alpha/beta regulates the development of intestinal lymphoid organs. The embryonic development of Peyer's patches, postnatal lamina propria B cell development, and isolated lymphoid follicle development all depend on lymphotoxin beta receptor interactions. Lymphotoxin alpha/beta signalling also contributes to the development of mesenteric lymph nodes. In addition, intestinal inflammation is suppressed by inhibition of lymphotoxin beta signalling, an observation which has initiated clinical studies using this treatment principal. Intestinal follicular lymphoid organs are sites of antigen presentation. Antigen presenting cells tune the delicate balance between intestinal immune tolerance and inflammation. Therefore, gut associated lymphatic organs and factors regulating their development are critical for the prevention of adverse immune reactions to intestinal antigens. This review provides an overview on the role of lymphotoxin and the gut associated lymphatic organs in the regulation of oral tolerance and intestinal inflammation.     

Immunomodulatory effects of prolactin and growth hormone in the tilapia,Oreochromis mossambicus

To clarify the roles of prolactin (PRL) and GH in the control of the immune system, the effects of environmental salinity, hypophysectomy, and PRL and GH administration on several immune functions were examined in tilapia (Oreochromis mossambicus). Transfer from fresh water (FW) to seawater (SW) did not alter plasma levels of immunoglobulin M (IgM) and lysozyme. The superoxide anion (O(2)(-)) production in head kidney leucocytes accompanied by phagocytosis was elevated in SW-acclimated fish over the levels observed in FW fish. Hypophysectomy of the fish in FW resulted in a reduction in O(2)(-) production in leucocytes isolated from the head kidney, whereas there was no significant change in plasma levels of IgM or lysozyme. Treatment with tilapia GH and PRLs (PRL(177) and PRL(188)) enhanced O(2)(-) production in vitro in head kidney leucocytes in a dose-related manner. Extrapituitary expression of two PRLs, GH and IGF-I mRNA was detected in lymphoid tissues and cells such as head kidney, spleen, intestine and leucocytes from peripheral blood and head kidney. PRL-receptor mRNA was detected in head kidney leucocytes, and the level of expression was higher in SW-acclimated fish than that in FW fish. Treatment with PRL(177) caused higher production of O(2)(-) in the head kidney leucocytes isolated from SW tilapia than that from FW fish. In view of the fact that PRL acts antagonistically to osmoregulation in SW, its immunomodulatory actions in this euryhaline fish would appear to be independent of its osmoregulatory action.     

Calcitonin expression in rat anterior pituitary gland is regulated by ovarian steroid hormones

Gonadotroph-derived calcitonin-like peptide (pit-CT) is a potent inhibitor of lactotroph function. We investigated the effect of ovarian hormones on pit-CT mRNA expression in the anterior pituitary (AP) gland of cycling female rats. Levels of mRNAs for pit-CT, CT receptor, prolactin (PRL), and beta-LH during 4-d estrous cycle were determined. In a second study, the effects of estrogens and progesterone on pit-CT and PRL mRNA levels were investigated. In a third group, the effect of estrogen or progesterone depletion on pit-CT mRNA expression was studied. In a fourth group, the effect of passive pit-CT immunization on PRL and LH mRNA expression was examined. Pit-CT mRNA levels varied during estrous cycle. They were highest in diestrus, but lowest in the evening of proestrus. CT-receptor mRNA levels displayed smaller fluctuations. Estrogen repletion caused a decline in pit-CT mRNA expression in ovariectomized rats, but progesterone produced a marked increase. ICI 182,780 prevented the decline of pit-CT mRNA levels during late proestrus-estrus, but RU 486 attenuated pit-CT mRNA levels. Passive CT immunization in diestrus altered PRL and LH mRNA expression, and advanced the estrus cycle. These results suggest that pit-CT mRNA expression is regulated by ovarian hormones, and depletion of pit-CT advances their estrous cycle.     

Prolactin potentiates insulin-stimulated leptin expression and release from differentiated brown adipocytes

The pituitary hormone prolactin (PRL) exerts pleiotropic effects, which are mediated by a membrane receptor (PRLR) present in numerous cell types including adipocytes. Brown adipose tissue (BAT) expresses uncoupling proteins (UCPs), involved in thermogenesis, but also secretes leptin, a key hormone involved in the control of body weight. To investigate PRL effects on BAT, we used the T37i brown adipose cell line, and demonstrated that PRLRs are expressed as a function of cell differentiation. Addition of PRL leads to activation of the JAK/STAT and MAP kinase signaling pathways, demonstrating that PRLRs are functional in these cells. Basal and catecholamine-induced UCP1 expression were not affected by PRL. However, PRL combined with insulin significantly increases leptin expression and release, indicating that PRL potentiates the stimulatory effect of insulin as revealed by the recruitment of insulin receptor substrates and the activation of phosphatidylinositol 3-kinase. To explore the in vivo physiological relevance of PRL action in BAT, we showed that leptin content was significantly increased in BAT of PRLR-null mice compared with wild-type mice, highlighting the involvement of PRL in the leptin secretion process. This study provides the first evidence for a functional link between PRL and energy balance via a cross-talk between insulin and PRL signaling pathways in brown adipocytes.     

Effects of Growth Hormone and Prolactin on Adipose Tissue Development and Function

GH and PRL are both implicated in adipose tissue development. Whilst direct effects of GH have been clearly demonstrated, direct effects of PRL have been subject to considerable debate. Recent studies have however provided compelling evidence for PRL receptors on adipocytes and in vitro effects on leptin and lipoprotein lipase activity have been demonstrated. Quantitatively however these effects of PRL are less significant than those of GH and the most pronounced effects of PRL on adipose tissue are indirect, for example, during lactation, when prolactin drives milk synthesis which results in a homeorhetic shift towards lipid mobilization from adipose tissue to support milk production. GH also exhibits such homeorhetic effects, most notably in ruminants, but also clearly has direct, insulin-antagonistic, metabolic effects. The roles of GH and PRL on adipocyte proliferation and differentiation have also been controversial, with GH stimulating adipocyte differentiation in vitro in cell lines whilst stimulating proliferation and inhibiting differentiation of primary cell cultures. Examination of adipose tissue development in PRLRko and GHRko mice has revealed roles for both hormones. PRLRko mice show impaired development of both internal and subcutaneous adipose tissue due to decreased numbers of adipocytes. In contrast, GHRko mice exhibit major decreases in the number of internal (parametrial) adipocytes whereas subcutaneous adipocytes develop almost normally. This leads to major changes in the sites of adipose tissue accretion and bears interesting parallels with the adipose tissue redistribution which occurs in HIV-induced lipodystrophy. Such individuals exhibit a central obesity which can be partially corrected by GH treatment. However, recent studies suggest that this may be a physiological response in which adipose tissue sites containing lymphoid tissue (such as mesenteric) show preservation of adipose tissue perhaps to support augmented immune responses.     

Recruitment of antibody-forming cells in the lung after local immunization is nonspecific

The deposition of particulate antigen into the dog lung induces a large accumulation of antigen-specific-forming cells in immunized lung lobes. The results of our previous studies with dogs indicate that these cells are recruited to the immunized lung lobes from the blood. The present study was designed to determine if the recruitment of immune cells to the lung is antigen-specific, or if changes occur in the lung after immunization that allow immune cells to enter the lung nonspecifically. Dogs were immunized in the left and right cardiac lung lobes with antigenically different particulate antigens. The number of lymphoid cells producing antibody to each antigen and the concentrations of antigen-specific antibody were measured in the immunized lung lobes and in a control lung lobe at 5 through 14 days after immunization. Similar numbers of antibody-forming cells and concentrations of antibody to both antigens were found in each immunized lung lobe, regardless of which antigen was used for immunization. These results indicate that antigen exposure alters the lung to allow a nonspecific recruitment of immune cells from the blood into the immunized lung lobes, regardless of antigen specificity. These data also provide further evidence that the antibody-forming cells found in the lung lavage fluid after localized immunization are produced in the thoracic lymph nodes or other systemic lymphoid tissues, rather than locally in lymphoid tissues in the deep lung.