Interferon-tau upregulates prolactin receptor mRNA in the ovine endometrium during the peri-implantation period.

Our objective was to determine the effect of ovine interferon-tau (IFN-tau) on prolactin receptor (PRL-R) gene expression in the ovine endometrium. IFN-tau is an embryonic cytokine which, via its paracrine anti-luteolytic activity, plays a critical role in maternal recognition of pregnancy in ruminants. Using ribonuclease protection assay procedures, we compared endometrial PRL-R mRNA levels in ewes that were intrauterine injected with either 2 mg bovine serum albumin or 2 mg recombinant ovine IFN-tau on day 10 of the oestrous cycle (day 0 = day of oestrus). IFN treatment significantly increased the abundance of both the long and short forms of PRL-R mRNA in the ovine uterus, but had no effect on the long:short form ratio. In situ hybridization experiments revealed that the increase in abundance of PRL-R mRNA in the uterus was localized to the glandular compartment of the endometrium. In pregnant ewes, a similar increase in PRL-R mRNA abundance was found to occur in ovine endometrium on days 14-15 post conception. Collectively, these data provided strong evidence that IFN-tau modulates the level of lactogenic hormone receptor mRNA in the ovine uterus. Whether the effect of IFN-tau on PRL-R expression is mediated directly or influenced, at least in part, by progesterone remains to be elucidated.

Establishment of feeder-independent cloned caprine trophoblast cell line which expresses placental lactogen and interferon tau.

A feeder-independent cloned trophoblast cell line, HTS-1, was established from a mature placenta of Shiba goat (Capra hircus). During the growth phase, single HTS-1 cells exhibited ruffled membranes or lamellipodia often accompanied by elongated cell shape, indicating highly motile nature of the cells. At or near confluence, HTS-1 cells formed monolayers with few sign of cellular overlapping. Binucleate cells were found at a high frequency especially in the peripheral regions of monolayers. In small colonies and the monolayers, majority of HTS-1 cells assumed polygonally shaped cobble-stone like morphology characteristic to epithelial cells, although considerable variations in cellular morphology were observed despite of repeated cloning. Time-lapse video recordings of HTS-1 cells during culture revealed that not only the small colonies but also the monolayers near or at confluence were remarkably motile, often causing extreme elongation of the cells within them. The extremely plastic nature of HTS-1 cells in vitro is likely to be the reflection of the extraordinary capacity of caprine trophoblast cells to be stretched to extreme thinness in vivo as shown by electron microscopy. HTS-1 cells cultured on matrigel are highly invasive, and express MT1-MMP which, in the mouse, has been known to be expressed at the invasive edge of trophoblast both in vitro and in vivo. HTS-1 cells express placental lactogen (PL) and interferon-tau (IFNtau), as confirmed by immunocytochemistry, Western blotting and RT-PCR analysis. Both PL and IFNtau expression in the cells appeared to be down-regulated by cell-cell contact. In the medium conditioned by HTS-1 cells, the presence of secretory form of PL and IFNtau was confirmed by Western blotting. The HTS-1 cell line will serve as a useful in vitro model for the analysis of the molecular and/or cellular mechanisms underlying synepitheliochorial placentation in bovidae animals.

Up-regulation of polymeric immunoglobulin receptor mRNA in mammary epithelial cells by IFN-gamma.

As shown in previous in vivo experiment, the amount of polymeric immunoglobulin receptor (pIgR), which mediates the transcytosis of pIgA across epithelial cells, is regulated by lactogenic hormones (PRL and cortisol) during the development of the mammary gland. In the present in vitro study, it appeared that these hormones were insufficient to induce the strong expression of the gene that we observed in vivo. Several papers have shown that IFN-gamma is a strong stimulator of pIgR gene expression in different models. In contrast, nothing is known of the effects of IFN-gamma on pIgR gene expression in the mammary gland. We report here that IFN-gamma strongly increased pIgR mRNA levels through a direct effect on mammary epithelial cells. We show that IFN-gamma activated not only Stat1 but also Stat5 and that expression of the pIgR and IRF-1 genes was strongly correlated following IFN-gamma stimulation in mammary epithelial cells. In conclusion, these experiments enabled the analysis of different types of regulation of pIgR gene expression in the mammary gland and suggest possible co-operation between circulating hormones and locally produced cytokines, leading to pIgR gene expression in the mammary gland.

Use of somatic cells from goat milk for dynamic studies of gene expression in the mammary gland.

Somatic cells are present in the milk throughout lactation and consist of leukocytes and epithelial cells exfoliated from the mammary epithelium. Our objective was to determine the efficacy of using somatic cells from goat milk for dynamic studies of gene expression in the mammary gland. Over a 4-wk interval, cells were isolated from daily morning milk samples and samples taken 30 min after milking. They were characterized by direct cell counts and by flow cytometry analysis after immunostaining with antibodies directed against cytokeratin and CD45, a common leukocyte antigen. Epithelial cell counts within the morning milk ranged from 15 to 45% of total milk somatic cells. After-milking samples contained twice as many cells as did morning milk samples. The RNA was extracted from the somatic cells of both types of milk samples with equivalent efficiency (a mean of 1.2 microg RNA/mL of milk). Four mRNA variants of the alpha-S1 casein gene were detected by Northern blot analysis and the amount of each mRNA in milk cells was related to protein concentration in milk. The comparison between mRNA from the mammary gland and from congruently collected milk cells showed that relative amounts of mRNA for each milk-protein (alpha-S1 and kappa-casein and alactalbumin) were conserved. In a third experiment, daily milk cell RNA preparations were extracted to assess the effect of growth hormone (GH) on mammary gene expression; four goats were separated into two groups in order to perform a switch-back design consisting of three treatment weeks: Control, GH-Control or GH-Control-GH. In this study, treatment of goats with GH increased milk yields by 5%. Throughout the control and GH treatments, the expression of the three milk-protein genes studied were highly and significantly correlated (r = 0.949 and r = 0.958, P < 0.001 for, respectively, alpha-S1 and kappa-casein and for alpha-S1 casein and alpha-lactalbumin). During GH treatment, the three milk-protein mRNA abundances increased with the same pattern. In conclusion, the opportunity to use milk somatic cells for RNA preparation and analysis provides a significant improvement over the use of biopsy samples in assessing gene activity in the mammary gland and allows easy and repetitive sampling without damaging mammary tissue. Furthermore, we propose that this method could be used to investigate the transcriptional status of the mammary gland of an animal in relation to its genotype, nutritional and pathologic status, and under influence by hormonal factors.

Identification of leptin receptors in human breast cancer: functional activity in the T47-D breast cancer cell line.

To evaluate whether leptin plays a putative role in breast tumorigenesis, we studied the expression of its long and short receptor isoforms in various tumoral breast tissues. We applied semiquantitative RT-PCR method to RNA extracted from 20 breast cancer biopsies and two human breast cancer cell lines (T47-D and MCF-7). Our results showed the expression of both leptin receptor transcripts in all tumoral tissues examined. By in situ hybridization experiments, we localized leptin receptors in proliferating epithelial cells. Study of leptin effects on human breast cancer cells growth was performed by [3H]-thymidine incorporation method and colorimetric MTT assay. We demonstrated that leptin (50-100 ng/ml) significantly stimulates proliferation of the human breast cancer cell line T47-D (P<0.05). Western blot analysis indicated that leptin induces a time-dependent activation of mitogen-activated protein kinases (MAPKinase) 1 and 2 in T47-D cell line. Moreover, the specific MAPK-inhibitor PD 98059 blocked cell proliferation induced by leptin. In conclusion, we demonstrate that leptin receptors are expressed in breast cancer and that leptin induces proliferation in the T47-D cell line via activation of the MAPKinases pathway. These data suggest that leptin and its receptors may be implicated in mammary cell proliferation and in breast cancer pathogenesis.

Leptin expression in the ovine mammary gland: putative sequential involvement of adipose, epithelial, and myoepithelial cells during pregnancy and lactation.

We examined the ability of the ovine mammary gland to synthesize leptin throughout pregnancy and lactation. Leptin gene expression was assayed by real-time reverse transcription and polymerase chain reaction in mammary gland from ewes at 15, 80, 106, 112, or 141 d of pregnancy and at 0 (30 min after parturition), 3, 48, or 70 d of lactation. Leptin mRNA level was high at the beginning (the first 80 d) and at the end of pregnancy and was lower at mid-pregnancy and throughout lactation. Furthermore, during these periods of mammary leptin expression, the location of leptin protein, as determined by immunohistochemical analysis, changed within mammary tissue. It was located in adipose cells during early stages of pregnancy, in epithelial cells after full cell differentiation just before parturition, and in myoepithelial cells after parturition. These data, compared with published data on leptin receptor gene expression, provide evidence that leptin could be produced by different cell types of the mammary gland and could act as a paracrine factor on mammary cell growth and differentiation via adipose-epithelial cells and myoepithelial-epithelial cell interactions.

Differential roles for signal transducers and activators of transcription 5a and 5b in PRL stimulation of ERalpha and ERbeta transcription.

PRL has been shown to stimulate mRNA expression of both ERalpha and ERbeta in the rat corpus luteum and decidua of pregnancy. To investigate whether PRL may stimulate ER expression at the level of transcription and which transcription factors may mediate this stimulation, we have cloned the 5'-flanking regions of both rat ER genes. A constitutively active PRL receptor (PRL-R(CA)) stimulated both ERalpha and ERbeta promoter activity, indicating that PRL is acting to stimulate ER transcription. Putative signal transducer and activator of transcription (Stat)5 response elements were identified at -189 in the ERalpha promoter and at -330 in the ERbeta promoter. Mutation of these response elements or overexpression of dominant negative Stat5 prevented stimulation of ERalpha and ERbeta promoter activity, indicating that PRL regulation of ER expression requires both intact Stat5 binding sites as well as functional Stat5. Interestingly, either Stat5a or Stat5b could stimulate ERalpha transcription while stimulation of ERbeta occurred only in the presence of Stat5b. Through mutational analysis, a single nucleotide difference between the ERalpha and ERbeta Stat5 response elements was shown to be responsible for the lack of Stat5a-mediated stimulation of ERbeta. These findings indicate that PRL stimulation of ER expression occurs at the level of transcription and that PRL regulation of ERalpha can be mediated by either Stat5a or Stat5b, while regulation of ERbeta appears to be mediated only by Stat5b.

Expression of BRCA1 gene in ewe mammary epithelial cells during pregnancy: regulation by growth hormone and steroid hormones.

OBJECTIVE: Steroid hormones (estradiol and progesterone) in association with prolactin and growth hormone are involved in lobulo alveolar development of the mammary gland during pregnancy. We hypothesized that the BRCA1 gene may be induced by these different hormones. METHODS AND RESULTS: In this study, we have demonstrated by Northern blot and in situ hybridization, that the expression of ovine (o) BRCA1 mRNA in mammary epithelial cells increased dramatically during a short period in the second half of pregnancy (days 70 to 112) and decreased at the end of pregnancy. The increase in oBRCA1 mRNA expression is concomitant with rapid lobulo alveolar growth. Using an in vivo protocol to artificially induce mammary gland development, we demonstrated by the real-time RT-PCR method that growth hormone in association with estrogen, progesterone and hydrocortisone induces an increase of BRCA1 mRNA expression in the ewe mammary gland. Moreover, we showed that estradiol and progesterone induce oBRCA1 expression in primary cultures of ewe mammary gland. CONCLUSIONS: These results suggest that BRCA1 is a potential regulator of the effects of steroid hormones and growth hormone in the induction of mammary epithelial cell proliferation.

Role of protein kinases in the prolactin-induced intracellular calcium rise in Chinese hamster ovary cells expressing the prolactin receptor.

There is still only limited understanding of the early steps of prolactin signal transduction in target cells. It has been shown that prolactin actions are associated with cell protein phosphorylation, Ca2+ increases, and so on. However, the link between the activation of kinases and calcium influx or intracellular Ca2+ mobilization has not yet been clearly established. Chinese hamster ovary (CHO) cells, stably transfected with the long form of rabbit mammary gland prolactin receptor (PRL-R) cDNA were used for PRL-R signal transduction studies. Spectrofluorimetric techniques were used to measure intracellular calcium ([Ca2+]i) in cell populations with Indo1 as a calcium fluorescent probe. We demonstrate that, although protein kinase C activation (PMA or DiC8) caused a calcium influx in CHO cells, prolactin-induced PKC activation was not responsible for the early effect of prolactin on [Ca2+]i. Activation of protein kinase A (PKA) or protein kinase G did not modify [Ca2+]i and inhibition of PKA pathway did not affect the prolactin response. In the same way, phosphatidylinositol-3 kinaseinhibition had no effect on the prolactin-induced Ca2+ increase. On the other hand, tyrosine kinase inhibitors (herbimycin A, lavendustin A, and genistein) completely blocked the effect of prolactin on [Ca2+]i (influx and release). W7, a calmodulin-antagonist, and a specific inhibitor of calmodulin kinases (KN-62), only blocked prolactin-induced Ca2+ influx but had no significant effect on Ca2+ release. Using pharmacological agents, we present new data concerning the involvement of protein phosphorylations in the early effects of prolactin on ionic channels in CHO cells expressing the long form of PRL-R. Our results suggest that, at least in the very early steps of prolactin signal transduction, serine-threonine phosphorylation does not participate in the prolactin-induced calcium increase. On the other hand, tyrosine phosphorylation is a crucial, very early step, since it controls K+ channel activation, calcium influx, and intracellular calcium mobilization. Calmodulin acts later, since its inhibition only blocks the prolactin-induced Ca2+ influx.

Increase in prolactin receptor (PRL-R) mRNA level in the mammary gland after hormonal induction of lactation in virgin ewes.

In order to examine the hormonal regulation of the prolactin-receptor (PRL-R) gene expression during mammary gland development, ewes were treated to induce lactation via an estrogen-progesterone-hydrocortisone and ovine growth hormone treatment. In situ hybridization analysis was used and revealed that sex steroids increased PRL-R mRNA levels in the mammary gland. Using RNase protection assay we showed that the estradiol + progesterone treatment increased both the levels of the long and the short forms of PRL-R mRNA. Addition of hydrocortisone increased the level of alphaS1-casein transcripts and the level of the ratio of the long to the short form of the PRL-R mRNA. This ratio can be further enhanced by addition of ovine growth hormone to the latter treatment. This suggests a role of hydrocortisone and ovine growth hormone in the alternative splicing that leads to the preferential expression of the long form of the PRL-R mRNA. In conclusion, the present experiments suggest that estrogen, progesterone and hydrocortisone are the major regulators of the PRL-R gene expression during pregnancy and prepare the mammary gland for its differentiation.

Characterization and modulation of a prolactin receptor mRNA isoform in normal and tumoral human breast tissues.

The role of prolactin (PRL) and its specific receptor (R-PRL) in human breast tumorigenesis remains unclear. We have investigated here the presence of extracellular-deleted hPRL-R isoforms in normal human breast, fibrocystic disease, primary breast carcinoma (ductal carcinoma, ductulo-lobular and lobular) and breast cancer cell lines (T47-D and MCF-7). RT-PCR and Southern blot analysis demonstrated the expression of full-length hPRL-R transcript in all samples tested. We also detected a hPRL-R transcript generated by alternative exon 6 splicing. This isoform has a 170 bp deletion in its extracellular sub-domain that induces a frameshift. Thus, the predicted amino-acid sequence should encode a putative soluble protein with the N-terminal sub-domain of the hPRL-R and 10 additional carboxy-terminal residues. This isoform should not bind PRL as previously demonstrated by other experiments. Moreover, the ratio of full-length to deleted form of hPRL-R transcripts differs from normal to tumoral breast tissue. This ratio is higher in tumoral mammary gland than in normal tissue. Our data suggest that the alternative splicing of the hPRL-R gene towards the deleted transcript may be a mechanism to down- or up-regulate the expression of the native transcript of hPRL-R in accordance to the physiological or pathological state of the mammary gland.

Functional heterodimerization of prolactin and growth hormone receptors by ovine placental lactogen.

Although homo- or heterodimerization are common mechanisms for activation of cytokine receptors, cross-talk between two distinct receptors in this superfamily has been never shown. Here we show a physiologically relevant example indicating that such an interaction does occurs, thus raising the hypothesis that heterodimerization between distinct cytokine receptors may be a novel mechanism contributing to the diversity of cytokine signaling. These findings were documented using both surface plasmon resonance and gel filtration experiments and show that ovine placental lactogen (PL) heterodimerizes the extracellular domains (ECDs) of ruminant growth hormone receptor (GHR) and prolactin receptor (PRLR). We also show that PL or PL analogues that exhibit little or no activity in cells transfected with PRLRs and no activity in cells transfected with ovine GHRs exhibit largely enhanced activity in cells cotransfected with both PRLRs and GHRs. Furthermore, chimeric receptors consisting of cytosolic and transmembrane part of ovine GHR or ovine PRLR and ECDs of human granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) alpha or beta were constructed. Upon transfection into Chinese hamster ovary cells along with reporter luciferase gene and stimulation by GM-CSF, a significant increase in luciferase activity occurred when GM-CSFR-alpha-PRLR and GM-CSFR-beta-GHR or GM-CSFR-alpha-GHR and GM-CSRR-beta-PRLR were cotransfected. In conclusion, we show that ovine PL is capable of functional heterodimerization of GHR and PRLR and that when their cytosolic parts, coupled to the ECD of GM-CSF receptors, are heterodimerized by GM-CSF, they are capable of transducing biological signal.

Detection and regulation of leptin receptor mRNA in ovine mammary epithelial cells during pregnancy and lactation.

Adipocyte-epithelial cell interactions and their secretions are critical determinants of mammary gland development. In this present study, we examined the possible involvement of leptin and its receptors in the process of mammogenesis/lactogenesis. We demonstrated by reverse transcription and polymerase chain reaction analysis that long and short forms of leptin receptors were expressed in the ovine mammary gland during pregnancy and lactation. Furthermore, quantitative determinations, via ribonuclease protection assays, provided evidence that the level of leptin receptor expression was greatest during mid-pregnancy when active growth of the mammary gland is initiated. Location of the leptin receptors, as determined by in situ hybridization analysis, revealed that leptin receptor transcripts were expressed specifically in mammary epithelial cells. These data provide evidence that leptin, with its receptors, could be an important mediator in regulating mammary gland growth and development.

In vitro expression of long and short ovine prolactin receptors: activation of Jak2/STAT5 pathway is not sufficient to account for prolactin signal transduction to the ovine beta-lactoglobulin gene promoter.

The recent finding that sheep had long (l-oPRLR) and short (s-oPRLR) prolactin receptors provided new tools to further explore prolactin signaling to target genes. Here we used CHO cells transfected with l-oPRLR or s-oPRLR cDNAs to compare the activation of known key steps of prolactin signaling by the two receptors. We found that prolactin stimulated l-oPRLR tyrosine phosphorylation, although it lacked the last tyrosine residue found in other long prolactin receptors. In addition, l-oPRLR and s-oPRLR both responded to prolactin stimulation by (1) Janus kinase 2 (Jak2) tyrosine phosphorylation, (2) DNA-binding activation of signal transducer and activator of transcription 5 (STAT5), (3) stimulation of transcription from a promoter made of six repeats of STAT5-responsive sequence. However, although it contains STAT5-binding consensus sequences, the ovine beta-lactoglobulin promoter (-4000 to +40) was transactivated by l-oPRLR, but not by s-oPRLR. Taken together, our results indicate that activation of Jak2/STAT5 pathway alone is not sufficient to account for prolactin-induced transcription of this milk protein gene, and that sequences of its promoter, other than STAT5-specific sequences, account for the opposite transcriptional activation capabilities of l-oPRLR and s-oPRLR.

[Impact of delta-9-tetrahydrocannabinol and its metabolites on the immune system]. / Impact du delta-9-tetrahydrocannabinol et ses métabolites sur le système immunitaire.

Many studies obvious impact of cannabinoids on the immune system. These studies follow the rapid advanced researches led in the immunology field. D9 Tetrahydrocannabinol and their metabolites decrease production of tumoral necrosis factor alpha. This decrease has for consequence a decrease of the apoptosis. Recent discovery of implication of cytokines in the phenomena of dependence, make the cannabis and their metabolites promoting agent induced dependence in association with drug abuse. The withdrawal of these products necessitates a intact immune system. D9 Tetrahydrocannabinnol and their metabolites inhibit production of IL-1 and gamma interferon. This inhibition has for consequence a decrease of 33% of the lymphocytes activity and an inhibition of 66% of the lymphocytes adenyl cyclase activity. The consumption of cannabis decreases immunological competence of macrophages, and alterate their essential role of trophicity of the nervous central system. Furthermore, inhibiting actions of cannabinoids on the cyclo-oxygenase, promote production of arachidonic acid degradation products. This compounds mimic the action of histamine, and inducing a raise of the vascular permeability and bronchospasm. These inolecules contributes at delayed reaction of anaphylaxia. However these actions of cannabinoids on the immune system promote their pull-back in cure of new pathology likes AIDS.

Cellular localization and evolution of prolactin receptor mRNA in ovine endometrium during pregnancy.

In this study, we have investigated the expression of the prolactin receptor gene in ovine endometrium during oestrus cycle and pregnancy. Using reverse transcription-PCR analysis, we provided evidence that the prolactin receptor gene is specifically transcribed in this tissue. As shown by Northern blot analysis, the level of the prolactin receptor transcripts increased dramatically during late pregnancy. In situ hybridization experiments revealed that prolactin receptor mRNA was specifically expressed in the glandular compartment and confirmed the dramatic increase of its expression that occurs at the end of pregnancy. Taken together, these findings are consistent with a putative role of prolactin and/or related molecules in the regulation of the proliferation of the glandular compartment and/or in the control of the secretory activity of the endometrium.

Treatment of Q fever endocarditis: comparison of 2 regimens containing doxycycline and ofloxacin or hydroxychloroquine.

BACKGROUND: Q fever endocarditis, caused by Coxiella burnetii, is fatal in 25% to 60% of patients. Currently, treatment with a long-term tetracycline and quinolone regimen for at least 4 years is recommended, although relapses are frequent. METHODS: Between January 1987 and December 1997, the reference treatment of Q fever endocarditis was compared with one of doxycycline and hydroxychloroquine sulfate. Patients were treated by conventional therapy until May 1991 and then by the new regimen. Microimmunofluorescence was used for antibody-level determination for diagnosis and follow-up. RESULTS: Thirty-five patients were included in the study, 26 males and 9 females. Of 14 patients treated with a doxycycline and quinolone combination, 1 died, 7 relapsed (3 were re-treated and 4 switched to the new regimen), 1 is still being treated, and 5 were considered cured using this regimen only. The mean duration of therapy for cure in this group was 55 months (median, 60 months). Twenty-one patients received the doxycycline and hydroxychloroquine regimen: 1 patient died of a surgical complication, 2 are still being treated, 17 were cured, and 1 is currently being evaluated. Two patients treated for 12 months but none of the patients treated for longer than 18 months relapsed. The mean duration of treatment in this group was 31 months (median, 26 months). No significant differences were observed between the 2 regimens in terms of death, valve surgery, or tolerance. The mortality rate for both regimens in this study was 5%. CONCLUSION: Prescription of the doxycycline and hydroxychloroquine combination for at least 18 months allows shortening of the duration of therapy and reduction in the number of relapses.

Novel recombinant analogues of bovine placental lactogen. G133K and G133R provide a tool to understand the difference between the action of prolactin and growth hormone receptors.

Two new analogues of bovine placental lactogen (bPL), bPL(G133K) and bPL(G133R), were expressed in Escherichia coli, refolded, and purified to a native form. Binding experiments, which are likely to represent the binding to site 1 only, to intact FDC-P1 cells transfected with rabbit (rb) growth hormone receptor (GHR) or with human (h) GHR, to Nb2 rat lymphoma cells, or to rabbit mammary gland membranes prolactin receptor (PRLR), revealed only small or no reduction in binding capacity. The complex formation between these analogues and receptor extracellular domains (R-ECD) of various hormones was determined by gel filtration. Wild type bPL yielded 1:2 complex with hGHR-ECD, rat PRLR-ECD, and rbPRLR-ECD, whereas both analogues formed only 1:1 complexes with all R-ECDs tested. Real time kinetics experiments demonstrated that the ability of the analogues to form homodimeric complexes was compromised in both PRLR- and GHR-ECDs. The biological activity transduced through lactogenic receptors in in vitro bioassays in rabbit mammary gland acini culture and in Nb2 cells was almost fully retained, whereas the activity transduced through somatogenic receptors in FDC-P1 cells transfected with rbGHRs or with hGHRs was abolished. Both analogues exhibited antagonistic activity in the latter cells. To explain the discrepancy between the effect of the mutation on the signal transduced by PLR versus GHRs we suggest that: 1) the mutation impairs the ability of site 2 of bPL to form a stable homodimeric complex with both lactogenic and somatogenic receptors by a drastic shortening of the half-life of 2:1 complex; 2) the transient existence of the homodimeric complex is still sufficient to initiate the signal transduced through lactogenic receptors but not through somatogenic receptors; and 3) one possible reason for this difference is that JAK2, which serves as a mediator of both receptors, is already associated with lactogenic receptors prior to hormone binding-induced receptor dimerization, whereas in somatogenic receptors the JAK2 receptor association occurs subsequently to receptor dimerization.

Regulation of mammary differentiation by extracellular matrix involves protein-tyrosine phosphatases.

Extracellular matrix and growth factors cooperate to regulate signaling pathways and gene transcription in adherent cells. However, the mechanism of extracellular matrix signaling is poorly defined. In mammary gland, the expression of milk protein genes is controlled by cross-talk between signals derived from the basement membrane protein, laminin, and the lactogenic hormone, prolactin. Signals from basement membrane are transduced by beta1 integrins and are required for prolactin to activate DNA binding of the milk protein gene transcription factor, Stat5. Here we show that basement membrane is necessary for tyrosine phosphorylation of the prolactin receptor and thus directly affects cytokine signaling and differentiation at the level of the plasma membrane. Prolactin does not induce tyrosine phosphorylation of its receptor, Jak2, or Stat5 in nondifferentiated breast epithelia cultured on collagen I, and we show that this is due to a vanadate-sensitive activity that inhibits the prolactin pathway. We suggest that protein-tyrosine phosphatases are novel targets for regulation by extracellular matrix and in mammary cells represent an additional control to the requirement of integrins for milk protein production.

In vivo study of prolactin (PRL) intracellular signalling during lactogenesis in the rat: JAK/STAT pathway is activated by PRL in the mammary gland but not in the liver.

The rat prolactin receptor (PRL-R) exists in two forms, which differ in the length of the cytoplasmic domains, tissue distribution, and biological activity. The short form predominates in liver while the long form is prevalent in mammary gland. We have compared activation by PRL of the JAK2-STAT pathway (protein tyrosine phosphorylation and STAT5 activation) in mammary gland and liver in an in vivo rat model of induction of lactogenesis by PRL injections, and we have studied the relative proportion of both forms of the receptor in these tissues by reverse transcription-polymerase chain reaction. Rats were ovario-hysterectomized on Day 19 of pregnancy, treated with bromocriptine, subsequently injected with 250 micrograms ovine PRL i.p. on Day 20, and killed 0-12 h after. Western blots of solubilized mammary gland and liver membranes immunoprecipitated with anti-PRL-R or anti-JAK2 antibodies showed that the PRL-R is constitutively associated with JAK2 and that the long form of the PRL-R is present in both tissues, while the short form was detected only in liver. Phosphorylated proteins corresponding to the long form of PRL-R and JAK2 appeared 15-60 min after ovine PRL injection in mammary extracts but not in liver. At these same times, an electrophoretic mobility shift assay, using a rat beta-casein probe specific for STAT5 binding, showed activated STAT5 in mammary gland cytosol and nuclear extracts. In the liver, low levels of activated STAT5 were detected in non-treated animals, which were not modified by PRL. Quantitative RT-PCR of liver and mammary PRL-R mRNA showed that the amount of the long form of PRL-R mRNA is roughly comparable in both tissues, while the short form is predominant in liver and in a minority in mammary tissue. Both forms were down-regulated by PRL only in mammary glands. Thus, during lactogenesis, mammary tissue responds to PRL by activation of JAK2 and STAT5, while the liver does not respond to PRL in spite of the presence of PRL-R associated with JAK2 and pre-existing activated STAT5. Thus, liver tissue may lack a critical component for activation of the PRL pathway, or the large quantities of the short form of the PRL-R may associate with the long form to constitute inactive heterodimers.