Biomedical subjects

J Djiane

Publications and source records attributed to J Djiane.

At least 55 records · Page 3Linked to original sources

PubMed1997

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.

Animals↗

PubMed1997

Site-directed mutagenesis of recombinant bovine placental lactogen at lysine-73 leads to selective attenuation of its somatogenic activity.

Bovine placental lactogen (bPL) is capable of binding and transducing biological activity via somatogenic and lactogenic receptors. To modify this capability, three analogs, bPL(K73D), bPL(K73F) and bPL(K73A), mutated at position 73, and corresponding to R64 in human GH (hGH), were produced in Escherichia coli. Circular dichroic spectrum analyses indicated proper refolding in all cases. Biological activity of these analogs was tested in vitro. In a lactogenic-receptor-mediated Nb2 rat lymphoma cell bioassay, bPL and its analogs acted similarly. In another lactogenic bioassay that measures beta-casein synthesis by HC-11 mouse mammary-gland cells, the analogs were 30-40% as potent as bPL. In contrast, somatogenic receptor-mediated bioactivity in FDC-P1 cells transfected with either rabbit (rb) or hGH receptor (R) was almost completely abolished in these analogs. In receptor binding assays, the effect was more conspicuous and the mutations affected not only somatogenic but also lactogenic binding. Binding to rat (r) and rabbit PRL receptor extracellular domains (ECDs) or membrane-embedded receptors was only slightly changed, except for bPL (K73D), which displayed very low affinity. In somatogenic binding assays to intact IM-9 human lymphocytes, hGHR-ECD or bovine liver membranes, bPL (K73D) did not bind at all, and bPL(K73F) or bPL(K73A) binding was drastically reduced. Binding experiments performed in real time using a BIAcore apparatus revealed that the decreased binding could be mainly attributed to increased k(off) rather than decreased k(on) values. The complex with hGHR-ECD revealed a 2:1 stoichiometry with bPL, bPL(K73F) and bPL(K73A), although the complex with these analogs was less stable than with bPL, whereas bPL(K73D) scarcely assembled a 1:1 complex. In contrast, bPL and the three analogs formed stable 1:2 complexes with rPRL-ECD. These results suggest that position 73 in bPL is more important for somatogenic than lactogenic properties and concurs with results from other groups, which have shown that R64, the analogous amino acid in hGH holds the same differential importance with respect to somatogenic binding.

Animals↗

PubMed1997

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.

Alternative Splicing↗

PubMed1997

Large-scale preparation and characterization of recombinant ovine placental lactogen.

To clone ovine placental lactogen (oPL) cDNA, total RNA from sheep placental cotyledon was reverse transcribed and the single-stranded cDNA was PCR-amplified with 5' and 3' primers containing, respectively, NcoI and PstI sites. The oPL cDNA fragment amplified between these two primers extended from A(-1) to the natural stop codon. The PCR product was gel-purified and subcloned into a Puc vector and the insert was sequenced on both strands, revealing several differences relative to the published sequence: S19N, S69N, D129E and R165Q. We assume that these differences can be accounted for by the high level of individual polymorphism, which has been described in detail for PLs of different species. The insert was subcloned into NcoI/ PstI-digested pTrc99A procaryotic expression plasmid and protein expression was induced by isopropyl-1-thio-beta-D-galactopyranoside. Because of low expression, oPL's cDNA was further subcloned into pET8 procaryotic expression plasmid. Its expression in E. coli strain BL21 transformed with this vector yielded 30-40 mg/l. The expressed protein, found in the inclusion bodies, was refolded into a monomer and purified on a Q-Sepharose column to homogeneity. Structural analysis using circular dichroism revealed a spectrum similar to that of human GH (hGH) thereby indicating proper refolding. Gel filtration and binding experiments, including real-time kinetic measurements using the surface plasmon resonance method revealed that oPL forms transient homodimeric complexes with extracellular domains of prolactin receptors from rabbit, rat and bovine and with hGH receptor. The purified oPL was biologically active in an Nb2-11C cell proliferation bioassay, in its ability to stimulate beta-casein synthesis in explants of ovine and rabbit mammary gland and fat synthesis in explants of bovine mammary gland, and in a proliferation assay using FDC-P1 cells transfected with rabbit or hGH receptors.

Animals↗

PubMed1997

Rapidly reversible binding of rabbit prolactin to the rabbit prolactin receptor accounts for the differences between homologous and heterologous binding.

The binding of radioiodinated rabbit (rb) prolactin (PRL) to rabbit mammary membranes is low and its affinity constant, 0.02 nM-1, calculated from heterologous inhibition assays, is about 300 times lower than that of ovine (o) PRL. Although the differences between homologous and heterologous binding are well documented in different species, the reasons for such differences are still unknown. Here we show that the low affinity of rbPRL for the native receptor does not affect its in vitro bioactivity compared with that of oPRL. We also show that rbPRL displays high specific binding to the baculovirus-expressed recombinant receptor and further establish that its lower affinity for binding to the homologous receptor is due to its faster and more complete dissociation compared with that of oPRL. Hormone binding affinity for full-length and carboxy-terminal truncated rbPRL receptor mutants expressed in mammalian or in baculovirus-infected cells was not affected by partial truncation of the cytoplasmic domain of the receptor, whereas the affinity for oPRL increased and that for rbPRL decreased upon truncation of both the cytoplasmic and membrane domains. The affinity of rbPRL for the native receptor is two orders of magnitude lower than that for the recombinant receptor. Affinity cross-linking and binding experiments showed that this difference in affinities is not related to selective cleavage of the native microsomal receptor during the binding reaction; however, this difference may be related to cell context-dependent differences in the oligomerization state of the receptor. Thus, obviously, the cloned receptor is alone sufficient for binding to rbPRL without requiring any receptor-associated protein. The lower affinity for rbPRL binding to its homologous receptor in comparison with higher affinity binding of oPRL to the same receptor is attributable to differences in their dissociation kinetics and in the conformational requirements of the receptor-hormone interaction site for binding to the two hormones.

Analysis of Variance↗

PubMed1997

Homology modeling of rabbit prolactin hormone complexed with its receptor.

The pituitary hormone prolactin (prl) is implicated in a number of biological functions, especially lactation, which is mediated through specific lactogenic receptors (PrlR). Human growth hormone (hGH) is also a pituitary hormone responsible for linear growth. While the growth hormone receptor (hGHR) binds only hGH, hPrlR can interact with both hGH and hPrl. Using structural information from the human growth hormone (hGH)/receptor (hGHR) complex, we modeled by homology a complex between rabbit prolactin hormone (rbPrl) and its receptor (rbPrlR). While the somatogenic hormone/somatogenic receptor (hGH/hGHR) and somatogenic hormone/lactogenic receptor (hGH/hPrlR) interactions are now known and well studied, here we propose a model for the interaction of the lactogenic hormone with its receptor (rbPrl/rbPrlR), and compare these three kinds of ligand/receptor interaction. We identified residues contributing to the active site and tested the potential dimerization of the receptor. Biochemical studies and information deduced from the modeled complex do not exclude a homodimeric form but point to a functional heterodimeric complex.

Amino Acid Sequence↗

PubMed1996

Real-time kinetic measurements of the interactions between lactogenic hormones and prolactin-receptor extracellular domains from several species support the model of hormone-induced transient receptor dimerization.

Interactions of recombinant soluble prolactin receptors-extracellular domains (PRLR-ECDs) from rabbit, rat, and cow and human growth hormone receptor ECD with immobilized human growth hormone, several prolactins, and bovine placental lactogen were studied utilizing surface plasmon resonance. This method enables real-time kinetic measurements of the interactions and calculations of kinetic constants and of the stoichiometry of interaction, even in cases where only transient interactions occur. In contrast to gel filtration or crystallographic studies, where in most cases the interaction of PRLR-ECDs with various lactogenic hormones indicated formation of 1:1 complexes, our surface plasmon resonance experiments indicated in all cases the transient formation of a 2:1 complex. In most of the interactions the 2:1 complex was very unstable and underwent rapid dissociation to a 1:1 complex. This situation was particularly characteristic of homologous interactions involving hormone and receptor from the same species and was mainly attributed to increased dissociation constants. We suggest that as in the case of growth hormone PRLR activation occurs via hormone-induced transient homodimerization of the receptor, lasting only a few seconds, and that this may be sufficient to initiate the biological signal. Once the signal is initiated, the receptor dimer is no longer required. Its rapid dissociation to a 1:1 complex or to its components may even be advantageous in that it permits activation of additional receptors.

Animals↗

PubMed1996

Involvement of a subset of tyrosine kinases and phosphatases in regulation of the beta-lactoglobulin gene promoter by prolactin.

This study used pharmacological intervention to provide support for a role of kinases and phosphatases in prolactin transactivation of a milk protein gene. It was based on transient cotransfection using a rabbit prolactin receptor expression plasmid and a beta-lactoglobulin promoter/CAT reporter construct. In cotransfected CHO cells, herbimycin A and tyrphostin, two tyrosine kinase inhibitors, were able to decrease the CAT response by over 50%, along with tyrosine phosphorylation of cellular proteins, whereas genistein and lavendustine were without effect on lactoglobulin transactivation. Orthovanadate, an inactivator of tyrosine phosphatases, was able to substitute for prolactin in inducing the CAT response. Staurosporine, a non-specific kinase inhibitor, was able, when used at low concentrations (10 nM), to augment the prolactin response strikingly. Threonine/serine kinases do not appear to be involved early in beta-lactoglobulin promoter transactivation, since four C-kinase inhibitors and okadaic acid a threonine/serine phosphatase inhibitor, were without substantive effect. We conclude that specific tyrosine kinases are responsible for most of the signal transduction from the prolactin receptor to the beta-lactoglobulin gene promoter.

Animals↗

PubMed1996

Selective modification of recombinant bovine placental lactogen by site-directed mutagenesis at its C terminus.

Five recombinant analogues of bovine placental lactogen (bPL) ((bPL(S184H), bPL(S187A), bPL(S187F), bPL(T188F), bPL(T188F,I190F)) were prepared, expressed in Escherichia coli, and purified to homogeneity. Circular dichroism analysis revealed no or minor structural changes, except in bPL(T188F,I190F). Binding and biological activities of bPL(T188F,I190F) were almost completely abolished, whereas bPL analogues mutated at position 187 retained their full activity. Point mutation T188F resulted in selective modification; binding to somatogenic receptors, their extracellular domains (ECDs), and to bPLR in the endometrium as well as somatogenic receptor-mediated biological activities were reduced or abolished, whereas binding to lactogenic receptors, their ECDs, and subsequent biological activity was fully or almost fully retained. This selective modification most likely results from a steric hindrance induced by a bulky Phe-188 chain of bPL which interacts with the Arg-43 of the human or Leu-43 of the non-human GHRs. Point mutation S184H abolished the interaction with hGHR, most likely due to the unfavorable charge-charge interaction, possibly accompanied by steric hindrance between Arg-43 of the receptor and the newly introduced His-184 and possible interference with the putative interaction between the alkyl portion of Thr-188 and Lys-185 of bPL with Trp-104 of hGHR. In contrast, bPL(S184H) retained its capacity to interact with nonhuman GHRs. Decrease in the biological activity of bPL(S184H) was also observed in two lactogenic receptor-mediated bioassays most likely due to the elimination of the intermolecular hydrogen bond of Ser-184 with a side chain of Tyr-127, which appears in all lactogenic receptors.

Amino Acid Sequence↗

PubMed1996

Growth hormone-binding protein in the goat: characterization, evolution under exogenous growth hormone treatment, and correlation with liver growth hormone receptor levels.

This report describes the identification and characterization of a specific, high-affinity growth hormone-binding protein (GHBP) in lactating goat serum. Serum samples were incubated with [125I]human GH as ligand and in the absence or in the presence of bovine GH as competitor. GH-GHBP complex formation was performed by high-performance liquid chromatography, and the radioactivity was recorded on-line with a Berthold LB detector connected to a computer. The results showed that a serum protein was able to bind specifically to human GH and bovine GH but not to ovine prolactin. Scatchard plots indicated an affinity constant of 4.5 x 10(8) M-1 and a maximum binding capacity of 4.8 x 10(-10) mol/l. In addition, we conducted a 4-wk study to determine the effects of recombinant bovine GH administration on milk production in lactating goats. The effects of recombinant bovine GH treatment on milk production and on the regulation of GHBP and hepatic GH receptor levels were studied. As expected, recombinant bovine GH injected daily increased yields of milk, fat, protein (40, 61, and 40%, respectively), and circulating insulin-like growth factor 1 concentrations compared with controls. During the pretreatment and treatment periods, the control goats exhibited a constant amount of GHBP in serum. No consistent effect of GH treatment on GHBP level was observed. The binding of [125I]bovine GH to hepatic microsomal membranes of GH-treated goats was significantly decreased compared with that of control goats. After MgCl2 desaturation of membranes, the results demonstrated that the down-regulation of GH hepatic receptors, observed for the treated goat group, was induced by receptor occupancy without modification of binding affinity. The GH receptor gene expression, analyzed by slot blot and hybridization with an [alpha-32P]GH receptor cDNA probe, was not modified by the GH treatment. In lactating goats, the galactopoietic effect of exogenous GH involved a hepatic receptor occupancy. The individual concentration of GHBP in serum cannot explain the individual variations of responses to GH treatment in goats.

Animals↗

PubMed1996

Development of a constitutively active mutant form of the prolactin receptor, a member of the cytokine receptor family.

The extracellular domain of the PRL receptor (PRL-R) is composed of two subdomains of approximately 100 amino acids, S1 and S2. To explore the functional significance of these subdomains in PRL binding and signal transduction, deletion mutants of S1 or/and S2 subdomains were constructed. We report here the inability of each of these mutant receptor forms to bind PRL after expression in COS-7 cells. We also studied the abilities of these different mutant receptors to respond to hormonal stimulation after transfection of each mutant complementary DNA into CHO-K1 cells along with a chimeric gene containing the promoter of a milk protein gene (beta-lactoglobulin) fused to chloramphenicol acetyltransferase coding sequence. Somewhat unexpectedly, a constitutively (PRL-independent) mutant form of the PRL-R was obtained after deletion of the S2 subdomain. Moreover, we analyzed, in CHO-K1 cells, the biological activity of chimeric receptors constructs in which each subdomain sequence was replaced by an unrelated, but coding, sequence of foreign protein, and we confirmed a specific requirement for the S1 sequence in the constitutive activity. In contrast, the S2 subdomain produced an inhibitory effect on S1 constitutive activity. Cotransfection experiments with the wild-type receptor and the constitutive mutant receptor provided evidence that the wild-type receptor was able to inhibit the constitutive activity of the deleted mutant. Furthermore, in the mouse mammary epithelial cell line HC11, the constitutive PRL-R form was able to induce transcription of the beta-casein gene in the absence of PRL. These results suggest a complex signal transduction process that implicates each extracellular PRL-R subdomain. Possible mechanisms for the constitutive effect are discussed.

Animals↗

PubMed1996

Ontogenesis of LH and FSH receptors in postnatal rabbit testes: age-dependent differential expression of long and short RNA transcripts.

The ontogeny of testicular LH and FSH receptors was studied in New Zealand rabbits from 20 to 180 days postpartum. The concentrations of free receptors (per mg total proteins) were very low at day 20. They increased steeply at day 30 for the LH receptor and at day 50 for the FSH receptor. Three RNA bands (1.2, 2.5 and 3 kb) were repeatedly detected on northern blots for the LH receptor and two bands (1.2 and 2.2 kb) were detected for the FSH receptor. The 1.2 kb band (which cannot give rise to full-length, membrane-anchored receptor) was present throughout the 20-180 day period for each receptor. However, the higher molecular mass bands were nearly undetectable at day 20. The 2.5 and 3 kb bands of the LH receptor increased twofold between day 20 and day 120, while the 2.2 kb band of the FSH receptor increased fivefold between day 20 and day 75. Thus the very low concentrations, or even absence, of the larger transcripts of both LH and FSH receptors were correlated with the inability to detect their cognate protein until 20 days of age. Subsequently, coordinated increases in high molecular mass transcripts and protein were observed for both receptors. Total LH receptor content increased in parallel to the previously reported increase in plasma testosterone between day 65 and day 100. FSH receptor density began to increase steeply at day 50, just at the onset of spermatogenesis. Thus, postnatal testicular development in the rabbit seems to entail the transcription of high molecular mass, translatable transcripts of the gonadotrophin receptors.

Animals↗

PubMed1996

Cloning of rabbit prolactin cDNA and prolactin gene expression in the rabbit mammary gland.

cDNA clones coding for rabbit prolactin were isolated from a pituitary library using a rat prolactin RNA probe. One cDNA contained 873 bases including the entire coding sequence of rabbit prolactin, its signal peptide and the 5' and 3' untranslated regions of 44 and 145 nucleotides respectively. The deduced amino acid sequence of the cloned prolactin cDNA presented a 93-78% identity with mink, porcine and human prolactins. The prolactin gene transcription was investigated by RT-PCR analysis in several organs of midlactating New Zealand White rabbits. The ectopic transcription of the prolactin gene was examined in more detail in the mammary gland. A strong PCR signal was detected in the mammary gland of virgin does and was also observed during pregnancy and at the beginning of lactation. This PCR signal was very weak in mid-lactating and absent in post-weaning mammary gland.

Amino Acid Sequence↗

PubMed1995

Preparation and characterization of recombinant prolactin receptor extracellular domain from rat.

Complementary (c)DNA of the extracellular domain of rat prolactin receptor (rPRLR-ECD) was cloned in the prokaryotic expression vector pTrc99A, and expressed in Escherichia coli following induction with isopropyl-b-D-thiogalactopyranoside. The expressed rPRLR-ECD protein, contained within the refractile body pellet was solubilized in 4.5 M urea, refolded and purified on a Q-Sepharose column by stepwise elution with NaCl. Only approximately 10% of the expressed protein refolded as a monomeric fraction, yielding 5-6 mg/l of induced culture. The purified protein was over 98% homogeneous, as shown by SDS-PAGE in the presence or absence of reducing agent, and by chromatography on a Superdex column. Its molecular mass, determined by SDS-PAGE in the absence of reducing agent, was 28 kDa, and by gel filtration, 25.6 kDa. Binding experiments indicated high affinity for bovine placental lactogen (bPL) and human growth hormone (hGH) as compared to ovine (o) or rat PRLs. Gel filtration was used to determine the stoichiometry of rPRLR-ECD's interaction with these hormones. At a 5 microM initial concentration of the hormones, formation of 2:1 (ECD:ligand) complexes was detected with bPL, hGH and oPRL whereas only 1:1 complex was formed with rPRL. Dilution (25-fold) of these complexes did not affect the stoichiometry with bPL, whereas with hGH a clear tendency towards dissociation of the initial 2:1 complex to 1:1 complex was observed. This tendency was even stronger in the case of oPRL. Although all four hormones exhibited nearly identical activities in the Nb2-11C lymphoma cell bioassay, the ability of the purified rat or rabbit PRLR-ECD to inhibit hormonal mitogenic activity generally reflected their affinity for the respective hormones. In view of these and former results, we suggest that unlike in the GH:GHR-ECD interaction, the inability of lactogenic hormones to form a 1:2 complex with soluble recombinant PRLR-ECDs does not necessarily predicts lack of biological activity.

Animals↗

PubMed1995

Role of tyrosine phosphorylation in potassium channel activation. Functional association with prolactin receptor and JAK2 tyrosine kinase.

Chinese hamster ovary (CHO) cells, stably transfected with the long form of the prolactin (PRL) receptor (PRL-R) cDNA, were used for PRL-R signal transduction studies. Patch-clamp technique in whole cell and cell-free configurations were employed. Exposure of transfected CHO cells to 5 nM PRL led to the increase of Ca(2+)- and voltage-dependent K+ channel (KCa) activity. The effect was direct as it was observed also in excised patch experiments. A series of tyrosine kinase inhibitors was studied to investigate the possible involvement of protein tyrosine kinases in KCa functioning and its stimulation by PRL. Genistein, lavendustin A, and herbimycin A decreased in a concentration and time-dependent manner the amplitude of the KCa current in whole cell and the open probability of KCa channels in cell-free experiments. The subsequent application of PRL was ineffective. The protein tyrosine phosphatase inhibitor orthovanadate (1 mM) stimulated KCa channel activity in excised patches, indicating that channels can be modulated in opposite directions by protein tyrosine kinase and protein tyrosine phosphatase. Moreover, in whole cell experiments as well as in excised patch recordings, anti-JAK2 tyrosine kinase antibody decreased the KCa conductance and the open probability of the KCa channels. Subsequent application of PRL was no longer able to stimulate KCa conductance. Immunoblotting studies using the same anti-JAK2 antibody, revealed the constitutive association of JAK2 kinase with PRL-R. Preincubation of anti-JAK2 antibody with the JAK2 Immunizing Peptide abolished the effects observed using anti-JAK2 antibody alone in both electrophysiological and immunoblotting studies. We conclude from these findings that these KCa channels are regulated through tyrosine phosphorylation/dephosphorylation; JAK2 tyrosine kinase, constitutively associated with PRL-R, is implicated in PRL stimulation of KCa channels.

Animals↗

PubMed1995

Polymeric-Ig receptor gene expression in rabbit mammary gland during pregnancy and lactation: evolution and hormonal regulation.

The polymeric immunoglobulin receptor (poly Ig-R) mediates transcytosis of IgA and IgM antibodies produced by local plasma cells across epithelial cells of mucosal and glandular tissues. Gene expression of the poly-Ig R was analyzed in rabbit mammary gland during pregnancy and lactation. The poly Ig-R was expressed as early as day 8 (G8) of gestation and mRNA accumulation remained low until about G18. From G21, the mRNA abundance increased and reached steady state levels approximately 5-fold higher at day 15 of lactation (L15) when compared to basal levels at G8. The hormonal regulation of poly-Ig receptor gene expression was assessed in mammary organ cultures. Poly-Ig R mRNA accumulation in mammary explants cultured for 24 or 48 h in the presence of ovine prolactin (oPRL) was significantly increased to a maximal 4-fold level at 1 microgram ml-1 of oPRL. Estradiol (100 pg ml-1) or progesterone (1 microgram ml-1) did not further stimulate poly-Ig R expression. In contrast, their combination resulted in a significant 30-50% decrease of poly-Ig-R mRNA levels. The addition of 1 microgram ml-1 of cortisol to medium in the absence or presence of estradiol or progesterone decreased the amount of poly-Ig-R mRNA. The results suggest that until mid-pregnancy, poly-Ig-R expression is inhibited by elevated progesterone-estradiol concentrations and that the subsequent increase is due to the concomitant decrease of the two circulating steroids and the increase of serum prolactin levels.

Animals↗

PubMed1995

The rabbit mammary gland prolactin receptor is tyrosine-phosphorylated in response to prolactin in vivo and in vitro.

We report the first in vivo study demonstrating tyrosine phosphorylation of mammary gland proteins including the prolactin receptor, in response to the injection of prolactin. Immunoblotting of mammary gland membrane extracts revealed that subunits of 200, 130, 115, 100, 90, 70, and 45 kDa display increased tyrosine phosphorylation within 5 min of prolactin administration. The 100-kDa component was identified as the full-length prolactin receptor by a variety of means including immunoprecipitation and immunoblotting with monoclonal (U5, 917, 110, and 82) and polyclonal (46) antibodies to the prolactin receptor. Maximal receptor phosphorylation was seen within 1 min of hormone injection, and to obtain a strong response it was necessary to deprive rabbits of their endogenous prolactin for 36 h. Rapid tyrosine phosphorylation of the full-length receptor was verified by its demonstration in Chinese hamster ovary cells stably transfected with rabbit prolactin receptor cDNA. Both in vivo and in vitro, the phosphorylation signal was transient, being markedly reduced within 10 min of exposure to prolactin. Tyrosine-phosphorylated receptor was shown to be associated with JAK 2 by immunoblotting of receptor immunoprecipitated from transfected Chinese hamster ovary cells with polyclonal 46. A 48-kDa ATP-binding protein was also shown to be associated with the mammary gland receptor by U5 or polyclonal 46 immunoprecipitation of receptor complexes following covalent labeling with [alpha-32P]azido-ATP. Our demonstration of prolactin receptor tyrosine phosphorylation raises the possibility of signaling pathways regulated by receptor/SH2 protein interaction, which would facilitate prolactin specific responses. The fact that a period of hormone deprivation is needed for significant hormone triggered receptor phosphorylation indicates that the mammary gland receptor exists in a largely desensitized state in vivo, analogous to the related growth hormone receptor.

Adenosine Triphosphate↗

PubMed1995

Prolactin induces growth inhibition and promotes differentiation of CHO cells stably transfected with prolactin receptor complementary DNA.

We have characterized a stable and functional transfectant of the rabbit prolactin receptor in Chinese hamster ovary cells, and investigated the action of prolactin (PRL) on the growth and differentiation of this transfectant (clone E32). PRL induced a significant inhibition of E32 cell proliferation. Growth inhibition correlated with gene induction of the molecular marker of ovarian differentiation cholesterol side chain cleavage P450 (P450scc). Both effects were inversely proportional to cell confluence. The limits and potential development of such transfected cellular systems are discussed.

Animals↗