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J Djiane

Publications and source records attributed to J Djiane.

At least 73 records · Page 4Linked to original sources
PubMed1995

The proximal milk protein binding factor binding site is required for the prolactin responsiveness of the sheep beta-lactoglobulin promoter in Chinese hamster ovary cells.

To identify cis-acting prolactin (PRL) response elements within the sheep beta-lactoglobulin (BLG) promoter, CHO cells were co-transfected with a rabbit PRL-receptor (PRL-R) expression plasmid and a number of BLG-CAT constructs. Resection through the 4200 bp BLG promoter diminished the PRL response. Mutation of the proximal binding site for milk protein binding factor (MPBF), a previously described mammary gland transcription factor, abolished the PRL inducibility of full length and shorter forms of the promoter. MPBF was shown to be similar to the Stat protein mammary gland factor (MGF) which has been shown to mediate PRL responsiveness of the rat beta-casein gene in mammary cells. MPBF binding activity was detected in the nucleus of CHO cells and was increased 2-6-fold in cells stably transfected with the PRL-R. The lactating mammary gland has high levels of MPBF binding activity and it is likely that this has an important role in the PRL induction of a variety of milk protein genes.

Animals↗
PubMed1995

Gonadotropins in lactating sows exposed to long or short days during pregnancy and lactation: serum concentrations and ovarian receptors.

The objective of this study was to determine the influence of day length on gonadotropin profiles and the expression of their ovarian receptors in lactating sows. Primiparous Large White gilts were exposed to either a gradual increase (from 12 to 16 h/day, LONG treatment, n = 13) or decrease (from 12 to 8 h/day, SHORT treatment, n = 12) in photoperiod during gestation. Weaning occurred at Day 21 of lactation. All 4 sows that were submitted to the SHORT light duration and checked for postpartum estrus demonstrated an estrus by 10 days postwearing in contrast to 2 of 5 sows submitted to the LONG light duration (p < 0.05). In the remaining 16 sows, day length had no significant effect on the number of LH pulses or on mean or basal concentrations of gonadotropins (FSH and LH) or estradiol-17 beta measured at Day 20 of lactation. Ovarian receptors for gonadotropins and prolactin (PRL) and their mRNA were measured through use of receptor-binding and slot-blot analyses, respectively, at Day 21 of lactation in these 16 sows. ALONG photoperiod duration had no influence on receptor number, binding or affinity, but it significantly increased LH receptor mRNA levels (p < 0.05). However, FSH receptor mRNA levels were similar in the two groups of sows. Plasma LH concentration was positively related to LH and FSH receptor content but not to their cognate mRNA levels. Plasma concentration of FSH was positively related to the level of its own receptor mRNA as well as to that of the PRL receptor mRNA. Although the LONG day length may have delayed the return to estrus, there was no effect on gonadotropin secretion. Our results show an effect of photoperiod only on the level of LH receptor mRNA. We suggest that not all transcripts of the LH receptor are translated and that nontranslatable mRNA accumulate in ovaries of sows exhibiting delayed estrus.

Animals↗
PubMed1995

Presence of the two growth hormone receptor messenger RNA isoforms in human breast cancer.

In the present study, we have investigated specific growth hormone (GH) receptor gene expression in breast cancer cell lines and tissues. By Northern blot analysis, using a human GH receptor cDNA probe, the classically observed 4.7-kilobase GH receptor mRNA was evidenced in 2 of 29 cancer biopsies and in the MCF7 and T47-D cell lines. Reverse transcription coupled to PCR was used to amplify the GH receptor sequence encompassing a part of the extracellular domain as well as the transmembrane domain. An amplification product of the expected size (456 base pairs) was observed in 28 of 29 breast biopsies and in all the breast cancer cell lines studied (T47-D, MCF7, MDA-MB-231, and BT-20). The reverse transcription-PCR product was shown to be specific by Southern blot hybridization with the GH receptor cDNA probe and by specific cleavage of the amplified products with restriction enzymes. For the first time, the expression of GH receptor gene in breast cancer cell lines and biopsies is demonstrated in this study, suggesting a GH-specific action in tumor development. Additionally, the two isoforms of the human GH receptor (hGHR) mRNA, one containing exon 3 (hGHR-wt mRNA) and one excluding exon 3 (hGHR-d3 mRNA), were found to be expressed independently or simultaneously (29 tumors analyzed). The presence of hGHR-d3 mRNA appears to be patient specific, as demonstrated by comparing the expression pattern of both mRNAs between tumor biopsy and lymphocytes of the same patient.

Base Sequence↗
PubMed1994

Evidence for N-glycosylation and ubiquitination of the prolactin receptor expressed in a baculovirus-insect cell system.

The molecular mass of the rabbit prolactin receptor (rbPRLR) deduced from cDNA cloning is 66 kDa. However, the molecular mass of the full-length receptor expressed in the insect Sf9 cells was found to be 94 kDa. In order to explain this discrepancy, we analyzed the possible post-translational modifications of the PRLR. Sf9 cells were infected with recombinant baculoviruses in the presence of tunicamycin, an inhibitor of N-glycosylation. Results showed that an additional approximately 9 kDa of the extracellular domain could be attributed to the N-glycosylation and another additional approximately 20 kDa covalent modification occurred in the cytoplasmic part of the receptor. Western blot analysis, using anti-ubiquitin antibodies, revealed that the rbPRLR was ubiquitinated in its cytoplasmic domain.

Animals↗
PubMed1994

Identification of JAK protein tyrosine kinases as signaling molecules for prolactin. Functional analysis of prolactin receptor and prolactin-erythropoietin receptor chimera expressed in lymphoid cells.

The mechanism of action of prolactin (PRL) was studied in murine lymphoid BAF-3 cells transfected with either the long form of the PRL receptor (PRL-R), or a chimeric receptor consisting of the extracellular domain of the PRL-R and the transmembrane and intracellular domain of the erythropoietin receptor (PRL/EPO-R). PRL sustained normal and long-term proliferation of BAF-3 cells expressing either the PRL-R or the hybrid PRL/EPO-R. Upon [125I]PRL cross-linking, both types of BAF-3 transfectants were shown to express two [125I]PRL cross-linked species differing in size by 20 kDa. These cross-linked complexes, after denaturation, were recognized by antibody against the PRL-R, indicating that they contain the transfected receptor. PRL induced rapid and transient tyrosine phosphorylation of both the PRL-R and the PRL/EPO-R in BAF-3 transfectants. Furthermore, PRL induced rapid tyrosine phosphorylation of the Janus kinase 2 (JAK2) which was already physically associated with the PRL-R or the PRL/EPO-R in the absence of ligand. JAK1 was also associated with PRL-R and PRL/EPO-R in the absence of ligand. However, only in BAF-3 cells expressing the PRL-R does PRL induce rapid and transient tyrosine phosphorylation of JAK1. These results demonstrate that JAK protein tyrosine kinases couple PRL binding to tyrosine phosphorylation and proliferation.

Animals↗
PubMed1994

Preparation of the extracellular domain of the rabbit prolactin receptor expressed in Escherichia coli and its interaction with lactogenic hormones.

The cDNA of the extracellular domain of the rabbit prolactin receptor (rbPRLR-ECD) was cloned in the prokaryotic expression vector pTrc99A to enable its expression in Escherichia coli after induction with isopropyl-1-thio-beta-D-galactopyranoside. The bacterially expressed rbPRLR-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. The bioactive monomeric fraction was eluted in 0.05 M NaCl, yielding 15-20 mg/8 liters of induced culture. The purified protein was > 98% homogeneous, as shown by SDS-polyacrylamide gel electrophoresis in the presence or absence of reducing agent and by chromatography on a Superdex column. Its molecular mass was 25 kDa as determined by SDS-polyacrylamide gel electrophoresis in the absence of reducing agent and 22 kDa as determined by gel filtration. Binding experiments revealed remarkable differences between rabbit and porcine prolactins (PRLs) and the other tested lactogenic hormones. Gel filtration was used to determine the stoichiometry of the rbPRLR-ECD interaction with ovine, rabbit, and porcine PRLs, with human growth hormone and its truncated des-7 analogue, and with bovine placental lactogen (bPL) and des-13-bPL. The formation of only 1:1 complexes was indicated, except with bPL, for which a 2:1 complex was detected. Identical stoichiometry was also obtained using excess radiolabeled rbPRLR-ECD in gel filtration experiments. Interaction of 125I-labeled ovine PRL with rbPRLR-ECD secreted into conditioned medium by rbPRLR-ECD cDNA-transfected COS 7 cells also indicated formation of 1:1 molar complexes. Despite the differences in binding potency and stoichiometries of the interaction with rbPRLR-ECD, all seven tested hormones were biologically active in inducing PRL receptor-mediated casein synthesis in explants of rabbit mammary gland. We therefore propose that the formation of the 1:2 complexes with soluble rbPRLR-ECD is not predictive of biological activity of the different lactogenic hormones.

Amino Acid Sequence↗
PubMed1994

Cytoplasmic sequences of the growth hormone receptor necessary for signal transduction.

To study structure-function relationships of the growth hormone (GH) receptor (GHR), two functional systems have been developed. CHO cells were transiently cotransfected with the cDNA encoding the full-length rat GHR and with a construct consisting of the 5' flanking region of one of two GH-dependent genes encoding ovine beta-lactoglobulin or serine protease inhibitor 2.1 (Spi 2.1, formerly Spi.1; the corresponding rat gene has recently been redesignated Spin2a) coupled to the bacterial reporter gene encoding chloramphenicol acetyltransferase (CAT). Transfected cells were grown in the absence and presence of human GH and dexamethasone for the Spi 2.1 gene construct. GH was able to activate each promoter (with approximately 4-fold induction of CAT activity) in a dose-dependent manner. For both tests, the maximal effect was observed at 20 nM human GH. These tests have been used to identify functional domains of the GHR. Two truncated (T) GHRs, lacking most or part of the cytoplasmic domain [called T276 (ending at residue 276) and T436 (ending at residue 436)], were unable to stimulate CAT activity. The GHR contains a proline-rich region, called "Box I," conserved in the cytokine/GH/prolactin receptor family. Alanine substitutions for the four prolines of GHR Box I were introduced. Single proline-to-alanine mutations did not affect the functional activity of the GHR. However, modification of the four prolines together or deletion of the Box I (15 amino acids between positions 279 and 293) resulted in the complete absence of GH stimulation. Thus, the proline-rich region, shown to be important for other members of this receptor superfamily, is also critical for GH signal transduction.

Amino Acid Sequence↗
PubMed1994

Endocytosis and degradation of prolactin and its receptor in Chinese hamster ovary cells stably transfected with prolactin receptor cDNA.

Molecular cloning of the prolactin (PRL) receptor cDNA has revealed different forms of the receptor: among them, the longest form encodes a transmembrane protein of 592-598 amino acids and was originally found in rabbit mammary gland as well as in human and rat tissues. It contains a cytoplasmic domain of 358 amino acids. In CHO cells transfected with the PRL receptor cDNA, PRL is able to induce the specific expression of a reporter gene provided with the promoter of the milk protein gene beta-lactoglobulin. The cDNA encoding this long receptor form has been expressed permanently after stable transfection of Chinese hamster ovary (CHO) cells. In these cells, we have determined the fate of the bound hormone and of the receptor. At 37 degrees C, transfected cells were able to endocytose 125I-labeled human growth hormone (hGH) or ovine prolactin (oPRL) at an initial rate of about 1 fmol/h at 100 pM labeled hormone and 10(6) cells/well. Lowering the temperature to 15 degrees C slowed the endocytosis of [125I]hGH by a factor of 5. These results were confirmed by electron microscopy with oPRL labeled with colloidal gold. At 37 degrees C, the receptor underwent rapid insertion to the cell surface and constitutive endocytosis (half-life 80 min). This rate of endocytosis was enhanced in the presence of 10 nM oPRL (half-life 8 min), leading to down-regulation of the receptor by exhaustion of the intracellular receptor pool. After down-regulation, the cell surface was replenished with newly synthesized PRL receptor with a half-time of 8-10 min. If cycloheximide was added, almost no receptors could be found on the cell surface. These results indicate that in transfected cells the PRL receptor behaved largely as in classical target cells. A "conveyor belt" endocytosis behavior was found, with degradation of the endocytosed receptors, and occupation by the hormone enhancing this process. Moreover, since the PRL receptor belongs to a family of receptors in which companion protein(s) seem to play important roles, transfected CHO cells appear to provide the expressed receptors with the necessary element(s) to function as in normal PRL target cells.

Animals↗
PubMed1994

A limited cytoplasmic region of the prolactin receptor critical for signal transduction.

Prolactin receptors (PRL-R) are members of the cytokine receptor superfamily, which have in common, an absence of any known consensus sequence for signal transduction in their cytoplasmic domains. Four areas of high sequence homology have been identified in the cytoplasmic domains of PRL and growth hormone (GH) receptors, which may be important for signal transduction. The aim of this study was to investigate the role of these cytoplasmic regions in the functional activity of the PRL-R. Several mutant forms of PRL-R were constructed either by truncation or by deletion of the cDNA. Biological activities of these mutant receptors were assayed in CHO cells using a functional assay consisting in the co-transfection of PRL-R cDNA, along with a PRL responsive promoter fused to the coding sequence of the chloramphenicol acetyl transferase (CAT) gene. Progressive truncation of the cytoplasmic domain led to a progressive loss of ability to transactivate the CAT gene. Fully active PRL-R could be obtained when 217 of 358 aa of the cytoplasmic domain were present. Deletion of the first region of homology with the GH-R (residues 245-267) abolished the functional activity of PRL-R, whereas deletion of the second region of homology (residues 322-333) was without effect. These results indicate that a critical cytoplasmic region of 23 residues proximal to the transmembrane domain is essential for PRL signal transduction. There is strong homology within an 8-residue segment of this region with other members of the cytokine receptor superfamily, suggesting it contains a sequence necessary for signal transduction.

Amino Acid Sequence↗
PubMed1994

Early effects of PRL on ion conductances in CHO cells expressing PRL receptor.

Chinese hamster ovary (CHO-K1) cells were stably transfected with prolactin (PRL) receptor cDNA. These cells (CHO-E32) expressed the long form of functional PRL receptor. Using microfluorimetric and patch-clamp techniques, we have investigated the effects of PRL on intracellular Ca2+ concentration ([Ca2+]i) and membrane ion conductances. Exposure of CHO-E32 cells to 5 nM PRL resulted in an increase in [Ca2+]i. Two types of response were observed: 1) a stimulation of Ca2+ entry and 2) an intracellular Ca2+ mobilization. As PRL inhibited voltage-activated Ca2+ current, the PRL-induced Ca2+ increase does not involve voltage-activated Ca2+ channels. PRL also increased a charybdotoxin-sensitive Ca(2+)-dependent K+ conductance. Simultaneous measurements showed that PRL hyperpolarized the membrane potential before increasing intracellular Ca2+ levels. In voltage clamp, hyperpolarizing voltage steps were associated with increased Ca2+ concentrations, whereas depolarizing voltage steps decreased [Ca2+]i. Cell-free patch-clamp experiments showed that PRL directly stimulates K+ channel activity. Our results suggest the existence of a regulatory complex involving a protein kinase tightly associated with the Ca(2+)-activated K+ channels and that PRL stimulates these channels by means of the activation of protein kinase. The resulting hyperpolarization stimulates Ca2+ entry, probably through voltage-insensitive nonspecific channels.

Animals↗
PubMed1994

Prolactin gene expression in ovine and caprine mammary gland.

The presence of prolactin (PRL) mRNA in the mammary gland of lactating goats and sheep was demonstrated by Northern analysis and RT-PCR. This provides evidence that the PRL gene is transcribed in this tissue. This ectopic expression is not restricted to the lactational period, as PRL transcripts were also found during the last third of pregnancy. Comparison of mammary and pituitary PRL mRNAs showed that they are similar in size but less abundant in mammary gland. In addition, an 847-bp cDNA fragment amplified from mammary retrotranscripts, containing the entire coding region and the major part of the 5' and 3' untranslated regions (UTRs), was found to be identical in sequence to its pituitary counterpart. Primer extension analysis, performed to obtain further information on the structure of the mammary PRL mRNA, has shown that the 5' UTR is 56 nucleotides (nt) long for both species. This is comparable with the size (53 nt) found using the caprine pituitary RNA as template. These results strongly suggest that the PRL gene is not transcribed from a different promoter in mammary gland, as has been demonstrated for placental and lymphocyte cells, but is more likely transcribed from the pituitary-specific promoter. Finally, the presence of PRL mRNA in polysomal fractions suggests that PRL is synthesized in mammary cells.

Amino Acid Sequence↗
PubMed1994

Selective modification of human growth hormone by site-directed mutagenesis: implications for the diversity of biological actions.

Human growth hormone (GH) is an anabolic hormone required for normal growth. In addition, human GH affects the metabolism of proteins, carbohydrates and fats and possesses lactogenic effects. Although the GH receptor has recently been cloned, it is not clear whether the diverse biological activities of human GH are transduced through a single or through several types of related receptors. To address this question, 15 recombinant analogues of human GH were prepared and tested in bioassays in vitro and in vivo, in which the hormone action is mediated through lactogen or somatogen receptors. The results clearly suggest that recombinant analogues of human GH that recognize either somatogen or lactogen receptors, or both, but have selectively modified post-receptor effects, are helpful in elucidating the diverse biological activities of GH. These differences are most probably due to minor structural variability in GH and lactogen receptors in different organs and/or species. Genetic engineering of human GH may lead to production of modified analogues with changed and narrower specificities. One of the possible applications would be for a human GH analogue devoid of diabetogenic activity.

Animals↗
PubMed1994

Short term effect of prolactin on intracellular calcium in Chinese hamster ovary cells stably transfected with prolactin receptor complementary deoxyribonucleic acid.

The mechanism of transduction of the PRL signal in target cells is poorly understood. We examined the effects of PRL on the intracellular free Ca2+ concentration in Chinese hamster ovary cells overexpressing functional PRL receptors. [Ca2+]i was determined by dual emission microspectrofluorimetry using indo-1 as the Ca2+ fluorescent probe. We demonstrate that at physiological concentrations (0.5-5 nM), PRL stimulates Ca2+ entry (type I) and/or induces a mobilization of calcium ions stored in intracellular compartments (type II). Two types of Ca2+ mobilization, distinguishable by their onset kinetics, were observed, a slow mobilization (type IIa; transition time to peak, approximately 10 sec) and a fast mobilization (type IIb; transition time to peak, < 2 sec). PRL responses were delayed (15-120 sec) compared to the well known activation by phosphatidylinositol 4,5 bisphosphate hydrolysis-coupled receptors. This suggests that inositol trisphosphate is not involved in PRL response or that phosphatidylinositol 4,5 bisphosphate hydrolysis is not directly coupled to the PRL receptor. The amplitude of the PRL-induced Ca2+ increases (300-1400 nM) would be sufficient to provoke several physiological responses, such as stimulation of secretion, cell proliferation, or gene activation. However, the relation between the increase in Ca2+ and activation of milk protein genes remains to be established.

Animals↗
PubMed1994

Effects of anoestrus and bromocryptine treatment on the expression of prolactin and LH receptors in the rabbit ovary during lactation.

The aim of the present study was to correlate the number of prolactin and LH receptors in the ovary with the changes in sexual behaviour that occur within a few days following parturition in rabbits. Multiparous New Zealand white rabbits at days 0, 3 and 10 of lactation were tested for their receptivity upon presentation to a male. Rabbits were classed as either receptive or nonreceptive at each stage of lactation; half of the animals in each class were treated with bromocryptine to examine the effects of prolactin deprivation. Ovarian receptors for LH and prolactin, as well as the concentration of their corresponding mRNA, were measured at each stage of lactation in every group. Results indicate that receptive behaviour is correlated with significantly more follicles on the rabbit ovary (diameter > 1 mm; P < 0.05) and an increase in the concentration of LH receptor mRNA (P < 0.001) and prolactin receptors (P < 0.05). In addition, on day 4 of lactation, there were significantly fewer follicles in nonreceptive rabbits (P < 0.05). LH receptor content remained constant on days 1 and 4 of lactation but increased on day 11 (P < 0.05). Bromocryptine treatment had no effect on the number of follicles or on the amount of LH receptor mRNA in does, but it significantly increased LH receptors (P < 0.01), and the concentration of prolactin receptor mRNA (P < 0.001), particularly on day 11 of lactation (P < 0.05), and prolactin receptor content (P < 0.001). Receptive rabbit ovaries therefore display more follicles that can respond to an LH surge via newly transcribed LH receptors than do nonreceptive.(ABSTRACT TRUNCATED AT 250 WORDS)

Anestrus↗
PubMed1994

Interaction of lactogenic hormones with the soluble extracellular domain of prolactin receptors.

Two variants of rabbit prolactin receptor extracellular domain (rbPRLR-ECD) were prepared using insect/baculovirus (amino acids 1-198) and E. coli (amino acids 4-210) expression systems. Bovine PRLR-ECD (bPRPL-ECD amino acids 1-210) and human growth hormone receptor ECD (hGHR-ECD amino acids 1-246) were also prepared using E. coli expression system. All four proteins were purified as monomers with > 95% homogeneity. Their affinity for various lactogenic and somatogenic hormones was determined by binding assays. The stoichiometry of complex formation with these hormones was studied by gel filtration on a Superdex 75 column, and bioactivity was determined by in vitro bioassays. The results summarized in this paper indicate that, in contrast to hGHR-ECD, in which the ability to form a 2:1 complex with hGH is indicative of the biological activity of the hormone, the ability or inability of prolactin and placental lactogen to form 2:1 complexes with rb or bPRLR-ECD cannot predict their biological activity. This conclusion does not preclude however, hormone- or antibody-induced dimerization of the membrane-embedded receptor.

Animals↗
PubMed1994

Prolactin receptor and signal transduction to milk protein genes.

After cloning of the mammary gland prolactin (PRL) receptor cDNA, a functional assay was established using co-transfection of PRL receptor cDNA together with a milk protein promoter/chloramphenicol acetyl transferase (CAT) construct in Chinese hamster ovary (CHO) cells. Different mutants of the PRL receptor were tested in this CAT assay to delimit the domains in the receptor necessary for signal transduction to milk protein genes. In CHO cells stably transfected with PRL receptor cDNA, high numbers of PRL receptor are expressed. By metabolic labeling and immunoprecipitation, expressed PRL receptor was identified as a single species of 100 kDa. Using these cells, we analyzed the effects of PRL on intracellular free Ca++ concentration. PRL stimulates Ca++ entry and induces secondary Ca++ mobilization. The entry of Ca++ is a result of an increase in K+ conductance that hyperpolarizes the membranes. We have also analyzed tyrosine phosphorylation induced by PRL. In CHO cells stably transfected with PRL receptor cDNA, PRL induced a very rapid and transient tyrosine phosphorylation of a 100-kDa protein which is most probably the PRL receptor. The same finding was obtained in mammary membranes after PRL injection to lactating rabbits. Whereas tyrosine kinase inhibitors genistein and lavendustin were without effect, PRL stimulation of milk protein gene promoters was partially inhibited by 2 microM herbimycin in CHO cells co-transfected with PRL receptor cDNA and the beta lactoglobulin CAT construct. Taken together these observations indicate that the cytoplasmic domain of the PRL receptor interacts with one or several tyrosine kinases, which may represent early postreceptor events necessary for PRL signal transduction to milk protein genes.

Animals↗
PubMed1993

Interaction of lactogenic hormones with purified recombinant extracellular domain of rabbit prolactin receptor expressed in insect cells.

The extracellular domain of rabbit prolactin receptor (rbPRLR-ECD) expressed in an insect/baculovirus expression system was purified by affinity chromatography on immobilized PRL followed by gel filtration. The purified protein was over 90% homogeneous as indicated 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 was 32 kDa, and by gel filtration, 27 kDa. Both values are higher than the 22.8 kDa deduced from the cDNA sequence, indicating extensive glycosylation. The Ka value for interaction with ovine (o) PRL was 25.4 nM-1, but even at high rbPRLR-ECD:hormone molar ratios, the stoichiometry of interaction with oPRL or human growth hormone indicated formation of only 1:1 complexes, in contrast to human growth hormone (hGH)-ECD which forms 2:1 complexes with hGH.

Animals↗