Jean Djiane

The growth hormone/prolactin receptor family.

Publisher Summary This chapter provides an overview of growth hormone (GH) and prolactin receptor (PRL) family. Sequence analysis has confirmed that GH and PRL have selected regions of strong homology and, along with placental lactogen (PL) or chorionic somatotropin (CS), form a family of polypeptide hormones that appear to have arisen by the duplication of an ancestral gene. Hormones act by first binding to a specific receptor on or inside the cell. Two general classes of hormone receptors exist, namely, DNA binding receptors and membrane receptors. The events responsible for the transfer of the hormonal message inside the cell occur just after the interaction of the ligand with the receptor; they remain, for the most part, unknown. In mammals, PRL is primarily responsible for the development of the mammary gland and lactogenesis. The best known effects of PRL are in mammary epithelial cells. PRL acts in association with insulin and glucocorticoids to stimulate milk protein gene expression at both the transcriptional and posttranscriptional levels, because the rate of gene transcription and the stability of milk protein mRNAs are increased under the influence of the hormone. GH, on the other hand, has a direct action on the differentiation of preadipocytes, where it is necessary for the initiation of the differentiation program for cells to become responsive to insulin-like growth factor-I and for its mitogenic effect.

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.

Structural symmetry of the extracellular domain of the cytokine/growth hormone/prolactin receptor family and interferon receptors revealed by hydrophobic cluster analysis.

Sequence comparison based on Hydrophobic Cluster Analysis procedures shows that the extracellular ≈ 200 amino acids domains of cytokines receptors belonging to the Cytokine/Growth hormone/Prolactin receptor family and to the Interferon one are organized in two homologous subdomains. Further, comparison of the subdomains of 32 independent sequences and of a lot of already recognized homologous domains with data bases could lead to the hypothesis that these ≈ 100 amino acids subdomains could possess the overall fold of the constant immunoglobulin domains and so could belong to the immunoglobulin superfamily.

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.

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.

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 Ca2+- 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.

Prolactin receptors in the rat hypothalamus: autoradiographic localization and characterization.

A precise mapping of prolactin (PRL) receptors in the rat brain has been achieved. Localization of binding sites for both 125I-human growth hormone (125I-hGH) and 125I-monoclonal anti-PRL receptor (125I-U5) was studied by in vitro autoradiography on brain sections in female rats (n = 7). The analysis of autoradiograms generated from 12 adjacent sections at 11 different brain levels (bregma 0.2 to -4.8 mm) revealed 9 distinctive localizations for 125I-hGH binding sites: preoptic suprachiasmatic nucleus, medial preoptic area, periventricular, supraoptic, paraventricular, arcuate and vetromedial nuclei and also the median eminence and the infundibulum. Specificity for PRL binding was assessed by competition experiment of 125I-hGH with unlabeled hGH and ovine PRL. Binding sites were similarly localized by 125I-U5 indicating the presence of PRL receptors moiety. The quantitative analysis with 0.6 nM 125I-hGH demonstrated maximal densities in the preoptic suprachiasmatic and arcuate nuclei and minimal densities in the median eminence and the infundibulum. Due to ample antero-posterior variations no significant changes were observed during the estrous cycle. Saturation analysis of binding in the arcuate nucleus indicated a single class of high affinity (Kd from 0.9 to 2.2 nM) receptors (Bmax from 34 to 44 fmol/mg of proteins). The present data provide the hypothalamic cartography of PRL receptors in the female rat brain and support all the physiological evidence for the existence of a direct action of PRL in the hypothalamus.

Rapid down-regulation of prolactin receptors in mammary gland and liver.

Summary In contrast with the well known delayed stimulatory effect of prolactin on the levels of its own receptors, a rapid and reversible decline of prolactin receptors was observed in vivo , both with rabbit mammary glands and rat liver, after i.v. injection of large doses of prolactin. This “down regulation” could be clearly distinguished from mere occupation by desaturating the occupied membrane receptors in vitro with 4M MgCl 2 . The experiments lend strong support to the ubiquitous occurrence of down regulation and to the involvement of compartimentalization and ultimate destruction of the receptor/hormone complex. They further suggest that “down regulation” and “up regulation” are not merely antagonistic regulatory events, but partake in the mechanism of hormone action on the target cell.

Messenger RNA Expression of Leptin and Leptin Receptors and their Prognostic Value in 322 Human Primary Breast Cancers

Purpose: Leptin and obesity are clearly related, and obesity is associated with an increased risk of breast cancer. We therefore measured the expression of leptin and its two main receptor isoforms, OBR-L and OBR-S, in 322 breast cancers. We analyzed their relations with the classical prognostic factors and with survival to establish their links with breast cancer. Experimental Design: The expression of leptin and its receptors was quantified by real-time reverse transcription-PCR, using TaqMan fluorogenic probes and an ABI PRISM 7700 sequence detector system (Applied Biosystems, Courtaboeuf, France). TATA box binding protein was used to normalize expression. The human breast cancer cell, SK-BR-3, expressing the three targets, was chosen as the calibrator sample (i.e., target expression = 1). Results: All the tumors expressed both receptors, and 318 of 322 expressed leptin. These three variables correlated positively with each other and with estradiol and progesterone receptors, whereas they correlated negatively with histoprognostic grading and tumor diameter. OBR-L/OBR-S expression was inversely correlated with progesterone receptors. Patients with elevated OBR-S expression had longer relapse-free survival (P = 0.008), whereas high OBR-L/OBR-S was associated with a shorter relapse-free survival (P = 0.05). In Cox multivariate analyses, OBR-S maintained its prognostic value (P = 0.02; relative risk, 0.51). Conclusions: This study shows that (a) almost all of the breast cancers coexpress leptin and its two main isoforms of receptors, suggesting that the human epithelial breast cancer cells respond to leptin acting via an autocrine pathway; (b) high expression levels of leptin and leptin receptors are biological markers of a more differentiated phenotype; and that (c) OBR-S is an independent prognostic factor.

Study of hypothalamic leptin receptor expression in low-birth-weight piglets and effects of leptin supplementation on neonatal growth and development

Low birth weight resulting from intrauterine growth retardation (IUGR) is a risk factor for further development of metabolic diseases. The pig appears to reproduce nearly all of the phenotypic pathological consequences of human IUGR and is likely to be more relevant than rodents in studies of neonatal development. In the present work, we characterized the model of low-birth-weight piglets with particular attention to the hypothalamic leptin-sensitive system, and we tested whether postnatal leptin supplementation can reverse the precocious signs of adverse metabolic programming. Our results demonstrated that 1) IUGR piglets present altered postnatal growth and increased adiposity; 2) IUGR piglets exhibit abnormal hypothalamic distribution of leptin receptors that may be linked to further disturbance in food-intake behavior; and 3) postnatal leptin administration can partially reverse the IUGR phenotype by correcting growth rate, body composition, and development of several organs involved in metabolic regulation. We conclude that IUGR may be characterized by altered leptin receptor distribution within the hypothalamic structures involved in metabolic regulation and that leptin supplementation can partially reverse the IUGR phenotype. These results open interesting therapeutic perspectives in physiopathology for the correction of defects observed in IUGR.