Vincent Goffin

A common polymorphism of the growth hormone receptor is associated with increased responsiveness to growth hormone

Growth hormone is used to increase height in short children who are not deficient in growth hormone, but its efficacy varies largely across individuals. The genetic factors responsible for this variation are entirely unknown. In two cohorts of short children treated with growth hormone, we found that an isoform of the growth hormone receptor gene that lacks exon 3 (d3-GHR) was associated with 1.7 to 2 times more growth acceleration induced by growth hormone than the full-length isoform (P < 0.0001). In transfection experiments, the transduction of growth hormone signaling through d3-GHR homo- or heterodimers was ∼30% higher than through full-length GHR homodimers (P < 0.0001). One-half of Europeans are hetero- or homozygous with respect to the allele encoding the d3-GHR isoform, which is dominant over the full-length isoform. These observations suggest that the polymorphism in exon 3 of GHR is important in growth hormone pharmacogenetics.

Bone homeostasis in growth hormone receptor–null mice is restored by IGF-I but independent of Stat5

Growth hormone (GH) regulates both bone growth and remodeling, but it is unclear whether these actions are mediated directly by the GH receptor (GHR) and/or IGF-I signaling. The actions of GH are transduced by the Jak/Stat signaling pathway via Stat5, which is thought to regulate IGF-I expression. To determine the respective roles of GHR and IGF-I in bone growth and remodeling, we examined bones of wild-type, GHR knockout (GHR(-/-)), Stat5ab(-/-), and GHR(-/-) mice treated with IGF-I. Reduced bone growth in GHR(-/-) mice, due to a premature reduction in chondrocyte proliferation and cortical bone growth, was detected after 2 weeks of age. Additionally, although trabecular bone volume was unchanged, bone turnover was significantly reduced in GHR(-/-) mice, indicating GH involvement in the high bone-turnover level during growth. IGF-I treatment almost completely rescued all effects of the GHR(-/-) on both bone growth and remodeling, supporting a direct effect of IGF-I on both osteoblasts and chondrocytes. Whereas bone length was reduced in Stat5ab(-/-) mice, there was no reduction in trabecular bone remodeling or growth-plate width as observed in GHR(-/-) mice, indicating that the effects of GH in bone may not involve Stat5 activation.

Osteoblasts Are a New Target for Prolactin: Analysis of Bone Formation in Prolactin Receptor Knockout Mice1

Bone development is a multistep process that includes patterning of skeletal elements, commitment of hematopoietic and/or mesenchymental cells to chondrogenic and osteogenic lineages, and further differentiation into three specialized cell types: chondrocytes in cartilage and osteoblasts and osteoclasts in bone. Although PRL has a multitude of biological actions in addition to its role in the mammary gland, very little is known about its effect on bone. Mice carrying a germline null mutation for the PRL receptor gene have been produced in our laboratory and used to study the role of PRL in bone formation. In− /− embryos, we observed an alteration in bone development of calvaria. In adults, histomorphometric analysis showed that the absence of PRL receptors leads to a decrease in bone formation rate using double calcein labeling and a reduction of bone mineral density, measured by dual energy x-ray absorptiometry. In addition, serum estradiol, progesterone, testosterone, and PTH levels were analyzed. We al...

The prolactin/growth hormone receptor family: structure/function relationships.

Prolactin (PRL) and growth hormone (GH)2 receptors are members of the cytokine receptor superfamily that are activated by ligand-induced homodimerization. On the basis of this mechanism of activation, hormone antagonists have been developed that block the receptors in an inactive conformation. PRL and GH receptors are non-kinase receptors whose activation of signaling pathways requires participation of receptor-associated kinases, such as Janus kinases or Src kinases. Signal transduction by these receptors mainly involves the JAK/Stat pathway. In this review, we discuss the mechanism of ligand binding and receptor homodimerization as well as the involvement of molecules transducing the hormonal signal. Whenever possible, we attempt to correlate cytoplasmic features of the receptors with association and/or activation of transducer molecules or with a given biological property.

New concepts in prolactin biology

Human prolactin (PRL) is currently viewed as a hormone of pituitary origin, whose production (i.e. serum levels) is controlled by dopamine, whose biological actions relate exclusively to lactation and reproductive functions, for which any genetic disorder is yet to be identified, and whose unique associated pathology is hyperprolactinemia. Both experimental studies and human sample/cohort-based investigations performed during the past decade have considerably widened our perception of PRL biology: i) there are now strong epidemiological arguments supporting the fact that circulating PRL is a risk factor for breast cancer, ii) in addition to the endocrine hormone, locally produced PRL has been documented in several human tissues; there is increasing evidence supporting the tumor growth potency of local PRL, acting via autocrine/paracrine mechanisms, in both rodent models, and human breast and prostate tumors, iii) the first functional germinal polymorphisms of the PRL receptor were recently identified in patients presenting with breast tumors, which involve single amino acid substitution variants exhibiting constitutive activity, iv) human PRL analogs have been engineered, which were shown in experimental models to down-regulate the effects triggered by local PRL (competitive antagonism) or by the constitutively active receptor variants (inverse agonism). The aim of this review is to discuss these novel concepts in PRL biology, including their potential pathophysiological outcomes.

Antagonistic Properties of Human Prolactin Analogs That Show Paradoxical Agonistic Activity in the Nb2 Bioassay

Based on the assumption that the prolactin receptor (PRLR) is activated by PRL-induced sequential dimerization, potential human PRL (hPRL) antagonists were designed that sterically interfere with binding site 2. We previously reported the unexpected agonistic properties of these hPRL analogs in the rat Nb2 bioassay (Goffin, V., Struman, I., Mainfroid, V., Kinet, S., and Martial, J. A. (1994) J. Biol. Chem. 269, 32598-32606). In order to investigate whether such paradoxical agonistic behavior might result from characteristic features of the Nb2 assay (e.g. species specificity), we transfected in the same cell system the cDNA encoding the PRLR from rat or human species along with reporter genes containing PRL-responsive DNA sequences. We characterized the agonistic, self-antagonistic and/or antagonistic effects of wild type rat PRL, wild type hPRL, and three hPRL analogs, mutated either at binding site 1 or at binding site 2. Our results clearly show that the agonistic/antagonistic properties of PRLs are species-specific. We thus propose different models of receptor activation, depending on the relative affinities of each hormonal binding site, which is directed by species specificity. Finally, this is the first report of hPRL binding site 2 analogs showing antagonistic properties on human and, to a lesser extent, rat receptors.

Human prolactin (hPRL) antagonists inhibit hPRL-activated signaling pathways involved in breast cancer cell proliferation.

The involvement of human prolactin (hPRL) in breast cancer has been recently reconsidered based on its autocrine/paracrine proliferative effect described in human mammary tumor epithelial cells. Therefore, there is growing interest in the development of potent hPRL antagonists that may inhibit this effect. We previously designed hPRL analogs displaying antagonistic properties in a human transcriptional bioassay. We now report that the most potent of those analogs, G129R-hPRL, antagonizes all hPRL-induced effects analysed in various breast cancer cell lines, including cell proliferation. The analog per se lacks intrinsic agonistic activity on PRL receptor-activated signaling cascades, cell proliferation and apoptosis, indicating that its mode of action only occurs through competitive inhibition of hPRL. We provide some molecular basis of this antagonistic effect by demonstrating that G129R-hPRL competitively inhibits hPRL-activation of the JAK-STAT and MAPK pathways, two signaling cascades involved in the mitogenic effect of hPRL in mammary epithelial cells. This competitive inhibition persists for at least 48 h, as evidenced by long term analysis of STAT5b activation or of progression through cell cycle. These results are the first demonstration at the molecular level that hPRL antagonists interfering with receptor dimerization disrupt signaling events in breast cancer cells, which prevents hPRL-induced cell proliferation.

Prolactin modulates TRPV1 in female rat trigeminal sensory neurons.

Sex dependency in pain perception is well documented and is thought to be attributable to the effect of reproductive hormones on nociceptive processing. In the present study, we evaluated whether estradiol alters gene transcription in the trigeminal ganglia (TG) of ovariectomized rats (OVX). These experiments demonstrated a dramatic (40-fold) upregulation of prolactin (PRL) expression in TG by 17-β-estradiol (E2). PRL expression was restricted to TG neurons and was highly overlapped with transient potential receptor vanilloid type 1 (TRPV1) (∼90%) in TG. Additionally, PRL is released from neurons during stimulation. Both forms of PRL receptors (PRLRs), short and long, were also present in TG neurons. Moreover, expression of the long PRLRs was under control of estradiol. We next evaluated the novel hypothesis that PRL acts as a neuromodulator of sensory neurons. PRL pretreatment significantly enhanced capsaicin-evoked inward currents, calcium influx, and immunoreactive calcitonin gene-related peptide release from cultured TG neurons. This PRL modulation of capsaicin responses was abolished by withdrawal of E2 from TG cultures. Biochemical analysis demonstrated that PRL increased (>50%) phosphorylation levels of TRPV1 in TG. In a behavioral test, PRL pretreatment significantly potentiated capsaicin-evoked nocifensive behavior in female rats at proestrous and in OVX rats after E2 treatment. The in vivo potentiating effect of PRL on capsaicin responses was also dependent on E2. Collectively, these data demonstrate that PRL is a novel modulator of sensory neurons tightly regulated by E2. These findings are consistent with the hypothesis that PRL could contribute to the development of certain pain disorders, possibly including those modulated by estrogen.

Impaired Islet Function in Commonly Used Transgenic Mouse Lines due to Human Growth Hormone Minigene Expression

The human growth hormone (hGH) minigene is frequently used in the derivation of transgenic mouse lines to enhance transgene expression. Although this minigene is present in the transgenes as a secondcistron, and thus not thought to be expressed, we found that three commonly used lines, Pdx1-Cre(Late), RIP-Cre, and MIP-GFP, each expressed significant amounts of hGH in pancreatic islets. Locally secreted hGH binds to prolactin receptors on β cells, activates STAT5 signaling, and induces pregnancy-like changes in gene expression, thereby augmenting pancreatic β cell mass and insulin content. In addition, islets of Pdx1-Cre(Late) mice have lower GLUT2 expression and reduced glucose-induced insulin release and are protected against the β cell toxin streptozotocin. These findings may be important when interpreting results obtained when these and other hGH minigene-containing transgenic mice are used.

From the molecular biology of prolactin and its receptor to the lessons learned from knockout mice models.

Prolactin (PRL), a polypeptide hormone secreted mainly by the pituitary and, to a lesser extent, by peripheral tissues, affects more physiological processes than all other pituitary hormones combined since it is involved in > 300 separate functions in vertebrates. Its main actions are related to lactation and reproduction. The initial step of PRL action is the binding to a specific membrane receptor, the PRLR, which belongs to the class 1 cytokine receptor superfamily. PRL-binding sites have been identified in a number of tissues and cell types in adult animals. Signal transduction by this receptor is mediated, at least in part, by two families of signaling molecules: Janus tyrosine kinases and signal transducers and activators of transcription (STATs). Disruption of the PRLR gene has provided a new mouse model with which to identify actions directly associated with PRL or any other PRLR ligands, such as placental lactogens. To date, several different phenotypes have been analyzed and are briefly described in this review. Coupled with the SAGE technique, this PRLR knockout model is being used to qualitatively and quantitatively evaluate the expression pattern of hepatic genes in two physiological situations: transcriptomes corresponding to livers from both wild type and PRLR KO mice are being compared, and following statistical analyses, candidate genes presenting a differential profile will be further characterized. Such a new approach will undoubtedly open future avenues of research for PRL targets. To date, no pathology linked to any mutation in the genes encoding PRL or its receptor have been identified. The development of genetic models provides new opportunities to understand how PRL can participate to the development of pathologies throughout life, as for example the initiation and progression of breast cancer.