Raymond Counis

Pituitary Adenylate Cyclase-activating Polypeptide and Cyclic Adenosine 3′,5′-Monophosphate Stimulate the Promoter Activity of the Rat Gonadotropin-releasing Hormone Receptor Gene via a Bipartite Response Element in Gonadotrope-derived Cells

Specific type I receptors for pituitary adenylate cyclase-activating polypeptide (PACAP) are present in gonadotrope cells of the anterior pituitary gland. By transient transfection of mouse gonadotrope-derived αT3-1 cells, which are direct targets for PACAP and express gonadotropin-releasing hormone receptor (GnRH-R), a marker of the gonadotrope lineage, we provide the first evidence that PACAP stimulates rat GnRH-R gene promoter activity. The EC50 of this stimulation is compatible with a mediation via activation of the cyclic AMP-dependent signaling pathway and, consistently, co-transfection of an expression vector expressing the protein kinase A inhibitor causes reduction in PACAP as well as cholera toxin-stimulated promoter activity. Deletion and mutational analyses indicate that PACAP activation necessitates a bipartite response element that consists of a first region (−272/−237) termed PACAP response element (PARE) I that includes a steroidogenic factor-1 (SF-1)-binding site and a second region (−136/−101) referred to as PARE II that contains an imperfect cyclic AMP response element. Gel shift experiments indicate the specific binding of the SF-1 and a potential SF-1-interacting factor to PARE I while a protein immunologically related to the cyclic AMP response element-binding protein interacts with PARE II. These findings suggest that PACAP might regulate the GnRH-R gene at the transcriptional level, providing novel insights into the regulation of pituitary-specific genes by hypothalamic hypophysiotropic signals.

EVIDENCE THAT GONADOTROPIN-RELEASING HORMONE STIMULATES GENE EXPRESSION AND LEVELS OF ACTIVE NITRIC OXIDE SYNTHASE TYPE I IN PITUITARY GONADOTROPHS, A PROCESS ALTERED BY DESENSITIZATION AND, INDIRECTLY, BY GONADAL STEROIDS

To determine the site and mechanism of action of gonadal steroids on pituitary nitric oxide synthase type I (NOS I), present in both gonadotrophs and folliculo-stellate cells, the effects of castration and steroids were examined in male rats, in the presence of a GnRH antagonist (Antarelix). Western analysis showed a rapid and substantial increase with time, after orchidectomy, of NOS I protein, the concentration doubling in 24 h and reaching a maximal 4- to 5-fold increase after 3–7 days, followed by a progressive decline after 2 weeks. Testosterone or estradiol replacement, or administration of GnRH antagonist, totally abolished the effects of castration, demonstrating a mediation of the steroid effects via GnRH. In noncastrated rats, steroids and the GnRH antagonist also caused a reduction in the levels of NOS I (by 50–60%), consistent with inhibition of endogenous GnRH stimulation. In marked contrast, administration of a potent GnRH agonist (Triptorelin) to intact rats increased the levels of NOS I. A...

Effect of gonadectomy on pituitary levels of mRNA encoding gonadotropin subunits and secretion of luteinizing hormone.

Using cell-free translation of pituitary mRNAs we have investigated how, following gonadectomy in rats, the translational capacity of the specific messages encoding precursors to gonadotropin subunits alpha, LH-beta and FSH-beta increases with time. In parallel, serum LH was assayed in order to compare release and synthesis patterns. We observed a rapid rise in the rate of synthesis of all three precursors, with a significant increase already detectable 4 days after gonadectomy, and a plateau reached after 21 days. The kinetics were similar in both males and females, but maximal translational values for alpha-subunit were slightly higher in males. During the same time period, serum LH rapidly increased in the males, while in the females the rise of circulating LH was somewhat delayed. Although no direct correlation seems to exist between synthesis and release processes of gonadotropins, it is evident from our previous findings and the present data that both phenomena are dependent on gonadal steroids. In this respect, estradiol has been shown to regulate negatively, via different routes, the synthesis as well as the secretion of pituitary gonadotropins.

GnRH-dependent up-regulation of nitric oxide synthase I level in pituitary gonadotrophs mediates cGMP elevation during rat proestrus

We have recently provided evidence that the concentration and activity of the enzyme nitric oxide synthase type I (NOS I) was stimulated in gonadotrophs by GnRH, suggesting a role for the NOS I/NO pathway in the GnRH control of cell functions. To further establish the GnRH regulation of pituitary NOS I under physiological circumstances, we have examined the expression of NOS I during the 4-day estrus cycle in rats. Western blot analysis demonstrated that NOS I was present in the anterior pituitary at low levels during diestrus I (DI) and diestrus II, then was subject to a significant increase on the afternoon of proestrus to reach a maximal 3-fold increase between 19:00 and 20:00 h, after which NOS I decreased to return, during estrus, to levels within the range detected in diestrus. No such variation was apparent in the posterior pituitary lobe. NADPH-diaphorase histochemistry combined with immuno-identification of the cells revealed that active NOS I was expressed in both the gonadotrophs and the folliculo-stellate cells throughout the whole cycle but only the gonadotrophs showed an up-regulation during proestrus. A high temporal correlation was observed in the profiles of NOS I, pituitary cGMP and serum luteinizing hormone (LH) suggesting an implication of GnRH. Consistently, the administration of a potent GnRH antagonist to rats totally abolished the rise in pituitary NOS I and cGMP, in addition to suppressing, as expected, the surge in the secretion of LH. A role of NOS I as a mediator in the GnRH-induced augmentation in cGMP was further established in vitro by incubating anterior pituitaries in the presence of the NOS inhibitor, L-NMMA (1 mM). Altogether, these data demonstrate that the level and activity of NOS I is up-regulated in gonadotrophs during proestrus, in a manner consistent with a major implication of GnRH over a period during which its release from the hypothalamus, as well as gonadotroph responsiveness, are at maximum. This effect is accompanied by a NOS/NO-mediated rise in cGMP. In the absence of obvious effect on gonadotropin release, the roles of NO and cGMP in the regulation of gonadotroph functions, especially during proestrus, remain to be established.

Estradiol regulates mRNAs encoding precursors to rat lutropin (LH) and follitropin (FSH) subunits

Anterior pituitary mRNA was prepared using a microscaled method and translated in a wheat-germ cell-free system in the presence of [35S] labeled cysteine and methionine. Translation products, immunologically related to LH beta, FSH beta and the common subunit alpha, were isolated as precursors with antisera to denatured subunits and characterized by SDS-polyacrylamide gel electrophoresis and fluorography. The radioactive bands were excised from the gel and counted for quantitative evaluation. Our data show that translation of pituitary mRNAs from ovariectomized (ovx) rats results in precursor levels increased by 10 fold for alpha and 14 fold for LH beta as compared to the levels in normal rats. The increase in FSH beta precursor was impossible to evaluate as a specific immunoprecipitation product was undetectable in the case of normal rats. Estradiol, but not progesterone, administered in vivo to ovariectomized rats, reversed the stimulatory effect of ovariectomy on the expression of mRNAs coding for gonadotropin subunit precursors 48 h after injection. These results suggest that estradiol, but not progesterone, negatively regulates the synthesis of the pituitary gonadotropins in the female rats via changes in specific mRNA levels. This hormonal control probably occurs at the transcriptional level.

Mechanisms of Action of Hormone-Sensitive Lipase in Mouse Leydig Cells: Its Role in the Regulation of the Steroidogenic Acute Regulatory Protein

Background: Hormone-sensitive lipase (HSL) is a multifunctional enzyme that is critically involved in regulating energy homeostasis. Results: HSL catalyzes the hydrolysis of cholesteryl esters and plays an indispensable role in the regulation of Bt2cAMP-induced steroidogenic acute regulatory protein (StAR) expression. Conclusion: The mode of action of HSL in cAMP/PKA-mediated regulation of steroidogenesis involves multiple signaling, including the LXR-regulatory pathway. Significance: The present findings can be useful for better understanding a number of physiopathological processes. Hormone-sensitive lipase (HSL) catalyzes the hydrolysis of cholesteryl esters in steroidogenic tissues and, thus, facilitates cholesterol availability for steroidogenesis. The steroidogenic acute regulatory protein (StAR) controls the rate-limiting step in steroid biosynthesis. However, the modes of action of HSL in the regulation of StAR expression remain obscure. We demonstrate in MA-10 mouse Leydig cells that activation of the protein kinase A (PKA) pathway, by a cAMP analog Bt2cAMP, enhanced expression of HSL and its phosphorylation (P) at Ser-660 and Ser-563, but not at Ser-565, concomitant with increased HSL activity. Phosphorylation and activation of HSL coincided with increases in StAR, P-StAR (Ser-194), and progesterone levels. Inhibition of HSL activity by CAY10499 effectively suppressed Bt2cAMP-induced StAR expression and progesterone synthesis. Targeted silencing of endogenous HSL, with siRNAs, resulted in increased cholesteryl ester levels and decreased cholesterol content in MA-10 cells. Depletion of HSL affected lipoprotein-derived cellular cholesterol influx, diminished the supply of cholesterol to the mitochondria, and resulted in the repression of StAR and P-StAR levels. Cells overexpressing HSL increased the efficacy of liver X receptor (LXR) ligands on StAR expression and steroid synthesis, suggesting HSL-mediated steroidogenesis entails enhanced oxysterol production. Conversely, cells deficient in LXRs exhibited decreased HSL responsiveness. Furthermore, an increase in HSL was correlated with the LXR target genes, steroid receptor element-binding protein 1c and ATP binding cassette transporter A1, demonstrating HSL-dependent regulation of steroidogenesis predominantly involves LXR signaling. LXRs interact/cooperate with RXRs and result in the activation of StAR gene transcription. These findings provide novel insight and demonstrate the molecular events by which HSL acts to drive cAMP/PKA-mediated regulation of StAR expression and steroidogenesis in mouse Leydig cells.

GnRH Receptor Gene Expression in the Developing Rat Hippocampus: Transcriptional Regulation and Potential Roles in Neuronal Plasticity

In the pituitary of mammals, the GnRH receptor (GnRHR) plays a primary role in the control of reproductive function. It is further expressed in the hippocampus, where its function, however, is not well defined. By quantitative RT-PCR analyses, we demonstrate herein that the onset of GnRHR gene (Gnrhr) expression in the rat hippocampus was unexpectedly delayed as compared to the pituitary and only occurred after birth. Using a previously described transgenic mouse model bearing the human placental alkaline phosphatase reporter gene under the control of the rat Gnrhr promoter, we established a positive correlation between the temporal pattern of Gnrhr mRNA levels and promoter activity in the hippocampal formation. The gradual appearance of human placental alkaline phosphatase transgene expression occurred simultaneously in the hippocampus and interconnected structures such as the lateral septum and the amygdala, coinciding with the establishment of hippocampo-septal projections. Analysis of transcription factors together with transient transfection assays in hippocampal neurons indicated that the combinatorial code governing the hippocampus-specific expression of the Gnrhr is distinct from the pituitary, likely involving transactivating factors such as NUR77, cyclic AMP response element binding protein, and Finkel-Biskis-Jinkins murine osteosarcoma virus oncogene homolog. A silencing transcription factor acting via the -3255/-1135 promoter region of the Gnrhr may be responsible for the transcriptional repression observed around birth. Finally, GnRH directly stimulated via activation of its receptor the expression of several marker genes of neuronal plasticity such as Egr1, synaptophysin, and spinophilin in hippocampal primary cultures, suggesting a role for GnRHR in neuronal plasticity. Further characterization of these mechanisms may help unravel important functions of GnRH/GnRHR signaling in the brain.

GnRH and PACAP action in gonadotropes: Cross-talk between phosphoinositidase C and adenylyl cyclase mediated signaling pathways

In order to respond appropriately to their environment, gonadotropes, like other cells, must integrate informational input from multiple ligands acting through multiple intracellular signaling pathways. In recent years, an increasing number of examples of functional interactions between the phosphoinositidase C (PIC) and adenylyl cyclase signaling pathways in gonadotropes have been described, and the discovery that these cells are targets for pituitary adenylyl cyclase activating peptide (PACAP) has provided a physiological context for earlier work on gonadotrope regulation by cAMP. It has become clear that gonadotropes possess multiple PIC-coupled receptor types, in addition to receptors activating adenylyl and guanylyl cyclases, so that the potential for both coincidence signaling and cross-talk in these cells is immense; examples of both are seen in the effects of PACAP and GnRH on Ca(2+) mobilization and adenylyl cyclase activation in alphaT3-1 cells. In these cells, GnRH, acting via PIC-coupled receptors, can dramatically inhibit adenylyl cyclase activated by PACAP, but can also alter cellular levels of protein kinase A subunits, providing a mechanism for coordinated regulation of both messenger and effector.

Mutations of the GnRH receptor gene: a new cause of autosomal-recessive hypogonadotropic hypogonadism.

Mutations in a few genes have been identified in hypogonadotropic hypogonadism (HH): the gene KAL-1 is involved in X-linked Kallmann syndrome associated with anosmia and mutations in transcription factors, namely, DAX-1 and Prop-1 were also evidenced when associated with other pituitary or endocrine defects. Recently, compound heterozygote mutations in the GnRH receptor gene were described both in males and females and hormonal resistance was confirmed in vitro. There is a wide spectrum of phenotype, ranging from complete HH with lack of pubertal development and cryptorchidism to partial hypogonadism with an arrest of pubertal development. In complete GnRH resistance, endogenous LH secretory patterns were abnormal, either apulsatile or characterized by a low-normal pulse frequency with small pulses or erratic pulses of low amplitude. In patients with partial resistance, basal LH plasma concentration was low, but FSH level was in the normal range. LH pulse analysis revealed normal frequency with decreased amplitude. Mutations are distributed along the coding sequence, as reported for other GPCRs. However, two hot-spots, Q106R and the R262Q, were observed, regardless of the geographic origin of the patients. In most cases, patients responded to GnRH administration, making the GnRH test inappropriate for screening GnRH resistance in IHH.

Cloning and sequence analysis of the cDNA for the pituitary glycoprotein hormone α-subunit of the European eel

A cDNA library constructed using mRNAs isolated from pituitary glands of estradiol-treated eels was screened with a cDNA fragment for the rat glycoprotein hormone alpha-subunit. Three out of 10,000 cDNA clones were revealed and subcloned in pUC13 for characterization and sequencing. All three had the same nucleotide sequence except for a single, silent change in the coding sequence for one of them, and for the location of the poly(A) tail. Analysis of the deduced amino acid sequence strongly suggests that these cDNA clones encode the precursor for the eel common glycoprotein hormone alpha-subunit. This precursor would therefore consist of a 93 amino acid apoprotein preceded by a 24 amino acid long signal peptide. Alignment with glycoprotein hormone alpha-subunits from fish and mammals reveals high homology, ranging from 60 to 90%. Particularly, the ten cysteines and the two putative N-linked glycosylation sites were at the same position. Comparison between fish and mammals shows also that two regions are highly conserved, comprising about half of the protein length. This high conservation rate through evolution argues for the importance of these regions in the conservation of biological properties of the alpha-subunits. In contrast, other regions are highly variable and could be responsible for the immunological specificity. Northern blot analysis of pituitary RNA from control and estradiol-treated eels showed that estradiol treatment strongly increases the pituitary content of mRNA encoding the glycoprotein hormone alpha-subunit.