Genetic mechanisms mediating kisspeptin regulation of GnRH gene expression.

Kisspeptins (Kiss) have been shown to be key components in the regulation of gonadotropin-releasing hormone (GnRH) secretion. In vitro studies have demonstrated an increase in GnRH gene expression by Kiss suggesting regulation of GnRH at both the secretory and pretranslational levels. Here, we define genetic mechanisms that mediate Kiss action on target gene expression. In vitro, sequential deletions of the mouse GnRH (mGnRH) gene promoter fused to the luciferase (LUC) reporter gene localized at kisspeptin-response element (KsRE) between -3446 and -2806 bp of the mGnRH gene. In vivo, transgenic mice bearing sequential deletions of the mGnRH gene promoter linked to the LUC reporter localized an identical KsRE. To define the mechanism of regulation, Kiss was first shown to induce nucleosome-depleted DNA within the KsRE, and a potential binding site for the transcription factor, Otx-2, was revealed. Furthermore, increased Otx-2 mRNA, protein, and binding to the KsRE after Kiss treatment were demonstrated. In conclusion, this work identified elements in GnRH-neuronal cell lines and in transgenic mice that mediate positive regulation of GnRH by Kiss. In addition, we show for the first time that Otx-2 is regulated by Kiss, and plays a role in mediating the transcriptional response of mGnRH gene.

A novel OTX2 mutation in a patient with combined pituitary hormone deficiency, pituitary malformation, and an underdeveloped left optic nerve.

Orthodenticle homolog 2 (OTX2) is a homeobox family transcription factor required for brain and eye formation. Various genetic alterations in OTX2 have been described, mostly in patients with severe ocular malformations. In order to expand the knowledge of the spectrum of OTX2 mutation, we performed OTX2 mutation screening in 92 patients with combined pituitary hormone deficiency (CPHD). We directly sequenced the coding regions and exon-intron boundaries of OTX2 in 92 CPHD patients from the Dutch HYPOPIT study in whom mutations in the classical CPHD genes PROP1, POU1F1, HESX1, LHX3, and LHX4 had been ruled out. Among 92 CPHD patients, we identified a novel heterozygous missense mutation c.401C>G (p.Pro134Arg) in a patient with CPHD, pituitary malformation, and an underdeveloped left optic nerve. Binding of both the wild-type and mutant OTX2 proteins to bicoid binding sites was equivalent; however, the mutant OTX2 exhibited decreased transactivation. We describe a novel missense heterozygous OTX2 mutation that acts as a dominant negative inhibitor of target gene expression in a patient with CPHD, pituitary malformation, and optic nerve hypoplasia. We provide an overview of all OTX2 mutations described till date, which show that OTX2 is a promising candidate gene for genetic screening of patients with CPHD or isolated GH deficiency (IGHD). As the majority of the OTX2 mutations found in patients with CPHD, IGHD, or short stature have been found in exon 5, we recommend starting mutational screening in those patients in exon 5 of the gene.

Genetic overlap in Kallmann syndrome, combined pituitary hormone deficiency, and septo-optic dysplasia.

CONTEXT: Kallmann syndrome (KS), combined pituitary hormone deficiency (CPHD), and septo-optic dysplasia (SOD) all result from development defects of the anterior midline in the human forebrain. OBJECTIVE: The objective of the study was to investigate whether KS, CPHD, and SOD have shared genetic origins. DESIGN AND PARTICIPANTS: A total of 103 patients with either CPHD (n = 35) or SOD (n = 68) were investigated for mutations in genes implicated in the etiology of KS (FGFR1, FGF8, PROKR2, PROK2, and KAL1). Consequences of identified FGFR1, FGF8, and PROKR2 mutations were investigated in vitro. RESULTS: Three patients with SOD had heterozygous mutations in FGFR1; these were either shown to alter receptor signaling (p.S450F, p.P483S) or predicted to affect splicing (c.336C>T, p.T112T). One patient had a synonymous change in FGF8 (c.216G>A, p.T72T) that was shown to affect splicing and ligand signaling activity. Four patients with CPHD/SOD were found to harbor heterozygous rare loss-of-function variants in PROKR2 (p.R85G, p.R85H, p.R268C). CONCLUSIONS: Mutations in FGFR1/FGF8/PROKR2 contributed to 7.8% of our patients with CPHD/SOD. These data suggest a significant genetic overlap between conditions affecting the development of anterior midline in the human forebrain.

The gonadotropin-releasing hormone cell-specific element is required for normal puberty and estrous cyclicity.

Appropriate tissue-specific gene expression of gonadotropin-releasing hormone (GnRH) is critical for pubertal development and maintenance of reproductive competence. In these studies, a common element in the mouse GnRH (mGnRH) promoter, between -2806 and -2078 bp, is shown to mediate differential regulation of hypothalamic and ovarian mGnRH expression. To further characterize this region, we generated a knock-out mouse (GREKO(-/-)) with a deletion of the mGnRH promoter fragment between -2806 and -2078 bp. GnRH mRNA expression in the brain of GREKO(-/-) was less than the expression in wild-type mice; however, immunohistochemical analysis revealed no difference between the numbers of GnRH neurons among groups. GnRH mRNA expression in the ovary was fivefold higher in GREKO(-/-). The immunohistochemical staining for GnRH in the ovary increased in surface epithelial and granulosa cells and also in the corpora lutea of GREKO(-/-) mice. The reproductive phenotype revealed that the mean day of vaginal opening was delayed, and additionally, there was a significant decrease in the length of proestrus and diestrus-metestrus phases of the estrous cycle, resulting in a shortened estrous cycle in GREKO(-/-) mice. This work supports the hypothesis that the region of the GnRH promoter contained between -2806 and -2078 bp acts as a cell-specific enhancer in the GnRH neuron and as a repressor in the ovary. Deletion of this region in vivo implicates the GnRH promoter in mediating pubertal development and periodic reproductive cycling, and forms the foundation to define the nuclear proteins important for puberty and estrous cycling in mammals.

Deletion of Otx2 in GnRH neurons results in a mouse model of hypogonadotropic hypogonadism.

GnRH is the central regulator of reproductive function responding to central nervous system cues to control gonadotropin synthesis and secretion. GnRH neurons originate in the olfactory placode and migrate to the forebrain, in which they are found in a scattered distribution. Congenital idiopathic hypogonadotropic hypogonadism (CIHH) has been associated with mutations or deletions in a number of genes that participate in the development of GnRH neurons and expression of GnRH. Despite the critical role of GnRH in mammalian reproduction, a comprehensive understanding of the developmental factors that are responsible for regulating the establishment of mature GnRH neurons and the expression of GnRH is lacking. orthodenticle homeobox 2 (OTX2), a homeodomain protein required for the formation of the forebrain, has been shown to be expressed in GnRH neurons, up-regulated during GnRH neuronal development, and responsible for increased GnRH promoter activity in GnRH neuronal cell lines. Interestingly, mutations in Otx2 have been associated with human hypogonadotropic hypogonadism, but the mechanism by which Otx2 mutations cause CIHH is unknown. Here we show that deletion of Otx2 in GnRH neurons results in a significant decrease in GnRH neurons in the hypothalamus, a delay in pubertal onset, abnormal estrous cyclicity, and infertility. Taken together, these data provide in vivo evidence that Otx2 is critical for GnRH expression and reproductive competence.

A novel dominant negative mutation of OTX2 associated with combined pituitary hormone deficiency.

CONTEXT: Combined pituitary hormone deficiency (CPHD) is characterized by deficiencies in more than one anterior pituitary hormone. Mutations in developmental factors responsible for pituitary cell specification and gene expression have been found in CPHD patients. OTX2, a bicoid class homeodomain protein, is necessary for both forebrain development and transactivation of the HESX1 promoter, but as of yet, has not been associated with CPHD. OBJECTIVE: The goal of this study was to identify and characterize novel mutations in pituitary specific transcription factors from CPHD patients. DESIGN: Genomic DNA was isolated from patients with hypopituitarism to amplify and sequence eight pituitary specific transcription factors (HESX1, LHX3, LHX4, OTX2, PITX2, POU1F1, PROP1, and SIX6). Characterization of novel mutations is based on structural and functional studies. RESULTS: We describe two unrelated children with CPHD who presented with neonatal hypoglycemia, and deficiencies of GH, TSH, LH, FSH, and ACTH. Magnetic resonance imaging revealed anterior pituitary hypoplasia with an ectopic posterior pituitary. A novel heterozygous OTX2 mutation (N233S) was identified. Wild-type and mutant OTX2 proteins bind equivalently to bicoid binding sites, whereas mutant OTX2 revealed decreased transactivation. CONCLUSIONS: A novel mutation in OTX2 binds normally to target genes and acts as a dominant negative inhibitor of HESX1 gene expression. This suggests that the expression of HESX1, required for spaciotemporal development of anterior pituitary cell types, when disrupted, results in an absent or underdeveloped anterior pituitary with diminished hormonal expression. These results demonstrate a novel mechanism for CPHD and extend our knowledge of the spectrum of gene mutations causing CPHD.

Repression of GLUT4 expression by the endoplasmic reticulum stress response in 3T3-L1 adipocytes.

Expression of GLUT4 is decreased in adipocytes in obesity and type 2 diabetes, contributing to the insulin resistance of these states. Recent investigations suggest a role for activation of the ER stress response in the pathophysiology of type 2 diabetes. We investigated activation of the ER stress response in 3T3-L1 adipocytes. We show that activation of the ER stress response decreased GLUT4 expression at the level of gene transcription. Activation of the ER stress response also increased the expression of CHOP10, an inhibitor of the activity and expression of C/EBPalpha. As expected, activation of the ER stress response decreased expression of C/EBPalpha, an activator of GLUT4 expression, providing a mechanism to account for the repression of GLUT4 by ER stress activation. Our studies identify repression of GLUT4 expression as another potential mechanism for obesity-induced activation of the ER stress response to contribute to the insulin resistance of obesity.