Development and Characterization of Novel Rat Anti-mERß Sera.

Estrogens regulate normal sexual and reproductive development in females. Their actions are mediated mainly by estrogen receptor (ER)α and ERß. Understanding the function of ERs necessitates knowing their cellular location and protein partners, which, in turn, requires reliable and specific antibodies. Several antibodies are available for ERα; however, discrepancies in immunoreactivity have been reported for ERß. Here, we have developed antisera for mouse ERß (mERß) using a specific C-terminal 18-amino acid peptide conjugated to mariculture keyhole limpet hemocyanin. Sprague Dawley rats were immunized, and the resulting antisera were characterized by Western blot analysis of nuclear extracts from tissues of wild-type (WT) mice, and mice genetically modified to lack either ERα (CERαKO) or ERß (CERßKO). An approximately 56-kDa protein was detected in the hypothalamus, uterus, ovary, mammary gland, testes, and epididymis of WT mice, consistent with the predicted molecular size of ERß. In addition, the same protein band was identified in in vitro synthesized mERß protein and in the mammary glands of CERαKO mice. The approximately 56-kDa protein was not observed in in vitro synthesized mERα protein or in any tissue examined in the CERßKO mice. Immunohistochemistry using the antisera revealed ERß staining in the granulosa cells of WT ovaries and in the mediobasal hypothalamus, paraventricular nucleus, and cerebral cortex in the WT adult mouse brain. These data suggest that the novel rat anti-mERß sera are specific to ERß to allow investigators to explore to cellular and physiological role of ERß in the brain and other mouse tissues.

GH safety workshop position paper: a critical appraisal of recombinant human GH therapy in children and adults.

Recombinant human GH (rhGH) has been in use for 30 years, and over that time its safety and efficacy in children and adults has been subject to considerable scrutiny. In 2001, a statement from the GH Research Society (GRS) concluded that 'for approved indications, GH is safe'; however, the statement highlighted a number of areas for on-going surveillance of long-term safety, including cancer risk, impact on glucose homeostasis, and use of high dose pharmacological rhGH treatment. Over the intervening years, there have been a number of publications addressing the safety of rhGH with regard to mortality, cancer and cardiovascular risk, and the need for long-term surveillance of the increasing number of adults who were treated with rhGH in childhood. Against this backdrop of interest in safety, the European Society of Paediatric Endocrinology (ESPE), the GRS, and the Pediatric Endocrine Society (PES) convened a meeting to reappraise the safety of rhGH. The ouput of the meeting is a concise position statement.

Kisspeptin Induces Dynamic Chromatin Modifications to Control GnRH Gene Expression.

In vitro studies have demonstrated an increase in GnRH gene expression associated with an elevated secretory response to kisspeptin administration, suggesting that kisspeptin mediates GnRH expression at both the secretory and pretranslational levels. However, the kisspeptin-mediated intracellular mechanisms associated with the dynamic chromatin modifications modulating GnRH gene expression are unclear. The studies in this manuscript describe specific histone modifications on the enhancer and promoter of the mouse GnRH (mGnRH) gene induced by kisspeptin in GnRH neuronal cell lines (GT1-7 cells). ChIP assays followed by quantitative real-time PCR (qPCR) demonstrate that 15 and 45 min of 10(-9) M kisspeptin significantly increased histone 3 acetylation (H3Ac) at the kisspeptin response element (KsRE) contained between -3446 and -2806 bp of the mGnRH enhancer (GnRHen) in GT1-7 cells, while no changes were observed in the downstream neuron-specific element (NSE). Moreover, kisspeptin specifically induced acetylation of H3AcK14 and K27 and trimethylation of H3 lysine 4 at the KsRE (markers of active chromatin) and no changes in dimethylation of H3K9 (a marker associated with gene repression). Occupancy of RNA Pol II (RNAPII) and a differential carboxyl-terminal domain (CTD) phosphorylation pattern was observed. An interaction between the NSE and the KsRE via a chromatin loop in the mGnRH gene by kisspeptin was detected by the chromosome conformation capture assay (3C). In conclusion, these results demonstrate that kisspeptin induces histone acetylation/methylation and consequently enhances the formation of a chromatin loop in the mGnRH gene which results in known increase in kisspeptin-dependent mGnRH expression.

Development of an immortalised, post-pubertal gonadotrophin-releasing hormone neuronal cell line.

Gonadotrophin-releasing hormone (GnRH) is important in reproduction, although some of the mechanisms for its synthesis and release remain elusive. Progress in understanding the GnRH neurone has been hampered by the limited number and diffuse distribution of the neurone in the mammalian brain. Several stable GnRH-expressing cell lines have been developed using in vivo expression of the simian virus 40 T Antigen (TAg), and they have been helpful for the study of gene expression and neuronal function. However, expression of an immortalising gene may interfere with normal cellular function. We developed a novel GnRH-secreting cell line transgenic mouse model suitable for targeted transformation in post-pubertal mice using a tetracycline-regulated TAg transgene. This clonal cell line, GRT, expresses neuronal markers and GnRH. GRT cells grown in medium containing tetracycline-free serum express increasing mRNA levels of GnRH associated with declining levels of TAg expression. The novelty and ultimately the usefulness of this cell line is that TAg expression, which could affect the GnRH neuronal phenotype, can be regulated by tetracycline.

Temporal and spatial regulation of CRE recombinase expression in gonadotrophin-releasing hormone neurones in the mouse.

Gonadotrophin-releasing hormone (GnRH) neurones located within the brain are the final neuroendocrine output regulating the reproductive hormone axis. Their small number and scattered distribution in the hypothalamus make them particularly difficult to study in vivo. The Cre/loxP system is a valuable tool to delete genes in specific cells and tissues. We report the production of two mouse lines that express the CRE bacteriophage recombinase in a GnRH-specific manner. The first line, the GnRH-CRE mouse, contains a transgene in which CRE is under the control of the murine GnRH promoter and targets CRE expression specifically to GnRH neurones in the hypothalamus. The second line, the GnRH-CRETeR mouse, uses the same murine GnRH promoter to target CRE expression to GnRH neurones, but is modified to be constitutively repressed by a tetracycline repressor (TetR) expressed from a downstream tetracycline repressor gene engineered within the transgene. GnRH neurone-specific CRE expression can therefore be induced by treatment with doxycycline which relieves repression by TetR. These GnRH-CRE and GnRH-CRETeR mice can be used to study the function of genes expressed specifically in GnRH neurones. The GnRH-CRETeR mouse can be used to study genes that may have distinct roles in reproductive physiology during the various developmental stages.

CREB binding protein recruitment to the transcription complex requires growth factor-dependent phosphorylation of its GF box.

Growth factors such as epidermal growth factor (EGF) and insulin regulate development and metabolism via genes containing both POU homeodomain (Pit-1) and phorbol ester (AP-1) response elements. Although CREB binding protein (CBP) functions as a coactivator on these elements, the mechanism of transactivation was previously unclear. We now demonstrate that CBP is recruited to these elements only after it is phosphorylated at serine 436 by growth factor-dependent signaling pathways. In contrast, p300, a protein closely related to CBP that lacks this phosphorylation site, binds only weakly to the transcription complex and in a growth factor-independent manner. A small region of CBP (amino acids 312-440), which we term GF box, contains a potent transactivation domain and mediates this effect. Direct phosphorylation represents a novel mechanism controlling coactivator recruitment to the transcription complex.

Combined pituitary hormone deficiency due to the F135C human Pit-1 (pituitary-specific factor 1) gene mutation: functional and structural correlates.

The pituitary-specific transcription factor Pit-1 (pituitary-specific factor 1) is known to play a key role in the differentiation of PRL-, GH-, and TSH-secreting cells, and in the regulation of expression of the corresponding genes. In recent years, 12 distinct mutations of the Pit-1 gene have been shown to be responsible for a phenotype of multiple congenital pituitary hormone deficiency involving PRL, GH, and TSH. We had previously identified, in four siblings with GH, PRL, and TSH deficiencies, a mutation (F135C) resulting in a single amino acid change within the POU-specific binding domain of the Pit-1 molecule. In the present report, we have explored the functional effect of the F135C mutation. In vitro activity tests performed by transfection in human HeLa cells showed decreased transactivation capacity on the PRL, GH, and Pit-1 genes. The DNA binding experiments performed by gel shift showed that the F135C mutation generated a protein capable of binding to DNA response elements. To analyze how the F135C mutation might affect functionality of the transcription factor despite a normal DNA binding, we used a structure modelization approach and also analyzed two other Pit-1 mutant proteins (F135A and F135Y). The loss of functionality in these two mutants was similar to that of F135C. This finding was in keeping with our molecular modeling studies. According to structural data derived from the crystallographic analysis of the DNA/Pit-1 POU domain complex, the conformation of the first helix of the F135C-mutated POU-specific domain could be perturbed to such an extent that any interaction with other transcription cofactors might be definitively prevented.

cAMP response element-binding protein-binding protein mediates thyrotropin-releasing hormone signaling on thyrotropin subunit genes.

Transcription of pituitary alpha-glycoprotein hormone subunit (alpha-GSU) and thyrotropin beta subunit (TSH-beta) genes is stimulated by thyrotropin-releasing hormone (TRH). Since cAMP response element-binding protein (CREB)-binding protein (CBP) integrates a number of cell signaling pathways, we investigated whether CBP is important for TRH stimulation of the TSH subunit genes. Cotransfection of E1A in GH(3) cells completely blocked TRH stimulation of the TSH subunit genes, suggesting that CBP is a key factor for TRH signaling in the pituitary. CBP and Pit-1 acted synergistically in TRH stimulation of the TSH-beta promoter, and amino acids 1-450 of CBP were sufficient for the TRH effect. In contrast, on the human alpha-GSU promoter, CREB and P-Lim mediated TRH signaling. Intriguingly, CREB was phosphorylated upon TRH stimulation, leading to CBP recruitment to the alpha-GSU promoter. CBP also interacted with P-Lim in a TRH-dependent manner, suggesting that P-Lim is an important factor for non-cAMP response element-mediated TRH stimulation of this promoter. Distinct domains of CBP were required for TRH signaling by CREB and P-Lim on the alpha-GSU promoter, amino acids 450-700 and 1-450, respectively. Thus, the amino terminus of CBP plays a critical role in TRH signaling in the anterior pituitary via both Pit-1-dependent and -independent pathways, yielding differential regulation of pituitary gene products.

CREB-independent regulation by CBP is a novel mechanism of human growth hormone gene expression.

Hypothalamic growth hormone-releasing hormone (GHRH) stimulates growth hormone (GH) gene expression in anterior pituitary somatotrophs by binding to the GHRH receptor, a G-protein-coupled transmembrane receptor, and by mediating a cAMP-mediated protein kinase A (PKA) signal-transduction pathway. Two nonclassical cAMP-response element motifs (CGTCA) are located at nucleotides -187/-183 (distal cAMP-response element; dCRE) and -99/-95 (proximal cAMP-response element; pCRE) of the human GH promoter and are required for cAMP responsiveness, along with the pituitary-specific transcription factor Pit-1 (official nomenclature, POU1F1). Although a role for cAMP-response element binding protein (CREB) in GH stimulation by PKA has been suggested, it is unclear how the effect may be mediated. CREB binding protein (CBP) is a nuclear cofactor named for its ability to bind CREB. However, CBP also binds other nuclear proteins. We determined that CBP interacts with Pit-1 and is a cofactor for Pit-1-dependent activation of the human GH promoter. This pathway appears to be independent of CREB, with CPB being the likely target of phosphorylation by PKA.

Divergent roles for thyroid hormone receptor beta isoforms in the endocrine axis and auditory system.

Thyroid hormone receptors (TRs) modulate various physiological functions in many organ systems. The TR alpha and TR beta isoforms are products of 2 distinct genes, and the beta 1 and beta 2 isoforms are splice variants of the same gene. Whereas TR alpha 1 and TR beta 1 are widely expressed, expression of the TR beta 2 isoform is mainly limited to the pituitary, triiodothyronine-responsive TRH neurons, the developing inner ear, and the retina. Mice with targeted disruption of the entire TR beta locus (TR beta-null) exhibit elevated thyroid hormone levels as a result of abnormal central regulation of thyrotropin, and also develop profound hearing loss. To clarify the contribution of the TR beta 2 isoform to the function of the endocrine and auditory systems in vivo, we have generated mice with targeted disruption of the TR beta 2 isoform. TR beta 2-null mice have preserved expression of the TR alpha and TR beta 1 isoforms. They develop a similar degree of central resistance to thyroid hormone as TR beta-null mice, indicating the important role of TR beta 2 in the regulation of the hypothalamic-pituitary-thyroid axis. Growth hormone gene expression is marginally reduced. In contrast, TR beta 2-null mice exhibit no evidence of hearing impairment, indicating that TR beta 1 and TR beta 2 subserve divergent roles in the regulation of auditory function.

Defective retinoic acid regulation of the Pit-1 gene enhancer: a novel mechanism of combined pituitary hormone deficiency.

Pit-1 is a pituitary-specific transcription factor responsible for pituitary development and hormone expression in mammals. Pit-1 contains two protein domains, termed POU-specific and POU-homeo, which are both necessary for DNA binding and activation of the GH and PRL genes and regulation of the PRL, TSH-beta subunit (TSH-beta), and Pit-1 genes. Pit-1 is also necessary for retinoic acid induction of its own gene during development through a Pit-1-dependent enhancer. Combined pituitary hormone deficiency is caused by defective transactivation of target genes in the anterior pituitary. In the present report, we provide in vivo evidence that retinoic acid induction of the Pit-1 gene can be impaired by a Pit-1 gene mutation, suggesting a new molecular mechanism for combined pituitary hormone deficiency in man.

A novel mechanism for cyclic adenosine 3',5'-monophosphate regulation of gene expression by CREB-binding protein.

The pituitary-specific transcription factor, Pit-1, is necessary to mediate protein kinase A (PKA) regulation of the GH, PRL, and TSH-beta subunit genes in the pituitary. Since these target genes lack classical cAMP DNA response elements (CREs), the mechanism of this regulation was previously unknown. We show that CREB binding protein (CBP), through two cysteine-histidine rich domains (C/H1 and C/H3), specifically and constitutively interacts with Pit-1 in pituitary cells. Pit-1 and CBP synergistically activate the PRL gene after PKA stimulation in a mechanism requiring both an intact Pit-1 amino-terminal and DNA-binding domain. A CBP construct containing the C/H3 domain [amino acids (aa) 1678-2441], but not one lacking the C/H3 domain (aa 1891-2441), is sufficient to mediate this response. Neither construct augments PKA regulation of CRE-containing promoters. Fusion of either CBP fragment to the GAL4 DNA-binding domain transferred complete PKA regulation to a heterologous promoter. These findings provide a mechanism for CREB-independent regulation of gene expression by cAMP.

Transcription Factors and Hypopituitarism.

Several homeodomain factors are found in the developing anterior pituitary lobe. The production of these developmental transcription factors has distinct temporal and spatial patterns. By interacting with each other, as well as with other extrinsic and intrinsic signals, they control cell determination and specification. Here, we discuss transcription factors that have been shown to have an in vivo role in pituitary cell-type specification.

Direct regulation of GnRH transcription by CRF-like peptides in an immortalized neuronal cell line.

The existence of a CRF-dependent inhibition of GnRH transcription was investigated using a neuronal GnRH-expressing cell line (Gn11) stably transfected with mouse (-611 bp) or chicken (-3000 bp) GnRH promoter/luciferase reporter constructs. The presence of the CRF-R1 receptor was established using a specific CRF-R1 antiserum. After 7 h of incubation, urotensin-I and sauvagine increased the mouse GnRH-reporter bioluminescence by 1.3- and 1.2-fold, respectively, compared with control cells. Subsequently, CRF, urotensin-I and sauvagine decreased luciferase reporter activity to about 60% of the control values after 14 h. Similar trends occurred with the chicken GnRH promoter with UI increasing reporter gene activity 2.4-fold over the controls after 14 h incubation. These data provide additional evidence for the direct regulation of GnRH transcription by CRF-like peptides.

Leptin levels in children with central precocious puberty.

Several studies have suggested that sufficient serum leptin levels may be involved in the initiation of puberty. To assess further the relationship between leptin and the onset of puberty in humans, we measured the serum leptin concentration in children with central precocious puberty (CPP). We studied 65 children with either idiopathic (IPP; n = 50 girls and 3 boys) or neurogenic central precocious puberty (NPP; n = 5 girls and 7 boys). The serum leptin levels in these patients were compared with normative data from healthy children and adolescents using SD scores that adjust for body mass index (BMI) and Tanner stage. The mean SD scores of IPP and NPP girls were +0.4 +/- 0.1 and +1.0 +/- 0.5, respectively, compared with that of age-matched prepubertal girls and +0.7 +/- 0.2 and +1.6 +/- 0.6 compared with that of girls matched for pubertal stage. The CPP girls with lower BMIs contributed larger SD scores, such that the leptin SD score was negatively correlated with BMI. A similar, modest increase in leptin levels in the CPP girls was evident when additional normative data were considered. The mean leptin SD scores of IPP and NPP boys were -0.9 +/- 0.5 and +0.7 +/- 0.3, respectively, compared with that of normal boys at Tanner stage 3-4. Serum leptin levels in the boys with CPP were not different from those in healthy boys in any of the normative studies. These data should be interpreted cautiously, but they suggest that girls with CPP have modestly elevated serum leptin concentrations compared with those in healthy children and adolescents. In addition, the negative correlation between the leptin SD score and BMI suggests that sufficient leptin levels may be associated with initiation of puberty in girls.

The impact of reversible gonadal sex steroid suppression on serum leptin concentrations in children with central precocious puberty.

Serum leptin concentrations increase during childhood in both sexes. During sexual maturation, levels rise further in girls, but decrease in boys. These data suggest that testosterone either directly suppresses leptin levels or induces changes in body composition that result in lower leptin concentrations. To examine further the relationship between sex steroids and leptin, we performed a longitudinal study in children with central precocious puberty (28 girls and 12 boys) before, during, and after discontinuation of GnRH agonist-induced pituitary-gonadal suppression. Nighttime and daytime leptin levels were measured to determine whether the activity of the pituitary-gonadal axis affects their diurnal variation. In the boys, suppression of testosterone increased leptin levels, whereas resumption of puberty was associated with decreased leptin levels [3.5 +/- 0.8 vs. 9.5 +/- 3.1 ng/dL (P = 0.005) and 12.2 +/- 4.5 vs. 7.0 +/- 2.6 ng/dL (P = 0.012), respectively]. Serum leptin levels did not change in the girls with alteration of the pituitary-ovarian axis and consistently exceeded those in boys. Nighttime levels were consistently greater than daytime values by an average of 38.3% in the girls and 29.4% in the boys. These serial observations during reversible pituitary-gonadal suppression suggest that testosterone decreases leptin concentrations, but that estrogen, at least in this childhood model, has no discernible effect. In addition, our data indicate that the presence of the diurnal rhythm in leptin concentrations is independent of the state of the reproductive axis.

The molecular basis of hypopituitarism.

Pit-1 is a pituitary-specific transcription factor responsible for pituitary development and hormone expression in mammals. My laboratory and others have recently described several patients with combined pituitary hormone deficiency (CPHD) due to point mutations in the pit-1 gene. In addition to pit-1, other nuclear factors appear to be necessary for full expression of pituitary genes. A zinc finger transcription factor, Zn-15, is responsible with pit-1 for synergistic activation of the GH gene. The Pr1 gene is regulated synergistically by pit-1 and the estrogen receptor. Finally, the pit-1 gene itself is regulated by an enhancer element located > 10 kb upstream of the transcriptional start. This element contains several pit-1 DNA binding sites and retinoic acid response elements (RAREs). On one of these elements, pit-1 and RAR interact functionally to mediate a synergistic response to RA. Recent data from our laboratory suggests that RA induction of the pit-1 gene can be impaired by pit-1 gene mutations. Study of pit-1 mutations and their diverse pathophysiological mechanisms should increase our understanding of anterior pituitary gland development and gene regulation in normal and disease states.

Regulation of gonadotropin-releasing hormone (GnRH) gene expression by insulin-like growth factor I in a cultured GnRH-expressing neuronal cell line.

A GnRH-expressing neuronal cell line (NLT) was used to determine whether insulin-like growth factor I (IGF-I) regulates GnRH gene expression. A receptor-binding assay demonstrated the expression of IGF-I receptors on NLT cells. Activation of IGF-I receptors induced the Ras/Raf-1/mitogen-activated protein kinase pathway and increased c-fos expression. NLT cells treated with IGF-I underwent cell proliferation and exhibited a growth-independent increase in mouse GnRH mRNA expression. In cells transfected with DNA constructs containing the human GnRH promoter, which includes a consensus AP-1 binding site fused to the luciferase reporter gene, a significant increase in reporter activities was induced by IGF-I, whereas mutation of this AP-1 site significantly reduced IGF-I-induced promoter activation. These results demonstrate that IGF-I serves as an important signal in the regulation of both human and rodent GnRH gene expression.

An alternative gonadotropin-releasing hormone (GnRH) RNA splicing product found in cultured GnRH neurons and mouse hypothalamus.

Gonadotropin-releasing hormone (GnRH) is encoded by the proGnRH gene which contains four exons and three introns. In this study, two immortalized GnRH-expressing cell lines (Gn11 and NLT) were characterized. The NLT and Gn11 cells, derived from a same brain tumor in a transgenic mouse, display neuronal morphology and neuron-specific markers. However, NLT cells secrete much higher levels of GnRH than Gn11 cells. To delineate the mechanism underlying this difference, reverse transcriptase-polymerase chain reaction and RNase protection assays were performed to examine proGnRH gene expression. While the mature proGnRH mRNA was predominately expressed in NLT cells, Gn11 cells express an abundant short transcript. Sequence analysis revealed that this short transcript contains exons 1, 3, and 4, but not exon 2, which encodes the GnRH decapeptide. RNase protection assays demonstrated that NLT cells express much higher levels of mature proGnRH mRNA than Gn11 cells. The lower level of GnRH secreting capacity in Gn11 cells is due, in part, to decreased expression of mature proGnRH mRNA. When proGnRH gene expression in the mouse brain was examined, the same short splicing variant was observed in the olfactory area and preoptic area-anterior hypothalamus. But the prevalent transcript in these regions was the mature proGnRH mRNA. In contrast, only the mature proGnRH mRNA was found in the caudal hypothalamus. These results suggest that alternative splicing may be one of the mechanisms regulating proGnRH gene expression in the animal brain.

Primary hypothyroidism presenting as severe vaginal bleeding in a prepubertal girl.

We describe a case of acquired primary hypothyroidism in a prepubertal girl who presented with profound bradycardia, precocious puberty, and severe vaginal bleeding. A pelvic ultrasound revealed markedly enlarged ovaries with numerous cysts. Cystic aspiration, in concert with L-thyroxine therapy, appeared to halt vaginal bleeding, and led to resolution of cysts. This is the first reported case of percutaneous cyst aspiration to treat ovarian hyperstimulation caused by hypothyroidism in a child.