Biomedical subjects

D D Belsham

Publications and source records attributed to D D Belsham.

14 recordsLinked to original sources

PubMed2004

Functional dimorphism of two hAgRP promoter SNPs in linkage disequilibrium.

The agouti related protein (AgRP) exerts its anabolic effects on food intake by antagonising the alpha-melanocyte stimulating hormone (alpha-MSH) at its receptors, melanocortin receptors 3 and 4 (MC3R and MC4R). A single nucleotide polymorphism (SNP) in the promoter of the human AgRP (hAgRP), -38C>T, was associated with low body fatness. The -38T allele that was associated with low body fatness also resulted in lower promoter activity. Here we report a novel SNP, -3019G>A, again in the promoter of hAgRP, which is in complete linkage disequilibrium (LD) with the -38C>T SNP (linked alleles: -3019A/-38T and -3019G/-38C). Functional analyses in a human adrenal and two mouse hypothalamus cell lines showed that the -3019A allele had significantly higher promoter activity. Hence, the two linked alleles (-3019A and -38T) had opposite effects on promoter function and yet they were both associated with low body fatness. The region encompassing the -38C>T SNP had approximately 1000-fold higher activity than the region encompassing the -3019G>A SNP, potentially determining the net functional effect between these two SNPs.

Agouti-Related Protein↗

PubMed2001

Cyclical regulation of GnRH gene expression in GT1-7 GnRH-secreting neurons by melatonin.

The pineal hormone melatonin plays an important role in the neuroendocrine control of reproductive physiology, but its effects on hypothalamic GnRH neurons are not yet known. We have found that GT1-7 GnRH-secreting neurons express membrane-bound G protein-coupled melatonin receptors, mt1 (Mel-1a) and MT2 (Mel-1b) as well as the orphan nuclear receptors ROR alpha and RZR beta. Melatonin (1 nM) significantly downregulates GnRH mRNA levels in a 24-h cyclical manner, an effect that is specifically inhibited by the melatonin receptor antagonist luzindole (10 microM). Repression of GnRH gene expression by melatonin appears to occur at the transcriptional level and can be mapped to the GnRH neuron-specific enhancer located within the 5' regulatory region of the GnRH gene. Using transient transfection of GT1-7 cells, downregulation of GnRH gene expression by melatonin was further localized to five specific regions within the GnRH enhancer including -1827/-1819, -1780/-1772, -1746/-1738, -1736/-1728, and -1697/-1689. Interestingly, the region located at -1736/-1728 includes sequences that correspond to two direct repeats of hexameric consensus binding sites for members of the ROR/RZR orphan nuclear receptor family. To begin to dissect the mechanisms involved in the 24-h cyclical regulation of GnRH transcription, we have found that melatonin (10 nM) induces rapid internalization of membrane-bound mt1 receptors through a beta-arrestin 1-mediated mechanism. These results provide the first evidence that melatonin may mediate its neuroendocrine control on reproductive physiology through direct actions on the GnRH neurons of the hypothalamus, both at the level of GnRH gene expression and through the regulation of G protein-coupled melatonin receptors.

Animals↗

PubMed2000

Transcription factors Oct-1 and C/EBPbeta (CCAAT/enhancer-binding protein-beta) are involved in the glutamate/nitric oxide/cyclic-guanosine 5'-monophosphate-mediated repression of mediated repression of gonadotropin-releasing hormone gene expression.

The physiological actions of nitric oxide (NO) as a signaling molecule in endothelial and brain cells and as a toxic molecule used by activated immune cells have been the focus of a wide range of studies. Nevertheless, the downstream effector molecules of this important neuromodulator are not well understood. We have previously demonstrated that expression of the gene for the reproductive neuropeptide, GnRH, is repressed by the glutamate/NO/cyclic GMP (cGMP) signal transduction pathway through cGMP-dependent protein kinase in the hypothalamic GnRH-secreting neuronal cell line GT1-7. This repression localized within a previously characterized 300-bp neuron-specific enhancer. Here, we find that mutation of either of two adjacent elements within the enhancer eliminates repression by this pathway. An AT-rich sequence located at -1695 has homology to the octamer motif known to bind POU-homeodomain proteins, while the adjacent element at -1676 has homology to the C/EBP (CCAAT/enhancer-binding protein) protein family consensus sequence. Antibody supershift assays reveal that one of the proteins bound at the -1695 sequence is Oct-1, and one of the proteins bound to the element at -1676 is C/EBPbeta. These two proteins can bind simultaneously to the adjacent -1695 and -1676 binding sites in vitro. In nuclear extracts of GT1-7 cells treated with an NO donor, the intensity of the Oct-1 complex is increased. However, although Western blot analysis indicates that neither Oct-1 nor C/EBPbeta protein levels are increased, the relative binding affinity of Oct-1 is increased. Dephosphorylation of the nuclear extracts decreases binding of the Oct-1 complex to the -1695 site only in NO donor-treated extracts. Thus, we conclude that Oct-1 and C/EBPbeta are both downstream transcriptional regulators involved in the repression of GnRH gene expression by the glutamate/NO/ cGMP signal transduction pathway.

Animals↗

PubMed1999

Estrogen directly respresses gonadotropin-releasing hormone (GnRH) gene expression in estrogen receptor-alpha (ERalpha)- and ERbeta-expressing GT1-7 GnRH neurons.

Estrogen has wide-ranging and complex effects on the reproductive axis, which are often difficult to interpret from in vivo studies. Estrogen negatively regulates tonic GnRH synthesis and also plays a pivotal role in the positive regulation of GnRH necessary for the preovulatory surge. To dissect the mechanisms by which these divergent effects occur, we attempted to observe the direct action of estrogen on the regulation of GnRH messenger RNA (mRNA) levels using the well characterized, GnRH-secreting, hypothalamic cell line, GT1-7. Using RT-PCR, we first investigated estrogen receptor transcript expression in GT1-7 neurons. We found that the GT1-7 cells express both estrogen receptor-alpha (ERalpha) and the recently described ERbeta mRNAs. We also detected the presence of both receptor subtypes in the GT1-7 neurons by Western blot analysis using specific ER antibodies. By Northern blot analysis of total GT1-7 RNA, we found that 17beta-estradiol (1 nM) down-regulates GnRH mRNA levels to approximately 55% of basal levels over a 48-h time course. This effect appears to occur specifically through an ER-mediated mechanism, as ICI 182,780, a complete ER antagonist, blocks the repression of GnRH mRNA levels by estradiol. The recently reported ERalpha-specific agonist/ERbeta-specific antagonist 2,2-bis-(p-hydroxyphenyl-1,1,1-trichloroethane (HPTE), a methoxychlor metabolite, also down-regulated GnRH gene expression. The repression of GnRH mRNA levels appears to occur at the transcriptional level, as simian virus 40 T antigen mRNA expression, which is under the control of 2.3 kb of the rat GnRH 5'-regulatory region, mimics the down-regulation of GnRH after treatment with estradiol. As the rat GnRH regulatory region in GT1-7 neurons does not appear to harbor a classic estrogen response element, the mechanism involved in the repression of GnRH has yet to be determined. These results suggest that estradiol directly regulates GnRH gene expression at the level of the GnRH neuron and may exert its neuroendocrine control through direct interaction with specific receptors expressed in these cells.

Animals↗

PubMed1998

Regulation of gonadotropin-releasing hormone (GnRH) gene expression by 5alpha-dihydrotestosterone in GnRH-secreting GT1-7 hypothalamic neurons.

Hypothalamic GnRH secretory neurons are precisely regulated by circulating gonadal steroids. However, the question of whether these cells are directly responsive to steroid hormones remains a central and controversial issue in reproductive science. In the present study, we demonstrate the expression of androgen receptor (AR) in a mouse hypothalamic GnRH-secreting cell line, GT1-7. AR messenger RNA was detected by Northern blot analysis of 10 microg total cellular RNA. Western blot analysis revealed a 110K AR immunoreactive band, and saturation binding analysis confirmed the presence of a high affinity low capacity androgen binding entity (Kd = 0.06 nM; Bmax = 12.4 fmol/mg protein). In addition, GT1-7 cells were found to express ARA70, an AR-specific coactivator that has been reported to enhance transactivational activity of the AR. GT1-7 cells transiently transfected with an androgen responsive MMTV-luciferase reporter construct displayed a 4.2-fold induction of luciferase reporter gene activity by 1 nM 5alpha-dihydrotestosterone (DHT), further demonstrating the presence of a functional AR. Treatment of GT1-7 cells with 1 or 10 nM DHT resulted in approximately 55% reduction in GnRH messenger RNA measured at 24 and 36 h after treatment. This repression was completely blocked by hydroxyflutamide, an AR antagonist. These results provide the first demonstration that androgen acts directly through an AR-mediated pathway to repress GnRH gene expression in hypothalamic GnRH-secreting neurons.

Animals↗

PubMed1996

NMDA and nitric oxide act through the cGMP signal transduction pathway to repress hypothalamic gonadotropin-releasing hormone gene expression.

The key roles of the excitatory neurotransmitter glutamate and its second messengers, nitric oxide (NO) and cGMP, in long-term potentiation and neural plasticity are well documented. However, complex functions such as memory are likely to require long term changes in synaptic efficacy which require gene expression and protein synthesis. Here we demonstrate that the glutamate receptor agonist, N-methyl-D-aspartic acid (NMDA), nitric oxide (NO) and cGMP each repress expression of the gonadotropin-releasing hormone (GnRH) gene in the hypothalamic cell line, GT1. This repression is dependent upon signals from NMDA receptors activating NO synthase to synthesize NO. In turn NO induces guanylyl cyclase to synthesize cGMP, activating cGMP- dependent protein kinase. Repression requires elevation of calcium because it only occurs in the presence of calcium ionophore or with release of intracellular calcium. Repression also requires protein synthesis. Activation of this pathway specifically represses expression of a reporter gene containing the regulatory region of the GnRH gene in transfected GT1 cells, indicating that repression occurs at the transcriptional level. Furthermore the target for transcriptional repression is a 300 bp neuron-specific enhancer found 1.5 kb upstream of the GnRH gene which is sufficient to confer repression to a heterologous promoter. Thus the NMDA/NO/cGMP neurotransmitter signal transduction pathway controls not only synaptic function but also neuron-specific gene expression.

Animals↗

PubMed1995

Leu-676-Pro mutation of the androgen receptor causes complete androgen insensitivity syndrome in a large Hutterite kindred.

A large Manitoba Hutterite kindred with X-linked receptor negative complete androgen insensitivity syndrome (CAIS) was studied. In attempts to identify all carriers of the syndrome in this kindred, using the androgen receptor (AR) cDNA, we have found a novel diagnostic MspI polymorphic pattern, which cosegregates with the disease. This polymorphism was not detected in 79 unrelated X-chromosomes of which 22 were from Hutterite controls. We were able to localize the polymorphism to exon 4, which is known to encode part of the androgen receptor hormone binding domain. A single base substitution (T-->C) was detected, which creates a new MspI site. This novel transition mutation replaces Leu-676 with Pro at a site which is conserved in numerous members of the steroid receptor gene family. Sequencing all 8 exons of the AR revealed the Leu-676-->Pro mutation as the only change in the primary structure of the receptor. Transfection of COS-1 cells with an expression vector of the mutant AR demonstrates that this point mutation of nucleotide 2558 abolishes receptor binding activity. The mutation can easily be detected by MspI digestion of the polymerase chain reaction (PCR) amplified exon 4 product.

Amino Acid Sequence↗

PubMed1995

A neuron-specific enhancer targets expression of the gonadotropin-releasing hormone gene to hypothalamic neurosecretory neurons.

The molecular mechanisms specifying gene expression in individual neurons of the mammalian central nervous system have been difficult to study due to the cellular complexity of the brain and the absence of cultured model systems representing differentiated central nervous system neurons. We have developed clonal, differentiated, neuronal tumor cell lines of the hypothalamic GnRH-producing neurons by targeting tumorigenesis in transgenic mice. These cells (GT1 cells) provide a model system for molecular studies of GnRH gene regulation. Here we present the identification and characterization of a neuron-specific enhancer responsible for directing expression of the rat GnRH gene in GT1 hypothalamic neurons. This approximately 300 base pair (bp) upstream region (-1571 to -1863) confers enhancer activity to a short -173-bp GnRH promoter or to a heterologous promoter only in GT1 cells. The enhancer is bound by multiple GT1 nuclear proteins over its entire length. Deletion of more than 30 bp from either end dramatically reduces activity, and even large internal fragments carrying seven of the eight DNAse I-protected elements show decreased activity. Scanning replacement mutations demonstrate that several of the internal elements are required for activity of the enhancer. Thus, the GnRH gene is targeted to hypothalamic neurons by a complex multicomponent enhancer that relies on the interaction of multiple nuclear-protein binding enhancer elements.

Animals↗

PubMed1993

Partial androgen insensitivity--are all tissues equal?

Nonaromatizable androgens, administered in high doses to an adult patient with partial androgen insensitivity, failed to result in a change in phallic size despite a clear decline in SHBG and gonadotropin levels. These findings raise the question of differential tissue sensitivity to androgens. Because ancillary laboratory testing does not predict reliably the genital response, a therapeutic trial should be advocated in such cases.

Adult↗

PubMed1992

Analysis of the CAG repeat region of the androgen receptor gene in a kindred with X-linked spinal and bulbar muscular atrophy.

Herein we describe a family with X-linked spinal and bulbar muscular atrophy (SBMA or Kennedy's disease), an adult onset neuromuscular disease characterized by slow progression, predominant proximal and bulbar muscle weakness. One frequent association is the appearance of gynecomastia. This disorder was previously shown to be linked to the locus DXYS1 on the proximal long arm of the X chromosome. Recently, a report implicated a mutation at the N-terminus of the androgen receptor gene involving amplification of CAG repeats as the cause of X-linked SBMA. We studied this region of the androgen receptor in a kindred clinically suspected but not confirmed of having X-linked SBMA by the polymerase chain reaction (PCR) followed by Southern analysis and DNA sequencing. The mutated allele was found to have an increased number of 51 CAG repeats confirming the clinical diagnosis of SBMA. Normal individuals revealed 23 repeat numbers within the normal range, while another unrelated X-linked SBMA patient had an enlarged CAG repeat region. The carrier or disease status could be established or confirmed in 12 individuals of this family on the basis of detecting normal and disease alleles reflected by the number of CAG repeats.

Adult↗

PubMed1992

Replacement of arginine 773 by cysteine or histidine in the human androgen receptor causes complete androgen insensitivity with different receptor phenotypes.

We have discovered two different point mutations in a single codon of the X-linked androgen-receptor (AR) gene in two pairs of unrelated families who have complete androgen insensitivity (resistance) associated with different AR phenotypes in their genital skin fibroblasts. One mutation is a C-to-T transition at a CpG sequence near the 5' terminus of exon 6; it changes the sense of codon 773 from arginine to cysteine, ablates specific androgen-binding activity at 37 degrees C, and eliminates a unique KpnI site at the intron-exon boundary. The other mutation is a G-to-A transition that changes amino acid 773 to histidine and eliminates an SphI site. This mutant AR has a normal androgen-binding capacity at 37 degrees C but has a reduced affinity for androgens and is thermolabile in their presence. Transient transfection of COS cells with cDNA expression vectors yielded little androgen-binding activity at 37 degrees C from Arg773Cys and abundant activity with abnormal properties from Arg773His, thereby providing the pathogenicity of both sequence alterations. This conclusion coincides with the following facts about evolutionary preservation of the position homologous to Arg773 in the AR: it is occupied by Arg or lysine in the progesterone, glucocorticoid, and mineralocorticoid receptors, and it is within a 14-amino-acid region of their steroid-binding domains that share approximately 85% amino acid identity.

Adolescent↗

PubMed1991

Amber mutation creates a diagnostic MaeI site in the androgen receptor gene of a family with complete androgen insensitivity.

We have discovered in the X-linked androgen receptor gene a single nucleotide substitution that is the putative cause of complete androgen insensitivity (resistance) in a family with affected individuals in 2 generations. Earlier studies on the family indicated co-segregation of mutant phenotype and the RFLPs at the loci DXS1 and DXYS1. The mutation is an adenine-to-thymine transversion in exon 8 that changes the sense of codon 882 from lysine to an amber (UAG) translation termination signal. The substitution creates a recognition sequence for the restriction endonuclease MaeI: this permits ready recognition of hemizygotes and heterozygotes after amplification of genomic exon 8 by the polymerase chain reaction. The mutation predicts the synthesis of a truncated receptor that lacks 36 amino acids at the carboxy terminus of its 252-amino acid androgen-binding domain. The cultured genital skin fibroblasts of the one affected patient examined have normal levels of androgen receptor mRNA, but negligible androgen-receptor binding activity. These results accord with a variety of data from spontaneous and artificial mutations indicating that all portions of the steroid binding domain contribute to normal steroid binding by a steroid receptor.

Amino Acid Sequence↗

PubMed1991

The 56/58 kDa androgen-binding protein in male genital skin fibroblasts with a deleted androgen receptor gene.

Human genital skin fibroblasts (GSF) make a relatively abundant 56/58 kDa protein that binds androgens. The protein shares many properties with the approximately 100 kDa androgen receptor that is encoded by a locus in the q12 region of the X chromosome. It does not appear to be androgen-induced, yet is absent in GSF of most patients with complete androgen insensitivity (CAI). A precursor-product relation with the androgen receptor (AR) protein has been largely excluded; that it may be an unorthodox product of the AR gene has not. The 56/58 kDa protein is made by the GSF of a mentally retarded subject who has CAI because of a complete deletion of the coding portion of the AR gene. Hence, the strong constitutional and statistical correlations that have been demonstrated between the two proteins cannot arise because they share the same gene. The subject's genomic DNA hybridizes normally with 11 single-copy probes from Xq11-Xq13. Therefore, we cannot attribute her mental retardation to a contiguous gene syndrome.

Androgen-Binding Protein↗

PubMed1989

The 56 kDa protein of human genital skin fibroblasts is identical to that radiolabelled by [3H]dihydrotestosterone 17 beta-bromoacetate.

Analysis of soluble proteins from human genital skin fibroblasts by two-dimensional polyacrylamide gel electrophoresis reveals an abundant protein doublet of mol. wt 56,000 with isoelectric points (pI) of 6.7 and 6.5. This protein is absent in non-genital skin fibroblasts as well as in genital skin fibroblasts of most patients with complete forms of androgen insensitivity. The protein specifically binds androgen. A protein of similar estimated molecular weight (58,000) from human genital skin fibroblasts has recently been found to be covalently radiolabelled by the affinity ligand dihydrotestosterone 17 beta-bromoacetate (DHT-BA). In the present study these proteins have been found to be indistinguishable on one- and two-dimensional gel electrophoresis. Antibodies raised against the 56 kDa pI 6.7/6.5 protein also recognized the protein covalently radiolabelled by DHT-BA. A third protein of estimated mol. wt 59,000 has been found to be associated with several steroid hormone receptor complexes but has no known ligand binding activity. This protein was found to be clearly separable from the 56/58 kDa protein on two-dimensional gel electrophoresis as it has a more acidic pI of approximately 5.4. Furthermore, antibodies against the 59 kDa protein do not recognize the 56 kDa species, and vice versa.

Affinity Labels↗