Sleep duration and bone health measures in older men.

The associations between objective measures of sleep duration and bone outcomes in older men are unknown. No consistent, significant association was identified between sleep duration and bone mineral density (BMD) in the current analysis. However, future research should determine if vitamin D status modifies this relationship. INTRODUCTION: Prior studies, predominantly in women, reported that long and short self-reported sleep duration are associated with lower BMD. Associations between actigraphy-determined sleep duration and BMD or bone turnover markers (BTMs) in older men are unknown. METHODS: Men in The Osteoporotic Fractures in Men (MrOS) Study with wrist actigraphy and concurrent BMD assessment but without comorbidities affecting bone health were included. Sleep duration was considered as a continuous (N = 1926) and dichotomized variable where men were classified as getting the recommended (7-8 h/night; N = 478) or short (< 6 h/night; N = 577) sleep. The cross-sectional association between BMD, BTMs, and sleep duration was examined using a t test or linear regression, where appropriate, in unadjusted and adjusted models. RESULTS: There were no clinically or statistically significant differences in BMD at the L-spine, total hip, or femoral neck between men getting the recommended vs. short sleep duration, using actigraphy or self-reported sleep duration (all p ≥ 0.07). When sleep duration was considered as a continuous variable, femoral neck BMD was higher in men with longer self-reported sleep duration (ß = 0.006 ±0.003, p = 0.02), but this was not significant after further adjustment. In men with low 25OHD (< 20 ng/mL), longer actigraphy-determined sleep duration was associated with higher total hip BMD (ß = 0.016 ± 0.008; p = 0.04). Sleep duration and BTMs were not associated. CONCLUSION: Sleep duration was not associated with hip or L-spine BMD or BTMs in older men. Future research should determine if vitamin D status or other factors modify this relationship.

Association between objective sleep duration and bone mineral density in older postmenopausal women from the Study of Osteoporotic Fractures (SOF).

Methodological limitations preclude determination of the association between sleep duration and bone mineral density (BMD) from existing literature. This was the first study to use objective sleep duration to determine its association with BMD. Nocturnal sleep duration, assessed objectively (actigraphy) or subjectively (questionnaire), was not independently associated with BMD in postmenopausal women. INTRODUCTION: Both long and short self-reported sleep durations are associated with low bone mineral density (BMD) in men and women. The association between sleep duration measured by actigraphy and BMD in postmenopausal women is unknown. METHODS: The Study of Osteoporotic Fractures (SOF) ancillary sleep study was used to determine the association between sleep duration and BMD at the total hip and femoral neck in postmenopausal women ≥ 75 years old. Sleep duration was assessed by wrist actigraphy (average 4 nights) and questionnaire. BMD was compared between postmenopausal women with short (< 6 h/night) vs. NIH-recommended (7-8 h/night) sleep durations. Data were analyzed using a 2-sample t test (unadjusted) and multivariate regression model (adjusted). Simple linear regression was used to estimate the difference in BMD per additional hour of sleep when sleep duration was considered as a continuous, rather than dichotomized, variable. RESULTS: Total hip BMD was higher in women with actigraphically assessed shorter sleep duration in unadjusted models only. No clinically or statistically significant differences in total hip or femoral neck BMD were observed according to nocturnal sleep duration after adjusting for body mass index (BMI) in dichotomized (N = 874) or continuous (N = 1624) sleep duration models or when subjective sleep duration was used. When sleep duration included daytime naps, longer sleep duration was associated with lower total hip BMD (ß = - 0.005, p = 0.04). CONCLUSIONS: Nocturnal sleep duration, whether assessed objectively (actigraphy) or subjectively (questionnaire), was not independently associated with BMD in older postmenopausal women.

Functional consequences of AXL sequence variants in hypogonadotropic hypogonadism.

CONTEXT: Prior studies showed that Axl /Tyro3 null mice have delayed first estrus and abnormal cyclicity due to developmental defects in GnRH neuron migration and survival. OBJECTIVE: The objective of the study was to test whether the absence of Axl would alter reproductive function in mice and that mutations in AXL are present in patients with Kallmann syndrome (KS) or normosmic idiopathic hypogonadotropic hypogonadism (nIHH). DESIGN AND SETTING: The sexual maturation of Axl null mice was examined. The coding region of AXL was sequenced in 104 unrelated, carefully phenotyped KS or nIHH subjects. Frequency of mutations was compared with other causes of GnRH deficiency. Functional assays were performed on the detected mutations. RESULTS: Axl null mice demonstrated delay in first estrus and the interval between vaginal opening and first estrus. Three missense AXL mutations (p.L50F, p.S202C, and p.Q361P) and one intronic variant 6 bp upstream from the start of exon 5 (c.586-6 C>T) were identified in two KS and 2 two nIHH subjects. Comparison of the frequencies of AXL mutations with other putative causes of idiopathic hypogonadotropic hypogonadism confirmed they are rare variants. Testing of the c.586-6 C>T mutation revealed no abnormal splicing. Surface plasmon resonance analysis of the p.L50F, p.S202C, and p.Q361P mutations showed no altered Gas6 ligand binding. In contrast, GT1-7 GnRH neuronal cells expressing p.S202C or p.Q361P demonstrated defective ligand dependent receptor processing and importantly aberrant neuronal migration. In addition, the p.Q361P showed defective ligand independent chemotaxis. CONCLUSIONS: Functional consequences of AXL sequence variants in patients with idiopathic hypogonadotropic hypogonadism support the importance of AXL and the Tyro3, Axl, Mer (TAM) family in reproductive development.

Hypogonadism on admission to acute rehabilitation is correlated with lower functional status at admission and discharge.

PRIMARY OBJECTIVE: To investigate the association between hormone levels and functional status during acute TBI rehabilitation. RESEARCH DESIGN: Retrospective cohort study of 43 men with moderate-to-severe TBI admitted to an acute rehabilitation unit during a 1 year period. METHODS AND PROCEDURES: Labs were drawn on admission, including total and free testosterone (T), prolactin, adrenocorticotropin hormone (ACTH), cortisol, thyroid stimulating hormone (TSH), free thyroxine (fT4) and insulin-like growth factor (IGF-1). Functional Independence Measure (FIM) scores were obtained at admission and discharge. MAIN OUTCOME AND RESULTS: Associations between admission hormone levels and the main outcomes, admission and discharge FIM scores, were assessed using linear regression. Lower total and free T-levels at admission were associated with lower total FIM scores at admission (p < 0.038) and discharge (p < 0.046). Higher cortisol levels at admission were significantly associated with lower admission (p = 0.012) and discharge (p = 0.036) scores on the cognitive-FIM. Prolactin, TSH, fT4 and IGF-1 were not correlated with functional status. CONCLUSIONS: In men, lower total and free T-levels at admission to acute rehabilitation correlate with lower admission and discharge FIM scores. These data support the need for studies to investigate the impact of physiological testosterone therapy on outcomes during and post-rehabilitation.

Gonadotropin-releasing hormone and its receptor in normal and malignant cells.

Gonadotropin-releasing hormone (GnRH) is the hypothalamic factor that mediates reproductive competence. Intermittent GnRH secretion from the hypothalamus acts upon its receptor in the anterior pituitary to regulate the production and release of the gonadotropins, LH and FSH. LH and FSH then stimulate sex steroid hormone synthesis and gametogenesis in the gonads to ensure reproductive competence. The pituitary requires pulsatile stimulation by GnRH to synthesize and release the gonadotropins LH and FSH. Clinically, native GnRH is used in a pump delivery system to create an episodic delivery pattern to restore hormonal defects in patients with hypogonadotropic hypogonadism. Agonists of GnRH are delivered in a continuous mode to turn off reproductive function by inhibiting gonadotropin production, thus lowering sex steroid production, resulting in medical castration. They have been used in endocrine disorders such as precocious puberty, endometriosis and leiomyomata, but are also studied extensively in hormone-dependent malignancies. The detection of GnRH and its receptor in other tissues, including the breast, ovary, endometrium, placenta and prostate suggested that GnRH agonists and antagonists may also have direct actions at peripheral targets. This paper reviews the current data concerning differential control of GnRH and GnRH receptor expression and signaling in the hypothalamic-pituitary axis and extrapituitary tissues. Using these data as a backdrop, we then review the literature about the action of GnRH in cancer cells, the utility of GnRH analogs in various malignancies and then update the research in novel therapies targeted to the GnRH receptor in cancer cells to promote anti-proliferative effects and control of tumor burden.

Myocyte enhancer factor 2A and 2D undergo phosphorylation and caspase-mediated degradation during apoptosis of rat cerebellar granule neurons.

Myocyte enhancer factor 2 (MEF2) proteins are important regulators of gene expression during the development of skeletal, cardiac, and smooth muscle. MEF2 proteins are also present in brain and recently have been implicated in neuronal survival and differentiation. In this study we examined the cellular mechanisms regulating the activity of MEF2s during apoptosis of cultured cerebellar granule neurons, an established in vitro model for studying depolarization-dependent neuronal survival. All four MEF2 isoforms (A, B, C, and D) were detected by immunoblot analysis in cerebellar granule neurons. Endogenous MEF2A and MEF2D, but not MEF2B or MEF2C, were phosphorylated with the induction of apoptosis. The putative sites that were phosphorylated during apoptosis are functionally distinct from those previously reported to enhance MEF2 transcription. The increased phosphorylation of MEF2A and MEF2D was followed by decreased DNA binding, reduced transcriptional activity, and caspase-dependent cleavage to fragments containing N-terminal DNA binding domains and C-terminal transactivation domains. Expression of the highly homologous N terminus of MEF2A (1-131 amino acids) antagonized the transcriptional activity and prosurvival effects of a constitutively active mutant of MEF2D (MEF2D-VP16). We conclude that MEF2A and MEF2D are prosurvival factors with high transcriptional activity in postmitotic cerebellar granule neurons. When these neurons are induced to undergo apoptosis by lowering extracellular potassium, MEF2A and MEF2D are phosphorylated, followed by decreased DNA binding and cleavage by a caspase-sensitive pathway to N-terminal fragments lacking the transactivation domains. The degradation of MEF2D and MEF2A and the generation of MEF2 fragments that have the potential to act as dominant-inactive transcription factors lead to apoptotic cell death.

Myocyte enhancer factors-2B and -2C are required for adhesion related kinase repression of neuronal gonadotropin releasing hormone gene expression.

Synthesis of the hypothalamic peptide, gonadotropin releasing hormone (GnRH), is paramount for reproductive function. GnRH neurons originate in the olfactory region and migrate into the forebrain during development. We recently implicated adhesion related kinase (Ark) in GnRH neuron development based on its differential expression in two GnRH producing cell lines, GT1-7 and Gn10. The Ark membrane receptor encodes an extracellular domain resembling cell adhesion molecules and an intracellular tyrosine kinase. Ark is expressed in Gn10 cells derived from migrating GnRH neurons but not GT1-7 cells of the post-migratory phenotype. Here, we show that Ark and GnRH transcripts are colocalized in the cribriform plate at midgestation, suggesting that Ark is expressed in migrating GnRH neurons in vivo. Furthermore, we have identified the GnRH gene as a downstream target of Ark signaling. Ark inhibits GnRH gene expression in GnRH neuronal cells via the coordinated binding of myocyte enhancer factor-2B and -2C (MEF-2B and -2C) and a putative homeoprotein within the proximal rat GnRH promoter. Given that MEF-2 proteins are widely expressed in the brain, these studies provide further evidence for MEF-2 action during neuronal development. Moreover, our studies elucidate a potential role for Ark in regulating GnRH gene expression during GnRH neuronal migration.

Growth arrest-specific gene 6 (Gas6)/adhesion related kinase (Ark) signaling promotes gonadotropin-releasing hormone neuronal survival via extracellular signal-regulated kinase (ERK) and Akt.

We identified Ark, the mouse homolog of the receptor tyrosine kinase Axl (Ufo, Tyro7), in a screen for novel factors involved in GnRH neuronal migration by using differential-display PCR on cell lines derived at two windows during GnRH neuronal development. Ark is expressed in Gn10 GnRH cells, developed from a tumor in the olfactory area when GnRH neurons are migrating, but not in GT1-7 cells, derived from a tumor in the forebrain when GnRH neurons are postmigratory. Since Ark (Ax1) signaling protects from programmed cell death in fibroblasts, we hypothesized that it may play an antiapoptotic role in GnRH neurons. Gn10 (Ark positive) GnRH cells were more resistant to serum withdrawal-induced apoptosis than GT1-7 (Ark negative) cells, and this effect was augmented with the addition of Gas6, the Ark (Ax1) ligand. Gas6/Ark stimulated the extracellular signal-regulated kinase, ERK, and the serine-threonine kinase, Akt, a downstream component of the phosphoinositide 3-kinase (PI3-K) pathway. To determine whether ERK or Akt activation is required for the antiapoptotic effects of Gas6/Ark in GnRH neurons, cells were serum starved in the absence or presence of Gas6, with or without inhibitors of ERK and PI3-K signaling cascades. Gas6 rescued Gn10 cells from apoptosis, and this effect was blocked by coincubation of the cells with the mitogen-activated protein/ERK kinase (MEK) inhibitor, PD98059, or wortmannin (but not rapamycin). These data support an important role for Gas6/Ark signaling via the ERK and PI3-K (via Akt) pathways in the protection of GnRH neurons from programmed cell death across neuronal migration.

Erectile dysfunction: a multifaceted disorder.

The cause may be vascular, neurogenic, hormonal, drug-related, psychogenic, or a combination thereof. In older men in particular, underlying causes may include life-threatening disorders. Evaluation requires directed questioning, since men often fail to volunteer important related symptoms. Clinical findings guide laboratory testing and treatment is tailored to the etiology.

Identification of novel factors that regulate GnRH gene expression and neuronal migration.

We used differential display PCR on two GnRH producing cell lines to identify genes involved in GnRH gene expression and neuronal migration. RNA from Gn10 cells (derived from a tumor in the olfactory area when GnRH neurons are migrating and make low levels of GnRH) and from GT1-7 cells (derived from a tumor in forebrain when GnRH neurons are postmigratory and make high levels of GnRH) was reverse transcribed into cDNA. The cDNA was amplified using three anchored primers and eight random primers from each cell line and products from duplicate reactions electrophoresed in parallel in a denaturing acrylamide gel. Differentially expressed cDNAs were excised, reamplified and used as probes in Northern analysis of total RNA from each cell line to confirm differentially expressed RNA. The cDNAs were sequenced and compared to the Genbank database. Four of five clones isolated from GT1-7 GnRH neurons are novel, while four of five clones isolated from Gn10 cells have homology to known DNA sequences. One clone, Gn8-01 encodes adhesion related kinase (Ark), a molecule that has an N-terminal domain characteristic of cell adhesion molecules and whose kinase domain may play a role in protection from apoptosis. Together these data support the usefulness of the technique to identify novel genes that play a role in the control of GnRH expression and neuronal migration.

Repression of gonadotropin-releasing hormone promoter activity by the POU homeodomain transcription factor SCIP/Oct-6/Tst-1: a regulatory mechanism of phenotype expression?

POU domain transcription factors are required for neuropeptide expression in selected subsets of hypothalamic neuroendocrine neurons. We now report that expression of the gonadotropin-releasing hormone (GnRH) gene, which controls sexual development, is regulated by the POU protein SCIP/Oct-6/Tst-1. Reverse transcriptase PCR cloning and RNase protection assays demonstrated the presence of SCIP/Oct-6/Tst-1 mRNA in the GnRH-producing neuronal cell line GT1-7. The physiological relevance of this regulatory activity was suggested by the detection of SCIP/Oct-6/Tst-1 mRNA in a subset of GnRH neurons in the hypothalamus of prepubertal female rats. Coexpression of SCIP/Oct-6/Tst-1 in neuronal cells inhibited rat GnRH (rGnRH) promoter activity via three regions of the proximal rGnRH promoter containing SCIP/Oct-6/Tst-1 binding sites. DNase I footprinting, gel shift assays, and DNA and protein mutagenesis studies indicated that both direct DNA binding and protein-protein interactions are required for SCIP/Oct-6/Tst-1 modulation of GnRH gene expression. Activation of SCIP/Oct-6/Tst-1 expression in terminally differentiated GnRH neurons may be a factor determining the ratio of phenotypically "inactive" versus "active" GnRH neurons during postnatal life.

Structure of the distal human gonadotropin releasing hormone (hGnrh) gene promoter and functional analysis in Gt1-7 neuronal cells.

To assess potential species-specific expression of gonadotropin releasing hormone (GnRH), the distal human (h) GnRH promoter was cloned, characterized and tested in gene transfer studies. The nucleotide sequence of approximately 3.8 kb of 5'-flanking region was determined. Homology to the rat (r) GnRH sequence was observed in the proximal promoter region between -551 h (-424 r) and the transcriptional start site and within multiple distal promoter regions. In contrast, there was little similarity in the sequences between -1131/-551 h and -1031/-424 r. A deletion panel of 5'-flanking hGnRH promoter constructs was made and tested in transient transfection assays in GnRH-producing mouse GT1-7 neuronal cells. The largest hGnRH promoter construct (-3832/+5 h) exhibited high levels of reporter activity, similar to that observed with the largest rGnRH construct (-3026/+116 r). However, in contrast to the rat gene, deletion of distal promoter sequences of the hGnRH promoter to -1971, -1131 or -551 did not result in a decrease in luciferase reporter activity. Further truncation to -350 resulted in a 3-fold decrease in luciferase activity. There was no preferential use of the putative upstream hGnRH start site in neuronal cells. DNase I protection assays showed unique protection patterns with nuclear extracts from GT1-7 and Gn10 neuronal cells and the hGnRH and rGnRH promoter fragments. These data suggest the presence of different cis-acting elements and transacting factors that mediate species-specific neuronal GnRH expression.

Direct binding of progesterone receptor to nonconsensus DNA sequences represses rat GnRH.

The mechanisms by which steroid receptors repress gene expression are not well understood. In this report, we show that progesterone receptor (PR), in the presence of progesterone (P) directly represses rat gonadotropin releasing hormone (rGnRH) gene transcription. Deletion analysis studies using transient transfection assays in GT1-7 neuronal cells mapped the effects of P to sequences in the proximal rGnRH promoter between -171 and -73. This DNA sequence lacks any consensus steroid response element binding sites. Cotransfection of a mutant progesterone receptor that lacks a functional DNA binding region (hPRcys) abolished repression of the rGnRH promoter by P. Gel mobility shift assays confirmed that PR directly binds to the DNA fragments -171/-126, -126/-73, and -111/-73, which encompass the negative progesterone response element (nPRE) of the rGnRH promoter. Mutagenesis of the rGnRH nPRE -171/-126 DNA fragment resulted in a loss of PR binding. Thus, direct DNA binding of PR to nonconsensus elements in the proximal rGnRH promoter inhibits rGnRH gene expression.

GnRH gene expression in neuronal cell lines.

Little is known about the mechanisms that subserve GnRH synthesis. The recent availability of clonal populations of GnRH neurons enables us for the first time to examine the structural organization of the GnRH locus. Studies are underway to dissect the manner in which neural specific expression and developmental regulation of GnRH are programmed. These studies will complement and provide direction to future animal studies designed to better understand the control of GnRH gene expression.

Phorbol ester inhibition of rat gonadotropin-releasing hormone promoter activity: role of Fos and Jun in the repression of transcription.

Treatment of GT1-7 neuronal cells with the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate (TPA), inhibits GnRH gene transcription. The present studies investigated the role of AP-1 (Fos and Jun) in this repression. Treatment of cells with TPA increased c-fos mRNA 20-fold with only a 2-fold increase in c-jun mRNA levels. In transient transfection studies, a luciferase expression vector containing fragments of the 5'-flanking DNA of the rat GnRH (rGnRH) promoter was cotransfected with Fos and Jun expression vectors to mimic the effects of TPA. A dose-dependent decrease in reporter activity was noted with increasing amounts of Fos but not with Jun overexpression. Deletion analysis mapped the region that mediates repression by AP-1 to the area between -126 and -73 base pairs (bp) of the rGnRH 5'-flanking region: the same area that mediates TPA-induced repression and contains an imperfect TPA response element sequence at -99. Gel retardation assays, however, showed that a DNA fragment from -111 to -73 of the rGnRH promoter does not directly interact with Fos in GT1-7 extracts. Coexpression of Fos proteins with mutations in the DNA-binding region, the dimerization domain, or carboxy terminus partially blocked inhibition of rGnRH promoter activity. These data support a novel mechanism of AP-1 repression of GnRH transcription that is mediated by Fos interaction with other protein(s) that directly bind to the proximal rGnRH promoter.

Regulation of gonadotropin-releasing hormone (GnRH) gene expression in hypothalamic neuronal cells.

1. Gonadotropin-releasing hormone (GnRH) is the hypothalamic releasing factor that controls pituitary gonadotropin subunit gene expression and indirectly gametogenesis and steroidogenesis from the gonad, which results in reproductive competence. 2. GnRH is synthesized in only about 1000 neurons in the hypothalamus and released in an episodic fashion down the median eminence to regulate gonadotropin biosynthesis. 3. Although much is known about the secretory dynamics of GnRH release, little is known about the pretranslational control of GnRH biosynthesis due to lack of appropriate model systems. The recent availability of immortalized neuronal cell lines that produce GnRH allows investigators for the first time to begin to dissect the factors that directly regulate GnRH gene expression. 4. This article reviews the current state of knowledge concerning the mechanisms that direct tissue-specific and peptide hormone control of GnRH biosynthesis.

Evidence for transcriptional inhibition of GnRH gene expression by phorbol ester at a proximal promoter region.

We previously showed that activation of protein kinase C (PKC) with the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) in GT1-7 hypothalamic cells decreases GnRH mRNA levels in a dose and time dependent fashion. In the present studies, we examined the mechanism of this effect. Analysis of the half-life of GnRH mRNA levels after transcriptional arrest with actinomycin-D (5 micrograms/ml) estimated the half-life of GnRH mRNA to be 22 h. TPA treatment did not alter the GnRH mRNA half-life directly, suggesting that the effects of TPA occur predominantly at the level of gene transcription. Exposure of cells transiently transfected with various deletion constructs of the rat (r)GnRH promoter to TPA resulted in a decrease of 60% in luciferase reporter activity. This repression was maintained in constructs deleted to position -126 and was lost with further deletion to position -73. In conclusion, these experiments suggest that phorbol esters repress GnRH expression at the level of transcription through DNA sequences in the proximal rGnRH promoter.