Diurnal profiling of neuroendocrine genes in murine heart, and shift in proopiomelanocortin gene expression with pressure-overload cardiac hypertrophy.

Neuroendocrine peptides express biologic activity relevant to the cardiovascular system, including regulating heart rate and blood pressure, though little is known about the mechanisms involved. Here, we investigated neuroendocrine gene expression underlying diurnal physiology of the heart. We first used microarray and RT-PCR analysis and demonstrate the simultaneous expression of neuroendocrine genes in normal murine heart, including POMC, GnRH, neuropeptide Y, leptin receptor, GH-releasing hormone, cocaine- and amphetamine-regulated transcript, proglucagon, and galanin. We examined diurnal gene expression profiles, with cosinar bioinformatics to evaluate statistically significant rhythms. The POMC gene exhibits a day/night, circadian or diurnal, pattern of expression in heart, and we postulated that this may be important to cardiac growth and renewal. POMC diurnal gene rhythmicity is altered in pressure-overload cardiac hypertrophy, when compared with control heart, and levels increased at the dark-to-light transition times. These findings are also consistent with the proposal that neuropeptides mediate adverse remodeling processes, such as occur in pathologic hypertrophy. To investigate cellular responses, we screened three cell lines representing fibroblasts, cardiac myocytes, and vascular smooth muscle cells (NIH3T3, heart line 1, and mouse vascular smooth muscle cell line 1 (Movas-1) respectively). POMC mRNA expression is the most notable in Movas-1 cells and, furthermore, exhibits rhythmicity with culture synchronization. Taken together, these results highlight the diverse neuroendocrine mRNA expression profiles in cardiovasculature, and provide a novel model vascular culture system to research the role these neuropeptides play in organ health, integrity, and disease.

IGF-I signaling prevents dehydroepiandrosterone (DHEA)-induced apoptosis in hypothalamic neurons.

Dehydroepiandrosterone (DHEA) is synthesized in the brain, but whether DHEA is involved in modulating neuronal cell survival is not yet fully understood. Herein we show that when deprived of trophic support, GT1-7 hypothalamic neurons undergo apoptosis following exposure to DHEA, as demonstrated both by morphological and biochemical criteria. This proapoptotic effect appeared to be specific to DHEA itself, and not through conversion of DHEA to other steroids such as androgen or estrogen. Importantly, we determined that IGF-I protects GT1-7 neurons from DHEA-induced cell death. DHEA-induced apoptosis was associated with increased activation of caspase 3 and decreased PARP, which were both attenuated with addition of IGF-I. Addition of DHEA prevented phosphorylation of both Akt and glycogen synthase kinase-3 beta (GSK-3beta), downstream effector molecules of the phosphatidylinositol 3-kinase (PI3K) pathway. Further IGF-I was able to sustain Akt activity and thus preventing GSK-3beta activation in the presence of DHEA. On the other hand, the MAP kinases, ERK, p38, and JNK, were not affected by DHEA. These findings suggest that in GT1-7 hypothalamic neurons, DHEA acts detrimentally to induce cell death and IGF-I is able to rescue the neurons by preserving the activity of Akt, and therefore maintaining the proapoptotic kinase GSK-3beta, in a phosphorylated catalytically inactive state.

Evidence that dehydroepiandrosterone, DHEA, directly inhibits GnRH gene expression in GT1-7 hypothalamic neurons.

Dehydroepiandrosterone (DHEA) has been reported to have diverse effects on overall physiology, although its mechanism of action and specific receptor are not yet known. We have used the immortalized, clonal GT1-7 hypothalamic neurons to study DHEA effects on gonadotropin-releasing hormone (GnRH) gene expression. DHEA (10(-4) M) downregulates GnRH transcription by 39, 70 and 83% at 24, 36, and 48 h, respectively, while DHEA-sulphate had no effect. Hydroxyflutamide a specific androgen receptor (AR) antagonist, and cyproterone acetate or trilostane, both inhibitors of 3 beta-hydroxysteroid dehydrogenase/delta 4,5 isomerase, the rate-limiting enzyme for the conversion of DHEA to sex steroids, did not affect the ability of DHEA to downregulate GnRH gene expression. We found that GT1-7 cells did not express aromatase, thereby precluding conversion to estrogen. Analysis of [(14)C] DHEA metabolism by thin layer chromatography indicates that the main metabolites produced are 7 alpha- and 7 beta-hydroxy DHEA, and 7-oxo DHEA, although these steroids were not able to repress GnRH gene expression alone. Cell viability studies indicated that the transcriptional repression observed is not due to GT1-7 cell death. Interestingly, SV40 T-antigen mRNA levels, under the control of 2.3 kb of the rat GnRH gene 5' regulatory region, are also repressed by DHEA. Our studies indicate that DHEA has direct effects on GnRH transcription that appear to be unique from those observed after conversion to other steroidogenic compounds.