Alcohol Dysregulates Corticotropin-Releasing-Hormone (CRH) Promoter Activity by Interfering with the Negative Glucocorticoid Response Element (nGRE)

Abstract

EtOH exposure in male rats increases corticotropin-releasing hormone (CRH) mRNA in the paraventricular nucleus of the hypothalamus (PVN), a brain region responsible for coordinating stress and anxiety responses. In this study we identified the molecular mechanisms involved in mediating these effects by examining the direct effects of EtOH on CRH promoter activity in a neuronal cell line derived from the PVN (IVB). In addition, we investigated the potential interactions of EtOH and glucocorticoids on the CRH promoter by concomitantly treating cells with EtOH and the glucocorticoid receptor (GR) antagonist RU486, and by sequentially deleting GR binding sites within glucocorticoid response element (GRE) on the CRH promoter. Cells were transiently transfected with a firefly luciferase reporter construct containing 2.5 kb of the rat wild type (WT) or mutated CRH promoter. Our results showed that EtOH treatment induced a biphasic response in CRH promoter activity. EtOH exposure for 0.5 h significantly decreased promoter activity compared to vehicle treated controls, whereas promoter activity was significantly increased after 2.0 h of EtOH exposure. Treatment with RU486, or deletion of the GR binding sites 1 and 2 within the GRE, abolished the EtOH-induced increase in the promoter activity, however did not affect EtOH-induced decrease in CRH promoter activity at an earlier time point. Overall, our data suggest that alcohol exposure directly regulates CRH promoter activity by interfering with the normal feedback mechanisms of glucocorticoids mediated by GR signaling at the GRE site of the CRH promoter.

Adolescent binge alcohol exposure increases risk assessment behaviors in male Wistar rats after exposure to an acute psychological stressor in adulthood

“…This is consistent with our previous observations in vivo (Przybycien-Szymanska et al, 2011b), and in vitro , in which we observed that alcohol inhibits GR binding to the CRF promoter in IVB cells, which are derived from rat PVN (Przybycien-Szymanska et al, 2011a). …”

“…Further behavioral studies are required to determine whether the observed differences in the amount of time spent in the intersection of the maze between rats exposed to alcohol as adolescents versus controls on repeated trials in the EPM are due to changes in learning abilities or the emotional state of the animal.To better examine the causal effects of adolescent binge alcohol consumption on the HPA axis and anxiety behavior, many different animal studies have been conducted with varying results. Our lab and others have established animal models demonstrating that adolescent binge alcohol exposure does indeed induce long-term effects on the regulation of the HPA axis (Allen et al, 2011; Przybycien-Szymanska et al, 2011a). However, observations that adolescent binge alcohol exposure induces changes in anxiety-like behaviors seem to vary based on the specific animal model used.…”

Ethanol-related alterations in gene expression patterns in the developing murine hippocampus

“…It is important to understand the molecular mechanism behind the harmful effects of ethanol gene expression profiling. Several investigations have already been reported which tried to unveil the mechanisms of ethanol-induced gene expression alteration in different aspects [17][18][19][20][21]. But the alcohol-regulated genes and their associated biological pathways have not yet been completely elucidated.…”

Chronic Unpredictable Mild Stress Induces Loss of GABA Inhibition in Corticotrophin-Releasing Hormone-Expressing Neurons through NKCC1 Upregulation

“…To efficiently identify CRH-expressing neurons in rat PVN, we constructed a viral plasmid containing rat Crh promoter [44] and eGFP and integrated this plasmid into an adeno-associated virus (AAV) vector construct. The full-length promoter fragment (−2125/+94) was subcloned into the AAV vector expression cassette by using short regulatory elements (woodchuck post regulatory element 2 [WPRE2] and short synthetic polyadenylation [SpA]).…”