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Ethanol regulation of serum glucocorticoid kinase 1 expression in DBA2/J mouse prefrontal cortex.

Costin BN, Dever SM, Miles MF - PLoS ONE (2013)

Bottom Line: Three separate results suggested that ethanol regulated Sgk1 via circulating glucocorticoids: acute ethanol increased glucocorticoid receptor binding to the Sgk1 promoter; adrenalectomy blocked ethanol induction of Sgk1 mRNA; and chronic ethanol exposure during locomotor sensitization down-regulated HPA axis activation and Sgk1 induction by acute ethanol.Chronic ethanol modifies both SGK1 protein and HPA-mediated induction of Sgk1 mRNA.These adaptive molecular responses of glucocorticoid-responsive gene expression and SGK1 in prefrontal cortex may contribute to mechanisms underlying behavioral responses to chronic ethanol exposure.

View Article: PubMed Central - PubMed

Affiliation: Virginia Commonwealth University Alcohol Research Center, Virginia Commonwealth University, Richmond, Virginia, USA.

ABSTRACT

Background: We previously identified a group of glucocorticoid-responsive genes, including Serum Glucocorticoid kinase 1 (Sgk1), regulated by acute ethanol in prefrontal cortex of DBA2/J mice. Acute ethanol activates the hypothalamic pituitary adrenal axis (HPA) causing release of glucocorticoids. Chronic ethanol dysregulates the HPA response in both humans and rodents, possibly contributing to important interactions between stress and alcoholism. Because Sgk1 regulates ion channels and learning and memory, we hypothesized that Sgk1 contributes to HPA-dependent acute and adaptive neuronal responses to ethanol. These studies characterized acute and chronic ethanol regulation of Sgk1 mRNA and protein and their relationship with ethanol actions on the HPA axis.

Results: Acute ethanol increased Sgk1 mRNA expression in a dose and time dependent manner. Three separate results suggested that ethanol regulated Sgk1 via circulating glucocorticoids: acute ethanol increased glucocorticoid receptor binding to the Sgk1 promoter; adrenalectomy blocked ethanol induction of Sgk1 mRNA; and chronic ethanol exposure during locomotor sensitization down-regulated HPA axis activation and Sgk1 induction by acute ethanol. SGK1 protein had complex temporal responses to acute ethanol with rapid and transient increases in Ser422 phosphorylation at 15 min. following ethanol administration. This activating phosphorylation had functional consequences, as suggested by increased phosphorylation of the known SGK1 target, N-myc downstream-regulated gene 1 (NDRG1). After repeated ethanol administration during locomotor sensitization, basal SGK1 protein phosphorylation increased despite blunting of Sgk1 mRNA induction by ethanol.

Conclusions: These results suggest that HPA axis and glucocorticoid receptor signaling mediate acute ethanol induction of Sgk1 transcription in mouse prefrontal cortex. However, acute ethanol also causes complex changes in SGK1 protein expression and activity. Chronic ethanol modifies both SGK1 protein and HPA-mediated induction of Sgk1 mRNA. These adaptive molecular responses of glucocorticoid-responsive gene expression and SGK1 in prefrontal cortex may contribute to mechanisms underlying behavioral responses to chronic ethanol exposure.

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Schematic of the SGK1 gene and N termini of Sgk1.1 versus Sgk1.(a) White boxes indicate 5’ and 3’ untranslated regions of Sgk1.1 and Sgk1, respectively; black boxes represent exons and the line represents introns. The SGK1 promoter contains a GRE. (b) Exons of the N termini of Sgk1.1. (c) Exons of the N termini of Sgk1.
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pone-0072979-g001: Schematic of the SGK1 gene and N termini of Sgk1.1 versus Sgk1.(a) White boxes indicate 5’ and 3’ untranslated regions of Sgk1.1 and Sgk1, respectively; black boxes represent exons and the line represents introns. The SGK1 promoter contains a GRE. (b) Exons of the N termini of Sgk1.1. (c) Exons of the N termini of Sgk1.

Mentions: Because our prior microarray studies and the studies of other labs would have used probes that recognize cDNA regions common to all Sgk1 isoforms (Figure 1a–c), we performed Q-rtPCR studies to determine which Sgk1 isoform was specifically regulated by ethanol, Sgk1.1 (Figure 1b) or Sgk1 (Figure 1c). Q-rtPCR was used to evaluate Sgk1 and Sgk1.1 levels 2, 4 and 8 hours following ethanol or saline administration. Prior to performing these studies on ethanol, we evaluated the effects of injection stress on Sgk1 expression as a crtical control. We compared Sgk1 levels in the PFC of D2 mice basally (0 hour time point) to D2 mice harvested 2, 4 and 8 hours following saline injections. Saline injections did not significantly alter Sgk1 levels at any time point compared to basal Sgk1 levels (Figure S1). Because we saw no significant effects of saline injections on Sgk1 expression, we did not include a 0 hour time point in the remainder of our studies. In evaluating Sgk1 levels, a two-way ANOVA showed an overall effect of treatment, ethanol versus saline (F1,17 = 16.44, p < 0.01), but no overall effect of time and no significant treatment x time interaction (Figure 2a). A one-way ANOVA showed a significant effect of treatment (F5,22 = 4.51, p < 0.01) and post-hoc analysis indicated that 4 hours following ethanol treatment Sgk1 levels were significantly increased compared to all saline treated animals and animals treated with ethanol 8 hours prior to harvest (Figure 2a). In addition, Sgk1 levels were significantly increased in animals harvested 2 hours following 4 g/kg ethanol administration compared to those harvested 8 hours following saline administration (Figure 2a). This indicates that Sgk1 levels were significantly increased 2 and 4 hours following 4 g/kg ethanol administration, returning to basal levels by 8 hours.


Ethanol regulation of serum glucocorticoid kinase 1 expression in DBA2/J mouse prefrontal cortex.

Costin BN, Dever SM, Miles MF - PLoS ONE (2013)

Schematic of the SGK1 gene and N termini of Sgk1.1 versus Sgk1.(a) White boxes indicate 5’ and 3’ untranslated regions of Sgk1.1 and Sgk1, respectively; black boxes represent exons and the line represents introns. The SGK1 promoter contains a GRE. (b) Exons of the N termini of Sgk1.1. (c) Exons of the N termini of Sgk1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750005&req=5

pone-0072979-g001: Schematic of the SGK1 gene and N termini of Sgk1.1 versus Sgk1.(a) White boxes indicate 5’ and 3’ untranslated regions of Sgk1.1 and Sgk1, respectively; black boxes represent exons and the line represents introns. The SGK1 promoter contains a GRE. (b) Exons of the N termini of Sgk1.1. (c) Exons of the N termini of Sgk1.
Mentions: Because our prior microarray studies and the studies of other labs would have used probes that recognize cDNA regions common to all Sgk1 isoforms (Figure 1a–c), we performed Q-rtPCR studies to determine which Sgk1 isoform was specifically regulated by ethanol, Sgk1.1 (Figure 1b) or Sgk1 (Figure 1c). Q-rtPCR was used to evaluate Sgk1 and Sgk1.1 levels 2, 4 and 8 hours following ethanol or saline administration. Prior to performing these studies on ethanol, we evaluated the effects of injection stress on Sgk1 expression as a crtical control. We compared Sgk1 levels in the PFC of D2 mice basally (0 hour time point) to D2 mice harvested 2, 4 and 8 hours following saline injections. Saline injections did not significantly alter Sgk1 levels at any time point compared to basal Sgk1 levels (Figure S1). Because we saw no significant effects of saline injections on Sgk1 expression, we did not include a 0 hour time point in the remainder of our studies. In evaluating Sgk1 levels, a two-way ANOVA showed an overall effect of treatment, ethanol versus saline (F1,17 = 16.44, p < 0.01), but no overall effect of time and no significant treatment x time interaction (Figure 2a). A one-way ANOVA showed a significant effect of treatment (F5,22 = 4.51, p < 0.01) and post-hoc analysis indicated that 4 hours following ethanol treatment Sgk1 levels were significantly increased compared to all saline treated animals and animals treated with ethanol 8 hours prior to harvest (Figure 2a). In addition, Sgk1 levels were significantly increased in animals harvested 2 hours following 4 g/kg ethanol administration compared to those harvested 8 hours following saline administration (Figure 2a). This indicates that Sgk1 levels were significantly increased 2 and 4 hours following 4 g/kg ethanol administration, returning to basal levels by 8 hours.

Bottom Line: Three separate results suggested that ethanol regulated Sgk1 via circulating glucocorticoids: acute ethanol increased glucocorticoid receptor binding to the Sgk1 promoter; adrenalectomy blocked ethanol induction of Sgk1 mRNA; and chronic ethanol exposure during locomotor sensitization down-regulated HPA axis activation and Sgk1 induction by acute ethanol.Chronic ethanol modifies both SGK1 protein and HPA-mediated induction of Sgk1 mRNA.These adaptive molecular responses of glucocorticoid-responsive gene expression and SGK1 in prefrontal cortex may contribute to mechanisms underlying behavioral responses to chronic ethanol exposure.

View Article: PubMed Central - PubMed

Affiliation: Virginia Commonwealth University Alcohol Research Center, Virginia Commonwealth University, Richmond, Virginia, USA.

ABSTRACT

Background: We previously identified a group of glucocorticoid-responsive genes, including Serum Glucocorticoid kinase 1 (Sgk1), regulated by acute ethanol in prefrontal cortex of DBA2/J mice. Acute ethanol activates the hypothalamic pituitary adrenal axis (HPA) causing release of glucocorticoids. Chronic ethanol dysregulates the HPA response in both humans and rodents, possibly contributing to important interactions between stress and alcoholism. Because Sgk1 regulates ion channels and learning and memory, we hypothesized that Sgk1 contributes to HPA-dependent acute and adaptive neuronal responses to ethanol. These studies characterized acute and chronic ethanol regulation of Sgk1 mRNA and protein and their relationship with ethanol actions on the HPA axis.

Results: Acute ethanol increased Sgk1 mRNA expression in a dose and time dependent manner. Three separate results suggested that ethanol regulated Sgk1 via circulating glucocorticoids: acute ethanol increased glucocorticoid receptor binding to the Sgk1 promoter; adrenalectomy blocked ethanol induction of Sgk1 mRNA; and chronic ethanol exposure during locomotor sensitization down-regulated HPA axis activation and Sgk1 induction by acute ethanol. SGK1 protein had complex temporal responses to acute ethanol with rapid and transient increases in Ser422 phosphorylation at 15 min. following ethanol administration. This activating phosphorylation had functional consequences, as suggested by increased phosphorylation of the known SGK1 target, N-myc downstream-regulated gene 1 (NDRG1). After repeated ethanol administration during locomotor sensitization, basal SGK1 protein phosphorylation increased despite blunting of Sgk1 mRNA induction by ethanol.

Conclusions: These results suggest that HPA axis and glucocorticoid receptor signaling mediate acute ethanol induction of Sgk1 transcription in mouse prefrontal cortex. However, acute ethanol also causes complex changes in SGK1 protein expression and activity. Chronic ethanol modifies both SGK1 protein and HPA-mediated induction of Sgk1 mRNA. These adaptive molecular responses of glucocorticoid-responsive gene expression and SGK1 in prefrontal cortex may contribute to mechanisms underlying behavioral responses to chronic ethanol exposure.

Show MeSH
Related in: MedlinePlus