CREB Protein Mediates Alcohol‐Induced Circadian Disruption and Intestinal Permeability

• Published in: Alcoholism, clinical and experimental research clinical and experimental research, 2017-12, Vol.41 (12), p.2007-2014
• Main Authors: Davis, Booker T, Voigt, Robin M., Shaikh, Maliha, Forsyth, Christopher B., Keshavarzian, Ali
• Format: Article
• Language: English
• Subjects: Alcohol; Alcohol use; Alcoholic beverages; Antioxidants; Calories; cAMP Response Element‐Binding; Circadian; Circadian rhythm; Circadian rhythms; Colon; Cyclic AMP response element-binding protein; Disruption; Endotoxemia; Endotoxins; Epithelial cells; Intestine; Liver; Liver diseases; Membrane permeability; Metabolism; Oxidative Stress; Period 2 protein; Permeability; Polymerase chain reaction; Protein kinase A; Proteins; Rodents; siRNA; Transcription
• ISSN: 0145-6008; 1530-0277
• DOI: 10.1111/acer.13513

Background Alcoholic liver disease (ALD) is commonly associated with intestinal permeability. An unanswered question is why only a subset of heavy alcohol drinkers develop endotoxemia. Recent studies suggest that circadian disruption is the susceptibility factor for alcohol‐induced gut leakiness to endotoxins. The circadian protein PER2 is increased after exposure to alcohol and siRNA knockdown of PER2 in vitro blocks alcohol‐induced intestinal barrier dysfunction. We have shown that blocking CYP2E1 (i.e., important for alcohol metabolism) with siRNA inhibits the alcohol‐induced increase in PER2 and suggesting that oxidative stress may mediate alcohol‐induced increase in PER2 in intestinal epithelial cells. The aim of this study was to elucidate whether a mechanism incited by alcohol‐derived oxidative stress mediates the transcriptional induction of PER2 and subsequent intestinal hyperpermeability. Methods Caco‐2 cells were exposed to 0.2% alcohol with or without pretreatment with modulators of oxidative stress or PKA activity. Permeability of the Caco‐2 monolayer was assessed by transepithelial electrical resistance. Protein expression was measured by Western blot and mRNA with real‐time polymerase chain reaction. Wild‐type C57BL/6J mice were fed with alcohol diet (29% of total calories, 4.5% v/v) for 8 weeks. Western blot was used to analyze PER2 expression in mouse proximal colon tissue. Results Alcohol increased oxidative stress, caused Caco‐2 cell monolayer dysfunction, and increased levels of the circadian clock proteins PER2 and CLOCK. These effects were mitigated by pretreatment of Caco‐2 cells with an antioxidant scavenger. Alcohol‐derived oxidative stress activated cAMP response element‐binding (CREB) via the PKA pathway and increased PER2 mRNA and protein. Inhibiting CREB prevented the increase in PER2 and Caco‐2 cell monolayer hyperpermeability. Conclusions Taken together, these data suggest that strategies to reduce alcohol‐induced oxidative stress may alleviate alcohol‐mediated circadian disruption and intestinal leakiness, critical drivers of ALD. CREB Mediates Alcohol‐Induced Circadian Disruption and Intestinal Permeability (A) CLOCK and BMAL1 initiate the transcription of circadian clock genes like PER2. PER2 forms a heterodimer with CRY1 inhibiting clock gene expression. This process takes approximately 24 h. (B) We hypothesize that Cyp2e1‐mediated alcohol metabolism activates cAMP response element‐binding (CREB) protein to induce circadi