CREB Protein Mediates Alcohol-Induced Circadian Disruption and Intestinal Permeability

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.

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.

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.

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.