Chronic and excessive ethanol consumption induces hepatocellular hyperproliferation, which is critical in all stages of hepatocarcinogenesis. Retinoic acid, the most active form of vitamin A, is known to play an important role in the regulation of cell proliferation. However, high dose of ethanol tr...
Chronic and excessive ethanol consumption induces hepatocellular hyperproliferation, which is critical in all stages of hepatocarcinogenesis. Retinoic acid, the most active form of vitamin A, is known to play an important role in the regulation of cell proliferation. However, high dose of ethanol treatment to rats significantly reduces hepatic retinoic acid concentrations. The present research examined whether the restoration of hepatic retinoic acid to a normal range by dietary supplementation of all-trans-retinoic acid in rats modulates ethanol-induced hepatocellular proliferation. This research further investigated the molecular mechanism of action of ethanol, as well as all-trans-retinoic acid on hepatic c-Jun N-terminal kinase (JNK)-dependent signaling pathways, which mediate the major regulatory role in cell proliferation. Male Sprague-Dawley rats were randomly divided into four groups: control, ethanol-fed, ethanol + 50 ㎍ all-trans-retinoic acid/ kg body weight, and ethanol + 100 ㎍ all-trans-retinoic acid/ kg body weight. Ethanol was given to rats at 6.2% (v/v) in a liquid diet to provide 36% of total caloric intake. Control animals received the same liquid diet with isocaloric maltodextrin in place of ethanol. One animal from each group were matched at the beginning of the study and pair-fed thereafter for either 1 or 6 months. Results showed that ethanol treatment significantly decreased hepatic retinoic acid concentration but significantly increased the mean number of proliferating cell nuclear antigen (PCNA)-positive hepatocytes by 17 fold (p<0.05), as compared to controls. Livers from ethanol-fed rats had greater levels of c-Jun (6.5-fold increase) and cyclin D1 (3.5-fold increase) than those from control rats. Further, activator protein 1 (AP-1) DNA binding activity was significantly higher in ethanol-fed rats than that in controls. Dietary all-trans-retinoic acid supplementation at both doses restored hepatic retinoic acid to normal levels in ethanol-fed rats. Moreover, all-trans-retinoic acid significantly suppressed the ethanol-induced PCNA-labeling in hepatocytes (∼80% inhibition as compared to controls, p<0.05). This inhibition of all-trans-retinoic acid was associated with the suppression of hepatic c-Jun and cyclin D1 protein levels and that of AP-1 DNA binding activities in ethanol-fed rats, which demonstrates that the restoration of hepatic retinoic acid suppresses ethanol-induced hepatocyte hyperproliferation via inhibiting c-Jun overexpression. I further examined effects of ethanol and all-transretinoic acid on hepatic JNK-dependent signaling cascades. Results showed that livers from ethanol-fed rats had greater levels of phosphorylated-MKK4, phosphorylated-JNK, and c-jun mRNA, as compared to controls. In contrast, all-trans-retinoic acid supplementation in ethanol-fed rats considerably attenuated the ethanol-induced phosphorylations of MKK4 and JNK, and overexpression of c-jun mRNA. Further, all-trans-retinoic acid greatly induced hepatic MKP-1 protein level in a dose-dependent manner. Taken together, this research demonstrates that all-trans-retinoic acid can suppress the stimulation of hepatic JNK-dependent signaling pathway, leading to inhibition of c-Jun overexpression and hepatocyte proliferation in ethanol-fed rats, and suggests that all-trans-retinoic acid plays a role in preventing or reversing certain types of ethanol-induced liver injury.
Chronic and excessive ethanol consumption induces hepatocellular hyperproliferation, which is critical in all stages of hepatocarcinogenesis. Retinoic acid, the most active form of vitamin A, is known to play an important role in the regulation of cell proliferation. However, high dose of ethanol treatment to rats significantly reduces hepatic retinoic acid concentrations. The present research examined whether the restoration of hepatic retinoic acid to a normal range by dietary supplementation of all-trans-retinoic acid in rats modulates ethanol-induced hepatocellular proliferation. This research further investigated the molecular mechanism of action of ethanol, as well as all-trans-retinoic acid on hepatic c-Jun N-terminal kinase (JNK)-dependent signaling pathways, which mediate the major regulatory role in cell proliferation. Male Sprague-Dawley rats were randomly divided into four groups: control, ethanol-fed, ethanol + 50 ㎍ all-trans-retinoic acid/ kg body weight, and ethanol + 100 ㎍ all-trans-retinoic acid/ kg body weight. Ethanol was given to rats at 6.2% (v/v) in a liquid diet to provide 36% of total caloric intake. Control animals received the same liquid diet with isocaloric maltodextrin in place of ethanol. One animal from each group were matched at the beginning of the study and pair-fed thereafter for either 1 or 6 months. Results showed that ethanol treatment significantly decreased hepatic retinoic acid concentration but significantly increased the mean number of proliferating cell nuclear antigen (PCNA)-positive hepatocytes by 17 fold (p<0.05), as compared to controls. Livers from ethanol-fed rats had greater levels of c-Jun (6.5-fold increase) and cyclin D1 (3.5-fold increase) than those from control rats. Further, activator protein 1 (AP-1) DNA binding activity was significantly higher in ethanol-fed rats than that in controls. Dietary all-trans-retinoic acid supplementation at both doses restored hepatic retinoic acid to normal levels in ethanol-fed rats. Moreover, all-trans-retinoic acid significantly suppressed the ethanol-induced PCNA-labeling in hepatocytes (∼80% inhibition as compared to controls, p<0.05). This inhibition of all-trans-retinoic acid was associated with the suppression of hepatic c-Jun and cyclin D1 protein levels and that of AP-1 DNA binding activities in ethanol-fed rats, which demonstrates that the restoration of hepatic retinoic acid suppresses ethanol-induced hepatocyte hyperproliferation via inhibiting c-Jun overexpression. I further examined effects of ethanol and all-transretinoic acid on hepatic JNK-dependent signaling cascades. Results showed that livers from ethanol-fed rats had greater levels of phosphorylated-MKK4, phosphorylated-JNK, and c-jun mRNA, as compared to controls. In contrast, all-trans-retinoic acid supplementation in ethanol-fed rats considerably attenuated the ethanol-induced phosphorylations of MKK4 and JNK, and overexpression of c-jun mRNA. Further, all-trans-retinoic acid greatly induced hepatic MKP-1 protein level in a dose-dependent manner. Taken together, this research demonstrates that all-trans-retinoic acid can suppress the stimulation of hepatic JNK-dependent signaling pathway, leading to inhibition of c-Jun overexpression and hepatocyte proliferation in ethanol-fed rats, and suggests that all-trans-retinoic acid plays a role in preventing or reversing certain types of ethanol-induced liver injury.
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