Dysregulated Autophagy and Lysosome Function Are Linked to Exosome Production by Micro‐RNA 155 in Alcoholic Liver Disease

Abstract

Cellular homeostais, that is normally maintained through autophagy, is disrupted in alcoholic liver disease (ALD). Because autophagy and exosome biogenesis share common elements, we hypothesized that increased exosome production in ALD may be linked to disruption of autophagic function. We found impaired autophagy both in ALD and alcoholic hepatitis (AH) mouse models and human livers with ALD as indicated by increased hepatic p62 and LC3‐II levels. Alcohol reduced autophagy flux in vivo in chloroquine‐treated mice as well as in vitro in hepatocytes and macrophages treated with bafilomycin A. Our results revealed that alcohol targets multiple steps in the autophagy pathway. Alcohol‐related decrease in mechanistic target of rapamycin (mTOR) and Ras homolog enriched in brain (Rheb), that initiate autophagy, correlated with increased Beclin1 and autophagy‐related protein 7 (Atg7), proteins involved in phagophore‐autophagosome formation, in ALD. We found that alcohol disrupted autophagy function at the lysosomal level through decreased lysosomal‐associated membrane protein 1 (LAMP1) and lysosomal‐associated membrane protein 2 (LAMP2) in livers with ALD. We identified that micro‐RNA 155 (miR‐155), that is increased by alcohol, targets mTOR, Rheb, LAMP1, and LAMP2 in the authophagy pathway. Consistent with this, miR‐155‐deficient mice were protected from alcohol‐induced disruption of autophagy and showed attenuated exosome production. Mechanistically, down‐regulation of LAMP1 or LAMP2 increased exosome release in hepatocytes and macrophages in the presence and absence of alcohol. These results suggested that the alcohol‐induced increase in exosome production was linked to disruption of autophagy and impaired autophagosome and lysosome function. Conclusion: Alcohol affects multiple genes in the autophagy pathway and impairs autophagic flux at the lysosome level in ALD. Inhibition of LAMP1 and LAMP2 promotes exosome release in ALD. We identified miR‐155 as a mediator of alcohol‐related regulation of autophagy and exosome production in hepatocytes and macrophages.

Autophagy, Mitophagy and MicroRNA Expression in Chronic Hepatitis C and Autoimmune Hepatitis

“…In agreement with this, miR-155 knock-out mice were observed to be protected from early and advanced fibrosis in a CCl 4 treatment model [48]. Regarding another regulatory aspect, miR-155 was not only found to initiate autophagy in ALD via downregulating mTOR, but also to impair the fusion of autophagosome with lysosome, leading to increased LC3 and p62 levels [21]. Our investigation is partly in agreement with this since high miR-155 expression (which was highest among the miRNAs) was observed in both CHC and AIH samples, but the levels of LC3 and p62 pointed to an impaired autophagy in case of AIH but not in CHC.…”

“…Thus, increased LC3 and p62 in AIH suggest that autophagosome formation has occurred without an increase in lysosomal degradation, as has been demonstrated in case of p62 in a recent study [33]. Like further supported by Babuta and co-workers, increased levels of p62 and LC3 were found as the result of chronic alcohol intake in ALD, leading to disruption of autophagy function at lysosome level (impaired autophagic flux) [21]. Decreased level of p62 was observed in CHC contrary to AIH, suggestive of an autophagic function occurring at a much higher rate.…”

“…Upon examining LC3 and p62 as indirect indicators of autophagy function, the present study revealed increased levels of these proteins in AIH as compared with CHC. The level of LC3 per se indicates that autophagy is initiated, but may not reflect upon whether autophagy has proceeded or become impaired [21,31]. A proceeding autophagy implies a low level of p62 since this protein becomes degraded together with the cargo in the autophago-lysosome [32].…”

“…Analysis of autophagy-associated miRNA expression may provide further knowledge about autophagic activity. Several miRNAs are known to downregulate autophagic activity (miR-101, -155 and 204) [21,22] or their expression is known to be regulated by autophagy (miR-224) [25]. Autophagyregulating miRNAs have been studied in HCV infection [40] but, to the best of our knowledge, no data is available on how these miRNAs are expressed in AIH.…”

“…Several microRNAs (miRNAs) have been implicated in the regulation of autophagy by interfering with gene expression at post-transcriptional level. For example, miR-155 induces the formation of autophagophores via downregulating mTOR signaling but it impairs lysosomal degradation, as shown in alcoholic liver disease (ALD) [21]. miR-101 is thought to be a key regulator of the autophagy process and is a potent inhibitor of rapamycin-induced autophagy [22,23].…”