Molecular Mechanism Of Phosphorylated Hsp27 And STAT3 Binding To Promote Autophagy Inhibition Of Lipid Degeneration In

As a major cause of chronic liver disease, nonalcoholic fatty liver disease (NAFLD) refers to redundant lipid depositing in the liver without excessive intake of alcohol and has become the major liver disease worldwide. However, the drug therapies are inefficient at present. Several studies identified autophagy, a ubiquitous lysosomal degradation system in eukaryotic cells, attenuated hepatic steatosis via stimulating lipid metabolism. Heat shock protein 27 (Hsp27), a cytoprotective cellular chaperone, can be phosphorylated in response to different stimuli, resulting in oligomerization and changes in biological functions. According to recent studies, Hsp27 is involved in autophagy. However, the underlying molecular mechanism for Hsp27, especially phosphorylated Hsp27, in autophagy activation and lipid droplets clearance remains to be clarified.In this work, we investigated the role of phosphorylated Hsp27 in autophagy and inhibiting hepatic steatosis. We found that phosphorylated Hsp27 stimulated autophagy and lipid droplet clearance and interacted with STAT3. Firstly, C57/BL6 mice were fed with high fat diet (HFD) to establish mice model with hepatic steatosis, and we found that both phosphorylation of hepatic Hsp25 (homologous protein of Hsp27 in mice) and autophagy were upregulated by HFD. In human hepatic cells L02, both Hsp27 phosphorylation and autophagy were induced upon palmitate administration. Intriguingly, knockdown of Hsp27 by shRNA, or suppressing Hsp27 phosphorylation by specific inhibitor KRIBB3, or overexpressing non-phosphorylatable mutant Hsp27-3A inhibited autophagy, and in turn, exacerbated the deposition of lipid droplets. In contrast, overexpression of Hsp27, either wild type Hsp27-WT or phosphorylatable mimetic mutant Hsp27-3D, promoted autophagy, subsequently, attenuated hepatocytes steatosis. In addition, Hsp27 induced lipid clearance can be abolished by autophagy inhibitor chloroquine. Moreover, suppression of hepatic Hsp25 phosphorylation by KRIBB3 inhibited autophagy, subsequently, exacerbated HFD induced hepatic steatosis.Next, we investigated the mechanism by which phosphorylated Hsp27 stimulated autophagy. We also found that phosphorylated Hsp27 interacted with STAT3 by co-immunoprecipitation. In addition, palmitate enhanced the interaction of Hsp27 and STAT3, meanwhile facilitated the STAT3/PKR complex disruption. Knockdown of Hsp27 by shRNA inhibited the disruption of STAT3/PKR complex. Suppression of Hsp27 phosphorylation by KRIBB3 not only reduced the formation of Hsp27/STAT3 complex, but also inhibited the disruption of STAT3/PKR complex. Over-expressing Hsp27, either wild type Hsp27-WT or phosphorylatable mimetic mutant Hsp27-3D, separated PKR from STAT3. However, over-expression of non-phosphorylatable mutant Hsp27-3A blocked the disassociation of STAT3/PKR complex. Moreover we found that Hsp27-3D had stronger ability in interacting with STAT3 than Hsp27-WT.Taken together, we demonstrate that phosphorylated Hsp27 interacts with STAT3, and then results in the disruption of STAT3/PKR complex, followed by PKR dependent eIF2a phosphorylation, which facilitates autophagy induction as a lipid scavenger. This study unravels an unsuspected mechanism of Hsp27 phosphorylation in autophagy activation and inhibiting hepatic steatosis. Our study provides a novel mechanism by which the phosphorylated Hsp27 promotes hepatic lipid clearance and suggests a new insight for therapy for steatotic diseases such as non-alcoholic fatty liver disease (NAFLD).