The autophagic protein p62 is a target of reactive aldehydes in human and murine cholestatic liver disease

Abstract

Inflammatory cholestatic liver diseases, including Primary Sclerosing Cholangitis (PSC), are characterized by periportal inflammation with progression to cirrhosis. The objective of this study was to examine interactions between oxidative stress and autophagy in cholestasis. Using hepatic tissue from male acute cholestatic (bile duct ligated) as well as chronic cholestatic (Mdr2KO) mice, localization of oxidative stress, the antioxidant response and induction of autophagy were analyzed and compared to human PSC liver. Concurrently, the ability of reactive aldehydes to post-translationally modify the autophagosome marker p62 was assessed in PSC liver tissue and in cell culture. Expression of autophagy markers was upregulated in human and mouse cholestatic liver. Whereas mRNA expression of Atg12, Lamp1, Sqstm1 and Map1lc3 was increased in acute cholestasis in mice, it was either suppressed or not significantly changed in chronic cholestasis. In human and murine cholestasis, periportal hepatocytes showed increased IHC staining of ubiquitin, 4-HNE, p62, and selected antioxidant proteins. Increased p62 staining colocalized with accumulation of 4-HNE-modified proteins in periportal parenchymal cells as well as with periportal macrophages in both human and mouse liver. Mechanistically, p62 was identified as a direct target of lipid aldehyde adduction in PSC hepatic tissue and in vitro cell culture. In vitro LS-MS/MS analysis of 4-HNE treated recombinant p62 identified carbonylation of His123, Cys128, His174, His181, Lys238, Cys290, His340, Lys341 and His385. These data indicate that dysregulation of autophagy and oxidative stress/protein damage are present in the same periportal hepatocyte compartment of both human and murine cholestasis. Thus, our results suggest that both increased expression as well as ineffective autophagic degradation of oxidatively-modified proteins contributes to injury in periportal parenchymal cells and that direct modification of p62 by reactive aldehydes may contribute to autophagic dysfunction.

Disrupting Poly(ADP-ribosyl)ating Pathway Creates Premalignant Conditions in Mammalian Liver

“…It is noteworthy that this type of regeneration, involving ductal response and differentiation of cells from progenitors or cholangioblasts to hepatocytes, is not commonly observed in all other scenarios of hepatocyte loss, such as partial hepatectomy, CCl4 or DDC diet-induced injury, but has been demonstrated in cases of choline-deficient-ethioninesupplemented diet, specific genetic perturbations, in cholestatic injury, and senescent hepatocytes-induced injury [60][61][62][63][64]. Ductal reactions play an increasingly vital role in understanding hepatic stem and progenitor cells during liver regeneration, the processes driving hepatic fibrosis, and the development of hepatobiliary carcinogenesis [65].…”

Expression of circadian regulatory genes is dysregulated by increased cytokine production in mice subjected to concomitant intestinal injury and parenteral nutrition

“…Western blots were performed liver homogenate using 30μg of protein per lane as previously described [ 37 , 38 ]. The antibodies used in this study are listed in Table 2 .…”