Simple epithelial keratins (SEKs) are found primarily in single-layered simple epithelia and include keratin 7 (K7), K8, K18-K20, and K23. Genetically engineered mice that lack SEKs or overexpress ...mutant SEKs have helped illuminate several keratin functions and served as important disease models. Insight into the contribution of SEKs to human disease has indicated that K8 and K18 are the major constituents of Mallory-Denk bodies, hepatic inclusions associated with several liver diseases, and are essential for inclusion formation. Furthermore, mutations in the genes encoding K8, K18, and K19 predispose individuals to a variety of liver diseases. Hence, as we discuss here, the SEK cytoskeleton is involved in the orchestration of several important cellular functions and contributes to the pathogenesis of human liver disease.
The pathogenesis of hepatitis C virus (HCV)-associated insulin resistance remains unclear. Therefore, we investigated mechanisms for HCV-associated insulin resistance. Homeostasis model assessment ...for insulin resistance was increased in patients with HCV infection. An increase in fasting insulin levels was associated with the presence of serum HCV core, the severity of hepatic fibrosis and a decrease in expression of insulin receptor substrate (IRS) 1 and IRS2, central molecules of the insulin-signaling cascade, in patients with HCV infection. Down-regulation of IRS1 and IRS2 was also seen in HCV core-transgenic mice livers and HCV core-transfected human hepatoma cells. Carbobenzoxy-
l-leucyl-
l-leucyl-
l-leucinal, a potent proteosomal proteolysis inhibitor, blocked down-regulation of IRS1 and IRS2 in HCV core-transfected hepatoma cells. In human hepatoma cells, HCV core up-regulated suppressor of cytokine signaling (SOCS) 3 and caused ubiquitination of IRS1 and IRS2. HCV core-induced down-regulation of IRS1 and IRS2 was not seen in SOCS3
−/− mouse embryonic fibroblast cells. Furthermore, HCV core suppressed insulin-induced phosphorylation of p85 subunit of phosphatidylinositol 3-kinase and Akt, activation of 6-phosphofructo-2-kinase, and glucose uptake. In conclusion, HCV infection changes a subset of hepatic molecules regulating glucose metabolism. A possible mechanism is that HCV core-induced SOCS3 promotes proteosomal degradation of IRS1 and IRS2 through ubiquitination.
Hepatitis C virus (HCV) infection is linked to greater insulin resistance. Although HCV itself is a candidate for the development of insulin resistance, the effects of antiviral treatment on impaired ...glucose metabolism remain unclear. The aim of this study is to examine the effects of clearance of HCV on insulin resistance, beta-cell function, and hepatic expression of insulin receptor substrate (IRS)1/2, central molecules for insulin signaling.
We analyzed 89 biopsy-proven patients with chronic HCV infection. Patients received interferon-alpha or interferon-alpha plus ribavirin for 6 months and were classified into three groups at 6 months after the conclusion of antiviral therapy according to their response to antiviral therapy: sustained responders (N = 29), relapsers (N = 12), and nonresponders (N = 48). Insulin resistance and beta-cell function were assessed by the homeostasis model assessment method (HOMA-IR and HOMA-%B, respectively). Hepatic expression of IRS1/2 was evaluated by immunoblotting and immunostaining in 14 sustained responders.
In nonresponders and relapsers, there were no significant changes in HOMA-IR and HOMA-%B values after antiviral therapy. On the other hand, in sustained responders, HOMA-IR values significantly decreased to 1.7 +/- 0.8 from 3.1 +/- 1.1 (P < 0.05) after antiviral therapy. Similarly, HOMA-%B values significantly decreased to 90.6 +/- 10.0 from 113.7 +/- 15.3 (P < 0.05). Immunoblotting showed a threefold increase in IRS1/2 expression after clearance of HCV. Immunostaining revealed that greater IRS1/2 expression was seen in hepatocytes.
We showed that clearance of HCV improves insulin resistance, beta-cell function, and hepatic IRS1/2 expression.
Mallory‐Denk bodies (MDBs) are hepatocyte inclusions that are associated with poor liver disease prognosis. The intermediate filament protein keratin 8 (K8) and its cross‐linking by ...transglutaminase‐2 (TG2) are essential for MDB formation. K8 hyperphosphorylation occurs in association with liver injury and MDB formation, but the link between keratin phosphorylation and MDB formation is unknown. We used a mutational approach to identify K8 Q70 as a residue that is important for K8 cross‐linking to itself and other liver proteins. K8 cross‐linking is markedly enhanced on treating cells with a phosphatase inhibitor and decreases dramatically on K8 S74A or Q70N mutation in the presence of phosphatase inhibition. K8 Q70 cross‐linking, in the context of synthetic peptides or intact proteins transfected into cells, is promoted by phosphorylation at K8 S74 or by an S74D substitution and is inhibited by S74A mutation. Transgenic mice that express K8 S74A or a K8 G62C liver disease variant that inhibits K8 S74 phosphorylation have a markedly reduced ability to form MDBs. Our findings support a model in which the stress‐triggered phosphorylation of K8 S74 induces K8 cross‐linking by TG2, leading to MDB formation. These findings may extend to neuropathies and myopathies that are characterized by intermediate filament‐containing inclusions.—Kwan, R., Hanada, S., Harada, M., Strnad, P., Li, D. H., Omary, M.B. Keratin 8 phosphorylation regulates its transamidation and hepatocyte Mallory‐Denk body formation. FASEB J. 26, 2318‐2326 (2012). www.fasebj.org
Background & Aims Mallory–Denk bodies (MDBs) are keratin (K)-rich cytoplasmic hepatocyte inclusions commonly associated with alcoholic steatohepatitis. Given the significant gender differences in ...predisposition to human alcohol-related liver injury, and the strain difference in mouse MDB formation, we hypothesized that sex affects MDB formation. Methods MDBs were induced in male and female mice overexpressing K8, which are predisposed to MDB formation, and in nontransgenic mice by feeding the porphyrinogenic compound 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). MDB presence was determined by histologic, immunofluorescence, and biochemical analyses and correlated to liver injury using serologic and pathologic markers. Cytoskeletal and metabolic liver protein analysis, in vitro metabolism studies, and measurement of oxidative stress markers and protoporphyrin-IX were performed. Results Male mice formed significantly more MDBs, which was attenuated modestly by estradiol. MDB formation was accompanied by increased oxidative stress. Female mice had significantly fewer MDBs and oxidative stress-related changes, but had increased ductular reaction protoporphyrin-IX accumulation, and MDB-preventive K18 induction. Evaluation of the microsomal cytochrome-P450 (CYP) enzymes revealed significant gender differences in protein expression and activity in untreated and DDC-fed mice, and showed that DDC is metabolized by CYP3A. The changes in CYPs account for the gender differences in porphyria and DDC metabolism. DDC metabolite formation and oxidative injury accumulate on chronic DDC exposure in males, despite more efficient acute metabolism in females. Conclusions Gender dimorphic formation of MDBs and porphyria associate with differences in CYPs, oxidative injury, and selective keratin induction. These findings may extend to human MDBs and other neuropathy- and myopathy-related inclusions.
Aim: Hepatitis C virus (HCV) core protein critically contributes to hepatocarcinogenesis, which is often observed in liver cirrhosis. Since the liver cirrhosis microenvironment is affected by ...hypoxia, we focused on the possible driving force of HCV core protein on signal relay from hypoxia‐inducible factor (HIF)‐1α to vascular endothelial growth factor (VEGF).
Methods: Human hepatocellular carcinoma cells stably overexpressing HCV core (Core cells) and NS5A (NS5A cells) were established; empty vector‐transfected (EV) cells were used as controls. Hypoxia was induced by oxygen deprivation or by using cobalt chloride (CoCl2). YC‐1 was used to inhibit HIF‐1α expression. Protein analyses for cultured cells and liver tissues obtained from CoCl2‐treated HCV core‐transgenic (Core‐Tg) mice were performed by western blot and/or immunocytochemistry. Cellular mRNA levels were evaluated by quantitative real‐time reverse transcription‐polymerase chain reaction.
Results: Under hypoxia, the sustained expression of HIF‐1α, but not HIF‐2α, was profoundly observed in Core cells but, was faint in EV and NS5A cells. Immunocytochemistry revealed increased HIF‐1α in the nucleus. HIF‐1α mRNA levels were significantly higher in Core cells than in EV cells under both normoxia and hypoxia. The HIF‐1α‐targeted VEGF and Bcl‐xL expressions were increased in Core cells under hypoxia and abolished by YC‐1 treatment. Hypoxic liver samples of Core‐Tg mice indicated significant increases in both HIF‐1α and VEGF expression compared with the wild type.
Conclusions: Hepatitis C virus core protein has the distinct potential to transcriptionally upregulate and sustain HIF‐1α expression under hypoxia, thereby contributing to increased VEGF expression, a key regulator in the hypoxic milieu of liver cirrhosis.
Mallory‐Denk bodies (MDBs) are hepatocyte inclusions found in several liver diseases and consist primarily of keratins 8 and 18 (K8/K18) and ubiquitin that are cross‐linked by transglutaminase‐2. We ...hypothesized that genetic variables contribute to the extent of MDB formation, because not all patients with an MDB‐associated liver disease develop inclusions. We tested this hypothesis using five strains of mice (FVB/N, C3H/He, Balb/cAnN, C57BL/6, 129X1/Sv) fed for three months (eight mice per strain) the established MDB‐inducing agent 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC). MDB formation was compared using hematoxylin‐and‐eosin staining, or immunofluorescence staining with antibodies to K8/K18/ubiquitin, or biochemically by blotting with antibodies to transglutaminase‐2/p62 proteins and to K8/K18/ubiquitin to detect keratin cross‐linking. DDC feeding induced MDBs in all mouse strains, but there were dramatic strain differences that quantitatively varied 2.5‐fold (P < 0.05). MDB formation correlated with hepatocyte ballooning, and most ballooned hepatocytes had MDBs. Immunofluorescence assessment was far more sensitive than hematoxylin‐and‐eosin staining in detecting small MDBs, which out‐numbered (by ∼30‐fold to 90‐fold) but did not parallel their large counterparts. MDB scores partially reflected the biochemical presence of cross‐linked keratin‐ubiquitin species but not the changes in liver size or injury in response to DDC. The extent of steatosis correlated with the total (large+small) number of MDBs, and there was a limited correlation between large MDBs and acidophil bodies. Conclusion: Mouse MDB formation has important genetic contributions that do not correlate with the extent of DDC‐induced liver injury. If extrapolated to humans, the genetic contributions help explain why some patients develop MDBs whereas others are less likely to do so. Detection and classification of MDBs using MDB‐marker‐selective staining may offer unique links to specific histological features of DDC‐induced liver injury. (HEPATOLOGY 2008.)
Genetic factors impact liver injury susceptibility and disease progression. Prominent histological features of some chronic human liver diseases are hepatocyte ballooning and Mallory-Denk bodies. In ...mice, these features are induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in a strain-dependent manner, with the C57BL and C3H strains showing high and low susceptibility, respectively. To identify modifiers of DDC-induced liver injury, we compared C57BL and C3H mice using proteomic, biochemical, and cell biological tools. DDC elevated reactive oxygen species (ROS) and oxidative stress enzymes preferentially in C57BL livers and isolated hepatocytes. C57BL livers and hepatocytes also manifested significant down-regulation, aggregation, and nuclear translocation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH knockdown depleted bioenergetic and antioxidant enzymes and elevated hepatocyte ROS, whereas GAPDH overexpression decreased hepatocyte ROS. On the other hand, C3H livers had higher expression and activity of the energy-generating nucleoside-diphosphate kinase (NDPK), and knockdown of hepatocyte NDPK augmented DDC-induced ROS formation. Consistent with these findings, cirrhotic, but not normal, human livers contained GAPDH aggregates and NDPK complexes. We propose that GAPDH and NDPK are genetic modifiers of murine DDC-induced liver injury and potentially human liver disease.
Background/Aims The precise mechanism of formation and significance of Mallory bodies (MBs) are poorly understood. The endoplasmic reticulum (ER) is the organelle responsible for proper folding and ...elimination of unfolded proteins. Therefore, failure of this function increases defective proteins in the cell. Methods We examined the effects of oxidative stress on induction of ER stress and keratin 8 and 18 (K8/18)-containing inclusion formation in cultured human hepatoma cells and hepatocytes by immunofluorescence and immunoblot analyses. Results Generation of H2 O2 was detected in glucose oxidase (GO)-treated cells by 2′,7′-dichlorodihydrofluorescein diacetate and co-treatment with GO and acetyl-leucyl-leucyl-norleucinal (ALLN), a proteasome inhibitor, induced formation of extensive keratin inclusions that were inhibited by pre-treatment with N -acetyl-cysteine. These inclusions shared similar features with MBs by immunofluorescence analysis. Electron microscopy showed that these structures appeared near the nuclei, surrounded by filamentous structures. GO and ALLN upregulated the expression of ER stress markers, however, 4-phenylbutyrate, a chemical chaperone, reduced formation of inclusions and expression of the ER stress markers. Conclusions The oxidative stress coupled with limited inhibition of the proteasome induces dysfunction of the ER and results in inclusion formation in cultured cells. This suggests that ER stress plays a role in MB formation in liver disease.
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