SummaryKeratin 8 and 18 (K8/K18) are major intermediate filament proteins of liver hepatocytes. They provide mechanical and nonmechanical stability, thereby protecting cells from stress. Hence, K8-null mice are highly sensitive to Fas-mediated liver cell apoptosis. However, the role of c-Flip protein in K8-null related susceptibility to liver injury is controversial. Here we analyzed c-Flip protein expression in various K8 or K18 null/mutant transgenic livers and show that they are similar in all analyzed transgenic livers and that previously reported c-Flip protein changes are due to antibody cross-reaction with mouse K18. Furthermore, analysis of various apoptosis- or cell survival-related proteins demonstrated that inhibition of phosphorylation of NF-κB and various stress activated protein kinases (SAPKs), such as p38 MAPK, p44/42 MAPK and JNK1/2, is related to the higher sensitivity of K8-null hepatocytes whose nuclear NF-κB is rapidly depleted through Fas-mediated apoptosis. Notably, we found that NF-κB and the studied protein kinases are associated with the K8/K18 complex and are released upon phosphorylation. Therefore, interaction of keratins with cell survival-related protein kinases and transcription factors is another important factor for hepatocyte survival.

Intermediate filament protein keratin 8 (K8) binds to heat shock protein 70 (Hsp70) and p38 MAPK, and is phosphorylated at Ser74 by p38α (MAPK14, hereafter p38). However, a p38 binding site on K8 and the molecular mechanism of K8-p38 interaction related to Hsp70 are unknown. Here, we identify a p38 docking site on K8 (Arg148/149 and Leu159/161) that is highly conserved in other intermediate filaments. A docking-deficient K8 mutation caused increased p38-Hsp70 interaction and enhanced p38 nuclear localization, indicating that the p38 dissociated from mutant K8 makes a complex with Hsp70, which is known as a potential chaperone for p38 nuclear translocation. Comparison of p38 MAPK binding with keratin variants associated with liver disease showed that the K18 I150V variant dramatically reduced binding with p38, which is similar to the effect of the p38 docking-deficient mutation on K8. Because the p38 docking site on K8 (Arg148/149 and Leu159/ 161) and the K18 Ile150 residue are closely localized in the parallel K8/K18 heterodimer, the K18 I150V mutation might interfere with K8-p38 interaction. These findings show that keratins, functioning as cytoplasmic anchors for p38, modulate p38 nuclear localization and thereby might affect a number of p38-mediated signal transduction pathways.

Keratin 8 (K8) and keratin 18 (K18) are the intermediate filament proteins whose phosphorylation/transamidation associate with their aggregation in Mallory‐Denk bodies found in patients with various liver diseases. However, the functions of other post‐translational modifications in keratins related to liver diseases have not been fully elucidated. Here, using a site‐specific mutation assay combined with nano‐liquid chromatography‐tandem mass spectrometry, we identified K8‐Lys108 and K18‐Lys187/426 as acetylation sites, and K8‐Arg47 and K18‐Arg55 as methylation sites. Keratin mutation (Arg‐to‐Lys/Ala) at the methylation sites, but not the acetylation sites, led to decreased stability of the keratin protein. We compared keratin acetylation/methylation in liver disease–associated keratin variants. The acetylation of K8 variants increased or decreased to various extents, whereas the methylation of K18‐del65‐72 and K18‐I150V variants increased. Notably, the highly acetylated/methylated K18‐I150V variant was less soluble and exhibited unusually prolonged protein stability, which suggests that additional acetylation of highly methylated keratins has a synergistic effect on prolonged stability. Therefore, the different levels of acetylation/methylation of the liver disease–associated variants regulate keratin protein stability. These findings extend our understanding of how disease–associated mutations in keratins modulate keratin acetylation and methylation, which may contribute to disease pathogenesis.—Jang, K.‐H., Yoon, H.‐N., Lee, J., Yi, H., Park, S.‐Y., Lee, S.‐Y., Lim, Y., Lee, H.‐J., Cho, J.‐W., Paik, Y.‐K., Hancock, W. S., Ku, N.‐O. Liver disease–associated keratin 8 and 18 mutations modulate keratin acetylation and methylation. FASEB J. 33, 9030–9043 (2019). http://www.fasebj.org

Although hepatocellular carcinoma (HCC) is developed with various etiologies, protection of hepatocytes seems basically essential to prevent the incidence of HCC. Keratin 8 and keratin 18 (K8/K18) are cytoskeletal intermediate filament proteins that are expressed in hepatocytes. They maintain the cell shape and protect cells under stress conditions. Their protective roles in liver damage have been described in studies of mouse models, and K8/K18 mutation frequency in liver patients. Interestingly, K8/K18 bind to signaling proteins such as transcription factors and protein kinases involved in HCC development. Since K8/K18 are abundant cytoskeletal proteins, K8/K18 binding with the signaling factors can alter the availability of the factors. Herein, we discuss the potential roles of K8/K18 in HCC development.

Keratin 8 (K8) is the cytoskeletal intermediate filament protein of simple-type epithelia. Mutations in K8 predispose the affected individual and transgenic mouse to liver disease. However, its role in lung has not been reported in mutant transgenic mouse models. Here, we investigated the susceptibility of two different transgenic mice expressing K8 gly-62-cys or ser-74-ala to lung injury. The mutant transgenic mice were highly susceptible to two independent acute and chronic lung injuries as compared with control mice. Both K8 gly-62-cys mice and K8 ser-74-ala mice showed the markedly enhanced mouse lethality (∼74% mutant mice vs. ∼34% control mice) and more severe lung damage with increased inflammation and apoptosis under L-arginine-mediated acute lung injury. Moreover, the K8 ser-74-ala mice had more severe lung damage with extensive hemorrhage and prominent fibrosis under bleomycin-induced chronic lung injury. Our study provides first direct evidence that the K8 mutations predispose to lung injury in transgenic mice.