Nam-On Ku

Keratins make up a major portion of epithelial intermediate filament proteins. The widely diverse keratins are found in both the small and large intestines. The human intestine mainly expresses keratins 8, 18, 19, and 20. Many of the common roles of keratins are for the integrity and stability of the epithelial cells. The keratins also protect the cells and tissue from stress and are biomarkers for some diseases in the organs. Although an increasing number of studies have been performed regarding keratins, the roles of keratin in the intestine have not yet been fully understood. This review focuses on discussing the roles of keratins in the intestine. Diverse studies utilizing mouse models and samples from patients with intestinal diseases in the search for the association of keratin in intestinal diseases have been summarized.

Keratin 8 and keratin 18 (K8/K18) are intermediate filament proteins that form the obligate heteropolymers in hepatocytes and protect the liver against toxins. The mechanisms of protection include the regulation of signaling pathway associated with cell survival. Previous studies show K8/K18 binding with Akt, which is a well-known protein kinase involved in the cell survival signaling pathway. However, the role of K8/K18 in the Akt signaling pathway is unclear. In this study, we found that K8/K18-Akt binding is downregulated by K8/K18 phosphorylation, specifically phosphorylation of K18 ser7/34/53 residues, whereas the binding is upregulated by K8 gly-62-cys mutation. K8/K18 expression in cultured cell system tends to enhance the stability of the Akt protein. A comparison of the Akt signaling pathway in a mouse system with liver damage shows that the pathway is downregulated in K18-null mice compared with nontransgenic mice. K18-null mice with Fas-induced liver damage show enhanced apoptosis combined with the downregulation of the Akt signaling pathway, i.e., lower phosphorylation levels of GSK3β and NFκB, which are the downstream signaling factors in the Akt signaling pathway, in K18-null mice compared with the control mice. Our study indicates that K8/K18 expression protects mice from liver damage by participating in enhancing the Akt signaling pathway.

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, the role of K8 in the lung has not been reported in mutant transgenic mouse models. Here, we investigated the susceptibility of two different transgenic mice expressing K8 Gly62–Cys (Gly62 replaced with Cys) or Ser74–Ala (Ser74 replaced with Ala) to lung injury. The mutant transgenic mice were highly susceptible to two independent acute and chronic lung injuries compared with control mice. Both K8 Gly62–Cys mice and K8 Ser74–Ala mice showed markedly increased mouse lethality (∼74% mutant mice versus ∼34% control mice) and more severe lung damage, with increased inflammation and apoptosis, under L-arginine-mediated acute lung injury. Moreover, the K8 Ser74–Ala mice had more severe lung damage, with extensive hemorrhage and prominent fibrosis, under bleomycin-induced chronic lung injury. Our study provides the first direct evidence that K8 mutations predispose to lung injury in transgenic mice.

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.

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). www.fasebj.org

Keratins are the largest subfamily of intermediate filament proteins. They are either type I acidic or type II basic keratins. Keratins form obligate heteropolymer in epithelial cells and their expression patterns are tissue-specific. Studies have shown that keratin mutations are the cause of many diseases in humans or predispose humans to acquiring them. Using mouse models to study keratin-associated human diseases is critical, because they allow researchers to get a better understanding of these diseases and their progressions, and so many such studies have been conducted. Acknowledging the importance, researches with genetically modified mice expressing human disease-associated keratin mutants have been widely done. Numerous studies using keratin knockout mice, keratin-overexpressed mice, or transgenic mice expressing keratin mutants have been conducted. This review summarizes the mouse models that have been used to study type I and type II keratin expression in the digestive organs, namely, the liver, pancreas, and colon.

A protein modification called O-linked glycosylation regulates the interactions between vimentin molecules under normal conditions, and the ability of Chlamydia bacteria to replicate after they infect cells.

Cytoskeletal keratin 18 (K18) undergoes caspase-mediated digestion during apoptosis, which leads to dramatic disassembly of keratin filaments. We studied the significance of K18 caspase digestion in a mouse model and generated transgenic mice expressing the human K18 caspase digestion-resistant double-mutant K18-D238/397E in a mouse (m) K18-null background, and compared their response to injury mediated by administration of antibody against tumor necrosis factor receptor superfamily member 6 (Fas), anti-FasAb. Notably, K18-D238/397E;mK18-null mice were significantly more resistant to anti-FasAb-induced injury as compared with K18-WT;mK18-null mice (23% vs 57% lethality, respectively; P<0.001). The same applied when the toxin microcystin-LR (MLR) was used to induce liver injury, i.e. lethality of K18-D238/397E;mK18-null mice in response to MLR treatment was reduced compared with the control mouse strain. The lesser rate of apoptosis in K18-D238/397E;mK18-null livers is associated with delayed degradation and, thus, sustained activation of cell-survival-related protein kinases, including stress-activated protein kinases and the NF-κB transcription factor, up to 6-8 h after administration of anti-FasAb. However, activation of the kinases and NF-κB in K18-WT-reconstituted livers decreases dramatically 8 h after anti-FasAb administration. In addition, the D238/397E double-mutation results in prolonged stability of K18 protein in transfected cells and transgenic livers. Therefore, our results show that the caspase digestion-resistant K18 helps to maintain keratin filament organization and delays apoptosis, thereby resulting in protection from liver injury.

Keratins, formerly known as cytokeratins, are the major epithelial‐specific subgroup of intermediate filament proteins. Adult hepatocytes express keratin polypeptides 8 and 18 (K8/K18), whereas cholangiocytes express K8/K18 and keratins 7 and 19 (K7/K19). Keratins function primarily to protect hepatocytes from apoptosis and necrosis, which was revealed using several genetic mouse models. This cytoprotective function was further clarified by the identification of natural human keratin variants that are normally silent, but become pathogenic by predisposing their carriers to apoptosis during acute or chronic liver injury mediated by toxins, virus infection, or metabolic stress. During apoptosis, caspases cleave K18 and K19 at conserved aspartates (human K18/K19:²³⁵Val‐Glu‐Val‐Asp^↓) and K18 at a unique aspartate (human K18:³⁹⁴Asp‐Ala‐Leu‐Asp^↓), with the latter exposed epitope becoming recognized by the M30 antibody in blood and tissues. Additional K18‐containing protein backbone epitopes are detected using the M6 and M5 (termed M65) antibodies. Intact K18 and its associated fragments, which are released into blood during apoptosis and necrosis in various diseases, have been analyzed by enzyme‐linked immunosorbent assay using the M30/M65 antibodies or their signal ratios. Furthermore, M30/M65 levels have been used as diagnostic and prognostic biomarkers in acute and chronic liver diseases, including nonalcoholic steatohepatitis and acute liver failure. Other keratin biomarkers include K8/K18/K19‐related tissue polypeptide antigen, K18‐related tissue polypeptide‐specific antigen, and K19‐related CYFRA‐21‐1, which have been evaluated mostly in patients with epithelial tumors.Conclusion: Keratins and their fragments are released into blood during liver and other epithelial tissue injury. The epithelial specificity of K18/K19, epitope unmasking upon caspase digestion, keratin abundance, and relative keratin stability render them useful biomarkers for hepatocyte and cholangiocyte apoptosis and necrosis. However, the precise biochemical nature and release mechanism of circulating keratins remain unknown. (Hepatology2016;64:966‐976)