Keratins: Biomarkers and modulators of apoptotic and necrotic cell death in the liver

Abstract

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), while 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 (for human K18/K19: 235Val-Glu-Val-Asp↓) and K18 at a unique aspartate (for 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 ELISA 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 K18-related tissue polypeptide antigen, K8-related tissue-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 injury. The epithelial specificity of K18/K19, epitope unmasking upon caspase digestion, abundance and relative 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.

Hepatic stellate cells as key target in liver fibrosis

“…A NOX1/NOX4 inhibitor, GKT137831, reduces hepatic apoptosis and fibrosis in CCl 4 and BDL mice [48]. Specific modes of hepatocyte and cholangiocyte death may be monitored by circulating cytokeratins as biomarkers [49]. Apoptosis inhibitors such as a pan-caspase inhibitor, emricasan (IDN-6556), aiming to reduce hepatocyte death, are under evaluation in clinical trials in post-transplant HCV and NASH patients (ClinicalTrials.gov: NCT02138253, NCT02686762, NCT02960204; ENCORE trials).…”

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

“…Methods that enable biochemical characterization of IF proteins can be useful to understand numerous pathophysiological phenomena in mammalian systems, since IF proteins are both markers and modulators of cellular and tissue stress 29 . The principle behind the current video method is based on the initial procedures developed in the 1970s and 1980s to isolate, separate and reconstitute IF proteins from cells and tissues, generally employing low and high salt solutions and Triton-X100 detergent 25,30-35 .…”

Serum keratin‐18 fragments as cell death biomarker in association with disease progression and prognosis in hepatitis B virus‐related cirrhosis

“…Therefore, the histological assessment of cell death in the liver is helpful in distinguishing ACLF from traditional AD . Alternative circulating biomarkers for hepatic cell death, keratin‐18 fragments, are also significantly elevated in ACLF comparing to patients with chronic liver disease …”

“…During the process of apoptosis, K18 is firstly cleaved at DALD (397) by caspase‐3, ‐7 or ‐9 then at VEVD (238) by caspase‐6 . The caspase‐cleaved K18 with unmasked cleavage site is released to extracellular space and circulated in serum/plasma in the form of cytoplasmic inclusion bodies within extracellular vesicles . During the process of necrotic cell death, K18 is released into circulation as an intact unit from the membrane of the destroyed cells (Figure ) .…”