Keratin 8 mutations in transgenic mice predispose to lung injury.

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.

Liver disease-associated keratin 8 and 18 mutations modulate keratin acetylation and methylation.

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.

Keratins: markers and modulators of liver disease.

PURPOSE OF REVIEW: Keratins are a subgroup of intermediate filaments expressed in the epithelia. Keratins emerged as important tissue-protecting genes and keratin variants cause/predispose to development of more than 50 human disorders. Our review focuses on the importance of keratins in context of liver disease. RECENT FINDINGS: K8/K18 variants are found in approximately 4% of white population and predispose to development and adverse outcome of multiple liver diseases. K8/K18 are major constituents of Mallory-Denk bodies, that is inclusions found in alcoholic and nonalcoholic steatohepatitis (NASH) and dysregulated keratin expression, K8 hyperphosphorylation, misfolding and crosslinking via transglutaminase 2 facilitate aggregate formation. Necrosis-generated and apoptosis-generated keratin serum fragments are emerging as important noninvasive markers of multiple liver diseases, particularly NASH. Keratins are established markers of tumor origin and in hepatocellular carcinoma, K19 expression is associated with poor prognosis. SUMMARY: Keratins are established tumor markers and are widely used as noninvasive markers of liver injury. In addition, the data that have become available in recent years have greatly advanced our understanding of keratins as modifiers of liver disease development.

Minimal Cube Explant Provides Optimal Isolation Condition of Mesenchymal Stem Cells from Umbilical Cord.

BACKGROUND: Enzymatic digestion and explant method have been widely used for isolating umbilical cord-derived mesenchymal stem cells (UC MSCs), although there is still a strong need for robust protocols for optimal isolation for large-scale stem cell banks. This study aims to establish an explant method for clinical scale production of MSCs from human UC tissue and to characterize UC MSCs isolated and cultured with the explant method. METHODS: UC MSCs were isolated by enzymatic digestion, minimal cube explant (MCE) 1-2, MCE 2-4, and MCE 10 and cultured, respectively. Also, human antibody array and basic fibroblast growth factor (bFGF) secretion in conditioned medium (CM) was analyzed. The cells were evaluated initial cell number, colony forming unit-fibroblast (CFU-F), proliferation capacity, CD marker expression, and multi-lineage differentiation. SA-ß-gal assay as well as expression of p16, p21 and p53 was performed by RT-PCR. RESULTS: MCE 2-4 is the most optimized method for isolation of small umbilical cord-derived fast proliferating cells (smumf cells) with the greatest number. MCE 2-4 had the highest secretion of various bioactive factors including bFGF. The MCE 2-4 provided significantly higher CD146 expression than enzymatic digestion, and that expression was maintained until P20. The gene expression of p16, p21, and p53 of smumf cells did not change until P10 and SA-ß-gal activity did not increase until P14. CONCLUSION: This study demonstrated that MCE 2-4 provided an optimal environment to isolate MSCs with quantity and quality from human whole UC tissue through secretion of various bioactive factors inherent to UC.

Predisposition to apoptosis in keratin 8-null liver is related to inactivation of NF-κB and SAPKs but not decreased c-Flip.

Keratin 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.