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.

Prognostic Value of Serum Caspase-Cleaved Cytokeratin-18 Levels before Liver Transplantation for One-Year Survival of Patients with Hepatocellular Carcinoma.

Cytokeratin (CK)-18 is the major intermediate filament protein in the liver and during hepatocyte apoptosis is cleaved by the action of caspases; the resulting fragments are released into the blood as caspase-cleaved cytokeratin (CCCK)-18. Higher circulating levels of CCCK-18 have been found in patients with hepatocellular carcinoma (HCC) than in healthy controls and than in cirrhotic patients. However, it is unknown whether serum CCCK-18 levels before liver transplantation (LT) in patients with HCC could be used as a prognostic biomarker of one-year survival, and this was the objective of our study with 135 patients. At one year after LT, non-survivors showed higher serum CCCK-18 levels than survivors (p = 0.001). On binary logistic regression analysis, serum CCCK-18 levels >384 U/L were associated with death at one year (odds ratio = 19.801; 95% confidence interval = 5.301-73.972; p < 0.001) after controlling for deceased donor age. The area under the receiver operating characteristic (ROC) curve of serum CCCK-18 levels to predict death at one year was 77% (95% CI = 69%-84%; p < 0.001). The new finding of our study was that serum levels of CCCK-18 before LT in patients with HCC could be used as prognostic biomarker of survival.

Keratin K18 increases cystic fibrosis transmembrane conductance regulator (CFTR) surface expression by binding to its C-terminal hydrophobic patch.

BACKGROUND: CFTR function is tightly regulated by many interacting proteins. RESULTS: Intermediate filament protein keratin 18 increases the cell surface expression of CFTR by interacting with the C-terminal hydrophobic patch of CFTR. CONCLUSION: K18 controls the function of CFTR. SIGNIFICANCE: These findings offer novel insights into the regulation of CFTR and suggest that K18 and its dimerization partner, K8, may be modifier genes in cystic fibrosis. Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to cystic fibrosis, but the regulation of CFTR is not fully understood. Here, we identified the intermediate filament protein keratin K18 (K18) as a CFTR-binding protein by various approaches. We mapped a highly conserved "hydrophobic patch" ((1413)FLVI(1416)) in the CFTR C-terminus, known to determine plasmalemmal CFTR stability, as the K18-binding site. On the other hand, the C-terminal tail of K18 was found to be a critical determinant for binding CFTR. Overexpression of K18 in cells robustly increased the surface expression of wild-type CFTR, whereas depletion of K18 through RNA interference specifically diminished it. K18 binding increased the surface expression of CFTR by accelerating its apical recycling rate without altering CFTR biosynthesis, maturation, or internalization. Importantly, CFTR surface expression was markedly reduced in duodenal and gallbladder epithelia of K18(-/-) mice. Taken together, our results suggest that K18 increases the cell surface expression of CFTR by interacting with the CFTR C-terminal hydrophobic patch. These findings offer novel insights into the regulation of CFTR and suggest that K18 and its dimerization partner, K8, may be modifier genes in cystic fibrosis.

Complex formation and kinetics of filament assembly exhibited by the simple epithelial keratins K8 and K18.

We have generated human recombinant keratins K8 and K18 and describe conditions to quantitatively follow their assembly into filaments. When renatured individually from 8M urea into a low ionic strength/high pH-buffer, K8 was present in a dimeric to tetrameric form as revealed by analytical ultracentrifugation. In contrast, K18 sedimented as a monomer. When mixed in 8 M urea and renatured together, K8 and K18 exhibited s-value profiles compatible with homogeneous tetrameric complexes. This finding was confirmed by sedimentation equilibrium centrifugation. Subsequently, these tetrameric starter units were subjected to assembly experiments at various protein concentrations. At low values such as 0.0025 g/l, unit-length filaments were abundantly present after 2s of assembly. During the following 5 min, filaments grew rapidly and by measuring the length of individual filaments we were able to generate time-dependent length profiles. These data revealed that keratins K8/K18 assemble several times faster than vimentin and desmin. In addition, we determined the persistence length l(p) of K8/K18 filaments to be in the range of 300 nm. Addition of 1 mM MgCl(2) increases l(p) to 480 nm indicating that magnesium ions affect the interaction of keratin subunits within the filament during assembly to some extent.

Cross ß-sheet conformation of keratin 8 is a specific feature of Mallory-Denk bodies compared with other hepatocyte inclusions.

BACKGROUND & AIMS: Mallory-Denk bodies (MDBs) are cytoplasmic protein aggregates in hepatocytes in steatohepatitis and other liver diseases. We investigated the molecular structure of keratin 8 (K8) and 18 (K18), sequestosome 1/p62, and ubiquitin, which are the major constituents of MDBs, to investigate their formation and role in disease pathogenesis. METHODS: Luminescent conjugated oligothiophenes (LCOs), h-HTAA, and p-FTAA are fluorescent amyloid ligands that specifically bind proteins with cross ß-sheet conformation. We used LCOs to investigate conformational changes in MDBs in situ in human and murine livers as well as in transfection studies. RESULTS: LCO analysis showed cross ß-sheet conformation in human MDBs from patients with alcoholic and nonalcoholic steatohepatitis or hepatocellular carcinoma, but not in intracellular hyaline bodies, α1-antitrypsin deficiency, or ground-glass inclusions. LCOs bound to MDBs induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine feeding of mice at all developmental stages. CHO-K1 cells transfected with various combinations of SQSTM1/p62, ubi, and Krt8/Krt18 showed that K8 was more likely to have cross ß-sheet conformation than K18, whereas p62 never had cross ß-sheet conformation. The different conformational properties of K8 and K18 were also shown by circular dichroism analysis. CONCLUSIONS: K8 can undergo conformational changes from predominantly α-helical to cross ß-sheet, which would allow it to form MDBs. These findings might account for the observation that krt8⁻/⁻ mice do not form MDBs, whereas its excess facilitates MDB formation. LCOs might be used in diagnosis of liver disorders; they can be applied to formalin-fixed, paraffin-embedded tissues to characterize protein aggregates in liver cells.

O-GlcNAcylation determines the solubility, filament organization, and stability of keratins 8 and 18.

Keratins 8 and 18 (K8/18) are intermediate filament proteins expressed specifically in simple epithelial tissues. Dynamic equilibrium of these phosphoglycoproteins in the soluble and filament pool is an important determinant of their cellular functions, and it is known to be regulated by site-specific phosphorylation. However, little is known about the role of dynamic O-GlcNAcylation on this keratin pair. Here, by comparing immortalized (Chang) and transformed hepatocyte (HepG2) cell lines, we have demonstrated that O-GlcNAcylation of K8/18 exhibits a positive correlation with their solubility (Nonidet P-40 extractability). Heat stress, which increases K8/18 solubility, resulted in a simultaneous increase in O-GlcNAc on these proteins. Conversely, increasing O-GlcNAc levels were associated with a concurrent increase in their solubility. This was also associated with a notable decrease in total cellular levels of K8/18. Unaltered levels of transcripts and the reduced half-life of K8 and K18 indicated their decreased stability on increasing O-GlcNAcylation. On the contrary, the K18 glycosylation mutant (K18 S29A/S30A/S48A) was notably more stable than the wild type K18 in Chang cells. The K18-O-GlcNAc mutant accumulated as aggregates upon stable expression, which possibly altered endogenous filament architecture. These results strongly indicate the involvement of O-GlcNAc on K8/18 in regulating their solubility and stability, which may have a bearing on the functions of these keratins.

New prognostic biomarkers of mortality in patients undergoing liver transplantation for hepatocellular carcinoma.

The outcome prediction of hepatocellular carcinoma (HCC) patients undergoing liver transplantation (LT) was classically established using various macromorphological factors and serum alpha-fetoprotein levels prior to LT. However, other biomarkers have recently been reported to be associated with the prognosis of HCC patients undergoing to LT. This review summarizes clinical data on these new biomarkers. High blood levels of malondialdehyde, total antioxidant capacity, caspase-cleaved cytokeratin-18, soluble CD40 ligand, substance P, C-reactive protein, and vascular endothelial growth factor, increased neutrophil to lymphocyte ratio and platelet to lymphocyte ratio in blood, high peripheral blood expression of human telomerase reverse transcriptase messenger ribonucleic acid, and high HCC expression of dickkopf-1 have recently been associated with decreased survival rates. In addition, high blood levels of des-gamma-carboxy prothrombin, and high HCC expression of glypican-3, E-cadherin and beta-catenin have been associated with increased HCC recurrence. Additional research is necessary to establish the prognostic role of these biomarkers in HCC prior to LT. Furthermore, some of these biomarkers are also interesting because their potential modulation could help to create new research lines for improving the outcomes of those patients.

NordiQC Assessments of Low Molecular Weight Keratin 8/18 Immunoassays.

This paper is number 2 in a series developed through a partnership between ISIMM and NordiQC for the purpose of reporting research assessing the performance characteristics of immunoassays in an external proficiency testing program.

Efficient generation of functional hepatocyte-like cells from menstrual blood-derived stem cells.

In recent years, the advantages of menstrual blood-derived stem cells (MenSCs), such as minimal ethical considerations, easy access and high proliferative ability, have inspired scientists to investigate the potential of MenSCs in cell therapy of different diseases. In order to characterize the potency of these cells for future cell therapy of liver diseases, we examined the potential of MenSCs to differentiate into hepatocytes, using different protocols. First, the immunophenotyping properties and potential of MenSCs to differentiate into osteoblasts, adipocytes and chondrocytes were evaluated. Thereafter, the differentiation protocols developed by two concentrations of hepatocyte growth factor (HGF) and oncostatin M (OSM), in combination with other components in serum-supplemented or serum-free culture media, were also investigated. The sequential differentiation was monitored by real-time PCR, immunostaining and functional assays. Our primary data revealed that the isolated MenSCs exhibited mesenchymal stem cell markers in parallel to OCT-4 as an embryonic marker. Regardless of differentiation procedures, the developed cells expressed mature hepatocyte markers, such as albumin, tyrosine aminotransferase and cytokeratin-18 at the mRNA and protein levels. They also showed functional properties of hepatocytes, including albumin secretion, glycogen storage and cytochrome P450 7A1 expression. However, the degree of differentiation was dependent on the concentrations of HGF and OSM. Indeed, omission of serum during the differentiation process caused typical improvement in hepatocyte-specific functions. This study is a novel report demonstrating the differentiation potential of MenSCs into hepatocyte-like cells. We recommend a complementary serum-free differentiation protocol for enrichment of in vitro production of functional MenSC-derived hepatocyte-like cells that could lead to a major step toward applied stem cell therapy of chronic liver diseases.

The cytokeratin-18 fragment level as a biomarker of nonalcoholic fatty liver disease in patients with type 2 diabetes mellitus.

BACKGROUND: The serum cytokeratin-18 fragment (CK-18) concentration has been suggested to be a biomarker of nonalcoholic fatty liver disease (NAFLD), although its usefulness in patients with type 2 diabetes mellitus (T2DM) is unknown. METHODS: The study was divided into two parts. In the first cross-sectional study, a total of 200 patients with T2DM and 58 healthy control subjects were recruited. NAFLD was diagnosed using ultrasonography. In the subsequent longitudinal study, we evaluated the three-month change (Δ) in the CK-18 concentration and other parameters in 40 T2DM patients with NAFLD. RESULTS: The serum CK-18 values were significantly higher in the NAFLD group than in the nonNAFLD group among both diabetic and nondiabetic subjects. The CK-18 concentration was found to be an independent determinant of NAFLD and was positively correlated with the ultrasonography score and AST and ALT concentrations in the T2DM patients. Positive correlations were also identified between the CK-18 and transaminase concentrations in the T2DM and NAFLD cohorts. ΔCK-18 was found to be significantly associated with ΔBMI in the T2DM patients with NAFLD. CONCLUSIONS: A dose effect between the CK-18 concentration and the severity of NAFLD was found in the T2DM patients; thus, the CK-18 concentration is a potentially useful biomarker for assessing the efficacy of treatment and the improvement in NAFLD in patients with T2DM.

Serum levels of caspase-cleaved cytokeratin-18 and mortality are associated in severe septic patients: pilot study.

OBJECTIVE: Apoptosis is increased in sepsis. Cytokeratin 18 (CK-18), a protein of the intermediate filament group present in most epithelial and parenchymal cells, is cleaved by the action of caspases and released into the blood as caspase-cleaved CK (CCCK)-18 during apoptosis. Circulating levels of CCCK-18 have scarcely been explored in septic patients. In one study with 101 severe septic patients, the authors reported higher serum CCCK-18 levels in non-survivors than in survivors; however, the sample size was too small to demonstrate an association between serum CCCK-18 levels and early mortality and whether they could be used as a biomarker to predict outcomes in septic patients. Thus, these were the objectives of this study with a large series of patients. METHODS: We performed a prospective, multicenter, observational study in six Spanish Intensive Care Units with 224 severe septic patients. Blood samples were collected at the time that severe sepsis was diagnosed to determine serum levels of CCCK-18, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-10. The end point was 30-day mortality. RESULTS: Non-surviving patients (n = 80) showed higher serum CCCK-18 levels (P<0.001) than survivors (n = 144). Multiple logistic regression analysis showed that serum CCCK-18 levels>391 u/L were associated with 30-day survival (Odds ratio = 2.687; 95% confidence interval = 1.449-4.983; P = 0.002), controlling for SOFA score, serum lactic acid levels and age. Kaplan-Meier survival analysis showed that the risk of death in septic patients with serum CCCK-18 levels >391 u/L was higher than in patients with lower values (Hazard Ratio = 3.1; 95% CI = 1.96-4.84; P<0.001). Serum CCCK-18 levels were positively associated with serum levels of IL-6 and lactic acid, and with SOFA and APACHE scores. CONCLUSIONS: The major novel finding of our study, the largest cohort of septic patients providing data on circulating CCCK-18 levels, was that serum CCCK-18 levels are associated with mortality in severe septic patients.

Parkin induces upregulation of 40S ribosomal protein SA and posttranslational modification of cytokeratins 8 and 18 in human cervical cancer cells.

Parkin was originally identified as a protein associated with Parkinson's disease. Recently, numerous research studies have suggested that parkin acts as a tumor suppressor. In accordance with these studies, we previously reported that overexpression of parkin in HeLa cells induced growth inhibition. To elucidate possible mechanisms by which parkin may inhibit cell growth, HeLa cells were infected with adenoviruses expressing either the parkin gene or adenovirus alone for 72 h and a total proteomic analysis was performed using 2-D gel electrophoresis followed by LC-MS/MS. We identified three proteins whose expression changed between the two groups: the 40S ribosomal protein SA (RPSA) was downregulated in parkin virus-infected cells, and cytokeratins 8 and 18 exhibited an acid shift in pI value without a change in molecular weight, suggesting that these proteins became phosphorylated in parkin virus-infected cells. The changes in these three proteins were first observed at 60 h postinfection and were most dramatic at 72 h postinfection. Because upregulation of RPSA and dephosphorylation of cytokeratins 8/18 have been linked with tumor progression, these data suggest that parkin may inhibit cell growth, at least in part, by decreasing RPSA expression and inducing phosphorylation of cytokeratin 8/18.

Diagnostic and prognostic role of plasma levels of two forms of cytokeratin 18 in patients with non-small-cell lung cancer.

PURPOSE: Cytokeratin 18 (CK18) can be used as a serum biomarker for carcinoma cell death, whereas caspase-cleaved (ccCK18) fragments reflect tumour apoptosis. We explored the potential diagnostic and prognostic role of circulating CK18 and ccCK18 in patients with non-small-cell lung cancer (NSCLC) in comparison with Cyfra 21.1, a fragment of cytokeratin 19. METHODS: Subject cohorts consisted of 200 healthy blood donors (HBD), 113 patients with benign lung diseases (BLD) and 179 NSCLC cases. Plasma levels of ccCK18, total CK18 and Cyfra 21.1 were determined with ELISA assays. RESULTS: Plasma levels of ccCK18 and total CK18 were higher in the NSCLC group compared to the HBD and BLD cohorts (p<0.0001). Using a cut-off of 104 U/L for ccCK18 and 302 U/L for total CK18 (95% specificity in the HBD group) the diagnostic accuracy of both CK18 forms to distinguish between NSCLC and BLD cases was 56%, whereas it was 94% for Cyfra 21.1. Multivariate survival analysis showed that total CK18 was a stronger prognostic factor than both ccCK18 and Cyfra 21.1 (HR 0.64 for low versus high total CK18 levels, 95% confidence interval (CI) 0.50-0.82; p=0.0004) in the entire NSCLC cohort and in 78 patients with locally advanced or metastatic disease treated with chemoradiotherapy or first-line chemotherapy (HR 0.70 95% CI 0.52-0.94; p=0.018). CONCLUSIONS: Cyfra 21.1 is a useful diagnostic biomarker for NSCLC. Total CK18 shows a promising potential as prognostic marker in NSCLC patients, independently of the therapeutical intervention. In contrast, ccCK18 was not of prognostic value in NSCLC, suggesting that tumour necrosis is of particular importance in this disease.

Caspase-cleaved fragments of cytokeratin 18 in patients with chronic hepatitis B.

BACKGROUND: During hepatocyte apoptosis, intermediate filament protein cytokeratin 18 is cleaved by caspases at Asp396 which can be specifically detected by the monoclonal antibody M30 (M30-antigen). In this study, we sought to determine whether serum M30-antigen levels can serve as a useful biomarker of liver injury in the clinical spectrum of HBV infection. METHODS: Serum M30-antigen levels were measured in inactive HBV carriers (n=54), patients with HBeAg-negative chronic hepatitis B (CHB, n=47), patients with HBeAg-positive CHB (n=42) and healthy controls (n=29). All subjects were treatment-naïve. RESULTS: There were significant differences in serum M30-antigen levels across the study groups (P<0.001; Kruskal-Wallis test). Post hoc analyses revealed that M30-antigen levels did not differ significantly between inactive HBV carriers (median 109.6 U/L) and healthy controls (median 106.1 U/L). However, both patients with HBeAg-negative (CHB, median 182.9 U/L, P<0.001) and HBeAg-positive CHB (median 158.3 U/L, P<0.001) had significantly higher levels of M30-antigen compared with inactive HBV carriers. CONCLUSIONS: Hepatocyte apoptotic activity--as reflected by serum M30-antigen levels--is increased in chronic active hepatitis B, but is not associated with the HBeAg status. In contrast, apoptosis does not appear to be a prominent feature of inactive HBV carriers.

M30 neoepitope expression in epithelial cancer: quantification of apoptosis in circulating tumor cells by CellSearch analysis.

PURPOSE: This study aimed to detect the M30 neoepitope on circulating tumor cells (CTC) as a tool for quantifying apoptotic CTC throughout disease course and treatment. EXPERIMENTAL DESIGN: An automated sample preparation and analysis platform for computing CTC (CellSearch) was integrated with a monoclonal antibody (M30) targeting a neoepitope disclosed by caspase cleavage at cytokeratin 18 (CK18) in early apoptosis. The assay was validated using cell lines and blood samples from healthy volunteers and patients with epithelial cancer. RESULTS: M30-positive CTC could be detected in >70% of CTC-positive carcinoma patients, which were free for both chemotherapy and radiologic treatments. The fraction of M30-positive CTC varied from 50% to 80%, depending on the histotype. To investigate the potential application of the M30 CTC assay for the evaluation of response in early phase trials, CTC and M30-positive CTC were enumerated in a small case series of breast cancer patients during treatment. Results indicate that changes in the balance of M30-negative/positive CTC may be used as a dynamic parameter indicating an active disease, as documented by consistent radiologic findings. CONCLUSIONS: M30 expression on CTC is detectable by immunofluorescence. The M30-integrated test has potential for monitoring dynamic changes in the quote of apoptotic CTC (in addition to CTC count) to evaluate response in clinical trials of molecularly targeted anticancer therapeutics as well as for translational research, in which there is a pressing need for informative circulating biomarkers.

High-mobility group box-1 protein and keratin-18, circulating serum proteins informative of acetaminophen-induced necrosis and apoptosis in vivo.

Drug-induced hepatotoxicity represents a major clinical problem and an impediment to new medicine development. Serum biomarkers hold the potential to provide information about pathways leading to cellular responses within inaccessible tissues, which can inform the medicinal chemist and the clinician with respect to safe drug design and use. Hepatocyte apoptosis, necrosis, and innate immune activation have been defined as features of the toxicological response associated with the hepatotoxin acetaminophen (APAP). Within this investigation, we have unambiguously identified and characterized by liquid chromatography-tandem mass spectrometry differing circulating molecular forms of high-mobility group box-1 protein (HMGB1) and keratin-18 (K18), which are linked to the mechanisms and pathological changes induced by APAP in the mouse. Hypoacetylated HMGB1 (necrosis indicator), caspase-cleaved K18 (apoptosis indicator), and full-length K18 (necrosis indicator) present in serum showed strong correlations with the histological time course of cell death and was more sensitive than alanine aminotransferase activity. We have further identified a hyperacetylated form of HMGB1 (inflammatory indicator) in serum, which indicated that hepatotoxicity was associated with an inflammatory response. The inhibition of APAP-induced apoptosis and K18 cleavage by the caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(OMe) fluoromethyl ketone are associated with increased hepatic damage, by a shift to necrotic cell death only. These findings illustrate the initial verification of K18 and HMGB1 molecular forms as serum-based sensitive tools that provide insights into the cellular dynamics involved in APAP hepatotoxicity within an inaccessible tissue. Based on these findings, potential exists for the qualification and measurement of these proteins to further assist in vitro, in vivo, and clinical bridging in toxicological research.

Nanoscale surface topography enhances cell adhesion and gene expression of madine darby canine kidney cells.

Substrate topography is one of the key factors that influence cell behavior, such as cell attachment, adhesion, proliferation and differentiation. In the present work, nanostructures were produced on polystyrene Petri dish by polarized laser irradiation with the wavelength of 266 nm and the energy of 3.0 mJ/cm2. Cell adhesion, growth and gene expression of Madine darby canine kidney (MDCK) cells cultured on smooth and nanogrooved substrates were investigated. The results indicated that cells preferred to adhere and grow on nanogrooved substrate. The distribution of cell cycle for cells on smooth substrates was different from that on nanogrooved substrate. The percentage of G1 phase cells on nanogrooved substrate (48.6 +/- 1.4%) was lower than that on smooth substrate (57.6 +/- 4.4%), while the percentage of cells on nanogrooved substrate in S (30.2 +/- 0.5%) and G2/M (21.2 +/- 1.1%) phase was higher than those on smooth substrate (25.1 +/- 1.5% and 17.3 +/- 3.3%, respectively). Moreover, the gene expression of cyclin D1 and keratin 18, which was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR), was significantly enhanced by nanogrooves, with an increase of cyclin D1 mRNA by 98% and an increase of keratin 18 mRNA by 75%. In conclusion, the nanogrooved surface features on polystyrene could alter cell cycle and enhance gene expression of cyclin D1 and keratin 18 in MDCK cells, which partly explained the increased cell adhesion and growth on nanogrooved substrate.

M-30 and 4HNE are sequestered in different aggresomes in the same hepatocytes.

M-30 and 4HNE adducts are two markers of active liver disease. M-30 is a serologic marker and 4HNE adducts are histologic markers. M-30 is a marker for apoptosis because it is a fragment of cytokeratin-18 left over from proteolysis by caspase 3. 4HNE is a marker of oxidative stress because it results from lipid peroxidation. Both markers are commonly found in nonalcoholic steatohepatitis and in alcoholic hepatitis. Liver biopsies from patients with steatohepatitis, 11 alcoholic and 11 non-alcoholics were stained for 4HNE and M-30. Almost all of the biopsies in both groups showed 4HNE- and M-30-positive aggresomes in hepatocytes. Mallory Denk bodies (MDB) stained variably positive for M-30, whereas 4HNE was present in aggresomes independent of MDBs. However, they were sometimes located in hepatocytes which also contained MDBs as shown by confocal microscopy of double stained biopsies. The results indicate that the formation of M-30 and 4HNE aggresomes occurs through different pathways of liver cell injury in both types of steatohepatitis.

A mutation of keratin 18 within the coil 1A consensus motif causes widespread keratin aggregation but cell type-restricted lethality in mice.

Mutations in genes encoding epidermal keratins cause skin disorders, while those in internal epithelial keratins, such as K8 and K18, are risk factors for liver diseases. The effect of dominant mutations in K8 or K18 during embryonic development and tissue homeostasis has not been examined so far. Here we demonstrate that the dominant mutation hK18 R89C, that is highly similar to hK14 R125C, causing EBS in humans, leads to cell type-specific lethality in mice, depending on the ratio of mutant to endogenous keratins. Mice expressing hK18 R89C in the absence of endogenous K19 and K18 died at mid-gestation from defects in trophoblast giant cells, accompanied by haematomas. A single, endogenous K18 allele rescued embryonic lethality but caused aggregation of keratins in all adult internal epithelia, surprisingly without spontaneous cell fragility. Closer analysis revealed that both filaments and aggregates coexisted in the same cell, depending on the ratio of mutant to endogenous keratins. Our results demonstrate that balanced overexpression of a wild-type keratin rescued the lethal consequences of a dominant-negative mutation. This has important implications for therapy approaches of keratinopathies, suggesting that suppressing the mutant allele is not necessary in vivo.

A conserved domain of the gp85/trans-sialidase family activates host cell extracellular signal-regulated kinase and facilitates Trypanosoma cruzi infection.

Chagas' disease is a chronic, debilitating and incapacitating illness, caused by the protozoan parasite Trypanosoma cruzi when infective trypomastigotes invade host cells. Although the mechanism of trypomastigotes interaction with mammalian cells has been intensively studied, a final and integrated picture of the signal transduction mechanisms involved still remains to be elucidated. Our group has previously shown that the conserved FLY domain (VTVXNVFLYNR), present in all members of the gp85/trans-sialidase glycoprotein family coating the surface of trypomastigotes, binds to cytokeratin 18 (CK18) on the surface of LLC-MK(2) epithelial cells, and significantly increases parasite entry into mammalian cells. Now it is reported that FLY, present on the surface of trypomastigotes or on latex beads binds to CK18, promotes dephosphorylation and reorganization of CK18 and activation of the ERK1/2 signaling cascade culminating in an increase of approximately 9-fold in the number of parasites/cell. Inhibition of ERK1/2 phosphorylation completely blocks the adhesion of FLY to cells and blocks by 57% the host cell infection by T. cruzi. Taken together our results indicate that the conserved FLY domain is an important tool that trypomastigotes have evolved to specific exploit the host cell machinery and guarantee a successful infection.