Wnt/ß-catenin signaling activation promotes lipogenesis in the steatotic liver via physical mTOR interaction.

Background and aims: Wnt/ß-catenin signaling plays an important role in regulating hepatic metabolism. This study is to explore the molecular mechanisms underlying the potential crosstalk between Wnt/ß-catenin and mTOR signaling in hepatic steatosis. Methods: Transgenic mice (overexpress Wnt1 in hepatocytes, Wnt+) mice and wild-type littermates were given high fat diet (HFD) for 12 weeks to induce hepatic steatosis. Mouse hepatocytes cells (AML12) and those transfected to cause constitutive ß-catenin stabilization (S33Y) were treated with oleic acid for lipid accumulation. Results: Wnt+ mice developed more hepatic steatosis in response to HFD. Immunoblot shows a significant increase in the expression of fatty acid synthesis-related genes (SREBP-1 and its downstream targets ACC, AceCS1, and FASN) and a decrease in fatty acid oxidation gene (MCAD) in Wnt+ mice livers under HFD. Wnt+ mice also revealed increased Akt signaling and its downstream target gene mTOR in response to HFD. In vitro, increased lipid accumulation was detected in S33Y cells in response to oleic acid compared to AML12 cells reinforcing the in vivo findings. mTOR inhibition by rapamycin led to a down-regulation of fatty acid synthesis in S33Y cells. In addition, ß-catenin has a physical interaction with mTOR as verified by co-immunoprecipitation in hepatocytes. Conclusions: Taken together, our results demonstrate that ß-catenin stabilization through Wnt signaling serves a central role in lipid metabolism in the steatotic liver through up-regulation of fatty acid synthesis via Akt/mTOR signaling. These findings suggest hepatic Wnt signaling may represent a therapeutic strategy in hepatic steatosis.

Butyrate induces development-dependent necrotizing enterocolitis-like intestinal epithelial injury via necroptosis.

BACKGROUND: The accumulation of short-chain fatty acids (SCFAs) from bacterial fermentation may adversely affect the under-developed gut as observed in premature newborns at risk for necrotizing enterocolitis (NEC). This study explores the mechanism by which specific SCFA fermentation products may injure the premature newborn intestine mucosa leading to NEC-like intestinal cell injury. METHODS: Intraluminal injections of sodium butyrate were administered to 14- and 28-day-old mice, whose small intestine and stool were harvested for analysis. Human intestinal epithelial stem cells (hIESCs) and differentiated enterocytes from preterm and term infants were treated with sodium butyrate at varying concentrations. Necrosulfonamide (NSA) and necrostatin-1 (Nec-1) were used to determine the protective effects of necroptosis inhibitors on butyrate-induced cell injury. RESULTS: The more severe intestinal epithelial injury was observed in younger mice upon exposure to butyrate (p = 0.02). Enterocytes from preterm newborns demonstrated a significant increase in sensitivity to butyrate-induced cell injury compared to term newborn enterocytes (p = 0.068, hIESCs; p = 0.038, differentiated cells). NSA and Nec-1 significantly inhibited the cell death induced by butyrate. CONCLUSIONS: Butyrate induces developmental stage-dependent intestinal injury that resembles NEC. A primary mechanism of cell injury in NEC is necroptosis. Necroptosis inhibition may represent a potential preventive or therapeutic strategy for NEC. IMPACT: Butyrate induces developmental stage-dependent intestinal injury that resembles NEC. A primary mechanism of cell injury caused by butyrate in NEC is necroptosis. Necroptosis inhibitors proved effective at significantly ameliorating the enteral toxicity of butyrate and thereby suggest a novel mechanism and approach to the prevention and treatment of NEC in premature newborns.

Metabolic model of necrotizing enterocolitis in the premature newborn gut resulting from enteric dysbiosis.

Necrotizing enterocolitis (NEC) is a leading cause of premature newborn morbidity and mortality. The clinical features of NEC consistently include prematurity, gut dysbiosis and enteral inflammation, yet the pathogenesis remains obscure. Herein we combine metagenomics and targeted metabolomics, with functional in vivo and in vitro assessment, to define a novel molecular mechanism of NEC. One thousand six hundred and forty seven publicly available metagenomics datasets were analyzed (NEC = 245; healthy = 1,402) using artificial intelligence methodologies. Targeted metabolomic profiling was used to quantify the concentration of specified fecal metabolites at NEC onset (n = 8), during recovery (n = 6), and in age matched controls (n = 10). Toxicity assays of discovered metabolites were performed in vivo in mice and in vitro using human intestinal epithelial cells. Metagenomic and targeted metabolomic analyses revealed significant differences in pyruvate fermentation pathways and associated intermediates. Notably, the short chain fatty acid formate was elevated in the stool of NEC patients at disease onset (P = 0.005) dissipated during recovery (P = 0.02) and positively correlated with degree of intestinal injury (r 2 = 0.86). In vitro, formate caused enterocyte cytotoxicity in human cells through necroptosis (P < 0.01). In vivo, luminal formate caused significant dose and development dependent NEC-like injury in newborn mice. Enterobacter cloacae and Klebsiella pneumoniae were the most discriminatory taxa related to NEC dysbiosis and increased formate production. Together, these data suggest a novel biochemical mechanism of NEC through the microbial production of formate. Clinical efforts to prevent NEC should focus on reducing the functional consequences of newborn gut dysbiosis associated metabolic pathways.

FAM83H is involved in the progression of hepatocellular carcinoma and is regulated by MYC.

Recently, the roles of FAM83H in tumorigenesis have been interested and increased expression of FAM83H and MYC in hepatocellular carcinoma (HCC) have been reported. Therefore, we investigated the expression and role of FAM83H in 163 human HCCs and further investigated the relationship between FAM83H and oncogene MYC. The expression of FAM83H is elevated in liver cancer cells, and nuclear expression of FAM83H predicted shorter survival of HCC patients. In HLE and HepG2 HCC cells, knock-down of FAM83H inhibited proliferation and invasive activity of HCC cells. FAM83H induced expression of cyclin-D1, cyclin-E1, snail and MMP2 and inhibited the expression of P53 and P27. In hepatic tumor cells derived from Tet-O-MYC mice, the expression of mRNA and protein of FAM83H were dependent on MYC expression. Moreover, a chromatin immunoprecipitation assay demonstrated that MYC binds to the promotor of FAM83H and that MYC promotes the transcription of FAM83H, which was supported by the results of a dual-luciferase reporter assay. In conclusion, we present an oncogenic role of FAM83H in liver cancer, which is closely associated with the oncogene MYC. In addition, our results suggest FAM83H expression as a poor prognostic indicator of HCC patients.

Impaired Activity of Blood Coagulant Factor XIII in Patients with Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is the most common gastrointestinal (GI) medical/surgical emergency of the newborn and a leading cause of preterm neonate morbidity and mortality. NEC is a challenge to diagnose since it often shares similar clinical features with neonatal sepsis. In the present study, plasma protein profiling was compared among NEC, sepsis and control cohorts using gel electrophoresis, immunoblot and mass spectrometry. We observed significant impairment in the formation of fibrinogen-γ dimers (FGG-dimer) in the plasma of newborns with NEC that could efficiently differentiate NEC and sepsis with a high level of sensitivity and specificity. Interestingly, the impaired FGG-dimer formation could be restored in NEC plasma by the addition of exogenous active factor XIII (FXIII). Enzymatic activity of FXIII was determined to be significantly lower in NEC subject plasma for crosslinking FGG when compared to sepsis. These findings demonstrate a potential novel biomarker and related biologic mechanism for diagnosing NEC, as well as suggest a possible therapeutic strategy.

PKC412 normalizes mutation-related keratin filament disruption and hepatic injury in mice by promoting keratin-myosin binding.

UNLABELLED: Keratins, among other cytoskeletal intermediate filament proteins, are mutated at a highly conserved arginine with consequent severe disease phenotypes due to disruption of keratin filament organization. We screened a kinase inhibitor library, using A549 cells that are transduced with a lentivirus keratin 18 (K18) construct, to identify compounds that normalize filament disruption due to K18 Arg90Cys mutation at the conserved arginine. High-throughput screening showed that PKC412, a multikinase inhibitor, ameliorated K18 Arg90Cys-mediated keratin filament disruption in cells and in the livers of previously described transgenic mice that overexpress K18 Arg90Cys. Furthermore, PKC412 protected cultured A549 cells that express mutant or wild-type K18 and mouse livers of the K18 Arg90Cys-overexpressing transgenic mice from Fas-induced apoptosis. Proteomic analysis of proteins that associated with keratins after exposure of K18-expressing A549 cells to PKC412 showed that nonmuscle myosin heavy chain-IIA (NMHC-IIA) partitions with the keratin fraction. The nonmuscle myosin-IIA (NM-IIA) association with keratins was confirmed by immune staining and by coimmunoprecipitation. The keratin-myosin association is myosin dephosphorylation-dependent; occurs with K8, the obligate K18 partner; is enhanced by PKC412 in cells and mouse liver; and is blocked by hyperphosphorylation conditions in cultured cells and mouse liver. Furthermore, NMHC-IIA knockdown inhibits PKC412-mediated normalization of K18 R90C filaments. CONCLUSION: The inhibitor PKC412 normalizes K18 Arg90Cys mutation-induced filament disruption and disorganization by enhancing keratin association with NM-IIA in a myosin dephosphorylation-regulated manner. Targeting of intermediate filament disorganization by compounds that alter keratin interaction with their associated proteins offers a potential novel therapeutic approach for keratin and possibly other intermediate filament protein-associated diseases.

Keratin 8 modulates ß-cell stress responses and normoglycaemia.

Keratin intermediate filament (IF) proteins are epithelial cell cytoskeletal components that provide structural stability and protection from cell stress, among other cellular and tissue-specific functions. Numerous human diseases are associated with IF gene mutations, but the function of keratins in the endocrine pancreas and their potential significance for glycaemic control are unknown. The impact of keratins on ß-cell organisation and systemic glucose control was assessed using keratin 8 (K8) wild-type (K8(+/+)) and K8 knockout (K8(-/-)) mice. Islet ß-cell keratins were characterised under basal conditions, in streptozotocin (STZ)-induced diabetes and in non-obese diabetic (NOD) mice. STZ-induced diabetes incidence and islet damage was assessed in K8(+/+) and K8(-/-) mice. K8 and K18 were the predominant keratins in islet ß-cells and K8(-/-) mice expressed only remnant K18 and K7. K8 deletion resulted in lower fasting glucose levels, increased glucose tolerance and insulin sensitivity, reduced glucose-stimulated insulin secretion and decreased pancreatic insulin content. GLUT2 localisation and insulin vesicle morphology were disrupted in K8(-/-) ß-cells. The increased levels of cytoplasmic GLUT2 correlated with resistance to high-dose STZ-induced injury in K8(-/-) mice. However, K8 deletion conferred no long-term protection from STZ-induced diabetes and prolonged STZ-induced stress caused increased exocrine damage in K8(-/-) mice. ß-cell keratin upregulation occurred 2 weeks after treatments with low-dose STZ in K8(+/+) mice and in diabetic NOD mice, suggesting a role for keratins, particularly in non-acute islet stress responses. These results demonstrate previously unrecognised functions for keratins in ß-cell intracellular organisation, as well as for systemic blood glucose control under basal conditions and in diabetes-induced stress.

Wnt/ß-catenin signaling protects mouse liver against oxidative stress-induced apoptosis through the inhibition of forkhead transcription factor FoxO3.

Numerous liver diseases are associated with extensive oxidative tissue damage. It is well established that Wnt/ß-catenin signaling directs multiple hepatocellular processes, including development, proliferation, regeneration, nutrient homeostasis, and carcinogenesis. It remains unexplored whether Wnt/ß-catenin signaling provides hepatocyte protection against hepatotoxin-induced apoptosis. Conditional, liver-specific ß-catenin knockdown (KD) mice and their wild-type littermates were challenged by feeding with a hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet to induce chronic oxidative liver injury. Following the DDC diet, mice with ß-catenin-deficient hepatocytes demonstrate increased liver injury, indicating an important role of ß-catenin signaling for liver protection against oxidative stress. This finding was further confirmed in AML12 hepatocytes with ß-catenin signaling manipulation in vitro using paraquat, a known oxidative stress inducer. Immunofluorescence staining revealed an intense nuclear FoxO3 staining in ß-catenin-deficient livers, suggesting active FoxO3 signaling in response to DDC-induced liver injury when compared with wild-type controls. Consistently, FoxO3 target genes p27 and Bim were significantly induced in ß-catenin KD livers. Conversely, SGK1, a ß-catenin target gene, was significantly impaired in ß-catenin KD hepatocytes that failed to inactivate FoxO3. Furthermore, shRNA-mediated deletion of FoxO3 increased hepatocyte resistance to oxidative stress-induced apoptosis, confirming a proapoptotic role of FoxO3 in the stressed liver. Our findings suggest that Wnt/ß-catenin signaling is required for hepatocyte protection against oxidative stress-induced apoptosis. The inhibition of FoxO through its phosphorylation by ß-catenin-induced SGK1 expression reduces the apoptotic function of FoxO3, resulting in increased hepatocyte survival. These findings have relevance for future therapies directed at hepatocyte protection, regeneration, and anti-cancer treatment.

SIRT1 and c-Myc promote liver tumor cell survival and predict poor survival of human hepatocellular carcinomas.

The increased expression of SIRT1 has recently been identified in numerous human tumors and a possible correlation with c-Myc oncogene has been proposed. However, it remains unclear whether SIRT1 functions as an oncogene or tumor suppressor. We sought to elucidate the role of SIRT1 in liver cancer under the influence of c-Myc and to determine the prognostic significance of SIRT1 and c-Myc expression in human hepatocellular carcinoma. The effect of either over-expression or knock down of SIRT1 on cell proliferation and survival was evaluated in both mouse and human liver cancer cells. Nicotinamide, an inhibitor of SIRT1, was also evaluated for its effects on liver tumorigenesis. The prognostic significance of the immunohistochemical detection of SIRT1 and c-Myc was evaluated in 154 hepatocellular carcinoma patients. SIRT1 and c-Myc regulate each other via a positive feedback loop and act synergistically to promote hepatocellular proliferation in both mice and human liver tumor cells. Tumor growth was significantly inhibited by nicotinamide in vivo and in vitro. In human hepatocellular carcinoma, SIRT1 expression positively correlated with c-Myc, Ki67 and p53 expression, as well as high á-fetoprotein level. Moreover, the expression of SIRT1, c-Myc and p53 were independent prognostic indicators of hepatocellular carcinoma. In conclusion, this study demonstrates that SIRT1 expression supports liver tumorigenesis and is closely correlated with oncogenic c-MYC expression. In addition, both SIRT1 and c-Myc may be useful prognostic indicators of hepatocellular carcinoma and SIRT1 targeted therapy may be beneficial in the treatment of hepatocellular carcinoma.

ß-Catenin regulates hepatic mitochondrial function and energy balance in mice.

BACKGROUND & AIMS: Wnt signaling regulates hepatic function and nutrient homeostasis. However, little is known about the roles of ß-catenin in cellular respiration or mitochondria of hepatocytes. METHODS: We investigated ß-catenin's role in the metabolic function of hepatocytes under homeostatic conditions and in response to metabolic stress using mice with hepatocyte-specific deletion of ß-catenin and their wild-type littermates, given either saline (sham) or ethanol (as a model of binge drinking and acute ethanol intoxication). RESULTS: Under homeostatic conditions, ß-catenin-deficient hepatocytes demonstrated mitochondrial dysfunctions that included impairments to the tricarboxylic acid cycle and oxidative phosphorylation (OXPHOS) and decreased production of adenosine triphosphate (ATP). There was no evidence for redox imbalance or oxidative cellular injury in the absence of metabolic stress. In mice with ß-catenin-deficient hepatocytes, ethanol intoxication led to significant redox imbalance in the hepatocytes and further deterioration in mitochondrial function that included reduced OXPHOS, fatty acid oxidation (FAO), and ATP production. Ethanol feeding significantly increased liver steatosis and oxidative damage, compared with wild-type mice, and disrupted the ratio of nicotinamide adenine dinucleotide. ß-catenin-deficient hepatocytes also had showed disrupted signaling of Sirt1/peroxisome proliferator-activated receptor-α signaling. CONCLUSIONS: ß-catenin has an important role in the maintenance of mitochondrial homeostasis, regulating ATP production via the tricarboxylic acid cycle, OXPHOS, and fatty acid oxidation; ß-catenin function in these systems is compromised under conditions of nutrient oxidative stress. Reagents that alter Wnt-ß-catenin signaling might be developed as a useful new therapeutic strategy for treatment of liver disease.

Intestinal involvement during 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced chronic liver injury in a mouse model.

PURPOSE: Although a physiologic relationship between intestinal mucosal integrity and hepatic function has been previously described, the effect of primary liver disease on intestinal mucosal homeostasis has not been previously well documented. In the current study, we studied the effects of chronic liver injury as a primary injury on enterocyte turnover (proliferation and apoptosis) in a mouse model. METHODS: The liver toxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-enriched diet was used to induce chronic cholestatic liver injury in mice. Livers and intestine were harvested after 3 weeks of dietary treatment of histologic analysis and a determination of cell proliferation (immunohistochemistry for Ki67), or apoptosis (immunohistochemistry for caspase-3), as well as a determination of Wnt/ß-catenin signaling activity. RESULTS: All DDC-fed animals exhibited histologic evidence of liver damage that was associated with the expansion of atypical ductal proliferation near the periportal areas and increased oxidative stress. In the intestine, DDC-induced liver damage was associated with decreased villus height, decreased enterocyte proliferation, and increased cell apoptosis compared with control animals. There was also evidence for decreased ß-catenin expression by immunostaining in crypt and villus cells of DDC-fed mice compared with control animals. CONCLUSION: Primary liver injury and cholestasis is associated with intestinal mucosal hypoplasia. Decreased cell proliferation and increased cell apoptosis may be responsible for decreased intestinal epithelial cell mass. The observed decrease in cell turnover is accompanied by an alteration in Wnt/ß-catenin signaling.

Wnt-ß-catenin signaling protects against hepatic ischemia and reperfusion injury in mice.

BACKGROUND & AIMS: Ischemia and reperfusion injury are common causes of oxidative tissue damage associated with many liver diseases and hepatic surgery. The Wnt-ß-catenin signaling pathway is an important regulator of hepatic development, regeneration, and carcinogenesis. However, the role of Wnt signaling in the hepatocellular response to ischemia-reperfusion (I/R) injury has not been determined. METHODS: Hepatic injury following ischemia or I/R was investigated in hepatocyte-specific, ß-catenin-deficient mice, as well as Wnt1-overexpressing and wild-type (control) mice. RESULTS: Wnt-ß-catenin signaling was affected by the cellular redox balance in hepatocytes. Following ischemia or I/R, mice with ß-catenin-deficient hepatocytes were significantly more susceptible to liver injury. Conversely, mice that overexpressed Wnt1 in hepatocytes were resistant to hepatic I/R injury. Hypoxia inducible factor (HIF)-1α signaling was reduced in ß-catenin-deficient liver but increased in hepatocytes that overexpressed Wnt1 under hypoxia and following I/R, indicating an interaction between ß-catenin and HIF-1α signaling in the liver. The mechanism by which Wnt signaling protects against liver injury involves the role of ß-catenin as a transcriptional coactivator of HIF-1α signaling, which promotes hepatocyte survival under hypoxic conditions. CONCLUSIONS: Cellular redox balance affects Wnt-ß-catenin signaling, which protects against hypoxia and I/R injury. These findings might be used to develop strategies for protection of hepatocytes, regeneration of liver, and inhibition of carcinogenesis.

Keratins modulate the shape and function of hepatocyte mitochondria: a mechanism for protection from apoptosis.

Absence or mutation of keratins 8 (K8) or 18 (K18) cause predisposition to liver injury and apoptosis. We assessed the mechanisms of hepatocyte keratin-mediated cytoprotection by comparing the protein expression profiles of livers from wild-type and K8-null mice using two-dimensional differential-in-gel-electrophoresis (2D-DIGE) and mass spectrometry. Prominent among the alterations were those of mitochondrial proteins, which were confirmed using 2D-DIGE of purified mitochondria. Ultrastructural analysis showed that mitochondria of livers that lack or have disrupted keratins are significantly smaller than mitochondria of wild-type livers. Immunofluorescence staining showed irregular distribution of mitochondria in keratin-absent or keratin-mutant livers. K8-null livers have decreased ATP content; and K8-null mitochondria have less cytochrome c, increased release of cytochrome c after exposure to Ca(2+) and oxidative stimulation, and a higher sensitivity to Ca(2+)-induced permeability transition. Therefore, keratins play a direct or indirect role in regulating the shape and function of mitochondria. The effects of keratin mutation on mitochondria are likely to contribute to hepatocyte predisposition to apoptosis and oxidative injury, and to play a pathogenic role in keratin-mutation-related human liver disease.

Keratin variants are overrepresented in primary biliary cirrhosis and associate with disease severity.

UNLABELLED: Keratins (K) 8 and 18 variants predispose carriers to the development of end-stage liver disease and patients with chronic hepatitis C to disease progression. Hepatocytes express K8/K18, whereas biliary epithelia express K8/K18/K19. K8-null mice, which are predisposed to liver injury, spontaneously develop anti-mitochondrial antibodies (AMA) and have altered hepatocyte mitochondrial size and function. There is no known association of K19 with human disease and no known association of K8/K18/K19 with human autoimmune liver disease. We tested the hypothesis that K8/K18/K19 variants associate with primary biliary cirrhosis (PBC), an autoimmune cholestatic liver disease characterized by the presence of serum AMA. In doing so, we analyzed the entire exonic regions of K8/K18/K19 in 201 Italian patients and 200 control blood bank donors. Five disease-associated keratin heterozygous variants were identified in patients versus controls (K8 G62C/R341H/V380I, K18 R411H, and K19 G17S). Four variants were novel and included K19 G17S/V229M/N184N and K18 R411H. Overall, heterozygous disease-associated keratin variants were found in 17 of 201 (8.5%) PBC patients and 4 of 200 (2%) blood bank donors (P < 0.004, odds ratio = 4.53, 95% confidence interval = 1.5-13.7). Of the K19 variants, K19 G17S was found in three patients but not in controls and all K8 R341H (eight patients and three controls) associated with concurrent presence of the previously described intronic K8 IVS7+10delC deletion. Notably, keratin variants associated with disease severity (12.4% variants in Ludwig stage III/IV versus 4.2% in stages I/II; P < 0.04, odds ratio = 3.25, 95% confidence interval = 1.02-10.40), but not with the presence of AMA. CONCLUSION: K8/K18/K19 variants are overrepresented in Italian PBC patients and associate with liver disease progression. Therefore, we hypothesize that K8/K18/K19 variants may serve as genetic modifiers in PBC.

"Toxic memory" via chaperone modification is a potential mechanism for rapid Mallory-Denk body reinduction.

UNLABELLED: The cytoplasmic hepatocyte inclusions, Mallory-Denk bodies (MDBs), are characteristic of several liver disorders, including alcoholic and nonalcoholic steatohepatitis. In mice, MDBs can be induced by long-term feeding with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) for 3 to 4 months or rapidly reformed in DDC-induced then recovered mice by DDC refeeding or exposure to a wide range of toxins for only 5 to 7 days. The molecular basis for such a rapid reinduction of MDBs is unknown. We hypothesized that protein changes retained after DDC priming contribute to the rapid MDB reappearance and associate with MDB formation in general terms. Two-dimensional differential-in-gel-electrophoresis coupled with mass spectrometry were used to characterize protein changes in livers from the various treatment groups. The alterations were assessed by real-time reverse-transcription polymerase chain reaction and confirmed by immunoblotting. DDC treatment led to pronounced charged isoform changes in several chaperone families, including Hsp25, 60, 70, GRP58, GRP75, and GRP78, which lasted at least for 1 month after discontinuation of DDC feeding, whereas changes in other proteins normalized during recovery. DDC feeding also resulted in altered expression of Hsp72, GRP75, and Hsp25 and in functional impairment of Hsp60 and Hsp70 as determined using a protein complex formation and release assay. The priming toward rapid MDB reinduction lasts for at least 3 months after DDC discontinuation, but becomes weaker after prolonged recovery. MDB reinduction parallels the rapid increase in p62 and Hsp25 levels as well as keratin 8 cross-linking that is normally associated with MDB formation. CONCLUSION: Persistent posttranslational modifications in chaperone proteins, coupled with protein cross-linking and altered chaperone expression and function likely contribute to the "toxic memory" of DDC-primed mice. We hypothesize that similar changes are important contributors to inclusion body formation in several diseases.

Keratin mutation predisposes to mouse liver fibrosis and unmasks differential effects of the carbon tetrachloride and thioacetamide models.

BACKGROUND & AIMS: Keratins 8 and 18 (K8/K18) are important hepatoprotective proteins. Animals expressing K8/K18 mutants show a marked susceptibility to acute/subacute liver injury. K8/K18 variants predispose to human end-stage liver disease and associate with fibrosis progression during chronic hepatitis C infection. We sought direct evidence for a keratin mutation-related predisposition to liver fibrosis using transgenic mouse models because the relationship between keratin mutations and cirrhosis is based primarily on human association studies. METHODS: Mouse hepatofibrosis was induced by carbon tetrachloride (CCl(4)) or thioacetamide. Nontransgenic mice, or mice that over express either human Arg89-to-Cys (R89C mice) or wild-type K18 (WT mice) were used. The extent of fibrosis was evaluated by quantitative real-time reverse-transcription polymerase chain reaction of fibrosis-related genes, liver hydroxyproline measurement, and Picro-Sirius red staining and collagen immunofluorescence staining. RESULTS: Compared with control animals, CCl(4) led to similar liver fibrosis but increased injury in K18 R89C mice. In contrast, thioacetamide caused more severe liver injury and fibrosis in K18 R89C as compared with WT and nontransgenic mice and resulted in increased messenger RNA levels of collagen, tissue inhibitor of metalloproteinase 1, matrix metalloproteinase 2, and matrix metalloproteinase 13. Analysis in nontransgenic mice showed that thioacetamide and CCl(4) have dramatically different molecular expression responses involving cytoskeletal and chaperone proteins. CONCLUSIONS: Over expression of K18 R89C predisposes transgenic mice to thioacetamide- but not CCl(4)-induced liver fibrosis. Differences in the keratin mutation-associated fibrosis response among the 2 models raise the hypothesis that keratin variants may preferentially predispose to fibrosis in unique human liver diseases. Findings herein highlight distinct differences in the 2 widely used fibrosis models.

Keratins let liver live: Mutations predispose to liver disease and crosslinking generates Mallory-Denk bodies.

Keratin polypeptides 8 and 18 (K8/K18) are the cytoskeletal intermediate filament proteins of hepatocytes while K8/K18/K19 are the keratins of hepatobiliary ductal cells. Hepatocyte K8/K18 are highly abundant and behave as stress proteins with injury-inducible expression. Human association studies show that K8/K18 germline heterozygous mutations predispose to end-stage liver disease of multiple etiologies ( approximately 3 fold increased risk), and to liver disease progression in patients with chronic hepatitis C infection. These findings are supported by extensive transgenic mouse and ex vivo primary hepatocyte culture studies showing that K8 or K18 mutations predispose the liver to acute or subacute injury and promote apoptosis and fibrosis. Mutation-associated predisposition to liver injury is likely related to mechanical and nonmechanical keratin functions including maintenance of cell integrity, protection from apoptosis and oxidative injury, serving as a phosphate sponge, regulation of mitochondrial organization/function and protein targeting. These functions are altered by mutation-induced changes in keratin phosphorylation, solubility and filament organization/reorganization. Keratins are also the major constituents of Mallory-Denk bodies (MDBs). A toxin-induced K8>K18 ratio, and keratin crosslinking by transglutaminase-2 play essential roles in MDB formation. Furthermore, intracellular or cell-released K18 fragments, generated by caspase-mediated proteolysis during apoptosis serve as markers of liver injury. Therefore, K8 and K18 are cytoprotective stress proteins that play a central role in guarding hepatocytes from apoptosis. Keratin involvement in liver disease is multi-faceted and includes modulating disease progression upon mutation, formation of MDBs in response to unique forms of injury, and serving as markers of epithelial cell death.

Reg-II is an exocrine pancreas injury-response product that is up-regulated by keratin absence or mutation.

The major keratins in the pancreas and liver are keratins 8 and 18 (K8/K18), but their function seemingly differs in that liver K8/K18 are essential cytoprotective proteins, whereas pancreatic K8/K18 are dispensable. This functional dichotomy raises the hypothesis that K8-null pancreata may undergo compensatory cytoprotective gene expression. We tested this hypothesis by comparing the gene expression profile in pancreata of wild-type and K8-null mice. Most prominent among the up-regulated genes in K8-null pancreas was mRNA for regenerating islet-derived (Reg)-II, which was confirmed by quantitative reverse transcription-polymerase chain reaction and by an anti-Reg-II peptide antibody we generated. Both K8-null and wild-type mice express Reg-II predominantly in acinar cells as determined by in situ hybridization and immunostaining. Analysis of Reg-II expression in various keratin-related transgenic mouse models showed that its induction also occurs in response to keratin cytoplasmic filament collapse, absence, or ablation of K18 Ser52 but not Ser33 phosphorylation via Ser-to-Ala mutation, which represent situations associated with predisposition to liver but not pancreatic injury. In wild-type mice, Reg-II is markedly up-regulated in two established pancreatitis models in response to injury and during the recovery phase. Thus, Reg-II is a likely mouse exocrine pancreas cytoprotective candidate protein whose expression is regulated by keratin filament organization and phosphorylation.

Analysis of keratin polypeptides 8 and 19 variants in inflammatory bowel disease.

BACKGROUND/AIMS: Keratin-8 (KRT8)-null mice develop spontaneous colitis and predisposition to liver injury. Human studies show that some KRT8 variants predispose to end-stage liver disease and progression and suggest that such variants might associate with UC or CD. We asked whether mutations in KRT8 or KRT19, the major intestinal keratins, are associated with UC/CD. METHODS: Exonic regions of the KRT8/KRT19 genes were polymerase chain reaction-amplified using genomic DNA from 2 independent groups. Group I included 91 unrelated patients with CD, 93 unrelated patients with UC, and 70 unrelated/unaffected volunteers. KRT8 variants were also tested with pyrosequencing in Group II that included 682 independent nuclear families with both parents and at least 1 CD/UC-affected offspring and 273 unaffected controls. Both cohorts were enriched for familial IBD. RESULTS: In Group I, KRT19 variants were identified in CD/UC patients within the promoter and exons 1+2, with similar mutation frequencies in the control/CD/UC groups. In contrast, 16 of 184 CD+UC patients harbored KRT8 heterozygous variants involving Gly62-to-Cys and Arg341-to-His and a novel Arg341-to-Cys, which were noted in 4 volunteers (Arg341-to-His) and correlated with extensive UC (P = .005). One family with unaffected parents had 3 pediatric-affected siblings with severe disease, 2 of whom are compound heterozygous (Gly62-to-Cys/Arg341-to-His). However, there was no significant departure from random transmission of the 3 alleles in Group II IBD families. CONCLUSIONS: KRT8 and KRT19 variants are not overtransmitted or associated with familial IBD, although a potential role in sporadic IBD cannot be excluded. A novel but rare keratin-8 Arg341-to-Cys is identified in IBD patients.

Reciprocal keratin 18 Ser48 O-GlcNAcylation and Ser52 phosphorylation using peptide analysis.

Phosphorylation and O-GlcNAcylation of keratin 18 (K18) are highly dynamic and involve primarily independent K18 populations. We used in vitro phosphorylation and O-GlcNAcylation of wild-type, phospho-Ser52, glyco-Ser48, and Ser-to-Ala mutant 17mer peptides (K18 amino acids 40-56), which include the major K18 glycosylation (Ser48) and phosphorylation (Ser52) sites, to address whether each modification blocks the other. The glyco-K18 peptide blocks Ser52 phosphorylation by protein kinase C, an in vivo K18 kinase, while the phospho-K18 peptide blocks its O-GlcNAcylation. Our findings support the reciprocity of these two post-translational modifications. Therefore, regulation of protein Ser/Thr phosphorylation and glycosylation at proximal sites can be interdependent and provides a potential mechanism of counter regulation.