Sujith Weerasinghe

Keratin hypersumoylation alters filament dynamics and is a marker for human liver disease and keratin mutation.

Keratin polypeptide 8 (K8) associates noncovalently with its partners K18 and/or K19 to form the intermediate filament cytoskeleton of hepatocytes and other simple-type epithelial cells. Human K8, K18, and K19 variants predispose to liver disease, whereas site-specific keratin phosphorylation confers hepatoprotection. Because stress-induced protein phosphorylation regulates sumoylation, we hypothesized that keratins are sumoylated in an injury-dependent manner and that keratin sumoylation is an important regulatory modification. We demonstrate that K8/K18/K19, epidermal keratins, and vimentin are sumoylated in vitro. Upon transfection, K8, K18, and K19 are modified by poly-SUMO-2/3 chains on Lys-285/Lys-364 (K8), Lys-207/Lys-372 (K18), and Lys-208 (K19). Sumoylation affects filament organization and stimulus-induced keratin solubility and is partially inhibited upon mutation of one of three known K8 phosphorylation sites. Extensive sumoylation occurs in cells transfected with individual K8, K18, or K19 but is limited upon heterodimerization (K8/K18 or K8/K19) in the absence of stress. In contrast, keratin sumoylation is significantly augmented in cells and tissues during apoptosis, oxidative stress, and phosphatase inhibition. Poly-SUMO-2/3 conjugates are present in chronically injured but not normal, human, and mouse livers along with polyubiquitinated and large insoluble keratin-containing complexes. Notably, common human K8 liver disease-associated variants trigger keratin hypersumoylation with consequent diminished solubility. In contrast, modest sumoylation of wild type K8 promotes solubility. Hence, conformational changes induced by keratin natural mutations and extensive tissue injury result in K8/K18/K19 hypersumoylation, which retains keratins in an insoluble compartment, thereby limiting their cytoprotective function.

Energy determinants GAPDH and NDPK act as genetic modifiers for hepatocyte inclusion formation.

Differential expression and activity of the cellular energy regulators GAPDH and NDPK underlie reactive oxygen species–induced damage in the mouse liver and may contribute to human liver disease progression.

Fibrinogen-γ proteolysis and solubility dynamics during apoptotic mouse liver injury: heparin prevents and treats liver damage.

Fas ligand (FasL)‐mediated hepatocyte apoptosis occurs in the context of acute liver injury that can be accompanied by intravascular coagulation (IC). We tested the hypothesis that analysis of selected protein fractions from livers undergoing apoptosis will shed light on mechanisms that are involved in liver injury that might be amenable to intervention. Proteomic analysis of the major insoluble liver proteins after FasL exposure for 4‐5 hours identified fibrinogen‐γ (FIB‐γ) dimers and FIB‐γ–containing high molecular mass complexes among the major insoluble proteins visible via Coomassie blue staining. Presence of the FIB‐γ–containing products was confirmed using FIB‐γ–specific antibodies. The FIB‐γ–containing products partition selectively and quantitatively into the liver parenchyma after inducing apoptosis. Similar formation of FIB‐γ products occurs after acetaminophen administration. The observed intrahepatic IC raised the possibility that heparin therapy may ameliorate FasL‐mediated liver injury. Notably, heparin administration in mice 4 hours before or up to 2 hours after FasL injection resulted in a dramatic reduction of liver injury—including liver hemorrhage, serum alanine aminotransferase, caspase activation, and liver apoptosis—compared with heparin‐untreated mice. Heparin did not directly interfere with FasL‐induced apoptosis in isolated hepatocytes, and heparin‐treated mice survived the FasL‐induced liver injury longer compared with heparin‐untreated animals. There was a sharp, near‐simultaneous rise in FasL‐induced intrahepatic apoptosis and coagulation, with IC remaining stable while apoptosis continued to increase. Conclusion: Formation of FIB‐γ dimers and their high molecular mass products are readily detectable within the liver during mouse apoptotic liver injury. Heparin provides a potential therapeutic modality, because it not only prevents extensive FasL‐related liver injury but also limits the extent of injury if given at early stages of injury exposure. (HEPATOLOGY 2011;)

Mutation of caspase-digestion sites in keratin 18 interferes with filament reorganization, and predisposes to hepatocyte necrosis and loss of membrane integrity

ABSTRACT Keratin 18 (K18 or KRT18) undergoes caspase-mediated cleavage during apoptosis, the significance of which is poorly understood. Here, we mutated the two caspase-cleavage sites (D238E and D397E) in K18 (K18-DE), followed by transgenic overexpression of the resulting mutant. We found that K18-DE mice develop extensive Fas-mediated liver damage compared to wild-type mice overexpressing K18 (K18-WT). Fas-stimulation of K18-WT mice or isolated hepatocytes caused K18 degradation. By contrast, K18-DE livers or hepatocytes maintained intact keratins following Fas-stimulation, but showed hypo-phosphorylation at a major stress-kinase-related keratin 8 (K8) phosphorylation site. Although K18-WT and K18-DE hepatocytes showed similar Fas-mediated caspase activation, K18-DE hepatocytes were more ‘leaky’ after a mild hypoosmotic challenge and were more susceptible to necrosis after Fas-stimulation or severe hypoosmotic stress. K8 hypophosphorylation was not due to the inhibition of kinase binding to the keratin but was due to mutation-induced inaccessibility to the kinase that phosphorylates K8. A stress-modulated keratin phospho-mutant expressed in hepatocytes phenocopied the hepatocyte susceptibility to necrosis but was found to undergo keratin filament reorganization during apoptosis. Therefore, the caspase cleavage of keratins might promote keratin filament reorganization during apoptosis. Interference with keratin caspase cleavage shunts hepatocytes towards necrosis and increases liver injury through the inhibition of keratin phosphorylation. These findings might extend to other intermediate filament proteins that undergo proteolysis during apoptosis.

Carbamoyl phosphate synthetase-1 is a rapid turnover biomarker in mouse and human acute liver injury

Several serum markers are used to assess hepatocyte damage, but they have limitations related to etiology specificity and prognostication. Identification of novel hepatocyte-specific biomarkers could provide important prognostic information and better pathogenesis classification. We tested the hypothesis that hepatocyte-selective biomarkers are released after subjecting isolated mouse hepatocytes to Fas-ligand-mediated apoptosis. Proteomic analysis of hepatocyte culture medium identified the mitochondrial matrix protein carbamoyl phosphate synthetase-1 (CPS1) among the most readily detected proteins that are released by apoptotic hepatocytes. CPS1 was also detected in mouse serum upon acute challenge with Fas-ligand or acetaminophen and in hepatocytes upon hypoosmotic stress, independent of hepatocyte caspase activation. Furthermore, CPS1 was observed in sera of mice chronically fed the hepatotoxin 3,5-diethoxycarbonyl-1,4-dihydrocollidine. Mouse CPS1 detectability was similar in serum and plasma, and its half-life was 126 ± 9 min. Immune staining showed that CPS1 localized to mouse hepatocytes but not ductal cells. Analysis of a few serum samples from patients with acute liver failure (ALF) due to acetaminophen, Wilson disease, or ischemia showed readily detectable CPS1 that was not observed in several patients with chronic viral hepatitis or in control donors. Notably, CPS1 rapidly decreased to undetectable levels in sera of patients with acetaminophen-related ALF who ultimately recovered, while alanine aminotransferase levels remained elevated. Therefore, CPS1 becomes readily detectable upon hepatocyte apoptotic and necrotic death in culture or in vivo. Its abundance and short serum half-life, compared with alanine aminotransferase, suggest that it may be a useful prognostic biomarker in human and mouse liver injury.

CD73 (ecto‐5′‐nucleotidase) hepatocyte levels differ across mouse strains and contribute to mallory‐denk body formation

Formation of hepatocyte Mallory‐Denk bodies (MDBs), which are aggregates of keratins 8 and 18 (K8/K18), ubiquitin, and the ubiquitin‐binding protein, p62, has a genetic predisposition component in humans and mice. We tested the hypothesis that metabolomic profiling of MDB‐susceptible C57BL and MDB‐resistant C3H mouse strains can illuminate MDB‐associated pathways. Using both targeted and unbiased metabolomic analyses, we demonstrated significant differences in intermediates of purine metabolism. Further analysis revealed that C3H and C57BL livers differ significantly in messenger RNA (mRNA) level, protein expression, and enzymatic activity of the adenosine‐generating enzyme, ecto‐5′‐nucleotidase (CD73), which was significantly lower in C57BL livers. CD73 mRNA levels were also dramatically decreased in human liver biopsies from hepatitis C and nonalcoholic fatty liver disease patients. Feeding mice with a diet containing the MDB‐inducing agent, 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC), significantly decreased CD73 protein and activity in C57BL livers and resulted in loss of plasma membrane CD73 expression and activity in isolated mouse hepatocytes. To further examine the role of CD73 in MDB formation in vivo, we fed wild‐type (WT) and CD73−/− mice a DDC‐containing diet. Liver enlargement, p62 induction, and disappearance of the K8/K18 cytoskeleton were attenuated in CD73−/−, compared to WT livers. MDB formation, as assessed by biochemical and immunofluorescence detection of keratin and ubiquitin complexes, was nearly absent in CD73−/− mice. Conclusion: Purine metabolism and CD73 expression are linked to susceptibility to MDB formation in livers of different mouse strains. Expression of the adenosine‐generating enzyme, CD73, contributes to experimental MDB induction and is highly regulated in MDB‐associated liver injury in mice and in chronic human liver disease. (Hepatology 2013;58:1790–1800)

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

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.(Hepatology 2015;62:1858–1869)

Hepatocyte-Specific Deletion of Mouse Lamin A/C Leads to Male-Selective Steatohepatitis

Background & Aims Lamins are nuclear intermediate filament proteins that comprise the major components of the nuclear lamina. Mutations in LMNA, which encodes lamins A/C, cause laminopathies, including lipodystrophy, cardiomyopathy, and premature aging syndromes. However, the role of lamins in the liver is unknown, and it is unclear whether laminopathy-associated liver disease is caused by primary hepatocyte defects or systemic alterations. Methods To address these questions, we generated mice carrying a hepatocyte-specific deletion of Lmna (knockout [KO] mice) and characterized the KO liver and primary hepatocyte phenotypes by immunoblotting, immunohistochemistry, microarray analysis, quantitative real-time polymerase chain reaction, and Oil Red O and Picrosirius red staining. Results KO hepatocytes manifested abnormal nuclear morphology, and KO mice showed reduced body mass. KO mice developed spontaneous male-selective hepatosteatosis with increased susceptibility to high-fat diet–induced steatohepatitis and fibrosis. The hepatosteatosis was associated with up-regulated transcription of genes encoding lipid transporters, lipid biosynthetic enzymes, lipid droplet-associated proteins, and interferon-regulated genes. Hepatic Lmna deficiency led to enhanced signal transducer and activator of transcription 1 (Stat1) expression and blocked growth hormone–mediated Janus kinase 2 (Jak2), signal transducer and activator of transcription 5 (Stat5), and extracellular signal–regulated kinase (Erk) signaling. Conclusions Lamin A/C acts cell-autonomously to maintain hepatocyte homeostasis and nuclear shape and buffers against male-selective steatohepatitis by positively regulating growth hormone signaling and negatively regulating Stat1 expression. Lamins are potential genetic modifiers for predisposition to steatohepatitis and liver fibrosis. The microarray data can be found in the Gene Expression Omnibus repository (accession number: GSE93643).

Mouse genetic background contributes to hepatocyte susceptibility to Fas-mediated apoptosis

Mouse strain–dependent selective loss of apoptosis enzymes, with consequent decrease in susceptibility to apoptosis, occurs upon short-term hepatocyte culture and in vivo. These susceptibility differences likely reflect genetic modifiers that provide resistance or predisposition to hepatocyte death.

Mo1772 Kinase Inhibition Normalizes Mutation-Mediated Keratin Filament Disruption and Predisposition to Apoptosis and Liver Injury

Objective: Mutations in members of the cytoskeletal intermediate filament proteins (IFs), keratin polypeptides 8 and 18 (K8/K18), predispose their carriers to acute and chronic liver disease progression. Keratin associated diseases are among .70 human diseases that are caused or predisposed to by IF mutations, with the defining feature in many of these diseases being disruption of the normal IF organization within the cytoplasm. In contrast to microfilaments and microtubules, no drugs are available for IFs that can modulate their organization and potentially provide a sorely lacking therapy. Methods: We used an imagingbased drug screening platform consisting of lung carcinoma A549 cells transduced with lentiviruses containing GFP-tagged K18 R90C and wild-type K8. The cells were screened against several compound libraries, and a select compound was further tested in cell culture and in livers of mice that express mutant K18 in order to define its mechanism of action. Results: Screening of a 320-compound kinase inhibitor library showed that the inhibitors of protein kinase C (PKC) and tyrosine kinases, particularly MIDOSTAURIN, led to significant correction of the defective organization of K18 R90C puncta towards normal filaments after 24 h with a maximal effect at 48 h. The findings were confirmed using multiple MIDOSTAURIN concentrations and in another cell line, Hacat, that expresses mutant K14. While MIDOSTAURIN diminished the expression of the mutant keratin 14 in Hacat cells, the effect in the A549 cells was independent of K18 expression but causes changes in K18 phosphorylation that are known to regulate filament formation. MIDOSTAURIN significantly prevented interferon and FAS ligand-induced apoptosis in A549 cells. The protective effect occurs after 48 h but not after 1 h treatment with MIDOSTAURIN, suggesting that the drug effect is more likely to be related to the correction of filament organization. In mice, administration of MIDOSTAURIN is well tolerated and its administration prior to exposure to FAS results in dramatic protection from liver injury. Conclusion: The kinase inhibitor MIDOSTAURIN alleviates cytoskeletal structural defects caused by K18 R90C mutation and also protects from apoptosis in cultured and in the livers of mice exposed to FAS ligand. MIDOSTAURIN may be a potential therapeutic for IF diseases and, notably, it is presently undergoing clinical trials in patients with acute myeloid leukemia and gastrointestinal stromal tumors.