Role of PINCH and Its Partner Tumor Suppressor Rsu-1 in Regulating Liver Size and Tumorigenesis

Particularly interesting new cysteine-histidine-rich protein (PINCH) protein is part of the ternary complex known as the IPP (integrin linked kinase (ILK)-PINCH-Parvin-α) complex. PINCH itself binds to ILK and to another protein known as Rsu-1 (Ras suppressor 1). We generated PINCH 1 and PINCH 2 Double knockout mice (referred as PINCH DKO mice). PINCH2 elimination was systemic whereas PINCH1 elimination was targeted to hepatocytes. The genetically modified mice were born normal. The mice were sacrificed at different ages after birth. Soon after birth, they developed abnormal hepatic histology characterized by disorderly hepatic plates, increased proliferation of hepatocytes and biliary cells and increased deposition of extracellular matrix. After a sustained and prolonged proliferation of all epithelial components, proliferation subsided and final liver weight by the end of 30 weeks in livers with PINCH DKO deficient hepatocytes was 40% larger than the control mice. The livers of the PINCH DKO mice were also very stiff due to increased ECM deposition throughout the liver, with no observed nodularity. Mice developed liver cancer by one year. These mice regenerated normally when subjected to 70% partial hepatectomy and did not show any termination defect. Ras suppressor 1 (Rsu-1) protein, the binding partner of PINCH is frequently deleted in human liver cancers. Rsu-1 expression is dramatically decreased in PINCH DKO mouse livers. Increased expression of Rsu-1 suppressed cell proliferation and migration in HCC cell lines. These changes were brought about not by affecting activation of Ras (as its name suggests) but by suppression of Ras downstream signaling via RhoGTPase proteins. In conclusion, our studies suggest that removal of PINCH results in enlargement of liver and tumorigenesis. Decreased levels of Rsu-1, a partner for PINCH and a protein often deleted in human liver cancer, may play an important role in the development of the observed phenotype. © 2013 Donthamsetty et al

Oct4 Is Crucial for Transdifferentiation of Hepatocytes to Biliary Epithelial Cells in an in Vitro Organoid Culture Model

BACKGROUND: Hepatocyte to biliary transdifferentiation has been documented in various models of bile duct injury. In this process, mature hepatocytes transform into mature biliary epithelial cells by acquiring biliary phenotypic markers. Several signaling pathways including PI3 kinase, Notch, Hes1, Sox9, and Hippo are shown to be involved in the process. However, if Oct4 is involved in hepatocyte to biliary transdifferentiation is unknown. METHODS: We investigated the role of Oct4 in hepatocyte to biliary transdifferentiation utilizing an in vitro organoid culture system as a model of transdifferentiation. Oct4 was inhibited using adenovirus containing Oct4 shRNA. Hepatocyte specific HNF-4α and biliary specific HNF-1β & CK19 expression were assessed to gauge the extent of transdifferentiation. RESULTS: Oct4 was induced during hepatocyte to biliary transdifferentiation. Oct4 inhibition significantly downregulated the appearance of biliary cells from hepatocytes. This was accompanied by a significant downregulation of signaling pathways including Notch, Sox9, and Hippo. CONCLUSION: Our findings suggest that Oct4 is crucial for hepatocyte to biliary transdifferentiation and maturation and that it acts upstream of Notch, Sox9, and Hippo signaling in this model. This finding identifies new signaling through Oct4 in plasticity between hepatocytes and biliary epithelial cells, which can be potentially utilized to identify new strategies in chronic biliary diseases

The Role of Secretory Phospholipase A2 (sPLA2) Inhibitor Varespladib in Mitigation of Acetaminophen(APAP) Induced Acute Liver Failure

INTRODUCTION:Progression of acute liver injury continues long after most of the toxicant has been eliminated from the body. sPLA2 has been previously shown to mediate this progressive phase of injury. OBJECTIVES:To evaluate the role of sPLA2 inhibitor Varespladib in mitigating progression of injury. We hypothesize that Varespladib confers protection against APAP-induced acute liver failure. METHODS:A pilot study suggested 420 mg/kg APAP(LD 80) to be appropriate for testing the hypothesis in C57/BL6J mice. We then went on to test our treatment with Varespladib, which required DMSO as its vehicle. However, inclusion of DMSO in our experiments conferred protection against liver injury. To overcome this hurdle, we carried out more studies to 1) determine a dose of APAP that would exhibit acute liver failure in the presence of DMSO, and 2) determine a suitable solvent cocktail for Varespladib using minimum DMSO and achieve a decent volume for ip injections. After a series of solubility and titration experiments, a dose of 60 uL/kg of DMSO+550 mg/kg APAP was finalized. Mice either received APAP+DMSO (Control) or APAP+Varespladib (treatment). A time course study will be done to collect serum and liver tissue at 0,3,6,12,24,36,48,72 and 96 hrs. Samples will be further analyzed for bioactivation of APAP (CYP2E1/CYP3A4, GSH, APAP-liver protein adducts). Liver injury will be assessed by ALT, AST, and histopathology. sPLA2 activity will be assessed for effective inhibition. Liver regeneration will be assessed by PCNA staining. Prostaglandins will be measured to investigate their role in mediating progression/regression downstream of sPLA2. RESULTS:Our data thus far demonstrate that sPLA2 inhibition via Varespladib confers protection against APAP-induced acute liver failure as evident by LD40 in control group and LD20 in the Varespladib-treated group. CONCLUSION:Varespladib seems to be conferring protection against progression of acute liver injury

Transdifferentiation of Hepatocytes to Biliary Epithelial Cells (BECs) Requires Expression of Reprogramming Factor OCT3/4

Transdifferentiation of liver epithelial cells (hepatocytes and biliary cells) into each other provides a rescue mechanism in liver disease under situations where either cell compartment fails to regenerate by itself. The mechanisms underlying such transdifferentiation of terminally differentiated epithelial cells are not known. Recently we reported that adult rat liver expresses reprogramming factors OCT3/4, Nanog, and KLF4, and their expression is important for hepatocyte proliferation and liver regeneration. To test if reprogramming factor OCT3/4 plays a role in transdifferentiation of hepatocytes to BECs, we used the hepatocyte organoid culture: an in vitro hepatocyte to biliary transdifferentiation model. In this model, primary hepatocytes when plated on collagen coated roller bottles and cultured in presence of HGF, EGF, and dexamethasone, transdifferentiate to form BECs between 6-15 days in culture. We found that OCT3/4 is upregulated at day 6 in this model as assessed by mRNA and protein levels suggesting its possible involvement in transdifferentiation. When expression of OCT3/4 was blocked in hepatocytes using adenovirus containing OCT3/4 shRNA, there was a significant decrease in appearance of biliary cells (compared to controls) as assessed by biliary marker HNF1-beta, strongly supporting the role of OCT3/4 in transdifferentiation of hepatocyte to biliary epithelial cells. Experiments to investigate mechanisms through which OCT3/4 might regulate transdifferentiation are under way

Transdifferentiation of hepatocytes to biliary epithelial cells requires reprogramming factor Oct4

Transdifferentiation of liver epithelial cells (hepatocytes and biliary cells) into each other provides a rescue mechanism in liver disease under situations where either cell compartment fails to regenerate by itself. The mechanisms underlying such transdifferentiation of terminally differentiated epithelial cells are not fully elucidated. Recently we reported that adult rat liver expresses reprogramming factorsOct4, Nanog, and KLF4, and their expression is important for hepatocyte proliferation and liver regeneration. To test if reprogramming factorOct4 plays a role in transdifferentiation of hepatocytes to BECs, we used the hepatocyte organoid culture: an in vitro hepatocyte to biliary transdifferentiation model. In this model, primary hepatocytes when plated on collagen coated roller bottles and cultured in presence of HGF, EGF, and dexamethasone, transdifferentiate to form BECs between 6-15 days in culture. We found that Oct3/4 is upregulated at day 6 in this model as assessed by mRNA and protein levels suggesting its possible involvement in transdifferentiation. When expression of Oct4 was blocked in hepatocytes using adenovirus containing Oct4 shRNA, there was a significant decrease in the number of biliary cells as compared to controls as assessed by biliary marker HNF1-beta, strongly supporting the role of Oct4 in transdifferentiation of hepatocyte to biliary epithelial cells. Moreover, Oct4 inhibited group shows significantly less SOX9, YAP, and Notch (also shown to be involved in transdifferentiation) expression as compared to controls. Experiments to investigate mechanisms through which Oct4 might regulate transdifferentiation are under way

The Role of Lipocalin -2 (Lcn2) in Acetaminophen Induced Acute Liver Failure

INTRODUCTION: Acetaminophen (APAP) toxicity is the number one cause of acute liver failure (ALF) and treatment options are limited. The initial toxicity is caused by the bioactivation of the drug to the reactive metabolite. However, progression of injury still occurs even after all of the drug has been eliminated from the body. APAP overdose involves sterile inflammation. Lipocalin-2 is an acute phase protein known to be upregulated in various disease models and has been observed to have a protective as well as a destructive role dependent on the model analyzed, via modulation of pro/anti-inflammatory signalling. The role of Lcn2 in a drug-induced acute liver failure model, when regeneration is inhibited, has not yet been investigated. OBJECTIVE: This study aims to investigate the role of Lcn2 in an APAP- induced ALF model. METHODS: An in vivo model was utilized and Lcn2KO mice were used as an intervention. Time course studies were done over a period of 0-96 hrs post APAP overdose. Liver and serum were harvested at each time point and the extent of injury was assessed via ALT/AST. Cyp2E1 and Cyp3A4 protein expression was analyzed via Western Blot. Lcn2 upregulation was analyzed via ELISA. RESULTS: Our data demonstrated that Lcn2KO mice are protected (LD20) against APAP-induced ALF as opposed to WT (LD60). Lcn2, ALT and AST was significantly upregulated in WT mice following APAP as compared to Lcn2KO mice. CYP2E1 and CYP3A4 protein expression was comparable between WT and KO mice indicating comparable bioactivation and initial injury by APAP. DISCUSSION: In conclusion, the protection of Lcn2KO mice from APAP overdose indicates that Lcn2 may play a role in mediating progression of injury in WT. Further studies including protein adduct formation, glutathione depletion and repletion, pro- and anti-inflammatory cytokine levels, histopathology, and proliferating cell nuclear antigen will be done to elucidate the mechanism of Lcn2 in mediating progression of injury

Metal-free Hydroamination of Alkynes: A Mild and Concise Synthesis of Thiazolo[3,2- a ]indoles and their Cytotoxic Activity

A metal-free, mild and efficient method for the synthesis of thiazolo[3,2-a]indoles has been developed starting from indoline-2-thiones. The reaction methodology involves first the formation of thermally labile 2-(prop-2-ynylthio)-1H-indole intermediates, which undergo base-mediated intramolecular hydroamination to produce the title compounds in excellent yields

Reprogramming factor Oct4 is crucial for transdifferentiation of hepatocytes to biliary epithelial cells

Transdifferentiation of liver epithelial cells (hepatocytes and biliary cells) into each other provides a rescue mechanism in liver disease under situations where either cell compartment fails to regenerate by itself. The mechanisms underlying such transdifferentiation of terminally differentiated epithelial cells are not fully elucidated. Recently we reported that adult rat liver expresses reprogramming factors Oct4, Nanog, and KLF4, and their expression is important for hepatocyte proliferation and liver regeneration. To test if reprogramming factor Oct4 plays a role in transdifferentiation of hepatocytes to BECs, we used the hepatocyte organoid culture: an in vitro hepatocyte to biliary transdifferentiation model. In this model, primary hepatocytes when plated on collagen coated roller bottles and cultured in presence of HGF, EGF, and dexamethasone, transdifferentiate to form BECs between 6-15 days in culture. We found that Oct3/4 is upregulated at day 6 in this model as assessed by mRNA and protein levels suggesting its possible involvement in transdifferentiation. When expression of Oct4 was blocked in hepatocytes using adenovirus containing Oct4 shRNA, there was a significant decrease in the number of biliary cells as compared to controls as assessed by biliary marker HNF1-beta, strongly supporting the role of Oct4 in transdifferentiation of hepatocyte to biliary epithelial cells. Moreover, Oct4 inhibited group shows significantly less SOX9, YAP, and Notch (also shown to be involved in transdifferentiation) expression as compared to controls. Experiments to investigate mechanisms through which Oct4 might regulate transdifferentiation are under way

Inhibition of cyclooxygenase-2 aggravates secretory phospholipase A2-mediated progression of acute liver injury

Our previous study [Bhave, V. S., Donthamsetty, S., Latendresse, J. R., Muskhelishvili, L., and Mehendale, H. M. 2008-this issue. Secretory phospholipase A2 mediates progression of acute liver injury in the absence of sufficient COX-2. Toxicol Appl Pharmacol] showed that in the absence of sufficient induction and co-presence of cyclooxygenase-2 (COX-2), secretory phospholipase A2 (sPLA2) appearing in the intercellular spaces for cleanup of post-necrotic debris seems to contribute to the progression of toxicant-initiated liver injury, possibly by hydrolysis of membrane phospholipids of hepatocytes in the perinecrotic areas. To further test our hypothesis on the protective role of COX-2, male Fisher-344 rats were administered a selective COX-2 inhibitor, NS-398, and then challenged with a moderately toxic dose of CCl4. This led to a 5-fold increase in the susceptibility of the COX-2 inhibited rats to CCl4 hepatotoxicity and mortality. The CCl4 bioactivating enzyme CYP2E1 protein, CYP2E1 enzyme activity, and the 14CCl4-derived radiolabel covalently bound to the liver proteins were unaffected by the COX-2 inhibitor suggesting that the increased hepatotoxic sensitivity of the COX-2 inhibited rats was not due to higher bioactivation of CCl4. Further investigation showed that this increased mortality was due to higher plasma and hepatic sPLA2 activities, inhibited PGE2 production, and progression of liver injury as compared to the non-intervened rats. In conclusion, inhibition of COX-2 mitigates the tissue protective mechanisms associated with COX-2 induction, which promotes sPLA2-mediated progression of liver injury in an acute liver toxicity model. Because increased sPLA2 activity in the intercellular space is associated with increased progression of injury, and induced COX-2 is associated with hepatoprotection, ratios of hepatic COX-2 and sPLA2 activities may turn out to be a useful tool in predicting the extent of hepatotoxicities. © 2007 Elsevier Inc. All rights reserved

Secretory phospholipase A2 mediates progression of acute liver injury in the absence of sufficient cyclooxygenase-2

Previous studies have shown that injury initiated by toxicants progresses even after most of the toxicant is eliminated from the body. One mechanism of progression of injury is the extracellular appearance of hydrolytic enzymes following leakage or upon cell lyses. Under normal conditions, after exposure to low to moderate doses of toxicants, secretory phospholipase A2 (sPLA2) and other hydrolytic enzymes are known to appear in the extracellular spaces in order to cleanup the post-necrotic debris in tissues. We tested the hypothesis that sPLA2 contributes to progression of toxicant-initiated liver injury because of hydrolysis of membrane phospholipids of hepatocytes in the perinecrotic areas in the absence of sufficient cyclooxygenase-2 (COX-2). Male Sprague-Dawley rats were administered either a moderately hepatotoxic dose (MD, 2 ml CCl4/kg, ip) or a highly hepatotoxic dose (HD, 3 ml CCl4/kg, ip) of CCl4. After MD, liver sPLA2 and COX-2 were co-localized in the necrotic and perinecrotic areas and their activities in plasma and liver increased before decreasing in tandem with liver injury (ALT and histopathology) leading to 100% survival. In contrast, after the HD, high extracellular and hepatic sPLA2 activities were accompanied by minimal COX-2 activity and localization in the liver throughout the time course. This led to progression of liver injury and 70% mortality. These data suggested a destructive role of sPLA2 in the absence of sufficient COX-2. Time- and dose-dependent destruction of hepatocytes by sPLA2 in isolated hepatocyte incubations confirmed the destructive ability of sPLA2 when present extracellularly, suggesting its ability to spread injury in vivo. These findings suggest that sPLA2, secreted for cleanup of necrotic debris upon initiation of hepatic necrosis, requires the co-presence of sufficiently induced COX-2 activity to prevent the run-away destructive action of sPLA2 in the absence of the tissue protective mechanisms afforded by COX-2 induction. © 2008 Elsevier Inc. All rights reserved