This study tested whether hepatic steatosis sensitizes liver to toxicant-induced injury and investigated the potential mechanisms of hepatotoxic sensitivity. Male Sprague-Dawley rats were fed a methionine-and choline-deficient diet for 31 days to induce steatosis. On the 32nd day, administration of a nonlethal dose of CCl 4 (2 mL/kg, intraperitoneally) yielded 70% mortality in steatotic rats 12-72 hours after CCl 4 administration, whereas all nonsteatotic rats survived. Neither CYP2E1 levels nor covalent binding of [ 14 C]CCl 4 -derived radiolabel differed between the groups, suggesting that increased bioactivation is not the mechanism for this amplified toxicity. Cell division and tissue repair, assessed by [ 3 H]thymidine incorporation and proliferative cell nuclear antigen assay, were inhibited in the steatotic livers after CCl 4 administration and led to progressive expansion of liver injury culminating in mortality. The hypothesis that fatty hepatocytes undergo cell cycle arrest due to (1) an inability to replenish ATP due to overexpressed uncoupling protein-2 (UCP-2) or (2) induction of growth inhibitor p21 leading to G 1 /S phase arrest was tested. Steatotic livers showed 10-fold lower ATP levels due to upregulated UCP-2 throughout the time course after N onalcoholic fatty liver disease (NAFLD) is defined as deposition of fat in the liver (hepatic steatosis) that is not caused by chronic ingestion of alcohol. The terms NAFLD and nonalcoholic steatohepatitis (NASH) are often used interchangeably; however, NASH specifically refers to that subset of patients with NAFLD who have evidence of inflammation on liver biopsy. NAFLD and NASH are highly prevalent among obese and type II diabetic patients suggesting that obesity and diabetes are predisposing factors for NAFLD and NASH. [1][2][3] The prevalence of obesity in Western populations is currently 10% to 25% and is expected to rise in the future; this increase will result in a parallel rise in NAFLD and NASH.Ob/Ob mice and Zucker fa/fa rats are genetic models for NAFLD. 4 Liver regeneration after two thirds partial hepatectomy is inhibited in obese diabetic Zucker fa/fa rats and ob/ob mice with fatty liver, indicating that ability of the liver to regenerate is decreased in hepatic steatosis. [4][5][6] These observations show that inhibition of cell division does not require inflammation associated with NASH and can occur in a nonalcoholic fatty liver (hepatic steatosis) devoid of the onset of inflammation. However, the response of nonalcoholic fatty liver to drug/chemicalinduced hepatotoxicity is still unknown.The purpose of this study was to investigate whether steatosis alone renders the liver susceptible to amplifica-

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.

Reprogramming factors have been used to induce pluripotent stem cells as an alternative to somatic cell nuclear transfer technology in studies targeting disease models and regenerative medicine. The neuronal repressor REST maintains self renewal and pluripotency in mouse embryonic stem cells by maintaining the expression Oct3/4, Nanog, and cMyc. We report that primary hepatocytes express REST and most of the reprogramming factors in culture. Their expression is upregulated by hepatocyte growth factor (HGF) and epidermal growth factor (EGF). REST inhibition results in downregulation of reprogramming factor expression, increased apoptosis, decreased proliferation, and cell death. The reprogramming factors are also upregulated after 70% partial hepatectomy in vivo. These findings show that genes inducing iPS phenotype, even though expressed at lower levels than embryonic stem cells, they nonetheless are associated with control of apoptosis and cell proliferation in hepatocytes in culture and may play a role for such processes during liver regeneration.

Acute liver failure induced by hepatotoxic drugs results from rapid progression of injury. Substantial research has shown that timely liver regeneration can prevent progression of injury leading to a favorable prognosis. However, the mechanism by which compensatory regeneration prevents progression of injury is not known. We have recently reported that calpain released from necrotic hepatocytes mediates progression of liver injury even after the hepatotoxic drug is cleared from the body. By examining expression of calpastatin (CAST), an endogenous inhibitor of calpain in three liver cell division models known to be resistant to hepatotoxicity, we tested the hypothesis that increased CAST in the dividing hepatocytes affords resistance against progression of injury. Liver regeneration that follows CCl 4 -induced liver injury, 70% partial hepatectomy, and postnatal liver development were used. In all three models, CAST was upregulated in the dividing/newly divided hepatocytes and declined to normal levels with the cessation of cell proliferation. To test whether CAST overexpression confers resistance against hepatotoxicity, CAST was overexpressed in the livers of normal SW mice using adenovirus before challenging them with acetaminophen (APAP) overdose. These mice exhibited markedly attenuated progression of liver injury and 57% survival. Whereas APAP-bioactivating enzymes and covalent binding of the APAP-derived reactive metabolites remained unaffected, degradation of calpain specific target substrates such as fodrin was significantly reduced in these mice. In conclusion, CAST overexpression could be used as a therapeutic strategy to prevent progression of liver injury where liver regeneration is severely hampered. (HEPATOLOGY 2006;44:379-388.)

Previous studies from our laboratory have found glypican 3 (GPC3) as a negative regulator of growth. CD81 was found to be a binding partner for GPC3, and its expression and co-localization with GPC3 increased at the end of hepatocyte proliferation. However, the mechanisms through which these two molecules might regulate liver regeneration are not known. We tested the hypothesis that GPC3 down-regulates the hedgehog (HH) signaling pathway by competing with patched-1 for HH binding. We found decreased GPC3-Indian HH binding at peak proliferation in mice followed by increase in glioblastoma 1 protein (effector of HH signaling). We performed a yeast two-hybrid assay and identified hematopoietically expressed homeobox (Hhex, a known transcriptional repressor) as a binding partner for CD81. We tested the hypothesis that Hhex binding to CD81 keeps it outside the nucleus. However, when GPC3 binds to CD81, CD81-Hhex binding decreases, resulting in nuclear translocation of Hhex and transcriptional repression. In support of this, we found decreased GPC3-CD81 binding at hepatocyte proliferation peak, increased CD81-Hhex binding, and decreased nuclear Hhex. GPC3 transgenic mice were used as an additional tool to test our hypothesis. Overall, our data suggest that GPC3 down-regulates cell proliferation by binding to HH and down-regulating the HH signaling pathway and binding with CD81, thus making it unavailable to bind to Hhex and causing its nuclear translocation.

No abstract

We have recently demonstrated that disruption of extracellular matrix (ECM)/integrin signaling via elimination of integrin-linked kinase (ILK) in hepatocytes interferes with signals leading to termination of liver regeneration. This study investigates the role of ILK in liver enlargement induced by phenobarbital (PB). Wild-type (WT) and ILK:liver-/- mice were given PB (0.1% in drinking water) for 10 days. Livers were harvested on 2, 5, and 10 days during PB administration. In the hepatocyte-specific ILK/liver-/- mice, the liver:body weight ratio was more than double as compared to 0 h at day 2 (2.5 times), while at days 5 and 10, it was enlarged three times. In the WT mice, the increase was as expected from previous literature (1.8 times) and seems to have leveled off after day 2. There were slightly increased proliferating cell nuclear antigen-positive cells in the ILK/liver-/- animals at day 2 as compared to WT after PB administration. In the WT animals, the proliferative response had come back to normal by days 5 and 10. Hepatocytes of the ILK/liver-/- mice continued to proliferate up until day 10. ILK/liver-/- mice also showed increased expression of key genes involved in hepatocyte proliferation at different time points during PB administration. In summary, ECM proteins communicate with the signaling machinery of dividing cells via ILK to regulate hepatocyte proliferation and termination of the proliferative response. Lack of ILK in the hepatocytes imparts prolonged proliferative response not only to stimuli related to liver regeneration but also to xenobiotic chemical mitogens, such as PB.

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.