Minoru Shigekawa

Increases in p53 expression induce CTGF synthesis by mouse and human hepatocytes and result in liver fibrosis in mice

The tumor suppressor p53 has been implicated in the pathogenesis of non-cancer-related conditions such as insulin resistance, cardiac failure, and early aging. In addition, accumulation of p53 has been observed in the hepatocytes of individuals with fibrotic liver diseases, but the significance of this is not known. Herein, we have mechanistically linked p53 activation in hepatocytes to liver fibrosis. Hepatocyte-specific deletion in mice of the gene encoding Mdm2, a protein that promotes p53 degradation, led to hepatocyte synthesis of connective tissue growth factor (CTGF; the hepatic fibrogenic master switch), increased hepatocyte apoptosis, and spontaneous liver fibrosis; concurrent removal of p53 completely abolished this phenotype. Compared with wild-type controls, mice with hepatocyte-specific p53 deletion exhibited similar levels of hepatocyte apoptosis but decreased liver fibrosis and hepatic CTGF expression in two models of liver fibrosis. The clinical significance of these data was highlighted by two observations. First, p53 upregulated CTGF in a human hepatocellular carcinoma cell line by repressing miR-17-92. Second, human liver samples showed a correlation between CTGF and p53-regulated gene expression, which were both increased in fibrotic livers. This study reveals that p53 induces CTGF expression and promotes liver fibrosis, suggesting that the p53/CTGF pathway may be a therapeutic target in the treatment of liver fibrosis.

The Bcl-xL inhibitor, ABT-737, efficiently induces apoptosis and suppresses growth of hepatoma cells in combination with sorafenib†

Tumor cells are characterized by uncontrolled proliferation, often driven by activation of oncogenes, and apoptosis resistance. The oncogenic kinase inhibitor sorafenib can significantly prolong median survival of patients with advanced hepatocellular carcinoma (HCC), although the response is disease‐stabilizing and cytostatic rather than one of tumor regression. Bcl‐xL (B cell lymphoma extra large), an antiapoptotic member of the B cell lymphoma‐2 (Bcl‐2) family, is frequently overexpressed in HCC. Here, we present in vivo evidence that Bcl‐xL overexpression is directly linked to the rapid growth of solid tumors. We also examined whether ABT‐737, a small molecule that specifically inhibits Bcl‐xL but not myeloid cell leukemia‐1 (Mcl‐1), could control HCC progression, especially when used with sorafenib. Administration of ABT‐737, even at an in vivo effective dose, failed to suppress Huh7 xenograft tumors in mice. ABT‐737 caused the levels of Mcl‐1 expression to rapidly increase by protein stabilization. This appeared to be related to resistance to ABT‐737, because decreasing Mcl‐1 expression levels to the baseline by a small interfering RNA–mediated strategy made hepatoma cells sensitive to this agent. Importantly, administration of ABT‐737 to Mcl‐1 knockout mice induced severe liver apoptosis, suggesting that tumor‐specific inhibition of Mcl‐1 is required for therapeutic purposes. Sorafenib transcriptionally down‐regulated Mcl‐1 expression specifically in tumor cells and abolished Mcl‐1 up‐regulation induced by ABT‐737. Sorafenib, not alone but in combination with ABT‐737, efficiently induced apoptosis in hepatoma cells. This combination also led to stronger suppression of xenograft tumors than sorafenib alone. Conclusion: Bcl‐xL inactivation by ABT‐737 in combination with sorafenib was found to be safe and effective for anti‐HCC therapy in preclinical models. Direct activation of the apoptosis machinery seems to unlock the antitumor potential of oncogenic kinase inhibitors and may produce durable clinical responses against HCC. (HEPATOLOGY 2010)

BH3-only Activator Proteins Bid and Bim Are Dispensable for Bak/Bax-dependent Thrombocyte Apoptosis Induced by Bcl-xL Deficiency: MOLECULAR REQUISITES FOR THE MITOCHONDRIAL PATHWAY TO APOPTOSIS IN PLATELETS

A pivotal step in the mitochondrial pathway of apoptosis is activation of Bak and Bax, although the molecular mechanism remains controversial. To examine whether mitochondrial apoptosis can be induced by just a lack of antiapoptotic Bcl-2-like proteins or requires direct activators of the BH3-only proteins including Bid and Bim, we studied the molecular requisites for platelet apoptosis induced by Bcl-xL deficiency. Severe thrombocytopenia induced by thrombocyte-specific Bcl-xL knock-out was fully rescued in a Bak and Bax double knock-out background but not with single knock-out of either one. In sharp contrast, deficiency of either Bid, Bim, or both did not alleviate thrombocytopenia in Bcl-xL knock-out mice. An in vitro study revealed that ABT-737, a Bad mimetic, induced platelet apoptosis in association with a conformational change of the amino terminus, translocation from the cytosol to mitochondria, and homo-oligomerization of Bax. ABT-737-induced Bax activation and apoptosis were also observed in Bid/Bim-deficient platelets. Human platelets, upon storage, underwent spontaneous apoptosis with a gradual decline of Bcl-xL expression despite a decrease in Bid and Bim expression. Apoptosis was attenuated in Bak/Bax-deficient or Bcl-xL-overexpressing platelets but not in Bid/Bim-deficient platelets upon storage. In conclusion, platelet lifespan is regulated by a fine balance between anti- and proapoptotic multidomain Bcl-2 family proteins. Despite residing in platelets, BH3-only activator proteins Bid and Bim are dispensable for Bax activation and mitochondrial apoptosis.

Mcl-1 and Bcl-xL regulate Bak/Bax-dependent apoptosis of the megakaryocytic lineage at multistages

Anti-apoptotic Bcl-2 family proteins, which inhibit the mitochondrial pathway of apoptosis, are involved in the survival of various hematopoietic lineages and are often dysregulated in hematopoietic malignancies. However, their involvement in the megakaryocytic lineage is not well understood. In the present paper, we describe the crucial anti-apoptotic role of Mcl-1 and Bcl-xL in this lineage at multistages. The megakaryocytic lineage-specific deletion of both, in sharp contrast to only one of them, caused apoptotic loss of mature megakaryocytes in the fetal liver and systemic hemorrhage, leading to embryonic lethality. ABT-737, a Bcl-xL/Bcl-2/Bcl-w inhibitor, only caused thrombocytopenia in adult wild-type mice, but further induced massive mature megakaryocyte apoptosis in the Mcl-1 knockout mice, leading to severe hemorrhagic anemia. All these phenotypes were fully restored if Bak and Bax, downstream apoptosis executioners, were also deficient. In-vitro study revealed that the Jak pathway maintained Mcl-1 and Bcl-xL expression levels, preventing megakaryoblastic cell apoptosis. Similarly, both were involved in reticulated platelet survival, whereas platelet survival was dependent on Bcl-xL due to rapid proteasomal degradation of Mcl-1. In conclusion, Mcl-1 and Bcl-xL regulate the survival of the megakaryocytic lineage, which is critically important for preventing lethal or severe hemorrhage in both developing and adult mice.

Bak deficiency inhibits liver carcinogenesis: a causal link between apoptosis and carcinogenesis.

BACKGROUND & AIMS Hepatocyte apoptosis is a key feature of chronic liver disease including viral hepatitis and steatohepatitis. A previous study demonstrated that absence of the Bcl-2 family protein Mcl-1 led to increased hepatocyte apoptosis and development of liver tumors in mice. Since Mcl-1 not only inhibits the mitochondrial pathway of apoptosis but can also inhibit cell cycle progression and promote DNA repair, it remains to be proven whether the tumor suppressive effects of Mcl-1 are mediated by prevention of apoptosis. METHODS We examined liver tumor development, fibrogenesis, and oxidative stress in livers of hepatocyte-specific knockout (KO) of Mcl-1 or Bcl-xL, another key antagonist of apoptosis in hepatocytes. We also examined the impact of additional KO of Bak, a downstream molecule of Mcl-1 towards apoptosis but not the cell cycle or DNA damage pathway, on tumor development, hepatocyte apoptosis, and inflammation. RESULTS Bcl-xL KO led to a high incidence of liver tumors in 1.5-year-old mice, similar to Mcl-1 KO. Bcl-xL- or Mcl-1-deficient livers showed higher levels of TNF-α production and oxidative stress than wild-type livers at as early as 6 weeks of age and oxidative DNA damage at 1.5 years. Deletion of Bak significantly inhibited hepatocyte apoptosis in Mcl-1 KO mice and reduced the incidence of liver cancer, coinciding with reduction of TNF-α production, oxidative stress, and oxidative DNA damage in non-cancerous livers. CONCLUSIONS Our findings strongly suggest that chronically increased apoptosis in hepatocytes is carcinogenic and offer genetic evidence that inhibition of apoptosis may suppress liver carcinogenesis in chronic liver disease.

STAT3 signaling within hepatocytes is required for anemia of inflammation in vivo.

BackgroundAnemia of inflammation, commonly observed in patients with chronic diseases, is associated with decreased serum iron. Hepcidin, mainly produced by hepatocytes in a STAT3- and/or SMAD-dependent manner, is involved in iron homeostasis. What remains to be established is whether or not the hepatic IL-6/STAT3 signal has a role in anemia of inflammation in vivo.MethodsTurpentine oil was subcutaneously injected into wild-type mice or hepatocyte-specific STAT3-deficient mice (L-STAT3KO) to induce inflammation.ResultsTurpentine injection increased serum IL-6 levels. It activated liver STAT3 in wild-type mice, but not in L-STAT3KO mice. In chronic inflammation, wild-type mice showed decreased serum iron levels and anemia with up-regulation of hepcidin levels in the liver. In contrast, L-STAT3KO mice showed no increase in hepatic hepcidin levels or anemia.ConclusionsLiver STAT3 is critically involved in the development of anemia of inflammation via the expression of hepcidin. The liver regulates anemia of inflammation through STAT3 signaling.

Pancreatic STAT3 protects mice against caerulein-induced pancreatitis via PAP1 induction.

The signal transducer and activator of transcription 3 (STAT3) is a transcription factor that controls expressions of several genes involved in cell survival, proliferation and differentiation, and tissue inflammation. However, the significance of pancreatic STAT3 in acute pancreatitis remains unclear. We generated conditional STAT3 knockout (stat3(Δ/Δ)) mice by crossing stat3(flox/flox) mice with Pdx1-promoter Cre transgenic mice. Caerulein administration activated pancreatic STAT3 and induced acute pancreatitis as early as 3 hours in wild-type mice, and full recovery from the induced pancreatic injury was observed within 7 days. The levels of serum amylase and lipase and histologic scores of pancreatic necrosis and inflammatory cell infiltration were significantly higher at 3 hours in stat3(Δ/Δ) mice than in stat3(flox/flox) mice. Pancreatic recovery after pancreatitis was significantly delayed in stat3(Δ/Δ) mice compared with stat3(flox/flox) mice. Although stat3(flox/flox) mice had marked production in the pancreas of pancreatitis-associated protein 1 (PAP1), a serum acute phase protein, this induction was completely abrogated in stat3(Δ/Δ) mice. Enforced production of PAP1 by a hydrodynamic procedure in the liver significantly suppressed pancreatic necrosis and inflammation and also promoted pancreatic regeneration and recovery in stat3(Δ/Δ) mice to levels similar to those observed in stat3(flox/flox) mice. In conclusion, pancreatic STAT3 is indispensable for PAP1 production, and this STAT3/PAP1 pathway plays a protective role in caerulein-induced pancreatitis.

Delayed‐onset caspase‐dependent massive hepatocyte apoptosis upon fas activation in bak/bax‐deficient mice

The proapoptotic Bcl‐2 family proteins Bak and Bax serve as an essential gateway to the mitochondrial pathway of apoptosis. When activated by BH3‐only proteins, Bak/Bax triggers mitochondrial outer membrane permeabilization leading to release of cytochrome c followed by activation of initiator and then effector caspases to dismantle the cells. Hepatocytes are generally considered to be type II cells because, upon Fas stimulation, they are reported to require the BH3‐only protein Bid to undergo apoptosis. However, the significance of Bak and Bax in the liver is unclear. To address this issue, we generated hepatocyte‐specific Bak/Bax double knockout mice and administered Jo2 agonistic anti‐Fas antibody or recombinant Fas ligand to them. Fas‐induced rapid fulminant hepatocyte apoptosis was partially ameliorated in Bak knockout mice but not in Bax knockout mice, and was completely abolished in double knockout mice 3 hours after Jo2 injection. Importantly, at 6 hours, double knockout mice displayed severe liver injury associated with repression of XIAP, activation of caspase‐3/7 and oligonucleosomal DNA breaks in the liver, without evidence of mitochondrial disruption or cytochrome c–dependent caspase‐9 activation. This liver injury was not ameliorated in a cyclophilin D knockout background nor by administration of necrostatin‐1, but was completely inhibited by administration of a caspase inhibitor after Bid cleavage. Conclusion: Whereas either Bak or Bax is critically required for rapid execution of Fas‐mediated massive apoptosis in the liver, delayed onset of mitochondria‐independent, caspase‐dependent apoptosis develops even in the absence of both. The present study unveils an extrinsic pathway of apoptosis, like that in type I cells, which serves as a backup system even in type II cells. (HEPATOLOGY 2011 )

Activation of the Mitochondrial Apoptotic Pathway Produces Reactive Oxygen Species and Oxidative Damage in Hepatocytes That Contribute to Liver Tumorigenesis

Chronic hepatitis, including viral hepatitis and steatihepatitis, is a well-known high-risk condition for hepatocellular carcinoma. We previously reported that continuous hepatocyte apoptosis drives liver tumors in hepatocyte-specific Bcl-xL or Mcl-1 knockout mice. In this study, we further examine the underlying cellular mechanisms of generating tumors in apoptosis-prone liver. In cultured hepatocytes, the administration of ABT-737, a Bcl-xL/-2/-w inhibitor, led to production of reactive oxygen species (ROS) as well as activation of caspases. Mitochondria isolated from murine liver, upon administration of truncated-Bid, a proapoptotic Bcl-2 family protein, released cytochrome c and produced ROS, which was dependent on mitochondrial respiration. Hepatic apoptosis, regeneration, accumulation of oxidative damages, and tumorigenesis observed in hepatocyte-specific Mcl-1 knockout mice were substantially attenuated by further deficiency of Bax or Bid, suggesting that a balance of mitochondrial Bcl-2 family proteins governs generation of oxidative stress and other pathologies. Whole-exome sequencing clarified that C>A/G>T transversion, which is often caused by oxidative DNA damage in proliferating cells, was a frequently observed mutation pattern in liver tumors of Mcl-1 knockout mice. The administration of antioxidant L-N-acetylcysteine did not affect apoptosis, compensatory regeneration, or fibrotic responses but significantly reduced oxidative DNA damage and incidence and multiplicity of live tumors in Mcl-1 knockout mice. In conclusion, activation of the mitochondrial apoptotic pathway in hepatocytes accumulates intracellular oxidative damages, leading to liver tumorigenesis, independently of liver regeneration or fibrosis. This study supports a concept that antioxidant therapy may be useful for suppressing liver carcinogenesis in patients with chronic liver disease. Cancer Prev Res; 8(8); 693–701. ©2015 AACR.

Involvement of STAT3-regulated hepatic soluble factors in attenuation of stellate cell activity and liver fibrogenesis in mice.

Glycoprotein 130 (gp130)/signal transducer and activator of transcription 3 (STAT3) signaling in hepatocytes controls a variety of physiological and pathological processes including liver regeneration, apoptosis resistance and metabolism. Recent research has shed light on the importance of acute phase proteins (APPs) regulated by hepatic gp130/STAT3 in host defense through suppression of innate immune responses during systemic inflammation. To examine whether these STAT3-regulated soluble factors directly affect liver fibrogenic responses during liver injury, hepatocyte-specific STAT3 knockout (L-STAT3 KO) mice and control littermates were subjected to bile duct ligation (BDL) and examined 10 days later. In contrast to controls, L-STAT3 KO mice failed to produce APPs, such as serum amyloid A and haptoglobin, after BDL. Whereas L-STAT3 KO mice displayed similar levels of cholestasis, inflammatory cell infiltration and regeneration in the liver, they developed exacerbated liver injury and fibrosis with significant increases in expression of alpha-smooth muscle actin and type I collagen genes. In vitro experiments revealed that attenuated expression of APPs in primary hepatocytes isolated from L-STAT3 KO mice with IL-6 exposure, compared to wild-type hepatocytes. The cultured supernatant from IL-6-treated wild-type hepatocytes inhibited expression of alpha-smooth muscle actin and type I collagen genes in activated hepatic stellate cells (HSCs), whereas this did not occur with the supernatant from IL-6-treated knockout hepatocytes or with control medium. In conclusion, the absence of STAT3 in hepatocytes leads to exacerbation of liver fibrosis during cholestasis. Soluble factors released from hepatocytes, dependent on STAT3, collectively play a protective role in liver fibrogenesis through an inhibitory effect on activated HSCs.