Anti-apoptotic members of the Bcl-2 family, including Bcl-2, Bcl-xL, Mcl-1, Bcl-w and Bfl-1, inhibit the mitochondrial pathway of apoptosis. Bcl-xL and Mcl-1 are constitutively expressed in the liver. Although previous research established Bcl-xL as a critical apoptosis antagonist in differentiated hepatocytes, the significance of Mcl-1 in the liver, especially in conjunction with Bcl-xL, has not been clear. To examine this question, we generated hepatocyte-specific Mcl-1– deficient mice by crossing mcl-1flox/flox mice and AlbCre mice and further crossed them with bcl-xflox/flox mice, giving Mcl-1/Bcl-xL– deficient mice. The mcl-1flox/flox AlbCre mice showed spontaneous apoptosis of hepatocytes after birth, as evidenced by elevated levels of serum alanine aminotransferase (ALT) and caspase-3/7 activity and an increased number of terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate nick-end labeling (TUNEL)-positive cells in the liver; these phenotypes were very close to those previously found in hepatocyte-specific Bcl-xL– deficient mice. Although mcl-1flox/+ AlbCre mice did not display apoptosis, their susceptibility to Fas-mediated liver injury significantly increased. Further crossing of Mcl-1 mice with Bcl-xL mice showed that bcl-xflox/+ mcl-1flox/+ AlbCre mice also showed spontaneous hepatocyte apoptosis similar to Bcl-xL– deficient or Mcl-1– deficient mice. In contrast, bcl-xflox/flox mcl-1flox/+ AlbCre, bcl-xflox/+ mcl-1flox/flox AlbCre, and bcl-xflox/flox mcl-1flox/flox AlbCre mice displayed a decreased number of hepatocytes and a reduced volume of the liver on day 18.5 of embryogenesis and rapidly died within 1 day after birth, developing hepatic failure evidenced by increased levels of blood ammonia and bilirubin. Conclusion: Mcl-1 is critical for blocking apoptosis in adult liver and, in the absence of Bcl-xL, is essential for normal liver development. Mcl-1 and Bcl-xL are two major anti-apoptotic Bcl-2 family proteins expressed in the liver and cooperatively control hepatic integrity during liver development and in adult liver homeostasis in a gene dose-dependent manner.

MHC class I-related chain A (MICA) is a ligand for the NKG2D-activating immunoreceptor that mediates activation of natural killer (NK) cells. The ectodomain of MICA is shed from tumor cells, which may be an important means of evading antitumor immunity. We previously reported that patients with hepatocellular carcinoma (HCC) display high levels of soluble MICA in circulation, which could be downregulated by chemotherapy. The present study shows that anti-HCC drugs suppress MICA ectodomain shedding by inhibiting expression of a disintegrin and metalloproteinase 10 (ADAM10). Both ADAM10 and CD44, a typical substrate of the ADAM10 protease, were expressed in human HCC tissues and HCC cells but not in normal liver tissues or cultured hepatocytes. Small interfering RNA-mediated knockdown experiments revealed that ADAM10 is a critical sheddase for both MICA and CD44 in HCC cells. Of interest is the finding that epirubicin clearly downregulated ADAM10 expression and MICA shedding in HCC cells; its suppressive effect on MICA shedding was abolished in ADAM10-depleted cells. Epirubicin treatment also enhanced the NKG2D-mediated NK sensitivity of HCC cells. Patients with HCC had significantly higher levels of serum-soluble CD44, which correlated well with serumsoluble MICA levels, thus suggesting a close link between ADAM10 activity and MICA shedding in these patients. Soluble MICA and CD44 levels were downregulated with a significant correlation in patients treated by transarterial chemoembolization using epirubicin. In conclusion, anticancer drugs can modulate expression of ADAM10, which is critically involved in MICA ectodomain shedding. Epirubicin therapy may have a previously unrecognized effect on antitumor immunity in HCC patients. [Cancer Res 2009;69(20):8050-7]

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Sepsis is an infection-induced syndrome with systemic inflammatory response leading to multiorgan failure and occasionally death. During this process, signal transducer and activator of transcription 3 (STAT3) is activated in the liver, but the significance of this molecule has not been established. We generated hepatocyte-specific STAT3-deficient mice (L-STAT3 KO) and examined the susceptibility of these mice to cecal ligation and puncture-induced peritonitis, a wellestablished septic model. L-STAT3 KO mice showed significantly higher mortality and produced lesser amounts of various acute phase proteins than control littermates. Although blood bacterial infection did not differ between L-STAT3 KO mice and control mice, the former showed deterioration of the systemic inflammatory response as evidenced by a significant increase in various cytokines such as tumor necrosis factor ␣, IFN-␥, IL-6, IL-10, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1␤. A similar hyperinflammatory response was observed in another septic model caused by lipopolysaccharide (LPS) injection. In vitro analysis revealed that soluble substances derived from hepatocytes and dependent on STAT3 were critical for suppression of cytokine production from LPS-stimulated macrophage and splenocytes. Conclusion: STAT3 activation in hepatocytes can attenuate a systemic hyperinflammatory response and lethality in sepsis, in part by suppressing immune cell overactivation, implying a critical role of hepatocyte STAT3 signaling in maintaining host homeostasis. (HEPATOLOGY 2007;46: 1564-1573.)

(1) In contrast to classical MHC class I molecules, MICA/B are expressed rarely on normal cells but frequently on tumor cells, including colon cancer, prostate cancer, HCC, and brain tumors.(2-5) The engagement of MICA/B and NKG2D strongly activates NK cells and costimulates T cells, enhancing their cytolytic ability and cytokine production. (6) Thus, the MICA/B-NKG2D pathway is an important mechanism by which the host immune system recognizes and kills transformed cells.(7) In addition to those membrane-bound forms, MICA/B are also cleaved proteolytically from tumor cells and appear as soluble forms in sera of patients with malignancy.(8-10) The levels of NKG2D expression tend to be decreased in patients with high levels of soluble MICA/B.(4) In addition, sera from those patients can downregulate NKG2D expression in vitro. (5,11) Hepatocellular carcinoma is one of the leading causes of cancer death worldwide. Chronic liver disease caused by hepatitis virus infection and non-alcoholic steatohepatitis leads to a predisposition for HCC; liver cirrhosis, in particular, is considered to be a premalignant condition. (14,15) With regard to treatment, surgical resection or percutaneous techniques such as ethanol injection and radiofrequency ablation are considered to be choices for curable treatment of localized HCC, whereas TAE is a wellestablished technique for unresectable HCC.(16) We reported previously that soluble MICA could be detected in sera of HCC patients.(17) However, the clinical significance of the soluble forms of NKG2D ligands in liver disease has not yet been established in a comprehensive manner, because the previous study was conducted on a small number of patients, did not include patients with premalignant conditions such as liver cirrhosis, and did not analyze its closely related molecule MICB. Furthermore, influences of therapeutic intervention on soluble NKG2D ligands in patients have been unclear. In the present study, we examined soluble MICA and soluble MICB in sera from a large number of patients with chronic liver diseases and HCC and their impact on NKG2D expression on immune cells during TAE therapy for HCC. 4 To whom correspondence should be addressed. E-mail: hayashin@gh.med.osaka-u.ac.jp 5 Keisuke Kohga and Tetsuo Takehara contributed equally to this work. Abbreviations: APC, allophycocyanin; ELISA, enzyme-linked immunosorbent assay; FITC, fluorescein isothiocyanate; HCC, hepatocellular carcinoma; MFI, mean fluorescence intensity; MICA/B, major histocompatibility complex (MHC) class I-related chain A and B; NK, natural killer; NKG2D, natural killer group 2, member D; PBMC, peripheral blood mononuclear cell; PE, phycoerythrin; TAE, transcatheter arterial embolization; TNM, tumor node metastasis.