cFLIP attenuates bile acid-induced apoptosis by directly inhibiting p38 MAP kinase activation

of control mice was 50% indicating that IKKI~ exhibits essential role in prevention of apoptosis in aduh hver,

cytochrome c release from mitochon&ia leading to apoptosis and HO1 expression has been shown to prevent apoptosis tbllowing some stresses. HO1 interacts physiologically with Bill as immunoprecipitation of HO1 results in co-immunoprecipitation of Bif-1 from wild type spleen. Importantly, no Bif-1 protein is observed following the immunoprecipitation from H014 spleen. Moreover, tire Bif-1 binding site on HO1 has been defined with series of truncation constructs of both proteins. Now, we are confrming the specific binding sites on each protein using GST pull-down assay and site-directed mutagenesis. In addition, we are investigating the physiologic importance this interaction by monitoring HO enzyme activity and HOl-mediated cellular protection, We hypothesize that~ follo,,~ng stress and induction of HO 1, translocation of a BiM/Bax complex from cytosol to HOI on endoplasmic reticulum may avert cell death by preventing Bax from entering the mitochondria, Thus, HO1 may prevent cell death through protein-protein interactions, in addition to modulating cellular iron levels, producing an antioxidant (bilirubin) and making carbon monoxide.

513 HCC Chemo-resistance to Camptothecin Is through NF-KB-lndependent Pathway Naoki Koizumi, Etanro Hatano, Takashi Nitta, Nobuko Harada, Nantaka Yamamoto, Yoshm Yan~'toka The transcription factor NF-KB has anti-apoptotic properties and may confer chemo-resistance to cancer cells, NF-KB is constitutively activated in bladder cancer, breast cancer, and melanoma, in which cancer cell lines, inhibition of NF-KB induces cell death in bepatocellular carcinoma (HCC), constitutive activation of NF-KB was tbund more frequently in tumor tissue compared with non-tumor tissue. Our AIM is to clarify the role of NF-KB in camptothecin-induced apoptosis of HCC. METHODS: Studies were performed in 3 hepatoma cell lines, Hep3B, HepG2 and Huh7. Camptothecin-induced apoptosis was evaluated wath DNA [addering assay" and DNA fragmentation ELISA. Western blot analysis for NF-KB (p65) and IKB were performed with nuclear extracts and whole lysates, respectively, to examine the activation of NF-KB by, camptothecin. To block NF-KB activation, adenov~rus expressing IKB super-repressor (Ad5IgBsf) was intected with ceils 12 hours befm'e the treatment of camptothecin We examined whether camptothecin (0.5-2 t~M)-iuduced apoptosis was enhanced under transt~ction of Ad5IKBsr (50-200 MOI) by" DNA fragmentation ELISA. RESULTS DNA laddering was clearly shown alter 1-2 p.M of camptnthecin treatment in all 3 HCC cells. DNA fragmentation ELISA demonstrated that the rate of apoptotic cell death in Hep3B was 20% lower than that of HepG2, indicating that Hep3B is resistant to camptothecininduced apoptosis. The pretreatment of cyclobeximide (to block the protein symthesis) with Hep3B did not enhance camptothecin-induced apoptosis. NF-KB was constitutively activated in Hep3B and camptothecni enhanced the translocation of NF-KB to the nucleus from 30 to 120min after camptothecin. After 12 hours of AdSIKBsr infection, we confirmed the expression of IKB super-repressor in Hep3B However, inactivation of NF-KB by Ad5lKBsr did not sensitize Hep3B to camptothecin-induced apoptosis in Hep3B in Conchision, HCC chemo-resistance to camptothecin is through NF-,~rB-independent pathway.

516

Angiotensin n Type la Receptor Deficient Mice Show Slow Progression of Liver Fibrosis Induced by Carbon Tetr~chloride: A Direct Evidence for Fibrogenetic Role of Renin-Angiotensin System Keishi Kanno, Susumu Tazuma, Tom@ Nishioka, Hideyuki Hyogo, Ynsushi Sunami, Kuniharu Nakai, Kazuhiko T~boi, Yasumasa Asamoto, Yoshihiro Numata, Atsushi Yamaguchi. Toshiya Kobuke. Daisuke Komichi, MieNhiro Nonaka, Kazuaki Chayama Background and Aim:. The renin angiotensin system (RAS) has been shown to contribute to fbrogenesis in a variety of organs, including the liver. In some animal models, blockers of the action of angiotensin (Aug). such as angtotensm-converting enzyme (ACE) inhibitors or Ang II receptor antagonists, have been shown to reduce regression or prevent (be development of hepatic fbrosis. The aim of the present study was to determine whether tire Ang 1I type 1A receptor (AT1A) is implicated in the devdopment of liver fibrosis through Aug II signaling, Methods: Male AT1A-deficient mice and wild-type (WI') mice (7w) were administered with carbon tetrachloride (CC14) (1 ml/kg) as a single intraperitoneal injection to assess the necrotic and inflammatory changes caused by' acute exposure to CC14. Liver fibrosis was induced by the subcutaneous injection of CC14 (1 ml/kg) twice weekly for 4 weeks. The extent of necrosis/inflammation or fibrosis was evaktated in AT1A-deficient mice and wild-type (WT) mice with analyses of fibrntic parameters; (1) Liver histology', (2) Hepatic hydoxyproline content, (3) lmmunohistocbemical expression of hepatic a-smooth muscle actin (a-SMA), (4) TGF-bl mRNA expression by RT-PCR. Results: After single dose of CC14, there were no significant differences between WT mice and AT1A-deficient mice with regard to serum transaminase and TNF-a levels, Histologically, the extent of necrosis and inflammatory infiltration was similar in the two groups. After chrome administration of CC14, histological examination revealed that AT1A-defcient mice showed less infiltration of monocytes and slower progression of fiver fibrosis when compared with WT mice. These findings were accompanied by an increased hepatic content of hydoxyproline, WT mice treated with chronic CCl4 showed a 6.1 -fold increment of the"hydoxyproline content, whereas ATIA-deficient mice showed a more modest 2,6-tbld increment lmmunnhistochemical expression of a-SMA was negligible in AT1A-deficient mice, while it was strongly detected in WT mice. The level of TGF-bl mRNA was markedly higher in WT mice when compared with AT1A-deficient mice. Conclusions: These results suggested that signaling via ATIA plays a pivotal role in hepatic fbrogenesis, while AT1A blockade reduces the progression of fiver fibrosis through the suppression of chronic inflammation. Thus, AT1Ais a potentiatedmolecular target for liver fibrosis treatment,

514 cFLIP Attenuates Bile Acid-Induced Apoptosis by Directly" Inhibiting p38 MAP Kinase Activation Annette Granibihler, Hajime Higuchi, Steven F. Bronk, Gregory J. Gores In cholestasis, toxic bile acids accumulate within the liver inducing death receptor-mediated hepatocyte apoptosis. The cellular tlice inhibitmy L)rotein (cFLIP), nihfbits death receptormediated apoptosis. Although initially thought to block caspase 8 activation, cFL1P has recently been shown to actually promote caspase 8 actisity (J BIOlChem 277:45162, 2002). Thus, the mechamsm by, which cFLIP blocks apoptosis has become controversial, Mitogen activated protein kitmses (ivLAPK) are also known to enhance death receptor mediated apoptosis, Therefore, we formulated the HYPOTHESIS that cFLIP may be antiapoptotic by inhibiting MAP kinase activity'. METHODS: HUH-7 cells (a human hepatonm cell line), were stabily transfected with cFLiP-L (HuH-cFLIP). Apoptosis was induced by treatment with deoxycholate (DC) and quantitated by DAP1 staining and fluorescent microscopy. Activation of MAPK was assessed by immunoblot analysis using phospho-specific antiboches for p42N4, JNK and p38. p38 and cFLIP binding interactions were assessed by eo-immunoprecipitation par'adigms, and further evaluated using a GST-cFL1P pulldomr assay. RESULTS: DC treatment msuhed in a tinge (0-24 hours) and dose dependent (0-200 ~M) induction of apoptosis in HuH7 cells; in contrast. HuH
517

A Cotransactivator Binds to a Homeudumain Core Sequence and Stimulates Smooth Muscle Alpha-actin Promoter Activity in Activated Hepatic Stellate Cells Zengdun Shi, Don C Rockey Following liver injury, hepatic stellate cells undergo a fnnctional and morpholoDcal transition to myofbrolast-fke cells, One of the prominent features in (his process is increased expression of the smooth muscle a-actin, which is thought to be linked to enhanced contraction and motility. However, the molecular mechanisms by which smooth muscle a-actin gene transcription is controlled are unclear. Although the smooth muscle a-actin gene promoter has three serum response elements (CArG-A, B and C) that allow serum response factor (SRF) binding, we hypothesized that a cotransactivator cooperates with SRF to stimulate smooth muscle a-actin promoter activity. Methods:A series of reporter constructs containing different smooth muscle a-actin gene promoter fragments or promoter constructs containing mutations were transduced into activated stefate cells; luciterase activity following transfection was assayed. Specific DNA binding proteins were identified by gel mobility shift assay (EMSA). Results: Luciferse assays showed that -671 + 5bp, -216 + 5bp (with CArG-A, B, and C boxes), and -158+ 5bp constructs (with CArG-A and B boxes) conierred a similar level of promoter activity. However, promoter activity was reduced by 5-10 fold with a -125 + 5bp construct (CArG-A and B boxes) or a -109 + 5bp construct (CArG-A box only') A -60 + 5bp construct (no CArG box) tailed to confer promoter activity Farther analysis of the region between d58bp and -125bp revealed that promoter activity was dramatically reduced when an ATTA motif (a DNA binding site for homeodomain proteins) was mutated. EMSA showed that two difterent sized proteins bound to a 32bp probe containing the ATTA motif in activated stellate cells, but only one specific protein bound to this probe in quiescent stellate ceils. No protein binding was detected v,ith the same probe containing a mutant ATTA motif in either quiescent or acin.,ated stellate cells. Condusion: The data indicate that serum response elements (CArG-B and A) and SRF are responsible for smooth musde a-actin promoter stimulation in activated stellate ceils. "the conserved ATTA core sequence and a homeodomain (cotransactivator) protein are required for maximal smooth muscle a-actin promoter activity; there is differential expression of homeodomain proteins in quiescent and acnvated sic{late cells. Finally, the data imply that these proteins play an important role in control of smooth muscle a-actin transcriptional actis'ation

515

Heme Oxygenase-1 specifically interacts with Bax-interactiug factor-1 (Bif-l) Koji Abe, Yingda Wang, Akira Sawa. Christopher D Ferns Heme oxygenase-1 (HOD catabolizes heine producing bdiverdin, carbon monooxide, and free ferrous iron Recently, we found that HO1 activity is linked to reduced cellular iron levels, thereby protecting ceils from stress induced cell death (Nature Cell Biology 1:152~ 157, 1999), Many of the molecules involved in cellular iron uptake are characterized, in contrast, little is known about how iron is released from cells. To elucidate the molecular mechanism linking HO 1 activity to celkdar iron elflux, we have identified an iron transporting ATPase that is co-distrlbuted with HO1 in tissues and physiologically induced by iron in cells and intact ainmals (J. Bid, Chem. 275: 15166-15173, 2000). These preliminary studies have identified new physiologic roles fur HO1 in cell death and iron homeostasis. To define the molecular mechanisms regulating these novel tuuctions, we have employed a yeast 2hybrid strategy' and screened a hunmn spleen hbrary with bait-constructs derived kom HO1. Employing a bait derived from the C-terminus of HO1 in >5.5 x 106clones, we obtained 139 yeast clones on selective media. We identified 6 distinct binding partners, confirmed interactions by' g-galactosidase analysis. One oi these binding partners, _Baxinteracting _tactor1 (Bif-1), directly interacts with and co-localizes with Bax. Bax is known to stimulate

AASLD Abstracts

A-716