αvβ3 integrin-mediated activation of p38 MAPK in human cholangiocarcinoma

system regulates cholangiocyte pmliterationAoss. Gastrin inhibits cholangiocarcinornagrowth by activation of PKC-a, Taurocholate stimulates cholangiocyte proliteration by activation of PKC-a. We pose these questions: (i) Does taurocholate protect against TNF-a-induced duct injury? and (ii) Are taurocholate protective effects against TNF~-induced duct damage regulated by the Ca-'+-dependent PKC pathway? Methods: Immediately after BDL, rats were fed 1% taumcholate or control diet. Seven days later control- or taurocholate-fed I~ts were treated by a single IP injection with actinomycin D (100 lag/kg body" weight) + TNF-c~ (50 ng/kg body weight). Twenty-four hours later we evaluated:0) apoptosis by TUNEL analysis in liver sections; (ii) proliferation by measurement of ductal mass and PCNA protein expression in cholangiocytes; and (iii) secretin-stimulated choleresis and cAMP levels. We evaluated protein expression of the Ca> -dependent PKCc~, 13-1,and 13-2isoforms. Purified cholangiocytes were treated in vuro for 18 hours with actinomycin D (2 ItM) + TNF-e~ (I0 ng/nd) in the absence or presence of taurocholate (20 p.M) with and without BAPTMAM (50 taM), or Go6976 (1 ~M). Subsequemly, we ureasured PCNA protein expression. Results: Administration of actinomycin D + TNF-a increased cholanglocyte apoptosis, which was associated with inhibition of cholangiocyte proliferation and secretin-stimulated ductal secretion. Taurocholate teeding prevented TNF-ot induced duct damage, which was associated with restoration o[ cholangiocyte proliferation and secretin-stimulated ductal secretion. Taurocholate feeding prevented TNF-et stimulation of PRC-tt-2 protein expression In vitro, taurocholate prevention of TNF-c~-inhibition of PCNA protein expression was blocked by BAPTA/AM and Go6976. Summary/Conclusions: Taurocholate protects the biliary tree against the injury caused by actinomycin D + TNF-ot by Ca~ - and PKC-dependent mechanisms. Taurocholate regulates the balance between cholangiocyte proliferation/loss in cholestatic liver diseases.

$930 Myeloid Cell Leukemia ( M d ) q Protein Mediates Apoptosis Resistance in Cholangioeytes Makiko Faniai, Hajime Higuchi, Steven F, Bronk, Gregory J, Gores Cholangiocytes and their transformed counterpart, cholangiocarcinomas, express high levels ot antiapoptotic proteins and are resistant to apoptosts. In particular, these ceils richly" express Mcl-l, a potent antiapoptotic protein oi the Bcl-2 family, Thus, our AlMS were to test the hypothesis that Mcl-1 is responsible tbr the resistance of cholangincytes and cholangiocarcinoma cells to apoptosis. We choose TNF-ct related apoptosts inducing ligand (TK4IL) to induce apoptosis as this death ligand is expressed by matural killer ceils and likeIy participates in imnmne mediated bile duct iNury, nod it is being evaluated as a chen~odterapeutk: agent tor human mahgnancms. METHODS: Cholanglocytes were isolated h-ore sham operated and two week bik duct ligated (BDL) mice. KMCH cells were used as representative human cholangtocarcinoma cell line. Mcl-1 expression was examined using real time PCR technology and immunoblot analysis. KMCH cells were stably transfected with small interfering RNA (siRNAs) construct to knock down Mcl-i expression, RMCHsiRNA. Hmnan recombinant Flag tagged TRAIL pre-ofigomenzed with M2 antisera was used to induce apoptosis. Apoptosis was qnantitated using the fluorescent dye DAP1 and fluorescence microscopy. Caspase activity was quantitated using CaspaTag and fluorescence microscopy RESULTS: Mcl-1 mRNA expression in cholangtocTtes from BDL mice was 100-told greater than in cholangiocytes from sham-operated animals. The enhanced Mcl-1 expression in cholangmc),les from BDL roice was confirmed by immunoblot analysis. TRAIL induced apoptosis was reduced in choalngiocytes from BDL animals vs sham operated mice. Having confirmed that Mcl-1 regulates TRAIL serLsttivity in primary, cells, we next evaluated the role of Mcl-1 in KMCH cells treated with TRAIL, These cells richly express Mcl-1; however, Mcl-1 expression was reduced 80% in the KMCH-siRNA cells as compared to the parent cells by immunoblot analysis. TRAIL-induced apoptosis was 3-fold greater in the KMCH-siRNA vs mock transfected cells. Caspase activity paralleled the morphologlc quantitative assessment of apoptosis. In CONCLUSION, these data indicate that Mcl-I is a potent anti-apoptotic prolein in cholangmQ'tes and cholang~ocarinoma cells, Up regulation of Mcl1 occurs in proliferating bile duct cells following BDL suggesting a role for this protein in bile dn;t proliieration Down regulation of Mcl-1 expression may be a potential therapeutic strategy for the treatment of cholangiocarcinonm

$933 Autocrine TGF-beta signaling in cholangiocytes reduces impaired bile duct repair due to TNF Xuefeng Xia, Shujnn Shentu, Marion Roundtree, Azmeena Najam, Gene Lesage Restitution of epithelial structure following injury" requites epithelial proliferation to replace loss cells and cell migration to restore epithelial structural integrity. We have previously shown that restitution of bile duct structure following injury requires growth factor dependent cholangtocyte proliferation and rmgration and activation of cholangiocyte ERK and PI3K We propose that dysregulation of bile duct repair mechanisms may lead to progression of duct loss and ductopema m human cholestauc liver diseases Since TNF and TGF-beta are upregulated in chronic cholestauc liver disease and our previous studies show that TNF promotes bile duct injury, we tested if TNF and/or TGF-beta alters bile duct repair. Methods: In primary cultures of rat cholangiocytes, bile duct repmr was assessed in vitro by"measuring cholangiocyte proliferation by bromodeoxyuridine labeling and cholangiocyte nugration from the velocity of cell movement into a cholangiocytes monolayer abrasion wound and in Boyden chambers. Phosphorylation of PI3K and ERK w'as measured by Western immunoblots. TGF-beta gene and protein expression were determined by PCR and Western imnmnoblots, respectively. Cholangiocyte apoptosis was determined by the presence of nuclear fragmentation. Results: EGF and TGF-beta (but not TNF) increased the rate of cholangiocyte migration and AKT and ERK phosphorylatinn. EGF stimulated but TGF-beta inhibited cholangiocyte proliferation. TNF alone partially inhibited but TNF combined with anti-TGF-beta antibody completely inhibited EGF-stimulated cholangiocyte proliferation and mtgt'ation and AKT and ERK phosphorylation. TNF increased both gene and protein expression of TGF-beta in cholangiocytes. TGF-beta induced apoptosis only in stationary cholanglocytes whereas TNF increased cholangiocyte apoptosis in both migrating and stationary cholangtocytes. Conclusions: TNF may promote bile duct loss following injury by increasing cholangiocyte apoptosis and by inhibition of cholangiocyte ERK, P13K, migration and proliferation. TGF-beta increases cholangioc'yte migration and does not induce apoptosis in migrating cholangtocytes. The TNF inhibition of bile duct repair is partially compensated by maintenance of cholangiocyte migration due to an increased autocrine release of TGF-beta from cholangiocytes, We speculate that ductopenia due to dysregulation of cholangiocyte repair mechanisms may be a consequence of an inadequate cholaugiocyte TGF-beta response.

$931 etv[~3 lntegrin-mediated Activation of p38 MAPK in Human Cholangiocarcinoma Yoko Yamagiwa, Carla MarienMd, Tushar Patel BACKGROUND:Tumor growth requires interactions between tumor cells and the extracellular matnx (ECM). Integrins are cell surl~ce receptors that stimulate intracellular signaling in response to ECM components Howewr, their role in cholangiocarcinoma growth is poorly understood We have recently shown that the p38 MAPK pathway is constitutively' activated and mediates transformed cell growth in malignant human cholangiocytes. Thus, our AIMS were to examine integrin expression aM their relatmnship to p38 MAPK signaling in malignant cholangiocTtes METttODS: c~ and [3 integnn expression was assessed in human cholangiocarcinoma cell lines (KMCH, I'FK-1 and Mz-ChA-1) by immunocytochemistry, lntegnn mediated signaling was activated by incubation m 1% ECM gel or 1% fibmnectin (FN) p38 MAPK activity was assessed by in vitro killase assays. Function blocking antibodies were used to inhibit integrin mediated signaling. Cell adhesion to 1% FN was quantitated using a viable cell assay. Horizontal cell migration was assessed as the spread of cells beyond an artificial wound and vertical cell migration was assessed using a chemotaxis chamber. RF_SUETS: NI cell lines expressed integnn crV, but not oil or c~4 The pattern of integrin expression was identical in all cell lines and differed from that reported in non-rnalignant cholangiocytes. ECM and FN increased p38 b'b~.PKactivity in Mz-Cl'ua,-1 in a time-dependent manner suggesting a role for integrin mediated signaling in p38 MAPK activity. Indeed, comstitutive p38 ~%.~PKactivW was almost comphtely inhibited in Mz-ChA-1 cells after incubation with function-blocking antibodies to uv~3 for 24 hours, but was not significantly ahercd by either ~4 or cd b[ockmg antibodies Incubauon of Mz-Ct'~-I ceils with the p38 MAPK inhibitor SB203580 decreased celt adhesion, horizontal and vertical migration in a concentration dependent maturer

$934 Unique anti-apoptotic action of biliary lecithin against bile acid-induced cholangiocyte apoptosis is based primarily upon the inhibition Asbt-mediated uptake and Mrp3-mediated wash-out of bile acids Daisuke Komichi, Susumu Taznma, Tomoji Nishioka, Hideyuki Hyogo, Yasushi Sunami, Yasumasa Asamoto, Kuniham Nakai, Kazuhiko Tsuboi, Keishi Kanno, Atsushi Yamaguchi, Yoshihiro Numata, Toshiya Kobuke, Michihiro Nonaka, Kazuaki Chayama, Mizuho Une

SUMMARY: (1) celi surface integnn expression is altered in human cholangmcarcinoma cell lines, (2) c~V~3 integrins are essential tbr cotkstitutive p38 MAPK activity m Mz-ChA-1 human rnalignant cholangiocytes, and (3) inhibition of p38 MAPK signaling decreases tumor cell adhesion and migration

Backgrounds & aims: We previously reported that glycochenodeoxycholate (GCDC) induced cholanglocyte apoptosis, and that such a cytotoxic action of GCDC was inhibited by tauroursodeoxycholate (TUDC) or lecthins (Gastroenterology 2002;122:.4407). However, the underlying mechanism(s) was undefined, and thus, the aim of this study was to clarify whether such an apoptotic action is induced by the intracellular (uptaken) or extracellular (biliary) bile acid with an attention to bile acid transporters. Methods: 1. Immortalized mouse cholanglocytes were mcnhated with bile acids in a series of different hydrophobicity, tbllowed by Caspase (3, 8, and 9) activity assay. 2. Expression of cholangiocyte bile acid transporters (apical sodinm-dependent bile acid transporter, Asbt; multidmg resistance associated protein 3, Mrp3) was examined by Western blotting and RT-PCR analyses. 3. Cholangiocyte bile acid uptake was determined using 14C- taurocholate (TC) and/or 3H-TUDC time-sequentially (0, 3, 6 or 24hrs). Results: 1. GCDC induced activities of Caspase 3 and 9, whereas no drastic" change was evident in Caspase 8 activity. 2. Expression of Asbt and Mrp3 was induced by bile acids at mRNA level in a time-dependent manner, whereas Mrp3 mRNA expression was suppressed by lecithins. Similar changes were evident m protein level of these transporters. 3. A time-dependent uptake of 14C-TC and 3H-TUDC by' cholangtocyte was evident, but this was suppressed by lecithins. When 14C-TC and 3H-TUDC were incubated concomitantly, both were taken up by cholangiocytes non-competitivdy. Summary' and conclusions: 1 EnhatKement of Asbt expression and bile acid uptake suggests that

CONCLUSlONS:Phenotypm features of cholangiocarcinoma celia that promote tumor spread can be mediated by ECM-tumor cell interactions through c~V~3 mtegrin dependent p38 MAPK activation. This link between p38 MAPK signaling, ECM signaling and tumor cell phenotypic features could be therapeutically exploited to reduce cholangiocarcinoma growth.

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Taurocholate prevents TNFmt-induced bile duct damage of bile duct ligated (BDL) rats by Ca2+- and PKC-dependent mediated mechanisms Silvia Taftetani, Toshar Patel, Yoshiynki Ueno, Julie Venter, Heather Francis, Brandy Baumann, Shannon Glaser, Carla MarienMd, Jo Lynne Phmizy, Marco Marzioni, Antonio genedetti, Giantmnco klpim While cholaugiocyte proliferation is associated with increased number of ducts and ductal secretion, ductopenia is coupled with decreased ductal mass and secretion. A single injection of actinomycin D + TNF-cr to BDL rats induced cholangiocyte injury" characterized by loss of cholanglocyte pmlderation, secretion and increased apoptosis. The Ca>-dependent PKC

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AASLD Abstracts