837 Transforming growth factor beta 1-induced hepatocyte apoptosis is mediated by SMAD3-dependent acute generation of reactive oxygen species

HEPATOLOGY, Vol. 38, No. 4, Suppl. 1, 2003

AASLD ABSTRACTS

supraoptic and paraventricular nuclei were activated. Hypothalamic c-Fos immunohistochemistry was indeed negative in shamoperated rats but strongly positive in hepatectomized rats one hour after surgery. Also, plasma vasopressin concentration raised sharply one hour after hepatectomy but not after sham surgery. Moreover, as shown by cytosolic Ca2+ m e a s u r e m e n t (spectrofluorimetry and videomicroscopy), isolated hepatocytes were fully responsive to vasopressin during the first hours of regeneration, and became desensitized thereafter, exhibiting slow oscillating Ca2+ responses to vasopressin on the following days. Two waves of hepatocyte desensitization occured, 24h and 120h, after partial hepatectomy, with restoration of hepatocyte sensitivity between the two waves, and at the sixth day. On the first day, hepatocyte vasopressin V l a receptor density decreased and its lobular gradient increased in hepatectomized rats, as shown by autohistoradiography and RTPCR. This remodelling of V l a receptor density and distribution was no more detected at 48h posthepatectomy. By specifically antagonizing the V l a receptor in vivo, we demonstrated that vasopressin contributes to NF-kapp aB (gel shift assay) and cyclin (D1 and A, western blot) activation, to hepatocyte progression in the cell cycle (BrdU hepatocyte incorporation) and to liver mass restoration. Finally, we found that vasopressin exerted a more p r o n o u n c e d choleretic effect in the isolated perfused liver from hepatectomized rats t h a n in liver from sham-operated animals. Also, in hepatectomized rats treated by a specific Vla receptor anatgonist, a more p r o n o u n c e d cholestasis occured as compared as n o n treated rats, as shown by plasma bile acids measurements. In conclusion, we provide compelling in vivo evidence that vasopressin contributes significantly to growth initiation and bile flow stimulation in the early stages of liver regeneration. Remodelling of Vla receptor expression and vasopressin-induced Ca2+ signalling are suggested to underlie these effects. Disclosures: G6rard Alonso - No relationships to disclose Sylviane Boucherie - No relationships to disclose Laurent Combettes - No relationships to disclose Gilles Guillon - No relationships to disclose Alexandra Nicou - No relationships to disclose Sylvie Prigent - No relationships to disclose Val6rie Serri~re - No relationships to disclose Thierry Tordjmann - No relationships to disclose

565A

Methods: Primary hepatocytes were isolated by collagenase perfusion from Smad3 wild-type ( + / + ) and knockout (-/-) mice and treated with TGF/3 (5 ng/ml) with or without pretreatment with the antioxidant, trolox (2 raM). Apoptosis was assessed morphologically with propidium iodide stained fluorescent microscopy and biochemically by caspase-8 and caspase-3 activities. Intracellular ROS was m e a s u r e d with the fluorometric probe H2DCFDA and the MPT was assessed by confocal microscopy with the dyes TMRM and calcein. Transfection experiments with wildtype (WT) Smad3 plasmid, dominant negative (DN) Smad3 plasmid, or control DNA vector were performed with Targafect F-1 (2.5 ul). TGF/3induced gene transcription was measured with the reporter plasmid p3TP-Luc. Results: Smad3 (-/-) hepatocytes were less sensitive to TGF/3induced apoptosis compared to ( + / + ) hepatocytes (8% -+ 1% vs 43% _+ 5%) at 48 hours. With TGF/3 treatment, Smad3 (-/-) hepatocytes did not generate caspase-8 and -3 activities (0.91- and 1.01-fold increase, respectively), but ( + / + ) cells had a 1.7 and 3.4-fold increase in caspase-8 and -3 activities, respectively. TGF/3 generated an acute (30 rain) increase in ROS activity (1.7-fold increase) in the ( + / + ) hepatocytes; but (-/-) cells exhibited no TGF/3-induced ROS activity (0.5-fold increase). Smad3 (-/-) hepatocytes also failed to undergo the MPT that was present 8-10 hours following TGF/3 treatment in ( + / + ) cells. Pretreatment with trolox inhibited TGF/3-induced ROS (2.8 fold decrease at one hour), apoptosis (6% _+3% vs 55% _+ 11%), and caspase-3 activity (2.2 fold decrease at 48 hours) in ( + / + ) hepatocytes. In ( + / + ) hepatocytes, transfection with DN Smad3 inhibited TGF/3-mediated transcription (7-fold decrease), whereas (-/-) cells transfected with WT Smad3 h a d TGF/3 transcriptional activity restored (2-fold increase). In ( + / + ) hepatocytes transfected with DN Smad3, TGF/3induced ROS was inhibited (65% decrease at 30 rain), in addition to the MPT (at 12 hours) and apoptosis (23% _+ 6% vs 57% _+ 19%); however, transfection of WT Smad3 plasmid in (-/-) hepatocytes permitted TGF/3-induced ROS activity (49% increase), MPT (at 12 hours), and apoptosis (41% _+ 5% vs 18% _+ 4%). Conclusions: TGF/3-induced primary hepatocyte apoptosis occurs through a Smad3-dependent signal transduction pathway. Smad3 is involved in the initiation of an acute generation of ROS. Downstream mediators of the TGF/3 apoptotic signaling pathway, including the MPT, caspase-8, and caspase-3, are secondarily d e p e n d e n t on Smad3.

837 T R A N S F O R M I N G G R O W T H F A C T O R BETA 1-INDUCED HEPATOCYTE APOPTOSIS IS MEDIATED BY S M A D 3 D E P E N D E N T ACUTE GENERATION OF REACTIVE OXYGEN

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SPECIES. Dalliah Black, Suzann e Lyman, Ting Qian, John J LeMasters, Richard Rippe, Kevin~ Behrns, University of North Carolina at Chapel Hill, Chapel Hill, NC

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Background: Transforming growth factor beta I (TGF/3) induces hepatocyte apoptosis and mediates other hepatocellular processes through Smad protein signal transduction pathways.The TGF/3 apoptotic pathway includes caspase activation, reactive oxygen species (ROS) generation, and the mitochondrial permeability transition (MPT). The mechanisms by which the TGF/3 apoptotic and Smad signal transduction pathways interact to cause apoptosis have not b e e n elucidated clearly. Purpose: Determine the mechanisms by which Smad3 mediates TGF/3-induced apoptosis in primary murine hepatocytes.

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Disclosures: Kevin E Behrns - No relationships to disclose Dalliah Black - No relationships to disclose John J LeMasters - No relationships to disclose Suzanne Lyman - No relationships to disclose Ting Qian - No relationships to disclose Richard Rippe - No relationships to disclose