HEPATOLOGY Vol. 22, No. 4, Pt . 2, 1995 A A S L D A B S T R A C T S 2 8 1 A

697 ROLE OF PLATELET-DERIVED GROWTH FACTOR (PDGF) IN HEPATIC FIBROSIS: EVALUATION IN A NOVEL TRANSGENIC MOUSE MODEL TJ Davern t, X Liao 2, L Ferrell 3, D Rockey t, SL Friedman l, JA Escabedo 2, LT Williams 2, and BF Scharschmidt t, UCSF Liver Center 1, Cardiovascular Research Inst. 2, and Depts of Pathology 3 and Medicine, Univ, of CA, San Francisco, CA 94143

PDGF is a potent in vitro mitogen for stellate cells, the main source of hepatic collagen and other matrix proteins. The regulatory role of PDGF in vivo, however, is uncertain. In vitro studies demonstrate that the soluble extracellular domain of the PDGFI3-receptor (XR) acts as a decoy which binds BB- and AB-PDGF isoforms and blocks PDGF- mediated mitogenesis (50% inhibition at ~2 p.g/ml; Duan et al., JBC 266: 413, 1991). Recently this work was extended (Liao et al., Clin Res 43: 378A, 1995) by developing transgenic mice in which XR is expressed in, and secreted by hepatocytes under the control of an albumin promoter. These mice secrete high (-5ug/ml) levels of XR in the peripheral circulation, with presumably even higher levels in the perisinusoidal fluid bathing stellate cells. The aim of this study was to assess the role of PDGF in normal (regeneration) and abnormal (cirrhosis) hepatic collagen production in this novel transgenic model. Methods: Regeneration was assessed by two-thirds hepatectomy and fibrosis was produced by six weekly doses of CC14 (5mg/kg in a 50% corn oil solution) given under light ether anesthesia by gavage. Liver sections, stained for H&E and trichrome, were graded blindly for distribution and severity of fibrosis. Stellate cell number was estimated by histochemical staining for desmin. Results: Hepatic regeneration (rate and histology) measured at one week after partial hepatectomy was equivalent in transgenic In=4) and normal In=3) mice. CCl4-treated transgenic and normal mice exhibited similar stellate cell proliferation. Both transgenic In=9) and normal (n=l 1) mice developed pericentral and scptal fibrosis on tfichrome stain, which tended (p=l}./15 and 0.25, respectively) to be less severe in transgenics. Conc lus ions : Blockade of PDGF had no effect on regeneration or stellate cell proliferation, but appeared to modestly attenuate hepatic collagen production in v ivo, suggesting an important regulatory role for other soluble factors or matrix. Confirmation of an effect of PDGF blockade on fibrosis in additional mice and models of fibrosis could nonetheless provide a rationale for therapeutic trials.

698 TRANSCRIPTIONAL ACTIVATION OF a2('I) COLLAGEN PROMOTER FOLLOWING CARBON TETRACHLORIDE I N J E C T I O N I N T O T R A N S G E N I C MICE. Y Ina~aki. P Greenwel*. S Truter*. T Nemoto. K Kobavashi. and F P, amirez*. National Kanazawa Hospital and the First Departlnem of Internal Medicine, 'Kanazawa University School of Medicine, Kanazawa, Japan, and *Brookdale Center for Molecular Biology, Mount Sinai School of Medicine, New York, New York.

Regulation of collagen gene expression plays critical roles in various physiopathological conditions in the liver. We have previously identified the promoter region of a2(I) collagen gene (COL1A2) that is essential for both the basal transcription and TGF-I~-elicited stimulation of the gene in dermal fibroblasts and fat-storing cells. In order to determine if the same promoter region is activated following acute and chronic liver injury in vivo. several transgenic mouse lines have been generated in which the -313 to +54 COL1A2 promoter sequence linked to the bacterial f,-galactosidase gene (LacZ) has been transmitted. Integration of the transgene was analyzed by Southern blot hybridization using tail DNA. Acute and chronic liver injury was introduced by injecting once a week 0.1ml/kg body weight of carbon tetrachloride (CC14) intmperitoneally. Liver tissues were excised at several time points alter CC14 injection, and serial sections were subjected to both the regular hemamxylin-eosin and Azan staining and the X-Gal staining that detects LacZ expression in the liver. Liver tissues from the mice treated with a single dose of CC14 showed eentrilobular zonal necrosis, and LacZ expression was detected only in the mesenchymal cells in the necrotic areas but not in the parenchymal hepatocytes around the portal tracts. In contrast, LacZ expression was markedly decreased 7 days alter a single dose of CC14 injection. In chronically treated mice, connective tissues began to accumulate in the centrilobular areas alter 4 weeks of CC14 treatment, and thicker fibrous septa were formed after 12 weeks of repeating CC14 injection. X-Gal staining of the serial sections clearly indicated that LacZ expression was observed in mesenchymal cells in and around the fibrous septa. Altogether these results indicated that the -313 to +54 COL1A2 promoter region containing the TGF-13-responsive element in vitro is utilized for stimulating the gene expression in vivo. It is also suggested that the continuous activation of this promoter may cause the progression of liver fibrosis.

699 RECOGNITION OF TWO Spl BINDING SITES UPSTREAM TO THE NUCLEAR PROTEIN I (NF-I) BINDING SITE IN THE MOUSE a2(I) COLLAGEN PROMOTER. K. Miao. J.J. Potter, F.A. Anania, L.L. Rennie-Tankersle¥ and E. Meze¥. Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD

Acetaldehyde (AC) activates the promoter of the mouse a2(I) collagen gene. This effect is mediated in part by increased binding of NF-I to the promoter. AC may act through additional mechanisms, since transient transfection studies with deletions in the promoter upstream to the NF-I binding site activated the promoter and prevented further enhancement by AC. The purpose of this study was to identify areas of nuclear protein binding to the promoter, and the identity, and transactivating function of these proteins. Nuclear proteins were extracted from NIH 3T3 cells and from rat myofibroblast~like cells in culture. Three major areas of protection were identified by DNAse I protection analysis upstream to the NF-I binding site which is located between -308 and -291 of the 5' flanking region of the cq(I) collagen promoter. Two of these regions, one located at -473 to -453 (region 1), and another located at -542 to -507 (region 2), have 70% and 80% homology, respectively, to the consensus binding sequence for the trans-aeting factor Spl. Oligonucleotide-protein complexes were identified by eleetrophoretic mobility shift assays with oligonueleotides specifying regions 1 and 2. Antibody to Spl supershifts one of three major complexes in region 1 and one of 2 complexes in region 2 conf'trrning that Spl protein binds to each of the two regions. Other oligonucleotide-protein complexes which are detected, and not supershifted by the Spl antibody, may represent other members of the Spl family. In conclusion, two areas of Spl binding have been identified upstream to the NF-I binding site in the a2(I) collagen promoter which may play a role in the regulation of the a2(I ) collagen gene.

700 INHIBITION OF PROTEIN SYNTHESIS IN LIVER STELLATE CELLS (LSC) BY AN ANTISENSE OLIGONUCLEOTIDE DERIVED FROM AN RNA-BINDING PROTEIN OF ACTIVATED

1 1 1 2 3 LSC. A Mat~¢i, I O2ata. H Ikeda. K Fuiiwara. P Greenwel and M • " 4 1 - Rolkmd. First Dept. of Internal Medicine, Faculty of Med. University

2 of Tokyo, Tokyo, Japan; Third Dept. of Internal Medicine, Saitama 3 Med. School, Saitama, Japan, Brookdale Ctr. for Mol. Biol., Mt Sinai

School of Med., New York, N.Y. and 4Marion Bessin Liver Res. Ctr. and Depts of Medicine and Pathology, Albert Einstein Cell of Med. Bronx, N.Y.

BACKGROUND: Clone #72 (C-72) was isolated from a eDNA li- brary prepared from LSC line, CFSC, after screening with an antibody prepared against the LSC line. C-72 mRNA which is weakly expressed in freshly isolated LSC, is increased after culturing the cells. The mRNA is also expressed in fibroblasts but not in hepatocytes, Kupffer and sinu- soidal endothelial cells. We now report further characterization of eDNA clone #72. METHODS: Primary cultures of LSC and the LSC clone derived from a cirrhotic rat liver, CFSC-3H, were used for these studies. Expression of protein encoded by C-72 was followed by western blot analysis. CFSC-3H was treated with either an antisense (AS) or a non- sense INS) oligonuclotide derived from the 3' end of C-72 and protein production was determined between 12 to 36 hours post-treatment. RESULTS: C-72 contained an open reading frame of 1404 bp that encodes for a protein of 468 amino acid residues. By western blot analy- sis, its m.w. was 55 kDa. Its derived C-terminus contains RNP-1 and RNP-2 domains, typical of RNA-binding proteins. This protein was not detected in one day LSC primary cultures but its expression increased in 4 and 7 day cultures. When CFSC-3H were incubated with 10 #M AS oligonuclotide, the expression of the 55 kDa protein was lost and colla- gen and non-collagenous protein production decreased to 36% and 27% respectively, of control cells incubated with NS oligonucleotide. AS oligonucleotide had no effect on the expression of or-smooth muscle actin or cell viability. CONCLUSION: Expression of C-72 is increased in cultured LSC when total RNA content increases 10-fold. Its down-regu- lation inhibits protein synthesis. We conclude that this putative RNA- binding protein may play an important role in LSC activation.