Scavenger receptors of the class B family: possible mediators of the biological actions of MAA adducts on hepatic stellate cells

Scavenger receptors of the class B family: possible mediators of the biological actions of MAA adducts on hepatic stellate cells

262A AASLD ABSTRACTS HEPATOLOGYO c t o b e r 2 0 0 1 359 360 OVEREXPRESSION OF DOMINANT-NEGATIVE RECEPTOR TYPE-II TO TGF-18 INHIBITS COLLAGEN TYP...

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262A

AASLD ABSTRACTS

HEPATOLOGYO c t o b e r 2 0 0 1

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OVEREXPRESSION OF DOMINANT-NEGATIVE RECEPTOR TYPE-II TO TGF-18 INHIBITS COLLAGEN TYPE I BY A DECREASE IN SMAD-2 A N D 3 ACTIVATION. J u a n Armendariz-Borunda, CUCS, University of Guadalajara, Guadalajara, Jalisco Mexico; Ivan Hernandez-Canaveral, Vidal DelgadoRizo, Jaime Gonzalez-Cuevas, University of Guadalajara, Guadalajara Mexico

PHOSPHATIDYLIN O SITOL-3 KINASE-DEPENDENT SIGNALING PATHWAYS MEDIATE BOTH CELL PROLIFERATION A N D PROTEIN P R O D U C T I O N IN HEPATOCYTES STIMULATED BY HEPATOCYTE GROWTH FACTOR. Tomoaki Tomiya, Yukiko Inoue, Department of Gastroenterology, University of Tokyo, Tokyo Japan; Miho Yamaoka, Third Departm e n t of Internal Medicine, Saitama Medical School, Saitama Japan; Mikio Yanase, Masahiro Arai, Hitoshi Ikeda, Kazuaki Tejima, Kayo Nagashima, Itsuro Ogata, Department of Gastroenterology, University of Tokyo, Tokyo Japan; Kenji Fujiwara, Third Department of Internal Medicine, Saitama Medical School, Saitama J a p a n

TGF-/3 is a pleiotropic protein w h i c h participate in cell differentiation, imm u n e response, a n d remodelling of extracellular matrix. TGF-13 induce the p r o d u c t i o n a n d deposition of collagen I, TIMP a n d PAI-I. TGF-]3 induces its effects by binding to its Ser/Thr Kinase receptor type-II a n d then recruiting a n d activating receptor type I, w h i c h is phosphorylated a n d activates Smads that transduce the signal to nucleus. In this work, we blocked the signal transduction p a t h w a y t h r o u g h specific delivery of cDNA of a dominant-negative receptor-II (ACytTI3RII) to TGF-/3 w h i c h lacks Ser/Thr kinase intracytoplasmic domain activity. To this end Cos-1 a n d Hepatic Steflate Cells (HSC) were co-transfected with pCMV5-ACytT/3RII a n d pAdTrack-GFP using lipofectamine. Fluorescence m i c r o s c o p y demonstrated a n average 10% transfection efficiency. An affinity 125I-TGF-13 radio-labelling was setup to verify the cell m e m b r a n e expression of truncated receptor-lI w h i c h overrode lzSI-TGFI3 binding by the endogenous wild type receptor type II in Cos-1 cells. ACytT/3RII expression in HSC was n o t e n o u g h by this transfection m e t h o d due to the high levels of endogenous wild type receptor in these cells. EMSA were performed using consensus sequence for AP-1 a n d Smad-2 a n d -3 rendering a 3-fold decrease in DNA-binding activity, reflecting a down-activation ill Smad complexes a n d AP-1 ill ACytT/3RII-transfected cells b u t no in pAdTrack-GFPcells. Identity of these transcriptional factors was confirmed using specific antibodies to compete for DNA binding. Collagen I mRNA expression was analyzed b y RT-PCR s h o w i n g a 5-fold decrease. Finally, all these events were reflected in a diminished Collagen type I p r o d u c t i o n in ACytT/3RII-transfected Cos-1 as measured by incorporation of [3H]Prohne a n d further SDS-PAGE electrophoresis. Thus, this could be a useful strategy to down-regulate or prevent exacerbated synthesis a n d deposition of extraceflular matrix in a given fibrotic process. Supported b y CONACYT grant # 33331-M.

While differentiated cellular functions are thought to be suppressed during ceil proliferation, recent studies have revealed that several mitogens stimulate protein production in hepatocytes as well as proliferation. At least two major signal transduction pathways, the mitogen-activated protein kinase (MAPK)pathway and the phosphatidylinositob3 kinase (PI3K) pathway,have been shown to be activatedin response to the stimulation of receptor tyrosine kinases by growth factors. However, the significanceof these pathways remains to be eIucidated in hepatocytes. In addition, we reported recently that endogenous transforming growth factor a (TGFa) is up-regulated by hepatocyte growth factor (HGF) and contributes to hepatocyte proliferation (AmJ Pathol, 157: 1693, 2000). Th/s suggests that the interactions of growth factors may play a role during signal transduction in hepatocytes. Aim: To study the significance of intracelluiar signal transduction during proliferation and protein production in hepatocytes stimulated by HGF. Methods and Results: Isolated rat hepatocytes were initially cultured at a density of 5x104cells/cm2(low density culture). The addition of HGF did not affect the concentration of albumin in the medium, but both DNA synthesis and TGFa content in these hepatocytes increased in a dose-dependent manner. Moreover,the addition ofa tyrosine kinase inhibitor, specificfor the EGF/TGFa receptor, reduced the stimulatory effect of HGF on DNA syntilesis. The addition of TGFa caused a similar increase in DNA synthesis by hepatocytes. In contrast, when hepatocytes were cultured at a density of 1.2x10~cells/cm2(high density culture), tile concentration of albumin in the medium but not DNA synthesis increased dose-dependently with the addition of either HGF or TGFR. Cellular TGFee content did not increase in the HGF-treatedhepatocytes;moreover, the addition of an inhibitor for the EGF/TGFcereceptor tyrosine kinase did not affect the stimulatory effect of HGF on albumin production. Hepatocytes were incubated in the medium containing specific inbibitors of various intracellular protein kinases. In hepatocytes cultured at the low density, the increased DNA synthesis observed in response to HGF treatment was blocked almost completelyby wortmannin, a PI3K inhibitor, and by rapamycin, a P70S6 kinase inhibitor, whereas PD98059, an inhibitor of MAPKkinase (extraceliular signal-regulatedkinase), was less potent and abrogated HGF-induced DNA synthesis by less than 20%. The p38 MAPKinhibitor, SB 203580, was without significant effect on the DNA synthesis. These protein kinase inhibitors had similar effects on the DNA synthesis induced in hepatocytes treated with TGFa. In hepatocytes cukured at the high density, wortmannin or rapamycin did not permit the HGF- or TGFa-induced increase in albumin concentration in the medium, whereas PD98059 or SB 203580 did not affect the increases of the albumin concentration. MTT assay showed that the mitochondrial function of the hepatocyteswas not impaired by wortmannin or rapamycin at the doses used in these experiments. Conclusions:In hepatocytes cultured at low density, the EGF/TGFa receptor activation and the PI3K signaling pathway, perhaps through the activation of p70S6 kinase, may contribute to the up-regulation of DNA synthesis by HGF. This growth factor also stimulates albumin production ill hepatocytes cultured at high density, also via tire activation of the P13Kpathway but independent of TGFa. These results suggest that, depending on the physiologicalconditions, PI3Kpathways can mediate either cell proliferation or protein production in hepatocytes stimulated by HGF.

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SCAVENGER RECEPTORS OF THE CLASS B FAMILY: POSSIBLE MEDIATORS OF THE BIOLOGICAL ACTIONS OF MAA A D D U C T S ON HEPATIC STELLATE CELLS. K u s u m K Kharbanda, Kris A Shubert, T h o m a s W Halverson, Michael F Sorrell, Dean J Tuma, Department of Veterans Affairs, Nebraska/Western Iowa Health Care System, Omaha, NE

REGULATION OF CAMP RESPONSIVE ELEMENT BINDING PROTEIN (CREB) EXPRESSION A N D ACTIVITY IN EXPERIMENTAL HEPATOCED LULAR CARCINOMA (HCC). Stephen J Kovach, University of Rochester Medical Center, Rochester, NY; Julie A Price, James V Sitzmann, Iain H McKiflop, University of Rochester, Rochester, NY

Key players in the pathogenesis of alcohol-induced injury are the hepatic stellate cells (HSCs). Many of the hepatotoxic effects of ethanol have been linked to the formation of adducts, resulting from the covalent binding of highly reactive aldehydes, acetaldehyde (ACH), malondialdehyde (MDA) or 4-hydroxy nonenal (HNE), produced in the liver during ethanol ingestion, to hepatic proteins. Studies from our laboratory have shown that ACH and MDA, can react together with proteins in a synergistic manner to form distinct hybrid adducts, designated as MAA adducts. Studies from our laboratory have also shown that MAA adducts are formed in the livers of ethanol-fed rats and that these adducts induce the secretion of two chemokines (macrophage chemotactic protein-i, macrophage inflammatory protein-2), increase the expression of intercellular adhesion molecule-1 and increase the synthesis of plasminogen activator by HSCs. A family of receptors, designated as scavenger receptors, are likely candidates mediating these effects of MAA adducts on HSCs, because these receptors have been shown to mediate the binding and endocytosis of a variety of aldehyde-modified proteins in other cell types. To test this hypothesis, the candidate binding protein(s) for MAA adducts on membrane preparations of HSCs were identified by ligand blotting and western blotting techniques. HSCs were isolated from male Wistar rats by sequential pronase and collagenase perfusion followed by density gradient centrifugation in Larcoll and cultured in 1:1 Dulbecco's modified Eagle's/F-12 medium supplemented with fetal calf serum, glutamine and antibiotics. The solubilized membrane proteins from HSCs in passages 2-6 were separated on SDS-PAGE under non-reducing conditions and transblotted on to nitrocellulose membrane filters. For ligand blotting, blots were incubated with 0.5 mg/ml BSA-MAA. The binding of BSA-MAA to HSC membrane proteins was visualized by sequentially incubating the blots with mouse anti-MAA and goat antimouse immunoglobulin G conjugated to peroxidase followed by enhanced chemiluminescence visualization. Under non-reducing conditions, major binding proteins with an estimated molecular mass of 55-80 kDa were detected on HSCs membranes. Competitive binding with a scavenger receptor-specific ligand or hexyl-MAA, a structural analog of the MAA adduct, significantly abrogated BSA-MAA binding, indicating the specificity of interaction of MAA adducts with the binding proteins on HSC membranes. Additional experiments determining whether these binding proteins on HSCs belong to the family of scavenger receptors was accomplished by western blotting technique using specific antibodies to SRBI/BII or CD36 scavenger receptor type. The results confirmed the presence of both receptor types in membrane preparations from HSCs. The apparent moIecular mass was estimated to be between 55-80 kDa. We conclude that 55-80 kDa binding proteins, belonging to class B family of scavenger receptors represent the most likely candidates for mediating the effects of MAA adducts on HSCs.

Previous reports from o u r laboratory have identified altered expression of G-proteins in h u m a n a n d animal models of HCC. Cyclic-AMP (cAMP) is a key second messenger in the regulation of diverse cell function including growth a n d differentiation, lntracellular cAMP levels control protein kinase A (PKA) activity and, in turn, regulate specific gene transcription factors termed cAMP responsive elements (CRE's). These CRE's are critical to cell function and differential gene transcription. The rat H C C cell line (H4IIE) was cultured in SWIMS-77 media supplemented with 5% FFBS to 75% confluency. Media was removed a n d cells were made quiescent for 24 h o u r s with s e r u m depleted media (0.1% FBS). Ceils were then treated with s e r u m (5% FBS), a cell permeable cAMP analog (8Br-cAMP) or the Gs-protein activator cholera toxin (CTx). Cell lysates were prepared at serial time points a n d analyzed for total CREB expression a n d CREB activity (phospho-CREB [pCREB]) expression. Cells were subsequently pretreated with either an adenylyl cyclase (AC) specific (MDL, [MDL-12 330A HC1]) or PKA specific inhibitor (H89, [N-(2-{pbromocinnamylamino}ethyl)-5-isoquinolinesulfonamide]) a n d treated with serum, 8Br-cAMP or CTx. Treatment with serum, 8Br-cAMP or CTx lead to significantly increased pCREB expression, a maximal increase being detected 15 rains after treatment, pCREB expression returned to baseline levels within 1 h o u r after addition. Treatment with MDL abrogated the effects of CTX a n d significantly blunted serum stimulated pCREB expression. In contrast, MDL failed to alter the effects of 8Br-cAMP on pCREB expression. H89 abolished the effects of b o t h 8Br-cAMP a n d CTx a n d significantly diminished the effects of s e r u m on pCREB expression. Expresson of total CREB remained u n c h a n g e d t h r o u g h o u t the course of all experiments. These data suggest cAMP m a y play a critical role in the progression of gene transcription a n d the increased cell mitogenesis characteristic of HCC.