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Intracellular signaling pathways activated by reactive oxygen species regulate proliferation and collagen synthesis of human hepatic stellate cells
Poster Sessions
84
I626
EFFECTS OF CYTOKINES ON THE PROMOTER ACTIVITIES OF TGFB_l AND LTBP-1 IN CIRRHOTIC RAT FAT STORING CELLS (CFSC)
K. Breitkopf, J.H. Westhoff, C.G. Tag, A.M. Gressner. Znstitute of Clinical Chemistry, University Hospital, Aachen, Germany
TGFb is an important cytokine during liver fibrosis. The major portion of it is secreted latent, complexed either only with LAP or also with LTBP. Due to the proposed functions of LTBP in the regulation of TGFb-bioavailability and -activity the effects of cytokines on the promoter-activities of TGW-1 and LTBP-1 were investigated in a comparative way in CFSC. This cell-line, that shares great homology with hepatic stellate cells, was obtained from cirrhotic rat liver (Greenwell et al., 1991). Luciferase-reporter gene assays for the promoters of TGFb-1 and LTBP-1 (L and S forms) were performed after stimulation of transfected CFSCs with TGFh-1, IL-6, PDGF and TNFa. All promoters showed basal activity, after 24 hours but PDGF and TNFa did not have a remarkable effect. TGFb-1 did not affect LTBP-promoter activity hut autocrine induction of the TGFh-promotor was found. Studies using a construct with nine copies of a TGFh-inducible DNA element (“CAGA”), confirmed that the cells can respond to TGFb-1 with maximal luciferase after six hours whereas highest promoter activity of TGFb was found after two days. IL-6 induces the promoters of LTBP-IL and S with a peak at six hours while it increases TGFb-promoter activity only marginal after 2 to 3 days. After stimulation with IL-6 and TGFb- 1 itself, LTBP and TGFb seem to be differentially regulated on the promoter level. Although basal activity of the TGFb-promoter was found, unstimulated cells did not reveal ongoing signalling. These results indicate that basally synthesized TGFb-1 is probably secreted in a latent form, bound to LTBP. The differences in the time courses of TGFb-1 action on CAGA and the promoter let us further speculate that the autoinduction of TGFb-1 involves the synthesis of additional molecules.
I688
CROSS-TALK BETWEEN PHOSPHATIDYLINOSITOL 3.KINASE (Pl3-K) AND THE NA+/H+ EXCHANGER IN HEPATIC STELLATE CELLS (HSC): EFFECT OF CANRENONE
A. Caligiuri, F. Marra, P. Failli, R.M.S. DeFranco, R.G. Romanelli, P. Gentilini, M. Pinzani. Medicina Interna, University di Firenze, Italy Canrenone (C), an aldosterone antagonist, is able to reduce PDGF-induced HSC proliferation and migration, key events in the hepatic fibrogenic process. These inhibitory effects are independent of the anti-aldosterone action of C and are, at least in part, mediated by an inhibition of PDGF-induced PD-K activity. Since PI3-K has been shown to modulate growth factor-induced intracellular pH changes through the Na+/H+ exchanger, we studied the cross-talk occurring between these signaling pathways and the effects of C in human activated HSC. Canrenone (10-25 uM) reduced PDGF-induced Na+/H+ exchanger activity similarly to the established inhibitor ethylisopropyilamiloride (EIPA) and the PI3-K inhibitor LY294002. However, while EIPA inhibited the basal activity of the exchanger after an acid load, no effects were observed with both LY294002 and C in the dose range employed, thus confirming that PI3-K is involved only in the regulation of PDGF-induced Na+/I-I+ exchanger activity. To confirm that a clear cross-talk occurs between these systems following stimulation with PDGF, the effect of EIPA on PI3-K was evaluated. EIPA markedly reduced PI3-K activity thus suggesting that inhibition of PI3-K activity by this compound contributes to the modulation of Na+/H+ exchanger activity. Both C and EIPA produced a dose-dependent inhibition of PDGF-induced focal adhesion kinase (FAK) phosphorylation, a signaling event occurring downstream of the activation PI3-K and the Na+/I-I+ exchanger and essential for growth factor-mediated cytoskeletal reorganization. These observations suggest a complex cross-talk involving PI3-K. Na+/H+ exchanger and
FAK in HSC and provide new basis for the antifibrogenic action of this drug.
I 716
INTRACELLULAR SIGNALING PATHWAYS ACTIVATED BY REACTIVE OXYGEN SPECIES REGULATE PROLIFERATION AND COLLAGEN SYNTHESIS OF HUMAN HEPATIC STELLATE CELLS
G. Svegliati-Baroni I, A. Galli ‘, E. Ceni ‘, F. Ridolfi ‘, R. Salzano ‘, E. Bendia’, A. Casini ‘. ‘Alcohol Research Center & GI Unit; 2 Department of Gastroenterology, University of Ancona, Italy
Reactive oxygen species (ROS) stimulate human hepatic stellate cells (HSC) proliferation and collagen synthesis. Intracellular signaling elements (such as ERKlR and PI3K) have a role in transducing signals from the cell surface to the nucleus and in mediating a fibrogenic behaviour in HSC. We thus verified which intracellular signaling pathways are activated by ROS and associated with HSC proliferation and collagen synthesis. The xanthine/xanthine oxidase system (K/X0) was used as a superoxide anion donor. Phosphorylation of ERKU2 and pp70S6K (a downstream component of PI3K) was evaluated by Western blot. HSC proliferation and matrix synthesis were evaluated by 3H-TdR incorporation and ELISA for type I collagen, respectively. HSC incubation with x/x0 was associated with increased ERK1/2 and pp70S6K phosphorylation, well evident at 10 and 30 min and maintained up to 60 min for ERKU2. These effects were completely abolished by tyrphostin A9, a selective inhibitor of the PDGF receptor tyrosine kinase, and PD98059, a MEK inhibitor. The PI3K inhibitor wortmannin reduced pp70S6K phosphorylation with no effect on ERKlR, while the PKC inhibitor calphostin C decreased protein phosphorylation only in the later phase. Inhibition of these mitogenic in a parallel reduction in X/X0-induced HSC proliferation, whereas X/KO-induced stimulation of type I collagen accumulation was inhibited only by calphostin C and PD98059. In conclusion, this study indicates that ROS activate several signaling pathways which differentially regulate HSC proliferation and collagen synthesis, thus providing new selective targets for the specific inhibition of these effects in human HSC.
I 719
EXPRESSION AND FUNCTION OF TETRASPANINS IN HEPATOCARCINOMA CELL LINES
C. Cordella, A. Mazzocca, V. Carloni. Medicina Intema, University di Firenze, Italy
Tetraspanins (TM4SF), are a large family of ubiquitously expressed membrane proteins that are implicated in cell proliferation and tumor cell invasion. Notably, TM4SF proteins form membrane complex with integrins. Recent data have clearly demonstrated that the function of integrinTM4SF protein complexes is relevant to cell migration. In this study we have characterized the TM4SF CD9, CD63, CD81, CD151 in two diffent hepatocarcinoma cell lines HepG2 and Huh7. Expression levels of TM4SF were evaluated by flow cytometry and immunofluorescence. HepG2 expressed CD63 and CD151, in contrast Huh7 showed high levels of CD9, CD63, CD8 1, CD15 1. Beta1 integrin co-immmunoprecipitd with TM4SF. mAbs against TM4SF were able to reduce haptotactic migration on laminin, collagen IV and collagen I without affecting cell adhesion on these matrix proteins. Take together, these results suggest that beta1 it&grin induces TM4SF-dependent cell motility in a matrix independent manner.
84
I626
EFFECTS OF CYTOKINES ON THE PROMOTER ACTIVITIES OF TGFB_l AND LTBP-1 IN CIRRHOTIC RAT FAT STORING CELLS (CFSC)
K. Breitkopf, J.H. Westhoff, C.G. Tag, A.M. Gressner. Znstitute of Clinical Chemistry, University Hospital, Aachen, Germany
TGFb is an important cytokine during liver fibrosis. The major portion of it is secreted latent, complexed either only with LAP or also with LTBP. Due to the proposed functions of LTBP in the regulation of TGFb-bioavailability and -activity the effects of cytokines on the promoter-activities of TGW-1 and LTBP-1 were investigated in a comparative way in CFSC. This cell-line, that shares great homology with hepatic stellate cells, was obtained from cirrhotic rat liver (Greenwell et al., 1991). Luciferase-reporter gene assays for the promoters of TGFb-1 and LTBP-1 (L and S forms) were performed after stimulation of transfected CFSCs with TGFh-1, IL-6, PDGF and TNFa. All promoters showed basal activity, after 24 hours but PDGF and TNFa did not have a remarkable effect. TGFb-1 did not affect LTBP-promoter activity hut autocrine induction of the TGFh-promotor was found. Studies using a construct with nine copies of a TGFh-inducible DNA element (“CAGA”), confirmed that the cells can respond to TGFb-1 with maximal luciferase after six hours whereas highest promoter activity of TGFb was found after two days. IL-6 induces the promoters of LTBP-IL and S with a peak at six hours while it increases TGFb-promoter activity only marginal after 2 to 3 days. After stimulation with IL-6 and TGFb- 1 itself, LTBP and TGFb seem to be differentially regulated on the promoter level. Although basal activity of the TGFb-promoter was found, unstimulated cells did not reveal ongoing signalling. These results indicate that basally synthesized TGFb-1 is probably secreted in a latent form, bound to LTBP. The differences in the time courses of TGFb-1 action on CAGA and the promoter let us further speculate that the autoinduction of TGFb-1 involves the synthesis of additional molecules.
I688
CROSS-TALK BETWEEN PHOSPHATIDYLINOSITOL 3.KINASE (Pl3-K) AND THE NA+/H+ EXCHANGER IN HEPATIC STELLATE CELLS (HSC): EFFECT OF CANRENONE
A. Caligiuri, F. Marra, P. Failli, R.M.S. DeFranco, R.G. Romanelli, P. Gentilini, M. Pinzani. Medicina Interna, University di Firenze, Italy Canrenone (C), an aldosterone antagonist, is able to reduce PDGF-induced HSC proliferation and migration, key events in the hepatic fibrogenic process. These inhibitory effects are independent of the anti-aldosterone action of C and are, at least in part, mediated by an inhibition of PDGF-induced PD-K activity. Since PI3-K has been shown to modulate growth factor-induced intracellular pH changes through the Na+/H+ exchanger, we studied the cross-talk occurring between these signaling pathways and the effects of C in human activated HSC. Canrenone (10-25 uM) reduced PDGF-induced Na+/H+ exchanger activity similarly to the established inhibitor ethylisopropyilamiloride (EIPA) and the PI3-K inhibitor LY294002. However, while EIPA inhibited the basal activity of the exchanger after an acid load, no effects were observed with both LY294002 and C in the dose range employed, thus confirming that PI3-K is involved only in the regulation of PDGF-induced Na+/I-I+ exchanger activity. To confirm that a clear cross-talk occurs between these systems following stimulation with PDGF, the effect of EIPA on PI3-K was evaluated. EIPA markedly reduced PI3-K activity thus suggesting that inhibition of PI3-K activity by this compound contributes to the modulation of Na+/H+ exchanger activity. Both C and EIPA produced a dose-dependent inhibition of PDGF-induced focal adhesion kinase (FAK) phosphorylation, a signaling event occurring downstream of the activation PI3-K and the Na+/I-I+ exchanger and essential for growth factor-mediated cytoskeletal reorganization. These observations suggest a complex cross-talk involving PI3-K. Na+/H+ exchanger and
FAK in HSC and provide new basis for the antifibrogenic action of this drug.
I 716
INTRACELLULAR SIGNALING PATHWAYS ACTIVATED BY REACTIVE OXYGEN SPECIES REGULATE PROLIFERATION AND COLLAGEN SYNTHESIS OF HUMAN HEPATIC STELLATE CELLS
G. Svegliati-Baroni I, A. Galli ‘, E. Ceni ‘, F. Ridolfi ‘, R. Salzano ‘, E. Bendia’, A. Casini ‘. ‘Alcohol Research Center & GI Unit; 2 Department of Gastroenterology, University of Ancona, Italy
Reactive oxygen species (ROS) stimulate human hepatic stellate cells (HSC) proliferation and collagen synthesis. Intracellular signaling elements (such as ERKlR and PI3K) have a role in transducing signals from the cell surface to the nucleus and in mediating a fibrogenic behaviour in HSC. We thus verified which intracellular signaling pathways are activated by ROS and associated with HSC proliferation and collagen synthesis. The xanthine/xanthine oxidase system (K/X0) was used as a superoxide anion donor. Phosphorylation of ERKU2 and pp70S6K (a downstream component of PI3K) was evaluated by Western blot. HSC proliferation and matrix synthesis were evaluated by 3H-TdR incorporation and ELISA for type I collagen, respectively. HSC incubation with x/x0 was associated with increased ERK1/2 and pp70S6K phosphorylation, well evident at 10 and 30 min and maintained up to 60 min for ERKU2. These effects were completely abolished by tyrphostin A9, a selective inhibitor of the PDGF receptor tyrosine kinase, and PD98059, a MEK inhibitor. The PI3K inhibitor wortmannin reduced pp70S6K phosphorylation with no effect on ERKlR, while the PKC inhibitor calphostin C decreased protein phosphorylation only in the later phase. Inhibition of these mitogenic in a parallel reduction in X/X0-induced HSC proliferation, whereas X/KO-induced stimulation of type I collagen accumulation was inhibited only by calphostin C and PD98059. In conclusion, this study indicates that ROS activate several signaling pathways which differentially regulate HSC proliferation and collagen synthesis, thus providing new selective targets for the specific inhibition of these effects in human HSC.
I 719
EXPRESSION AND FUNCTION OF TETRASPANINS IN HEPATOCARCINOMA CELL LINES
C. Cordella, A. Mazzocca, V. Carloni. Medicina Intema, University di Firenze, Italy
Tetraspanins (TM4SF), are a large family of ubiquitously expressed membrane proteins that are implicated in cell proliferation and tumor cell invasion. Notably, TM4SF proteins form membrane complex with integrins. Recent data have clearly demonstrated that the function of integrinTM4SF protein complexes is relevant to cell migration. In this study we have characterized the TM4SF CD9, CD63, CD81, CD151 in two diffent hepatocarcinoma cell lines HepG2 and Huh7. Expression levels of TM4SF were evaluated by flow cytometry and immunofluorescence. HepG2 expressed CD63 and CD151, in contrast Huh7 showed high levels of CD9, CD63, CD8 1, CD15 1. Beta1 integrin co-immmunoprecipitd with TM4SF. mAbs against TM4SF were able to reduce haptotactic migration on laminin, collagen IV and collagen I without affecting cell adhesion on these matrix proteins. Take together, these results suggest that beta1 it&grin induces TM4SF-dependent cell motility in a matrix independent manner.