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Modulation of cellular fibronectin (cFN) in mononuclear phagocytes (MP) from CCL4-treated rats. in vitro and in vivo study
83
1P/CO3/0091
1 P/CO3/011 1
NON-TRANSFERRIN-BOUND TRANSFERRtN
RECEPTORS
IRON ARE
(NTBI)
TRANSPORT
DIFFERENTLY
AND REGULATED
IN HepG2 CELLS. D. Barisani, G. Cairo* Cattedra di Gastroenterologia IRCCS Ospedale Maggiore, and *Centro Studi Patologia Cellulare, Milano, Italy. Background: Nitric oxide (NO) regulates several cellular
BY NO GENERATORS
and D. Come.
processes, including iron transport. NO generators (s-nitroso-dSNP) modulate iron penicillamine, SNAP and sodium nitroprusside, regulatory protein (IRP) activity and thus transferrin receptor mRNA. No data are available on the effect of NO on NTBI transport Aim: To evaluate NTBI transport in HepG2 cells treated with NO generators. Methods: HepG2 cells were incubated with 0.5 and I
mM SNP and SNAP for 6-24 hours. j5Fe-NTA was employed as a model for NTBI, and Fe3’and Fe2- uptake were measured at 5 and I5 min. Transfer-tin receptor mRNA was evaluated by Northern Blot and IRP activity by gel retardation assay. Results: NO effect was timedependent and reached a plateau between 24-30 hours. Both Fe3’ and Fe2’ uptake were significantly decreased by treatment with NO
generators in a dose dependent manner. The effect of I mM SNP and SNAP was mantained at all the Fe concentration tested, and doublereciprocal plot revealed a reduction in Vmax (50, 36 and I4 tinol/ug protein/min in control, SNP- and SNAP-treated cells, respectively), whereas no Km variation was observed. These data indicate a reduction in the number of receptor on the cell surface, and were not attributable to cell death, since no reduction in viability or apoptosis could be detected. IRP activity and transferrin receptor mRNA were reduced by SNP treatment, whereas SNAP caused a modest increase. Conclusions: Both SNP and SNAP significantly reduce NTBI transport in HepG2 cells, whereas they have a dual effect on IRP activity and transfertin receptor mRNA. These data suggest that NO acts on NTBI transport through a IRP independent mechanism, probably chelating free intracellular iron.
MODULATION OF CELLULAR FIBRONECTIN (CM) IN MONONUCLEAR PHAGOCYTES (MP) FROM CCL,-TREATED RATS. IN VITRO AND IN VW0 STUDY.
Th. Armbmst. M.KreiOie. H. Herbst*. G. Ramadori. Zentrum lnnere Medizin,Abt. Gastroenterologie & Endokrinologie, Universitat GOttingen;*lnstitut 8lr Pathologie, Universit8tsklinikum Hamburg-Eppendorff, Germany Fibronectin (IN) is supposed to be an important glycoprotein involved in pathogenese of hepatic fibrosis. After acute liver injury, cl% gene expression is strongly increased in the area of damage. As the number of MP is increased in acutely damaged liver, we studied the contribution of these MP to hepatic FNsyn:hesis. Methods: In situ hybridization combined with immunohistochemistry was performed on cryostat sections obtained 36h after the application of CCII. Liver MP were isolated and characterized by means of the moAb EDl. Protein synthesis was studied by biosynthetic labeling, immunoprecipitation, and SDS-PAGE. RNA was extracted and analyzed by Nothem Blotting using a cFN specific probe. Results: Only few ofthe EDI+ cells on the normal liver sections contained cFN transcripts. They were equally distributed within the liver. In necrotic areas of the acutely injured liver, EDI+ cells which were abundant in these areas showed strongly increased cFN expression. In contrast, C I q was detected in most of ED I + cells of the normal liver. In the acutely damaged liver, only those ED I + cells contained C I q transcripts which were located outside necrotic areas. Spontaneous synthesis of cFN in cultured EDI+ cells from the acutely damaged liver was strongly increased as compared to cultures obtained from normal liver. Stimulation of the cultures with dexamethasone, interferon-y and lipopolysachtide decreased FN production, stimulation with TGF-8 increased M gene expression. Conclusions: Our results indicate that immigrating MP may play a major role in FN metabolism at the site of cellular necrosis during acute liver injury.
Early in an acute liver injury deposited cFN may be provided by immigrating MP well before activation of mesenchymal cells. M synthesis in MP of the acutely damaged liver appears to be a highly regulated process.
1 P/CO3/012
POTENTIAL ROLE OF NF-kB IN THE NEGATIVE REGULATION OF HUMAN HEPATIC STELLATE CELL PROLIFERATION. C. Gallois. A. Habib*.T. Jian&ne. J. Maclouf*p_A. Mallat and S, Lotersztain. INSERM U99, Hop. H. Mondor, 94010. CrCteil, France. *lNSERM U348, IFR Lariboisibre, Hop. Lariboisiere, 75010 Paris, France. During chronic liver diseases, hepatic stellate cells (HSC) acquire an activated myofibroblast-like phenotype, proliferate and synthetize fibrosis components. Since increasing evidences support a role for NF-kB in cell growth arrest, we investigated whether activation of NF-LB may be involved in the negative control of human HSC uroliferation. Endothelin-1 @T-l) and tumor necrosis factor-a (TNF-a) which both inhibited the growth of human HSC. increased the formation of two NFkB binding complexes. Activation of NF-LB was associated with the degradation of the inhibitory cytosolic protein IkB-a). These two complexes were identified as the p5O/p50 and p5O/p65 NF-kB dimers. Activation of NF-kB and degradation of IkB-a were prevented by the antiinflammatory agent sodium salicylate and by the proteasome inhibitor MG132. ET-l and TNF-a) up-regulated the inducible form of cyclooxygenase (COX-2). by inducing COX-2 mRNA and COX-2 protein, and did not affect the constitutive COX-1. Up-regulation of COX-2 by ET1 and TNF- a was blocked by sodium salicylate and MG132, suggesting that activation of NF-kB by both neutides is needed for stimulation of COX-2. Finally, the COX-2 inhibitbr’dexametbasone blunted the growth inhibitory effect of ET-l and TNF-a , indicating that activation of COX-2 is reauired for inhibition of HSC uroliferation. We conclude that in human activated HSC, ET-l and TNF-a activate NF-LB, resulting in induction of COX-2 and in subsequent inhibition of HSC growth. Our results suggest that NF-kB, by inducing COX-2 expression, may play an important role in the negative regulation of human activated HSC proliferation.
1
GENERATION OF REACTIVE OXYGEN SPECIES ANB NO’ IN THE EARLY PHASE OF FIBROSIS: EFFECT OF INTERFERONG. Sveabati-Baroni, A. de Jaaer-K&ken”. S. Saccomannd. P.L.M. Jansen’, A.M. Jezeauel’. H. Moshaae’. Dept. ofGastroentero1. & HeDatol. *Institute ofExperimental Pathol., University ~$Ancona, Italy & TX&t. of Gastroenterol & Hepatol., University Hospital Groningen, the Netherlad Background: In liver fibrosis hepatic stellate cells (HSCs) are activated. Reactrve oxygen species (ROS) are known to activate HSCs and induce collagen gene transcription in vitro. Infiltrating neutrophils are a possible source of ROS. ROS can be scavenged by nitric oxide (NO) radicals, yielding peroxynitrite and nitrotyrosine. Interferon-y (IFN-y) has antifibrotic actions in viva and is an important inducer of iNOS in vitro. Aim: to investigate neutrophil infiltration, the generation of ROS and mtrotyrosine and the expression of iNOS in the early phase of liver fibrosis and to study the effect of IFN-1 on these parameters Methods: liver injury was induced by three consecutive DMN injections. Animals were sacrificed 6hrs, 24hrs, 48hrs and 7 days after the last injection. Immunohisto chemistry was performed for neutrophils (His48), iNOS, nitrotyrosine and ROS producing cells. In v&o, the effect of ROS scavengers (SOD + catalase) and the NO-donor SNAP (1mM) on proliferation of HSCs was investigated using brdmodeoxyuridine incorporation. Results: The generation of ROS and nitrotyrosine was evident at 6hr and declined thereafter. Infiltrating neutrophils were abundant at 6hrs and 24hrs and hardly detectable after 1 week. iNOS expression was detectable in some infhunmatoly cells at 6hrs and decreased thereafter. IFN-y treatment decreased nitrotyrosine generation but did not affect iNOS expression. In vlho, proliferation of HSCs was increased by the ROS donor iron/ascorbate. This effect was abolished by the NO donor SNAP and by ROS scavengers. Conclusion ROS are generated m the early phase of experimental liver fibrosis and contributes to HSC activation. Neutrophils may be an important source of ROS. A role for NO as ROS scavenger is suggested since NO inhibits ROS-induced HSC proliferation and iNOS is weakly present in the early phase of liver injury. IFN-1 decreases nitrotyrosine generation but has no significant effect on iNOS expression.
1P/CO3/0091
1 P/CO3/011 1
NON-TRANSFERRIN-BOUND TRANSFERRtN
RECEPTORS
IRON ARE
(NTBI)
TRANSPORT
DIFFERENTLY
AND REGULATED
IN HepG2 CELLS. D. Barisani, G. Cairo* Cattedra di Gastroenterologia IRCCS Ospedale Maggiore, and *Centro Studi Patologia Cellulare, Milano, Italy. Background: Nitric oxide (NO) regulates several cellular
BY NO GENERATORS
and D. Come.
processes, including iron transport. NO generators (s-nitroso-dSNP) modulate iron penicillamine, SNAP and sodium nitroprusside, regulatory protein (IRP) activity and thus transferrin receptor mRNA. No data are available on the effect of NO on NTBI transport Aim: To evaluate NTBI transport in HepG2 cells treated with NO generators. Methods: HepG2 cells were incubated with 0.5 and I
mM SNP and SNAP for 6-24 hours. j5Fe-NTA was employed as a model for NTBI, and Fe3’and Fe2- uptake were measured at 5 and I5 min. Transfer-tin receptor mRNA was evaluated by Northern Blot and IRP activity by gel retardation assay. Results: NO effect was timedependent and reached a plateau between 24-30 hours. Both Fe3’ and Fe2’ uptake were significantly decreased by treatment with NO
generators in a dose dependent manner. The effect of I mM SNP and SNAP was mantained at all the Fe concentration tested, and doublereciprocal plot revealed a reduction in Vmax (50, 36 and I4 tinol/ug protein/min in control, SNP- and SNAP-treated cells, respectively), whereas no Km variation was observed. These data indicate a reduction in the number of receptor on the cell surface, and were not attributable to cell death, since no reduction in viability or apoptosis could be detected. IRP activity and transferrin receptor mRNA were reduced by SNP treatment, whereas SNAP caused a modest increase. Conclusions: Both SNP and SNAP significantly reduce NTBI transport in HepG2 cells, whereas they have a dual effect on IRP activity and transfertin receptor mRNA. These data suggest that NO acts on NTBI transport through a IRP independent mechanism, probably chelating free intracellular iron.
MODULATION OF CELLULAR FIBRONECTIN (CM) IN MONONUCLEAR PHAGOCYTES (MP) FROM CCL,-TREATED RATS. IN VITRO AND IN VW0 STUDY.
Th. Armbmst. M.KreiOie. H. Herbst*. G. Ramadori. Zentrum lnnere Medizin,Abt. Gastroenterologie & Endokrinologie, Universitat GOttingen;*lnstitut 8lr Pathologie, Universit8tsklinikum Hamburg-Eppendorff, Germany Fibronectin (IN) is supposed to be an important glycoprotein involved in pathogenese of hepatic fibrosis. After acute liver injury, cl% gene expression is strongly increased in the area of damage. As the number of MP is increased in acutely damaged liver, we studied the contribution of these MP to hepatic FNsyn:hesis. Methods: In situ hybridization combined with immunohistochemistry was performed on cryostat sections obtained 36h after the application of CCII. Liver MP were isolated and characterized by means of the moAb EDl. Protein synthesis was studied by biosynthetic labeling, immunoprecipitation, and SDS-PAGE. RNA was extracted and analyzed by Nothem Blotting using a cFN specific probe. Results: Only few ofthe EDI+ cells on the normal liver sections contained cFN transcripts. They were equally distributed within the liver. In necrotic areas of the acutely injured liver, EDI+ cells which were abundant in these areas showed strongly increased cFN expression. In contrast, C I q was detected in most of ED I + cells of the normal liver. In the acutely damaged liver, only those ED I + cells contained C I q transcripts which were located outside necrotic areas. Spontaneous synthesis of cFN in cultured EDI+ cells from the acutely damaged liver was strongly increased as compared to cultures obtained from normal liver. Stimulation of the cultures with dexamethasone, interferon-y and lipopolysachtide decreased FN production, stimulation with TGF-8 increased M gene expression. Conclusions: Our results indicate that immigrating MP may play a major role in FN metabolism at the site of cellular necrosis during acute liver injury.
Early in an acute liver injury deposited cFN may be provided by immigrating MP well before activation of mesenchymal cells. M synthesis in MP of the acutely damaged liver appears to be a highly regulated process.
1 P/CO3/012
POTENTIAL ROLE OF NF-kB IN THE NEGATIVE REGULATION OF HUMAN HEPATIC STELLATE CELL PROLIFERATION. C. Gallois. A. Habib*.T. Jian&ne. J. Maclouf*p_A. Mallat and S, Lotersztain. INSERM U99, Hop. H. Mondor, 94010. CrCteil, France. *lNSERM U348, IFR Lariboisibre, Hop. Lariboisiere, 75010 Paris, France. During chronic liver diseases, hepatic stellate cells (HSC) acquire an activated myofibroblast-like phenotype, proliferate and synthetize fibrosis components. Since increasing evidences support a role for NF-kB in cell growth arrest, we investigated whether activation of NF-LB may be involved in the negative control of human HSC uroliferation. Endothelin-1 @T-l) and tumor necrosis factor-a (TNF-a) which both inhibited the growth of human HSC. increased the formation of two NFkB binding complexes. Activation of NF-LB was associated with the degradation of the inhibitory cytosolic protein IkB-a). These two complexes were identified as the p5O/p50 and p5O/p65 NF-kB dimers. Activation of NF-kB and degradation of IkB-a were prevented by the antiinflammatory agent sodium salicylate and by the proteasome inhibitor MG132. ET-l and TNF-a) up-regulated the inducible form of cyclooxygenase (COX-2). by inducing COX-2 mRNA and COX-2 protein, and did not affect the constitutive COX-1. Up-regulation of COX-2 by ET1 and TNF- a was blocked by sodium salicylate and MG132, suggesting that activation of NF-kB by both neutides is needed for stimulation of COX-2. Finally, the COX-2 inhibitbr’dexametbasone blunted the growth inhibitory effect of ET-l and TNF-a , indicating that activation of COX-2 is reauired for inhibition of HSC uroliferation. We conclude that in human activated HSC, ET-l and TNF-a activate NF-LB, resulting in induction of COX-2 and in subsequent inhibition of HSC growth. Our results suggest that NF-kB, by inducing COX-2 expression, may play an important role in the negative regulation of human activated HSC proliferation.
1
GENERATION OF REACTIVE OXYGEN SPECIES ANB NO’ IN THE EARLY PHASE OF FIBROSIS: EFFECT OF INTERFERONG. Sveabati-Baroni, A. de Jaaer-K&ken”. S. Saccomannd. P.L.M. Jansen’, A.M. Jezeauel’. H. Moshaae’. Dept. ofGastroentero1. & HeDatol. *Institute ofExperimental Pathol., University ~$Ancona, Italy & TX&t. of Gastroenterol & Hepatol., University Hospital Groningen, the Netherlad Background: In liver fibrosis hepatic stellate cells (HSCs) are activated. Reactrve oxygen species (ROS) are known to activate HSCs and induce collagen gene transcription in vitro. Infiltrating neutrophils are a possible source of ROS. ROS can be scavenged by nitric oxide (NO) radicals, yielding peroxynitrite and nitrotyrosine. Interferon-y (IFN-y) has antifibrotic actions in viva and is an important inducer of iNOS in vitro. Aim: to investigate neutrophil infiltration, the generation of ROS and mtrotyrosine and the expression of iNOS in the early phase of liver fibrosis and to study the effect of IFN-1 on these parameters Methods: liver injury was induced by three consecutive DMN injections. Animals were sacrificed 6hrs, 24hrs, 48hrs and 7 days after the last injection. Immunohisto chemistry was performed for neutrophils (His48), iNOS, nitrotyrosine and ROS producing cells. In v&o, the effect of ROS scavengers (SOD + catalase) and the NO-donor SNAP (1mM) on proliferation of HSCs was investigated using brdmodeoxyuridine incorporation. Results: The generation of ROS and nitrotyrosine was evident at 6hr and declined thereafter. Infiltrating neutrophils were abundant at 6hrs and 24hrs and hardly detectable after 1 week. iNOS expression was detectable in some infhunmatoly cells at 6hrs and decreased thereafter. IFN-y treatment decreased nitrotyrosine generation but did not affect iNOS expression. In vlho, proliferation of HSCs was increased by the ROS donor iron/ascorbate. This effect was abolished by the NO donor SNAP and by ROS scavengers. Conclusion ROS are generated m the early phase of experimental liver fibrosis and contributes to HSC activation. Neutrophils may be an important source of ROS. A role for NO as ROS scavenger is suggested since NO inhibits ROS-induced HSC proliferation and iNOS is weakly present in the early phase of liver injury. IFN-1 decreases nitrotyrosine generation but has no significant effect on iNOS expression.