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Expression of plasminogen activators and inhibitors in normal and fibrotic human liver
58
Working
purties
EXPRESSION OF PLASMINOGEN ACTIVATORS AND INHIBITORS IN NORMAL AND FIBROTIC HUMAN LIVER C. Grannone. G. Pellegrini. G. Fibbi ‘, R. Salzano, A. Casini. M. Del Rosso’, S. Milani. Gastroenterology Unit, Department of Clinical Pathophysiology, University of Florence, Italy. ‘General Pathology Institute, University of Florence, Italy. Plasmin activation has a key role in the regulation of extracellular matrix turn-over and growth factor metabolism. Plasmin is generated from plasminogen by urokinase (uPA) or tissue plasminogen activator. The activity of uPA is mediated by binding to specific membrane receptor (uPAR) and it is regulated by specific inhibitors such as PAI-1. We investigated the expression of uPA, uPAR and PAI- in acute and chronic liver injury, by immunohistochemistry, in situ hybridization, and RNase protection assay. In normal human liver, uPA, uPAR, and PAI- proteins and mRNAs were expressed at very low levels in sinusoidal cells. In fulminant hepatitis, uPA and uPAR expression was markedly upregulated in non-parenchymal cells, while PAI- expression was moderately increased in both mesenchymal cells and hepatocytes. The cirrhotic liver showed a prevalent expression of PAI- in hepatocytes and mesenchymal cells, associated with a moderate increase of uPA and uPAR expression in sinusoidal and inflammatory cells. uPAR expression was also observed in bile duct-like cells. We conclude that the plasminogen generating system is activated and differentially regulated during acute and chronic liver damage, with a prevalent expression if its inhibitors such as PAI- 1, during chronic fibrogenesis. Different hepatic cell populations cooperate to the regulation of this system.
(TIMPs) play an essential ‘role in liver injury- associated with tissue remodeling like cirrhosis. Since the precise origin of MMPs and TIM& within the liver still remaiis to be cl&fied, hepatocytes (HEP), Kupffer cells (KC), hepatic stellate cells (HSC) and rat liver myofibroblasts (rMF) were analysed with respect to their MMP / TIMP expression by RT-PCR, Northern blot and zymography and, in the case of HSC, following exposure to cytokines. Results: Although MMP and TIMP coding transcripts were detectable in all liver cell types, the cellular expression levels were markedly different. Gelatinase-B was predominantly expressed in KC, while Gelatinase-A synthesis was mostlv nresent in HSC and rMF. Stromelvsins-1. -2 and collagenase here predominantly expressed in Il’SC. Mkmbrane tvne-1 MMP (MMP-14) was found in significant amounts in all li;er cells. ThP-1 coding m-RNAs we&present mainly in HSC and rMF, TIMP-2 additionally in KC, while TIMP-3 expression was detectable bv Northern blot onlv in HEP. During in vitro activation of HS(: MMP expression &as mostly downiegulated, while TIMP exnression was enhanced. With the exception of MMP-14 TNF-a’ stimulated the expression of every M‘MP and TIMP, while TGF-Bl induced MMP-2, -14 and TIMP coding transcripts only. Conclusions: The data suggest that all resident liver cells are involved in matrix degradation to some extent. HSC exnress significant amounts of ati matrix degrading enzymes ill&trating”that HSC play an important role in iatrixIbre&own in addition to matrix svnthesis. The activation-dependent expression pattern of MMqs and TIMPs as well as its regulation by TNF-a and TGF-B might indicate that HSC are involved in initial matrix breakdown following acute liver injury and matrix accumulation colocalized with these cells in cirrhotic livers.
ADHESION OF HUMAN LIVER MYOFIBROBLASTS TO TYPE I COLLAGEN SPECIFICALLY INDUCES ACTIVATION OF GELATINASE A P. Mavier. A.M. Prkaux. M.P. d’Ortho*. A. Mallat. INSERM U99, U296*,, HBpital Henri Mondor, Cr&eil, France.
FUNCTlONAL AND REGULATORY ANALYSIS OF NFK6 PROTEINS DURING CULTURE ACTIVATION OF HEPATIC STELLATE CELLS awv. RT Hav t . MJP Arthur and DA
The mati metalloproteinase (MMP) gelatinase A (gel A) degrades type IV collagen. Gel A is secreted in the proenzyme form (progel A) and activated following proteolytic cleavage mediated by transmembrane-MMPs (MT-MMP), such as MTlMMP. Gel A is highly expressed in fibrotic liver where it is produced by myofibroblasts (MF). However, its activation is poorly understood. Activation might be regulated by cell-matrix interactions since only progel is secreted by MF cultured on plastic. Human hepatic MF were cultured on plastic, fibronectin, laminin, or on a type I collagen gel (collI). Gel A was detected in cell supematant, by zymography. Wheras progel A alone (66 kD) was produced by MF cultured on plastic, fibronectin or laminin, active gel A (59 kd) was de&ted in the supematant of MF cultured-on ~0111.Membrane fractions derived from MF cultured on collI markedlv activated purified gel A, wheras membranes from MF cultivated on plastic showed almost no activating effect. Activation was blunted by two specific MMP inhibitors, TIMP-2 and BB-3103. These results demonstrate that adhesion of MF to coll1 induces progel A activation through a MT-MMP. MTl-MMP mRNA expression was not increased in MF adhering to ~0111. Western blotting showed an increased expression of MTl-MMP protein (63 kD) in cells cultured on collI, as compared to cells cultured on plastic. These results demonstrate that MF adhesion to coll1 specif~ally induces activation of gel A secreted by these cells. Activation is in part due to an increased expression of MTl-MMP by a
translational or post-translational mechanism.
EXPRESSION PATTERNS OF MMPs AND TIMPs IN LIVER CELLS IN VITRO: REGULATION BY TNF-a AND TGF-I31 T. Knittel. M. Mehde. D. Kobold. B. Saile. G.Ramadori. Dept. of Internal Medicine, University of [email protected], Germany. Matrixmetallooroteinases (MMPs) and their soecific inhibitors
University Medicine, University of Southampton, UK “Dept of Biochemistry, University of St Andrews, UK In this study we demonstrate that NF-KB is constitutively active in culture activated rat hepatic stellate cells (HSC). Nuclear extracts from activated HSCs expressed three sequence specific NF-KB proteinDNA binding complexes. Complexes 1 and 2 were rapidly induced following isolation with peak activity at 24ht-s that fell to a lower but constitutive level thereafter. Supershift studies with anti-Rel antisera revealed that complexes 1 and 2 consist of p5O/p65 and p65/p65 dimers. A third complex was induced with later kinetics and reached a plateux of expression at day 3. Supershift studies failed to identify the constituents of complex 3. As complex formation was inhibited by recombinant IKB and excess competitor oligonucleotides we suggest that ,ectivated HSCs may express novel NF-KB proteins. Constitutive NF-KB was shown to bs mediated by a novel mechanism involving long-term down-regulation of nuclear IKB expression. Function of NF-KB was studied by treatment of HSCs with inhibitors of NF-KB activation. Results to date indicate that induction of NF-KB may be a requirement for either prevention of HSC apoptosis or adherance of HSCs to tissue culture plastic.
Working
purties
EXPRESSION OF PLASMINOGEN ACTIVATORS AND INHIBITORS IN NORMAL AND FIBROTIC HUMAN LIVER C. Grannone. G. Pellegrini. G. Fibbi ‘, R. Salzano, A. Casini. M. Del Rosso’, S. Milani. Gastroenterology Unit, Department of Clinical Pathophysiology, University of Florence, Italy. ‘General Pathology Institute, University of Florence, Italy. Plasmin activation has a key role in the regulation of extracellular matrix turn-over and growth factor metabolism. Plasmin is generated from plasminogen by urokinase (uPA) or tissue plasminogen activator. The activity of uPA is mediated by binding to specific membrane receptor (uPAR) and it is regulated by specific inhibitors such as PAI-1. We investigated the expression of uPA, uPAR and PAI- in acute and chronic liver injury, by immunohistochemistry, in situ hybridization, and RNase protection assay. In normal human liver, uPA, uPAR, and PAI- proteins and mRNAs were expressed at very low levels in sinusoidal cells. In fulminant hepatitis, uPA and uPAR expression was markedly upregulated in non-parenchymal cells, while PAI- expression was moderately increased in both mesenchymal cells and hepatocytes. The cirrhotic liver showed a prevalent expression of PAI- in hepatocytes and mesenchymal cells, associated with a moderate increase of uPA and uPAR expression in sinusoidal and inflammatory cells. uPAR expression was also observed in bile duct-like cells. We conclude that the plasminogen generating system is activated and differentially regulated during acute and chronic liver damage, with a prevalent expression if its inhibitors such as PAI- 1, during chronic fibrogenesis. Different hepatic cell populations cooperate to the regulation of this system.
(TIMPs) play an essential ‘role in liver injury- associated with tissue remodeling like cirrhosis. Since the precise origin of MMPs and TIM& within the liver still remaiis to be cl&fied, hepatocytes (HEP), Kupffer cells (KC), hepatic stellate cells (HSC) and rat liver myofibroblasts (rMF) were analysed with respect to their MMP / TIMP expression by RT-PCR, Northern blot and zymography and, in the case of HSC, following exposure to cytokines. Results: Although MMP and TIMP coding transcripts were detectable in all liver cell types, the cellular expression levels were markedly different. Gelatinase-B was predominantly expressed in KC, while Gelatinase-A synthesis was mostlv nresent in HSC and rMF. Stromelvsins-1. -2 and collagenase here predominantly expressed in Il’SC. Mkmbrane tvne-1 MMP (MMP-14) was found in significant amounts in all li;er cells. ThP-1 coding m-RNAs we&present mainly in HSC and rMF, TIMP-2 additionally in KC, while TIMP-3 expression was detectable bv Northern blot onlv in HEP. During in vitro activation of HS(: MMP expression &as mostly downiegulated, while TIMP exnression was enhanced. With the exception of MMP-14 TNF-a’ stimulated the expression of every M‘MP and TIMP, while TGF-Bl induced MMP-2, -14 and TIMP coding transcripts only. Conclusions: The data suggest that all resident liver cells are involved in matrix degradation to some extent. HSC exnress significant amounts of ati matrix degrading enzymes ill&trating”that HSC play an important role in iatrixIbre&own in addition to matrix svnthesis. The activation-dependent expression pattern of MMqs and TIMPs as well as its regulation by TNF-a and TGF-B might indicate that HSC are involved in initial matrix breakdown following acute liver injury and matrix accumulation colocalized with these cells in cirrhotic livers.
ADHESION OF HUMAN LIVER MYOFIBROBLASTS TO TYPE I COLLAGEN SPECIFICALLY INDUCES ACTIVATION OF GELATINASE A P. Mavier. A.M. Prkaux. M.P. d’Ortho*. A. Mallat. INSERM U99, U296*,, HBpital Henri Mondor, Cr&eil, France.
FUNCTlONAL AND REGULATORY ANALYSIS OF NFK6 PROTEINS DURING CULTURE ACTIVATION OF HEPATIC STELLATE CELLS awv. RT Hav t . MJP Arthur and DA
The mati metalloproteinase (MMP) gelatinase A (gel A) degrades type IV collagen. Gel A is secreted in the proenzyme form (progel A) and activated following proteolytic cleavage mediated by transmembrane-MMPs (MT-MMP), such as MTlMMP. Gel A is highly expressed in fibrotic liver where it is produced by myofibroblasts (MF). However, its activation is poorly understood. Activation might be regulated by cell-matrix interactions since only progel is secreted by MF cultured on plastic. Human hepatic MF were cultured on plastic, fibronectin, laminin, or on a type I collagen gel (collI). Gel A was detected in cell supematant, by zymography. Wheras progel A alone (66 kD) was produced by MF cultured on plastic, fibronectin or laminin, active gel A (59 kd) was de&ted in the supematant of MF cultured-on ~0111.Membrane fractions derived from MF cultured on collI markedlv activated purified gel A, wheras membranes from MF cultivated on plastic showed almost no activating effect. Activation was blunted by two specific MMP inhibitors, TIMP-2 and BB-3103. These results demonstrate that adhesion of MF to coll1 induces progel A activation through a MT-MMP. MTl-MMP mRNA expression was not increased in MF adhering to ~0111. Western blotting showed an increased expression of MTl-MMP protein (63 kD) in cells cultured on collI, as compared to cells cultured on plastic. These results demonstrate that MF adhesion to coll1 specif~ally induces activation of gel A secreted by these cells. Activation is in part due to an increased expression of MTl-MMP by a
translational or post-translational mechanism.
EXPRESSION PATTERNS OF MMPs AND TIMPs IN LIVER CELLS IN VITRO: REGULATION BY TNF-a AND TGF-I31 T. Knittel. M. Mehde. D. Kobold. B. Saile. G.Ramadori. Dept. of Internal Medicine, University of [email protected], Germany. Matrixmetallooroteinases (MMPs) and their soecific inhibitors
University Medicine, University of Southampton, UK “Dept of Biochemistry, University of St Andrews, UK In this study we demonstrate that NF-KB is constitutively active in culture activated rat hepatic stellate cells (HSC). Nuclear extracts from activated HSCs expressed three sequence specific NF-KB proteinDNA binding complexes. Complexes 1 and 2 were rapidly induced following isolation with peak activity at 24ht-s that fell to a lower but constitutive level thereafter. Supershift studies with anti-Rel antisera revealed that complexes 1 and 2 consist of p5O/p65 and p65/p65 dimers. A third complex was induced with later kinetics and reached a plateux of expression at day 3. Supershift studies failed to identify the constituents of complex 3. As complex formation was inhibited by recombinant IKB and excess competitor oligonucleotides we suggest that ,ectivated HSCs may express novel NF-KB proteins. Constitutive NF-KB was shown to bs mediated by a novel mechanism involving long-term down-regulation of nuclear IKB expression. Function of NF-KB was studied by treatment of HSCs with inhibitors of NF-KB activation. Results to date indicate that induction of NF-KB may be a requirement for either prevention of HSC apoptosis or adherance of HSCs to tissue culture plastic.