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P70S6K in hepatic stellate cells: An in vivo and in vitro study
~bstr~cts
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REGULATION AND CROSSTALK OF ERK/JNKIP70S6K 1N HEPATIC STELLATE CELLS: AN IN VIVO AND IN VITRO STUDY.
INAPPROPRIATELY LOW ANGIOTENS1N H PRODUCTION: A FACTOR DETERMINING IMPAIRED RENAL FUNCTION AND REDUCED SURVIVAL IN PATIENTS W I T H DECOMPENSATED LIVER CIRRHOSIS. Sanso6 G, ]Silvano S, 2M~gozzi G, tTodros L, LSmedile A, ITouscoz A, 1Rizzetto M. Gastroenterology Unit, Gradenigo Hospital, Torino, Italy;
F. Ridolfi, G. Svegllati BaroN, S. Sancomanno, C. Candelaresi, P. Sterpetti, A. Benedetti. Deparauent of Gastroenterology, University of Ancona, Ancona, Italy.
1Department of Gastroenterology, Molinette Hospital, University of Torino, Tormo, 1rely and 2Institute of General Pathology, Molinette Hospital, Tonno,
Liver fibrosis is characterized by hepatic stallate cell (HSC) activation. Intraoellular kinases play a pivotal role in transducing extraoellular signals and ERK, INK mad p70S6K regulates cell proliferation and collagen synthesis in HSC in vitro. Activation of these kinases was thus evaluated in HSC isolated from rat liver using two different models of hepatic fibrosis, i.e. dimethylnitrosamme administration (DMN) and bile duct ligution (BDL). After DMN, kinases activation showed a specular behaviour increasing early at 6 h and Non peaking again at 1 week. After BDL, a constant activation of ERK and p70S6k was observed, while INK phosphorylation peaked after 7 days. Kinases activation in HSC in vivo preceded cell proliferation and alpha-smooth muscle actin (ctSMA) appearance, as a marker of HSC transformation in myofibroblastlike cells which was well evident at 7 days only (4.5_+0.7% for DMN model and 10.7-+1.I% for BDL model vs. 0.5_+0.I% for control animals of aSMA positive parenchyma, p<0.05) . To identify eventual relationship between these klaases HSC were stimulated in vitro with 10% serum that induced ERKI/2, .INK, and p70g6K pbospborylation, in addition to an almost 18-fold increase in HSC proliferation, evaluated as bromodeoxyuridine incorporation by ELISA (0.513_+0.01 vs. 0.033+0.005 relative absorbanoe units, p<0.01). SP600125, a INK specific inhibitor, mpamycin, a p70S6K specific inhibitor, and PD98059, a MEK specific inhibitor, reduced serum-induced HSC proliferation (respectively 0.128_+0.005, 0.086+0.01 and 0.33_+0.02 relative absorbance units vs. 10% serum alone, p<0.05). Furthermore, by using these specific kinase inhibiters, JNK and p70S6K were able to induce ERK phosphorylation which thus represents a downstream component in this cascade leading to cell proliferation. To confirm this, HSC transientIy transfected with a dominant negative form of ERKI (K71R) were incubated with a different stimulus (PDGF) and showed a reduced proliferation rate compared to non-transfected cells (0.231_+0.005 vs. 0.109_+0.003 relative absorbanoe units, p<0.05). This study thus indicates that intracellular klaases activation precedes HSC transformation into myofibroblast in vivo. Specific intraoellalar targets, such as ERKI/2 which represent a nmm regulating HSC proliferation, could be inhibited by selective delivery of lahibitors or dornmant negative mutant te reduce hepatic fibrosis in chronic liver diseases.
~taly. Angietensin II is the main mediator in vasoconmiction of the glomemlar vas efferens, which maintains glomemlar perfusiou pressure and glomemhr filtration rate (GFR) in renal hypoperinsion. This study explores the hypothesis that depressed angiotensin II ganerafiou, due to inapalred hepatic production of angiotensinogen or to reduced angiotansin-converdng enzyme (ACE) plasma levels, causes deranged r ~ a l fimetion in patients with advanced eirdaosis. We studied and prospectively followed up 21 patients with diuretic-free cirrhosis and overt ascites, through the following determinations: a) systemic plasma levels of active renin (AR), renin activity (PRA), angintensin U, angiotensinconverting enzyme (ACE) and aldosterone; b) renal clearances of sodium, potassium, inulin and para-aminohippuratu (PAH); c) antipyrine lap) systemic clearance and plasma half-life. 15 healthy subjects also underwent hormonal and renal function determinations as control group. Distribution of the GFR values was bimodal: 10 patients had low GFR values (I-GFR group) and I 1 had normal GFR values (n-GFR group) (below and above 105 ml/min/1.73 m: b.s.a., respectively). Crentinine plasma levels did not differ in the two groups (respectively, 1.32 _+0.83 vs. 0.95 + 0.34 mg/dl, P=0.08), nor did PAIl or Ap clearances (respectively, P=0.18 and P---0.47) and Child-Pugh score (P=0.48). Compared with the n-GFR group, the I-GFR group had significantly higher AR and PRA values (all P<0.01), sigui:fienntty lower levels of plasma ACE (P<0.03) and a significantly higher AR/Angiote~sla II ratio (1.66 4" 0.97 vs. 0.48 + 0.38, P<0.003).. Compared with healthy conta-ols all 21 patients showed markedly laerensed AR/PRA ratio (both P<0.03), indirect evidence of reduced renin subs~ate circulating levels. The respective 18-mouth survival rates of IGFR and n-GFR groups were 41% and 81% (P<0.02). In conclusion, among decompensated cirrhotic patients those with reduced GFR had a worse survival rate associated with more severe contraction of the effective arterial blood volume and lower capacity to cope with this phenomenon (higher AR/Ang lI ratio), possibly due to lower ACE plasma levels. ACE plasma levels were not related to the degree of liver insufficiency, but may be genetically determined.
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ANp73 regulates NFkB-dependent proliferation and counteracts p53 and TAp73induced growth arrest of hepatoeytes. Stefania Vossio^, Emanuele PescandoloA. Francesca Moreni^, Marcella Fulco^. Antonio Costanzo^, Clara Balsano*^, Massimo Levrero^$. ^Fondazione Andrea Cesalpino, University of Rome "La Sapienza", Rome, italy: * Dept. Internal Medicine, University of L'Aquila. L'Aquila, Italy; $ Dept. Internal Medicine. University of Cagliari, Cagliari. Italy.
D I F F E R E N T I A L R E G U L A T I O N OF MCP-1 E X P R E S S I O N IN HEPATIC STELLATE CELLS BY MEMBERS OF THE MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) FAMILY. IIaria Petrai, Wanda Delogu Sabrina Pastacaldi, Andrea Bonacchi. Eva Efsen, Paolo Gentilini. Fabio Morro Dipartimento di Medicine lnetrna, University o f Florence
Mature hepatocytes re-entry into tile cell cycle and proliferate to compensate loss of hepatic parenchyma due to surgery, chemical toxicity or viral infections. At~er partial hepateetomy (PHx) liver mass is restored in about a week by one or two rounds o hepatocytes cell division. Extensive study of liver regeneration after PHx has revealed the role of growth factors (such as HGF and its receptor Met, EGF, TGF-alpha), of cytokines (such as the TNF-aIpha and IL-6) and of transcription factors (such as E2FI. APt, NFkB and Star3) in the regulation of hepatocyte proliferation whereas TGF beta/activin, p53 and the cyclin-dependent kinase (CDK) inhibitor p21 prevent uncontrolled hepatocytes proliferation. Although p53 levels greatIy increase in the regenerating liver after PHx and suppression of its expression alters liver regeneration in vivo. p21 activation and hepatocytes growth arrest in response to a variety of stimuli also occur in a p53 deficient genetic background, suggesting a role tbr the p53-related proteins p63 and p73. The p73 gene is transcribed from two promoters. Pl and P2. that direct the expression of the TA (transactivation competent, pro-apoptotic and anti proliferative) and the DN (transactivation deficient, anti-apoptotic and pro-proliferative) p73 proteins, respectively. We have previously demonstrated that, in response to a no apoptotic DNA damage the P2-p73 promoter is transcriptionally activated in a p53 dependent manner, leading to the expression and accumuIation of DN-p73 proteins, that [n turn selectively downregulate their own promoter as well as the p21 promoter to ensure the cell-cycle reentry of cells that have repaired their DNA damage. We show here that liver DN-p73 mRNA levels increase in chronic hepatitis and cirrhosis and correlate with hepatocytes loss and proliferation. Using slrlall interfering RNAs specific for DN-p73 to selectively abrogate its function, we found that DN-p73 is required fo TNF-alpha induced proliferation of hepatocytes and that, in this context DN-p73 expression is controlled by NFkB. To study the regulation of DN-p73 expression at th transcriptional level we cloned the DN-p73 alternative promoter (P2p73Pr). Differently from the Pip73 promoter, that directs the expression of TA-p73 proteins and is regulated by E2FI. the P2p73 promoter is activated by TNF-alpha and IFN-gamma through NFkB and IRF-I sites and downregulated by retinoids and lFN-alpha in hepatocarcinoma cell lines and in immortalized Met Murine Hepatocytes D3 (MMH D3) cells. Functional studies showed that DN-p73 counteracts both p53 and TA-p73 induced growth arrest and apoptosis in MMH-D3 cells. In conclusion we have identified a role of DN-p73 variants in controlling hepatocytes growth in the context o an inflammatory liver enviromnent.
Hepatic stellate cells (HSC) coordinate the liver wound-healing response through secretion of several cytokines and chemokines, including MCP1. In this study, we evaluated the role of different proteins of the M A P K family (ERK, p38, and JNK) in the regulation of monocyte chemoattractant rprotein-I (MCP-1) expression by HSC, as an index of their pro-inflammatory activity, Several mediators activated all three M A P K , including TNF, IL-I and PDGF, although with different potency. While TNF and 1L-1 were very effective in inducing MCP-1 expression, PDGF was only modestly active. To assess the relative role o f the different MAPKs, specific pharmacologic inhibitors were used, namely SB203580 (p38), SP600125 (JNK), and PD98059 (MEK/ERK). The efficacy and specificity of the different inhibitors in our cellular system was verified analyzing the enzymatic activity o f the different MAPKs using in vitro kinase assays and/or testing the inhibition of phosphorylation o f downstream substrates. S B 2 0 3 5 8 0 dose-dependently inhibited MCP-1 secretion and gone expression induced by IL-1 or TNF. This effect occurred independently of reduced DNA binding of NFkappaB. Similarly, inhibition of JNK activity by SP600125 120 microM) blocked MCP-1 secretion. In contrast, inhibition of ERK did not affect the up-regulation of MCP-1 induced by IL-1 or TNF. Additionally. Activin A, which stimulates MCP-1 expression, activated JNK and p38. but not ERK. Conversely, when M A P K inhibitors were compared in their ability to block DNA synthesis in response to PDGF. PD98059 (ERK inhibitor) was the most effective. We conclude that members of the M A P K family differentially regulate the biologic response of HSC to soluble mediators, p38 and .INK are necessary for chemokine secretion, while ERK is more relevant for mitogenesis.
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REGULATION AND CROSSTALK OF ERK/JNKIP70S6K 1N HEPATIC STELLATE CELLS: AN IN VIVO AND IN VITRO STUDY.
INAPPROPRIATELY LOW ANGIOTENS1N H PRODUCTION: A FACTOR DETERMINING IMPAIRED RENAL FUNCTION AND REDUCED SURVIVAL IN PATIENTS W I T H DECOMPENSATED LIVER CIRRHOSIS. Sanso6 G, ]Silvano S, 2M~gozzi G, tTodros L, LSmedile A, ITouscoz A, 1Rizzetto M. Gastroenterology Unit, Gradenigo Hospital, Torino, Italy;
F. Ridolfi, G. Svegllati BaroN, S. Sancomanno, C. Candelaresi, P. Sterpetti, A. Benedetti. Deparauent of Gastroenterology, University of Ancona, Ancona, Italy.
1Department of Gastroenterology, Molinette Hospital, University of Torino, Tormo, 1rely and 2Institute of General Pathology, Molinette Hospital, Tonno,
Liver fibrosis is characterized by hepatic stallate cell (HSC) activation. Intraoellular kinases play a pivotal role in transducing extraoellular signals and ERK, INK mad p70S6K regulates cell proliferation and collagen synthesis in HSC in vitro. Activation of these kinases was thus evaluated in HSC isolated from rat liver using two different models of hepatic fibrosis, i.e. dimethylnitrosamme administration (DMN) and bile duct ligution (BDL). After DMN, kinases activation showed a specular behaviour increasing early at 6 h and Non peaking again at 1 week. After BDL, a constant activation of ERK and p70S6k was observed, while INK phosphorylation peaked after 7 days. Kinases activation in HSC in vivo preceded cell proliferation and alpha-smooth muscle actin (ctSMA) appearance, as a marker of HSC transformation in myofibroblastlike cells which was well evident at 7 days only (4.5_+0.7% for DMN model and 10.7-+1.I% for BDL model vs. 0.5_+0.I% for control animals of aSMA positive parenchyma, p<0.05) . To identify eventual relationship between these klaases HSC were stimulated in vitro with 10% serum that induced ERKI/2, .INK, and p70g6K pbospborylation, in addition to an almost 18-fold increase in HSC proliferation, evaluated as bromodeoxyuridine incorporation by ELISA (0.513_+0.01 vs. 0.033+0.005 relative absorbanoe units, p<0.01). SP600125, a INK specific inhibitor, mpamycin, a p70S6K specific inhibitor, and PD98059, a MEK specific inhibitor, reduced serum-induced HSC proliferation (respectively 0.128_+0.005, 0.086+0.01 and 0.33_+0.02 relative absorbance units vs. 10% serum alone, p<0.05). Furthermore, by using these specific kinase inhibiters, JNK and p70S6K were able to induce ERK phosphorylation which thus represents a downstream component in this cascade leading to cell proliferation. To confirm this, HSC transientIy transfected with a dominant negative form of ERKI (K71R) were incubated with a different stimulus (PDGF) and showed a reduced proliferation rate compared to non-transfected cells (0.231_+0.005 vs. 0.109_+0.003 relative absorbanoe units, p<0.05). This study thus indicates that intracellular klaases activation precedes HSC transformation into myofibroblast in vivo. Specific intraoellalar targets, such as ERKI/2 which represent a nmm regulating HSC proliferation, could be inhibited by selective delivery of lahibitors or dornmant negative mutant te reduce hepatic fibrosis in chronic liver diseases.
~taly. Angietensin II is the main mediator in vasoconmiction of the glomemlar vas efferens, which maintains glomemlar perfusiou pressure and glomemhr filtration rate (GFR) in renal hypoperinsion. This study explores the hypothesis that depressed angiotensin II ganerafiou, due to inapalred hepatic production of angiotensinogen or to reduced angiotansin-converdng enzyme (ACE) plasma levels, causes deranged r ~ a l fimetion in patients with advanced eirdaosis. We studied and prospectively followed up 21 patients with diuretic-free cirrhosis and overt ascites, through the following determinations: a) systemic plasma levels of active renin (AR), renin activity (PRA), angintensin U, angiotensinconverting enzyme (ACE) and aldosterone; b) renal clearances of sodium, potassium, inulin and para-aminohippuratu (PAH); c) antipyrine lap) systemic clearance and plasma half-life. 15 healthy subjects also underwent hormonal and renal function determinations as control group. Distribution of the GFR values was bimodal: 10 patients had low GFR values (I-GFR group) and I 1 had normal GFR values (n-GFR group) (below and above 105 ml/min/1.73 m: b.s.a., respectively). Crentinine plasma levels did not differ in the two groups (respectively, 1.32 _+0.83 vs. 0.95 + 0.34 mg/dl, P=0.08), nor did PAIl or Ap clearances (respectively, P=0.18 and P---0.47) and Child-Pugh score (P=0.48). Compared with the n-GFR group, the I-GFR group had significantly higher AR and PRA values (all P<0.01), sigui:fienntty lower levels of plasma ACE (P<0.03) and a significantly higher AR/Angiote~sla II ratio (1.66 4" 0.97 vs. 0.48 + 0.38, P<0.003).. Compared with healthy conta-ols all 21 patients showed markedly laerensed AR/PRA ratio (both P<0.03), indirect evidence of reduced renin subs~ate circulating levels. The respective 18-mouth survival rates of IGFR and n-GFR groups were 41% and 81% (P<0.02). In conclusion, among decompensated cirrhotic patients those with reduced GFR had a worse survival rate associated with more severe contraction of the effective arterial blood volume and lower capacity to cope with this phenomenon (higher AR/Ang lI ratio), possibly due to lower ACE plasma levels. ACE plasma levels were not related to the degree of liver insufficiency, but may be genetically determined.
20
18
ANp73 regulates NFkB-dependent proliferation and counteracts p53 and TAp73induced growth arrest of hepatoeytes. Stefania Vossio^, Emanuele PescandoloA. Francesca Moreni^, Marcella Fulco^. Antonio Costanzo^, Clara Balsano*^, Massimo Levrero^$. ^Fondazione Andrea Cesalpino, University of Rome "La Sapienza", Rome, italy: * Dept. Internal Medicine, University of L'Aquila. L'Aquila, Italy; $ Dept. Internal Medicine. University of Cagliari, Cagliari. Italy.
D I F F E R E N T I A L R E G U L A T I O N OF MCP-1 E X P R E S S I O N IN HEPATIC STELLATE CELLS BY MEMBERS OF THE MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) FAMILY. IIaria Petrai, Wanda Delogu Sabrina Pastacaldi, Andrea Bonacchi. Eva Efsen, Paolo Gentilini. Fabio Morro Dipartimento di Medicine lnetrna, University o f Florence
Mature hepatocytes re-entry into tile cell cycle and proliferate to compensate loss of hepatic parenchyma due to surgery, chemical toxicity or viral infections. At~er partial hepateetomy (PHx) liver mass is restored in about a week by one or two rounds o hepatocytes cell division. Extensive study of liver regeneration after PHx has revealed the role of growth factors (such as HGF and its receptor Met, EGF, TGF-alpha), of cytokines (such as the TNF-aIpha and IL-6) and of transcription factors (such as E2FI. APt, NFkB and Star3) in the regulation of hepatocyte proliferation whereas TGF beta/activin, p53 and the cyclin-dependent kinase (CDK) inhibitor p21 prevent uncontrolled hepatocytes proliferation. Although p53 levels greatIy increase in the regenerating liver after PHx and suppression of its expression alters liver regeneration in vivo. p21 activation and hepatocytes growth arrest in response to a variety of stimuli also occur in a p53 deficient genetic background, suggesting a role tbr the p53-related proteins p63 and p73. The p73 gene is transcribed from two promoters. Pl and P2. that direct the expression of the TA (transactivation competent, pro-apoptotic and anti proliferative) and the DN (transactivation deficient, anti-apoptotic and pro-proliferative) p73 proteins, respectively. We have previously demonstrated that, in response to a no apoptotic DNA damage the P2-p73 promoter is transcriptionally activated in a p53 dependent manner, leading to the expression and accumuIation of DN-p73 proteins, that [n turn selectively downregulate their own promoter as well as the p21 promoter to ensure the cell-cycle reentry of cells that have repaired their DNA damage. We show here that liver DN-p73 mRNA levels increase in chronic hepatitis and cirrhosis and correlate with hepatocytes loss and proliferation. Using slrlall interfering RNAs specific for DN-p73 to selectively abrogate its function, we found that DN-p73 is required fo TNF-alpha induced proliferation of hepatocytes and that, in this context DN-p73 expression is controlled by NFkB. To study the regulation of DN-p73 expression at th transcriptional level we cloned the DN-p73 alternative promoter (P2p73Pr). Differently from the Pip73 promoter, that directs the expression of TA-p73 proteins and is regulated by E2FI. the P2p73 promoter is activated by TNF-alpha and IFN-gamma through NFkB and IRF-I sites and downregulated by retinoids and lFN-alpha in hepatocarcinoma cell lines and in immortalized Met Murine Hepatocytes D3 (MMH D3) cells. Functional studies showed that DN-p73 counteracts both p53 and TA-p73 induced growth arrest and apoptosis in MMH-D3 cells. In conclusion we have identified a role of DN-p73 variants in controlling hepatocytes growth in the context o an inflammatory liver enviromnent.
Hepatic stellate cells (HSC) coordinate the liver wound-healing response through secretion of several cytokines and chemokines, including MCP1. In this study, we evaluated the role of different proteins of the M A P K family (ERK, p38, and JNK) in the regulation of monocyte chemoattractant rprotein-I (MCP-1) expression by HSC, as an index of their pro-inflammatory activity, Several mediators activated all three M A P K , including TNF, IL-I and PDGF, although with different potency. While TNF and 1L-1 were very effective in inducing MCP-1 expression, PDGF was only modestly active. To assess the relative role o f the different MAPKs, specific pharmacologic inhibitors were used, namely SB203580 (p38), SP600125 (JNK), and PD98059 (MEK/ERK). The efficacy and specificity of the different inhibitors in our cellular system was verified analyzing the enzymatic activity o f the different MAPKs using in vitro kinase assays and/or testing the inhibition of phosphorylation o f downstream substrates. S B 2 0 3 5 8 0 dose-dependently inhibited MCP-1 secretion and gone expression induced by IL-1 or TNF. This effect occurred independently of reduced DNA binding of NFkappaB. Similarly, inhibition of JNK activity by SP600125 120 microM) blocked MCP-1 secretion. In contrast, inhibition of ERK did not affect the up-regulation of MCP-1 induced by IL-1 or TNF. Additionally. Activin A, which stimulates MCP-1 expression, activated JNK and p38. but not ERK. Conversely, when M A P K inhibitors were compared in their ability to block DNA synthesis in response to PDGF. PD98059 (ERK inhibitor) was the most effective. We conclude that members of the M A P K family differentially regulate the biologic response of HSC to soluble mediators, p38 and .INK are necessary for chemokine secretion, while ERK is more relevant for mitogenesis.
A7