Knockdown of SPARC (Secreted Protein Acidic and Rich in Cysteine) attenuates the profibrogenic response induced by TGF-£]1 and PDGF-BB in hepatic stellate cells

ATORRASAGASTI C; HOFMAN L; AQUINO JB; ALANIZ L; MALVICINI M; PICCIONI F; BAYO J; GARCIA MG; PODHAJCER O; SILVA M; MAZZOLINI G

Congreso; 61 Reunion Anual de la Asociacion Americana para el Estudio de las Enferemedades del Higado (AASLD); 2010

Background: SPARC is an extracellular matrix (ECM) protein involved in many biological processes and over-expressed in cirrhosis. We demonstrated that in vivo down-regulation of SPARC ameliorates fibrogenic response to chronic thioacetamide intoxication in rats (Camino et al., 2008). Nevertheless cellular mechanisms involved remain largely unknown Aim: to assess if down-regulation of SPARC could affect TGF-B1 and PDGF-mediated profibrogenic activity of hepatic stellate cells (HSC) M&M: Experiments were carried out in CFSC-2G and LX2 cells, an immortalized HSC line from cirrhotic rats and a spontaneous immortalized human HSC line, respectively. SPARC inhibition was assessed by a synthetic specific small interfering RNA constructs siRNA in CFSC-2G cells and by a plasmid contained a specific siRNA for LX2 cells. The efficacy of SPARC knock-down was assessed by real time PCR (qPCR). For transwell cell migration assays, non-modified or SPARC down-regulated HSC were seeded on the upper chambers of 48-well chamber plates; in the lower chamber, TGF-B1 or PDGF were added as the chemoattractants. Adhesion to plastic was also performed. TGF-B1 production was quantified by ELISA on cultured HSC supernatants. Comparative analyses of rat ECM and adhesion molecules by qPCR arrays of non-modified HSC versus SPARC down-regulated HSC were performed. Differentially expressed genes were confirmed by qPCR Results: Both CFSC-2G and LX2 cells showed a significant inhibition SPARC mRNA expression at 72 h post-transfection (60% and 80%, respectively). Down-regulation of SPARC by specific siRNA did not affect proliferation and have minor effects on HSC apoptosis. However, SPARC knockdown increased the adhesion of HSC to fibronectin and prevents HSC chemotaxis in response to FBS, PDFG and TGF-B1. In addition, SPARC siRNA transfected cells were found to secrete significantly lower TGF-B1 levels than controls. Chemotaxis deficiency in SPARC siRNA-treated HSC was partially reversed by exogenous application of TGF-B1 suggesting its involvement downstream of SPARC effects. Comparative analyses by qPCR arrays of non-modified HSC versus SPARC down-regulated HSC revealed an important number of genes (26) whose expression levels were modified more than 1.5-fold. Importantly, SPARC knockdown increased E-cadherin expression and a concomitant decrease in N-cadherin expression Conclusions: These data indicate that a reduction in PARC levels alters the adhesion/detachment balance in HSC and/or increases their adhesion and this likely impairs the ability of HSC to move and migrate in response to PDGF-BB. Our results further suggest SPARC as a novel target for the treatment of liver fibrosis.1 and PDGF-mediated profibrogenic activity of hepatic stellate cells (HSC) M&M: Experiments were carried out in CFSC-2G and LX2 cells, an immortalized HSC line from cirrhotic rats and a spontaneous immortalized human HSC line, respectively. SPARC inhibition was assessed by a synthetic specific small interfering RNA constructs siRNA in CFSC-2G cells and by a plasmid contained a specific siRNA for LX2 cells. The efficacy of SPARC knock-down was assessed by real time PCR (qPCR). For transwell cell migration assays, non-modified or SPARC down-regulated HSC were seeded on the upper chambers of 48-well chamber plates; in the lower chamber, TGF-B1 or PDGF were added as the chemoattractants. Adhesion to plastic was also performed. TGF-B1 production was quantified by ELISA on cultured HSC supernatants. Comparative analyses of rat ECM and adhesion molecules by qPCR arrays of non-modified HSC versus SPARC down-regulated HSC were performed. Differentially expressed genes were confirmed by qPCR Results: Both CFSC-2G and LX2 cells showed a significant inhibition SPARC mRNA expression at 72 h post-transfection (60% and 80%, respectively). Down-regulation of SPARC by specific siRNA did not affect proliferation and have minor effects on HSC apoptosis. However, SPARC knockdown increased the adhesion of HSC to fibronectin and prevents HSC chemotaxis in response to FBS, PDFG and TGF-B1. In addition, SPARC siRNA transfected cells were found to secrete significantly lower TGF-B1 levels than controls. Chemotaxis deficiency in SPARC siRNA-treated HSC was partially reversed by exogenous application of TGF-B1 suggesting its involvement downstream of SPARC effects. Comparative analyses by qPCR arrays of non-modified HSC versus SPARC down-regulated HSC revealed an important number of genes (26) whose expression levels were modified more than 1.5-fold. Importantly, SPARC knockdown increased E-cadherin expression and a concomitant decrease in N-cadherin expression Conclusions: These data indicate that a reduction in PARC levels alters the adhesion/detachment balance in HSC and/or increases their adhesion and this likely impairs the ability of  HSC to move and migrate in response to PDGF-BB. Our results further suggest SPARC as a novel target for the treatment of liver fibrosis.