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

CATALINA ATORRASAGASTI, LEONARDO HOFFMAN, JORGE AQUINO, LAURA ALANIZ, MARIANA MALVICINI, FLAVIA PICCIONI, JUAN BAYO, MARIANA GARCIA, OSVALDO PODHAJCER , MARCELO SILVA AND GUILLERMO MAZZOLINI.

Congreso; The Liver Meeting 2010; 2010

American Asociation for Study of the Liver Diseases

Introduction: SPARC is an extracellular matrix (ECM) protein involved in many biological processes and over-expressed in cirrhotic livers. We recently 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: The aim of the present investigation was to assess if down-regulation of SPARC could affect TGF-1 and PDGF-BB-mediated profibrogenic activity of hepatic stellate cells (HSC). Material & Methods: Experiments were carried out in CFSC-2G and LX2 cells, an immortalized HSC line derived from cirrhotic rats and a spontaneous immortalized human HSC line, respectively. SPARC inhibition was assessed by a synthetic specific small interfering RNA constructs siRNA (Dharmacon) 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 (Neuroprobe); in the lower chamber, TGF-1 or PDGF-BB were added as the chemoattractants. Adhesion assays were performed on fibronectin coated plates. TGF-1 production was quantified by ELISA on cultured HSC supernatants. Comparative analyses of rat extracellular matrix and adhesion molecules by qPCR arrays (SABiosciences) 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 HSCs to fibronectin and prevents HSC chemotaxis in response to fetal bovine serum (FBS), PDFG-BB and TGF-1. In addition, SPARC siRNA transfected cells were found to secrete significantly lower TGF-1 levels than control cells. Chemotaxis deficiency in SPARC siRNA-treated HSCs was partially reversed by exogenous application of TGF-1 suggesting the involvement of this cytokine 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. In conclusion: These data indicate that a reduction in SPARC 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.