Molecular insights into connective tissue growth factor action in rat pancreatic stellate cells Anna Karger, Brit Fitzner, Peter Brock, Gisela Sparmann, Jörg Emmrich, Stefan Liebe, Robert Jaster ⁎ Department of Medicine, Division of Gastroenterology, Medical Faculty, University of Rostock, E.-Heydemann-Str. 6,18057 Rostock, Germany ABSTRACT ARTICLE INFO Article history: Received 7 May 2008 Received in revised form 25 June 2008 Accepted 27 June 2008 Available online 2 July 2008 Keywords: Connective tissue growth factor Interleukin-6 Pancreatic stellate cells Pancreatic fibrosis, a key feature of chronic pancreatitis and pancreatic cancer, is mediated by activated pancreatic stellate cells (PSC). Connective tissue growth factor (CTGF) has been suggested to play a major role in fibrogenesis by enhancing PSC activation after binding to α5β1 integrin. Here, we have focussed on molecular determinants of CTGF action. Inhibition of CTGF expression in PSC by siRNA was associated with decreased proliferation, while application of exogenous CTGF stimulated both cell growth and collagen synthesis. Real-time PCR studies revealed that CTGF target genes in PSC not only include mediators of matrix remodelling but also the proinflammatory cytokines interleukin (IL)-1β and IL-6. CTGF stimulated binding of NF-κB to the IL-6 promoter, and siRNA targeting the NF-κB subunit RelA interfered with CTGF-induced IL-6 expression, implicating the NF-κB pathway in the mediation of the CTGF effect. In further studies, we have analyzed regulation of CTGF expression in PSC. Transforming growth factor-β1, activin A and tumor necrosis factor-α enhanced expression of the CTGF gene, while interferon-γ displayed the opposite effect. The region from - 74 to - 125 of the CTGF promoter was revealed to be critical for its activity in PSC as well as for the inhibitory effect of interferon-γ. Taken together, our results indicate a tight control of CTGF expression in PSC at the transcriptional level. CTGF promotes fibrogenesis both directly by enhancing PSC proliferation and matrix protein synthesis, and indirectly through the release of proinflammatory cytokines that may accelerate the process of chronic inflammation. © 2008 Elsevier Inc. All rights reserved. 1. Introduction The CCN family of secreted proteins comprises six structurally related members that specifically associate with the extracellular matrix (ECM). CCN proteins act as regulators of diverse cellular functions, including mitogenesis, apoptosis, ECM production, adhesion and migration, as well as complex biological processes such as angiogenesis, chondrogenesis and osteogenesis [1,2]. As a major principle of CCN protein action, modification of signal transduction pathways induced by other molecules (which vary depending on the particular biological system) has emerged. Recent studies have linked the CCN family member connective tissue growth factor (CTGF; also known as CCN2) to the induction of fibrogenesis and, under pathological conditions, the development of fibroproliferative diseases [3]. At least in part, CTGF exerts its profibrotic effects in direct synergy with transforming growth factor-beta (TGF-β), e.g. by mediating downstream actions of this cytokine. Furthermore, TGF-β is a well-established inductor of CTGF gene expression [3]. In the pancreas, pancreatic stellate cells (PSC) have been identified both as a source of CTGF and a target of CTGF action [4,5]. PSC are the main ECM protein-synthesizing cells in the organ [6,7]. The cells have recently been shown to display a transcriptional phenotype that closely resembles the one of hepatic stellate cells, the principle effector cells in liver fibrosis [8]. Dysregulated production of ECM by activated PSC has been implicated in the progression of chronic pancreatitis and pancreatic cancer (PC); two diseases that are characterized by an extended organ fibrosis [9,10]. In detail, CTGF has been shown to enhance collagen synthesis and proliferation of PSC, and to promote both adhesion and migration of the cells [4]. CTGF effects on PSC are mediated, at least in part, by the cell surface integrin α5β1 and heparan sulfate proteoglycan receptors [4,5]. With respect to PC, it is important to note that CTGF has also been suggested to promote cancer growth directly by stimulating proliferation and migration of the tumor cells, while antibodies to CTGF exert antiproliferative, antiangiogenetic and antimetastatic effects [11,12]. Cellular Signalling 20 (2008) 1865–1872 Abbreviations: ECM, extracellular matrix; CTGF, connective tissue growth factor; TGF-β, transforming growth factor-beta; PSC, pancreatic stellate cell; PC, pancreatic cancer; PDGF, platelet-derived growth factor; ERK, extracellular signal-regulated kinase; STAT, signal transducer and activator of transcription; IL, interleukin; IMDM, Iscove's modified Dulbecco's medium; ECL, enhanced chemoluminescence; IFN, interferon; BrdU, 5-bromo-2′-deoxyuridine; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of matrix metalloproteinase; HPRT, hypoxanthine-guanine phosphor- ibosyl transferase; NF, nuclear factor; SEM, standard error of the mean; TNF, tumor necrosis factor. ⁎ Corresponding author. Tel.: +49 381 4947349; fax: +49 381 4947482. E-mail address: jaster@med.uni-rostock.de (R. Jaster). 0898-6568/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cellsig.2008.06.016 Contents lists available at ScienceDirect Cellular Signalling journal homepage: www.elsevier.com/locate/cellsig