JOURNAL OF CELLULAR PHYSIOLOGY 208:336–343 (2006) Upregulation of Secretory Connective Tissue Growth Factor (CTGF) in Keratinocyte-Fibroblast Coculture Contributes to Keloid Pathogenesis YING TING KHOO, 1 CHEE TIAN ONG, 1 ANANDAROOP MUKHOPADHYAY, 4 HWAN CHOUR HAN, 5 DANG VINH DO, 1 IVOR JIUN LIM, 1 AND TOAN THANG PHAN 1,2,3 * 1 Department of Surgery, National University of Singapore, Singapore 2 Department of Bioengineering, National University of Singapore, Singapore 3 NUS Tissue Engineering and Stem Cell Research Program, National University of Singapore, Singapore 4 Department of Pharmacy, National University of Singapore, Singapore 5 Department of Pathology, National University of Singapore, Translational Interface Group, Oncology Research Institute, NUMI, Singapore Connective tissue growth factor (CTGF) plays a critical role in keloid pathogenesis by promoting collagen synthesis and deposition. Previous work suggested epithelial-mesenchymal interactions as a plausible factor affecting the expression of various growth factors and cytokines by both the epithelial and dermal mesenchymal cells. The aim of this study is to explore the role of epithelial- mesenchymal interactions in modulating CTGF expression. Immunohistochemistry was employed to check CTGF localization in skin tissue. Western blot assay was performed on total protein extracts from skin tissue, cell lysates and conditioned media to detect the basal/expression levels of CTGF. Study groups were subjected to serum stimulation (fibroblast-single cell culture) and pharmacological inhibitors targeted against mTOR (Rapamycin), Sp1 (WP631 and Mitoxanthrone), Smad3 (SB431542), and PI3K (LY294002). Increased localization of CTGF in the basal layer of keloid epidermis and higher expression of CTGF was observed in the keloid tissue extract. Interestingly, lower basal levels of CTGF was observed in fibroblast cell lysates cocultured with keloid keratinocytes compared to normal keratinocytes, while the conditioned media from the former culture consistently demonstrated a higher expression of secreted CTGF as compared to the latter group. These results demonstrate an important role of epithelial- mesenchymal interactions in the regulation of CTGF expression. Fibroblasts treated with inhibitors against mTOR, Sp1, Smad3, and PI3K demonstrated a reduced expression of CTGF, suggesting these signaling pathways to be important in the regulation of CTGF expression. Thus, revealing the therapeutic potentials for inhibitors that are selective for these factors in controlling CTGF expression in fibrotic conditions. J. Cell. Physiol. 208: 336 – 343, 2006. ß 2006 Wiley-Liss, Inc. Keloid is a dermal fibroproliferative disorder char- acterized by the overgrowth of dense fibrous tissue coupled with excessive deposition of extracellular matrix (ECM) components such as collagen, glycopro- teins, and fibronectin (Messadi et al., 2004), resulting in excess protrusive scarring of skin, which only occurs in humans. Numerous published studies have showed that CTGF is present and frequently over-expressed and co- expressed with transforming growth factor-beta (TGF- b) in keloid tissues (Igarashi et al., 1996) and that it could play a pathologically significant role in this fibrotic disorder (Leask et al., 2002). Although CTGF is causally implicated in dermal fibrosis via its apparent stimula- tion by TGF-b, very little is known about the level and control of its expression in keloid pathogenesis. A detailed study of the functions of CTGF is required to increase our understanding of its role in normal tissue formation and development, and also to elucidate its participation in tissue regeneration and repair in human fibrotic disorders. CTGF is a cysteine-rich peptide that exhibits platelet- derived growth factor-like activities, it also appears to be antigenically related to platelet derived growth factor (PDGF) (Bradham et al., 1991). A myriad of different cell types including fibroblasts, epithelial cells, endothelial cells, vascular smooth muscle cells, glioblastoma cells, and chondrocytes have been demonstrated to express CTGF (Moussad and Brigstock, 2000). CTGF exhibits diverse biological actions in vitro depending on the cell type, these include mitogenesis, matrix production, angiogenesis, and apoptosis (Gupta et al., 2000; Crean et al., 2001). CTGF was noted to have mitogenic and chemotactic effects on fibroblasts (Bradham et al., 1991). In fibroblasts, CTGF has been shown to be expressed ß 2006 WILEY-LISS, INC. Abbreviations: CTGF, connective tissue growth factor; NS, normal skin; KS, keloid scar; KK, keloid keratinocytes; KF, keloid fibroblasts; NF, normal fibroblasts; NK, normal keratinocytes; DMEM, Dulbecco’s modified Eagle’s medium; HBSS, hanks balanced salt solution; FCS, fetal calf serum; KGM, keratinocyte growth medium; PBS, phosphate buffered saline; TBS, tris buffered saline; KGF, keratinocyte growth factor; HGF, hepato- cyte growth factor; EGF, epidermal growth factor; FGF, fibroblast growth factor; IGF, insulin-like growth factor; TGF-b, transform- ing growth factor-beta; TNF-a, tumor necrosis factor-alpha; ECM, extracellular matrix; PDGF, platelet derived growth factor; SDS– PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophor- esis; BSA, bovine serum albumin; PI3K, phosphatidylinositol 3- kinase; ALK, activin receptor-like kinase; JNK, jun N-terminal kinase; MAPK, mitogen-activated protein kinase; ERK, extra- cellular signal-regulated kinase; MEK, MAPK/ERK kinase. Contract grant sponsor: Biomedical Research Council, Singapore; Contract grant number: 03/1/21/19/251; Contract grant sponsor: National HealthCare Group Research Fund; Contract grant number: NHG-STP 04027; Contract grant sponsor: Biomedical Research Council, Singapore; Contract grant number: 04/1/21/19/ 338. *Correspondence to: T.T. Phan, Department of Surgery, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260. E-mail: surptt@nus.edu.sg Received 13 February 2006; Accepted 10 March 2006 DOI: 10.1002/jcp.20668