Increased CTGF transcription in keloid fibroblasts requires cooperativity between AP-1 and SMAD binding sites

Abstract

Abstract Introduction: Keloids are proliferative dermal growths representing a pathological wound-healing response. We have previously demonstrated increased serum induced transcription of connective tissue growth factor (CTGF) in keloid fibroblasts (KF) versus normal fibroblasts (NF). We present here an analysis of the mechanisms for increased CTGF transcription in KFs. Methods: A series of 5’ CTGF promoter deletions were cloned into PGL3-basic luciferase reporter vector. CTGF promoter constructs and pSV-beta-galactosidase vectors were co-transfected into KFs and NFs that were serum stimulated. Luciferase activity was normalized to beta-galactosidase activity. Nuclear extracts were harvested before and after 1 hour of serum stimulation and used for gel mobility shift assays. Site-directed mutagenesis of CTGF promoter/reporter gene constructs was performed using the QuikChange Site-directed Mutagenesis Kit. Results: Promoter analysis demonstrated the fragment from -625/-140 conferred increased serum responsiveness. Mutational analysis of an AP-1 and Smad binding site in this fragment showed both were necessary for serum responsiveness. Gel shift analysis showed a specific band binding to the AP-1 site in KFs but not in NFs. A similar binding pattern is seen using the Smad binding site in KFs versus NFs. Supershift assay demonstrated c-Jun, which was a downstream transcription factor of JNK pathway, was able to bind the AP-1 site. Conclusions: Increased transcription of CTGF following serum stimulation in KFs requires both the AP-1 and SMAD binding site. Mutation of either of these two sites is sufficient to abolish the serum response of CTGF in KFs. These studies may identify new targets for the therapy of keloids.