Increased CCN2 Transcription in Keloid Fibroblasts Requires Cooperativity Between AP-1 and SMAD Binding Sites

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Abstract
Objective: We examined the transcriptional response to serum stimulation as an in vitro model of wound healing in keloid fibroblasts to identify molecular mechanisms leading to their aberrant growth. Summary Background Data: Keloids are proliferative dermal growths representing a pathologic wound healing response. Although several groups have shown increased expression of profibrotic factors in keloids, there is little known about why they are expressed at higher levels than normal. Methods: Fibroblasts derived from keloids and normal scar were subjected to serum stimulation as an in vitro model to mimic a component of the wound microenvironment to examine differential gene expression in keloid derived fibroblasts versus normal human fibroblasts. A promoter analysis was performed to identify specific enhancers involved in mediating the differential response of connective tissue growth factor (CTGF, CCN2). Point mutations in the enhancers were performed to confirm their role. Finally, we examined activation of transcription factors known to bind the targeted enhancers. Results: Transcription of CCN2 after serum stimulation was significantly higher in keloid versus normal fibroblasts. Promoter analysis demonstrates the fragment from −625/−140 conferred increased serum responsiveness. Mutational analysis showed an AP-1 and SMAD binding site were both necessary for serum responsiveness. Preventing activation of either transcriptional complex will block CCN2 transcription. Additional experiments suggest that a single complex that includes components of the AP-1 and SMAD binding complexes is responsible for transactivation in response to serum. The key difference between keloid and normal fibroblasts appears to be the degree of activation of c-Jun. Conclusions: We suggest that altered responsiveness to cellular stress, based upon current data using serum stimulation and past data on response to mechanical strain, is a key defect leading to keloid formation.

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