CYP24A1 is overexpressed in keloid keratinocytes and its inhibition alters profibrotic gene expression

Abstract

Background Keloids are disfiguring, fibrotic scar-like lesions that are challenging to treat and commonly recur after therapy. A deeper understanding of the mechanisms driving keloid formation is necessary for the development of more effective therapies. Reduced vitamin D receptor (VDR) expression has been observed in keloids, implicating vitamin D signaling in keloid pathology. Vitamin D exhibits anti-proliferative and anti-inflammatory properties, suggesting it could have therapeutic utility in keloid disorder. The current study investigated vitamin D-regulated gene expression in keloid keratinocytes and the effects of inhibiting an enzyme involved in vitamin D metabolism on the phenotype of keloid-derived keratinocytes. Methods Normal and keloid-derived primary keratinocytes were isolated from normal skin and keloid lesions, respectively, and were cultured in the absence or presence of vitamin D. In some experiments, inhibitors of the vitamin D metabolizing enzyme CYP24A1, ketoconazole or VID400 were added in the absence or presence of vitamin D. Cellular proliferation, migration and gene expression were measured. Results We observed significant overexpression of CYP24A1 mRNA in keloid versus normal keratinocytes and increased CYP24A1 protein levels in keloids versus normal skin. CYP24A1 encodes 24 hydroxylase and is induced by vitamin D in a feedback loop that regulates vitamin D levels; thus, inhibition of CYP24A1 activity may locally increase active vitamin D levels. Ketoconazole, a non-specific cytochrome P-450 inhibitor, reduced proliferation of keloid and normal keratinocytes, but VID400, a specific CYP24A1 inhibitor, only significantly affected keloid keratinocyte proliferation. Neither inhibitor significantly reduced keratinocyte migration. The two inhibitors had different effects on vitamin D target gene expression in keratinocytes. Specifically, ketoconazole treatment reduced CYP24A1 expression in normal and keloid keratinocytes, whereas VID400 increased CYP24A1 expression. Both inhibitors decreased expression of profibrotic genes, including periostin and hyaluronan synthase 2, in keloid-derived cells. Combined treatment of keloid keratinocytes with vitamin D and ketoconazole or VID400 increased the effects of vitamin D treatment on target genes, although the effects were gene- and cell type-specific. Conclusions The data suggest that reduction of vitamin D inactivation with CYP24A1 inhibitors may reduce profibrotic gene expression in keloid-derived cells. Therefore, CYP24A1 inhibitors may serve as adjunctive therapies to suppress keloid-associated gene expression changes.