(WHS-P2.05) INHIBITION OF CYP24A1, AN ENZYME INVOLVED IN VITAMIN D METABOLISM, ALTERS PROFIBROTIC GENE EXPRESSION IN KELOID-DERIVED KERATINOCYTES
Friday, May 17, 2024
10:30 AM – 11:30 AM East Coast USA Time
Introduction: Keloids are disfiguring fibroproliferative lesions that can occur following injury to skin in susceptible individuals. Keloids are challenging to treat and recurrence after treatment is common. A deeper understanding of the molecular mechanisms driving keloid formation is necessary for development of more effective therapies for keloid suppression. We previously identified reduced expression and decreased nuclear localization of the vitamin D receptor (VDR) in keloid epidermis, implicating vitamin D signaling in keloid pathology. Here we report that CYP24A1 is overexpressed in keloid keratinocytes compared with normal keratinocytes. The CYP24A1 gene encodes 24 hydroxylase, a vitamin D metabolizing enzyme that degrades 1,25-dihydroxyvitamin D3 (1,25-D3), the active form of vitamin D. The CYP24A1 gene is itself induced by vitamin D in a feedback loop that regulates 1,25-D3 levels. In this study, we investigated the effects of CYP24A1 inhibition in normal and keloid-derived keratinocytes.
Methods: Normal and keloid keratinocytes (N=3 donors each) were cultured +/- 1,25-D3 and +/- inhibitors of CYP24A1, which included ketoconazole, a non-specific inhibitor of cytochrome P-450 enzymes, and VID-400, a specific inhibitor of CYP24A1. Proliferation was measured using an MTT assay, and gene expression was analyzed by quantitative PCR. Statistical analyses were performed using t test (2 groups) or One Way ANOVA (>2 groups) using SigmaPlot 15.0.
Results: CYP24A1 mRNA was expressed at 3.5X higher levels in keloid keratinocytes compared with normal keratinocytes. Ketoconazole inhibited proliferation of keloid and normal keratinocytes, but VID-400 had no significant effect on keratinocyte proliferation. The two inhibitors had different effects on expression of vitamin D target genes in keratinocytes. For example, ketoconazole treatment alone reduced expression of CYP24A1 in normal and keloid keratinocytes, whereas VID-400 treatment increased CYP24A1 expression. Both inhibitors decreased expression of the profibrotic genes Periostin (POSTN) and Hyaluronan synthase 2 (HAS2), previously shown to be upregulated in keloid-derived cells. Combined treatment of keloid-derived keratinocytes with 1,25-D3 and ketoconazole or VID-400 increased the effects of 1,25-D3 treatment on vitamin D target genes and profibrotic gene expression, although the effects were gene- and cell type-specific.
Conclusions: The data suggest that reduction of 1,25-D3 inactivation with inhibitors of CYP24A1 may reduce profibrotic gene expression in keloid-derived cells. Because vitamin D has numerous anti-inflammatory and antifibrotic activities, inhibitors of CYP24A1 may serve as adjunctive therapies to suppress keloid-associated gene expression changes.