131 CYP24A1 Is Overexpressed in Keloid Keratinocytes and Its Inhibition Reduces Profibrotic Gene Expression

TL;DR

This study finds that CYP24A1 is overexpressed in keloid skin cells and that inhibiting it reduces fibrosis-related gene activity, suggesting a new treatment approach.

Contribution

The study identifies CYP24A1 overexpression in keloid keratinocytes and demonstrates that its inhibition reduces profibrotic gene expression.

Findings

Keloid keratinocytes show significantly higher CYP24A1 expression compared to normal keratinocytes.

CYP24A1 inhibition with ketoconazole reduces profibrotic genes like HAS2 and POSTN.

Ketoconazole treatment decreases keratinocyte proliferation and enhances 1,25-D3 effects on target genes.

Abstract

Keloids are aggressive fibroproliferative lesions that can occur following skin injuries, including burns. Keloids negatively impact quality of life and are extremely challenging to treat. A better understanding of the underlying molecular mechanisms is needed for development of improved keloid therapies. We previously identified reduced vitamin D receptor (VDR) expression in keloid epidermis, implicating vitamin D and VDR-mediated signaling in keloid pathology. To investigate vitamin D signaling in keloid-derived cells, the current study analyzed expression of VDR and VDR target genes cathelicidin antimicrobial peptide (CAMP), cluster of differentiation 14 (CD14), endothelin 1 (EDN1), and 24 hydroxylase (encoded by CYP24A1) in normal and keloid keratinocytes. CAMP and CD14 are involved in innate immunity, EDN1 plays a role in endothelial dysfunction and fibrosis, and CYP24A1 is a vitamin D metabolizing enzyme that limits the amount of 1,25-dihydroxyvitamin D3 (1,25-D3), the active form of vitamin D. Additionally, the effects of 1,25-D3 treatment and CYP24A1 inhibition on expression of these genes, and other genes known to be aberrantly expressed in keloids, were investigated.

Normal skin (N=6) and keloid (N=8) samples were obtained with IRB approval and primary keratinocyte cultures were established. For some experiments, keloid keratinocytes (N=3) were cultured +/- 1,25-D3, and +/- ketoconazole, which inhibits CYP24A1 enzyme activity. Gene expression was measured using quantitative PCR. Proliferation was measured via MTT assay. Statistical analyses were performed using t test (2 groups) or One Way ANOVA (>2 groups) using SigmaPlot 15.0.

Despite reduced VDR in keloid epidermis in vivo, keloid keratinocytes (KKs) in vitro expressed VDR mRNA at levels similar to normal keratinocytes (NKs) and responded to 1,25-D3 treatment with appropriate changes in target gene expression. Notably, in the absence of 1,25-D3 treatment, CYP24A1 expression in KKs was significantly higher than in NKs. Inhibition of CYP24A1 activity in KKs via ketoconazole treatment enhanced the effects of 1,25-D3 on expression of some target genes: levels of CYP24A1 and CD14 were increased, whereas EDN1 was significantly decreased. Ketoconazole treatment alone decreased expression of hyaluronan synthase 2 (HAS2) and periostin (POSTN), two profibrotic genes known to be overexpressed in keloid cells. Further, ketoconazole significantly reduced keratinocyte proliferation.

The data suggest that CYP24A1 inhibition with ketoconazole may reduce 1,25-D3 inactivation, thereby enhancing the effects of 1,25-D3 on VDR target gene expression.

Vitamin D has numerous anti-inflammatory, antiproliferative, and antifibrotic properties. Ketoconazole may potentiate the antifibrotic effects of 1,25-D3 and may thereby serve as an adjunctive therapy for suppression of keloid-associated gene expression changes.