Dissection of an impact of VDR and RXRA on the genomic activity of 1,25(OH)2D3 in A431 squamous cell carcinoma

Abstract

Human skin is the natural source, place of metabolism, and target for vitamin D₃. The classical active form of vitamin D₃, 1,25(OH)₂D₃, expresses pluripotent properties and is intensively studied in cancer prevention and therapy. To define the specific role of vitamin D₃ receptor (VDR) and its co-receptor retinoid X receptor alpha (RXRA) in genomic regulation, VDR or RXRA genes were silenced in the squamous cell carcinoma cell line A431 and treated with 1,25(OH)₂D₃ at long incubation time points 24 h/72 h. Extending the incubation time of A431 WT (wild-type) cells with 1,25(OH)₂D₃ resulted in a two-fold increase in DEGs (differentially expressed genes) and a change in the amount of downregulated from 37% to 53%. VDR knockout led to a complete loss of 1,25(OH)₂D₃-induced genome-wide gene regulation at 24 h time point, but after 72 h, 20 DEGs were found, of which 75% were downregulated, and most of them belonged to the gene ontology group "immune response". This may indicate the existence of an alternative, secondary response to 1,25(OH)₂D₃. In contrast, treatment of A431 ΔRXRA cells with 1,25(OH)₂D₃ for 24 h only partially affected DEGs, suggesting RXRA-independent regulation. Interestingly, overexpression of classic 1,25(OH)₂D₃ targets, like CYP24A1 (family 24 of subfamily A of cytochrome P450 member 1) or CAMP (cathelicidin antimicrobial peptide) was found to be RXRA-independent. Also, immunofluorescence staining of A431 WT cells revealed partial VDR/RXRA colocalization after 24 h and 72 h 1,25(OH)₂D₃ treatment. Comparison of transcriptome changes induced by 1,25(OH)₂D₃ in normal keratinocytes vs. cancer cells showed high cell type specific expression pattern with only a few genes commonly regulated by 1,25(OH)₂D₃. Activation of the genomic pathway at least partially reversed the expression of cancer-related genes, forming a basis for anti-cancer activates of 1,25(OH)₂D₃. In summary, VDR or RXRA independent genomic activities of 1,25(OH)₂D₃ suggest the involvement of alternative factors, opening new challenges in this field.

Keywords: Calcitriol; Keratinocytes; Retinoid X receptor; Squamous cell carcinoma; Vitamin D(3); Vitamin D(3) receptor.