HaCaT cell line as a model system for vitamin D3 metabolism in human skin

Synthesis and catabolism of calcitriol (1,25(OH)2D3) were studied using HaCaT cell line as a cell culture model. Our results indicate that stimulation of HaCaT cells with epidermal growth factor (EGF) or transforming growth factor-alpha (TGF-alpha) within 16 h just prior to reaching confluence amplified the production of calcitriol when calcidiol (3H-25OHD3) was used as a substrate. EGF- and TGF-alpha-induced (0.1-10 nM) 1-hydroxylation of 3H-25OHD3 was concentration-dependent but showed different kinetics. Synthesis of calcitriol induced by EGF was inversely related to the degree of cellular confluence. Stimulation by EGF was an actinomycin D- and cycloheximide-sensitive process. Independently of the growth factor used, the production of 3H-24R,25(OH)2D3 and the catabolism of 3H-1,25(OH)2D3 to 3H-1,24,25(OH)3D3 were unexpectedly low (< or = 5% and < or = 2%/), as compared to the amount of calcitriol generated. Exogenous addition of unlabeled 1,25(OH)2D3, 1,24R(OH)2D3, calcipotriol, or 24R,25(OH)2D3 at concentrations as low as 10(-11) M, potently inhibited the 3H-1,25(OH)2D3 production. These results suggest that EGF-treated HaCaT keratinocytes could serve for further studies of the vitamin D3 pathway and its relationship to proliferation and differentiation, but differences in calcitriol synthesis and catabolism from those in cultured primary keratinocytes or other cell lines must be considered.