HACAT CELL-LINE AS A MODEL SYSTEM FOR VITAMIN-D-3 METABOLISM IN HUMANSKIN

Synthesis and catabolism of calcitriol (1,25(OH)(2)D-3) were studied u sing HaCaT cell line as a cell culture model, Our results indicate tha t stimulation of HaCaT cells with epidermal growth factor (EGF) or tra nsforming growth factor-alpha (TGF-alpha) within 16 h just prior to re aching confluence amplified the production of calcitriol when calcidio l (H-3-25OHD(3)) was used as a substrate. EGF- and TGF-alpha-induced ( 0.1-10 nM) 1-hydroxylation of H-3-25OHD(3) was concentration-dependent but showed different kinetics, Synthesis of calcitriol induced by EGF was inversely related to the degree of cellular confluence. Stimulati on by EGF was an actinomycin D- and cycloheximide-sensitive process. I ndependently of the growth factor used, the production of H-3-24R,25(O H)(2)D-3 and the catabolism of H-3-1,25(OH)(2)D-3 to H-3-1,24,25(OH)(3 )D-3 were unexpectedly low (less than or equal to 5% and less than or equal to 2%, as compared to the amount of calcitriol generated. Exogen ous addition of unlabeled 1,25(OH)(2)D-3, 1,24R(OH)(2)D-3, calcipotrio l, or 24R,25(OH)(2)D-3 at concentrations as low as 10(-11) M, potently inhibited the H-3-1,25(OH)(2)D-3 production. These results suggest th at EGF-treated HaCaT keratinocytes could serve for further studies of the vitamin D-3 pathway and its relationship to proliferation and diff erentiation, but differences in calcitriol synthesis and catabolism fr om those in cultured primary keratinocytes or other cell Lines must be considered.