TRANSCRIPTIONAL ACTIVATION BY 1,25-DIHYDROXYVITAMIN-D-3 AND SYNTHETICVITAMIN-D-3 ANALOGS IN TRANSFECTED CULTURES OF HUMAN KERATINOCYTES

Analogues of vitamin Dg have recently been introduced for the topical treatment of psoriasis. Their therapeutic effects are thought to be me diated by interaction with the vitamin D-3 receptor (VDR) in epidermal keratinocytes (KCs). The purpose of the present study was to investig ate the trans-acting activity of the endogenous VDR in human KCs trans fected with a vitamin D response element (VDRE) in response to 1,25-di hydroxyvitamin D-3 [1,25(OH)(2)D-3] and the synthetic vitamin D-3 anal ogues GS 1500, EB 1213, MC 903 (calcipotriol) and KH 1060. Cultured KC s obtained from normal human adults were transfected with a VDRE consi sting of a direct repeat (DR) of 2 hexanucleotides separated by 3 nucl eotides (AGGTCAaggAGGTCA) cloned as a triple copy into the chloramphen icol acetyltransferase (CAT) reporter plasmid pBLCAT2. This DR3 respon se element is preferentially activated by heterodimers of the VDR and the retinoid X receptor. Twenty-four hours after transfection, 1,25(OH )(2)D-3, vitamin D-3 analogues or 9-cis-retinoic acid (9-cis-RA) were added, and, after an additional 24 h, cells were harvested and assayed for CAT activity. 1,25(OH)(2)D-3 dose-dependently induced CAT activit y in VDRE-transfected KCs and co-transfection with exogenous human VDR enhanced the response to 1,25(OH)(2)D-3. Induction of CAT activity by 1,25(OH) D-2(3) was enhanced in the presence of the endogenous ligand for retinoid X receptor, 9-cis-RA. The synthetic vitamin D-3 analogue s dose-dependently stimulated CAT activity. Compared to 1,25(OH)(2)D-3 , KCs were less sensitive to stimulation with MC 903? equally sensitiv e to EB 1213 and more sensitive to GS 1500 and KH 1060. In conclusion, the endogenous VDR in KCs is responsive to 1,25(OH)(2)D-3 and its syn thetic analogues in stimulating gene transcription. To the extent that the biological actions of vitamin Dg are dependent on its ability to induce gene transcription through the endogenous VDR, this transfectio n model may be used in the screening of novel vitamin Dg analogues for biological activity.