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Involucrin is a major protein of the cornified envelope of keratinocytes that provides much of the structural integrity of skin. Its expression is stimulated by a number of agents including calcium and 1,25-dihydroxy-vitamin D3 that promote the differentiation process in keratinocytes. Within the distal regulatory region of the involucrin promoter lies an AP-1 site and an element homologous to other vitamin D response elements. In previous studies mutation of the AP-1 site was found to reduce basal activity and block calcium stimulation of the involucrin promoter, whereas the vitamin D response element was not critical for calcium regulation. In this study both elements proved to be important for 1,25-dihydroxyvitamin D3 stimulation of the involucrin promoter. Mutation of the AP-1 site reduced basal activity and blocked 1,25-dihydroxyvitamin D3 stimulation of the involucrin promoter. In contrast, mutation of the vitamin D response element did not reduce basal expression of the involucrin promoter or prevent calcium stimulation of involucrin gene expression, but blocked 1,25-dihydroxyvitamin D3 stimulation. The vitamin D response element from the involucrin gene bound the vitamin D receptor and the retinoid X receptor, but not the retinoic acid receptor, in a specific manner. We conclude that the AP-1 site and the vitamin D response element in the involucrin promoter play important roles in mediating the action of 1,25-dihydroxyvitamin D3 on involucrin expression, but the vitamin D response element provides specificity for the 1,25-dihydroxyvitamin D3 response lacking at the AP-1 site.

Squamous cell carcinomas (SCC) derived from human epidermis fail to differentiate normally under the influence of 1,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 [ despite the presence of the vitamin D receptor. Previous studies from our laboratory showed that phospholipase C-γ1 (PLC-γ1) was upregulated transcriptionally by 1,25(OH) 2 D 3 in normal human keratinocytes, and a vitamin D responsive element (VDRE) in its promoter region has been identified. To examine the inducibility of human PLC-γ1 transcription by 1,25(OH) 2 D 3 and/or retinoic acid in SCC cell lines, we transiently transfected SCC4 and SCC12B2 cells with human PLC-γ1 promoter-luciferase constructs containing the VDRE and tested the response of these constructs to 1,25(OH) 2 D 3 and/or all-trans retinoic acid. The induction of the human PLC-γ1 VDRE by 1,25(OH) 2 D 3 was synergistic with all-trans retinoic acid in normal human keratinocytes, but none of the constructs was induced by 1,25(OH) 2 D 3 and/or all-trans retinoic acid in SCC4 and SCC12B2 cells. In contrast, the construct containing the VDRE of the human 24-hydroxylase gene was induced several fold by 1,25(OH) 2 D 3 in normal human keratinocytes and by both 1,25(OH) 2 D 3 and all-trans retinoic acid in SCC4 and SCC12B2 cells. DNA mobility shift assays showed that both the vitamin D receptor and the retinoic acid receptor in SCC4 and SCC12B2 cells bound the human PLC-γ1 VDRE similarly to that seen in normal keratinocytes. The data indicate that the VDRE in the human PLC-γ1 gene is not functional in SCC4 and SCC12B2 cells, unlike normal human keratinocytes, even though vitamin D receptors bind normally to it. Failure of transcriptional control of the PLC-γ1 gene by 1,25(OH) 2 D 3 suggests the lack of a cofactor(s) linking the VDRE to the transcriptional machinery.