1α,25-dihydroxyvitamin D3 induced differentiation of cultured human keratinocytes is accompanied by a PKC-independent regulation of AP-1 DNA binding activity

Abstract

1α,25(OH)2D3 and its analogs are potent mediators of keratinocyte differentiation in vitro. The precise mechanism of this action is still unknown. The nuclear transcription factor activator protein I seems to play an important role in keratinocyte differentiation. The purpose of this study was to investigate the effect of 1α,25(OH)2D3 on activator protein 1 DNA binding activity in cultured human keratinocytes. In a time-course study of human keratinocytes incubated with 1α,25(OH)2D3 (10-7-10-11 M) a significant dose-dependent increase in activator protein 1 DNA binding activity as determined by electrophoretic mobility shift assay was seen after 36 h. This increase was followed by a significant dose-dependent decrease in activator protein 1 DNA binding activity after 72 h. When differentiation was induced by raising the calcium concentration in the culture medium from 0.09 to 0.3 mM a similar increase in activator protein 1 DNA binding activity was observed after incubation for 48 h. Pharmacologic down-modulation of the protein kinase C activity with GF 109203X reversed the calcium-induced increase in activator protein 1 DNA binding activity and abolished keratinocyte differentiation as determined by a transglutaminase assay. In contrast, activator protein 1 DNA binding activity and keratinocyte differentiation were not affected when protein kinase C activity was down- modulated in the experiments with 1α,25(OH)2D3. The activator protein I complex in human keratinocytes consists of dimers of Fra-1, Fra-2, c-Jun, JunD, and c-Fos. Our results demonstrate that 1α,25(OH)2D3- and calcium- induced keratinocyte differentiation are accompanied by changes in activator protein 1 DNA binding activity. Protein kinase C activation appears to be essential for the calcium-dependent induction of keratinocyte differentiation, whereas a protein-kinase-C-independent activation of activator protein 1 DNA binding and keratinocyte differentiation is responsible for the 1α,25(OH)2D3-induced effects.