Vitamin D represses retinoic acid-dependent transactivation of the retinoic acid receptor-beta 2 promoter: The AF-2 domain of the vitamin D receptor is required for transrepression

Retinoic acid (RA)-dependent activation of the RA receptor beta 2 (RAR beta 2) gene in embryonal carcinoma cells is mediated by binding of retinoid re ceptor heterodimers (RAR/RXR) to a RE response element (RARE) located close ly to the TATA box. We have analyzed the effect of vitamin D on the respons e of the RAR beta 2 promoter to RA in pituitary GH4C1 cells that coexpress receptors for retinoids and vitamin D. Incubation with vitamin D markedly r educed the response to RA caused by transcriptional interference of the vit amin D receptor (VDR) on the RARE. This DNA element binds VDR/RXR heterodim ers with high affinity, and these inactive heterodimers can displace active RAR/RXR from the RARE. Overexpression of RXR in GH4C1 cells, as well as in cubation with BMS649 (a RXR-specific ligand), increased the inhibitory effe ct of vitamin D, suggesting that the VDR/RXR heterodimer is the repressive species and that titration of RXR is not responsible for this inhibition. A lthough DNA binding could be required for full potency of the inhibitory ac tivity of VDR, it is not absolutely required because a truncated receptor ( VDR Delta 1-111), lacking the DNA binding domain, also displays repressor a ctivity. Furthermore, the ability to mediate transrepression by vitamin D w as strongly decreased when a mutant VDR in which the last 12 C-terminal ami noacids have been deleted (VDR Delta AF-2) was used. Because this region co ntains the domain responsible for ligand-dependent recruitment of coactivat ors, titration of common coactivators for VDR and RAR could be involved in the inhibitory effect of vitamin D. In agreement with this hypothesis, over expression of E1A, which can act as a RAR beta 2 promoter-specific coactiva tor, significantly reversed repression by vitamin D.