We investigated the role of Heat shock protein 90 (Hsp90) in vitamin D action in Caco-2 cells using geldanamycin (GA) to block Hsp90 function and RNA interference to reduce Hsp90 beta expression. When cells were exposed to GA, vitamin D-mediated gene expression and transcriptional activity were inhibited by 69% and 54%, respectively. Gel shift analysis indicated that GA reduced vitamin D-mediated DNA binding activity of the vitamin D receptor (VDR). We tested the specific role of Hsp90 beta by knocking down its expression with stably expressed short hairpin RNA. Vitamin D-induced gene expression and transcriptional activity were reduced by 90% and 80%, respectively, in Hsp90 beta-deficient cells. Nuclear protein for VDR and RXR alpha, its heterodimer partner, were not reduced in Hsp90 beta-deficient cells, These findings indicate that Hsp90 beta is needed for optimal vitamin D responsiveness in the enterocyte and demonstrate a specific role for Hsp90 beta in VDR signaling. (C) 2008 Elsevier Inc. All rights reserved.