A novel interaction between insulin-like growth factor binding protein-6 and the vitamin D receptor inhibits the role of vitamin D3 in osteoblast differentiation.

Insulin-like growth factor binding protein-6 (IGFBP-6) is a secreted glycoprotein that reduces the bioavailability of IGFs. It has both IGF-dependent and -independent effects on cell growth, however the mechanisms responsible for its IGF-independent actions of IGFBP-6 are not fully understood. In previous studies, we have shown that recombinant IGFBP-6 can be internalized and translocated to the nucleus. The present study shows that IGFBP-6 interacts with the vitamin D receptor (VDR). Physical interactions between IGFBP-6 and the VDR were confirmed by GST pulldown and co-immunoprecipitation assays. We also determined that the interaction binding sites were on the C-terminal region of the VDR. This interaction can influence retinoid X receptor (RXR):VDR heterodimerization. Furthermore, immunofluorescence colocalization studies showed that IGFBP-6 colocalized with the VDR predominantly in the cell's nucleus. Inductions of osteocalcin and growth hormone promoter activities by 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) were significantly decreased when cells were co-transfected with IGFBP-6 and the VDR compared with cells transfected with the VDR only. Moreover, we found that alkaline phosphatase activity (ALP, a general marker of osteoblast differentiation) was significantly decreased in osteoblast-like cells when they were transfected with IGFBP-6 in the presence of 1,25(OH)(2)D(3). No obvious difference in ALP activity was observed when cells were transfected with IGFBP-6 and endogenous VDR was knocked down by siRNA. These results demonstrate that IGFBP-6 inhibits osteoblastic differentiation mediated by 1,25(OH)(2)D(3) and the VDR through interacting with the VDR and inhibiting its function. This is a novel mechanism for IGFBP-6.