Igfbp-6 And The Interaction And Function Of Vdr

Insulin-like growth factor binding protein (IGFBP)-6 is a member of a family containing six high affinity IGFBPs that regulate IGF-â…¡activity and also show IGF-independent effects. IGFBP-6 is unique among the IGFBPs for its high IGF-â…¡binding specificity. It can inhibit IGF-â…¡actions in vitro and in vivo by preventing IGF from binding to cell surface receptors. Although IGFBP-6'smajor action appears to be inhibiting IGF-â…¡activity, a number of studies suggest that it may also have IGF-independent actions, such as growth inhibition and apoptosis. In additon, many studies have suggest that IGFBP-6 may regulate osteoblast differentiation but the mechanisms are still unkown.The vitamin D receptor (VDR) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. It regulates the expression of target genes by binding to 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). The classic roles of VDR and its ligands are to stimulate calcium absorption in the intestines to maintain normal calcium levels and to indirectly regulate bone mineralization. Studies using mice that lack the VDR demonstrate its essential functions in bone formation andmetabolism. In growing animals, the primary role of the VDR is to maintain high levels of intestinal calcium absorption, and a loss of the VDR results in rickets. Transgenic mice that over-express the VDR in mature osteoblasts, which are the bone-forming cells that deposit bone extracellularmatrix have higher rates of bone formation. It has been reported that 1,25-(OH)2D3 promotes differentiation and maturation of the osteoblast. Treating osteoblasts with 1,25-(OH)2D3 results in inhibiting proliferation, upregulating osteoblast-associated genes, such as osteocalcin and osteopontin, and stimulating calcium accumulation.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 ite 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 D3 (1,25(OH)2D3) were significantly decreased when cells were co-transfected with IGFBP-6 and the VDR compared with the 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)2D3. 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)2D3 and the VDR through its interacting with the VDR. This is a novel mechanism for IGF-independent effects of IGFBP-6.