Sclerostin, the SOST gene product, is a negative regulator of bone formation and a positive regulator of bone resorption. In a screen to identify novel regulators of SOST expression, we found that treatment of human primary osteoblasts with 1α,25-dihydroxyvitaminD3 (1,25D) resulted in increased expression of SOST mRNA and sclerostin protein. This effect was also evident in the human osteosarcoma/osteocyte-like cell line SAOS2. Effects on SOST mRNA levels occurred as early as 3 hours post-stimulation, consistent with a direct effect of 1,25D on the SOST promoter. Sequence analysis of the published human SOST gene revealed a single putative vitamin D response element (VDRE) upstream of the transcription start site (TSS). Cloning of this sequence into a luciferase reporter construct upstream of the constitutive thymidine kinase (TK) promoter, and transfection into HEK-293T cells, identified the presence of a 1,25D responsive element with activity equivalent to that of the well characterised mouse osteopontin VDRE. Electrophoretic mobility shift analysis (EMSA) of HEK-293T nuclear extracts revealed a 1,25D dependent gel-shift, consistent with binding of the VDR/RXR heterodimeric complex. Sequence substitution in the VDR/RXR half-sites abolished VDRE reporter activity and binding of nuclear proteins. In addition, transient expression of a 6.3 kb fragment of the proximal SOST promoter ahead of the TSS in a luciferase expression vector demonstrated a promoter responsive to 1,25D. However, addition of a known bone specific enhancer region ECR5 ahead of the cloned promoter fragment did not increase the level of responsiveness to 1,25D. Mutation or deletion of the predicted VDRE resulted in the 6.3 kb SOST promoter being unresponsive to 1,25D. We conclude that 1,25D is a direct regulator of SOST gene expression, extending the pathways of control of sclerostin expression.
Disclosure: The authors declared no competing interests. This work was supported by the National Health and Medical Research Council of Australia (grant numbers APP1004871, APP1047796).