Sphingosine-1-phosphate (S1P) is an anabolic clastokine. Colony Stimulating Factor 1 (CSF1) induces expression of the rate limiting enzyme required for S1P synthesis, sphingosine kinase 1 (SPHK1) in bone in vivo, and in osteoclasts in vitro. To study the mechanism of CSF1-induced SPHK1 gene expression, a 2608 bp fragment of the murine SPHK1 gene (- 2497 to + 111 bp relative to the transcription start site) was cloned and transfected into pZen cells (murine fibroblasts engineered to express c-fms). SPHK1 promoter activity was assessed using a dual-luciferase reporter assay system. By analyzing a series of 5'-deletions, a CSF1-responsive region was identified in the region - 1250 to - 1016 bp. To define putative DNA binding site(s) in this fragment, two biotin-labeled fragments that completely overlapped this region were generated, one 163 bp in length (- 1301 to - 1139) and one 169 bp in length (- 1157 to - 989). EMSAs revealed the 163 bp fragment as the target for protein binding. Using EMSAs, the nuclear protein binding region was further narrowed to an 85 bp fragment, (- 1223 to - 1139). Using a series of unlabeled DNA sequences as "cold competitors" in EMSAs, a 22 bp sequence is identified as the smallest fragment that could successfully compete away protein binding. The same 22 bp sequence also competed DNA binding in EMSAs using nuclear protein isolated from primary murine osteoclasts. A full-length wild-type SPHK1 promoter and an SPHK1 promoter in which the ATGGGGG motif was mutated were subsequently expressed in pZen cells. Mutating this ATGGGGG motif nearly completely abrogated the ability of CSF1 to activate the promoter. Although two transcription factors, KLF6 and Sp1 have been reported to bind to this sequence, supershift EMSAs failed to detect either among the proteins bound to the 85 bp DNA fragment.
Keywords: Bone formation; CSF1; Osteoclasts; SPHK1; Transcription factor.