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.

1-磷酸鞘氨醇 (S1P) 是一种合成代谢因子。集落刺激因子 1 (CSF1) 诱导体内骨骼和体外破骨细胞中 S1P 合成所需的限速酶、鞘氨醇激酶 1 (SPHK1) 的表达。为了研究 CSF1 诱导 SPHK1 基因表达的机制，小鼠 SPHK1 基因的 2608 bp 片段（相对于转录起始位点为 − 2497 至 + 111 bp）被克隆并转染至 pZen 细胞（经过工程改造的小鼠成纤维细胞，可表达 c-调频管理系统）。使用双荧光素酶报告基因测定系统评估 SPHK1 启动子活性。通过分析一系列 5' 缺失，在 − 1250 至 − 1016 bp 区域中鉴定出 CSF1 响应区域。为了定义该片段中假定的 DNA 结合位点，生成了两个与该区域完全重叠的生物素标记片段，一个长度为 163 bp（− 1301 至 − 1139），另一个长度为 169 bp（− 1157 至 − 989） ）。 EMSAs 揭示了 163 bp 片段作为蛋白质结合的靶标。使用 EMSA，核蛋白结合区域进一步缩小为 85 bp 片段（− 1223 至 − 1139）。使用一系列未标记的 DNA 序列作为 EMSA 中的“冷竞争者”，22 bp 的序列被鉴定为可以成功竞争蛋白质结合的最小片段。相同的 22 bp 序列还使用从原代鼠破骨细胞中分离的核蛋白在 EMSA 中竞争 DNA 结合。随后在pZen细胞中表达全长野生型SPHK1启动子和ATGGGGGG基序突变的SPHK1启动子。突变这个 ATGGGGG 基序几乎完全消除了 CSF1 激活启动子的能力。 尽管据报道两种转录因子 KLF6 和 Sp1 与该序列结合，但 supershift EMSAs 未能检测到与 85 bp DNA 片段结合的蛋白质中的任何一种。