Ito Hiromi, Yoshida Kayo, Murakami Masashi, Hagiwara Kazumi, Sasaki Noriko, Kobayashi Misa, Takagi Akira, Kojima Tetsuhito, Sobue Sayaka, Suzuki Motoshi, Tamiya-Koizumi Keiko, Nakamura Mitsuhiro, Banno Yoshiko, Nozawa Yoshinori, Murate Takashi
Department of Medical Technology, Nagoya University Graduate School of Health Sciences, Nagoya, Japan.
Biochim Biophys Acta. 2011 Mar;1811(3):119-28. doi: 10.1016/j.bbalip.2010.12.005. Epub 2010 Dec 22.
The role of sphingolipid metabolic pathway has been recognized in determining cellular fate. Although sphingolipid degradation has been extensively studied, gene expression of human sphingosine 1-phosphate lyase (SPL) catalyzing sphingosine 1-phosphate (S1P) remains to be determined. Among 5 human lung cancer cell lines examined, SPL protein levels paralleled the respective mRNA and enzyme activities. Between H1155 and H1299 cells used for further experiments, higher cellular S1P was observed in H1155 with higher SPL activity compared with H1299 with low SPL activity. GATA-4 has been reported to affect SPL transcription in Dictyostelium discoideum. GATA-4 was observed in H1155 but not in other cell lines. Overexpression of GATA-4 in H1299 increased SPL expression. However, promoter analysis of human SPL revealed that the most important region was located between -136bp and -88bp from the first exon, where 2 Sp1 sites exist but no GATA site. DNA pull-down assay of H1155 showed increased DNA binding of Sp1 and GATA-4 within this promoter region compared with H1299. Electrophoresis mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) assay, reporter assay using mutated binding motif, and mithramycin A, a specific Sp1 inhibitor, suggest the major role of Sp1 in SPL transcription and no direct binding of GATA-4 with this 5' promoter region. The collaborative role of GATA-4 was proved by showing coimmunoprecipitation of Sp1 and GATA-4 using GST-Sp1 and overexpressed GATA-4. Thus, high SPL transcription of H1155 cells was regulated by Sp1 and GATA-4/Sp1 complex formation, both of which bind to Sp1 sites of the 5'-SPL promoter.
鞘脂代谢途径在决定细胞命运方面的作用已得到认可。尽管鞘脂降解已得到广泛研究，但催化1-磷酸鞘氨醇（S1P）的人1-磷酸鞘氨醇裂解酶（SPL）的基因表达仍有待确定。在所检测的5种人肺癌细胞系中，SPL蛋白水平与各自的mRNA及酶活性平行。在用于进一步实验的H1155和H1299细胞之间，与SPL活性低的H1299相比，SPL活性高的H1155中观察到更高的细胞内S1P。据报道，GATA-4可影响盘基网柄菌中SPL的转录。在H1155中观察到GATA-4，但在其他细胞系中未观察到。H1299中GATA-4的过表达增加了SPL的表达。然而，人SPL的启动子分析显示，最重要的区域位于距第一个外显子-136bp至-88bp之间，此处存在2个Sp1位点但无GATA位点。与H1299相比，H1155的DNA下拉实验显示该启动子区域内Sp1和GATA-4的DNA结合增加。电泳迁移率变动分析（EMSA）、染色质免疫沉淀（ChIP）分析、使用突变结合基序的报告基因分析以及特异性Sp1抑制剂光神霉素A表明，Sp1在SPL转录中起主要作用，且GATA-4与该5'启动子区域无直接结合。使用GST-Sp1和过表达的GATA-4进行Sp1和GATA-4的共免疫沉淀，证明了GATA-4的协同作用。因此，H1155细胞中SPL的高转录受Sp1以及GATA-4/Sp1复合物形成的调节，二者均与5'-SPL启动子的Sp1位点结合。
