Involvement and abnormality of lysosphingolipid metabolic enzymes in malignant diseases

人鞘氨醇1-磷酸裂解酶(SPL)催化1-磷酸鞘氨醇(S1P)的基因表达尚不清楚。在检测的5个人肺癌细胞系中，SPL蛋白水平与各自的mRNA和酶活性平行。在用于进一步实验的H1155和H1299细胞之间，观察到具有较高SPL活性的H1155细胞S1P低于具有较低SPL活性的H1299细胞。前人的研究表明，GATA-4影响盘基网柄菌中SPL的转录。在H1155中观察到GATA-4，而在其他细胞系中未观察到GATA-4的表达。对人SPL基因的启动子分析表明，最重要的区域位于第一外显子的-136~-88 bp之间，其中存在2个Sp1位点，但没有GATA位点。凝胶迁移率改变分析(EMSA)、染色质免疫沉淀分析(CHIP)、结合基序突变的报告报告分析表明Sp1在SPL转录中起主要作用，GATA-4不与该5‘启动子区域直接结合。Sp1和GATA-4的免疫共沉淀证明了GATA-4的共生作用。因此，H1155细胞的高SPL转录受Sp1和GATA-4/Sp1复合体形成的调控，这两个复合体都与5‘-SPL启动子的Sp1位点结合。

The 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, lower cellular S1P was observed in H1155 with higher SPL activity compared with H1299 with lower SPL activity. Previous reprots by others have shown that GATA-4 affect SPL transcription in Dictyostelium discoideum. GATA-4 was observed in H1155 but not in other cell lines. Promoter analysis of human SPL revealed that the most important region was located between -136 bp and -88 bp from the first exon, where 2 Sp1 sites exist but no GATA site. Electrophoresis mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) assay, reporter assay using reporter with mutated binding motif suggest the major role of Sp1 in SPL transcription and no direct binding of GATA-4 with this 5' promoter region. The collive role of GATA-4 wasproved by showing co-immunoprecipitation of Sp1 and 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.