Involvement and abnormality of lysosphingolipid metabolic enzymes in malignant diseases

催化1-磷酸鞘氨醇（S1 P）的人1-磷酸鞘氨醇裂解酶（SPL）的基因表达仍有待确定。在检测的5种人肺癌细胞系中，SPL蛋白水平与各自的mRNA和酶活性相关。在用于进一步实验的H1155和H1299细胞之间，与具有较低SPL活性的H1299相比，在具有较高SPL活性的H1155中观察到较低的细胞S1 P。其他人的前期报道表明，加塔-4影响盘基网柄藻SPL的转录。在H1155中观察到加塔-4，但在其它细胞系中未观察到。对人SPL基因启动子序列的分析表明，SPL基因的最重要的启动子区域位于第一外显子的-136 ~-88 bp之间，存在2个Sp1位点，但没有加塔位点。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表明，Sp1在SPL转录中起主要作用，而加塔-4与该5'启动子区域没有直接结合。GATA-4与Sp1的免疫共沉淀证实了加塔-4的协同作用。因此，H1155细胞的SPL高转录受Sp1和加塔-4/Sp1复合物形成的调节，这两者都结合到5' -SPL启动子的Sp1位点。

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.