Heterogeneous sphingosine-1-phosphate lyase gene expression and its regulatory mechanism in human lung cancer cell lines
Heterogeneous sphingosine-1-phosphate lyase gene expression and its regulatory mechanism in human lung cancer cell lines
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人肺癌细胞系中异质1-磷酸鞘氨醇裂解酶基因表达及其调控机制
DOI：
10.1016/j.bbalip.2010.12.005
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发表时间：
2011
期刊：
影响因子：
--
通讯作者：
Murate T.
中科院分区：
文献类型：
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作者：
Ito H.;Yoshida K.;Murakami M.;Hagiwara K.;Sasaki N.;Kobayashi M.;Takagi A.;Kojima T.;Sobue S.;Suzuki M.;Tamiya-Koizumi K.;Nakamura M.;Banno Y.;Nozawa Y.;Murate T.
鞘脂代谢途径在决定细胞命运中的作用已被公认。虽然鞘脂降解已被广泛研究，但催化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，与具有低SPL活性的H1299相比，在具有高SPL活性的H1155中观察到更高的细胞S1 P。据报道，加塔-4在盘基网柄藻中影响SPL转录。在H1155中观察到加塔-4，但在其它细胞系中未观察到。在H1299中过表达加塔-4增加SPL表达。然而，人类SPL的启动子分析显示，最重要的区域位于第一外显子的-136 bp和-88 bp之间，其中存在2个Sp1位点，但没有加塔位点。H1155的DNA pull-down分析显示，与H1299相比，该启动子区域内Sp1和加塔-4的DNA结合增加。电泳迁移率变动分析（EMSA）、染色质免疫沉淀分析（ChIP）、突变结合基序的报告基因分析以及特异性Sp1抑制剂光辉霉素A（Mithramycin A）的检测结果表明，Sp1在SPL转录中起主要作用，而加塔-4与该5′启动子区没有直接结合。通过使用GST-Sp1和过表达的加塔-4显示Sp1和加塔-4的共免疫沉淀，证明了加塔-4的协同作用。因此，H1155细胞SPL的高转录受Sp1和加塔-4/Sp1复合物的形成的调节，这两者都结合到5′-SPL启动子的Sp1位点。
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.