title: Heterogeneous sphingosine-1-phosphate lyase gene expression and its regulatory mechanism in human lung cancer cell lines.
abstract: 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.
authors.affiliation: Department of Medical Technology, Nagoya University Graduate School of Health Sciences, Nagoya, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Ito
authors.forename: Kayo
authors.initials: K
authors.lastname: Yoshida
authors.forename: Masashi
authors.initials: M
authors.lastname: Murakami
authors.forename: Kazumi
authors.initials: K
authors.lastname: Hagiwara
authors.forename: Noriko
authors.initials: N
authors.lastname: Sasaki
authors.forename: Misa
authors.initials: M
authors.lastname: Kobayashi
authors.forename: Akira
authors.initials: A
authors.lastname: Takagi
authors.forename: Tetsuhito
authors.initials: T
authors.lastname: Kojima
authors.forename: Sayaka
authors.initials: S
authors.lastname: Sobue
authors.forename: Motoshi
authors.initials: M
authors.lastname: Suzuki
authors.forename: Keiko
authors.initials: K
authors.lastname: Tamiya-Koizumi
authors.forename: Mitsuhiro
authors.initials: M
authors.lastname: Nakamura
authors.forename: Yoshiko
authors.initials: Y
authors.lastname: Banno
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Nozawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Murate
mesh_terms: D000446:Aldehyde-Lyases / Q000096:biosynthesis* / Q000235:genetics; D045744:Cell Line, Tumor; D004023:Dictyostelium / Q000235:genetics; D050991:GATA4 Transcription Factor / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008175:Lung Neoplasms / Q000201:enzymology* / Q000235:genetics; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000235:genetics; D020218:Response Elements; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D050991:GATA4 Transcription Factor; C484170:GATA4 protein, human; D009363:Neoplasm Proteins; D015800:Protozoan Proteins; D016329:Sp1 Transcription Factor; D000446:Aldehyde-Lyases; C022649:sphingosine 1-phosphate lyase (aldolase)