Cell-specific transcription of leukotriene C-4 synthase involves a Kruppellike transcription factor and Sp1

Leukotriene C-4 synthase (LTC4S) is responsible for the biosynthesis of cys teinyl leukotrienes that participate in allergic and asthmatic inflammation . We analyzed 2.1 kilobases of the 5'-flanking region of the human LTC4S ge ne, which contains three DNase I hypersensitivity sites, for its transcript ional activity when fused to a promoterless and enhancerless luciferase gen e. Deletion analysis revealed a nonspecific basal promoter region between n ucleotides -122 and -56 upstream of the translation start site which contai ns a consensus Spl binding site and a putative initiator element (Inr) and cell-specific enhancer regions further upstream. A single mutation of eithe r the Spl binding site between nucleotides -120 and -115 or the Inr (CAGAC) between nucleotides -66 and -62 reduced the expression of the reporter gen e by similar to 60%, whereas double mutations decreased the expression by s imilar to 80%. The incubation of nuclear extracts from THP-1 and K562 cells with a P-32-labeled oligonucleotide containing the Spl site or the Inr seq uence gave gel-shifted complexes that mere blocked by their respective cold oligonueleotides, and antisera specific for Spl and Sp3 provided supershif ts for the former. Linker-scanning mutations of a cell-specific regulatory region revealed that mutations from nucleotides -165 to -125 reduced report er activity. This region contains a tandem CACCC repeat (at nucleotides -14 9 to -145 and -139 to -135), An oligonucleotide containing the distal CACCC motif was gel shifted by THP-1 cell nuclear extract and was supershifted b y antisera to Spl and Sp3, Cotransfection of an Spl expression plasmid into Drosophila SL2 cells with a -228 to -3 LTC4S reporter construct transactiv ated the reporter gene, whereas mutations at the CACCC repeat region reduce d Spl transactivation by similar to 66%. Similarly, the Kruppel-like factor Zf9/CPBP (core promoter-binding protein) transactivated the -228 construct in COS cells but not its CACCC mutant construct. These findings indicate t he involvement of Spl and an Inr in non-cell-specific regulation and a Krup pel-like transcription factor and Spl in the cell-specific regulation of th e LTC4S gene. These are the first such analyses of a member of a newly reco gnized superfamily of membrane-associated proteins involved in eicosanoid a nd glutathione metabolism, which contains key proteins involved in the gene ration of both prostanoids and cysteinyl leukotrienes.