Cell-specific Transcription of Leukotriene C4Synthase Involves a Kruppel-like Transcription Factor and Sp1

Abstract

Leukotriene C 4 synthase (LTC 4 S) is responsible for the biosynthesis of cysteinyl leukotrienes that participate in allergic and asthmatic inflammation. We analyzed 2.1 kilobases of the 5-flanking region of the human LTC 4 S gene, which contains three DNase I hypersensitivity sites, for its transcriptional activity when fused to a promoterless and enhancerless luciferase gene. Deletion analysis revealed a nonspecific basal promoter region between nucleotides ؊122 and ؊56 upstream of the translation start site which contains a consensus Sp1 binding site and a putative initiator element (Inr) and cell-specific enhancer regions further upstream. A single mutation of either the Sp1 binding site between nucleotides ؊120 and ؊115 or the Inr (CA-GAC) between nucleotides ؊66 and ؊62 reduced the expression of the reporter gene by ϳ60%, whereas double mutations decreased the expression by ϳ80%. The incubation of nuclear extracts from THP-1 and K562 cells with a 32 P-labeled oligonucleotide containing the Sp1 site or the Inr sequence gave gel-shifted complexes that were blocked by their respective cold oligonucleotides, and antisera specific for Sp1 and Sp3 provided supershifts for the former. Linker-scanning mutations of a cell-specific regulatory region revealed that mutations from nucleotides ؊165 to ؊125 reduced reporter activity. This region contains a tandem CACCC repeat (at nucleotides ؊149 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 by antisera to Sp1 and Sp3. Cotransfection of an Sp1 expression plasmid into Drosophila SL2 cells with a ؊228 to ؊3 LTC 4 S reporter construct transactivated the reporter gene, whereas mutations at the CACCC repeat region reduced Sp1 transactivation by ϳ66%. 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 the involvement of Sp1 and an Inr in noncell-specific regulation and a Kruppel-like transcription factor and Sp1 in the cell-specific regulation of the LTC 4 S gene. These are the first such analyses of a member of a newly recognized superfamily of membraneassociated proteins involved in eicosanoid and glutathione metabolism, which contains key proteins involved in the generation of both prostanoids and cysteinyl leukotrienes.LTC 4 synthase (LTC 4 S) 1 catalyzes the conjugation of leukotriene A 4 and glutathione (GSH) to form LTC 4 . LTC 4 is implicated in the pathobiology of bronchial asthma by the efficacy of inhibitors of its biosynthesis and of receptor blockers for its constrictor metabolites in the treatment of this condition. LTC 4 S is an 18-kDa perinuclear membrane protein that functions as a dimer (1, 2) and is expressed predominantly in mast cells, basophils, and eosinophils (3-5). LTC 4 S was the first catalytic protein identified in a newly recognized superfamily of membrane-associated proteins involved in eicosanoid and GSH metabolism (M...

Transcriptional activation of endoglin and transforming growth factor-β signaling components by cooperative interaction between Sp1 and KLF6: their potential role in the response to vascular injury

“…40 Similarly, KLF6 may cooperate with other coactivators in binding to the proximal GC box of the leukotriene C 4 synthase promoter. 60 Neither of these other studies, however, has provided evidence of either physical interaction or coimmunoprecipitation in nuclear extracts, as we demonstrate here.Our data suggest a model whereby Sp1 and KLF6 have similar DNA-binding properties but different biologic roles in vascular injury. Both proteins may potentially bind DNA at the same Sp1 consensus in the endoglin promoter, CCCAGCCC (Ϫ37/Ϫ29).…”

“…40 Similarly, KLF6 may cooperate with other coactivators in binding to the proximal GC box of the leukotriene C 4 synthase promoter. 60 Neither of these other studies, however, has provided evidence of either physical interaction or coimmunoprecipitation in nuclear extracts, as we demonstrate here.…”

The Kruppel-like Factor Zf9 and Proteins in the Sp1 Family Regulate the Expression of HSP47, a Collagen-specific Molecular Chaperone

“…Cooperative but distinctive functions between members of the two families have been shown in various cases in addition to the activation of the COL1A1 gene and TGF-␤ and TGF-␤ receptor genes (25,26). The core promoter of leukotriene C4 synthase, which catalyzes the conjugation of glutathione with leukotriene A4 to form leukotriene C4, is composed of the CACCC motif and a downstream GC box (42). The basal expression of leukotriene C4 synthase is regulated by Sp1 bound to the GC box, and cell type-specific expression in THP-1 cells is regulated by both Sp1 and Zf9 bound to the CACCC motif.…”

A new role for the Krüppel-like transcription factor KLF6 as an inhibitor of c-Jun proto-oncoprotein function

“…Interestingly, several independent reports described mutations or downregulation of KLF6 gene in human malignancies such as in prostate cancer, astrocytic gliomas, colorectal cancer and non-small-cell lung cancer, thus KLF6 has been proposed as a potential candidate tumor suppressor gene (Narla et al, 2001;Chen et al, 2003;Jeng and Hsu, 2003;Reeves et al, 2004;Ito et al, 2004). Additionally, KLF6 regulates the transcription of several genes such as those encoding the pregnancyspecific glycoprotein 5 (PSG) (Koritschoner et al, 1997), transforming growth factor (TGF)-b1, types I and II TGF-b receptors (Kim et al, 1998), leukotriene C 4 synthase (LTC 4 ) (Zhao et al, 2000), urokinase plasminogen activator (uPA) (Kim et al, 1998) and inducible Nitric Oxide Synthase (Warke et al, 2003), most of which are implicated in proliferation and development of the tissues where they are expressed.…”