Cell-specific transcription of leukotriene C(4) synthase involves a Kruppel-like transcription factor and Sp1

J Biol Chem. 2000 Mar 24;275(12):8903-10. doi: 10.1074/jbc.275.12.8903.


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 (CAGAC) between nucleotides -66 and -62 reduced the expression of the reporter gene by approximately 60%, whereas double mutations decreased the expression by approximately 80%. 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 approximately 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 the involvement of Sp1 and an Inr in non-cell-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 membrane-associated proteins involved in eicosanoid and glutathione metabolism, which contains key proteins involved in the generation of both prostanoids and cysteinyl leukotrienes.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • DNA-Binding Proteins / metabolism*
  • Deoxyribonuclease I / metabolism
  • Genes, Reporter
  • Glutathione Transferase / genetics*
  • Humans
  • Kruppel-Like Factor 6
  • Kruppel-Like Transcription Factors
  • Molecular Sequence Data
  • Mutagenesis
  • Promoter Regions, Genetic
  • Protein Binding
  • Proto-Oncogene Proteins*
  • Repressor Proteins*
  • Sequence Deletion
  • Sp1 Transcription Factor / metabolism*
  • Trans-Activators / metabolism*
  • Transcription Factors / metabolism*
  • Transcription, Genetic
  • Transcriptional Activation*


  • DNA-Binding Proteins
  • KLF6 protein, human
  • Kruppel-Like Factor 6
  • Kruppel-Like Transcription Factors
  • Proto-Oncogene Proteins
  • Repressor Proteins
  • Sp1 Transcription Factor
  • Trans-Activators
  • Transcription Factors
  • Glutathione Transferase
  • Deoxyribonuclease I
  • leukotriene-C4 synthase