GC-rich sequences in the 5-lipoxygenase gene promoter are required for expression in Mono Mac 6 cells, characterization of a novel Sp1 binding site

Biochim Biophys Acta. 2005 Dec 30;1738(1-3):37-47. doi: 10.1016/j.bbalip.2005.11.008. Epub 2005 Dec 27.

Abstract

5-lipoxygenase (5LO) catalyzes formation of leukotrienes, mediators with roles in several inflammatory disorders, including atherosclerosis. The human 5LO gene promoter contain multiple GC-boxes. The relevance of these for expression of 5LO in the human monocytic cell line Mono Mac 6 (MM6) was studied. A downregulating effect of the GC-box binding compound mithramycin indicated that GC-rich sequences in the 5LO gene promoter are important for expression of native 5LO. In DNase I footprinting, mithramycin and Sp1 protected known GC-boxes, but also a novel Sp1 binding site was found, comprising 20 bp upstream of the major transcription initiation site, beside an Initiator-like sequence. Mutation of this site reduced Sp1 binding and expression of reporter genes in MM6 cells, compatible with a function as basal promoter element for the TATA-less 5LO gene. When differentiation was induced by TGFbeta and vitamin D(3), 5LO expression became prominent, but expression levels of Sp1/3 and Egr-1 were the same as for control cells. Also, 5LO reporter gene activity in transiently transfected MM6 cells was insensitive to differentiation. Thus, the GC-rich part of the 5LO gene promoter, including a novel Sp1 site, appear important for basal (rather than upregulated) transcription of 5LO in MM6 cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arachidonate 5-Lipoxygenase / genetics*
  • Arachidonate 5-Lipoxygenase / metabolism
  • Binding Sites
  • Cell Differentiation / drug effects
  • Cell Differentiation / genetics
  • Cell Line
  • Early Growth Response Protein 1 / genetics
  • Early Growth Response Protein 1 / metabolism
  • Electrophoretic Mobility Shift Assay
  • GC Rich Sequence*
  • Gene Expression Regulation
  • Gene Expression Regulation, Enzymologic / drug effects
  • Humans
  • Monocytes / cytology
  • Monocytes / drug effects
  • Monocytes / physiology*
  • Plicamycin / pharmacology
  • Promoter Regions, Genetic / genetics*
  • Sp1 Transcription Factor / genetics
  • Sp1 Transcription Factor / metabolism*
  • Sp3 Transcription Factor / genetics
  • Sp3 Transcription Factor / metabolism
  • Transcription Initiation Site

Substances

  • Early Growth Response Protein 1
  • SP3 protein, human
  • Sp1 Transcription Factor
  • Sp3 Transcription Factor
  • Arachidonate 5-Lipoxygenase
  • Plicamycin