Functional analysis of the lysyl oxidase promoter in myofibroblast-like clones of 3T6 fibroblast

C. Jourdan-Le Saux, C. Gleyzal, M. Raccurt, P. Sommer

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

Abstract

Lysyl oxidase (LO), an extracellular enzyme catalysing the first step of collagen and elastin cross-linking, is transiently expressed by myofibroblasts during fibrosis. A cell model with features of myofibroblast was thus established for studying the regulation of LO. Two clones of the 3T6 fibroblast cell line were selected because 1) they produced a relatively high steady-state level of the three lysyl oxidase mRNAs with the same relative ratio similar to fibrotic tissue and 2) they stably displayed certain features of myofibroblast (α-smooth muscle actin cytoskeleton, bundles of cytoskeletal filaments beneath the cytoplasmic membranes). These clones synthesized predominantly type I collagen fibers and a small amount of type II collagen. Neither type IV collagen nor elastin were observed. The cloning and sequencing of 2,073 bp of the mouse Balb/C LO promoter was performed, allowing the identification around the initiation of transcription of consensus sequences which are found on the COL1 promoters. A series of deletion constructs containing the LO 5'-flanking region ligated to the luciferase gene were transiently transfected into 3T6-5 fibroblasts. The region allowing the maximal activity was found between positions -416 to 192, while the more upstream region negatively regulated the promoter. The -898 to -865 sequence (called LOcol1) displayed 79% of homology with a conserved sequence of murine, rat, and human COL1A1 promoters. This sequence participated to the binding of several nuclear factors within a region (-970 to -784) allowing 50% of inhibition of the LO promoter. Therefore, the level of LO transcription is regulated in 3T6-5 fibroblast by positive and negative cis-acting regulatory elements which might have common features with the COL1A1 promoter.

Original languageEnglish (US)
Pages (from-to)328-341
Number of pages14
JournalJournal of Cellular Biochemistry
Volume64
Issue number2
DOIs
StatePublished - Feb 1 1997
Externally publishedYes

Keywords

  • Lysyl oxidase
  • fibrosis
  • mRNA
  • myofibroblast
  • type I collagen

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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