Functional analysis of the lysyl oxidase promoter in myofibroblast-like clones of 3T6 fibroblast.
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 (alpha-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 III 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.
Jourdan-Le Saux
C
C
Institut Pasteur de Lyon, Unité de Pathologie des Fibroses, France.
Gleyzal
C
C
Raccurt
M
M
Sommer
P
P
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002999:Clone Cells; D003001:Cloning, Molecular; D003094:Collagen / Q000096:biosynthesis; D004247:DNA; D004549:Elastin / Q000096:biosynthesis; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009132:Muscles / Q000166:cytology / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D008249:Protein-Lysine 6-Oxidase / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity; D014158:Transcription, Genetic
D009687:Nuclear Proteins; D003094:Collagen; D004247:DNA; D004549:Elastin; D008249:Protein-Lysine 6-Oxidase