Down-regulation of human type II collagen gene expression by transforming growth factor-beta 1 (TGF-beta 1) in articular chondrocytes involves SP3/SP1 ratio.
Although transforming growth factor beta1 (TGF-beta1) is generally considered as a stimulator of type I collagen production in smooth organs, we found that it can inhibit type II collagen biosynthesis in primary rabbit articular chondrocytes (RAC) at transcriptional levels. Constructs of promoter and first intron sequences associated with the luciferase reporter gene were used to delineate the gene sequences involved in TGF-beta1 control of human COL2A1 gene transcription. Cotransfection of these DNA fragments with a TbetaRII/I cDNA hybrid receptor, capable of inducing a TGF-beta1 dominant negative effect, showed that TGF-beta1 inhibits specifically COL2A1 gene transcription in RAC by a 63-bp proximal promoter. Footprint and gel retardation analyses revealed that the TGF-beta1-induced inhibition effect exerted through the 63-bp promoter sequence implies a multimeric complex that binds to the -41/-33 sequence and involves Sp1 and Sp3 transcription factors. Transfection of decoy Sp-binding oligonucleotides corroborated the implication of the proximal promoter in the TGF-beta1-induced inhibition of COL2A1 gene transcription. In addition, TGF-beta1 was found to increase the expression of Sp3 without significant changes to its binding level, but repressed both the biosynthesis and binding activity of Sp1. In functional assays, Sp3 inhibited the 63-bp promoter activity and prevented Sp1 induction of transcription. These findings suggest that TGF-beta1 inhibition of COL2A1 gene transcription in RAC is mediated by an increase of the Sp3/Sp1 ratio and by the repression of Sp1 transactivating effects on that gene.
Laboratoire de Biochimie du Tissu Conjonctif, Faculté de Médecine, CHU niveau 3, Avenue de Côte de Nacre, 14032, Caen Cedex, France.
Christos
C
Chadjichristos
Chafik
C
Ghayor
Jean-Francois
JF
Herrouin
Leena
L
Ala-Kokko
Gunthram
G
Suske
Jean-Pierre
JP
Pujol
Philippe
P
Galéra
D000818:Animals; D015153:Blotting, Western; D002356:Cartilage / Q000196:embryology*; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism; D019902:Chondrocytes / Q000378:metabolism*; D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D024043:Collagen Type II / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015536:Down-Regulation; D017930:Genes, Reporter; D007438:Introns; D008156:Luciferases / Q000378:metabolism; D008957:Models, Genetic; D009841:Oligonucleotides / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D053773:Transforming Growth Factor beta1
C499177:COL2A1 protein, human; D024043:Collagen Type II; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; C508960:TGFB1 protein, human; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D051705:Sp3 Transcription Factor; D003094:Collagen; D004247:DNA; D008156:Luciferases