Abstract
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.
Highlights
transforming growth factor 1 (TGF-1),1 a multifunctional regulatory cytokine, generally stimulates cell growth and production of extracellular matrix in mesenchymal cells [1,2,3,4,5,6]
Type II Collagen mRNA Levels of Proliferating Chondrocytes Are Decreased by TGF-1—To determine whether TGF-1decreased type II collagen synthesis was accompanied by a similar effect at the transcriptional level, steady-state levels of mRNA were estimated by semiquantitative reverse transcriptase-PCR on total RNA extracts of proliferating primary rabbit articular chondrocytes (RAC) treated or not with TGF-1, in Dulbecco’s modified Eagle’s medium (DMEM) with or without 10% fetal calf serum (FCS)
We addressed the molecular mechanisms that control type II collagen expression in RAC under the influence of TGF-1, a cytokine that may play a major role in osteoarticular diseases and in cartilage repair
Summary
Cell Cultures—RAC were prepared from the shoulders and knees of 3-week old rabbits, as previously described [46, 50]. Reporter plasmids were cotransfected with Sp1 and/or Sp3 expression vectors (pEVR2/Sp1 and/or pRC/CMV/Sp3, respectively) as previously described [40, 52]. The corresponding insertless expression vectors were used as controls (pEVR2 and pRC/CMV) In these experiments, the pSV40-gal plasmid has not been cotransfected because Sp1 is able to increase SV40 promoter activity, because of the reported presence of several Sp1 DNA-binding motifs in this promoter [40]. The reporter plasmids have been cotransfected with a pCMV5 expression vector containing the cDNA coding for a hybrid TGF-1 receptor called TRII/I, which induces a dominant negative effect on growth factor signaling [53]. After photography of the gels, the intensity of the corresponding bands was quantified by densitometric scanning using the ImageQuant software (Molecular Dynamics) and normalized to GAPDH cDNA levels. The electrophoregram was scanned with an image scanner and the relative intensity of detected signals was measured and analyzed with a computerized image analysis program (ImageQuant, Molecular Dynamics)
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