Transforming growth factor-beta induced collagenase-3 production in human osteoarthritic chondrocytes is triggered by smad proteins: Cooperation between activator protein-1 and PEA-3 binding sites

Objective. To examine the signaling pathways leading to transforming growth factor-beta (TCF-beta) induced collagenase-3 production in human osteoarth ritic (OA) chondrocytes, as well as the transcription factors and their bin ding sites involved in the transcriptional control of collagenase-3 gene. Methods. Identification of the TGF-beta signaling pathway was by Western im munoblotting using specific antibodies for the phosphorylated forms of p44/ 42 and p38 MAPK, SAPK/JNK, and the Smad2 protein. Electromobility shift ass ays (EMSA) were carried out for activator protein-1 (AP-1), polyomavirus en hancer A (PEA-3), activin-response-element-like, Smad-binding-element-like, and TGF-beta inhibitory element oligonucleotides, Supershift assays using antibodies to the Jun, Fos, and Smad families of proteins were used for ide ntification of transcription factors. Chondrocyte transfections were also p erformed using the -133CAT collagenase-3 promoter plasmid (containing PEA-3 , AP-1, and TATA sites) and mutated AP-1 and PEA-3 sites. Results. The primary target of TGF-beta induced collagenase-3 in OA chondro cytes was the Smad2 protein, with significant phosphorylation within 5 min. Contrasting with the Smad2, the untreated OA chondrocytes already had dete ctable levels of the phosphorylated forms of p38 and p44/42 MAPK. Of the ol igonucleotides tested, EMSA revealed that TGF-beta treated OA chondrocyte p roteins bound only to the AP-1 and PEA-3, Supershifts with the AP-I oligonu cleotide showed the presence of the Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) proteins in the untreated and TGF-beta treated OA chon drocytes, whereas only Smad proteins (Smad2, 3, 4) were present in the AP-I binding proteins from the TGF-beta treated chondrocytes. The AP-1 mutation decreased both basal (95%) and TGF-beta induced (99%) collagenase-3 produc tion, whereas the PEA-3 mutation decreased the basal(15%) but more signific antly (50%) the TGF-beta induced transcription. Conclusion. Smad proteins are the main cytoplasmic signaling pathways in TG F-beta stimulated collagenase-3 in OA chondrocytes. The AP-1 sire appears c ritical for upregulation of collagenase-3 production. but TGF-beta stimulat ion requires both AP-I and PEA-3 sites for optimal response.