TGF-beta-induced expression of tissue inhibitor of metalloproteinases-3 gene in chondrocytes is mediated by extracellular signal-regulated kinase pathway and Sp1 transcription factor.
Transforming growth factor (TGF-beta1) is a potent inducer of chondrogenesis and stimulant of cartilage extracellular matrix (ECM) synthesis. Tissue inhibitor of metalloproteinases-3 (TIMP-3) is located in ECM and is the major inhibitor of matrix metalloproteinases (MMPs) and aggrecanase, the principal enzymes implicated in collagen and aggrecan degradation in arthritis. We investigated the role of extracellular-signal-regulated kinase (ERK)-mitogen-activated protein kinases (MAPK) and Sp1 transcription factor in TGF-beta-induced TIMP-3 gene in chondrocytes and chondrosarcoma cells. TGF-beta time-dependently induced a sustained phosphorylation of ERK-MAPKs in primary human or bovine chondrocytes. Inhibitors of this pathway, PD98059 and U0126, downregulated TGF-beta-induced expression of TIMP-3 RNA and protein. Since the ERKs can phosphorylate Sp1, and the promoter of human TIMP-3 gene contains four Sp1-binding sites, we investigated whether Sp1 is a downstream target of this pathway. Mithramycin and WP631, the agents that prevent binding of Sp1 to its consensus site, downregulated TGF-beta-inducible TIMP-3 expression. Indeed, mithramycin blocked TGF-beta-stimulated Sp1 binding activity. Transfection of cytomegalovirus (CMV) promoter-Sp1 plasmid increased TIMP-3 promoter (-940 to +376)-driven luciferase activity. Depletion of Sp1 by transfection of an antisense phosphorothioate oligonucleotide suppressed TGF-beta-induced TIMP-3 protein expression, while its sense homolog had no effect. These results suggest that activation of ERK-MAPK pathway and Sp1 transcription factor play a pivotal role in the induction of TIMP-3 by TGF-beta in chondrocytes.
Department of Medicine and Centre de Recherche du Centre Hospitalier de l'Université de Montréal (CRCHUM), Montreal, Quebec, Canada.
Hamid Yaqoob
HY
Qureshi
Judith
J
Sylvester
Mohammed
M
El Mabrouk
Muhammad
M
Zafarullah
D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D002356:Cartilage / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism*; D020219:Chondrogenesis / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005109:Extracellular Matrix / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D011401:Promoter Regions, Genetic / Q000235:genetics; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012038:Regeneration / Q000187:drug effects / Q000502:physiology; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D020319:Oligodeoxyribonucleotides, Antisense; D011500:Protein Synthesis Inhibitors; D016329:Sp1 Transcription Factor; D019717:Tissue Inhibitor of Metalloproteinase-3; D016212:Transforming Growth Factor beta; D048049:Extracellular Signal-Regulated MAP Kinases