Smad signaling pathway is a pivotal component of tissue inhibitor of metalloproteinases-3 regulation by transforming growth factor beta in human chondrocytes.
Transforming growth factor beta (TGF-beta1) promotes cartilage matrix synthesis and induces tissue inhibitor of metalloproteinases-3 (TIMP-3), which inhibits matrix metalloproteinases, aggrecanases and TNF-alpha-converting enzyme implicated in articular cartilage degradation and joint inflammation. TGF-beta1 activates Akt, ERK and Smad2 pathways in chondrocytes. Here we investigated previously unexplored roles of specific Smads in TGF-beta1 induction of TIMP-3 gene by pharmacological and genetic knockdown approaches. TGF-beta1-induced Smad2 phosphorylation and TIMP-3 protein expression could be inhibited by the Smad2/3 phosphorylation inhibitors, PD169316 and SB203580 and by Smad2-specific siRNA. Specific inhibitor of Smad3 (SIS3) and Smad3 siRNA abolished TGF-beta induction of TIMP-3. Smad2/3 siRNAs also down regulated TIMP-3 promoter-driven luciferase activities, suggesting transcriptional regulation. SiRNA-driven co-Smad4 knockdown abrogated TIMP-3 augmentation by TGF-beta. TIMP-3 promoter deletion analysis revealed that -828 deletion retains the original promoter activity while -333 and -167 deletions display somewhat reduced activity suggesting that most of the TGF-beta-responsive, cis-acting elements are found in the -333 fragment. Chromatin Immunoprecipitation (ChIP) analysis confirmed binding of Smad2 and Smad4 with the -940 and -333 promoter sequences. These results suggest that receptor-activated Smad2 and Smad3 and co-Smad4 critically mediate TGF-beta-stimulated TIMP-3 expression in human chondrocytes and TIMP-3 gene is a target of Smad signaling pathway.
Qureshi
Hamid Yaqoob
HY
Department of Medicine, University of Montreal and Research Centre of Centre Hospitalier de l'Université de Montréal (CR-CHUM), K-5255 Mailloux, Notre-Dame Hospital, 1560 Sherbrooke E., Montreal, Quebec, Canada H2L 4M1.
Ricci
Gemma
G
Zafarullah
Muhammad
M
D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011725:Pyridines / Q000494:pharmacology; D011758:Pyrroles / Q000494:pharmacology; D034622:RNA Interference; D015398:Signal Transduction; D051785:Smad Proteins / Q000378:metabolism*; D051899:Smad2 Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051900:Smad3 Protein / Q000037:antagonists & inhibitors / Q000235:genetics; D051901:Smad4 Protein / Q000037:antagonists & inhibitors / Q000235:genetics; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000096:biosynthesis / Q000235:genetics*; D016212:Transforming Growth Factor beta / Q000037:antagonists & inhibitors / Q000494:pharmacology*
C508504:6,7-dimethyl-2-(2E)-3-(1-methyl-2-phenyl-1H-pyrrolo(2,3-b)pyridin-3-yl-prop-2-enoyl)-1,2,3,4-tetrahydroisoquinoline hydrochloride; D007546:Isoquinolines; D011725:Pyridines; D011758:Pyrroles; D051785:Smad Proteins; D051899:Smad2 Protein; D051900:Smad3 Protein; D051901:Smad4 Protein; D019717:Tissue Inhibitor of Metalloproteinase-3; D016212:Transforming Growth Factor beta