Transforming growth factor beta (TGF-beta1) induces cartilage extracellular matrix synthesis and tissue inhibitor of metalloproteinases-3 (TIMP-3), an important natural inhibitor of matrix metalloproteinases, aggrecanases and TNF-alpha-converting enzyme, which are implicated in cartilage degradation and joint inflammation. This study tested the hypothesis that Akt/protein kinase B signaling pathway could mediate TGF-beta1 induction of TIMP-3 in human articular chondrocytes. TGF-beta activated phosphorylation of Akt in a delayed and sustained fashion that correlated with TIMP-3 mRNA induction. Phosphatidylinositol kinase (PI3K) inhibitors, Wortmannin and LY294002 and Akt inhibitor (NL-71-101) significantly inhibited TGF-beta-induced Akt phosphorylation, TIMP-3 expression, TIMP-3 promoter (-940 to +376)-driven luciferase activity and Sp1 transcription factor binding. PI3K p85, Akt and Sp1 small interfering RNA (siRNA)-driven knockdown of the respective gene products significantly suppressed TGF-beta-induced TIMP-3 gene expression. TGF-beta-stimulated phosphorylation of p70S6 Kinase and TIMP-3 protein induction was inhibited by rapamycin. Thus TGF-beta induces TIMP-3 gene expression in human chondrocytes partly through PI3K/Akt pathway and Sp1 transcription factor and by translational mechanisms via mammalian target of rapamycin (mTOR) signaling. TGF-beta induction of pro-survival Akt cascade and TIMP-3 may be related to strengthening of cartilage extracellular matrix, increased chondrocyte viability and maintenance of joint tissue integrity.