Elsevier

Cellular Signalling

Volume 19, Issue 8, August 2007, Pages 1643-1651
Cellular Signalling

Requirement of phosphatidylinositol 3-kinase/Akt signaling pathway for regulation of tissue inhibitor of metalloproteinases-3 gene expression by TGF-β in human chondrocytes

https://doi.org/10.1016/j.cellsig.2007.02.007Get rights and content

Abstract

Transforming growth factor beta (TGF-β1) 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-β1 induction of TIMP-3 in human articular chondrocytes. TGF-β 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-β-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-β-induced TIMP-3 gene expression. TGF-β-stimulated phosphorylation of p70S6 Kinase and TIMP-3 protein induction was inhibited by rapamycin. Thus TGF-β 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-β 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.

Introduction

A hallmark of rheumatoid arthritis (RA) and osteoarthritis (OA) is resorption of cartilage extracellular matrix (ECM). This is partly due to impaired endogenous repair processes induced by an imbalance between anabolic growth factors and catabolic proinflammatory cytokines, interleukin-1 (IL-1), IL-17 and tumor necrosis factor (TNF-α), which inhibit the ECM synthesis and induce matrix metalloproteinases (MMPs) production [1], [2]. Adult cartilage has limited capacity to regenerate and transforming growth factor beta (TGF-β) family members have the potential to stimulate its repair. Human OA cartilage responds poorly to TGF-β due to decreased receptor II [3]. Inhibition of endogenous TGF-β causes impaired cartilage repair and excessive TGF-β leads to the formation of osteophytes in OA [4]. TGF-β, a multi-functional factor produced by monocytes–macrophages, platelets and chondrocytes, induces chondrogenesis and ECM synthesis [5]. TGF-β1 is elevated in human RA synovial fluid and tissue, has immunosuppressive properties, [6] and is a major growth factor for maintaining chondrocyte phenotype and homeostasis [7]. It suppresses inflammatory cell infiltration, pannus formation and joint erosion during acute and chronic arthritis by counteracting the effects of IL-1 [8].

MMPs and aggrecanases (ADAMTS, a disintegrin and metalloproteinase with thrombospondin motif) digest major cartilage ECM components including type II collagen and aggrecan as well as several non-ECM substrates during physiological and pathological remodeling [9], [10]. Tissue inhibitors of metalloproteinases (TIMPs) are 4 natural inhibitors of MMPs with growth promoting, pro-apoptotic, anti-apoptotic and anti-angiogenic functions [11], [12]. Excessive MMPs and ADAMTSs over TIMPs cause loss of articular cartilage. TGF-β inhibits the expression of most MMPs but induces TIMP-1 and TIMP-3 in chondrocytes [7]. TIMP-3 is uniquely located in ECM where its N-terminal domain binds to chondroitin- and heparan sulfate [13] and also inhibits MMP-13, ADAMTS4 and ADAMTS5, the principal cartilage-degrading enzymes [14], [15]. It blocks aggrecan degradation in cartilage explants [16] and inhibits proinflammatory, TNF-α converting enzyme (TACE/ADAM-17) activity [17]. TIMP-3 can thus reduce inflammation in arthritis. TIMP-3 inhibits angiogenesis by blocking the binding of VEGF to its receptor and could reduce rheumatoid pannus formation [reviewed in [11]]. Such unique features make TIMP-3 a potentially therapeutic protein in arthritis [11], [17]. Indeed, TIMP-3 overexpression in proliferating rheumatoid synovial fibroblasts induces apoptosis [18] and prevents invasion of cartilage by pannus [19]. TIMP-3 knockout mice display an increased initial inflammation and serum TNF-α level in antigen-induced arthritis, supporting its protective function against inflammatory arthritis [20]. In other systems, TGF-β binding to cell surface associates types I and II receptors leading to phosphorylation of type I receptor kinase domain, transmission of signal via stimulatory Smads and transcription of the target genes [21]. In chondrocytes, Smad, PKA, PKC and Wnt pathways are induced by TGF-β relative to various cartilage functions [22]. We previously showed the involvement of Smad and extracellular-signal-regulated kinase (ERK1/2)-mitogen-activated protein pathways in TGF-β-induced TIMP-3 in chondrocytes [23], [24], however, role of phosphoinositide 3-kinase (PI3K/Akt) pathway and its target transcription factors implicated in this induction are unknown. PI3K-Akt/protein kinase B (PKB) pathway is stimulated by insulin-like growth factor leading to cell proliferation, survival and inhibition of apoptosis [25]. Although PI3K/Akt pathway is activated by TGF-β in human rheumatoid synovial fibroblasts in association with their proliferation [26], its role in chondrocytes and regulation of specific genes is not known. Here, we show the previously unknown and critical role of PI3K/Akt pathway and Sp1 transcription factor in TGF-β-stimulated increase of TIMP-3 in human knee articular chondrocytes.

Section snippets

Culture of chondrocytes and treatments

The normal human knee articular chondrocytes (Cambrex; Walkerville, MD) were grown to confluence as high-density passage 2 monolayer cultures in Differentiation Bullekit medium for maintaining their differentiated phenotype (Cambrex). These cells do not express type I collagen but express type II collagen, a marker of differentiated chondrocytes as determined by Northern and Western blot analysis (Fig. 1A). After trypsinization, the cells were grown in 6-well plates in Dulbecco's modified

Induction of Akt phosphorylation and TIMP-3 mRNA by TGF-β1 in human articular chondrocytes

We first examined the differentiated phenotype of human chondrocytes under our experimental conditions. As determined by Western blotting, these cells at passage 3 do not express 115 kDa type I collagen band but do express high levels of Collagen II mRNA and 100–210 kDa type II collagen bands, a chondrocyte-specific marker (Fig. 1A). To examine if TGF-β1 stimulates Akt phosphorylation in human chondrocytes, quiescent cells were exposed to this factor for different time periods. TGF-β induced

Discussion

TGF-β is an important pleiotropic factor involved in chondrogenesis, cartilage repair and matrix synthesis. Due to the versatile ability of TIMP-3 to inhibit cartilage-degrading MMPs and ADAMTS, and TNF-α-activating ADAM-17, it is an important therapeutic protein for arthritis. We have shown here for the first time by several pharmacological and genetic approaches that PI3K/Akt pathway mediates TGF-β-induced TIMP-3 gene expression primarily at the promoter level via Sp1 transcription factor

Conclusions

In conclusion, TGF-β1 could exert its growth-promoting effects on chondrocytes by activating PI3 kinase/Akt pathway and TIMP-3 expression, which by binding to heparan sulfate and chondroitin sulfate may result in strengthening of cartilage ECM, increased chondrocyte viability and maintenance of joint tissue integrity (Fig. 6B). The abilities of TIMP-3 to block MMP and ADAMTS activities could be an added benefit. It is also interesting to note that in contrast with the cell proliferation and

Acknowledgements

This work was supported by the Canadian Institutes of Health Research (CIHR) and Canadian Arthritis Network (CAN) of Centers of Excellence grants. We are grateful to Dr Jean Bennett for the TIMP-3 promoter plasmid.

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