Cell surface-bound TIMP3 induces apoptosis in mesenchymal Cal78 cells through ligand-independent activation of death receptor signaling and blockade of survival pathways.

BACKGROUND: The matrix metalloproteinases (MMPs) and their endogenous regulators, the tissue inhibitor of metalloproteinases (TIMPs 1-4) are responsible for the physiological remodeling of the extracellular matrix (ECM). Among all TIMPs, TIMP3 appears to play a unique role since TIMP3 is a secreted protein and, unlike the other TIMP family members, is tightly bound to the ECM. Moreover TIMP3 has been shown to be able to induce apoptotic cell death. As little is known about the underlying mechanisms, we set out to investigate the pro-apoptotic effect of TIMP3 in human mesenchymal cells. METHODOLOGY/PRINCIPAL FINDINGS: Lentiviral overexpression of TIMP3 in mesenchymal cells led to a strong dose-dependent induction of ligand-independent apoptosis as reflected by a five-fold increase in caspase 3 and 7 activity compared to control (pLenti6/V5-GW/lacZ) or uninfected cells, whereas exogenous TIMP3 failed to induce apoptosis. Concordantly, increased cleavage of death substrate PARP and the caspases 3 and 7 was observed in TIMP3 overexpressing cultures. Notably, activation of caspase-8 but not caspase-9 was observed in TIMP3-overexpressing cells, indicating a death receptor-dependent mechanism. Moreover, overexpression of TIMP3 led to a further induction of apoptosis after stimulation with TNF-alpha, FasL and TRAIL. Most interestingly, TIMP3-overexpression was associated with a decrease in phosphorylation of cRaf, extracellular signal-regulated protein kinase (Erk1/2), ribosomal S6 kinase (RSK1) and Akt and serum deprivation of TIMP3-overexpressing cells resulted in a distinct enhancement of apoptosis, pointing to an impaired signaling of serum-derived survival factors. Finally, heparinase treatment of heparan sulfate proteoglycans led to the release of TIMP3 from the surface of overexpressing cells and to a significant decrease in apoptosis indicating that the binding of TIMP3 is necessary for apoptosis induction. CONCLUSION: The results demonstrate that exclusively cell surface-bound endogenous TIMP3 induces apoptosis in mesenchymal Cal78 cells through ligand-independent activation of death receptor signaling and blockade of survival signaling pathways

TIMP3-induced apoptosis is influenced by serum factors.

<p><b>A:</b> Influence of TIMP3 on activation of cRaf, ERK1/2, RSK1 and Akt was determined in Cal78 cells and transduced Cal78 cells with LacZ or TIMP3 by spot array measurements. <b>B:</b> Phosphorylation of cRaf, ERK1/2, RSK1 and Akt was confirmed by western blotting. <b>C:</b> Influence of serum withdrawal on apoptosis rates after 24 hours. Apoptosis in lentiviral transduced cells was measured relative to Cal78 cells cultured with 10% FCS. Values less than p<0.05 (*or °) were considered statistically significant. <b>D:</b> Influence of specific growth factors on apoptosis rates under serum-free conditions. Prior to the assessment of apoptosis, Cal78 cells and transduced Cal78 cells with LacZ or TIMP3 were stimulated with 100 ng/ml EGF, TGF-ß or FGF-2 and cultured for 24 hours. Apoptosis was defined by caspase 3 and 7 activities. *Indicates statistical significance (p<0.05). <b>E:</b> Influence of serum withdrawal on autophagocytosis in Cal78 cells after 24 hours. Autophagocytosis in TIMP3 overexpressing CAL78 cells was determined relative to Cal78 cells transduced with a control construct (LacZ) cultured with 10% FCS, 1% FCS or ITS by Western blot analysis of the typical marker proteins LC3 and Beclin.</p

Proteoglycan-bound TIMP3 induces apoptosis.

<p><b>A:</b> Cal78 or Cal78 transduced with TIMP3 or LacZ were cultured and subsequently treated with heparinase I and III for 2 hours. Supernatants from non-treated and treated cells were transferred onto Cal78 cells for 72 hours prior assessment of apoptosis <b>B:</b> Cal78 or Cal78 transduced with TIMP3 or LacZ were incubated with or without heparinase I and III for 72 hours until evaluation of apoptotic cell death. Inactive heparinases (without CaCl₂) were used as a treatment control. Apoptosis was assessed by measurement of caspase 3 and 7 activities. Values less than p<0.05 (*) were considered statistically significant. <b>C:</b> Evaluation of TIMP3 release from cell surface of transduced cells by heparinase. Western Blot analysis of TIMP3-V5 from cell extracts and corresponding supernatants 4 hours after treatment with heparinase. <b>D:</b> Quantification of the Western Blot bands of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070709#pone-0070709-g003" target="_blank">figure 3C</a>. The band of soluble TIMP3-V5 from supernatants was shown versus bounded TIMP3-V5 from the cell lysates. <b>E:</b> Western blot analysis of the supernatants shown in figure C with a specific TIMP3 antibody. 75 ng/ml rh TIMP3 serves as an indication of the amount of TIMP3 in supernatants of Cal78 cells.</p

Dose-dependent effect of TIMP3 on apoptosis.

<p><b>A–C:</b> Determination of the apoptosis response in different cell clones (clone 1 to 4) by caspase-3/7 activity and corresponding TIMP3 expression in these cell clones. <b>D,E:</b> Activation of initiator caspases-8 and -9 in transduced Cal78 cells with LacZ or TIMP3 cultured for 72 h. <b>F:</b> The effect of exogeneous TIMP3 on apoptosis after stimulation of Cal78 cells with recombinant human (rh) TIMP3 up to 200 nM for 96 h. Values less than p<0.05 (*) were considered statistically significant.</p

Effect of TIMP3 on apoptosis.

<p><b>A:</b> Death receptor dependent apoptosis was analyzed in Cal78 cells and Cal78 cells transduced with LacZ or TIMP3 cultured for 24 h and stimulated with 100 ng/ml FasL, TNF-a or TRAIL for 16 hours by measurement of caspase 3 and 7 activities and <b>B,C:</b> cleavage of caspase 3 and PARP in western blot analyses. GAPDH serves as an internal control. <b>D:</b> TIMP3-induced apoptosis was evaluated in Cal78 cells and transduced Cal78 cells with LacZ or TIMP3 cultured for 24 to 72 h. Apoptosis was assessed by measurement of caspase 3 and 7 activities and <b>E:</b> by histone fragmentation assay.</p

Sclerostin inhibition promotes TNF-dependent inflammatory joint destruction

Sclerostin, an inhibitor of the Wnt/β-catenin pathway, has anti-anabolic effects on bone formation by negatively regulating osteoblast differentiation. Mutations in the human sclerostin gene (SOST) lead to sclerosteosis with progressive skeletal overgrowth, whereas sclerostin-deficient (Sost(-/-)) mice exhibit increased bone mass and strength. Therefore, antibody-mediated inhibition of sclerostin is currently being clinically evaluated for the treatment of postmenopausal osteoporosis in humans. We report that in chronic TNFα (tumor necrosis factor α)-dependent arthritis, fibroblast-like synoviocytes constitute a major source of sclerostin and that either the lack of sclerostin or its antibody-mediated inhibition leads to an acceleration of rheumatoid arthritis (RA)-like disease in human TNFα transgenic (hTNFtg) mice with enhanced pannus formation and joint destruction. Inhibition of sclerostin also failed to improve clinical signs and joint destruction in the partially TNFα-dependent glucose-6-phosphate isomerase-induced arthritis mouse model, but ameliorated disease severity in K/BxN serum transfer-induced arthritis mouse model, which is independent of TNF receptor signaling, thus suggesting a specific role for sclerostin in TNFα signaling. Sclerostin effectively blocked TNFα- but not interleukin-1-induced activation of p38, a key step in arthritis development, pointing to a previously unrealized protective role of sclerostin in TNF-mediated chronic inflammation. The possibility of anti-sclerostin antibody treatment worsening clinical RA outcome under chronic TNFα-dependent inflammatory conditions in mice means that caution should be taken both when considering such treatment for inflammatory bone loss in RA and when using anti-sclerostin antibodies in patients with TNFα-dependent comorbidities