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

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

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

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

Regulation of matrixmetalloproteinase-3 and matrixmetalloproteinase-13 by SUMO-2/3 through the transcription factor NF-κB

OBJECTIVE: Based on previous data that have linked the small ubiquitin-like modifier-1 (SUMO-1) to the pathogenesis of rheumatoid arthritis (RA), we have investigated the expression of the highly homologous SUMO family members SUMO-2/3 in human RA and in the human tumour necrosis factor α transgenic (hTNFtg) mouse model of RA and studied their role in regulating disease specific matrixmetalloproteinases (MMPs). METHODS: Synovial tissue was obtained from RA and osteoarthritis (OA) patients and used for histological analyses as well as for the isolation of synovial fibroblasts (SFs). The expression of SUMO-2/3 in RA and OA patients as well as in hTNFtg and wild type mice was studied by PCR, western blot and immunostaining. SUMO-2/3 was knocked down using small interfering RNA in SFs, and TNF-α induced MMP production was determined by ELISA. Activation of nuclear factor-κB (NF-κB) was determined by a luciferase activity assay and a transcription factor assay in the presence of the NF-κB inhibitor BAY 11-7082. RESULTS: Expression of SUMO-2 and to a lesser extent of SUMO-3 was higher in RA tissues and RASFs compared with OA controls. Similarly, there was increased expression of SUMO-2 in the synovium and in SFs of hTNFtg mice compared with wild type animals. In vitro, the expression of SUMO-2 but not of SUMO-3 was induced by TNF-α. The knockdown of SUMO-2/3 significantly increased the TNF-α and interleukin (IL)-1β induced expression of MMP-3 and MMP-13, accompanied by increased NF-κB activity. Induction of MMP-3 and MMP-13 was inhibited by blockade of the NF-κB pathway. TNF-α and IL-1β mediated MMP-1 expression was not regulated by SUMO-2/3. CONCLUSIONS: Collectively, we show that despite their high homology, SUMO-2/3 are differentially regulated by TNF-α and selectively control TNF-α mediated MMP expression via the NF-κB pathway. Therefore, we hypothesise that SUMO-2 contributes to the specific activation of RASF

Synovial fibroblasts spread rheumatoid arthritis to unaffected joints

Active rheumatoid arthritis originates from few joints but subsequently affects the majority of joints. Thus far, the pathways of the progression of the disease are largely unknown. As rheumatoid arthritis synovial fibroblasts (RASFs) which can be found in RA synovium are key players in joint destruction and are able to migrate in vitro, we evaluated the potential of RASFs to spread the disease in vivo. To simulate the primary joint of origin, we implanted healthy human cartilage together with RASFs subcutaneously into severe combined immunodeficient (SCID) mice. At the contralateral flank, we implanted healthy cartilage without cells. RASFs showed an active movement to the naive cartilage via the vasculature independent of the site of application of RASFs into the SCID mouse, leading to a marked destruction of the target cartilage. These findings support the hypothesis that the characteristic clinical phenomenon of destructive arthritis spreading between joints is mediated, at least in part, by the transmigration of activated RASFs