title: Cell surface-bound TIMP3 induces apoptosis in mesenchymal Cal78 cells through ligand-independent activation of death receptor signaling and blockade of survival pathways.
abstract: 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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute of Experimental Musculoskeletal Medicine, University Hospital Muenster, Muenster, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Koers-Wunrau
authors.forename: Corinna
authors.initials: C
authors.lastname: Wehmeyer
authors.forename: Anja
authors.initials: A
authors.lastname: Hillmann
authors.forename: Thomas
authors.initials: T
authors.lastname: Pap
authors.forename: Berno
authors.initials: B
authors.lastname: Dankbar
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics / Q000502:physiology; D053148:Caspase 3 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D004285:Dogs; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism; D019763:Heparin Lyase / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008648:Mesoderm / Q000378:metabolism; D010766:Phosphorylation; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics / Q000378:metabolism*
chemical_list: D019812:Heparan Sulfate Proteoglycans; D019717:Tissue Inhibitor of Metalloproteinase-3; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D019763:Heparin Lyase
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