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Tissue inhibitor of metalloproteinase-3 (TIMP3) promotes endothelial apoptosis via a caspase-independent mechanism

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Abstract

Tissue inhibitor of metalloproteinases-3 (TIMP3) is a tumor suppressor and a potent inhibitor of angiogenesis. TIMP3 exerts its anti-angiogenic effect via a direct interaction with vascular endothelial growth factor (VEGF) receptor-2 (KDR) and inhibition of proliferation, migration and tube formation of endothelial cells (ECs). TIMP3 has also been shown to induce apoptosis in some cancer cells and vascular smooth muscle cells via MMP inhibition and caspase-dependent mechanisms. In this study, we examined the molecular mechanisms of TIMP3-mediated apoptosis in endothelial cells. We have previously demonstrated that mice developed smaller tumors with decreased vascularity when injected with breast carcinoma cells overexpressing TIMP3, than with control breast carcinoma cells. TIMP3 overexpression resulted in increased apoptosis in human breast carcinoma (MDA-MB435) in vivo but not in vitro. However, TIMP3 could induce apoptosis in ECs in vitro. The apoptotic activity of TIMP3 in ECs appears to be independent of MMP inhibitory activity. Furthermore, the equivalent expression of functional TIMP3 promoted apoptosis and caspase activation in ECs expressing KDR (PAE/KDR), but not in ECs expressing PDGF beta-receptor (PAE/β-R). Surprisingly, the apoptotic activity of TIMP3 appears to be independent of caspases. TIMP3 inhibited matrix-induced focal adhesion kinase (FAK) tyrosine phosphorylation and association with paxillin and disrupted the incorporation of β3 integrin, FAK and paxillin into focal adhesion contacts on the matrix, which were not affected by caspase inhibitors. Thus, TIMP3 may induce apoptosis in ECs by triggering a caspase-independent cell death pathway and targeting a FAK-dependent survival pathway.

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Acknowledgments

This work was supported in part by US National Institute of Health EY016490 (BA-A), EY020861 (BA-A), EY022768 (JHQ), Foundation Fighting Blindness Center Grant (BA-A), Research to Prevent Blindness (RPB) Challenge Grant and RPB Lew Wasserman award to BA-A. We are grateful to Mariya Ali and Alecia Cutler for providing technical assistance. We wish to extend a sincere apology to colleagues whose work was not cited because of space limitations.

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Authors and Affiliations

  1. Department of Ophthalmology, Cole Eye Institute, Cleveland Clinic Lerner College of Medicine, Case Western Reserve University, Cleveland, OH, USA

    Jian Hua Qi & Bela Anand-Apte

  2. Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine, Case Western Reserve University, Cleveland, OH, USA

    Bela Anand-Apte

  3. Department of Ophthalmology, Cole Eye Institute i3-161, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH, 44195, USA

    Bela Anand-Apte

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  1. Jian Hua Qi
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Correspondence to Bela Anand-Apte.

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10495_2014_1076_MOESM1_ESM.tif

DEVD and Z-IETD inhibit Caspase 3 and Caspase 8 respectively in PAE cell lysates. Analysis of Caspase 3 and Caspase 8 activity in cell lysates from PAE-KDR/V (V) and PAE-KDR/T3 (W1 and W3) cells treated with DEVD (10 μM), Z-IETD (30 μM) or DMSO as a vehicle control. Supplementary material 1 (TIFF 454 kb)

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Qi, J.H., Anand-Apte, B. Tissue inhibitor of metalloproteinase-3 (TIMP3) promotes endothelial apoptosis via a caspase-independent mechanism. Apoptosis 20, 523–534 (2015). https://doi.org/10.1007/s10495-014-1076-y

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