Title: Tissue Inhibitor of Metalloproteinase-3 Induces a Fas-associated Death Domain-dependent Type II Apoptotic Pathway
Abstract: Tissue inhibitors of metalloproteinases (TIMPs) are important regulators of matrix metalloproteinase (MMP) and adamalysin metalloproteinase activity. We previously reported that overexpression of TIMP-3 inhibits MMPs and induces apoptotic cell death in a variety of cell types and demonstrated that apoptosis is mediated through the N terminus of TIMP-3, which harbors the MMP inhibitory domain. However, little is known about the mechanisms underlying TIMP-3-induced apoptosis. Here we demonstrate that overexpression of TIMP-3 induced activation of initiator caspase-8 and -9 and promoted caspase-mediated cleavage of the death substrates poly(ADP-ribose) polymerase and focal adhesion kinase. Furthermore, TIMP-3 induced mitochondrial activation as demonstrated by loss of mitochondrial membrane potential and release of cytochrome c. Intervention studies demonstrated that overexpression of Bcl-2, the anti-apoptotic mitochondrial membrane protein, or CrmA, a viral serpin inhibitor of caspase-8, completely inhibited TIMP-3-induced apoptosis. Furthermore, a dominant-negative Fas-associated death domain mutant inhibited TIMP-3-induced death substrate cleavage and apoptotic death. Taken together, these results indicate that TIMP-3 overexpression induces a type II apoptotic pathway initiated via a Fas-associated death domain-dependent mechanism. Tissue inhibitors of metalloproteinases (TIMPs) are important regulators of matrix metalloproteinase (MMP) and adamalysin metalloproteinase activity. We previously reported that overexpression of TIMP-3 inhibits MMPs and induces apoptotic cell death in a variety of cell types and demonstrated that apoptosis is mediated through the N terminus of TIMP-3, which harbors the MMP inhibitory domain. However, little is known about the mechanisms underlying TIMP-3-induced apoptosis. Here we demonstrate that overexpression of TIMP-3 induced activation of initiator caspase-8 and -9 and promoted caspase-mediated cleavage of the death substrates poly(ADP-ribose) polymerase and focal adhesion kinase. Furthermore, TIMP-3 induced mitochondrial activation as demonstrated by loss of mitochondrial membrane potential and release of cytochrome c. Intervention studies demonstrated that overexpression of Bcl-2, the anti-apoptotic mitochondrial membrane protein, or CrmA, a viral serpin inhibitor of caspase-8, completely inhibited TIMP-3-induced apoptosis. Furthermore, a dominant-negative Fas-associated death domain mutant inhibited TIMP-3-induced death substrate cleavage and apoptotic death. Taken together, these results indicate that TIMP-3 overexpression induces a type II apoptotic pathway initiated via a Fas-associated death domain-dependent mechanism. Tissue inhibitors of metalloproteinases (TIMPs) 1The abbreviations used are: TIMPstissue inhibitors of metalloproteinasesMMPmatrix metalloproteinaseTNFtumor necrosis factorFADDFas-associated death domainVSMCsvascular smooth muscle cellsSMCssmooth muscle cellsPARPpoly(ADP-ribose) polymeraseFAKfocal adhesion kinaserAdrecombinant adenovirusDNdominant-negativepfuplaque-forming unitsPIPES1,4-piperazinediethanesulfonic acidare a family of four secreted proteins that collectively regulate the activity of the matrix metalloproteinases (MMPs) and at least some of the adamalysin proteases (1.Birkedal-Hansen H. Moore W.G.I. Bodden M.K. Windsor L.J. Birkedal-Hansen B. De Carlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2639) Google Scholar, 2.Amour A. Slocombe P.M. Webster A. Butler M. Knight C.G. Smith B.J. Stephens P.E. Shelley C. Hutton M. Knauper V. Docherty A.J.P. Murphy G. FEBS Lett. 1998; 435: 39-44Crossref PubMed Scopus (546) Google Scholar). TIMPs are thought to fold into a similar two-domain structure, with each domain folded into three loops constrained by three disulfide bonds (3.Docherty A.J.P. Lyons A. Smith B.J. Wright E.M. Stephens P.E. Harris T.J.R. Murphy G. Reynolds J.J. Nature. 1985; 318: 66-69Crossref PubMed Scopus (585) Google Scholar, 4.Apte S.S. Mattei M.G. Olsen B.R. Genomics. 1994; 19: 86-90Crossref PubMed Scopus (199) Google Scholar, 5.Boone T.C. Johnson M.J. DeClerck Y.A. Langley K.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2800-2804Crossref PubMed Scopus (180) Google Scholar, 6.Greene J. Wang M.S. Liu Y.L.E. Raymond L.A. Rosen C. Shi Y.N.E. J. Biol. Chem. 1996; 271: 30375-30380Abstract Full Text Full Text PDF PubMed Scopus (478) Google Scholar). The N-terminal domain contains a highly conserved CXC motif deemed responsible for MMP inhibition, whereas the smaller C-terminal domain confers specific functions such as the ability of TIMP-1 to interact with pro-MMP-9 and of TIMP-2 to interact with pro-MMP-2 (7.Gomez D.E. Alonso D.F. Yoshiji H. Thorgeirsson U.P. Eur. J. Cell Biol. 1997; 74: 111-122PubMed Google Scholar, 8.Langton K.P. Barker M.D. McKie N. J. Biol. Chem. 1998; 273: 16778-16781Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 9.Yu W.H. Brew K. Yu S.S. Meng Q. Woessner F. Mol. Biol. Cell. 1998; 9: 1024Google Scholar, 10.Leco K.J. Khokha R. Pavloff N. Hawkes S.P. Edwards D.R. J. Biol. Chem. 1994; 269: 9352-9360Abstract Full Text PDF PubMed Google Scholar, 11.Goldberg G.I. Marmer B.L. Grant G.A. Eisen A.Z. Wilhelm S. He C.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8207-8211Crossref PubMed Scopus (546) Google Scholar, 12.Wilhelm S.M. Collier I.E. Marmer B.L. Eisen A.Z. Grant G.A. Goldberg G.I. J. Biol. Chem. 1989; 264: 17213-17221Abstract Full Text PDF PubMed Google Scholar, 13.Stetler-Stevenson W.G. Krutzsch H.C. Liotta L.A. J. Biol. Chem. 1989; 264: 17374-17378Abstract Full Text PDF PubMed Google Scholar). tissue inhibitors of metalloproteinases matrix metalloproteinase tumor necrosis factor Fas-associated death domain vascular smooth muscle cells smooth muscle cells poly(ADP-ribose) polymerase focal adhesion kinase recombinant adenovirus dominant-negative plaque-forming units 1,4-piperazinediethanesulfonic acid TIMPs regulate MMP activity. Thus, TIMPs play important roles in regulating cellular functions that are dependent on matrix composition such as invasion, migration, differentiation, and proliferation. Several additional functions have also been attributed to individual TIMPs such as regulation of angiogenesis, activation of pro-MMP-2, and mitogenesis (14.Takigawa M. Nishida Y. Suzuki F. Kishi J. Yamashita K. Hayakawa T. Biochem. Biophys. Res. Commun. 1990; 171: 1264-1271Crossref PubMed Scopus (130) Google Scholar, 15.Stetler-Stevenson W.G. Bersch N. Golde D.W. FEBS Lett. 1992; 296: 231-234Crossref PubMed Scopus (171) Google Scholar, 16.Strongin A. Collier I.E. Bannikov G. Marmer B. Grant G. Goldberg G. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1438) Google Scholar, 17.Corcoran M.L. Stetler-Stevenson W.G. J. Biol. Chem. 1995; 270: 13453-13459Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 18.Hayakawa T. Yamashita K. Ohuchi E. Shinagawa A. J. Cell Sci. 1994; 107: 2373-2379Crossref PubMed Google Scholar). Using adenovirus-mediated gene transfer, we recently demonstrated that overexpression of TIMP-3 in human vascular smooth muscle cells and cancer cell cultures reduces cell migration and promotes apoptotic cell death, the latter being evoked through an unknown mechanism (19.Baker A.H. George S.J. Zaltsman A.B. Murphy G. Newby A.C. Br. J. Cancer. 1999; 79: 1347-1355Crossref PubMed Scopus (250) Google Scholar, 20.Ahonen M. Baker A.H. Kahari V.M. Cancer Res. 1998; 58: 2310-2315PubMed Google Scholar). Furthermore, this unique phenotype induced by TIMP-3 translates to a beneficial reduction in vascular neointima formation in a human model of late vein graft failure (21.George S.J. Lloyd C.T. Angelini G.D. Newby A.C. Baker A.H. Circulation. 2000; 101: 296-304Crossref PubMed Scopus (190) Google Scholar). The pro-death domain of TIMP-3 has recently been localized to the N terminus, the region associated with MMP inhibitory activity (22.Bond M. Murphy G. Bennett M. Amour A. Knauper V. Newby A.C. J. Biol. Chem. 2000; 275: 41358-41363Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar); and it has been proposed, at least in colon cancer lines, that TIMP-3 promotes death through stabilization of tumor necrosis factor-α (TNF-α) receptors on the cell surface, leading to increased susceptibility to apoptosis (23.Smith M.R. Kung H.F. Durum S.K. Colburn N.H. Sun Y. Cytokine. 1997; 9: 770-780Crossref PubMed Scopus (175) Google Scholar). Interestingly, deficiency of TIMP-3 in homozygous knockout mice results in enhanced apoptosis during mammary gland involution (24.Fata J.E. Leco K.J. Voura E.B. Yu H.Y.E. Waterhouse P. Murphy G. Moorehead R.A. Khokha R. J. Clin. Invest. 2001; 108: 831-841Crossref PubMed Scopus (146) Google Scholar), suggesting that the physiological levels of TIMP-3 play an important role in regulating apoptosis during a number of physiological and pathological processes. It is therefore important to define the mechanism(s) through which TIMP-3 regulates apoptotic mediators when overexpressed. Apoptotic cell death can be induced by a variety of stimuli, including death ligands such as TNF-α, Fas ligand, and TRAIL; DNA-damaging agents; and loss of matrix attachment in adherence-dependent cells. Many of these stimuli activate the apoptotic cascade through the action of two distinct initiator caspases, caspase-8 and -9. Caspase-9 is an important regulator of downstream effector caspases (caspase-3, -6, and -7) in response to signals generated by the mitochondria (25.Budihardjo I. Oliver H. Lutter M. Luo X. Wang X.D. Annu. Rev. Cell Dev. Biol. 1999; 15: 269-290Crossref PubMed Scopus (2268) Google Scholar,26.Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X.D. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6230) Google Scholar). Its activation requires the formation of a cytosolic complex with apoptosis protease activating factor, ATP, and cytochromec released from the mitochondria (26.Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X.D. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6230) Google Scholar). Caspase-8 is typically recruited to activated death receptors via interactions with adaptor proteins such as FADD, where they form part of the death-inducing signaling complex (27.Ashkenazi A. Dixit V. Science. 1998; 277: 1305-1308Crossref Scopus (5151) Google Scholar). Two caspase-8-initiated apoptotic signaling pathways have been described (28.Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2629) Google Scholar). A strong activation of caspase-8 in response to death receptor activation can directly activate downstream effector caspases (type I pathway), whereas a weaker activation of caspase-8 can initiate mitochondrial activation, resulting in mitochondrial membrane depolarization, release of cytochrome c into the cytosol, and subsequent activation of caspase-9 and effector caspases (type II pathway) (28.Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2629) Google Scholar). During a type II pathway, the mitochondria act as amplifiers, resulting in additional caspase-8 activation (28.Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2629) Google Scholar). Here we investigated the cellular mechanism activated by overexpression of TIMP-3 in primary cultures of both vascular smooth muscle cells (VSMCs) and a HeLa carcinoma cell line. We show that TIMP-3 induces a FADD-dependent type II apoptotic signaling pathway. Human embryonic kidney 293 cells were purchased from Microbix (Toronto, Canada), and HeLa cells were from the European Collection of Animal Cell Cultures (Salisbury, United Kingdom). All chemicals were purchased from Sigma (Poole, UK) and were of the highest quality available. Culture media and additives were obtained from Invitrogen (Paisley, Scotland). Rabbit anti-human TIMP-3 polyclonal antibody was purchase from Chemicon International, Inc. (Harrow, UK). Recombinant human TNF-α and rabbit anti-cleaved poly(ADP-ribose) polymerase (PARP) (p85) antibody were purchased from Roche Molecular Biochemicals (Lewes, UK). Anti-CrmA, anti-Bcl-2, anti-focal adhesion kinase (FAK), and anti-cytochrome c antibodies and caspase substrates and inhibitors were obtained from CN Biosciences (Nottingham, UK). Anti-TNF-α antibody was purchased from R & D Systems. MitoTracker® Orange CMTMRos was obtained from Molecular Probes, Inc. (Leiden, The Netherlands). Human embryonic kidney 293 cells were maintained in minimal essential medium supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% (v/v) fetal calf serum. Rat smooth muscle cells (SMCs) were prepared from thoracic aortas as described previously (29.Bennett M.R. Anglin S. McEwan J.R. Jagoe R. Newby A.C. Evan G.I. J. Clin. Invest. 1994; 93: 820-828Crossref PubMed Scopus (231) Google Scholar). Rat SMCs and HeLa cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. All cells were maintained at 37 °C in an atmosphere of 95% air and 5% carbon dioxide. The adenoviral construct rAd/TIMP-3 has been described previously (30.Baker A.H. Zaltsman A.B. George S.J. Newby A.C. J. Clin. Invest. 1998; 101: 1478-1487Crossref PubMed Scopus (416) Google Scholar). The control adenoviral construct rAd/β-galactosidase was a gift from Dr. G. W. G. Wilkinson (University of Wales College of Medicine, Heath Park, Cardiff, UK). rAd/DN-FADD was a gift from Dr. C. Trautwein (Department of Gastroenterology and Hepatology, Mediziniche Hochschule Hannover, Hannover, Germany). The rAd/CrmA adenoviral construct was a gift from Dr. G. Nabel, and the rAd/Bcl-2 adenoviral construct was a gift from Dr. J. Uney. Primary rat aortic SMCs (passages 2–4) and HeLa cells were cultured in six-well plates or on sterile glass coverslips until 80% confluent. An accurate cell number was determined pre-infection by trypsinization of three wells and counting using a Neubauer hemocytometer. The remaining wells were infected at 300 (rat SMCs) and 100 (HeLa cells) plaque-forming units (pfu)/cell in 1 ml of fresh complete medium for 2 h. The medium was then replaced with 2 ml of fresh complete medium and left for the required length of time prior to analysis. For adenoviral co-infections, cells were pre-infected with either the control adenovirus or the test adenovirus at 50 pfu/cell for 2 h. Cells were then incubated in growth medium for an additional 2 h and subsequently infected with the second adenovirus at 100 pfu/cell for an additional 2 h, after which the medium was replaced with fresh complete medium. Total cell lysates were prepared in SDS lysis buffer (50 mm Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 1× Complete protease inhibitor mixture (Roche Molecular Biochemicals)). Protein concentration was determined using a Bio-Rad micro-BCA protein assay according to the manufacturer's instructions. Proteins (150 μg) were separated by SDS-PAGE under reducing conditions and transferred to Bio-Rad polyvinylidene difluoride nylon membranes. Membranes were blocked in Tris-buffered saline/Tween (200 mm Tris-HCl (pH 7.4), 137 mm NaCl, and 0.2% Tween 20) containing 5% skim milk powder, followed by incubation with primary antibody. Following washing with Tris-buffered saline/Tween, blots were incubated with horseradish peroxidase-conjugated secondary antibody for 60 min, and immunoreactive proteins were visualized using the enhanced chemiluminescence system (ECL, Amersham Biosciences). Cells were grown on glass coverslips and fixed in ice-cold methanol 72 h post-infection. After air drying, cells were washed twice with 1× TE buffer (10 mm Tris-HCl (pH 8.0) and 1 mm EDTA) and incubated in labeling mixture (50 mm Tris-HCl (pH 7.2), 10 mm MgSO4, 0.1 mm dithiothreitol, 0.01 mm dATP, 0.01 mm dCTP, 0.01 mmdGTP, 0.01 mm biotin-dUTP, and 8 units/ml DNA polymerase I (Klenow)) for 15 min at room temperature. Cells were rinsed in 1× TE buffer, and endogenous peroxidase activity was reduced by incubation in 2% H2O2 for 5 min. After further washing, biotin was labeled with ExtrAvidinTM-peroxidase conjugate (1:200). Incubation with diaminobenzidine and subsequent counterstaining with hematoxylin were carried out to distinguish between positive apoptotic nuclei containing nicked DNA and non-apoptotic nuclei. Apoptotic cell death was quantified using a photometric enzyme-linked immunosorbent assay for the detection of cytoplasmic histo-associated DNA fragments (Roche Molecular Biochemicals). Briefly, cell lysates were incubated with a biotin-conjugated anti-histone antibody and a peroxidase-conjugated anti-DNA antibody for 2 h. Complexes were captured on streptavidin-coated microtiter plates and quantified using ABTS colorimetric substrate. Mitochondrial membrane potential (Ψm) was measured using the MitoTracker® Orange CMTMRos fluorescent dye (31.Macho A. Decaudin D. Castedo M. Hirsch T. Susin S. Zamzami N. Kroemer G. Cytometry. 1996; 25: 333-340Crossref PubMed Scopus (157) Google Scholar). Briefly, cells were incubated with MitoTracker®Orange CMTMRos (150 nm) for 30 min in culture medium at 37 °C and 5% CO2. As a positive control for Ψm loss, cells were incubated with 2 μmstaurosporine for 2 h. Cells were washed once with phosphate-buffered saline, collected by centrifugation at 200 ×g, and fixed in 2 ml of 4% paraformaldehyde in phosphate-buffered saline (pH 7.4) for 10 min at 4 °C. Fixed cells were analyzed by flow cytometry (BD PharMingen FACScan) using CellQuest acquisition software. Cells were collected by trypsinization and centrifuged at 200 × g for 5 min at 4 °C. Cells were then washed twice with ice-cold phosphate-buffered saline (pH 7.4), followed by an additional centrifugation at 200 × g for 5 min. The cell pellet was resuspended in 600 μl of extraction buffer containing 220 mm mannitol, 68 mm sucrose, 50 mm PIPES-KOH (pH 7.4), 50 mm KCl, 5 mm EGTA, 2 mmMgCl2, 1 mm dithiothreitol, and Complete protease inhibitor mixture. After 30 min of incubation on ice, cells were homogenized with a glass Dounce homogenizer and a small clearance pestle until the majority of the cells had been disrupted. Cell homogenates were spun at 14,000 × g for 15 min, and supernatants were removed and analyzed by Western blotting for cytochrome c. In agreement with previously published data (19.Baker A.H. George S.J. Zaltsman A.B. Murphy G. Newby A.C. Br. J. Cancer. 1999; 79: 1347-1355Crossref PubMed Scopus (250) Google Scholar, 21.George S.J. Lloyd C.T. Angelini G.D. Newby A.C. Baker A.H. Circulation. 2000; 101: 296-304Crossref PubMed Scopus (190) Google Scholar, 22.Bond M. Murphy G. Bennett M. Amour A. Knauper V. Newby A.C. J. Biol. Chem. 2000; 275: 41358-41363Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), infection of rat VSMCs and HeLa cells with a replication-deficient adenovirus expressing human TIMP-3 (rAd/TIMP-3; 300 and 100 pfu/cell, respectively), but not with the control adenovirus (rAd/β-galactosidase), induced apoptotic cell death 48–72 h post-infection (data not shown) (22.Bond M. Murphy G. Bennett M. Amour A. Knauper V. Newby A.C. J. Biol. Chem. 2000; 275: 41358-41363Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 30.Baker A.H. Zaltsman A.B. George S.J. Newby A.C. J. Clin. Invest. 1998; 101: 1478-1487Crossref PubMed Scopus (416) Google Scholar). TIMP-3-induced apoptosis was characterized by cell shrinkage, cell rounding, membrane blebbing, and cell detachment from the substratum. In situ end labeling staining of rAd/TIMP-3-infected cells revealed intense brown nuclear staining indicative of fragmented DNA, with nuclear condensation and fragmentation (data not shown). Cells expressing TIMP-3 also exhibited phosphatidylserine externalization as indicated by strong annexin V staining without uptake of propidium iodide (data not shown). To characterize the intracellular downstream mechanism(s) activated by TIMP-3, we first measured the effect of TIMP-3 overexpression on caspase activity. Primary cultures of both rat VSMCs and HeLa cells infected with rAd/TIMP-3 exhibited increased effector caspase activity (50.1 ± 0.5- and 4.5 ± 0.2-fold (n = 3), respectively; p < 0.05) as measured by cleavage of the synthetic fluorescent substrate DEVD-7-amino-4-trifluoromethylcoumarin 72 h post-infection (Fig. 1A). Activation of initiator caspase-8 and -9 was also analyzed to identify possible mechanisms through which TIMP-3 initiates the apoptotic cascade. The activity of caspase-8 as measured by cleavage of IETD-7-amino-4-trifluoromethylcoumarin was elevated 18.61 ± 1.36- and 2.95 ± 0.36-fold (n = 3), respectively (p < 0.05) in rat VSMCs and HeLa cells, whereas cleavage of the caspase-9-specific substrate LEHD-7-amino-4-trifluoromethylcoumarin was elevated 10.88 ± 1.76- and 2.53 ± 0.35-fold (n = 3), respectively (p < 0.05) (Fig. 1A). Anti-activated caspase-8 and -9 antibodies also detected activated caspase-8 (p14) and caspase-9 (p37) in total HeLa cell lysates infected with rAd/TIMP-3, but not with rAd/β-galactosidase (Fig. 1B). We performed a detailed time course experiment to evaluate caspase-8 and -9 cleavage following TIMP-3 overexpression. The activated fragments of caspase-8 and -9 were first detected 36 h post-infection and were both detected throughout the remaining time course of the experiment (Fig. 2A).Figure 2Time course of TIMP-3-induced caspase activation and death substrate cleavage.A, HeLa cells were infected with rAd/TIMP-3 (100 pfu/cell), and total cell lysates were prepared at the times indicated. Cell lysates (150 μg) were analyzed for activated caspase-8 and -9 and cleavage of PARP and FAK by Western blotting. B, cells infected with rAd/TIMP-3 were cultured in the presence of 100 μmbenzyloxycarbonyl-VAD-fluoromethyl ketone (ZVAD) or 100 μmN-acetyl-Leu-Leu-norleucinal (ALLN). Cell lysates were prepared 72 h post-infection and analyzed for PARP and FAK cleavage by Western blotting.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Caspase-mediated cleavage of death substrates is an important event during the commitment to and execution of apoptotic death. Cleavage of PARP and FAK has previously been reported in numerous cell types undergoing apoptosis (32.Margolin N. Raybuck S.A. Wilson K.P. Chen W. Fox T. Gu Y. Livingston D.J. J. Biol. Chem. 1997; 272: 7223-7228Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 33.Levkau B. Herren B. Koyama H. Ross R. Raines E.W. J. Exp. Med. 1998; 187: 579-586Crossref PubMed Scopus (226) Google Scholar). We therefore analyzed PARP and FAK cleavage status in TIMP-overexpressing cells. Infection of HeLa cells with rAd/TIMP-3 (but not with rAd/β-galactosidase) resulted in the formation of two prominent lower molecular mass FAK fragments between 70 and 80 kDa and the appearance of an 85-kDa fragment of PARP detected with an anti-cleaved PARP antibody (Fig. 2A). Cleaved forms of FAK and PARP were first detectable 32–36 h after rAd/TIMP-3 infection and increased through the time course of the experiment (Fig. 2A). As expected, overexpression of TIMP-3 preceded the appearance of PARP and FAK cleavage by ∼20 h (Fig. 2A). The time course of PARP and FAK cleavage mirrored that of initiator caspase-8 and -9 activation, suggesting that TIMP-3 induces caspase-mediated cleavage of FAK and PARP. To test this hypothesis, we incubated HeLa cells overexpressing TIMP-3 with either the pan-caspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone or the calpain peptide inhibitorN-acetyl-Leu-Leu-norleucinal. Incubation with benzyloxycarbonyl-VAD-fluoromethyl ketone (but not withN-acetyl-Leu-Leu-norleucinal) completely inhibited the formation of the lower molecular mass forms of FAK and the p85 PARP fragment, demonstrating caspase-mediated cleavage of these proteins in response to TIMP-3 overexpression (Fig. 2B). Activation of the mitochondria and release of cytochrome c into the cytosol have been shown to participate in activation of caspase-9 (26.Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X.D. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6230) Google Scholar). As we observed activation of caspase-9 in response to TIMP-3, we first measured changes in mitochondrial membrane potential (ΔΨm) using CMTMRos and release of cytochrome c into the cytosol in rat VSMCs and HeLa cells infected with rAd/TIMP-3. rAd/β-galactosidase-infected rat VSMCs and HeLa cells showed polarized Ψm as indicated by a high level of CMTMRos fluorescence (Fig. 3A). As expected, treatment with 2 μm staurosporine, a potent stimulus for mitochondrial activation, resulted in a marked reduction in CMTMRos fluorescence in both rat SMCs and HeLa cells, demonstrating a reduction in Ψm (Fig. 3A). Infection with rAd/TIMP-3 resulted in a similar reduction in Ψm (Fig. 3A). Interestingly, TIMP-3 overexpression resulted in a larger reduction in CMTMRos fluorescence than that evoked by staurosporine. To confirm these observations, efflux of cytochromec into the cytosol, another indicator of mitochondrial activation, was evaluated (28.Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2629) Google Scholar, 34.Yang J. Liu X.S. Bhalla K. Kim C.N. Ibrado A.M. Cai J.Y. Peng T.I. Jones D.P. Wang X.D. Science. 1997; 275: 1129-1132Crossref PubMed Scopus (4410) Google Scholar). Low levels of cytochromec were detectable in cytosolic extracts from HeLa cells (but not from rat VSMCs) infected with the control virus rAd/β-galactosidase (Fig. 3B). However, infection of both VSMCs and HeLa cells with rAd/TIMP-3 dramatically increased the levels of cytosolic cytochrome c (Fig. 3B). Numerous reports have described the role of Bcl-2 as a negative regulator of apoptosis-specific mitochondrial functions (reviewed in Refs. 35.Adams J.M. Cory S. Trends Biochem. Sci. 2001; 26: 61-66Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar and 36.Antonsson B. Martinou J.C. Exp. Cell Res. 2000; 256: 50-57Crossref PubMed Scopus (628) Google Scholar). The ratio between pro- and anti-apoptotic Bcl-2 family members is thought to regulate such functions as permeability transition pore formation and release of apoptosis inducing factor and cytochrome c into the cytosol and ultimately activation of caspase-9 (26.Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X.D. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6230) Google Scholar). To determine whether mitochondrial activation is an important step in the apoptotic cascade initiated by TIMP-3, we used an adenoviral vector to up-regulate Bcl-2. Infection of HeLa cells with rAd/Bcl-2 (but not with rAd/β-galactosidase) strongly elevated levels of Bcl-2 (Fig. 4A). For coexpression studies, we pretreated cells with rAd/Bcl-2 prior to rAd/TIMP-3 infection to allow sufficient inhibitor expression to precede expression of TIMP-3. Pre-infection with rAd/β-galactosidase prior to co-infection with rAd/TIMP-3 failed to rescue cleavage of PARP and FAK (Fig. 4B). Pre-infection of HeLa cells with rAd/Bcl-2 prior to subsequent infection 3 h later with rAd/TIMP-3 blocked TIMP-3-induced cleavage of PARP and FAK death substrates (Fig. 4B). Additionally, overexpression of Bcl-2 inhibited activation of caspase-9, implying that the mitochondria function as regulators of caspase-9 activation in response to TIMP-3 (Fig. 4B). Furthermore, Bcl-2 co-overexpression also inhibited activation of caspase-8, suggesting that TIMP-3-induced caspase-8 activation occurs downstream of mitochondrial activation (Fig. 4B). Although we have demonstrated activation of caspase-8 following adenovirus-mediated overexpression of TIMP-3, we further assessed the direct involvement of this caspase by co-overexpression of CrmA, a viral serpin inhibitor of caspase-8 (37.Bennett M. Macdonald K. Chan S.W. Luzio J.P. Simari R. Weissberg P. Science. 1998; 282: 290-293Crossref PubMed Scopus (655) Google Scholar), using adenovirus-mediated gene transfer. As expected, infection of HeLa cells with rAd/CrmA (but not with rAd/β-galactosidase) resulted in high level expression of CrmA (Fig. 5A). Pre-infection of HeLa cells with rAd/CrmA (but not with rAd/β-galactosidase) blocked cleavage of PARP and FAK induced by TIMP-3 and activation of caspase-8 (Fig. 5B). Furthermore, CrmA expression also inhibited activation of caspase-9 (Fig. 5B), implying that TIMP-3-induced caspase-9 activation occurs downstream of caspase-9. Caspase-8 is typically activated in response to engagement of death receptors, which contain cytoplasmic death domains (38.Muzio M. Chinnaiyan A.M. Kischkel F.C. O'Rourke K. Shevchenko A. Ni J. Scaffidi C. Bretz J.D. Zhang M. Gentz R. Mann M. Krammer P.H. Peter M.E. Dixit V.M. Cell. 1996; 85: 817-827Abstract Full Text Full Text PDF PubMed Scopus (2741) Google Scholar, 39.Srinivasula S.M. Ahmad M. Fernandes-Alnemri T. Litwack G. Alnemri E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14486-14491Crossref PubMed Scopus (482) Google Scholar). Caspase-8 is recruited to activated death receptors through interactions with the adaptor protein FADD, which contains a C-terminal death domain and an N-terminal death effector domain and which is responsible for recruitment and activation of caspase-8 (27.Ashkenazi A. Dixit V. Science. 1998; 277: 1305-1308Crossref Scopus (5151) Google Scholar). To test whether TIMP-3 induces death receptor-