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Tissue Inhibitor of Metalloproteinase-1 Protects Human Breast Epithelial Cells Against Intrinsic Apoptotic Cell Death via the Focal Adhesion Kinase/Phosphatidylinositol 3-Kinase and MAPK Signaling Pathway

2003; Elsevier BV; Volume: 278; Issue: 41 Linguagem: Inglês

10.1074/jbc.m302999200

ISSN

1083-351X

Autores

Xuwen Liu, M. Margarida Bernardo, Rafael Fridman, Hyeong-Reh Choi Kim,

Tópico(s)

Cell death mechanisms and regulation

Resumo

Tissue inhibitor of metalloproteinase (TIMP-1) is a natural protease inhibitor of matrix metalloproteinases (MMPs). Recent studies revealed a novel function of TIMP-1 as a potent inhibitor of apoptosis in mammalian cells. However, the mechanisms by which TIMP-1 exerts its anti-apoptotic effect are not understood. Here we show that TIMP-1 activates cell survival signaling pathways involving focal adhesion kinase, phosphatidylinositol 3-kinase, and ERKs in human breast epithelial cells to TIMP-1. TIMP-1-activated cell survival signaling down-regulates caspase-mediated classical apoptotic pathways induced by a variety of stimuli including anoikis, staurosporine exposure, and growth factor withdrawal. Consistently, down-regulation of TIMP-1 expression greatly enhances apoptotic cell death. In a previous study, substitution of the second amino acid residue threonine for glycine in TIMP-1, which confers selective MMP inhibition, was shown to obliterate its anti-apoptotic activity in activated hepatic stellate cells suggesting that the anti-apoptotic activity of TIMP-1 is dependent on MMP inhibition. Here we show that the same mutant inhibits apoptosis of human breast epithelial cells, suggesting different mechanisms of TIMP-1 regulation of apoptosis depending on cell types. Neither TIMP-2 nor a synthetic MMP inhibitor protects breast epithelial cells from intrinsic apoptotic cell death. Furthermore, TIMP-1 enhances cell survival in the presence of the synthetic MMP inhibitor. Taken together, the present study unveils some of the mechanisms mediating the anti-apoptotic effects of TIMP-1 in human breast epithelial cells through TIMP-1-specific signal transduction pathways. Tissue inhibitor of metalloproteinase (TIMP-1) is a natural protease inhibitor of matrix metalloproteinases (MMPs). Recent studies revealed a novel function of TIMP-1 as a potent inhibitor of apoptosis in mammalian cells. However, the mechanisms by which TIMP-1 exerts its anti-apoptotic effect are not understood. Here we show that TIMP-1 activates cell survival signaling pathways involving focal adhesion kinase, phosphatidylinositol 3-kinase, and ERKs in human breast epithelial cells to TIMP-1. TIMP-1-activated cell survival signaling down-regulates caspase-mediated classical apoptotic pathways induced by a variety of stimuli including anoikis, staurosporine exposure, and growth factor withdrawal. Consistently, down-regulation of TIMP-1 expression greatly enhances apoptotic cell death. In a previous study, substitution of the second amino acid residue threonine for glycine in TIMP-1, which confers selective MMP inhibition, was shown to obliterate its anti-apoptotic activity in activated hepatic stellate cells suggesting that the anti-apoptotic activity of TIMP-1 is dependent on MMP inhibition. Here we show that the same mutant inhibits apoptosis of human breast epithelial cells, suggesting different mechanisms of TIMP-1 regulation of apoptosis depending on cell types. Neither TIMP-2 nor a synthetic MMP inhibitor protects breast epithelial cells from intrinsic apoptotic cell death. Furthermore, TIMP-1 enhances cell survival in the presence of the synthetic MMP inhibitor. Taken together, the present study unveils some of the mechanisms mediating the anti-apoptotic effects of TIMP-1 in human breast epithelial cells through TIMP-1-specific signal transduction pathways. Cell interactions with extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; FAK, focal adhesion kinase; polyHEMA, polyhydroxyethylmethacrylate; mAb, monoclonal antibody; pAb, polyclonal antibody; JNK, c-Jun N-terminal kinase; PI, phosphatidylinositol; AS, antisense; WT, wild type; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; rTIMP, recombinant tissue inhibitor of metalloproteinase.1The abbreviations used are: ECM, extracellular matrix; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; FAK, focal adhesion kinase; polyHEMA, polyhydroxyethylmethacrylate; mAb, monoclonal antibody; pAb, polyclonal antibody; JNK, c-Jun N-terminal kinase; PI, phosphatidylinositol; AS, antisense; WT, wild type; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; rTIMP, recombinant tissue inhibitor of metalloproteinase. greatly influence cell survival, and removal of anchorage-dependent cells from their association with the ECM results in apoptotic cell death, known as anoikis (1Frisch S.M. 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FASEB J. 1998; 12: 1075-1095Crossref PubMed Scopus (695) Google Scholar, 8McCawley L.J. Matrisian L.M. Mol. Med. Today. 2000; 6: 149-156Abstract Full Text Full Text PDF PubMed Scopus (575) Google Scholar, 9Nelson A.R. Fingleton B. Rothenberg M.L. Matrisian L.M. J. Clin. Oncol. 2000; 18: 1135-1149Crossref PubMed Google Scholar) known to accomplish the degradation of ECM components. Four members of the tissue inhibitor of metalloproteinase family (TIMP-1 to -4) have been identified as natural inhibitors of MMPs. Previous studies in our laboratory (10Li G. Fridman R. Kim H.R. Cancer Res. 1999; 59: 6267-6275PubMed Google Scholar) showed that bcl-2 overexpression is associated with enhanced levels of TIMP-1 expression in human breast epithelial cells, suggesting a role for TIMP-1 in apoptosis. Indeed, apoptosis studies showed that TIMP-1 protects against a variety of apoptotic stimuli including anoikis, hydrogen peroxide, x-ray irradiation, and adriamycin treatment (10Li G. Fridman R. Kim H.R. Cancer Res. 1999; 59: 6267-6275PubMed Google Scholar). Furthermore, TIMP-1 inhibition of apoptosis in human breast epithelial cells involves focal adhesion kinase (FAK)-mediated cell survival signaling rather than regulation of cell-ECM interactions via MMP activity. During the past several years, investigators have shown the role of TIMPs in apoptosis regulation. TIMP-1 inhibits apoptosis in many cell types including activated hepatic stellate cells (11Murphy F.R. Issa R. Zhou X. Ratnarajah S. Nagase H. Arthur M.J. Benyon C. Iredale J.P. J. Biol. Chem. 2002; 277: 11069-11076Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 12Yoshiji H. Kuriyama S. Yoshii J. Ikenaka Y. Noguchi R. Nakatani T. Tsujinoue H. Yanase K. Namisaki T. Imazu H. Fukui H. Hepatology. 2002; 36: 850-860Crossref PubMed Google Scholar), TIMP-1-negative Burkitt's lymphoma cell lines (13Guedez L. Stetler-Stevenson W.G. Wolff L. Wang J. Fukushima P. Mansoor A. Stetler-Stevenson M. J. Clin. Invest. 1998; 102: 2002-2010Crossref PubMed Scopus (359) Google Scholar, 14Guedez L. Courtemanch L. Stetler-Stevenson M. Blood. 1998; 92: 1342-1349Crossref PubMed Google Scholar), human breast epithelial cells (10Li G. Fridman R. Kim H.R. Cancer Res. 1999; 59: 6267-6275PubMed Google Scholar), and mammary epithelial cell in transgenic mice (15Alexander C.M. Howard E.W. Bissell M.J. Werb Z. J. Cell Biol. 1996; 135: 1669-1677Crossref PubMed Scopus (203) Google Scholar), whereas TIMP-3 enhances apoptosis in retinal pigment epithelial and MCF-7 cells (16Majid M.A. Smith V.A. Easty D.L. Baker A.H. Newby A.C. Br. J. Ophthalmol. 2002; 86: 97-101Crossref PubMed Scopus (25) Google Scholar, 17Majid M.A. Smith V.A. Easty D.L. Baker A.H. Newby A.C. FEBS Lett. 2002; 529: 281-285Crossref PubMed Scopus (22) Google Scholar), human DLD colon carcinoma cells (18Smith M.R. Kung H. Durum S.K. Colburn N.H. Sun Y. Cytokine. 1997; 9: 770-780Crossref PubMed Scopus (175) Google Scholar, 19Bian J. Wang Y. Smith M.R. Kim H. Jacobs C. Jackman J. Kung H.F. Colburn N.H. Sun Y. Carcinogenesis. 1996; 17: 1805-1811Crossref PubMed Scopus (139) Google Scholar), rat vascular smooth muscle cells (20Baker A.H. Zaltsman A.B. George S.J. Newby A.C. J. Clin. Invest. 1998; 101: 1478-1487Crossref PubMed Scopus (411) Google Scholar, 21Bond M. Murphy G. Bennett M.R. Newby A.C. Baker A.H. J. Biol. Chem. 2002; 277: 13787-13795Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), melanoma SK-Mel-5 and A2058 cells (22Ahonen M. Baker A.H. Kahari V.M. Cancer Res. 1998; 58: 2310-2315PubMed Google Scholar), and cancer cell lines such as HeLa (21Bond M. Murphy G. Bennett M.R. Newby A.C. Baker A.H. J. Biol. Chem. 2002; 277: 13787-13795Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) and HT1080 cells (23Baker A.H. George S.J. Zaltsman A.B. Murphy G. Newby A.C. Br. J. Cancer. 1999; 79: 1347-1355Crossref PubMed Scopus (244) Google Scholar). TIMP-3 induction of human colon carcinoma cell apoptosis is mediated by protecting tumor necrosis factor-α receptor on the cell surface against MMP-mediated cleavage (18Smith M.R. Kung H. Durum S.K. Colburn N.H. Sun Y. Cytokine. 1997; 9: 770-780Crossref PubMed Scopus (175) Google Scholar, 19Bian J. Wang Y. Smith M.R. Kim H. Jacobs C. Jackman J. Kung H.F. Colburn N.H. Sun Y. Carcinogenesis. 1996; 17: 1805-1811Crossref PubMed Scopus (139) Google Scholar). In contrast, a recent study (24Fata J.E. Leco K.J. Voura E.B. Yu H.Y. Waterhouse P. Murphy G. Moorehead R.A. Khokha R. J. Clin. Invest. 2001; 108: 831-841Crossref PubMed Scopus (144) Google Scholar) showed increased epithelial cell apoptosis in TIMP-3-deficient mice during mammary gland involution, suggesting an anti-apoptotic activity of TIMP-3. Both pro- and anti-apoptotic activities of TIMP-2 and TIMP-4 have been reported depending on the cell type. For example, TIMP-2 and TIMP-4 enhance apoptosis in human T lymphocytes and cardiac fibroblasts, respectively (25Lim M.S. Guedez L. Stetler-Stevenson W.G. Stetler-Stevenson M. Ann. N. Y. Acad. Sci. 1999; 878: 522-523Crossref PubMed Scopus (33) Google Scholar, 26Tummalapalli C.M. Heath B.J. Tyagi S.C. J. Cell. Biochem. 2001; 80: 512-521Crossref PubMed Scopus (80) Google Scholar), whereas TIMP-2 enhances cell survival of metanephric mesenchymes (27Barasch J. Yang J. Qiao J. Tempst P. Erdjument-Bromage H. Leung W. Oliver J.A. J. Clin. Invest. 1999; 103: 1299-1307Crossref PubMed Scopus (94) Google Scholar) and B16F10 melanoma cells (28Valente P. Fassina G. Melchiori A. Masiello L. Cilli M. Vacca A. Onisto M. Santi L. Stetler-Stevenson W.G. Albini A. Int. J. Cancer. 1998; 75: 246-253Crossref PubMed Scopus (249) Google Scholar), and TIMP-4 protects human breast cancer cells, as well as mammary tumor xenografts, from apoptotic cell death (29Jiang Y. Wang M. Celiker M.Y. Liu Y.E. Sang Q.X. Goldberg I.D. Shi Y.E. Cancer Res. 2001; 61: 2365-2370PubMed Google Scholar). Although it has become clear that TIMPs are critical determinants for cell survival, the signaling mechanisms by which TIMPs control cell survival in a cell type-specific manner remain undefined. Also, it is still controversial whether the anti-apoptotic effect of TIMP-1 depends on its ability to inhibit MMP activity. In Burkitt's lymphoma cell lines TIMP-1 inhibition of apoptosis was shown to be independent of its inhibitory activity (13Guedez L. Stetler-Stevenson W.G. Wolff L. Wang J. Fukushima P. Mansoor A. Stetler-Stevenson M. J. Clin. Invest. 1998; 102: 2002-2010Crossref PubMed Scopus (359) Google Scholar, 14Guedez L. Courtemanch L. Stetler-Stevenson M. Blood. 1998; 92: 1342-1349Crossref PubMed Google Scholar). However, a mutant TIMP-1 devoid of inhibitory activity against certain MMPs had no anti-apoptotic effect in hepatic stellate cells (11Murphy F.R. Issa R. Zhou X. Ratnarajah S. Nagase H. Arthur M.J. Benyon C. Iredale J.P. J. Biol. Chem. 2002; 277: 11069-11076Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar). In this study, we further examined the TIMP-1-mediated cell survival pathways and its significance in TIMP-1 regulation of apoptosis using breast epithelial MCF10A cells engineered to express high or low levels of TIMP-1. We also investigated whether TIMP-1 inhibition of apoptosis in these cells was dependent or independent of MMP inhibitory activity. Cell Culture—Immortalized nonmalignant human breast epithelial MCF10A cells were cultured in Dulbecco's modified Eagle's medium/F-12 medium supplemented with 5% horse serum, 0.5 μg/ml hydrocortisone, 10 μg/ml insulin, 20 ng/ml epidermal growth factor, 0.1 μg/ml cholera enterotoxin, 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mml-glutamine, and 0.5 μg/ml Fungizone in a 95% air and 5% CO2 incubator at 37 °C (10Li G. Fridman R. Kim H.R. Cancer Res. 1999; 59: 6267-6275PubMed Google Scholar). The human embryonic kidney cell line 293 (30Graham F.L. Smiley J. Russell W.C. Nairn R. J. Gen. Virol. 1977; 36: 59-74Crossref PubMed Scopus (3446) Google Scholar) was maintained in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. Reagents—Anti-TIMP-1 Ab-2 (clone 102 D1) monoclonal antibodies (mAb) were purchased from NeoMarkers, Inc. (Fremont, CA). Anti-human β-actin mAb, anti-mouse IgG peroxidase conjugate, and anti-Rabbit IgG peroxidase conjugate antibodies were purchased from Sigma. Anti-p44/42 MAPK, anti-phospho-p44/42 MAPK (Thr-202/Tyr-204), and anti-p38 MAPK polyclonal antibodies (pAb) were purchased from Cell Signaling Technology, Inc. Anti-active JNK pAb (pTPpY) was purchased from Promega. Anti-JNK1 (C-17) goat affinity purified pAb and anti-PI 3-kinase p85α (Z-8) pAb were purchased from Santa Cruz Biotechnology, Inc. Anti-phosphotyrosine (clone 4G10) mAb was purchased from Upstate USA Inc. MAPK kinase inhibitor PD98059, SB202474 (PD-, a negative control for PD98059), PI 3-kinase inhibitor wortmannin (Wor), LY294002 (LY), and LY303511 (LY-, a negative control for LY294002) were purchased from Calbiochem-Novabiochem. Recombinant Proteins, Enzymes, and Protein Inhibitors—Human recombinant TIMP-1, TIMP-2, pro-MMP-2, and pro-MMP-9 were produced using a vaccinia mammalian cell expression system as described previously (10Li G. Fridman R. Kim H.R. Cancer Res. 1999; 59: 6267-6275PubMed Google Scholar, 31Fridman R. Fuerst T.R. Bird R.E. Hoyhtya M. Oelkuct M. Kraus S. Komarek D. Liotta L.A. Berman M.L. Stetler-Stevenson W.G. J. Biol. Chem. 1992; 267: 15398-15405Abstract Full Text PDF PubMed Google Scholar, 32Olson M.W. Gervasi D.C. Mobashery S. Fridman R. J. Biol. Chem. 1997; 272: 29975-29983Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). Heat-activated human stromelysin-1 (MMP-3) was a generous gift from Dr. Paul Cannon (Center for Bone and Joint Research, Palo Alto, CA). Recombinant human active matrilysin (MMP-7) was obtained from Chemicon. Batimastat (BB-94), a hydroxamate-based broad spectrum MMP inhibitor (33Rasmussen H.S. McCann P.P. Pharmacol Ther. 1997; 75: 69-75Crossref PubMed Scopus (348) Google Scholar), was obtained from British Biotech (Annapolis, MD). Plasmid Constructs and Transfection—Antisense TIMP-1 construct (AS TIMP-1) was generated by EcoRI digestion and re-ligation from its sense construct containing the human full-length TIMP-1 cDNA and the neomycin resistance gene, under control of the long terminal repeats of the Moloney murine sarcoma virus (kindly provided by Dr. M. Johnson at Northwestern University, Chicago, IL). To construct a TIMP-1/FLAG fusion cDNA, BamHI sites were introduced to both ,5′ and 3′ ends of the human full-length TIMP-1 cDNA fragment by the polymerase chain reaction using primers TIMP-1AA BamHI, 5′-AAG GAT CCA TGG CCC CCT TTG AGC CCC TGG-3′, and TIMP1-207AA BamHIA, 5′-AAG GAT CCG GCT ATC TGG GAC CGC AGG GAC-3′. The fragment was digested with BamHI and cloned into the BamHI site in the p3XFLAG-CMV-14 vector (Sigma). To substitute Thr-2 with Gly at the mature TIMP-1 protein (after cleavage of the signal peptide), site-directed mutagenesis was performed using primers T1T2G, 5′-CCA GCA GGG CCT GCG GGT GTG TCC CAC CCC A-3′, and T1T2GAA, 5′-TGG GGT GGG ACA CAC CCG CAG GCC CTG CTG G-3′, and a TIMP-1/FLAG construct as a template as instructed by the manufacturer (Stratagene). DNA sequencing analysis confirmed the fidelity of the constructs. Hereafter, the wild type TIMP-1/FLAG and T2G mutated TIMP-1/FLAG constructs are referred to as WT TIMP-1/FLAG and T2G TIMP-1/FLAG. Generation of TIMP-1 overexpressing MCF10A clones was described previously (10Li G. Fridman R. Kim H.R. Cancer Res. 1999; 59: 6267-6275PubMed Google Scholar). AS TIMP-1 construct was transfected into MCF10A cells as previously described (10Li G. Fridman R. Kim H.R. Cancer Res. 1999; 59: 6267-6275PubMed Google Scholar), and WT TIMP-1/FLAG, T2G TIMP-1/FLAG, and FLAG control constructs were transfected, respectively, into both MCF10A cells and human embryonic kidney 293 cells using LipofectAMINE2000 (Invitrogen) according to the manufacturer's protocol. The cells were subjected to 400 μg/ml G418 antibiotic selection for 14 days, and at least six colonies from each transfection were isolated for further analysis. Immunoblot Analysis—Cell lysates were obtained by lysing the cell monolayer with SDS lysis buffer (2% SDS, 125 mm Tris-HCl, pH 6.8, and 20% glycerol). The lysates were boiled for 5 min and then clarified by a 20-min centrifugation at 4 °C. Protein concentration was measured using the BCA protein assay reagent (Pierce). Equal amount of protein samples in SDS sample buffer (1% SDS, 62.5 mm Tris-HCl, pH 6.8, 10% glycerol, 5% β-mercaptoethanol, and 0.05% bromphenol blue) were boiled for 5 min and subjected to reducing SDS-PAGE. After electrophoresis, the proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 5% nonfat dry milk in T-TBS (100 mm Tris-HCl, pH 7.5, 150 mm NaCl, 0.02% NaN3, and 0.2% Tween 20) for 1 h at room temperature. The membranes were incubated with T-TBS containing 5% milk and in the appropriate antibodies. After three washes with T-TBS, the blot was incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies. The antigen was detected using the Western blot Chemiluminescence Reagent Plus (PerkinElmer Life Sciences), according to the manufacturer's instruction. Apoptosis Induction—Apoptosis was induced by culturing cells in serum-free medium (growth factor withdrawal) or by treatment with staurosporine. Anoikis (apoptosis induced by loss of cell adhesion) was induced by culturing cells in polyHEMA-coated 6-well plates, which prevents cell adhesion (34Kim H.R. Lin H.M. Biliran H. Raz A. Cancer Res. 1999; 59: 4148-4154PubMed Google Scholar). Evaluation of TIMP-1 Effects on Cell Survival Using MTT Assay—Cell number was determined by an indirect colorimetric immunoassay (MTT assay). In brief, the cells (4 × 103 cells/well) were plated in a 96-well culture plate overnight followed by culture in serum-free medium for 48 h in the absence or presence of 500 ng/ml TIMP-1, 500 ng/ml TIMP-2, 10 μm PD98059, 10 μm SB202474, 200 nm wortmannin, 50 nm LY294002, 50 nm LY303511, 5 μm BB-94, or vehicle (Me2SO). MTT (0.5 mg/ml) was then added, and the plates were incubated for 4 h at 37 °C. The cellular formazan was extracted with acidic isopropanol, and the absorbance of the converted dye was measured at a wavelength of 570 nm, with background subtraction at 650 nm, using a Bio-Rad Benchmark microplate reader (Bio-Rad). DEVDase Activity Assay—Cells were lysed in cell extract buffer (150 mm NaCl, 50 mm Tris-HCL, pH 7.5, 0.5 mm EDTA, and 0.5% Nonidet P-40). Lysates were incubated on ice for 30 min and centrifuged at 15,000 × g for 10 min. Fifty μl of the cytosolic fraction were incubated for 60 min at 37 °C in a total volume of 200 μl of caspase buffer (20 mm HEPES, pH 7.5, 50 mm NaCl, and 2.5 mm dithiothreitol), containing 25 μm capase-3 substrate acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) (BioSource International, Inc., Camarillo, CA). 7-Amino-4-methylcoumarin fluorescence, released by active caspase, was measured at 460 nm using 360-nm excitation wavelength by a Spectra Maxi Germini fluorescence plate reader (Molecular Devices, Menlo Park, CA). Caspase activity was normalized per microgram of protein determined by the BCA protein assay kit (Pierce). Detection of PI 3-Kinase Phosphorylation—Cells were lysed in a radioimmune precipitation assay buffer (20 mm HEPES, pH 7.4, 100 mm NaCl, 0.1% deoxycholic acid, 10% Nonidet P-40, 1 mm EDTA, 1 mm EGTA, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 mm sodium vanadate, and 50 mm sodium fluoride) at 4 °C for 30 min. The lysates were centrifuged for 15 min at 12,000 × g to remove debris and immunoprecipitated using an anti-phosphotyrosine mAb (clone 4G10; Upstate USA Inc.) and immobilized protein G-agarose beads (Pierce). Immunoprecipitates were washed three times with radioimmune precipitation assay buffer and resolved by 8% reducing SDS-PAGE. Tyrosine-phosphorylated PI 3-kinase proteins were detected by immunoblotting using an anti-PI 3-kinase p85α pAb (Santa Cruz Biotechnology, Inc.). Purification of FLAG-tagged TIMP-1 Proteins—Human embryonic kidney 293 cells that stably expressed WT TIMP-1/FLAG or T2G TIMP-1/FLAG, were cultured in phenol red-free conditioned medium for 48 h. The conditioned medium was clarified by centrifugation and concentrated 5-fold using Centricon YM-10 (Millipore Corp.). Twenty ml of the conditioned medium were incubated with 120 μl of EZview Red anti-FLAG M2 affinity gel (Sigma) at 4 °C. After the resin was collected and washed with TBS, the proteins were eluted with TBS buffer containing 3× FLAG peptide (150 μg/ml). Proteins were then analyzed by reducing SDS-PAGE and stained with Simply Blue SafeStain (Invitrogen). The fidelity of the T → G TIMP-1 mutation protein was confirmed by N-terminal microsequencing of affinity purified T2G TIMP-1/FLAG (Proseq, Inc., Boxford, MA). The concentrations of wild type TIMP-1/FLAG and T2G TIMP-1/FLAG were estimated by densitometry after immunoblot analysis and by titration with a MMP-9 solution with known concentration. MMP Inhibition Studies—The inhibitory activity of WT TIMP-1/FLAG, T2G TIMP-1/FLAG, and TIMP-1 produced by the vaccinia expression system was tested with MMP-2, MMP-9, MMP-3, and MMP-7. The active enzymes (MMP-2 and MMP-9) were obtained by incubating pro-MMP-2 and pro-MMP-9 with 1 mm p-aminophenylmercuric acetate dissolved in 200 mm Tris at 37 °C in Buffer A (50 mm HEPES, 150 mm NaCl, 5 mm CaCl2, 0.01% Brij-35, pH 7.5). The activation reaction was monitored with the fluorescence-quenched peptide MOCAcPLGLA2pr(Dnp)AR-NH2, as will be described below. p-Aminophenylmercuric acetate was removed by dialysis against collagenase buffer (50 mm Tris, 150 mm NaCl, 5 mm CaCl2, 0.02% Brij-35, pH 7.5) at 4 °C. The enzyme concentrations were determined by active-site titration with TIMP-1 solutions of known concentration. The concentration of MMP-3 and MMP-7 was determined by titration with TIMP-1 and TIMP-2, respectively. The enzymatic assays were carried out at 25 °C, using a Photon Technology International spectrofluorometer, equipped with the RadioMaster™ and FeliX™ hardware and software, respectively. Excitation and emission band passes of 1 and 3 nm, respectively, were used. Fluorescence measurements were taken every 4 s. MMP-2, MMP-9, and MMP-7 enzymatic activities were monitored with the fluorogenic synthetic substrate MOCAcPLGLA2pr(Dnp)AR-NH2, at excitation and emission wavelengths of 328 and 393 nm, respectively. MOCAcRPKPVE(Nva)WRK(Dnp)NH2 was the substrate used to monitor MMP-3 activity at λex = 325 and λem = 393 nm. All fluorogenic substrates were obtained from Peptides International Inc., Louisville, KY. MMP inhibition was monitored after incubating the enzyme (0.2-0.7 nm) with increasing concentrations of the inhibitor at 37 °C in Buffer A for at least 1 h. The remaining enzymatic activity was monitored with the appropriate fluorogenic substrate (5-7 μm) at 25 °C. The initial rates of the enzyme reaction with the fluorogenic substrate were determined by linear regression analysis of the fluorescence versus time traces using FeliX™. These rates were analyzed according to a competitive model of inhibition (35Segel I.H. Enzyme Kinetics. John Wiley & Sons, Inc., New York1993: 100-120Google Scholar) using the program SCIENTIST (MicroMath Scientific Software, Salt Lake City, UT), yielding K i(app) values, because TIMP-1 has been shown to be a tight, slow binding inhibitor of the MMPs (32Olson M.W. Gervasi D.C. Mobashery S. Fridman R. J. Biol. Chem. 1997; 272: 29975-29983Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar, 36Olson M.W. Bernardo M.M. Pietila M. Gervasi D.C. Toth M. Kotra L.P. Massova I. Mobashery S. Fridman R. J. Biol. Chem. 2000; 275: 2661-2668Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), and the association and dissociation rate constants (k on and k off, respectively) were not determined. Down-regulation of TIMP-1 Expression in MCF10A Cells and Its Effects on Apoptosis—Our previous study (10Li G. Fridman R. Kim H.R. Cancer Res. 1999; 59: 6267-6275PubMed Google Scholar) showed that TIMP-1 overexpression protects MCF10A cells from apoptosis induced by a variety of stimuli. To further investigate TIMP-1 regulation of human breast epithelial cell survival, we generated MCF10A clones in which endogenous TIMP-1 expression is down-regulated by an antisense construct (AS TIMP-1 MCF10A). Immunoblot analysis confirmed significant down-regulation of TIMP-1 in AS TIMP-1 MCF10A cells and up-regulation of TIMP-1 in TIMP-1 overexpressing MCF10A clone 29 (T29) cells, when compared with the neo-control MCF10A cells (Fig. 1, A and B). To test the consequences of different levels of TIMP-1 expression on cell death, we compared the percentage of cell survival of neo-MCF10A (Neo), AS TIMP-1 MCF10A (AS), and TIMP-1 overexpressing MCF10A clone 29 (T29) cells, following loss of cell adhesion. As shown in Fig. 1C, only ∼25% of AS TIMP-1 MCF10A cells remained viable after 18 h of anoikis, whereas ∼80% of control vector-transfected and ∼95% of T29 MCF10A cells were viable. After 48 h, almost none of AS TIMP-1 MCF10A and only ∼15% neo-MCF10A cells survived, whereas ∼85% of T29 MCF10A cells remained viable. These results further confirmed our previous finding (10Li G. Fridman R. Kim H.R. Cancer Res. 1999; 59: 6267-6275PubMed Google Scholar) that TIMP-1 levels are critical determinants for apoptosis sensitivity in human breast epithelial cells. We next examined whether exogenously added TIMP-1 could protect AS TIMP-1 MCF10A cells from cell death. We also tested TIMP-2, a close homologue of TIMP-1, for its effect on apoptosis. As shown in Fig. 1D, TIMP-1 significantly enhanced AS TIMP-1 MCF10A cell survival (∼5-fold increase) following growth factor withdrawal, whereas TIMP-2 had little effect. Compared with AS TIMP-1 MCF10A cells, TIMP-1 effect on neo-MCF10A cell survival was less drastic (∼2-fold). This is likely to be because of the fact that the neo-MCF10A cells express higher levels of endogenous TIMP-1 than AS TIMP-1 MCF10A cells (Fig. 1, A and B). Considering that both TIMP-1 and TIMP-2 inhibit MMP activity, these results suggest that inhibition of MMP activity is unlikely to be a key mechanism for the anti-apoptotic effects of TIMP-1. TIMP-1 Regulates Caspase Activity—We showed previously (10Li G. Fridman R. Kim H.R. Cancer Res. 1999; 59: 6267-6275PubMed Google Scholar) that TIMP-1 inhibits cleavage of poly(ADP-ribose) polymerase following anoikis induction, suggesting that TIMP-1 regulates caspase-mediated apoptotic pathways. Because caspases, including caspase-3 and -7, cleave poly(ADP-ribose) polymerase at the DEVD216G site, we measured DEVDase activity. As shown in Fig. 2A, DEVDase activity greatly increased in AS TIMP-1 MCF10A cells after 18 h of anoikis induction, whereas neo-MCF10A cells had only a mild induction of DEVDase activity. At 24 and 48 h of anoikis, both neo-MCF10A and AS TIMP-1 MCF10A cells exhibited high levels of activity. In contrast, there was no significant induction of DEVDase activity in the TIMP-1 overexpressing T29 MCF10A cells. We then examined whether TIMP-1 could inhibit caspase activity induced by staurosporine, an apoptotic agent that rapidly decreases the transmembrane potential of the mitochondria, resulting in activation of the intrinsic caspase cascade (37Nomura K. Imai H. Koumura T. Arai M. Nakagawa Y. J. Biol. Chem. 1999; 274: 29294-29302Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar, 38Samali A. Cai J. Zhivotovsky B. Jones D.P. Orrenius S. EMBO J. 1999; 18: 2040-2048Crossref PubMed Scopus (453) Google Scholar). Down-regulation of TIMP-1 expression significantly enhanced staurosporine-induced DEVDase activity in AS TIMP-1 MCF10A cells, and TIMP-1 overexpression effectively prevented DEVDase activity in T29 MCF10A cells (Fig. 2B). These studies demonstrate that TIMP-1 regulates classical apoptotic pathways involving caspases. TIMP-1 Activation of PI 3-Kinase and MAPK Survival Signaling Pathways Are Critical for Its Apopt

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