Cartilage Oligomeric Matrix Protein Protects Cells against Death by Elevating Members of the IAP Family of Survival Proteins

Cartilage oligomeric matrix protein (COMP) is a component of cartilage, synovium, ligament, and tendon, yet its normal function is largely unknown. To identify its function we have expressed it in 293 and HeLa cell lines and in primary human chondrocytes. We find that COMP protects these cells against death, either in the presence or absence of tumor necrosis factor α and is able to block activation of caspase 3, a critical effector caspase. This effect appears to be mediated by the IAP (inhibitor of apoptosis protein) family of anti-apoptotic proteins because the levels of XIAP, survivin, cIAP1 and cIAP2 are significantly elevated in the COMP-expressing cells and down-regulation of survivin and XIAP protein levels by small interfering RNAs blocks the ability of COMP to enhance survival. The mRNAs for most of the IAP family members were not increased by COMP, indicating that a translational/post-translational mechanism was involved in their induction. However, in both HeLa cells and chondrocytes, COMP induced survivin mRNA by 5-fold. Thus survivin is the first gene identified to be up-regulated transcriptionally by COMP. The carboxyl-terminal half of the protein comprising the type 3 repeats and the RGD sequence (CaCTD domain) was sufficient to promote survival and to elevate the IAPs. Further, an RGD peptide was able to block the prosurvival effect of COMP and the induction of XIAP and survivin, indicating that survival is likely mediated through integrin signaling. These data point to a new role for COMP in protecting cells against death. Cartilage oligomeric matrix protein (COMP) is a component of cartilage, synovium, ligament, and tendon, yet its normal function is largely unknown. To identify its function we have expressed it in 293 and HeLa cell lines and in primary human chondrocytes. We find that COMP protects these cells against death, either in the presence or absence of tumor necrosis factor α and is able to block activation of caspase 3, a critical effector caspase. This effect appears to be mediated by the IAP (inhibitor of apoptosis protein) family of anti-apoptotic proteins because the levels of XIAP, survivin, cIAP1 and cIAP2 are significantly elevated in the COMP-expressing cells and down-regulation of survivin and XIAP protein levels by small interfering RNAs blocks the ability of COMP to enhance survival. The mRNAs for most of the IAP family members were not increased by COMP, indicating that a translational/post-translational mechanism was involved in their induction. However, in both HeLa cells and chondrocytes, COMP induced survivin mRNA by 5-fold. Thus survivin is the first gene identified to be up-regulated transcriptionally by COMP. The carboxyl-terminal half of the protein comprising the type 3 repeats and the RGD sequence (CaCTD domain) was sufficient to promote survival and to elevate the IAPs. Further, an RGD peptide was able to block the prosurvival effect of COMP and the induction of XIAP and survivin, indicating that survival is likely mediated through integrin signaling. These data point to a new role for COMP in protecting cells against death. Cartilage oligomeric matrix protein (COMP), 2The abbreviations used are:COMPcartilage oligomeric matrix proteinPSACHpseudoachondroplasiaTNFtumor necrosis factorsiRNAsmall interfering RNAPBSphosphate-buffered salineTUNELdeoxynucleotidyltransferase-mediated dUTP nick end labelingRTreverse transcriptionActDactinomycin D. also known as thrombospondin 5, is a 524-kDa pentameric glycoprotein expressed primarily in cartilage, tendon, ligament, and synovium (1Adams J.C. Annu. Rev. Cell Dev. Biol. 2001; 17: 25-51Crossref PubMed Scopus (328) Google Scholar, 2Lawler J. Chen H. Encyclopedia of Molecular Medicine. John Wiley & Sons, Inc., New York2002: 481-484Google Scholar). It is an extracellular matrix protein that has been shown to bind collagens type I, II, and IX, as well as fibronectin, the matrilins, and aggrecan (3Rosenberg K. Olsson H. Morgelin M. Heinegard D. J. Biol. Chem. 1998; 273: 20397-20403Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 4Holden P. Meadows R.S. Chapman K.L. Grant M.E. Kadler K.E. Briggs M.D. J. Biol. Chem. 2001; 276: 6046-6055Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 5DiCesare P.A. Chen F.S. Morgelin M. Carlson C.S. Leslie M.P. Perris R. Fang C. Matrix Biol. 2002; 21: 461-470Crossref PubMed Scopus (96) Google Scholar, 6Mann H.H. Ozbek S. Engel J. Paulsson M. Wagener R. J. Biol. Chem. 2004; 279: 25294-25298Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 53Chen F.H. Herndon M.E. Patel N. Hecht J.T. Tuan R.S. Lawler J.J. J. Biol. Chem. 2007; 282: 24591-24598Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The mature protein is a pentamer of identical subunits where each monomer is linked to its neighbor via a disulfide bond located at the very amino terminus of the protein (7Hedbom E. Antonsson P. Hjerpe A. Aeschlimann D. Paulsson M. Rosa-Pimente E. Sommarin Y. Wendel M. Oldberg A. Heinegard D. J. Biol. Chem. 1992; 267: 6132-6136Abstract Full Text PDF PubMed Google Scholar, 8Morgelin M. Heinegard D. Engel J. Paulsson M. J. Biol. Chem. 1992; 267: 6137-6141Abstract Full Text PDF PubMed Google Scholar, 9Efimov V.P. Lustig A. Engel J. FEBS Lett. 1994; 341: 54-58Crossref PubMed Scopus (72) Google Scholar). Interestingly, the amino-terminal domain of each monomer is also α-helical, and each helix also interacts with its neighbor via a coiled-coil structure (9Efimov V.P. Lustig A. Engel J. FEBS Lett. 1994; 341: 54-58Crossref PubMed Scopus (72) Google Scholar, 10Malashkevich V.N. Kammerer R.A. Efimov V.P. Schulthess T. Engel J. Science. 1996; 274: 761-765Crossref PubMed Scopus (271) Google Scholar). This interaction results in the formation of a hydrophobic pore within this oligomerization region, which is large enough to bind small aliphatic compounds such as retinol or vitamin D (11Guo Y. Bozic D. Malashkevich V.N. Kammerer R.A. Schulthess T. Engel J. EMBO J. 1998; 17: 5265-5272Crossref PubMed Scopus (64) Google Scholar), although the role of this binding is not yet clear. cartilage oligomeric matrix protein pseudoachondroplasia tumor necrosis factor small interfering RNA phosphate-buffered saline deoxynucleotidyltransferase-mediated dUTP nick end labeling reverse transcription actinomycin D The central domain of COMP contains both epidermal growth factor-like repeats (type 2 repeats) and a calcium-binding region (type 3 repeats), which are present in all the thrombospondins (1Adams J.C. Annu. Rev. Cell Dev. Biol. 2001; 17: 25-51Crossref PubMed Scopus (328) Google Scholar, 2Lawler J. Chen H. Encyclopedia of Molecular Medicine. John Wiley & Sons, Inc., New York2002: 481-484Google Scholar). Missense mutations in the calcium-binding region of COMP gene are responsible for at least two forms of skeletal dysplasias in humans, multiple epiphysial dysplasia (MED) and pseudoachondroplasia (PSACH) (12Briggs M.D. Hoffman S.M. King L.M. Olsen A.S. Mohrenweiser H. Leroy J.G. Mortier G.R. Rimoin D.L. Lachman R.S. Gaines E.S. Cekleniak J.A. Knowlton R.G. Cohn D.H. Nat. Genet. 1995; 10: 330-336Crossref PubMed Scopus (431) Google Scholar, 13Briggs M.D. Mortier G.R. Cole G.W. King L.M. Golik S.S. Bonaventure J. Nuytinck L. De Paepe A. Leroy J.G. Biesecker L. Lipson M. Wilcox W.R. Lachman R.S. Rimoin D.L. Knowlton R.G. Cohn D.H. Am. J. Hum. Genet. 1998; 62: 311-319Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 14Hecht J.T. Nelson L.D. Crowder E. Wang Y. Elder F.B. Harrison W.R. Francomano C.A. Prange C.K. Lennon G.G. Deere M. Lawler J. Nat. Genet. 1995; 10: 325-329Crossref PubMed Scopus (315) Google Scholar, 15Deere M. Sanford T. Ferguson H.L. Daniels K. Hecht J.T. Am. J. Med. Genet. 1998; 80: 510-513Crossref PubMed Scopus (60) Google Scholar). As a corollary to these genetic studies, it has been demonstrated that COMP is subjected to cleavage via extracellular proteases in the cartilage of patients suffering from various forms of arthritis (16Clark A.G. Jordan J.M. Vilim V. Renner J.B. Dragomir A.D. Luta G. Kraus V.B. Arthritis Rheum. 1999; 42: 2356-2364Crossref PubMed Scopus (232) Google Scholar, 17Lohmander L.S. Ionescu M. Jugessur H. Poole A.R. Arthritis Rheum. 1999; 42: 534-544Crossref PubMed Scopus (217) Google Scholar, 18Petersson I.F. Boegard T. Svensson B. Heinegard D. Saxne T. Br. J. Rheumatol. 1998; 37: 46-50Crossref PubMed Scopus (170) Google Scholar, 19Neidhart M. Hauser N. Paulsson M. Dicesare P.E. Michel B.A. Hauselmann H.J. Br. J. Rheumatol. 1997; 36: 1151-1160Crossref PubMed Scopus (214) Google Scholar). Some of the resulting fragments of COMP that are produced from this cleavage appear to be stable and can be easily identified in both the synovival fluid and serum of these patients (16Clark A.G. Jordan J.M. Vilim V. Renner J.B. Dragomir A.D. Luta G. Kraus V.B. Arthritis Rheum. 1999; 42: 2356-2364Crossref PubMed Scopus (232) Google Scholar, 17Lohmander L.S. Ionescu M. Jugessur H. Poole A.R. Arthritis Rheum. 1999; 42: 534-544Crossref PubMed Scopus (217) Google Scholar, 18Petersson I.F. Boegard T. Svensson B. Heinegard D. Saxne T. Br. J. Rheumatol. 1998; 37: 46-50Crossref PubMed Scopus (170) Google Scholar, 19Neidhart M. Hauser N. Paulsson M. Dicesare P.E. Michel B.A. Hauselmann H.J. Br. J. Rheumatol. 1997; 36: 1151-1160Crossref PubMed Scopus (214) Google Scholar), yet it is not known what role these fragments may play, if any, in the pathology of arthritis. In cartilage, COMP is expressed in both the developing and mature tissue (7Hedbom E. Antonsson P. Hjerpe A. Aeschlimann D. Paulsson M. Rosa-Pimente E. Sommarin Y. Wendel M. Oldberg A. Heinegard D. J. Biol. Chem. 1992; 267: 6132-6136Abstract Full Text PDF PubMed Google Scholar, 20DiCesare P.E. Morgelin M. Carlson C.S. Pasumarti S. Paulsson M. J. Ortho. Res. 1995; 13: 422-428Crossref PubMed Scopus (94) Google Scholar, 21Kipnes J. Carlberg A.L. Loredo G.A. Lawler J. Tuan R.S. Hall D.J. Osteoarthr. Cartil. 2003; 11: 442-454Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In adult cartilage, COMP has been shown to be located primarily in the interterritorial matrix between chondrocytes (22Murphy J.M. Heinegard D. McIntosh A. Sterchi D. Barry F.P. Matrix Biol. 1999; 18: 487-497Crossref PubMed Scopus (49) Google Scholar). As such it has been proposed that COMP may directly interact with cells. COMP has been shown to aid in the attachment of cells to surfaces (23Riessen R. Fenchel M. Chen H. Axel D.I. Karsch K.R. Lawler J. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 47-54Crossref PubMed Scopus (104) Google Scholar, 24Chen F.H. Thomas A.O. Hecht J.T.T. Goldring M.B. Lawler J. J. Biol. Chem. 2005; 280: 32655-32661Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) and does so via an interaction with integrins (24Chen F.H. Thomas A.O. Hecht J.T.T. Goldring M.B. Lawler J. J. Biol. Chem. 2005; 280: 32655-32661Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). A COMP mutation in the type 3 repeat, which occurs in 30% of patients with PSACH, abolishes the ability of COMP to aid in attachment (24Chen F.H. Thomas A.O. Hecht J.T.T. Goldring M.B. Lawler J. J. Biol. Chem. 2005; 280: 32655-32661Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). It has also been shown that COMP supports the migration of cells in vitro (23Riessen R. Fenchel M. Chen H. Axel D.I. Karsch K.R. Lawler J. Arterioscler. Thromb. Vasc. Biol. 2001; 21: 47-54Crossref PubMed Scopus (104) Google Scholar). In terms of the extracellular matrix protein, it is presumed that COMP may play a role in the structural integrity of cartilage via its interaction with other extracellular matrix proteins such as the collagens and fibronectin. However, it is not yet known whether COMP plays any role in maintaining the stability of cartilage. Additional studies have indicated that COMP can inhibit cell proliferation while enhancing in vitro chondrogenesis (21Kipnes J. Carlberg A.L. Loredo G.A. Lawler J. Tuan R.S. Hall D.J. Osteoarthr. Cartil. 2003; 11: 442-454Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Finally, expression of COMP in the presence of BMP2 stimulation can lead to a late increase in apoptosis (21Kipnes J. Carlberg A.L. Loredo G.A. Lawler J. Tuan R.S. Hall D.J. Osteoarthr. Cartil. 2003; 11: 442-454Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). This latter finding is consistent with the fact that the mutant COMP proteins in multiple epiphysial dysplasia and PSACH appear to mediate apoptosis of chondrocytes in cartilage in vivo (25Hecht J.T. Montufar-Solis D. Decker G. Lawler J. Daniels K. Duke P.J. Matrix Biol. 1998; 1: 625-633Crossref Scopus (81) Google Scholar, 26Hecht J.T. Makitie O. Hayes E. Haynes R. Susic M. Montufar-Solis D. Duke P.J. Cole W.G. J. Ortho. Res. 2004; 22: 759-767Crossref PubMed Scopus (70) Google Scholar, 27Hashimoto Y. Tomiyama T. Yamano Y. Mori H. Am. J. Pathol. 2003; 163: 101-110Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). To better understand the function of COMP, we began transient expression studies in both transformed and nontransformed human cells with the goal of assessing its effects on cell viability. We find that COMP is a potent suppressor of apoptosis in both primary human chondrocytes and transformed cells and that it accomplishes this function by inducing the IAP family of survival proteins. Cell Culture, Transfections, Cell Counts, and Flow Cytometry—HeLa and 293 cells were obtained from the ATCC, whereas human chondrocytes were isolated from patients undergoing total knee arthroplasty. The knee samples were provided by National Disease Research Interchange (NDRI) (Philadelphia, PA). The average age was 62 years old (range 52-71 years). To isolate chondrocytes, areas of undamaged cartilage were shaved off the femoral condyles or the tibial plateau. The shavings were washed three times in PBS and then digested with 0.1% trypsin-EDTA in serum-free culture medium for 1 h at 37 °C under agitation. After washing in PBS, the cartilage pieces were minced and then digested with collagenase 2 (Sigma) at 50 μg/ml in serum-free medium overnight at 37 °C under agitation. The chondrocyte suspension was filtered through a 40-μm cell strainer, washed in PBS, and plated at 1.5 × 105 cells/cm2 in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) plus antibiotic-antimycotic solution. The cells were allowed to adhere, spread, and proliferate for 7-10 days. Chondrocytes were used for experiments between passage 1 and 4, during which time cartilage marker genes were expressed. The actinomycin D was purchased from Sigma-Aldrich, and the TNFα was from R & D Systems, Inc. (Merrisville, NC). Monolayer cultures were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 50 units/ml penicillin, and 50 μg/ml streptomycin. The cultures were incubated in a humidified atmosphere at 37 °C and 5% CO2. The medium was changed every 3 days. All of the transfection experiments were initiated on 50% confluent monolayer cultures. Plasmids (20 μg) were transfected either by the calcium phosphate procedure for 293 cells or by Amaxa nucleofection for human chondrocytes and HeLa cells (Amaxa, Gaithersburg, MD) according to the manufacturer's protocol. The human COMP cDNA expression plasmid was obtained from Origene Technologies (Rockville, MD). The empty vector was pSec2B (Invitrogen). The cells were rinsed with PBS, trypsinized, resuspended in 10 ml of growth medium, and counted in the presence of trypan blue using a hemocytometer. For cell cycle analysis (flow cytometry), the cells were rinsed once in chilled PBS then trypsinized and resuspended in 10 ml of Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. The cells were pelleted and resuspended in 70% ethanol. The cells were kept on ice for 10 min, pelleted, and then treated with RNase A (180 μg; Sigma) for 30 min at room temperature. Propidium iodide (Sigma) was added to a final concentration of 75 mg/ml. Cell cycle analysis was performed on a Coulter Profile 2 flow cytometer. For the TUNEL assay, monolayer cells were fixed in paraformaldehyde and then processed as per the manufacturer's recommendation (DeadEnd Tunel System, Promega, Madison, WI). The RGD (cycloRGDFV) and control (cycloRADFV) peptides were purchased from Bachem and used at a concentration of 1 mm. The RGD is a potent inhibitor of integrin signaling, whereas the control peptide is not (40Hammes H.P. Brownlee M. Jonczyk A. Sutter A. Preissner K.T. Nat. Med. 1996; 2: 529-533Crossref PubMed Scopus (314) Google Scholar). siRNA for human COMP was purchased from Ambion, whereas the siRNAs for survivin and XIAP were obtained from Cell Signaling Technology (Danvers, MA). The COMP, XIAP, and survivin siRNAs (100 nm) were transfected into 293 cells, HeLa cells, and chondrocytes as per each manufacturer's recommendation. RNA Isolation and RT/PCR Analysis—Total RNA was isolated from cells by the TRIzol method (Invitrogen). Oligo(dT) was used as the primer in the reverse transcription reaction which was followed by a PCR with the appropriate primers. For the RT/PCRs, one mg of total RNA from the cells was used in each reaction. All of the RNA samples were DNase I-treated prior to the PCRs. Additionally, as a control, PCR done in the absence of reverse transcriptase was negative for any ethidium bromide-stained bands (data not shown). Protein Analysis and Immunoblotting—The cells were lysed on ice in 0.1% Nonidet P-40, 10 mm Tris-HCl, pH 7.9, 10 mm MgCl2, 15 mm NaCl, 0.2 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, and a protease inhibitor mixture (Roche Applied Science). The nuclei were pelleted by centrifugation at 800 × g for 5 min, and the supernatant fraction was termed “cytosol.” The nuclei were resuspended in extraction buffer consisting of 0.5 m NaCl, 20 mm Hepes, pH 7.9, 20% glycerol, 0.2 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Roche Applied Science) and incubated on ice for 10 min. The nuclei were then spun at 14,000 × g for 5 min to pellet the residual nuclear material, and the supernatant fraction was termed “nuclear extract.” For analysis of secreted COMP proteins, the cells were cultured in serum-free medium for 24 h. The medium was harvested, and the cellular debris was removed by centrifugation. Aliquots of the medium were added to two volumes of ice-cold acetone, and the proteins were precipitated at -20 °C, as by Jordan-Sciutto et al. (28Jordan-Sciutto K.L. Logan T.J. Norton P. Derfoul A. Dodge G.R. Hall D.J. Exp. Cell Res. 1997; 236: 527-536Crossref PubMed Scopus (14) Google Scholar). The protein precipitate was pelleted by centrifugation at 14,000 × g for 10 min at 4 °C. The protein precipitate was then boiled in SDS-PAGE sample buffer in either the presence or absence of the reducing agent β-mercaptoethanol. Additionally, an enzyme-linked immunosorbent assay plate reader assay kit (Serotec, Seattle, WA) for COMP was used to determine COMP protein levels secreted into the medium. For generation of conditioned medium, 293 cells were transfected with the COMP plasmid, and 24 h post-transfection the cells were washed, and serum free medium was added for an additional 48 h. COMP concentrations were determined by enzyme-linked immunosorbent assay. For immunoblotting, the proteins were electrophoretically resolved by SDS-PAGE (40 μg of protein/lane) and transferred onto nitrocellulose membranes. The blots were then washed in TBST buffer (10 mm Tris, pH 8, 150 mm NaCl, 0.05% Tween 20), blocked with 5% bovine serum albumin in TBST for 1 h at room temperature, and incubated with the primary antibodies overnight at 4 °C in TBST. Anti-human COMP antibody (Serotec, Raleigh, NC) was used at a concentration of 1:500. The blot was then washed three times, 10 min each, in TBST and then incubated with a 1:2,500 dilution of secondary antibody conjugated to either alkaline phosphatase (Protoblot System) or to horseradish peroxidase (Chemiluminescence) for 1 h at room temperature in TBST. The blots were then processed using either the Protoblot system (Promega) using 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazoleum (BCIP/NBT) as a color developer or using the Supersignal West Pico Chemiluminescent Substrate System (Pierce) and exposed to x-ray film. COMP Enhances Cell Survival in 293 Cells—It has been previously demonstrated that expression of COMP, as either a wild type or mutant form, can affect the level of apoptosis both in vivo and in vitro (21Kipnes J. Carlberg A.L. Loredo G.A. Lawler J. Tuan R.S. Hall D.J. Osteoarthr. Cartil. 2003; 11: 442-454Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 25Hecht J.T. Montufar-Solis D. Decker G. Lawler J. Daniels K. Duke P.J. Matrix Biol. 1998; 1: 625-633Crossref Scopus (81) Google Scholar, 26Hecht J.T. Makitie O. Hayes E. Haynes R. Susic M. Montufar-Solis D. Duke P.J. Cole W.G. J. Ortho. Res. 2004; 22: 759-767Crossref PubMed Scopus (70) Google Scholar, 27Hashimoto Y. Tomiyama T. Yamano Y. Mori H. Am. J. Pathol. 2003; 163: 101-110Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). To explore an effect of COMP on apoptosis, we began transient transfection experiments using a COMP expression plasmid in 293 cells. These cells represent an excellent cell line to test the biological effects of COMP because they are effectively null for COMP expression (they express no detectable COMP by either RT/PCR analysis of RNA or by immunoblotting of cell extracts or conditioned medium). Also, very high transfection efficiencies can be obtained in 293 cells by the calcium phosphate method (50-80%, based on 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) staining), which makes them very useful in transient expression studies. 293 cells were transfected with a human COMP expression vector and then monitored for expression of the COMP protein. Immunoblots of cytosolic and secreted proteins from the cells showed detectable levels of the protein (Fig. 1A). When these same analyses were performed in the absence of β-mercaptoethanol in the Laemmli sample buffer, the COMP protein migrated as a very large molecular complex (>500 kDa), indicating that it had likely formed a pentamer in solution (Fig. 1B). When the concentration of COMP was assessed in 293 cell medium following the transfection, the levels were on average about 3.5 μg/ml. Given that its pentameric molecular mass is 524 kDa, it would suggest an 8 nm concentration of COMP in the media. To determine whether the expression of COMP affected the cells in any way, total viable adherent cell numbers were assessed at 24 h post-transfection. From Fig. 1C, it is clear that the calcium phosphate-mediated transfection led to a decrease in viable cell numbers, a phenomena that is well known (29Renzing J. Lane D.P. Oncogene. 1995; 4: 1865-1868Google Scholar, 30Rodriguez A. Flemington E.K. Anal. Biochem. 1999; 272: 171-181Crossref PubMed Scopus (28) Google Scholar, 31Carvalho-Bittencourt M. Saas P. Resnay S. Yerly-Motta V. Ferrand C. Perruche S. Duperrier A. Herve P. Tiberghien P. Chalmers D.E. J. Gene. Med. 2002; 4: 14-24Crossref PubMed Scopus (7) Google Scholar, 32Ebert O. Ropke G. Marten A. Lefterova P. Micka B. Buttgereit P. Niemitz S. Trojaneck B. Schmidt-Wolf G. Huhn D. Wittig B. Schmidt-Wolf I.G. Cytokines Cell. Mol. Ther. 1999; 5: 165-173PubMed Google Scholar). This toxicity appears independent of the plasmid used to transfect the cells in that transfection of a number of vectors leads to a significant decrease in cell number (Fig. 1C). However, as seen in Fig. 1C, COMP expression in these cells resulted in an increase in viable cell number (4-7-fold) at 24 h post-transfection compared with the vector controls. We next assessed whether the increase in cell numbers following COMP expression was due to a reduction in apoptosis. As seen in Fig. 1D, there is a 3-fold decrease in apoptosis in the COMP expressing cultures at 24 h post-transfection, as assessed by either flow cytometry analysis, evident as cells with less than a 2 n DNA content, or by TUNEL assay. These data indicate that the increase in cell number following COMP expression is due at least in part to a decrease in apoptosis. As a control for these studies, a COMP siRNA obtained from Ambion was cotransfected with the COMP expression plasmid. As seen in the Western blot (Fig. 2A), the COMP siRNA reduces the level of COMP protein in these cells compared with the cells transfected with the control siRNA. Correspondingly, the COMP siRNA blocks the ability of the COMP expression plasmid to enhance cell survival as measured either by total viable cells (Fig. 2B) or by a percentage of apoptotic cells (Fig. 2C). These data indicate that simple transfection of the COMP expression plasmid into cells is not sufficient to aid in survival but that production of the COMP protein is required. Thus ectopic expression of COMP in these cells appears to aid in cell survival. To determine whether the survival effect is specific for COMP, an additional secreted protein was expressed in these 293 cells (prostate-specific antigen). We found that prostate-specific antigen was not able to protect the cells against apoptosis when compared with cell expressing COMP (data not shown). COMP Induces the IAP Family of Prosurvival Proteins in 293 Cells—To more thoroughly explore the nature of the apoptosis inhibited by COMP, we examined the levels of a number of active caspases, the mediators of the apoptotic response. We assessed active effector caspases 3, 6, and 7, the TNFα-activated caspase 8, and the stress-responsive/endoplasmic reticulum caspases 10 and 12. As seen in the immunoblot (Fig. 3A), active caspase 3 is present in the control-transfected cells, yet its level is reduced in the COMP-expressing cells. Further, levels of procaspase 3 (uncleaved) are slightly increased in the COMP-expressing cells. An examination of the other caspases (6, 7, 8, 9, 10, and 12) did not reveal any COMP-mediated changes following transfection (data not shown). Therefore, these data show that COMP inhibits the activation of caspase 3. To identify the mechanism by which COMP blocks the activation of caspase 3 and protects cells from apoptosis, we examined the levels of a number of apoptotic and antiapoptotic proteins. It would be expected that COMP would affect the levels of some of these pro- and antiapoptotic factors, thereby mediating an effect on cell survival. These factors comprise mitochondrial (cytochromeC/Bcl2) and non-mitochondrial (Akt) mediators of apoptosis. As seen in Fig. 3B, a number of these proteins are not affected by COMP, which includes Akt, Akt1, phosphoAkt(Ser), cytochrome C, Bcl2, phosphoBcl2(Ser), BclXL, and Mcl1. These data indicate that the block to caspase 3 activation and apoptosis is not through the regulation of these factors. The IAP family of survival proteins was then examined because they act to directly block caspase 3 activation (33Holcik M. Gibson H. Korneluk R.G. Apoptosis. 2001; 6: 253-261Crossref PubMed Scopus (359) Google Scholar, 34LaCasse E.C. Baird S. Korneluk R.G. MacKenzie A.E. Oncogene. 1998; 17: 3247-3259Crossref PubMed Scopus (947) Google Scholar). The levels of XIAP, cIAP1, cIAP2, and survivin were significantly increased in the COMP-expressing cells (Fig. 3C, left panels). To determine whether the increase in levels of these proteins was due to an increase in their cognate mRNAs, RT/PCR analysis was performed. As seen in Fig. 3C (right panels), the levels of their corresponding mRNAs were not affected by COMP. Because it has been demonstrated that NF-κB transcriptionally induces the IAP family of proteins (33Holcik M. Gibson H. Korneluk R.G. Apoptosis. 2001; 6: 253-261Crossref PubMed Scopus (359) Google Scholar, 34LaCasse E.C. Baird S. Korneluk R.G. MacKenzie A.E. Oncogene. 1998; 17: 3247-3259Crossref PubMed Scopus (947) Google Scholar), we examined levels of NF-κB in the control and COMP-expressing cells. We found no increase in NF-κB or phosphoNF-κB in the COMP-expressing cells (data not shown), which is consistent with the fact that the IAP transcripts are not up-regulated by COMP. These data would indicate that the elevation in cIAP1, cIAP2, survivin, and XIAP protein levels are likely regulated at the translational/post-translational level. XIAP and Survivin Induction Mediates the Survival Effect of COMP in 293 Cells—To determine whether these IAP family members mediate the survival effect of COMP in 293 cells, siRNA experiments were performed in an attempt to reduce their levels. XIAP and survivin were chosen as targets because they are the most potent inhibitors of caspase 3 (33Holcik M. Gibson H. Korneluk R.G. Apoptosis. 2001; 6: 253-261Crossref PubMed Scopus (359) Google Scholar). Following transfection of the XIAP and survivin siRNAs, their cellular protein levels were reduced by 3-4-fold compared with the control siRNA plasmid (Fig. 4, A and B). When the cells were analyzed for levels of apoptosis, as shown in Fig. 4C, COMP was not able to optimally enhance cell survival in the presence of either the XIAP or survivin siRNAs. The survivin siRNA was not as effective as the XIAP siRNA in blocking the antiapoptotic effect of COMP, suggesting differences in the way these two proteins mediate cell survival. These data indicate that COMP mediates cell survival at least in part through an elevation the XIAP and survivin proteins. COMP Expression in 293 and HeLa Cells Leads to Resistance to TNFα—From the data above it appears that expression of COMP reduces the level of transfection-mediated cell death in 293 cells. It was next important to determine whether induction of cell death by the addition o