Portal de Periódicos da CAPES

Integrin-mediated Adhesion and Soluble Ligand Binding Stabilize COX-2 Protein Levels in Endothelial Cells by Inducing Expression and Preventing Degradation

2004; Elsevier BV; Volume: 280; Issue: 2 Linguagem: Inglês

10.1074/jbc.m410006200

1083-351X

Jelena Zarić, Curzio Rüegg,

Monoclonal and Polyclonal Antibodies Research

Cyclooxygenase-2 (COX-2), a key enzyme in prostaglandin synthesis, is highly expressed during inflammation and cellular transformation and promotes tumor progression and angiogenesis. We have previously demonstrated that endothelial cell COX-2 is required for integrin αVβ3-dependent activation of Rac-1 and Cdc-42 and for endothelial cell spreading, migration, and angiogenesis (Dormond, O., Foletti, A., Paroz, C., and Ruegg, C. (2001) Nat. Med. 7, 1041–1047; Dormond, O., Bezzi, M., Mariotti, A., and Ruegg, C. (2002) J. Biol. Chem. 277, 45838–45846). In this study, we addressed the question of whether integrin-mediated cell adhesion may regulate COX-2 expression in endothelial cells. We report that cell detachment from the substrate caused rapid degradation of COX-2 protein in human umbilical vein endothelial cells (HUVEC) independent of serum stimulation. This effect was prevented by broad inhibition of cellular proteinases and by neutralizing lysosomal activity but not by inhibiting the proteasome. HUVEC adhesion to laminin, collagen I, fibronectin, or vitronectin induced rapid COX-2 protein expression with peak levels reached within 2 h and increased COX-2-dependent prostaglandin E2 production. In contrast, nonspecific adhesion to poly-l-lysine was ineffective in inducing COX-2 expression. Furthermore, the addition of matrix proteins in solution promoted COX-2 protein expression in suspended or poly-l-lysine-attached HUVEC. Adhesion-induced COX-2 expression was strongly suppressed by pharmacological inhibition of c-Src, phosphatidylinositol 3-kinase, p38, extracellular-regulated kinase 1/2, and, to a lesser extent, protein kinase C and by the inhibition of mRNA or protein synthesis. In conclusion, this work demonstrates that integrin-mediated cell adhesion and soluble integrin ligands contribute to maintaining COX-2 steady-state levels in endothelial cells by the combined prevention of lysosomal-dependent degradation and the stimulation of mRNA synthesis involving multiple signaling pathways. Cyclooxygenase-2 (COX-2), a key enzyme in prostaglandin synthesis, is highly expressed during inflammation and cellular transformation and promotes tumor progression and angiogenesis. We have previously demonstrated that endothelial cell COX-2 is required for integrin αVβ3-dependent activation of Rac-1 and Cdc-42 and for endothelial cell spreading, migration, and angiogenesis (Dormond, O., Foletti, A., Paroz, C., and Ruegg, C. (2001) Nat. Med. 7, 1041–1047; Dormond, O., Bezzi, M., Mariotti, A., and Ruegg, C. (2002) J. Biol. Chem. 277, 45838–45846). In this study, we addressed the question of whether integrin-mediated cell adhesion may regulate COX-2 expression in endothelial cells. We report that cell detachment from the substrate caused rapid degradation of COX-2 protein in human umbilical vein endothelial cells (HUVEC) independent of serum stimulation. This effect was prevented by broad inhibition of cellular proteinases and by neutralizing lysosomal activity but not by inhibiting the proteasome. HUVEC adhesion to laminin, collagen I, fibronectin, or vitronectin induced rapid COX-2 protein expression with peak levels reached within 2 h and increased COX-2-dependent prostaglandin E2 production. In contrast, nonspecific adhesion to poly-l-lysine was ineffective in inducing COX-2 expression. Furthermore, the addition of matrix proteins in solution promoted COX-2 protein expression in suspended or poly-l-lysine-attached HUVEC. Adhesion-induced COX-2 expression was strongly suppressed by pharmacological inhibition of c-Src, phosphatidylinositol 3-kinase, p38, extracellular-regulated kinase 1/2, and, to a lesser extent, protein kinase C and by the inhibition of mRNA or protein synthesis. In conclusion, this work demonstrates that integrin-mediated cell adhesion and soluble integrin ligands contribute to maintaining COX-2 steady-state levels in endothelial cells by the combined prevention of lysosomal-dependent degradation and the stimulation of mRNA synthesis involving multiple signaling pathways. Tumor angiogenesis, i.e. the formation of new blood vessels in response to angiogenic stimuli, promotes tumor progression by stimulating tumor cell survival, tumor invasion, and metastasis formation (1Carmeliet P. Jain R.K. Nature. 2000; 407: 249-257Crossref PubMed Scopus (7350) Google Scholar). Many molecules mediating or regulating angiogenesis have been identified (2Yancopoulos G.D. Davis S. Gale N.W. Rudge J.S. Wiegand S.J. Holash J. Nature. 2000; 407: 242-248Crossref PubMed Scopus (3253) Google Scholar). They include vascular endothelial growth factors and their cell surface receptors, matrix-degrading enzymes, vascular remodeling ligands (i.e. angiopoietins), and their receptors and adhesion molecules of the integrin and cadherin families. Integrins are the main receptors for extracellular matrix (ECM) 1The abbreviations used are: ECM, extracellular matrix; COX, cyclooxygenase; HUVEC, human umbilical vein endothelial cell(s); PLL, poly-l-lysine; PKC, protein kinase C; ERK, extracellular-regulated kinase; PI3-K, phosphatidylinositol 3-kinase; PKB, protein kinase B; JNK, c-Jun N-terminal kinase; TNF, tumor necrosis factor; PGE2, prostaglandin E2; BSA, bovine serum albumin; mAb, monoclonal antibody; PBS, phosphate-buffered saline; FCS, fetal calf serum; Act D, actinomycin D; CHX, cyclohexamide; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; IL, interleukin.1The abbreviations used are: ECM, extracellular matrix; COX, cyclooxygenase; HUVEC, human umbilical vein endothelial cell(s); PLL, poly-l-lysine; PKC, protein kinase C; ERK, extracellular-regulated kinase; PI3-K, phosphatidylinositol 3-kinase; PKB, protein kinase B; JNK, c-Jun N-terminal kinase; TNF, tumor necrosis factor; PGE2, prostaglandin E2; BSA, bovine serum albumin; mAb, monoclonal antibody; PBS, phosphate-buffered saline; FCS, fetal calf serum; Act D, actinomycin D; CHX, cyclohexamide; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; IL, interleukin. proteins, consisting of two non-covalently associated α and β subunits (3Hynes R.O. Trends Cell Biol. 1999; 9: M33-M37Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 4Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8966) Google Scholar). Besides promoting physical adhesion, integrins also transduce signaling events ("outside-in" signaling), which are essential for cell spreading, migration, survival, proliferation, and differentiation. Signaling molecules downstream of integrins include Src family kinases, integrin-linked kinase, focal adhesion kinase, protein kinase C (PKC), extracellular-regulated kinase (ERK), stress-regulated kinases (e.g. p38, and JNK), phosphatidylinositol 3-kinase (PI3-K), and protein kinase B (PKB/Akt) (5Giancotti F.G. Ruoslahti E. Science. 1999; 285: 1028-1032Crossref PubMed Scopus (3785) Google Scholar, 6Ruegg C. Mariotti A. Cell Mol. Life Sci. 2003; 60: 1135-1157Crossref PubMed Scopus (159) Google Scholar). These signaling pathways are also activated by growth factor receptors and the cross-talk between integrins, and growth factor receptors provide enhanced signaling efficacy, specificity, and control (6Ruegg C. Mariotti A. Cell Mol. Life Sci. 2003; 60: 1135-1157Crossref PubMed Scopus (159) Google Scholar, 7Schwartz M.A. Trends Cell Biol. 2001; 11: 466-470Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 8Smyth S.S. Patterson C. J. Cell Biol. 2002; 158: 17-21Crossref PubMed Scopus (52) Google Scholar).The cyclooxygenase (COX) isoenzymes COX-1 and-2 catalyze the first two steps in prostanoid biosynthesis (9Marnett L.J. Kalgutkar A.S. Trends Pharmacol. Sci. 1999; 20: 465-469Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). COX-1 expression is constitutive in many tissues, whereas COX-2 expression is induced by inflammatory cytokines, such as tumor necrosis factor (TNF), transforming growth factor β, or interleukin (IL)-1 (10Diaz A. Chepenik K.P. Korn J.H. Reginato A.M. Jimenez S.A. Exp. Cell Res. 1998; 241: 222-229Crossref PubMed Scopus (137) Google Scholar) and by oncogenic mutations in several proto-oncogenes genes including c-Src (11Simmons D.L. Levy D.B. Yannoni Y. Erikson R.L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1178-1182Crossref PubMed Scopus (296) Google Scholar), Ras (12Sheng H. Williams C.S. Shao J. Liang P. DuBois R.N. Beauchamp R.D. J. Biol. Chem. 1998; 273: 22120-22127Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar), ErbB2 (13Vadlamudi R. Mandal M. Adam L. Steinbach G. Mendelsohn J. Kumar R. Oncogene. 1999; 18: 305-314Crossref PubMed Scopus (206) Google Scholar), or tumor suppressor genes such as p53 (14Subbaramaiah K. Altorki N. Chung W.J. Mestre J.R. Sampat A. Dannenberg A.J. J. Biol. Chem. 1999; 274: 10911-10915Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar) or APC (15Dimberg J. Hugander A. Sirsjo A. Soderkvist P. Anticancer Res. 2001; 21: 911-915PubMed Google Scholar) (for review see Refs. 16Coyne D.W. Nickols M. Bertrand W. Morrison A.R. Am. J. Physiol. 1992; 263: F97-F102PubMed Google Scholar and 17Dubois R.N. Abramson S.B. Crofford L. Gupta R.A. Simon L.S. Van De Putte L.B. Lipsky P.E. FASEB J. 1998; 12: 1063-1073Crossref PubMed Scopus (2207) Google Scholar). High levels of COX-2 expression are often observed in many human cancers including colon, breast, lung, and skin. Experimental studies have demonstrated that COX-2 overexpression promotes tumorigenesis, whereas non-steroidal anti-inflammatory drugs and COX-2-specific inhibitors suppress tumorigenesis and tumor progression (reviewed in Refs. 17Dubois R.N. Abramson S.B. Crofford L. Gupta R.A. Simon L.S. Van De Putte L.B. Lipsky P.E. FASEB J. 1998; 12: 1063-1073Crossref PubMed Scopus (2207) Google Scholar and 18Ruegg C. Zaric J. Stupp R. Ann. Med. 2003; 35: 476-487Crossref PubMed Scopus (64) Google Scholar). For example, in a murine model of human familial adenomatous polyposis coli, genetic inactivation of COX-2 dramatically reduced the number and size of intestinal polyps (19Oshima M. Dinchuk J.E. Kargman S.L. Oshima H. Hancock B. Kwong E. Trzaskos J.M. Evans J.F. Taketo M.M. Cell. 1996; 87: 803-809Abstract Full Text Full Text PDF PubMed Scopus (2271) Google Scholar). Transgenic overexpression of COX-2 in the skin and in the breast promoted tumorigenesis and tumor progression (20Muller-Decker K. Neufang G. Berger I. Neumann M. Marks F. Furstenberger G. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 12483-12488Crossref PubMed Scopus (230) Google Scholar, 21Liu C.H. Chang S.H. Narko K. Trifan O.C. Wu M.T. Smith E. Haudenschild C. Lane T.F. Hla T. J. Biol. Chem. 2001; 276: 18563-18569Abstract Full Text Full Text PDF PubMed Scopus (734) Google Scholar). Celecoxib was shown to reduce the risk of developing polyps and colon cancer in patients with familial adenomatous polyposis coli (22Steinbach G. Lynch P.M. Phillips R.K. Wallace M.H. Hawk E. Gordon G.B. Wakabayashi N. Saunders B. Shen Y. Fujimura T. Su L.K. Levin B. N. Engl. J. Med. 2000; 342: 1946-1952Crossref PubMed Scopus (2263) Google Scholar). COX-2 and its main metabolite prostaglandin (PG) E2 promote tumor progression through two distinct but complementary mechanisms. First, they stimulate tumor cell survival, proliferation, migration, and invasion, and second, they induce tumor angiogenesis, which in turn favors local tumor progression and metastatic spreading. COX-2 stimulates angiogenesis by inducing vascular endothelial growth factor expression in tumor and stromal cells (23Tsujii M. Kawano S. Tsuji S. Sawaoka H. Hori M. DuBois R.N. Cell. 1998; 93: 705-716Abstract Full Text Full Text PDF PubMed Scopus (2205) Google Scholar, 24Williams C.S. Tsujii M. Reese J. Dey S.K. DuBois R.N. J. Clin. Investig. 2000; 105: 1589-1594Crossref PubMed Scopus (643) Google Scholar), by enhancing the mitogenic activity of vascular endothelial growth factor on endothelial cells (25Jones M.K. Wang H. Peskar B.M. Levin E. Itani R.M. Sarfeh I.J. Tarnawski A.S. Nat. Med. 1999; 5: 1418-1423Crossref PubMed Scopus (793) Google Scholar), and by promoting integrin αVβ3- and cAMP/PKA-dependent activation of the small GTPases, Rac-1 and Cdc42 (26Dormond O. Foletti A. Paroz C. Ruegg C. Nat. Med. 2001; 7: 1041-1047Crossref PubMed Scopus (269) Google Scholar, 27Dormond O. Bezzi M. Mariotti A. Ruegg C. J. Biol. Chem. 2002; 277: 45838-45846Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar), thereby resulting in increased endothelial cell proliferation, survival, and migration (reviewed in Refs 28Ruegg C. Dormond O. Mariotti A. Biochim. Biophys. Acta. 2004; 1654: 51-67PubMed Google Scholar and 29Dormond O. Ruegg C. Thromb. Haemostasis. 2003; 90: 577-585Crossref PubMed Google Scholar). COX-2 expression is largely controlled at the transcriptional and post-transcriptional level (mRNA stability and translation) (30Ramsay R.G. Ciznadija D. Vanevski M. Mantamadiotis T. Int. J. Immunopathol. Pharmacol. 2003; 16: 59-67PubMed Google Scholar, 31Dixon D.A. Curr. Pharm. Des. 2004; 10: 635-646Crossref PubMed Scopus (76) Google Scholar). Although much is known regarding the role of growth factors and cytokines in the induction of COX-2 expression, little is known regarding the contribution of cell adhesion.In this study, we investigated the role of cell adhesion in modulating COX-2 protein level in human umbilical vein endothelial cells (HUVEC). Here we report that COX-2 protein is rapidly degraded through a lysosomal-dependent pathway upon HUVEC detachment from the substrate and that integrin-mediated adhesion to ECM proteins induced de novo COX-2 synthesis involving signaling through multiple pathways (i.e. p38, mitogen-activated protein kinase, PI3-K, PKC, and c-Src).EXPERIMENTAL PROCEDURESReagents and Antibodies—Bovine gelatin, bovine plasma fibronectin, human plasma vitronectin, rat collagen I, BSA, murine laminin, poly-l-lysine (PLL), ammonium chloride, and LY294002 were purchased from Sigma. CGP77675, RAD001, PKC412, and STI571 were obtained from Novartis AG (Basel, Switzerland). SB203580, PD98059, NS-398, and SC-560 were purchased from Biomol (Plymouth Meeting, PA). The DNA molecular weight marker was from Roche Applied Science. The anti-human COX-2 antibody was purchased from Cayman Chemical (Ann Arbor, MI). The anti-COX-1 rabbit polyclonal antibody (H-62) was purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). The anti-human actin antibody was from Sigma. Function-blocking mAbs: FB12 (anti-α1), P1E6 (anti-α2), P1B5 (anti-α3), and LM609 (anti-αVβ3) were from Chemicon (Temecula, CA); mAbs Lia1/2 (anti-β1), mAb GI9 (anti-α2), Sam-1 (anti-α5), and GoH3 (anti-α6) were from Beckman Coulter (Nyon, Switzerland). Celecoxib was kindly provided by Pfizer AG (Dübendorf, Switzerland). Human recombinant TNF (5 × 107 units/mg) was a gift of Dr. G. Adolf (Boehringer Ingelheim, Vienna, Austria). Protease inhibitor mixture containing 4-(2-aminoethyl)-benzenesulfonyl fluoride, aprotinin, leupeptin, bestatin, pepstatin A, and E-64 were from Sigma. Lactacystin was a generous gift of Dr. F. Levy (Ludwig Institute for Cancer Research, Epalinges s/Lausanne, Switzerland).Cell Culture and Treatments—HUVEC were prepared and cultured as previously described (32Ruegg C. Yilmaz A. Bieler G. Bamat J. Chaubert P. Lejeune F.J. Nat. Med. 1998; 4: 408-414Crossref PubMed Scopus (424) Google Scholar) except for the use of M199 (Invitrogen) as a basal medium. Basal medium was supplemented with 12 μg/ml bovine brain extract (Clonetics, Walkersville, MA), 10 ng/ml human recombinant epidermal growth factor (Genzyme, Cambridge, MA), 25 units/ml heparin, and 1 μg/ml hydrocortisone (Sigma). For de novo cell attachment experiments, HUVEC were collected by trypsin treatment (Invitrogen), resuspended in serum-free M199 medium, incubated for 2 h at 37 °C in suspension, and subsequently plated at 2 × 105 cells/well on 12-well plates (Evergreen Scientific, Los Angeles, CA) pre-coated with fibronectin (3 μg/ml), gelatin (0.5%), collagen I (10 μg/ml), laminin (10 μg/ml), or vitronectin (1 μg/ml) in PBS for 1 h at 37 °C. For EDTA-mediated release, cells cultured for 12 h on gelatin-coated plates were washed twice with PBS and incubated with 3 mm EDTA for various periods of time. In experiments where ECM proteins were added in solution, cells were collected by trypsin treatment, washed with PBS, and resuspended in serum-free M199 medium and incubated for 2 h at 37 °C. Laminin, collagen I, fibronectin, or vitronectin (10 μg each) then were added, and cells were further incubated in suspension for another 30 and 60 min. Cells were observed and photographed using an inverted microscope (Axiovert 40 CFL, Carl Zeiss AG, Zürich, Switzerland) equipped with a PowerShot G5 digital camera (Canon, Dietlikon, Switzerland). Cell counting and viability determination was performed by trypan blue exclusion.COX-2 Degradation Studies—Cells cultured for 12 h on gelatin-coated plates were washed twice with PBS, detached by trypsin treatment, and immediately exposed to different inhibitors: protease inhibitor mixture at 1:1000 dilution (33Stinson M.W. Alder S. Kumar S. Infect. Immun. 2003; 71: 2365-2372Crossref PubMed Scopus (61) Google Scholar); ammonium chloride (34Seglen P.O. Reith A. Exp. Cell Res. 1976; 100: 276-280Crossref PubMed Scopus (133) Google Scholar) at 40 mm; or lactacystin (35Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1493) Google Scholar) at a 10 μm final concentration. Cells were left in suspension for different periods of time and subsequently analyzed for COX-2 protein by Western blotting.Assays with Pharmacological Inhibitors—Cells were serum-starved for 2 h before plating at 2 × 105 cells/well on 12-well plates coated with ECMs (see above). Pharmacological inhibitors were added at the time of plating at the following concentrations: LY294002, 20 μm; SB203580, 20 μm; PD98059, 20 μm; RAD001, 0.1 μm; PKC 412, 1 μm; STI571, 10 μm; and CGP77675, 5 μm. Cells were collected 2 h later, and total protein extracts were analyzed for COX-2 protein by Western blotting.Cell Adhesion Assays—Maxisorp II Nunc enzyme-linked immunosorbent assay plates (Roskilde, Denmark) were coated with 10 μg/ml laminin, collagen I, fibronectin, or vitronectin overnight at 4 °C and blocked with BSA for 2 h at room temperature. Assays were done as previously described (32Ruegg C. Yilmaz A. Bieler G. Bamat J. Chaubert P. Lejeune F.J. Nat. Med. 1998; 4: 408-414Crossref PubMed Scopus (424) Google Scholar). HUVEC were collected by trypsin digestion and seeded in serum-free M199 medium at 2 × 104 cells/well. Blocking anti-integrin-mAbs were added at this step (at 10 μg/ml). After 1 h at 37°C, wells were gently washed with PBS and attached cells were fixed in 4% paraformaldehyde (Fluka Chemie, Buchs, Switzerland) and stained with 0.5% crystal violet. Absorbance of each well was read at 620 nm in a plate reader (Packard Spectra Count, Meriden, CT). Results are expressed as the mean value ± S.D of triplicate determinations.PGE2 Determinations—PGE2 was measured in conditioned culture medium using an enzyme immunoassay system (Amersham Biosciences). Results are expressed as pg of PGE2/ml of a 2 × 105 cell culture supernatant and represent the mean ± S.D. of duplicate determinations.Reverse Transcription-Polymerase Chain Reaction—Total RNA was prepared from HUVEC using RNAsol B (Biogenesis) and following manufacturer's instructions. 1 μg of total RNA was reverse-transcribed (Superscript II, Invitrogen), and cDNA was subjected to PCR amplifications using primer pairs specific for human COX-2 (forward, 5′-TTCAAATGAGATTGTGGGAAAATTGCT-3′; reversed, 5′-AGATCATCTCTGCCTGAGTATCTT-3′) and glyceraldehyde-3-phosphate dehydrogenase (forward, 5′-CAACTACATGGTTTACATGTTC-3′; reversed, 5′-CATGGTGGTGAAGACGCCAG-3′ (Microsynth, Balgach, Switzerland).SDS-PAGE and Western Blot Analysis—Total cell lysates were resolved by SDS-PAGE, blotted onto Immobilon-P membranes (Millipore, Volketswil, Switzerland), and incubated in 1% BSA with anti-COX-2 mAb or anti-COX-1 antibody and anti-actin mAb followed by incubation with 1 μg/ml horseradish peroxidase-labeled secondary antibody (DAKO, Zug, Switzerland). The ECL system was used for detection (Amersham Biosciences). Unless otherwise indicated, experiments were carried out at a minimum of three times to yield similar results. Representative experiments are shown.RESULTSEndothelial Cell Detachment Causes COX-2 Degradation—We have previously shown that endothelial cell COX-2 activity is required for αVβ3 integrin-mediated endothelial cell spreading and migration in vitro and fibroblast growth factor 2-induced angiogenesis in vivo (26Dormond O. Foletti A. Paroz C. Ruegg C. Nat. Med. 2001; 7: 1041-1047Crossref PubMed Scopus (269) Google Scholar, 27Dormond O. Bezzi M. Mariotti A. Ruegg C. J. Biol. Chem. 2002; 277: 45838-45846Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). These observations raised the possibility that adhesion itself may be involved in regulating COX-2 protein expression and activity (36Dormond O. Rüegg C. Drug Res. Updates. 2001; 4: 314-321Crossref PubMed Scopus (53) Google Scholar). To experimentally address this hypothesis, we analyzed COX-2 protein levels by Western blotting in HUVEC cultured under adhesive conditions and in HUVEC detached by trypsin treatment and cultured for up to 3 h in suspension. HUVEC were serum-starved for 2 h before detachment, and upon detachment, they were further cultured in the absence of FCS to avoid any possible effects of serum on COX-2 expression. Adherent HUVEC expressed significant amounts of COX-2 protein, and this level rapidly and dramatically dropped after detachment (Fig. 1A). In contrast, HUVEC expressed only low levels of COX-1, and this was not altered by cell detachment (Fig. 1A). Within the experimental conditions tested throughout these experiments (i.e. detachment and culture in suspension up to 3 h), HUVEC viability remained above 90% (data not shown). To directly test for the effects of serum on COX-2 expression, HUVEC were maintained in complete medium (i.e. medium supplemented with FCS and growth factors) before detachment and during cultures in suspension. FCS and growth factors did not prevent the drop in COX-2 protein level observed after cell detachment (Fig. 1B). To obtain additional evidence that adhesion was required for maintaining COX-2 protein expression, we induced HUVEC detachment by adding 3 mm EDTA to confluent HUVEC cultures. Consistently, we observed a decrease in COX-2 protein level but with a slower kinetic compared with trypsin treatment (Fig. 1C).COX-2 protein is mostly localized on the luminal side of the rough endoplasmic reticulum and Golgi complex (37Hashimoto Y. Kondo Y. Kimura G. Matsuzawa I. Sato S. Ishizaki M. Imura N. Akimoto M. Hara S. Histopathology. 2004; 44: 353-359Crossref PubMed Scopus (41) Google Scholar) through asymmetric insertion into the membrane via a monotopic membrane binding domain (38Spencer A.G. Thuresson E. Otto J.C. Song I. Smith T. DeWitt D.L. Garavito R.M. Smith W.L. J. Biol. Chem. 1999; 274: 32936-32942Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 39Nina M. Berneche S. Roux B. Eur. Biophys. J. 2000; 29: 439-454Crossref PubMed Scopus (48) Google Scholar). This localization suggested the possibilities that the decrease in COX-2 protein induced by cell detachment could be either due to cell surface transport and extracellular secretion or to intracellular proteolytic degradation. To test the first possibility, we separately monitored COX-2 protein content in cells and conditioned culture supernatant following EDTA-induced cell detachment. Whereas cell-associated COX-2 protein levels progressively decreased, there was no detectable COX-2 protein accumulation in the culture supernatant (Fig. 2A). To address the second possibility, HUVEC were detached by trypsin and cultured in suspension in the presence or absence of a mixture of protease inhibitors (see "Experimental Procedures"). Broad inhibition of protease activity suppressed COX-2 degradation in HUVEC cultured in suspension (Fig. 2B). We then repeated the experiment in the presence of ammonium chloride, an inhibitor of lysosomal proteolytic activity (34Seglen P.O. Reith A. Exp. Cell Res. 1976; 100: 276-280Crossref PubMed Scopus (133) Google Scholar), or lactacystin, a drug that inhibits trypsin-like, chymotrypsin-like, and peptidylglutamyl peptide-hydrolyzing activities of the proteasome (35Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1493) Google Scholar). Ammonium chloride, but not lactacystin treatment, prevented the decrease in COX-2 protein observed upon cell detachment (Fig. 2, C and D).Fig. 2COX-2 is degraded via the lysosmal pathway upon HUVEC detachment.A, confluent HUVEC were washed with PBS, and EDTA (3 mm) was added at t = 0 to induce cell detachment. Cells (P) and conditioned culture supernatants (S) were collected at the indicated time points and analyzed by Western blotting for COX-2 protein expression. Adh, adherent cells before the addition of EDTA. B–D, HUVEC were collected by trypsin digestion and maintained in suspension in the absence (– PI) or presence (+ PI) of a mixture of protease inhibitors (B), in the absence (– AC) or presence (+ AC) of ammonium chloride (40 mm) to inhibit the proteolytic lysosomal activity (C), or in the absence (– LC) or presence (+ LC) of lactacystin (10 μm), a proteasome inhibitor (D). Total cell lysates were analyzed by Western blotting for COX-2 protein at the indicated time points. Blots were concomitantly probed for actin to demonstrate equal protein loading.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Taken together these data demonstrate that the HUVEC detachment from the substrate causes a rapid decrease in COX-2 protein level, independently of serum stimulation, and that this decrease is due to lysosomal-mediated degradation.HUVEC Adhesion to Multiple Extracellular Matrix Proteins Induces COX-2 Protein Synthesis—We next tested whether integrin-mediated HUVEC adhesion to ECM proteins did induce COX-2 protein expression. To this purpose, HUVEC were detached and maintained in suspension for 2 h to deplete COX-2 protein and then plated on laminin, collagen I, fibronectin, vitronectin, PLL, a substrate that promotes nonspecific integrin-dependent cell attachment to the substrate (40Bershadsky A. Chausovsky A. Becker E. Lyubimova A. Geiger B. Curr. Biol. 1996; 6: 1279-1289Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar), or on BSA (a non-adhesive substrate). The integrin specificity of the adhesion was confirmed in short-term cell adhesion experiments by adding function blocking anti-integrin mAbs to HUVEC at the time of plating on the different substrates. Adhesion to laminin was mostly mediated by α6β1. Adhesion to collagen I was largely mediated by α2β1 with a minor contribution by α1β1 and α3β1. Adhesion to fibronectin was largely mediated by α5β1 with a minor contribution of αVβ3, whereas adhesion to vitronectin was mediated by integrin αVβ3 (Fig. 3). HUVEC adhesion to each of the four ECM proteins induced a time-dependent increase in COX-2 protein level, whereas integrin-independent adhesion to PLL or lack of adhesion (i.e. HUVEC on BSA) did not increase the level (Fig. 4A). Increase in COX-2 protein levels were observed as soon as 30 min after plating, whereas peak levels were observed at 2 h. At this time point, we observed some differences in the relative induction of COX-2 by with collagen I and laminin being the most potent inducers (Fig. 4B).Fig. 3Integrin specificity of HUVEC adhesion to ECM proteins. HUVEC were collected and seeded into wells previously coated with laminin (LM), collagen I (Col I), fibronectin (FN), vitronectin (VN), PLL, or BSA in the absence or presence of integrin function-blocking mAbs (10 μg/ml) for 1 h at 37 °C. Antibody specificity: FB12, anti-α1; P1B5, anti-α3; LM609, anti-αV β3; Lia1/2, anti-β; mAb GI9 and P1E6, anti-α2; Sam-1, anti-α5; and GoH3, anti-α6. Attached cells were fixed and stained with crystal violet and quantified by absorbance reading. Results are expressed as mean value ± S.D. of triplicate determinations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4HUVEC adhesion to different ECM proteins induces COX-2 expression.A, serum-starved HUVEC were plated onto wells coated with laminin (LM), collagen I (Col I), fibronectin (FN), vitronectin (VN), PLL, or BSA. At the indicated times, cells were collected and COX-2 and actin proteins were determined by Western blotting. B, serum-starved HUVEC were plated in ECM-protein-coated wells as in panel A. After 2 h of culture, cells were collected, (total cell lysates were run on the same SDS-PAGE run), and COX-2 and actin proteins were determined by Western blotting.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To demonstrate that the increase in COX-2 protein level upon adhesion translated into increased enzymatic activity, we monitored the generation of PGE2, one of the main products of COX-2 enzymatic activity, in the culture supernatant of HUVEC plated on different ECMs. An increase of 2–3-