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Actin Cytoskeletal Architecture Regulates Nitric Oxide-induced Apoptosis, Dedifferentiation, and Cyclooxygenase-2 Expression in Articular Chondrocytes via Mitogen-activated Protein Kinase and Protein Kinase C Pathways

2003; Elsevier BV; Volume: 278; Issue: 43 Linguagem: Inglês

10.1074/jbc.m304887200

1083-351X

Song-Ja Kim, Sang‐Gu Hwang, Il-Chul Kim, Jang‐Soo Chun,

Inflammatory mediators and NSAID effects

Nitric oxide (NO) in articular chondrocytes regulates differentiation, survival, and inflammatory responses by modulating ERK-1 and -2, p38 kinase, and protein kinase C (PKC) α and ζ. In this study, we investigated the effects of the actin cytoskeletal architecture on NO-induced dedifferentiation, apoptosis, cyclooxygenase (COX)-2 expression, and prostaglandin E2 production in articular chondrocytes, with a focus on ERK-1/-2, p38 kinase, and PKC signaling. Disruption of the actin cytoskeleton by cytochalasin D (CD) inhibited NO-induced apoptosis, dedifferentiation, COX-2 expression, and prostaglandin E2 production in chondrocytes cultured on plastic or during cartilage explants culture. CD treatment did not affect ERK-1/-2 activation but blocked the signaling events necessary for NO-induced dedifferentiation, apoptosis, and COX-2 expression such as activation of p38 kinase and inhibition of PKCα and -ζ. CD also suppressed activation of downstream signaling of p38 kinase and PKC, such as NF-κB activation, p53 accumulation, and caspase-3 activation, which are necessary for NO-induced apoptosis. NO production in articular chondrocytes caused down-regulation of phosphatidylinositol (PI) 3-kinase and Akt activities. The down-regulation of PI 3-kinase and Akt was blocked by CD treatment, and the CD effects on apoptosis, p38 kinase, and PKCα and -ζ were abolished by the inhibition of PI 3-kinase with LY294002. Our results collectively indicate that the actin cytoskeleton mediates NO-induced regulatory effects in chondrocytes by modulating down-regulation of PI 3-kinase and Akt, activation of p38 kinase, and inhibition of PKCα and -ζ Nitric oxide (NO) in articular chondrocytes regulates differentiation, survival, and inflammatory responses by modulating ERK-1 and -2, p38 kinase, and protein kinase C (PKC) α and ζ. In this study, we investigated the effects of the actin cytoskeletal architecture on NO-induced dedifferentiation, apoptosis, cyclooxygenase (COX)-2 expression, and prostaglandin E2 production in articular chondrocytes, with a focus on ERK-1/-2, p38 kinase, and PKC signaling. Disruption of the actin cytoskeleton by cytochalasin D (CD) inhibited NO-induced apoptosis, dedifferentiation, COX-2 expression, and prostaglandin E2 production in chondrocytes cultured on plastic or during cartilage explants culture. CD treatment did not affect ERK-1/-2 activation but blocked the signaling events necessary for NO-induced dedifferentiation, apoptosis, and COX-2 expression such as activation of p38 kinase and inhibition of PKCα and -ζ. CD also suppressed activation of downstream signaling of p38 kinase and PKC, such as NF-κB activation, p53 accumulation, and caspase-3 activation, which are necessary for NO-induced apoptosis. NO production in articular chondrocytes caused down-regulation of phosphatidylinositol (PI) 3-kinase and Akt activities. The down-regulation of PI 3-kinase and Akt was blocked by CD treatment, and the CD effects on apoptosis, p38 kinase, and PKCα and -ζ were abolished by the inhibition of PI 3-kinase with LY294002. Our results collectively indicate that the actin cytoskeleton mediates NO-induced regulatory effects in chondrocytes by modulating down-regulation of PI 3-kinase and Akt, activation of p38 kinase, and inhibition of PKCα and -ζ Chondrocytes are differentiated from mesenchymal cells during embryo development (1Sandell L.J. Adler P. Front. Biosci. 1999; 4: 731-742Crossref PubMed Google Scholar, 2DeLise A.M. Fisher L. Tuan R.S. Osteoarthritis Cartilage. 2000; 8: 309-334Abstract Full Text PDF PubMed Scopus (657) Google Scholar). The phenotypes of differentiated chondrocytes in articular cartilage are characterized by the synthesis and maintenance of cartilage-specific extracellular matrix molecules, including type II collagen and proteoglycans such as aggrecan. However, the differentiated phenotype is unstable both in vivo and in vitro and thus lost by a process designated "dedifferentiation" upon exposure of cells to interleukin-1β (3Goldring M.B. Birkhead J.R. Suen L.F. Yamin R. Mizuno S. Glowacki J. Arbiser J.L. Apperley J.F. J. Clin. Invest. 1994; 94: 2307-2316Crossref PubMed Scopus (387) Google Scholar, 4Demoor-Fossard M. Redini F. Boittin M. Pujol J.P. Biochim. Biophys. Acta. 1998; 1398: 179-191Crossref PubMed Scopus (41) Google Scholar), nitric oxide (NO) 1The abbreviations used are: NO, nitric oxide; ATF, activating transcription factor; CD, cytochalasin D; DMEM, Dulbecco's modified Eagle's medium; ERK, extracellular signal-regulated protein kinase; IGF-1, insulin-like growth factor-1; I-κB, inhibitory κB; MAP kinase, mitogen-activated protein kinase; NF-κB, nuclear factor κB; PI, phosphatidylinositol; PKC, protein kinase C; SNP, sodium nitroprusside; TUNEL, terminal deoxynucleotidyl transfer-mediated nick-end labeling; PG, prostaglandin; COX, cyclooxygenase; PBS, phosphate-buffered saline; EGF, epidermal growth factor; PMA, phorbol 12-myristae 13-acetate; LNMMA, N-monomethyl-l-arginine. (5Amin A.R. Abramson S.B. Curr. Opin. Rheumatol. 1998; 10: 263-268Crossref PubMed Scopus (209) Google Scholar), or retinoic acid (6Cash D.E. Bock C.B. Schughart K. Linney E. Underhill T.M. J. 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We showed previously that direct production of NO by treatment of primary culture articular chondrocytes with a NO donor, sodium nitroprusside (SNP), led to apoptosis, dedifferentiation, and cyclooxygenase (COX-2) expression via a complex protein kinase signaling cascade involving mitogen-activated protein (MAP) kinase and protein kinase C (PKC) (12Kim S.-J. Ju J.-W. Oh C.-D. Yoon Y.-M. Song W.-K. Kim J.-H. Yoo Y.-J. Bang O.-S. Kang S.-S. Chun J.-S. J. Biol. Chem. 2002; 277: 1332-1339Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 13Kim S.-J. Kim H.-G. Oh C.-D. Hwang S.-G. Song W.-K. Yoo Y.-J. Kang S.-S. Chun J.-S. J. Biol. Chem. 2002; 277: 30375-30381Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 14Kim S.-J. Hwang S.-G. Shin D.-Y. Kang S.-S. Chun J.-S. J. Biol. Chem. 2002; 277: 33501-33508Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 15Kim S.-J. Chun J.-S. Biochem. Biophys. Res. Commun. 2003; 303: 206-211Crossref PubMed Scopus (44) Google Scholar). For example, NO-induced activation of extracellular signal-regulated protein kinase (ERK) promotes dedifferentiation, COX-2 expression, and inhibition of apoptosis, whereas NO-induced p38 kinase activation triggers apoptosis, COX-2 expression, and maintains differentiated phenotypes (12Kim S.-J. Ju J.-W. Oh C.-D. Yoon Y.-M. Song W.-K. Kim J.-H. Yoo Y.-J. Bang O.-S. Kang S.-S. Chun J.-S. J. Biol. Chem. 2002; 277: 1332-1339Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 15Kim S.-J. Chun J.-S. Biochem. Biophys. Res. Commun. 2003; 303: 206-211Crossref PubMed Scopus (44) Google Scholar). NO additionally inhibits protein kinase C (PKC) α and -ζ activities (13Kim S.-J. Kim H.-G. Oh C.-D. Hwang S.-G. Song W.-K. Yoo Y.-J. Kang S.-S. Chun J.-S. J. Biol. Chem. 2002; 277: 30375-30381Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). PKCα activity is inhibited due to blockage of expression independent of MAP kinase signaling, whereas PKCζ activity is suppressed as a result of p38 kinase activation that follows proteolytic cleavage by caspase-3. Inhibition of PKCα and -ζ is necessary for NO-induced dedifferentiation and nuclear factor (NF)-κB activation (15Kim S.-J. Chun J.-S. Biochem. Biophys. Res. Commun. 2003; 303: 206-211Crossref PubMed Scopus (44) Google Scholar). Activated NF-κB has dual functions, specifically induction of COX-2 expression and subsequent PGE2 production, and apoptosis by stimulating p53 transcription. A previous study by our group (14Kim S.-J. Hwang S.-G. Shin D.-Y. Kang S.-S. Chun J.-S. J. Biol. Chem. 2002; 277: 33501-33508Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar) also reveals that p38 kinase stimulates NO-induced apoptosis via p53 accumulation as a result of stabilization due to Ser-15 phosphorylation. The actin cytoskeletal architecture is believed to be an important modulator of chondrocyte phenotype. Dedifferentiation of chondrocytes by retinoic acid or serial monolayer culture is accompanied by significant changes in actin cytoskeletal architecture. Disruption of the actin cytoskeleton by dihydrocytochalasin B promotes redifferentiation of chondrocytes (16Benya P.D. Brown P.D. Padilla S.R. J. Cell Biol. 1988; 106: 161-170Crossref PubMed Scopus (167) Google Scholar, 17Brown P.D. Benya P.D. J. Cell Biol. 1988; 106: 171-179Crossref PubMed Scopus (136) Google Scholar). The actin cytoskeleton additionally mediates changes in articular chondrocyte phenotype induced by bone morphogenetic protein (18Vinall R.L. Lo S.H. Reddi A.H. Exp. Cell Res. 2002; 272: 32-44Crossref PubMed Scopus (55) Google Scholar) or NO (19Clancy R. Osteoarthritis Cartilage. 1999; 7: 399-400Abstract Full Text PDF PubMed Scopus (28) Google Scholar). Furthermore, disruption of the actin cytoskeleton by cytochalasin D (CD) triggers chondrogenesis in limb mesenchymal cells (20Zanetti N. Solursh M. J. Cell Biol. 1984; 99: 115-123Crossref PubMed Scopus (223) Google Scholar, 21Loty S. Forest N. Boulekbache H. Sautier J.M. Biol. Cell. 1995; 83: 149-161Crossref PubMed Scopus (109) Google Scholar, 22Lim Y.-B. Kang S.-S. Park T.-K. Lee Y.-S. Chun J.-S. Sonn J.-K. Biochem. Biophys. Res. Commun. 2000; 273: 609-613Crossref PubMed Scopus (65) Google Scholar, 23Lim Y.-B. Kang S.-S. An W.-G. Lee Y.-S. Chun J.-S. Sonn J.-K. J. Cell. Biochem. 2003; 88: 713-718Crossref PubMed Scopus (32) Google Scholar), indicating a function in the regulation of chondrogenic differentiation of mesenchymal cells and maintenance of differentiated chondrocyte phenotype. However, to date, the molecular mechanism underlying the regulation of chondrocyte phenotype by actin cytoskeletal architecture has yet to be fully elucidated. In addition to regulating chondrocyte phenotype, the actin cytoskeleton modulates apoptosis either positively or negatively in a variety of cell types, depending on the experimental system (24Huot J. Houle F. Rousseau S. Deschesnes R.G. Shah G.M. Landry J. J. Cell Biol. 1998; 143: 1361-1373Crossref PubMed Scopus (266) Google Scholar, 25Posey S.C. Bierer B.E. J. Biol. Chem. 1999; 274: 4259-4265Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 26Korichneva I. Hammerling U. J. Cell Sci. 1999; 112: 2521-2528PubMed Google Scholar). For instance, disruption of the actin cytoskeleton causes apoptosis of capillary endothelial cells (27Flusberg D.A. Numaguchi Y. Ingber D.E. Mol. Biol. Cell. 2001; 12: 3087-3094Crossref PubMed Scopus (96) Google Scholar), airway epithelial cells (28White S.R. Williams P. Wojcik K.R. Sun S. Hiemstra P.S. Rabe K.F. Dorscheid D.R. Am. J. Respir. Cell Mol. Biol. 2001; 24: 282-294Crossref PubMed Scopus (104) Google Scholar), T cells (29Suria H. Chau L.A. Negrou E. Kelvin D.J. Madrenas J. Life Sci. 1999; 65: 2697-2707Crossref PubMed Scopus (59) Google Scholar), megakaryoblastic leukemia cells (30Yamazaki Y. Tsuruga M. Zhou D. Fujita Y. Shang X. Dang Y. Kawasaki K. Oka S. Exp. Cell Res. 2000; 259: 64-78Crossref PubMed Scopus (96) Google Scholar), and dentate granule cells (31Kim J.A. Mitsukawa K. Yamada M.K. Nishiyama N. Matsuki N. Ikegaya Y. Neuropharmacology. 2002; 42: 1109-1118Crossref PubMed Scopus (47) Google Scholar) but inhibits apoptosis in B lymphocytes (32Melamed I. Gelfand E.W. Cell. Immunol. 1999; 194: 136-142Crossref PubMed Scopus (19) Google Scholar) and lymphoma cells (33Bando M. Miyake Y. Shiina M. Wachi M. Nagai K. Kataoka T. Biochem. Biophys. Res. Commun. 2002; 290: 268-274Crossref PubMed Scopus (27) Google Scholar). However, it is currently unknown whether actin cytoskeleton also regulates apoptosis of articular chondrocytes. In view of the evident significance of the actin cytoskeletal architecture in the physiology of chondrocytes and cartilage, we initially investigated its role in NO-induced dedifferentiation, apoptosis, and inflammatory responses, such as cyclooxygenase (COX)-2 and prostaglandin E2 (PGE2) production in primary culture rabbit articular chondrocytes. We additionally characterized the molecular mechanism of regulation of chondrocyte function by the actin cytoskeleton, focusing on the roles of MAP kinase and PKC. Here we report that disruption of the actin cytoskeleton by CD results in the inhibition of NO-induced dedifferentiation, apoptosis, COX-2 expression, and PGE2 production in articular chondrocytes via modulation of MAP kinase activation and inhibition of PKCα and -ζ signaling. Culture of Articular Chondrocytes—Articular chondrocytes were isolated from knee joint cartilage slices of 2-week-old New Zealand White rabbits by enzymatic digestion with collagenase type II in Dulbecco's modified Eagle's medium (DMEM) (9Yoon Y.-M. Kim S.-J. Oh C.-D. Ju J.-W. Song W.-K. Yoo Y.-J. Huh T.-L. Chun J.-S. J. Biol. Chem. 2002; 277: 8412-8420Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Cells were maintained in DMEM supplemented with 10% fetal bovine calf serum, 50 μg/ml streptomycin, and 50 units/ml penicillin by plating on culture dishes at a density of 5 × 104 cells/cm2 with medium replacement every 2 days. At 3.5 days in culture, cells were treated with the specified pharmacological reagents for 1 h prior to SNP treatment, including CD to disrupt the actin cytoskeleton, jasplakinolide to stabilize the actin cytoskeleton, PD98059 (Calbiochem) to inhibit MEK-1/-2 (34Alessi D.R. Cuenda A. Cohen P. Dudley D.T. Saltiel A.R. J. Biol. Chem. 1995; 270: 27489-27494Abstract Full Text Full Text PDF PubMed Scopus (3256) Google Scholar), SB203580 (Calbiochem) to inhibit p38 kinase (35Cuenda A. Rouse J. Doza Y.N. Meier R. Cohen P. Gallagher T.F. Young P.R. Lee J.C. FEBS Lett. 1995; 364: 229-233Crossref PubMed Scopus (1980) Google Scholar), benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone (benzyloxycarbonyl-DEVD; Bachem, Heidelberg, Germany) to inhibit caspase-3 (36Varghese J. Chattopadhaya S. Sarin A. J. Immunol. 2001; 166: 6570-6577Crossref PubMed Scopus (44) Google Scholar), and SN-50 peptide (Biomol, Plymouth Meeting, PA) to inhibit nuclear translocation of NF-κB (37Lin Y.Z. Yao S.Y. Veach R.A. Torgerson T.R. Hawiger J. J. Biol. Chem. 1995; 270: 14255-14258Abstract Full Text Full Text PDF PubMed Scopus (854) Google Scholar). Where indicated, chondrocytes at day 3 in culture were infected with either control adenovirus or adenovirus coding for wild-type rabbit PKCα or mouse PKCζ inserted into a cosmid cassette. Infected cells were cultured in complete medium for 24 h and treated with 1 mm SNP for an additional 24 h. Cartilage Explants Culture—Cartilage slices (∼125 mm3) were obtained from rabbit knee joints and maintained in DMEM in the absence or presence of various pharmacological reagents specified in individual experiments. Cartilage explants were fixed in 4% paraformaldehyde for 24 h at 4 °C, dehydrated with graded ethanol, embedded in paraffin, and sectioned into 4-μm slices. Sections were stained with Alcian blue to detect accumulation of sulfated proteoglycan using standard procedures as described previously (38Ryu J.-H. Kim S.-J. Kim S.-H. Oh C.-D. Hwang S.-G. Chun C.-H. Oh S.-H. Seong J.-K. Huh T.-L. Chun J.-S. Development. 2002; 129: 5541-5550Crossref PubMed Scopus (131) Google Scholar). Apoptotic cells were determined using the method described below. Immunofluorescence Microscopy—Immunofluorescence microscopy was used to determine F-actin organization in primary culture articular chondrocytes or cartilage explants, which were either left untreated or treated with various pharmacological reagents. Briefly, primary culture chondrocytes were fixed with 3.5% paraformaldehyde in PBS for 10 min at room temperature. Cells were permeabilized and blocked in PBS containing 0.1% Triton X-100 and 5% fetal calf serum for 30 min. Fixed cells were washed with PBS and incubated for 1 h with rhodamine-conjugated phalloidin, re-washed, and then observed under a standard fluorescence microscope (38Ryu J.-H. Kim S.-J. Kim S.-H. Oh C.-D. Hwang S.-G. Chun C.-H. Oh S.-H. Seong J.-K. Huh T.-L. Chun J.-S. Development. 2002; 129: 5541-5550Crossref PubMed Scopus (131) Google Scholar). Determination of Cell Death—NO induces apoptosis in chondrocytes, as demonstrated previously by DNA fragmentation and terminal deoxynucleotidyl transfer-mediated nick-end labeling (TUNEL) assays (12Kim S.-J. Ju J.-W. Oh C.-D. Yoon Y.-M. Song W.-K. Kim J.-H. Yoo Y.-J. Bang O.-S. Kang S.-S. Chun J.-S. J. Biol. Chem. 2002; 277: 1332-1339Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Apoptotic cell death in cartilage explants was determined by the TUNEL method using an assay kit (Roche Applied Science). Apoptotic cells were additionally quantified using ∼1 × 104 cells on a FACSort flow cytometer with the Cellquest analysis program (BD Biosciences), as described previously (12Kim S.-J. Ju J.-W. Oh C.-D. Yoon Y.-M. Song W.-K. Kim J.-H. Yoo Y.-J. Bang O.-S. Kang S.-S. Chun J.-S. J. Biol. Chem. 2002; 277: 1332-1339Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Assay of Caspase-3 Activity—Caspase-3 activation in SNP-treated chondrocytes was determined by measuring the absorbance of a cleaved synthetic substrate of caspase-3, Ac-Asp-Glu-Val-Asp-chromophore p-nitroaniline (12Kim S.-J. Ju J.-W. Oh C.-D. Yoon Y.-M. Song W.-K. Kim J.-H. Yoo Y.-J. Bang O.-S. Kang S.-S. Chun J.-S. J. Biol. Chem. 2002; 277: 1332-1339Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Briefly, chondrocytes were lysed on ice for 10 min in cell lysis buffer provided in the Clontech A ApoAlert™ CPP32 colorimetric assay kit. Lysates were reacted with 50 μm Ac-Asp-Glu-Val-Asp-chromophore p-nitroaniline in reaction buffer (0.1 m HEPES, 20% glycerol, 10 mm dithiothreitol, and protease inhibitors (pH 7.4)). Mixtures were maintained at 37 °C for 1 h in a water bath and subsequently analyzed in an enzyme-linked immunosorbent assay reader. Enzyme activity was calculated from a standard curve prepared using p-nitroaniline. The relative levels of p-nitroaniline were normalized against the protein concentration of each extract. NF-κB Reporter Gene Assay—NF-κB activation was examined indirectly by analyzing the degradation of inhibitor protein κB (I-κB) by using Western blot analysis and directly with a reporter gene assay. For a reporter gene assay, chondrocytes were transfected with a plasmid containing luciferase and three tandem repeats of serum-response element or a control vector, using LipofectAMINE PLUS (12Kim S.-J. Ju J.-W. Oh C.-D. Yoon Y.-M. Song W.-K. Kim J.-H. Yoo Y.-J. Bang O.-S. Kang S.-S. Chun J.-S. J. Biol. Chem. 2002; 277: 1332-1339Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Transfected cells were cultured in complete medium for 24 h, either left untreated or treated with various pharmacological reagents, and used to determine luciferase activity with an assay kit from Promega (Madison, WI). Luciferase activity was normalized against β-galactosidase activity. Immunoprecipitation and Kinase Assays—To determine the activities of PKCα, PKCζ and p38 kinase, cell lysates were prepared in lysis buffer containing 20 mm Tris-HCl (pH 7.5), 1 mm EDTA, 1 mm EGTA, 150 mm NaCl, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mm β-glycerol phosphate, protease inhibitors (10 μg/ml leupeptin, 10 μg/ml pepstatin A, 10 μg/ml aprotinin, and 1 mm 4-(2-aminoethyl) benzenesulfonyl fluoride), and phosphatase inhibitors (1 mm NaF and 1 mm Na3VO4). Cell lysates were precipitated with polyclonal antibody against p38 kinase, PKCζ (Santa Cruz Biotechnology Inc., Santa Cruz, CA), or PKCα (Transduction Laboratories). Immune complexes were collected using protein A-Sepharose beads, and the kinase reaction was performed in 20 μl of reaction buffer containing 25 mm Tris-HCl (pH 7.5), 5 mm β-glycerol phosphate, 2 mm dithiothreitol, 0.1 mm sodium orthovanadate, 10 mm MgCl2, [γ-32P]ATP, and 1 μg of substrate (activating transcription factor-2 (ATF-2)) for p38 kinase or myelin basic protein for PKCα and -ζ. Substrate phosphorylation was detected by autoradiography (13Kim S.-J. Kim H.-G. Oh C.-D. Hwang S.-G. Song W.-K. Yoo Y.-J. Kang S.-S. Chun J.-S. J. Biol. Chem. 2002; 277: 30375-30381Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). PGE 2 Assay—PGE2 production in articular chondrocytes was determined by measuring the levels of cellular and secreted PGE2 with an assay kit purchased from Amersham Biosciences, as reported previously (39Huh Y.-H. Kim S.-H. Kim S.-J. Chun J.-S. J. Biol. Chem. 2003; 278: 9691-9697Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Briefly, chondrocytes were seeded in standard 96-well microtiter plates and treated with various pharmacological reagents. Total cell lysates were used to quantify the amount of PGE2 according to the manufacturer's protocol. PGE2 levels were calculated against a standard curve. Western Blot Analysis—Whole cell lysates were prepared by extracting proteins using a buffer containing 50 mm Tris-HCl (pH 7.4), 150 mm NaCl, 1% Nonidet P-40, and 0.1% SDS supplemented with protease inhibitors and phosphatase inhibitors, as described above. Proteins were size-fractionated by SDS-PAGE and transferred to a nitrocellulose membrane. Proteins were detected using the following antibodies: anti-type II collagen from Chemicon (Temecula, CA), polyclonal anti-p53 and phosphorylation-specific antibody for ERK from New England Biolabs (Beverly, MA), monoclonal PKCα, PKCζ, and ERK-1/-2 from Transduction Laboratories, and polyclonal p38 kinase from Santa Cruz Biotechnology Inc. Blots were developed using a peroxidase-conjugated secondary antibody and visualized with an ECL system. Disruption of the Actin Cytoskeleton Inhibits NO-induced Apoptosis of Articular Chondrocytes—We have shown previously that direct production of NO by SNP in primary culture articular chondrocytes results in apoptosis and dedifferentiation (12Kim S.-J. Ju J.-W. Oh C.-D. Yoon Y.-M. Song W.-K. Kim J.-H. Yoo Y.-J. Bang O.-S. Kang S.-S. Chun J.-S. J. Biol. Chem. 2002; 277: 1332-1339Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 13Kim S.-J. Kim H.-G. Oh C.-D. Hwang S.-G. Song W.-K. Yoo Y.-J. Kang S.-S. Chun J.-S. J. Biol. Chem. 2002; 277: 30375-30381Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 14Kim S.-J. Hwang S.-G. Shin D.-Y. Kang S.-S. Chun J.-S. J. Biol. Chem. 2002; 277: 33501-33508Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 15Kim S.-J. Chun J.-S. Biochem. Biophys. Res. Commun. 2003; 303: 206-211Crossref PubMed Scopus (44) Google Scholar). Prior to determining the role of the actin cytoskeleton in NO-induced apoptosis, we examined the organization of actin filaments in articular chondrocytes. The actin cytoskeleton in primary culture chondrocytes at passage 0 comprised a stress fiber-like structure or thick struts of actin fiber extended across the length of the chondrocytes (Fig. 1A). NO induced a typical peripheral distribution of actin filaments that is consistent with their mechanical supporting function for shrinking apoptotic cells. Treatment with CD led to the collapse of the filamentous actin structure to amorphous clots of depolymerized protein and a more round shape of cells (Fig. 1A). The effects of actin cytoskeleton disruption on NO-induced apoptosis were determined by staining cell nuclei with propidium iodide and TUNEL assay. As shown in Fig. 1B, NO induced apoptotic cell death, and disruption of the actin cytoskeleton by CD significantly reduced the number of TUNEL-positive cells. Flow cytometric analyses revealed that CD inhibits NO-induced apoptosis in a dose-dependent manner (Fig. 1C). The role of the actin cytoskeleton in NO-induced apoptosis was further investigated using jasplakinolide, which is a potent inducer of actin polymerization and selectively locks actin in a polymeric state. Treatment with jasplakinolide led to the formation of relatively fine stress fiber and large aggregations of actin filaments (Fig. 2A). Jasplakinolide also increased the number of cells that show NO-induced peripheral distribution of actin filaments (Fig. 2A). Indeed, jasplakinolide dramatically enhanced NO-induced apoptosis in a dose-dependent manner (Fig. 2B). Taken together, the above results clearly indicate that disruption of actin filaments reduces NO-induced apoptosis, whereas stabilization of actin cytoskeleton enhances apoptotic cell death.Fig. 2Stabilization of actin filaments with jasplakinolide potentiates NO-induced apoptosis. A, articular chondrocytes were treated for 24 h with Me2SO as a control, 50 nm jasplakinolide, 1 mm SNP without (SNP) or with 50 nm jasplakinolide. Cells were stained for F-actin with rhodamine-conjugated phalloidin. B, articular chondrocytes were left untreated (Control) or treated with the indicated concentrations of jasplakinolide and 1 mm SNP for 24 h. Apoptosis was determined by flow cytometry. Data are presented as results of a typical experiment (A) and as mean values with standard deviation (B) (n = 4).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the next series of experiments, we investigated the molecular mechanism of apoptosis regulation by the actin cytoskeleton. Because NO induces chondrocyte apoptosis via NF-κB-dependent accumulation of p53 that in turn leads to caspase-3 activation (12Kim S.-J. Ju J.-W. Oh C.-D. Yoon Y.-M. Song W.-K. Kim J.-H. Yoo Y.-J. Bang O.-S. Kang S.-S. Chun J.-S. J. Biol. Chem. 2002; 277: 1332-1339Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 13Kim S.-J. Kim H.-G. Oh C.-D. Hwang S.-G. Song W.-K. Yoo Y.-J. Kang S.-S. Chun J.-S. J. Biol. Chem. 2002; 277: 30375-30381Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 15Kim S.-J. Chun J.-S. Biochem. Biophys. Res. Commun. 2003; 303: 206-211Crossref PubMed Scopus (44) Google Scholar), the effects of actin cytoskeleton disruption on the activation of caspase-3 and NF-κB and on the accumulation of p53 were determined. CD treatment blocked NO-induced activation of caspase-3 (Fig. 3A) and NF-κB (as demonstrated by a NF-κB reporter gene assay and inhibition of I-κB degradation (Fig. 3, B and C)) and accumulation of pro-apoptotic p53 protein (Fig. 3C) in a dose-dependent manner. Because treatment with SN-50 (a NF-κB inhibitor) and benzyloxycarbonyl-DEVD peptide (a caspase-3 inhibitor) blocked NO-induced apoptosis (Fig. 3D), the above results indicate that inhibition of these activities by disruption of the actin cytoskeleton is responsible for the suppression of cell death. We previously (13Kim S.-J. Kim H.-G. Oh C.-D. Hwang S.-G. Song W.-K. Yoo Y.-J. Kang S.-S. Chun J.-S. J. Biol. Chem. 2002; 277: 30375-30381Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) demonstrated that protein levels and activities of PKCα and -ζ, signaling molecules upstream of NF-κB activation and p53 accumulation, decrease upon NO production. Moreover, these events are necessary for apoptosis and dedifferentiation of chondrocytes. We additionally showed that PKCζ activity is inhibited as a result of p38 kinase activation, whereas suppression of PKCα activity is independent of MAP kinase signaling (12Kim S.-J. Ju J.-W. Oh C.-D. Yoon Y.-M. Song W.-K. Kim J.-H. Yoo Y.-J. Bang O.-S. Kang S.-S. Chun J.-S. J. Biol. Chem. 2002; 277: 1332-1339Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 13Kim S.-J. Kim H.-G. Oh C.-D. Hwang S.-G. Song W.-K. Yoo Y.-J. Kang S.-S. Chun J.-S. J. Biol. Chem. 2002; 277: 30375-30381Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Accordingly, we examined whether disruption of the actin cytoskeleton blocks NO-induced apoptosis via the modulation of MAP kinase and PKCα and -ζ signaling. As depicted in Fig. 4A, expression and activity of PKCα and -ζ decreased in SNP-treated chondrocytes. Disruption of the actin cytoskeleton abolished the observed decrease in PKCα and -ζ expression and activity. In addition, ectopic expression of PKCα or -ζ by adenovirus infection suppressed NO-induced apoptosis (Fig. 4B), indicating that the inhibitory effects of CD on NO-induced apoptosis are a result of blockage of inhibition of PKCα and -ζ activity. As expected, SNP treatment in articular chondrocytes also activated ERK-1/-2 and p38 kinase, as determined by Western blotting and kinase assays, respectively (Fig. 4C). Disruption of the actin cytoskeleton did not significantly affect NO-induced ERK-1/-2 activation (Fig. 4C, upper panel), but activation of p38 kinase by NO was completely blocked (Fig. 4C, lower panel). Treatment with the ERK-1/-2 inhibitor,