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. 1997 Oct 1;100(7):1789–1796. doi: 10.1172/JCI119706

Outside-in signaling in the chondrocyte. Nitric oxide disrupts fibronectin-induced assembly of a subplasmalemmal actin/rho A/focal adhesion kinase signaling complex.

R M Clancy 1, J Rediske 1, X Tang 1, N Nijher 1, S Frenkel 1, M Philips 1, S B Abramson 1
PMCID: PMC508364  PMID: 9312179

Abstract

Elevated levels of fibronectin (Fn) in articular cartilage have been linked to the progression of both rheumatoid and osteoarthritis. In this study, we examined intracellular events which follow ligation of Fn to its receptor, the integrin alpha5beta1. In addition, we examined the regulatory influence of nitric oxide on these events, since this free radical has been implicated in cartilage degradation. Exposure of chondrocytes to Fn-coated beads resulted in the circumferential clustering of the alpha5beta1 integrin receptor, which was accompanied by the subplasmalemmal assembly of a focal activation complex comprised of F-actin, the tyrosine kinase, focal adhesion kinase (FAK), the ras related G protein rho A, as well as tyrosine-phosphorylated proteins. Treatment with exogenous nitric oxide (NO) or catabolic cytokines which induce nitric oxide synthase blocked the assembly of F-actin, FAK, rho A and tyrosine-phosphorylated proteins while not affecting the total number of beads bound per cell nor the clustering of alpha5beta1 integrin. Use of a cGMP antagonist (Rp-8-Br cGMPS) or cGMP agonist (Sp-cGMPS) either abolished or mimicked the NO effect, respectively. Adherence of chondrocytes to fibronectin enhanced proteoglycan synthesis by twofold (vs. albumin). In addition, basic fibroblast growth factor (FGF) and insulin growth factor (IGF-1) induced proteoglycan synthesis in chondrocytes adherent to Fn but not albumin suggesting a costimulatory signal transduced by alpha5betal and the FGF receptor. Both constitutive and FGF stimulated proteoglycan synthesis were completely inhibited by nitric oxide. These data indicate that the ligation of alpha5beta1 in the chondrocyte induced the intracellular assembly of an activation complex comprised of the cytoplasmic tail of alpha5beta1 integrin, actin, and the signaling molecules rho A and FAK. We show that NO inhibits the assembly of the intracellular activation complex and the synthesis of proteoglycans, but has no effect on the extracellular aggregation of alpha5beta1 integrin. These observations provide a basis by which nitric oxide can interfere with chondrocyte functions by affecting chondrocyte-matrix interactions.

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Selected References

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  1. Albelda S. M., Buck C. A. Integrins and other cell adhesion molecules. FASEB J. 1990 Aug;4(11):2868–2880. [PubMed] [Google Scholar]
  2. Amin A. R., Di Cesare P. E., Vyas P., Attur M., Tzeng E., Billiar T. R., Stuchin S. A., Abramson S. B. The expression and regulation of nitric oxide synthase in human osteoarthritis-affected chondrocytes: evidence for up-regulated neuronal nitric oxide synthase. J Exp Med. 1995 Dec 1;182(6):2097–2102. doi: 10.1084/jem.182.6.2097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burton-Wurster N., Butler M., Harter S., Colombo C., Quintavalla J., Swartzendurber D., Arsenis C., Lust G. Presence of fibronectin in articular cartilage in two animal models of osteoarthritis. J Rheumatol. 1986 Feb;13(1):175–182. [PubMed] [Google Scholar]
  4. Butt E., Pöhler D., Genieser H. G., Huggins J. P., Bucher B. Inhibition of cyclic GMP-dependent protein kinase-mediated effects by (Rp)-8-bromo-PET-cyclic GMPS. Br J Pharmacol. 1995 Dec;116(8):3110–3116. doi: 10.1111/j.1476-5381.1995.tb15112.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chiquet-Ehrismann R., Kalla P., Pearson C. A., Beck K., Chiquet M. Tenascin interferes with fibronectin action. Cell. 1988 May 6;53(3):383–390. doi: 10.1016/0092-8674(88)90158-4. [DOI] [PubMed] [Google Scholar]
  6. Clancy R. M., Abramson S. B. Nitric oxide: a novel mediator of inflammation. Proc Soc Exp Biol Med. 1995 Nov;210(2):93–101. doi: 10.3181/00379727-210-43927aa. [DOI] [PubMed] [Google Scholar]
  7. Clancy R. M., Miyazaki Y., Cannon P. J. Use of thionitrobenzoic acid to characterize the stability of nitric oxide in aqueous solutions and in porcine aortic endothelial cell suspensions. Anal Biochem. 1990 Nov 15;191(1):138–143. doi: 10.1016/0003-2697(90)90400-4. [DOI] [PubMed] [Google Scholar]
  8. Clancy R., Leszczynska J., Amin A., Levartovsky D., Abramson S. B. Nitric oxide stimulates ADP ribosylation of actin in association with the inhibition of actin polymerization in human neutrophils. J Leukoc Biol. 1995 Aug;58(2):196–202. doi: 10.1002/jlb.58.2.196. [DOI] [PubMed] [Google Scholar]
  9. Clark E. A., Brugge J. S. Integrins and signal transduction pathways: the road taken. Science. 1995 Apr 14;268(5208):233–239. doi: 10.1126/science.7716514. [DOI] [PubMed] [Google Scholar]
  10. Curtis A. S., McGrath M., Gasmi L. Localised application of an activating signal to a cell: experimental use of fibronectin bound to beads and the implications for mechanisms of adhesion. J Cell Sci. 1992 Feb;101(Pt 2):427–436. doi: 10.1242/jcs.101.2.427. [DOI] [PubMed] [Google Scholar]
  11. Danen E. H., Aota S., van Kraats A. A., Yamada K. M., Ruiter D. J., van Muijen G. N. Requirement for the synergy site for cell adhesion to fibronectin depends on the activation state of integrin alpha 5 beta 1. J Biol Chem. 1995 Sep 15;270(37):21612–21618. doi: 10.1074/jbc.270.37.21612. [DOI] [PubMed] [Google Scholar]
  12. Farrell A. J., Blake D. R., Palmer R. M., Moncada S. Increased concentrations of nitrite in synovial fluid and serum samples suggest increased nitric oxide synthesis in rheumatic diseases. Ann Rheum Dis. 1992 Nov;51(11):1219–1222. doi: 10.1136/ard.51.11.1219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Frenkel S. R., Clancy R. M., Ricci J. L., Di Cesare P. E., Rediske J. J., Abramson S. B. Effects of nitric oxide on chondrocyte migration, adhesion, and cytoskeletal assembly. Arthritis Rheum. 1996 Nov;39(11):1905–1912. doi: 10.1002/art.1780391118. [DOI] [PubMed] [Google Scholar]
  14. Gopalakrishna R., Chen Z. H., Gundimeda U. Nitric oxide and nitric oxide-generating agents induce a reversible inactivation of protein kinase C activity and phorbol ester binding. J Biol Chem. 1993 Dec 25;268(36):27180–27185. [PubMed] [Google Scholar]
  15. Hall A. The cellular functions of small GTP-binding proteins. Science. 1990 Aug 10;249(4969):635–640. doi: 10.1126/science.2116664. [DOI] [PubMed] [Google Scholar]
  16. Hynes R. O. Integrins: a family of cell surface receptors. Cell. 1987 Feb 27;48(4):549–554. doi: 10.1016/0092-8674(87)90233-9. [DOI] [PubMed] [Google Scholar]
  17. Jun C. D., Han M. K., Kim U. H., Chung H. T. Nitric oxide induces ADP-ribosylation of actin in murine macrophages: association with the inhibition of pseudopodia formation, phagocytic activity, and adherence on a laminin substratum. Cell Immunol. 1996 Nov 25;174(1):25–34. doi: 10.1006/cimm.1996.0290. [DOI] [PubMed] [Google Scholar]
  18. Loeser R. F., Carlson C. S., McGee M. P. Expression of beta 1 integrins by cultured articular chondrocytes and in osteoarthritic cartilage. Exp Cell Res. 1995 Apr;217(2):248–257. doi: 10.1006/excr.1995.1084. [DOI] [PubMed] [Google Scholar]
  19. Loeser R. F. Integrin-mediated attachment of articular chondrocytes to extracellular matrix proteins. Arthritis Rheum. 1993 Aug;36(8):1103–1110. doi: 10.1002/art.1780360811. [DOI] [PubMed] [Google Scholar]
  20. Maier R., Bilbe G., Rediske J., Lotz M. Inducible nitric oxide synthase from human articular chondrocytes: cDNA cloning and analysis of mRNA expression. Biochim Biophys Acta. 1994 Sep 21;1208(1):145–150. doi: 10.1016/0167-4838(94)90171-6. [DOI] [PubMed] [Google Scholar]
  21. Miyamoto S., Akiyama S. K., Yamada K. M. Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function. Science. 1995 Feb 10;267(5199):883–885. doi: 10.1126/science.7846531. [DOI] [PubMed] [Google Scholar]
  22. Miyamoto S., Teramoto H., Gutkind J. S., Yamada K. M. Integrins can collaborate with growth factors for phosphorylation of receptor tyrosine kinases and MAP kinase activation: roles of integrin aggregation and occupancy of receptors. J Cell Biol. 1996 Dec;135(6 Pt 1):1633–1642. doi: 10.1083/jcb.135.6.1633. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Murphy-Ullrich J. E., Lightner V. A., Aukhil I., Yan Y. Z., Erickson H. P., Hök M. Focal adhesion integrity is downregulated by the alternatively spliced domain of human tenascin. J Cell Biol. 1991 Nov;115(4):1127–1136. doi: 10.1083/jcb.115.4.1127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Murphy-Ullrich J. E., Pallero M. A., Boerth N., Greenwood J. A., Lincoln T. M., Cornwell T. L. Cyclic GMP-dependent protein kinase is required for thrombospondin and tenascin mediated focal adhesion disassembly. J Cell Sci. 1996 Oct;109(Pt 10):2499–2508. doi: 10.1242/jcs.109.10.2499. [DOI] [PubMed] [Google Scholar]
  25. Murrell G. A., Doland M. M., Jang D., Szabo C., Warren R. F., Hannafin J. A. Nitric oxide: an important articular free radical. J Bone Joint Surg Am. 1996 Feb;78(2):265–274. [PubMed] [Google Scholar]
  26. Nobes C. D., Hall A. Rho, rac, and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell. 1995 Apr 7;81(1):53–62. doi: 10.1016/0092-8674(95)90370-4. [DOI] [PubMed] [Google Scholar]
  27. Osborn K. D., Trippel S. B., Mankin H. J. Growth factor stimulation of adult articular cartilage. J Orthop Res. 1989;7(1):35–42. doi: 10.1002/jor.1100070106. [DOI] [PubMed] [Google Scholar]
  28. Pennypacker J. P., Hassell J. R., Yamada K. M., Pratt R. M. The influence of an adhesive cell surface protein on chondrogenic expression in vitro. Exp Cell Res. 1979 Jul;121(2):411–415. doi: 10.1016/0014-4827(79)90022-3. [DOI] [PubMed] [Google Scholar]
  29. Prins A. P., Lipman J. M., McDevitt C. A., Sokoloff L. Effect of purified growth factors on rabbit articular chondrocytes in monolayer culture. II. Sulfated proteoglycan synthesis. Arthritis Rheum. 1982 Oct;25(10):1228–1238. doi: 10.1002/art.1780251012. [DOI] [PubMed] [Google Scholar]
  30. Rediske J. J., Koehne C. F., Zhang B., Lotz M. The inducible production of nitric oxide by articular cell types. Osteoarthritis Cartilage. 1994 Sep;2(3):199–206. doi: 10.1016/s1063-4584(05)80069-x. [DOI] [PubMed] [Google Scholar]
  31. Rees J. A., Ali S. Y., Brown R. A. Ultrastructural localisation of fibronectin in human osteoarthritic articular cartilage. Ann Rheum Dis. 1987 Nov;46(11):816–822. doi: 10.1136/ard.46.11.816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ridley A. J., Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell. 1992 Aug 7;70(3):389–399. doi: 10.1016/0092-8674(92)90163-7. [DOI] [PubMed] [Google Scholar]
  33. Ridley A. J., Paterson H. F., Johnston C. L., Diekmann D., Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell. 1992 Aug 7;70(3):401–410. doi: 10.1016/0092-8674(92)90164-8. [DOI] [PubMed] [Google Scholar]
  34. Ruoslahti E., Reed J. C. Anchorage dependence, integrins, and apoptosis. Cell. 1994 May 20;77(4):477–478. doi: 10.1016/0092-8674(94)90209-7. [DOI] [PubMed] [Google Scholar]
  35. Stadler J., Stefanovic-Racic M., Billiar T. R., Curran R. D., McIntyre L. A., Georgescu H. I., Simmons R. L., Evans C. H. Articular chondrocytes synthesize nitric oxide in response to cytokines and lipopolysaccharide. J Immunol. 1991 Dec 1;147(11):3915–3920. [PubMed] [Google Scholar]
  36. Stefanovic-Racic M., Stadler J., Evans C. H. Nitric oxide and arthritis. Arthritis Rheum. 1993 Aug;36(8):1036–1044. doi: 10.1002/art.1780360803. [DOI] [PubMed] [Google Scholar]
  37. Taskiran D., Stefanovic-Racic M., Georgescu H., Evans C. Nitric oxide mediates suppression of cartilage proteoglycan synthesis by interleukin-1. Biochem Biophys Res Commun. 1994 Apr 15;200(1):142–148. doi: 10.1006/bbrc.1994.1426. [DOI] [PubMed] [Google Scholar]
  38. Tremble P., Chiquet-Ehrismann R., Werb Z. The extracellular matrix ligands fibronectin and tenascin collaborate in regulating collagenase gene expression in fibroblasts. Mol Biol Cell. 1994 Apr;5(4):439–453. doi: 10.1091/mbc.5.4.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Waldman S. A., Murad F. Biochemical mechanisms underlying vascular smooth muscle relaxation: the guanylate cyclase-cyclic GMP system. J Cardiovasc Pharmacol. 1988;12 (Suppl 5):S115–S118. [PubMed] [Google Scholar]
  40. Woods V. L., Jr, Schreck P. J., Gesink D. S., Pacheco H. O., Amiel D., Akeson W. H., Lotz M. Integrin expression by human articular chondrocytes. Arthritis Rheum. 1994 Apr;37(4):537–544. doi: 10.1002/art.1780370414. [DOI] [PubMed] [Google Scholar]
  41. Wurster N. B., Lust G. Fibronectin in osteoarthritic canine articular cartilage. Biochem Biophys Res Commun. 1982 Dec 31;109(4):1094–1101. doi: 10.1016/0006-291x(82)91889-7. [DOI] [PubMed] [Google Scholar]
  42. Wyatt T. A., Lincoln T. M., Pryzwansky K. B. Vimentin is transiently co-localized with and phosphorylated by cyclic GMP-dependent protein kinase in formyl-peptide-stimulated neutrophils. J Biol Chem. 1991 Nov 5;266(31):21274–21280. [PubMed] [Google Scholar]
  43. Xie D. L., Meyers R., Homandberg G. A. Fibronectin fragments in osteoarthritic synovial fluid. J Rheumatol. 1992 Sep;19(9):1448–1452. [PubMed] [Google Scholar]

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