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. 2004 Mar 1;378(Pt 2):539–547. doi: 10.1042/BJ20030794

Calcium mobilization and Rac1 activation are required for VCAM-1 (vascular cell adhesion molecule-1) stimulation of NADPH oxidase activity.

Joan M Cook-Mills 1, Jacob D Johnson 1, Tracy L Deem 1, Atsuo Ochi 1, Lei Wang 1, Yi Zheng 1
PMCID: PMC1223954  PMID: 14594451

Abstract

VCAM-1 (vascular cell adhesion molecule-1) plays an important role in the regulation of inflammation in atherosclerosis, asthma, inflammatory bowel disease and transplantation. VCAM-1 activates endothelial cell NADPH oxidase, and this oxidase activity is required for VCAM-1-dependent lymphocyte migration. We reported previously that a mouse microvascular endothelial cell line promotes lymphocyte migration that is dependent on VCAM-1, but not on other known adhesion molecules. Here we have investigated the signalling mechanisms underlying VCAM-1 function. Lymphocyte binding to VCAM-1 on the endothelial cell surface activated an endothelial cell calcium flux that could be inhibited with anti-alpha4-integrin and mimicked by anti-VCAM-1-coated beads. VCAM-1 stimulation of calcium responses could be blocked by an inhibitor of intracellular calcium mobilization, a calcium channel inhibitor or a calcium chelator, resulting in the inhibition of NADPH oxidase activity. Addition of ionomycin overcame the calcium channel blocker suppression of VCAM-1-stimulated NADPH oxidase activity, but could not reverse the inhibitory effect imposed by intracellular calcium blockage, indicating that both intracellular and extracellular calcium mobilization are required for VCAM-1-mediated activation of NADPH oxidase. Furthermore, VCAM-1 specifically activated the Rho-family GTPase Rac1, and VCAM-1 activation of NADPH oxidase was blocked by a dominant negative Rac1. Thus VCAM-1 stimulates the mobilization of intracellular and extracellular calcium and Rac1 activity that are required for the activation of NADPH oxidase.

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Selected References

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  1. Allen W. E., Jones G. E., Pollard J. W., Ridley A. J. Rho, Rac and Cdc42 regulate actin organization and cell adhesion in macrophages. J Cell Sci. 1997 Mar;110(Pt 6):707–720. doi: 10.1242/jcs.110.6.707. [DOI] [PubMed] [Google Scholar]
  2. Balogh Péter, Aydar Yüksel, Tew John G., Szakal Andras K. Appearance and phenotype of murine follicular dendritic cells expressing VCAM-1. Anat Rec. 2002 Oct 1;268(2):160–168. doi: 10.1002/ar.10148. [DOI] [PubMed] [Google Scholar]
  3. Baron J. L., Madri J. A., Ruddle N. H., Hashim G., Janeway C. A., Jr Surface expression of alpha 4 integrin by CD4 T cells is required for their entry into brain parenchyma. J Exp Med. 1993 Jan 1;177(1):57–68. doi: 10.1084/jem.177.1.57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Best A., Ahmed S., Kozma R., Lim L. The Ras-related GTPase Rac1 binds tubulin. J Biol Chem. 1996 Feb 16;271(7):3756–3762. doi: 10.1074/jbc.271.7.3756. [DOI] [PubMed] [Google Scholar]
  5. Blaheta R. A., Hailer N. P., Brude N., Wittig B., Leckel K., Oppermann E., Bachmann M., Harder S., Cinatl J., Scholz M. In vitro analysis of verapamil-induced immunosuppression: potent inhibition of T cell motility and lymphocytic transmigration through allogeneic endothelial cells. Transplantation. 2000 Feb 27;69(4):588–597. doi: 10.1097/00007890-200002270-00021. [DOI] [PubMed] [Google Scholar]
  6. Boivin D., Bilodeau D., Béliveau R. Regulation of cytoskeletal functions by Rho small GTP-binding proteins in normal and cancer cells. Can J Physiol Pharmacol. 1996 Jul;74(7):801–810. [PubMed] [Google Scholar]
  7. Chin J. E., Hatfield C. A., Winterrowd G. E., Brashler J. R., Vonderfecht S. L., Fidler S. F., Griffin R. L., Kolbasa K. P., Krzesicki R. F., Sly L. M. Airway recruitment of leukocytes in mice is dependent on alpha4-integrins and vascular cell adhesion molecule-1. Am J Physiol. 1997 Feb;272(2 Pt 1):L219–L229. doi: 10.1152/ajplung.1997.272.2.L219. [DOI] [PubMed] [Google Scholar]
  8. Cook-Mills J. M., Gallagher J. S., Feldbush T. L. Isolation and characterization of high endothelial cell lines derived from mouse lymph nodes. In Vitro Cell Dev Biol Anim. 1996 Mar;32(3):167–177. doi: 10.1007/BF02723682. [DOI] [PubMed] [Google Scholar]
  9. Cook-Mills Joan M. VCAM-1 signals during lymphocyte migration: role of reactive oxygen species. Mol Immunol. 2002 Dec;39(9):499–508. doi: 10.1016/s0161-5890(02)00206-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Crawford L. E., Milliken E. E., Irani K., Zweier J. L., Becker L. C., Johnson T. M., Eissa N. T., Crystal R. G., Finkel T., Goldschmidt-Clermont P. J. Superoxide-mediated actin response in post-hypoxic endothelial cells. J Biol Chem. 1996 Oct 25;271(43):26863–26867. doi: 10.1074/jbc.271.43.26863. [DOI] [PubMed] [Google Scholar]
  11. Dahlgren C., Johansson A., Lundqvist H., Bjerrum O. W., Borregaard N. Activation of the oxygen-radical-generating system in granules of intact human neutrophils by a calcium ionophore (ionomycin). Biochim Biophys Acta. 1992 Oct 27;1137(2):182–188. doi: 10.1016/0167-4889(92)90200-u. [DOI] [PubMed] [Google Scholar]
  12. Doan T. N., Gentry D. L., Taylor A. A., Elliott S. J. Hydrogen peroxide activates agonist-sensitive Ca(2+)-flux pathways in canine venous endothelial cells. Biochem J. 1994 Jan 1;297(Pt 1):209–215. doi: 10.1042/bj2970209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dorseuil O., Quinn M. T., Bokoch G. M. Dissociation of Rac translocation from p47phox/p67phox movements in human neutrophils by tyrosine kinase inhibitors. J Leukoc Biol. 1995 Jul;58(1):108–113. doi: 10.1002/jlb.58.1.108. [DOI] [PubMed] [Google Scholar]
  14. Duplàa C., Couffinhal T., Dufourcq P., Llanas B., Moreau C., Bonnet J. The integrin very late antigen-4 is expressed in human smooth muscle cell. Involvement of alpha 4 and vascular cell adhesion molecule-1 during smooth muscle cell differentiation. Circ Res. 1997 Feb;80(2):159–169. doi: 10.1161/01.res.80.2.159. [DOI] [PubMed] [Google Scholar]
  15. Fraker P. J., King L. E., Lill-Elghanian D., Telford W. G. Quantification of apoptotic events in pure and heterogeneous populations of cells using the flow cytometer. Methods Cell Biol. 1995;46:57–76. doi: 10.1016/s0091-679x(08)61924-x. [DOI] [PubMed] [Google Scholar]
  16. Geijsen N., van Delft S., Raaijmakers J. A., Lammers J. W., Collard J. G., Koenderman L., Coffer P. J. Regulation of p21rac activation in human neutrophils. Blood. 1999 Aug 1;94(3):1121–1130. [PubMed] [Google Scholar]
  17. Genot E. M., Arrieumerlou C., Ku G., Burgering B. M., Weiss A., Kramer I. M. The T-cell receptor regulates Akt (protein kinase B) via a pathway involving Rac1 and phosphatidylinositide 3-kinase. Mol Cell Biol. 2000 Aug;20(15):5469–5478. doi: 10.1128/mcb.20.15.5469-5478.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Goodman E. B., Tenner A. J. Signal transduction mechanisms of C1q-mediated superoxide production. Evidence for the involvement of temporally distinct staurosporine-insensitive and sensitive pathways. J Immunol. 1992 Jun 15;148(12):3920–3928. [PubMed] [Google Scholar]
  19. Granfeldt Daniel, Samuelsson Marie, Karlsson Anna. Capacitative Ca2+ influx and activation of the neutrophil respiratory burst. Different regulation of plasma membrane- and granule-localized NADPH-oxidase. J Leukoc Biol. 2002 Apr;71(4):611–617. [PubMed] [Google Scholar]
  20. Gurtner G. C., Davis V., Li H., McCoy M. J., Sharpe A., Cybulsky M. I. Targeted disruption of the murine VCAM1 gene: essential role of VCAM-1 in chorioallantoic fusion and placentation. Genes Dev. 1995 Jan 1;9(1):1–14. doi: 10.1101/gad.9.1.1. [DOI] [PubMed] [Google Scholar]
  21. Henderson L. M., Chappell J. B. Dihydrorhodamine 123: a fluorescent probe for superoxide generation? Eur J Biochem. 1993 Nov 1;217(3):973–980. doi: 10.1111/j.1432-1033.1993.tb18328.x. [DOI] [PubMed] [Google Scholar]
  22. Hess K. L., Tudor K. S., Johnson J. D., Osati-Ashtiani F., Askew D. S., Cook-Mills J. M. Human and murine high endothelial venule cells phagocytose apoptotic leukocytes. Exp Cell Res. 1997 Nov 1;236(2):404–411. doi: 10.1006/excr.1997.3745. [DOI] [PubMed] [Google Scholar]
  23. Huang A. J., Manning J. E., Bandak T. M., Ratau M. C., Hanser K. R., Silverstein S. C. Endothelial cell cytosolic free calcium regulates neutrophil migration across monolayers of endothelial cells. J Cell Biol. 1993 Mar;120(6):1371–1380. doi: 10.1083/jcb.120.6.1371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Iiyama K., Hajra L., Iiyama M., Li H., DiChiara M., Medoff B. D., Cybulsky M. I. Patterns of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression in rabbit and mouse atherosclerotic lesions and at sites predisposed to lesion formation. Circ Res. 1999 Jul 23;85(2):199–207. doi: 10.1161/01.res.85.2.199. [DOI] [PubMed] [Google Scholar]
  25. Imamichi T., Koyama J. Two distinct types of Fc receptor for IgG on guinea pig macrophages activate the NADPH oxidase through different signal-transduction pathways. Biochem Biophys Res Commun. 1990 Oct 15;172(1):223–228. doi: 10.1016/s0006-291x(05)80197-4. [DOI] [PubMed] [Google Scholar]
  26. Lang M. L., Kerr M. A. Characterization of FcalphaR-triggered Ca(2+) signals: role in neutrophil NADPH oxidase activation. Biochem Biophys Res Commun. 2000 Sep 24;276(2):749–755. doi: 10.1006/bbrc.2000.3542. [DOI] [PubMed] [Google Scholar]
  27. Leusen J. H., de Klein A., Hilarius P. M., Ahlin A., Palmblad J., Smith C. I., Diekmann D., Hall A., Verhoeven A. J., Roos D. Disturbed interaction of p21-rac with mutated p67-phox causes chronic granulomatous disease. J Exp Med. 1996 Oct 1;184(4):1243–1249. doi: 10.1084/jem.184.4.1243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lorenzon P., Vecile E., Nardon E., Ferrero E., Harlan J. M., Tedesco F., Dobrina A. Endothelial cell E- and P-selectin and vascular cell adhesion molecule-1 function as signaling receptors. J Cell Biol. 1998 Sep 7;142(5):1381–1391. doi: 10.1083/jcb.142.5.1381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Matheny H. E., Deem T. L., Cook-Mills J. M. Lymphocyte migration through monolayers of endothelial cell lines involves VCAM-1 signaling via endothelial cell NADPH oxidase. J Immunol. 2000 Jun 15;164(12):6550–6559. doi: 10.4049/jimmunol.164.12.6550. [DOI] [PubMed] [Google Scholar]
  30. May M. J., Entwistle G., Humphries M. J., Ager A. VCAM-1 is a CS1 peptide-inhibitable adhesion molecule expressed by lymph node high endothelium. J Cell Sci. 1993 Sep;106(Pt 1):109–119. doi: 10.1242/jcs.106.1.109. [DOI] [PubMed] [Google Scholar]
  31. McPhail L. C., Clayton C. C., Snyderman R. The NADPH oxidase of human polymorphonuclear leukocytes. Evidence for regulation by multiple signals. J Biol Chem. 1984 May 10;259(9):5768–5775. [PubMed] [Google Scholar]
  32. Mueller J. P., Evans M. J., Cofiell R., Rother R. P., Matis L. A., Elliott E. A. Porcine vascular cell adhesion molecule (VCAM) mediates endothelial cell adhesion to human T cells. Development of blocking antibodies specific for porcine VCAM. Transplantation. 1995 Dec 15;60(11):1299–1306. [PubMed] [Google Scholar]
  33. Murphy H. S., Shayman J. A., Till G. O., Mahrougui M., Owens C. B., Ryan U. S., Ward P. A. Superoxide responses of endothelial cells to C5a and TNF-alpha: divergent signal transduction pathways. Am J Physiol. 1992 Jul;263(1 Pt 1):L51–L59. doi: 10.1152/ajplung.1992.263.1.L51. [DOI] [PubMed] [Google Scholar]
  34. Okuda M., Takahashi M., Suero J., Murry C. E., Traub O., Kawakatsu H., Berk B. C. Shear stress stimulation of p130(cas) tyrosine phosphorylation requires calcium-dependent c-Src activation. J Biol Chem. 1999 Sep 17;274(38):26803–26809. doi: 10.1074/jbc.274.38.26803. [DOI] [PubMed] [Google Scholar]
  35. Palicz A., Foubert T. R., Jesaitis A. J., Marodi L., McPhail L. C. Phosphatidic acid and diacylglycerol directly activate NADPH oxidase by interacting with enzyme components. J Biol Chem. 2000 Nov 1;276(5):3090–3097. doi: 10.1074/jbc.M007759200. [DOI] [PubMed] [Google Scholar]
  36. Patel J. M., Li Y. D., Zhang J., Gelband C. H., Raizada M. K., Block E. R. Increased expression of calreticulin is linked to ANG IV-mediated activation of lung endothelial NOS. Am J Physiol. 1999 Oct;277(4 Pt 1):L794–L801. doi: 10.1152/ajplung.1999.277.4.L794. [DOI] [PubMed] [Google Scholar]
  37. Ricard I., Payet M. D., Dupuis G. Clustering the adhesion molecules VLA-4 (CD49d/CD29) in Jurkat T cells or VCAM-1 (CD106) in endothelial (ECV 304) cells activates the phosphoinositide pathway and triggers Ca2+ mobilization. Eur J Immunol. 1997 Jun;27(6):1530–1538. doi: 10.1002/eji.1830270632. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Saito H., Minamiya Y., Kitamura M., Saito S., Enomoto K., Terada K., Ogawa J. Endothelial myosin light chain kinase regulates neutrophil migration across human umbilical vein endothelial cell monolayer. J Immunol. 1998 Aug 1;161(3):1533–1540. [PubMed] [Google Scholar]
  40. Soriano A., Salas A., Salas A., Sans M., Gironella M., Elena M., Anderson D. C., Piqué J. M., Panés J. VCAM-1, but not ICAM-1 or MAdCAM-1, immunoblockade ameliorates DSS-induced colitis in mice. Lab Invest. 2000 Oct;80(10):1541–1551. doi: 10.1038/labinvest.3780164. [DOI] [PubMed] [Google Scholar]
  41. Tudor K. S., Deem T. L., Cook-Mills J. M. Novel alpha 4-integrin ligands on an endothelial cell line. Biochem Cell Biol. 2000;78(2):99–113. [PubMed] [Google Scholar]
  42. Tudor K. S., Hess K. L., Cook-Mills J. M. Cytokines modulate endothelial cell intracellular signal transduction required for VCAM-1-dependent lymphocyte transendothelial migration. Cytokine. 2001 Aug 21;15(4):196–211. doi: 10.1006/cyto.2001.0922. [DOI] [PubMed] [Google Scholar]
  43. Van Aelst L., Joneson T., Bar-Sagi D. Identification of a novel Rac1-interacting protein involved in membrane ruffling. EMBO J. 1996 Aug 1;15(15):3778–3786. [PMC free article] [PubMed] [Google Scholar]
  44. Wahl M., Lucherini M. J., Gruenstein E. Intracellular Ca2+ measurement with Indo-1 in substrate-attached cells: advantages and special considerations. Cell Calcium. 1990 Aug;11(7):487–500. doi: 10.1016/0143-4160(90)90081-5. [DOI] [PubMed] [Google Scholar]
  45. Wójciak-Stothard B., Potempa S., Eichholtz T., Ridley A. J. Rho and Rac but not Cdc42 regulate endothelial cell permeability. J Cell Sci. 2001 Apr;114(Pt 7):1343–1355. doi: 10.1242/jcs.114.7.1343. [DOI] [PubMed] [Google Scholar]
  46. Yakubu Momoh A., Leffler Charles W. L-type voltage-dependent Ca2+ channels in cerebral microvascular endothelial cells and ET-1 biosynthesis. Am J Physiol Cell Physiol. 2002 Aug 22;283(6):C1687–C1695. doi: 10.1152/ajpcell.00071.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Yamaguchi M., Suwa H., Miyasaka M., Kumada K. Selective inhibition of vascular cell adhesion molecule-1 expression by verapamil in human vascular endothelial cells. Transplantation. 1997 Mar 15;63(5):759–764. doi: 10.1097/00007890-199703150-00024. [DOI] [PubMed] [Google Scholar]
  48. Yang F. C., Atkinson S. J., Gu Y., Borneo J. B., Roberts A. W., Zheng Y., Pennington J., Williams D. A. Rac and Cdc42 GTPases control hematopoietic stem cell shape, adhesion, migration, and mobilization. Proc Natl Acad Sci U S A. 2001 Apr 24;98(10):5614–5618. doi: 10.1073/pnas.101546898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zeitler H., Ko Y., Glodny B., Totzke G., Appenheimer M., Sachinidis A., Vetter H. Cell-cycle arrest in G0/G1 phase of growth factor-induced endothelial cell proliferation by various calcium channel blockers. Cancer Detect Prev. 1997;21(4):332–339. [PubMed] [Google Scholar]
  50. Zent R., Ailenberg M., Waddell T. K., Downey G. P., Silverman M. Puromycin aminonucleoside inhibits mesangial cell-induced contraction of collagen gels by stimulating production of reactive oxygen species. Kidney Int. 1995 Mar;47(3):811–817. doi: 10.1038/ki.1995.123. [DOI] [PubMed] [Google Scholar]
  51. Zhu Y., Liao H. L., Lin J. H., Verna L., Stemerman M. B. Low-density lipoprotein augments interleukin-1-induced vascular adhesion molecule expression in human endothelial cells. Atherosclerosis. 1999 Jun;144(2):357–365. doi: 10.1016/s0021-9150(99)00005-2. [DOI] [PubMed] [Google Scholar]
  52. van Buul Jaap D., Voermans Carlijn, van den Berg Veronique, Anthony Eloise C., Mul Frederik P. J., van Wetering Sandra, van der Schoot C. Ellen, Hordijk Peter L. Migration of human hematopoietic progenitor cells across bone marrow endothelium is regulated by vascular endothelial cadherin. J Immunol. 2002 Jan 15;168(2):588–596. doi: 10.4049/jimmunol.168.2.588. [DOI] [PubMed] [Google Scholar]
  53. van Wetering Sandra, van Buul Jaap D., Quik Safira, Mul Frederik P. J., Anthony Eloise C., ten Klooster Jean-Paul, Collard John G., Hordijk Peter L. Reactive oxygen species mediate Rac-induced loss of cell-cell adhesion in primary human endothelial cells. J Cell Sci. 2002 May 1;115(Pt 9):1837–1846. doi: 10.1242/jcs.115.9.1837. [DOI] [PubMed] [Google Scholar]
  54. van Wetering Sandra, van den Berk Nadia, van Buul Jaap D., Mul Frederik P. J., Lommerse Ingrid, Mous Rogier, ten Klooster Jean-Paul, Zwaginga Jaap-Jan, Hordijk Peter L. VCAM-1-mediated Rac signaling controls endothelial cell-cell contacts and leukocyte transmigration. Am J Physiol Cell Physiol. 2003 Apr 16;285(2):C343–C352. doi: 10.1152/ajpcell.00048.2003. [DOI] [PubMed] [Google Scholar]
  55. von Weikersthal S. F., Barrand M. A., Hladky S. B. Functional and molecular characterization of a volume-sensitive chloride current in rat brain endothelial cells. J Physiol. 1999 Apr 1;516(Pt 1):75–84. doi: 10.1111/j.1469-7793.1999.075aa.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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