Histochemistry and Cell Biology

, Volume 121, Issue 5, pp 361–369

Actin microdomains on endothelial cells: association with CD44, ERM proteins, and signaling molecules during quiescence and wound healing

Original Paper

Abstract

During studies of the actin cytoskeleton in cultured endothelial cells we have observed that the luminal side of many cells contains F-actin microdomains that are rich in the hyaluronan receptor CD44 and in ezrin-radixin-moesin (ERM) proteins. A small subpopulation of the domains are also enriched in tyrosine phosphorylated proteins and signaling molecules. Confocal microscopy of rat aortic endothelial cells in situ demonstrated that similar microdomains occur in vivo. During healing of endothelial wounds, characteristic alterations of the actin cytoskeleton occurred. Thus, in many cells close to the wound, focal F-actin branching points appeared. The branching points were similar to the microdomains in that they colocalized with CD44 and ERM proteins, but, in addition, they formed centers for actin filament branching and were associated with phosphorylated protein kinase C α/βII. These colocalization data are consonant with the view that activated PKC is responsible for activating ERM-mediated crosslinking between CD44 and the actin cytoskeleton. Importantly, inhibition of PKC activity decreased staining for phosphorylated ERM proteins, decreased the frequency of F-actin branching points, and inhibited monolayer wound healing. Together, our data show that endothelial cells contain a novel actin cytoskeletal structure, the F-actin microdomain, and suggest that during wound healing such structures become associated with activated signaling molecules and thereby enhance actin cytoskeletal remodeling.

Keywords

Endothelial cells Wound healing Actin cytoskeleton CD44 ERM proteins PKC 

References

  1. Algrain M, Turunen O, Vaheri A, Louvard D, Arpin M (1993) Ezrin contains cytoskeletal and membrane binding domains accounting for its proposed role as a membrane-cytoskeletal linker. J Cell Biol 120:129–139PubMedGoogle Scholar
  2. Battegay EJ (1995) Angiogenesis: mechanistic insights, neovascular diseases, and therapeutic prospects. J Mol Med 73:333–346PubMedGoogle Scholar
  3. Bernardini G, Ribatti D, Spinetti G, Morbidelli L, Ziche M, Santoni A, Capogrossi MC, Napolitano M (2003) Analysis of the role of chemokines in angiogenesis. J Immunol Methods 273:83–101CrossRefPubMedGoogle Scholar
  4. Bray D, Levin MD, Morton-Firth CJ (1998) Receptor clustering as a cellular mechanism to control sensitivity. Nature 393:85–88PubMedGoogle Scholar
  5. Bretscher A, Edwards K, Fehon RG (2002) ERM proteins and merlin: integrators at the cell cortex. Nat Rev Mol Cell Biol 3:586–599CrossRefPubMedGoogle Scholar
  6. Daviet I, Herbert JM, Maffrand M (1989) Tumor-promoting phorbol esters stimulate bovine cerebral cortex capillary endothelial cell growth in vitro. Biochem Biophys Res Commun 158:584–589PubMedGoogle Scholar
  7. Doctrow SR, Folkmann J (1987) Protein kinase C activators suppress stimulation of capillary endothelial cell growth by angiogenic endothelial mitogens. J Cell Biol 104:679–687PubMedGoogle Scholar
  8. Folkman J (1995) Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1:27–31PubMedGoogle Scholar
  9. Folkman J (2000) Tumor angiogenesis. In: Holland JF, Frei E, Bast RC, Kufe DW, Pollock RE, Weichselbaum RR (eds) Cancer medicine, 5th edn. Decker, Hamilton, Ontario, pp 132–152Google Scholar
  10. Gautreau A, Louvard D, Arpin M (2000) Morphogenic effects of ezrin require a phosphorylation-induced transition from oligomers to monomers at the plasma membrane. J Cell Biol 150:193–203CrossRefPubMedGoogle Scholar
  11. Habib A, Vezza R, Créminon C, Maclouf J, Fitzgerald GA (1997) Rapid, agonist-dependent phosphorylation in vivo of human thromboxane receptor isoforms. J Biol Chem 272:7191–7200CrossRefPubMedGoogle Scholar
  12. Higaki T, Sawada S, Kono Y, Imamura H, Tada Y, Yamasaki S, Toratani A, Sato T, Komatsu S, Akamatsu N, Tamagaki T, Tsuda Y, Tsuji H, Nakagawa M (1999) A role of protein kinase C in the regulation of cytosolic phospholipase A2 in bradykinin-induced PGI2 synthesis by human vascular endothelial cells. Microvasc Res 58:144–155CrossRefPubMedGoogle Scholar
  13. Hunter T (1998) The Croonian Lecture 1997. The phosphorylation of proteins on tyrosine: its role in cell growth and disease. Philos Trans R Soc Lond B Biol Sci 353:583–605CrossRefPubMedGoogle Scholar
  14. Ingber DE, Prusty D, Sun Z, Betensky H, Wang N (1995) Cell shape, cytoskeletal mechanics, and cell cycle control in angiogenesis. J Biomech 28:1471–1484CrossRefPubMedGoogle Scholar
  15. Kent KC, Mii S, Harrington EO, Chang JD, Mallette S, Ware JA (1995) Requirement for protein kinase C activation in basic fibroblast growth factor-induced human endothelial cell proliferation. Circ Res 77:231–238PubMedGoogle Scholar
  16. Kim SH, Wang W, Kim KK (2002) Dynamic and clustering model of bacterial chemotaxis receptors: structural basis for signaling and high sensitivity. Proc Natl Acad Sci USA 99:11611–11615CrossRefPubMedGoogle Scholar
  17. Larsson L-I (1988) Immunocytochemistry: theory and practice, 1st edn. CRC Press, Boca RatonGoogle Scholar
  18. Legg JW, Isacke CM (1998) Identification and functional analysis of the ezrin-binding site in the hyaluronan receptor, CD44. Curr Biol 8:705–708PubMedGoogle Scholar
  19. Lehoux S, Tedgui A (2003) Cellular mechanics and gene expression in blood vessels. J Biomech 36:631–643CrossRefPubMedGoogle Scholar
  20. Li H, Wallerath T, Förstermann U (2002) Physiological mechanisms regulating the expression of endothelial-type NO synthase. Nitric Oxide 7:132–147CrossRefPubMedGoogle Scholar
  21. Li J, Zhang Y-P, Kirsner RS (2003) Angiogenesis in wound repair: angiogenic growth factors and the extracellular matrix. Microsc Res Tech 60:107–114CrossRefPubMedGoogle Scholar
  22. Liotta LA, Steeg PS, Stetler-Stevenson WG (1991) Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell 64:327–336PubMedGoogle Scholar
  23. Liu WS, Heckman CA (1998) The sevenfold way of PKC regulation. Cell Signal 10:529–542CrossRefPubMedGoogle Scholar
  24. Matsui T, Maeda M, Doi Y, Yonemura S, Amano M, Kaibuchi K, Tsukita S, Tsukita S (1998) Rho-kinase phosphorylates COOH-terminal threonines of ezrin/radixin/moesin (ERM) proteins and regulates their head-to-tail association. J Cell Biol 140:647–657CrossRefPubMedGoogle Scholar
  25. Nakamura F, Amieva MR, Furhtmayr H (1995) Phosphorylation of threonine 558 in the carboxyl-terminal actin-binding domain of moesin by thrombin activation of human platelets. J Biol Chem 270:31377–31385CrossRefPubMedGoogle Scholar
  26. Ng T, Parsons M, Hughes WE, Monypenny J, Zicha D, Gautreau A, Arpin M, Gschmeissner S, Verveer PJ, Bastiaens PIH, Parker PJ (2001) Ezrin is a downstream effector of trafficking PKC-integrin complexes involved in the control of cell motility. EMBO J 20:2723–2741CrossRefPubMedGoogle Scholar
  27. Nguyen LL, D’Amore PA (2001) Cellular interactions in vascular growth and differentiation. Int Rev Cytol 204:1–48PubMedGoogle Scholar
  28. Pietromonaco SF, Simons PC, Altman A, Elias L (1998) Protein kinase C-theta phosphorylation of moesin in the actin-binding sequence. J Biol Chem 273:7594–7603CrossRefPubMedGoogle Scholar
  29. Presta M, Tiberio L, Rusnati M, Dell’Era P, Ragnotti G (1991) Basic fibroblast growth factor requires a long-lasting activation of protein kinase C to induce cell proliferation in transformed fetal bovine aortic endothelial cells. Cell Regul 2:719–726PubMedGoogle Scholar
  30. Resnick N, Yahav H, Shay-Salit A, Shushy M, Schubert S, Zilberman LCM, Wofovitz E (2003) Fluid shear stress and the vascular endothelium: for better and for worse. Prog Biophys Mol Biol 81:177–199CrossRefPubMedGoogle Scholar
  31. Rubanyi GM, Johns A, Kauser K (2002) Effect of estrogen on endothelial function and angiogenesis. Vasc Pharmacol 38:89–98CrossRefGoogle Scholar
  32. Simons PC, Pietromonaco SF, Reczek D, Bretscher A, Elias L (1998) C-terminal threonine phosphorylation activates ERM proteins to link the cell’s cortical lipid bilayer to the cytoskeleton. Biochem Biophys Res Commun 253:561–565CrossRefPubMedGoogle Scholar
  33. Slevin M, Kumar S, Gaffney J (2002) Angiogenic oligosaccharides of hyaluronan induce multiple signaling pathways affecting vascular endothelial cell mitogenic and wound healing responses. J Biol Chem 277:41046–41059CrossRefPubMedGoogle Scholar
  34. Tang S, Morgan KG, Parker C, Ware JA (1997) Requirement for protein kinase C theta for cell cycle progression and formation of actin stress fibers and filopodia in endothelial vascular cells. J Biol Chem 272:28704–28711CrossRefPubMedGoogle Scholar
  35. Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane M, Baudet V, Boissin P, Boursier E, Loriolle F, Duhamel L, Charon D, Kirilowsky J (1991) The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem 266:15771–15781PubMedGoogle Scholar
  36. Tsukita S, Oishi K, Sato N, Sagara J, Kawai A, Tsukita S (1994) ERM family members as molecular linkers between the cell surface glycoprotein CD44 and actin-based cytoskeletons. J Cell Biol 126:391–401PubMedGoogle Scholar
  37. Uchida S, Watanabe G, Shimada Y, Maeda M, Kawabe A, Mori A, Arii S, Uehata M, Kishimoto T, Oikawa T, Imamura M (2000) The suppression of small GTPase rho signal transduction pathway inhibits angiogenesis in vitro and in vivo. Biochem Biophys Res Commun 269:633–640CrossRefPubMedGoogle Scholar
  38. Villard E, Alonso A, Agrapart M, Challah M, Soubrier F (1998) Induction of angiotensin I-converting enzyme transcription by a protein kinase C-dependent mechanism in human endothelial cells. J Biol Chem 273:25191–25197CrossRefPubMedGoogle Scholar
  39. Vincent MA, Montagnani M, Quon MJ (2003) Molecular and physiologic actions of insulin related to production of nitric oxide in vascular endothelium. Curr Diabetes Rep 3:279–288Google Scholar
  40. Walch L, Brink C, Norel X (2001) The muscarinic receptor subtypes in human blood vessels. Therapie 56:223–226PubMedGoogle Scholar
  41. Wang A, Nomura M, Patan S, Ware JA (2002) Inhibition of protein kinase Calpha prevents endothelial cell migration and vascular tube formation in vitro and myocardial neovascularization in vivo. Circ Res 90:609–616CrossRefPubMedGoogle Scholar
  42. Yamamura S, Nelson PR, Kent KC (1996). Role of protein kinase C in attachment, spreading and migration of human endothelial cells. J Surg Res 63:349–354CrossRefPubMedGoogle Scholar
  43. Yonemura S, Hirao M, Doi Y, Takahashi N, Kondo T, Tsukita S, Tsukita S (1998) Ezrin/radixin/moesin (ERM) proteins bind to a positively charged amino acid cluster in the juxta-membrane cytoplasmic domain of CD44, CD43, and ICAM-2. J Cell Biol 140:885–895CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  1. 1.Division of Cell Biology, Department of Anatomy and PhysiologyThe Royal Veterinary and Agricultural UniversityFrederiksberg CDenmark

Personalised recommendations