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Involvement of vasodilator-stimulated phosphoprotein in UDP-induced microglial actin aggregation via PKC- and Rho-dependent pathways

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Abstract

Microglia are major immunocompetent cells in the central nervous system and retain highly dynamic motility. The processes which allow these cells to move, such as chemotaxis and phagocytosis, are considered part of their functions and are closely related to purinergic signaling. Previously, we reported that the activation of the P2Y6 receptor by UDP stimulation in microglia evoked dynamic cell motility which enhanced their phagocytic capacity, as reported by Koizumi et al. (Nature 446(7139):1091–1095, 2007). These responses require actin cytoskeletal rearrangement, which is seen after UDP stimulation. However, the intracellular signaling pathway has not been defined. In this study, we found that UDP in rat primary microglia rapidly induced the transient phosphorylation at Ser157 of vasodilator-stimulated phosphoprotein (VASP). VASP, one of actin binding protein, accumulated at the plasma membrane where filamentous (F)-actin aggregated in a time-dependent manner. The phosphorylation of VASP was suppressed by inhibition of PKC. UDP-induced local actin aggregations were also abrogated by PKC inhibitors. The Rho inhibitor CT04 and the expression of p115-RGS, which suppresses G12/13 signaling, attenuated UDP-induced phosphorylation of VASP and actin aggregation. These results indicate that PKC- and Rho-dependent phosphorylation of VASP is involved in UDP-induced actin aggregation of microglia.

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References

  1. Koizumi S, Shigemoto-Mogami Y, Nasu-Tada K, Shinozaki Y, Ohsawa K, Tsuda M, Joshi BV, Jacobson KA, Kohsaka S, Inoue K (2007) UDP acting at P2Y6 receptors is a mediator of microglial phagocytosis. Nature 446(7139):1091–1095. doi:10.1038/nature05704

    Article  PubMed  CAS  Google Scholar 

  2. Davalos D, Grutzendler J, Yang G, Kim JV, Zuo Y, Jung S, Littman DR, Dustin ML, Gan WB (2005) ATP mediates rapid microglial response to local brain injury in vivo. Nat Neurosci 8(6):752–758. doi:10.1038/nn1472

    Article  PubMed  CAS  Google Scholar 

  3. Nimmerjahn A, Kirchhoff F, Helmchen F (2005) Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science 308(5726):1314–1318. doi:10.1126/science.1110647

    Article  PubMed  CAS  Google Scholar 

  4. Farber K, Kettenmann H (2006) Purinergic signaling and microglia. Pflugers Arch 452(5):615–621. doi:10.1007/s00424-006-0064-7

    Article  PubMed  Google Scholar 

  5. Honda S, Sasaki Y, Ohsawa K, Imai Y, Nakamura Y, Inoue K, Kohsaka S (2001) Extracellular ATP or ADP induce chemotaxis of cultured microglia through Gi/o-coupled P2Y receptors. J Neurosci 21(6):1975–1982

    PubMed  CAS  Google Scholar 

  6. Nasu-Tada K, Koizumi S, Inoue K (2005) Involvement of beta1 integrin in microglial chemotaxis and proliferation on fibronectin: different regulations by ADP through PKA. Glia 52(2):98–107. doi:10.1002/glia.20224

    Article  PubMed  Google Scholar 

  7. Irino Y, Nakamura Y, Inoue K, Kohsaka S, Ohsawa K (2008) Akt activation is involved in P2Y12 receptor-mediated chemotaxis of microglia. J Neurosci Res 86(7):1511–1519. doi:10.1002/jnr.21610

    Article  PubMed  CAS  Google Scholar 

  8. Ohsawa K, Irino Y, Sanagi T, Nakamura Y, Suzuki E, Inoue K, Kohsaka S. P2Y12 receptor-mediated integrin-beta1 activation regulates microglial process extension induced by ATP. Glia 58(7):790–801. doi:10.1002/glia.20963

  9. Haynes SE, Hollopeter G, Yang G, Kurpius D, Dailey ME, Gan WB, Julius D (2006) The P2Y12 receptor regulates microglial activation by extracellular nucleotides. Nat Neurosci 9(12):1512–1519. doi:10.1038/nn1805

    Article  PubMed  CAS  Google Scholar 

  10. Bear JE, Gertler FB (2009) Ena/VASP: towards resolving a pointed controversy at the barbed end. J Cell Sci 122(Pt 12):1947–1953. doi:10.1242/jcs.038125

    Article  PubMed  CAS  Google Scholar 

  11. Lee S, Chung CY (2009) Role of VASP phosphorylation for the regulation of microglia chemotaxis via the regulation of focal adhesion formation/maturation. Mol Cell Neurosci 42(4):382–390. doi:10.1016/j.mcn.2009.08.010

    Article  PubMed  CAS  Google Scholar 

  12. Nakajima K, Shimojo M, Hamanoue M, Ishiura S, Sugita H, Kohsaka S (1992) Identification of elastase as a secretory protease from cultured rat microglia. J Neurochem 58(4):1401–1408

    Article  PubMed  CAS  Google Scholar 

  13. Tsuda M, Shigemoto-Mogami Y, Koizumi S, Mizokoshi A, Kohsaka S, Salter MW, Inoue K (2003) P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. Nature 424(6950):778–783. doi:10.1038/nature01786

    Article  PubMed  CAS  Google Scholar 

  14. Benz PM, Blume C, Seifert S, Wilhelm S, Waschke J, Schuh K, Gertler F, Munzel T, Renne T (2009) Differential VASP phosphorylation controls remodeling of the actin cytoskeleton. J Cell Sci 122(Pt 21):3954–3965. doi:10.1242/jcs.044537

    Article  PubMed  CAS  Google Scholar 

  15. Krause M, Dent EW, Bear JE, Loureiro JJ, Gertler FB (2003) Ena/VASP proteins: regulators of the actin cytoskeleton and cell migration. Annu Rev Cell Dev Biol 19:541–564. doi:10.1146/annurev.cellbio.19.050103.103356

    Article  PubMed  CAS  Google Scholar 

  16. Chitaley K, Chen L, Galler A, Walter U, Daum G, Clowes AW (2004) Vasodilator-stimulated phosphoprotein is a substrate for protein kinase C. FEBS Lett 556(1–3):211–215

    Article  PubMed  CAS  Google Scholar 

  17. von Kugelgen I, Wetter A (2000) Molecular pharmacology of P2Y-receptors. Naunyn Schmiedebergs Arch Pharmacol 362(4–5):310–323

    Google Scholar 

  18. Slater SJ, Seiz JL, Stagliano BA, Stubbs CD (2001) Interaction of protein kinase C isozymes with Rho GTPases. Biochemistry 40(14):4437–4445

    Article  PubMed  CAS  Google Scholar 

  19. Profirovic J, Gorovoy M, Niu J, Pavlovic S, Voyno-Yasenetskaya T (2005) A novel mechanism of G protein-dependent phosphorylation of vasodilator-stimulated phosphoprotein. J Biol Chem 280(38):32866–32876. doi:10.1074/jbc.M501361200

    Article  PubMed  CAS  Google Scholar 

  20. Maruyama Y, Nishida M, Sugimoto Y, Tanabe S, Turner JH, Kozasa T, Wada T, Nagao T, Kurose H (2002) Galpha(12/13) mediates alpha(1)-adrenergic receptor-induced cardiac hypertrophy. Circ Res 91(10):961–969

    Article  PubMed  CAS  Google Scholar 

  21. Arai K, Maruyama Y, Nishida M, Tanabe S, Takagahara S, Kozasa T, Mori Y, Nagao T, Kurose H (2003) Differential requirement of G alpha12, G alpha13, G alphaq, and G beta gamma for endothelin-1-induced c-Jun NH2-terminal kinase and extracellular signal-regulated kinase activation. Mol Pharmacol 63(3):478–488

    Article  PubMed  CAS  Google Scholar 

  22. Nishida M, Tanabe S, Maruyama Y, Mangmool S, Urayama K, Nagamatsu Y, Takagahara S, Turner JH, Kozasa T, Kobayashi H, Sato Y, Kawanishi T, Inoue R, Nagao T, Kurose H (2005) G alpha 12/13- and reactive oxygen species-dependent activation of c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase by angiotensin receptor stimulation in rat neonatal cardiomyocytes. J Biol Chem 280(18):18434–18441. doi:10.1074/jbc.M409710200

    Article  PubMed  CAS  Google Scholar 

  23. Nishida M, Sato Y, Uemura A, Narita Y, Tozaki-Saitoh H, Nakaya M, Ide T, Suzuki K, Inoue K, Nagao T, Kurose H (2008) P2Y6 receptor-Galpha12/13 signalling in cardiomyocytes triggers pressure overload-induced cardiac fibrosis. EMBO J 27(23):3104–3115. doi:10.1038/emboj.2008.237

    Article  PubMed  CAS  Google Scholar 

  24. von Kugelgen I (2006) Pharmacological profiles of cloned mammalian P2Y-receptor subtypes. Pharmacol Ther 110(3):415–432. doi:10.1016/j.pharmthera.2005.08.014

    Article  Google Scholar 

  25. El-Tayeb A, Qi A, Muller CE (2006) Synthesis and structure–activity relationships of uracil nucleotide derivatives and analogues as agonists at human P2Y2, P2Y4, and P2Y6 receptors. J Med Chem 49(24):7076–7087. doi:10.1021/jm060848j

    Article  PubMed  CAS  Google Scholar 

  26. Halbrugge M, Friedrich C, Eigenthaler M, Schanzenbacher P, Walter U (1990) Stoichiometric and reversible phosphorylation of a 46-kDa protein in human platelets in response to cGMP- and cAMP-elevating vasodilators. J Biol Chem 265(6):3088–3093

    PubMed  CAS  Google Scholar 

  27. Blume C, Benz PM, Walter U, Ha J, Kemp BE, Renne T (2007) AMP-activated protein kinase impairs endothelial actin cytoskeleton assembly by phosphorylating vasodilator-stimulated phosphoprotein. J Biol Chem 282(7):4601–4612. doi:10.1074/jbc.M608866200

    Article  PubMed  CAS  Google Scholar 

  28. Wong AM, Chow AW, Au SC, Wong CC, Ko WH (2009) Apical versus basolateral P2Y(6) receptor-mediated Cl(−) secretion in immortalized bronchial epithelia. Am J Respir Cell Mol Biol 40(6):733–745. doi:10.1165/rcmb.2008-0020OC

    Article  PubMed  CAS  Google Scholar 

  29. Larsson C (2006) Protein kinase C and the regulation of the actin cytoskeleton. Cell Signal 18(3):276–284. doi:10.1016/j.cellsig.2005.07.010

    Article  PubMed  CAS  Google Scholar 

  30. Martiny-Baron G, Kazanietz MG, Mischak H, Blumberg PM, Kochs G, Hug H, Marme D, Schachtele C (1993) Selective inhibition of protein kinase C isozymes by the indolocarbazole Go 6976. J Biol Chem 268(13):9194–9197

    PubMed  CAS  Google Scholar 

  31. Gschwendt M, Dieterich S, Rennecke J, Kittstein W, Mueller HJ, Johannes FJ (1996) Inhibition of protein kinase C mu by various inhibitors. Differentiation from protein kinase c isoenzymes. FEBS Lett 392(2):77–80

    Article  PubMed  CAS  Google Scholar 

  32. Hofmann J (1997) The potential for isoenzyme-selective modulation of protein kinase C. FASEB J 11(8):649–669

    PubMed  CAS  Google Scholar 

  33. Slepko N, Patrizio M, Levi G (1999) Expression and translocation of protein kinase C isoforms in rat microglial and astroglial cultures. J Neurosci Res 57(1):33–38. doi:10.1002/(SICI)1097-4547(19990701)57:1<33::AID-JNR4>3.0.CO;2-6

    Article  PubMed  CAS  Google Scholar 

  34. Hart MJ, Jiang X, Kozasa T, Roscoe W, Singer WD, Gilman AG, Sternweis PC, Bollag G (1998) Direct stimulation of the guanine nucleotide exchange activity of p115 RhoGEF by Galpha13. Science 280(5372):2112–2114

    Article  PubMed  CAS  Google Scholar 

  35. Fukuhara S, Murga C, Zohar M, Igishi T, Gutkind JS (1999) A novel PDZ domain containing guanine nucleotide exchange factor links heterotrimeric G proteins to Rho. J Biol Chem 274(9):5868–5879

    Article  PubMed  CAS  Google Scholar 

  36. Fukuhara S, Chikumi H, Gutkind JS (2000) Leukemia-associated Rho guanine nucleotide exchange factor (LARG) links heterotrimeric G proteins of the G(12) family to Rho. FEBS Lett 485(2–3):183–188

    Article  PubMed  CAS  Google Scholar 

  37. Ridley AJ (2006) Rho GTPases and actin dynamics in membrane protrusions and vesicle trafficking. Trends Cell Biol 16(10):522–529. doi:10.1016/j.tcb.2006.08.006

    Article  PubMed  CAS  Google Scholar 

  38. Klages B, Brandt U, Simon MI, Schultz G, Offermanns S (1999) Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets. J Cell Biol 144(4):745–754

    Article  PubMed  CAS  Google Scholar 

  39. Yuan J, Slice LW, Rozengurt E (2001) Activation of protein kinase D by signaling through Rho and the alpha subunit of the heterotrimeric G protein G13. J Biol Chem 276(42):38619–38627. doi:10.1074/jbc.M105530200M105530200

    Article  PubMed  CAS  Google Scholar 

  40. Xie Z, Ho WT, Spellman R, Cai S, Exton JH (2002) Mechanisms of regulation of phospholipase D1 and D2 by the heterotrimeric G proteins G13 and Gq. J Biol Chem 277(14):11979–11986. doi:10.1074/jbc.M109751200

    Article  PubMed  CAS  Google Scholar 

  41. Sinnett-Smith J, Santiskulvong C, Duque J, Rozengurt E (2000) [d-Arg(1), d-Trp(5,7,9), Leu(11)]Substance P inhibits bombesin-induced mitogenic signal transduction mediated by both G(q) and G(12) in Swiss 3T3cells. J Biol Chem 275(39):30644–30652. doi:10.1074/jbc.M003702200

    Article  PubMed  CAS  Google Scholar 

  42. Nonaka H, Tanaka K, Hirano H, Fujiwara T, Kohno H, Umikawa M, Mino A, Takai Y (1995) A downstream target of RHO1 small GTP-binding protein is PKC1, a homolog of protein kinase C, which leads to activation of the MAP kinase cascade in Saccharomyces cerevisiae. EMBO J 14(23):5931–5938

    PubMed  CAS  Google Scholar 

  43. Hippenstiel S, Kratz T, Krull M, Seybold J, von Eichel-Streiber C, Suttorp N (1998) Rho protein inhibition blocks protein kinase C translocation and activation. Biochem Biophys Res Commun 245(3):830–834

    Article  PubMed  CAS  Google Scholar 

  44. Ohsawa K, Irino Y, Nakamura Y, Akazawa C, Inoue K, Kohsaka S (2007) Involvement of P2X4 and P2Y12 receptors in ATP-induced microglial chemotaxis. Glia 55(6):604–616. doi:10.1002/glia.20489

    Article  PubMed  Google Scholar 

  45. Bear JE, Svitkina TM, Krause M, Schafer DA, Loureiro JJ, Strasser GA, Maly IV, Chaga OY, Cooper JA, Borisy GG, Gertler FB (2002) Antagonism between Ena/VASP proteins and actin filament capping regulates fibroblast motility. Cell 109(4):509–521

    Article  PubMed  CAS  Google Scholar 

  46. Gertler FB, Niebuhr K, Reinhard M, Wehland J, Soriano P (1996) Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamics. Cell 87(2):227–239

    Article  PubMed  CAS  Google Scholar 

  47. Lacayo CI, Pincus Z, VanDuijn MM, Wilson CA, Fletcher DA, Gertler FB, Mogilner A, Theriot JA (2007) Emergence of large-scale cell morphology and movement from local actin filament growth dynamics. PLoS Biol 5(9):e233. doi:10.1371/journal.pbio.0050233

    Article  PubMed  Google Scholar 

  48. Harbeck B, Huttelmaier S, Schluter K, Jockusch BM, Illenberger S (2000) Phosphorylation of the vasodilator-stimulated phosphoprotein regulates its interaction with actin. J Biol Chem 275(40):30817–30825. doi:10.1074/jbc.M005066200

    Article  PubMed  CAS  Google Scholar 

  49. Benz PM, Blume C, Moebius J, Oschatz C, Schuh K, Sickmann A, Walter U, Feller SM, Renne T (2008) Cytoskeleton assembly at endothelial cell–cell contacts is regulated by alphaII-spectrin-VASP complexes. J Cell Biol 180(1):205–219. doi:10.1083/jcb.200709181

    Article  PubMed  CAS  Google Scholar 

  50. Lambrechts A, Kwiatkowski AV, Lanier LM, Bear JE, Vandekerckhove J, Ampe C, Gertler FB (2000) cAMP-dependent protein kinase phosphorylation of EVL, a Mena/VASP relative, regulates its interaction with actin and SH3 domains. J Biol Chem 275(46):36143–36151. doi:10.1074/jbc.M006274200

    Article  PubMed  CAS  Google Scholar 

  51. Ferron F, Rebowski G, Lee SH, Dominguez R (2007) Structural basis for the recruitment of profilin–actin complexes during filament elongation by Ena/VASP. EMBO J 26(21):4597–4606. doi:10.1038/sj.emboj.7601874

    Article  PubMed  CAS  Google Scholar 

  52. Coppolino MG, Krause M, Hagendorff P, Monner DA, Trimble W, Grinstein S, Wehland J, Sechi AS (2001) Evidence for a molecular complex consisting of Fyb/SLAP, SLP-76, Nck, VASP and WASP that links the actin cytoskeleton to Fcgamma receptor signalling during phagocytosis. J Cell Sci 114(Pt 23):4307–4318

    PubMed  CAS  Google Scholar 

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Acknowledgements

We thank Prof. Hitoshi Kurose for providing the plasmid coding for the RGS domain of p115-RhoGEF (p115-RGS), the mutant of the RGS domain of p115-RhoGEF (mut-RGS). This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to H.S.-T., M.T. and K.I.).

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  1. Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi, Fukuoka, 812–8582, Japan

    Ayako Kataoka, Yui Koga, Ayumi Uesugi, Hidetoshi Tozaki-Saitoh, Makoto Tsuda & Kazuhide Inoue

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  1. Ayako Kataoka
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Correspondence to Kazuhide Inoue.

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Kataoka, A., Koga, Y., Uesugi, A. et al. Involvement of vasodilator-stimulated phosphoprotein in UDP-induced microglial actin aggregation via PKC- and Rho-dependent pathways. Purinergic Signalling 7, 403–411 (2011). https://doi.org/10.1007/s11302-011-9237-8

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