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Na/H exchanger NHE1 acts upstream of rho GTPases to promote neurite outgrowth

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Abstract

Na+/H+ exchanger NHE1, a major determinant of intracellular pH (pHi) in mammalian central neurons, promotes neurite outgrowth under both basal and netrin-1-stimulated conditions. The small GTP binding proteins and their effectors have a dominant role in netrin-1-stimulated neurite outgrowth. Since NHE1 has been shown previously to work downstream of the Rho GTPases-mediated polarized membrane protrusion in non-neuronal cells, we examined whether NHE1 has a similar relationship with Cdc42, Rac1 and RhoA in neuronal morphogenesis. Interestingly, our results suggest the possibility that NHE1 acting upstream of Rho GTPases to promote neurite outgrowth induced by netrin-1. First, we found that netrin-1-induced increases in the activities of Rho GTPases using FRET (Forster Resonance Energy Transfer) analyses in individual growth cones; furthermore, their increased activities were abolished by cariporide, a specific NHE1 inhibitor. Second, NHE1 inhibition had no effect on neurite retraction induced by L-α-Lysophosphatidic acid (LPA), a potent RhoA activator. The regulation of Rho GTPases by NHE1 was further evidenced by reduced Rac1, Cdc42 and RhoA activities in NHE1-null neurons. Taken together, our findings suggest that NHE1-dependent neuronal morphogenesis involves the activation of Rho-family of small GTPases.

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References

  • Abramoff MD, Magelhaes PJ, Ram SJ (2004) Image Processing with Image. J Biophotonics International 11:36–42

    Google Scholar 

  • Aoki K, Matsuda M (2009) Visualization of small GTPase activity with fluorescence resonance energy transfer-based biosensors. Nat Protoc 4:1623–1631

    CAS  PubMed  Google Scholar 

  • Barallobre MJ, Pascual M, Del Rio JA, Soriano E (2005) The Netrin family of guidance factors: emphasis on Netrin-1 signalling. Brain Res Brain Res Rev 49:22–47

    CAS  PubMed  Google Scholar 

  • Baumgartner M, Patel H, Barber DL (2004) Na(+)/H(+) exchanger NHE1 as plasma membrane scaffold in the assembly of signaling complexes am. J Physiol Cell Physiol 287:C844–C850. https://doi.org/10.1152/ajpcell.00094.2004

    Article  CAS  Google Scholar 

  • Baxter KA, Church J (1996) Characterization of acid extrusion mechanisms in cultured fetal rat hippocampal neurones. J Physiol 493(Pt 2):457–470

    CAS  PubMed  PubMed Central  Google Scholar 

  • Benard V, Bohl BP, Bokoch GM (1999) Characterization of rac and cdc42 activation in chemoattractant-stimulated human neutrophils using a novel assay for active GTPases. J Biol Chem 274:13198–13204

    CAS  PubMed  Google Scholar 

  • Bernstein BW, Painter WB, Chen H, Minamide LS, Abe H, Bamburg JR (2000) Intracellular pH modulation of ADF/cofilin proteins. Cell Motil Cytoskeleton 47:319–336

    CAS  PubMed  Google Scholar 

  • Bito H et al. (2000) A critical role for a rho-associated kinase, p160ROCK, in determining axon outgrowth in mammalian CNS neurons Neuron 26:431-441

  • Briancon-Marjollet A et al (2008) Trio mediates netrin-1-induced Rac1 activation in axon outgrowth and guidance. Mol Cell Biol 28:2314–2323

    CAS  PubMed  PubMed Central  Google Scholar 

  • Cardone RA et al (2005a) Protein kinase a gating of a pseudopodial-located RhoA/ROCK/p38/NHE1 signal module regulates invasion in breast cancer cell lines. Mol Biol Cell 16:3117–3127

    CAS  PubMed  PubMed Central  Google Scholar 

  • Cardone RA, Casavola V, Reshkin SJ (2005b) The role of disturbed pH dynamics and the Na+/H+ exchanger in metastasis. Nat Rev Cancer 5:786–795

    CAS  PubMed  Google Scholar 

  • Casey JR, Grinstein S, Orlowski J (2010) Sensors and regulators of intracellular pH. Nat Rev Mol Cell Biol 11:50–61

    CAS  PubMed  Google Scholar 

  • Causeret F, Hidalgo-Sanchez M, Fort P, Backer S, Popoff MR, Gauthier-Rouviere C, Bloch-Gallego E (2004) Distinct roles of Rac1/Cdc42 and rho/Rock for axon outgrowth and nucleokinesis of precerebellar neurons toward netrin 1. Development 131:2841–2852

    CAS  PubMed  Google Scholar 

  • Chen Y, Elangovan M, Periasamy A (2005) FRET data analysis: the algorithm. In: Periasamy AD, R.N. (eds) Molecular imaging - FRET microscopy and spectrocopy. Oxford University Press, New York, pp 126–145

    Google Scholar 

  • Chesler M (2003) Regulation and modulation of pH in the brain. Physiol Rev 83:1183–1221

    CAS  PubMed  Google Scholar 

  • Cook DR, Rossman KL, Der CJ (2014) Rho guanine nucleotide exchange factors: regulators of Rho GTPase activity in development and disease. Oncogene 33:4021–4035. https://doi.org/10.1038/onc.2013.362

    Article  CAS  PubMed  Google Scholar 

  • da Silva JS, Dotti CG (2002) Breaking the neuronal sphere: regulation of the actin cytoskeleton in neuritogenesis. Nat Rev Neurosci 3:694–704

    PubMed  Google Scholar 

  • Denker SP, Barber DL (2002a) Cell migration requires both ion translocation and cytoskeletal anchoring by the Na-H exchanger NHE1. J Cell Biol 159:1087–1096

    CAS  PubMed  PubMed Central  Google Scholar 

  • Denker SP, Barber DL (2002b) Ion transport proteins anchor and regulate the cytoskeleton. Curr Opin Cell Biol 14:214–220

    CAS  PubMed  Google Scholar 

  • Denker SP, Huang DC, Orlowski J, Furthmayr H, Barber DL (2000) Direct binding of the Na--H exchanger NHE1 to ERM proteins regulates the cortical cytoskeleton and cell shape independently of H(+) translocation. Mol Cell 6:1425–1436

    CAS  PubMed  Google Scholar 

  • Frantz C, Karydis A, Nalbant P, Hahn KM, Barber DL (2007) Positive feedback between Cdc42 activity and H+ efflux by the Na-H exchanger NHE1 for polarity of migrating cells. J Cell Biol 179:403–410

    CAS  PubMed  PubMed Central  Google Scholar 

  • Govek EE, Newey SE, Van Aelst L (2005) The role of the Rho GTPases in neuronal development. Genes Dev 19:1–49

    CAS  PubMed  Google Scholar 

  • Grinstein S, Woodside M, Waddell TK, Downey GP, Orlowski J, Pouyssegur J, Wong DC, Foskett JK (1993) Focal localization of the NHE-1 isoform of the Na+/H+ antiport: assessment of effects on intracellular pH. EMBO J 12:5209–5218

    CAS  PubMed  PubMed Central  Google Scholar 

  • Hawkins M, Pope B, Maciver SK, Weeds AG (1993) Human actin depolymerizing factor mediates a pH-sensitive destruction of actin filaments. Biochemistry 32:9985–9993

    CAS  PubMed  Google Scholar 

  • Heasman SJ, Ridley AJ (2008) Mammalian Rho GTPases: new insights into their functions from in vivo studies. Nat Rev Mol Cell Biol 9:690–701

    CAS  PubMed  Google Scholar 

  • Hirose M et al (1998) Molecular dissection of the rho-associated protein kinase (p160ROCK)-regulated neurite remodeling in neuroblastoma N1E-115 cells. J Cell Biol 141:1625–1636

    CAS  PubMed  PubMed Central  Google Scholar 

  • Koivusalo M et al (2010) Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling. J Cell Biol 188:547–563

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kranenburg O, Poland M, van Horck FP, Drechsel D, Hall A, Moolenaar WH (1999) Activation of RhoA by lysophosphatidic acid and Galpha12/13 subunits in neuronal cells: induction of neurite retraction. Mol Biol Cell 10:1851–1857

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kurokawa K, Matsuda M (2005) Localized RhoA activation as a requirement for the induction of membrane ruffling. Mol Biol Cell 16:4294–4303

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kurokawa K, Nakamura T, Aoki K, Matsuda M (2005) Mechanism and role of localized activation of Rho-family GTPases in growth factor-stimulated fibroblasts and neuronal cells. Biochem Soc Trans 33:631–634

    CAS  PubMed  Google Scholar 

  • Lagana A, Vadnais J, Le PU, Nguyen TN, Laprade R, Nabi IR, Noel J (2000) Regulation of the formation of tumor cell pseudopodia by the Na(+)/H(+) exchanger NHE1. J Cell Sci 113(Pt 20):3649–3662

    CAS  PubMed  Google Scholar 

  • Lai Wing Sun K, Correia JP, Kennedy TE (2011) Netrins: versatile extracellular cues with diverse functions. Development 138:2153–2169. https://doi.org/10.1242/dev.044529

    Article  CAS  PubMed  Google Scholar 

  • Li X, Meriane M, Triki I, Shekarabi M, Kennedy TE, Larose L, Lamarche-Vane N (2002a) The adaptor protein Nck-1 couples the netrin-1 receptor DCC (deleted in colorectal cancer) to the activation of the small GTPase Rac1 through an atypical mechanism. J Biol Chem 277:37788–37797

    CAS  PubMed  Google Scholar 

  • Li X, Saint-Cyr-Proulx E, Aktories K, Lamarche-Vane N (2002b) Rac1 and Cdc42 but not RhoA or rho kinase activities are required for neurite outgrowth induced by the Netrin-1 receptor DCC (deleted in colorectal cancer) in N1E-115 neuroblastoma cells. J Biol Chem 277:15207–15214

    CAS  PubMed  Google Scholar 

  • Li X, Gao X, Liu G, Xiong W, Wu J, Rao Y (2008) Netrin signal transduction and the guanine nucleotide exchange factor DOCK180 in attractive signaling. Nat Neurosci 11:28–35

    CAS  PubMed  Google Scholar 

  • Luo L (2002) Actin cytoskeleton regulation in neuronal morphogenesis and structural plasticity. Annu Rev Cell Dev Biol 18:601–635

    CAS  PubMed  Google Scholar 

  • Luo J, Sun D (2007) Physiology and pathophysiology of Na(+)/H(+) exchange isoform 1 in the central nervous system. Curr Neurovasc Res 4:205–215

    CAS  PubMed  Google Scholar 

  • Malo ME, Fliegel L (2006) Physiological role and regulation of the Na+/H+ exchanger. Can J Physiol Pharmacol 84:1081–1095

    CAS  PubMed  Google Scholar 

  • Masereel B, Pochet L, Laeckmann D (2003) An overview of inhibitors of Na(+)/H(+) exchanger. Eur J Med Chem 38:547–554

    CAS  PubMed  Google Scholar 

  • Meima ME, Mackley JR, Barber DL (2007) Beyond ion translocation: structural functions of the sodium-hydrogen exchanger isoform-1. Curr Opin Nephrol Hypertens 16:365–372

    CAS  PubMed  Google Scholar 

  • Moore SW, Correia JP, Lai Wing Sun K, Pool M, Fournier AE, Kennedy TE (2008) Rho inhibition recruits DCC to the neuronal plasma membrane and enhances axon chemoattraction to netrin 1. Development 135:2855–2864

    CAS  PubMed  Google Scholar 

  • Nakamura T, Aoki K, Matsuda M (2005) FRET imaging in nerve growth cones reveals a high level of RhoA activity within the peripheral domain. Brain Res Mol Brain Res 139:277–287

    CAS  PubMed  Google Scholar 

  • Ohnami S, Endo M, Hirai S, Uesaka N, Hatanaka Y, Yamashita T, Yamamoto N (2008) Role of RhoA in activity-dependent cortical axon branching. J Neurosci 28:9117–9121

    CAS  PubMed  PubMed Central  Google Scholar 

  • Orlowski J (1993) Heterologous expression and functional properties of amiloride high affinity (NHE-1) and low affinity (NHE-3) isoforms of the rat Na/H exchanger. J Biol Chem 268:16369–16377

    CAS  PubMed  Google Scholar 

  • Orlowski J, Grinstein S (2004) Diversity of the mammalian sodium/proton exchanger SLC9 gene family. Pflugers Arch 447:549–565

    CAS  PubMed  Google Scholar 

  • Orlowski J, Grinstein S (2007) Emerging roles of alkali cation/proton exchangers in organellar homeostasis. Curr Opin Cell Biol 19:483–492

    CAS  PubMed  PubMed Central  Google Scholar 

  • Paradiso A et al (2004) The Na+-H+ exchanger-1 induces cytoskeletal changes involving reciprocal RhoA and Rac1 signaling, resulting in motility and invasion in MDA-MB-435 cells. Breast Cancer Res 6:R616–R628

    CAS  PubMed  PubMed Central  Google Scholar 

  • Pertz O, Hodgson L, Klemke RL, Hahn KM (2006) Spatiotemporal dynamics of RhoA activity in migrating cells. Nature 440:1069–1072

    CAS  PubMed  Google Scholar 

  • Picard M, Petrie RJ, Antoine-Bertrand J, Saint-Cyr-Proulx E, Villemure JF, Lamarche-Vane N (2009) Spatial and temporal activation of the small GTPases RhoA and Rac1 by the netrin-1 receptor UNC5a during neurite outgrowth. Cell Signal 21:1961–1973

    CAS  PubMed  Google Scholar 

  • Rajasekharan S, Baker KA, Horn KE, Jarjour AA, Antel JP, Kennedy TE (2009) Netrin 1 and dcc regulate oligodendrocyte process branching and membrane extension via Fyn and RhoA. Development 136:415–426

    CAS  PubMed  Google Scholar 

  • Rajasekharan S, Bin JM, Antel JP, Kennedy TE (2010) A central role for RhoA during oligodendroglial maturation in the switch from netrin-1-mediated chemorepulsion to process elaboration. J Neurochem 113:1589–1597. https://doi.org/10.1111/j.1471-4159.2010.06717.x

    Article  CAS  PubMed  Google Scholar 

  • Riento K, Ridley AJ (2003) Rocks: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol 4:446–456

    CAS  PubMed  Google Scholar 

  • Round J, Stein E (2007) Netrin signaling leading to directed growth cone steering. Curr Opin Neurobiol 17:15–21

    CAS  PubMed  Google Scholar 

  • Sander EE, ten Klooster JP, van Delft S, van der Kammen RA, Collard JG (1999) Rac downregulates rho activity: reciprocal balance between both GTPases determines cellular morphology and migratory behavior. J Cell Biol 147:1009–1022

    CAS  PubMed  PubMed Central  Google Scholar 

  • Shekarabi M, Kennedy TE (2002) The netrin-1 receptor DCC promotes filopodia formation and cell spreading by activating Cdc42 and Rac1 Mol cell. Neurosci 19:1–17

    CAS  Google Scholar 

  • Shekarabi M, Moore SW, Tritsch NX, Morris SJ, Bouchard JF, Kennedy TE (2005) Deleted in colorectal cancer binding netrin-1 mediates cell substrate adhesion and recruits Cdc42, Rac1, Pak1, and N-WASP into an intracellular signaling complex that promotes growth cone expansion. J Neurosci 25:3132–3141

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sin WC, Moniz DM, Ozog MA, Tyler JE, Numata M, Church J (2009) Regulation of early neurite morphogenesis by the Na+/H+ exchanger NHE1. J Neurosci 29:8946–8959

    CAS  PubMed  PubMed Central  Google Scholar 

  • Slepkov ER, Rainey JK, Sykes BD, Fliegel L (2007) Structural and functional analysis of the Na+/H+ exchanger. Biochem J 401:623–633

    CAS  PubMed  PubMed Central  Google Scholar 

  • Stock C, Schwab A (2006) Role of the Na/H exchanger NHE1 in cell migration. Acta Physiol (Oxf) 187:149–157

    CAS  Google Scholar 

  • Tigyi G, Fischer DJ, Sebok A, Yang C, Dyer DL, Miledi R (1996) Lysophosphatidic acid-induced neurite retraction in PC12 cells: control by phosphoinositide-Ca2+ signaling and rho. J Neurochem 66:537–548

    CAS  PubMed  Google Scholar 

  • Tominaga T, Barber DL (1998) Na-H exchange acts downstream of RhoA to regulate integrin-induced cell adhesion and spreading. Mol Biol Cell 9:2287–2303

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tominaga T, Ishizaki T, Narumiya S, Barber DL (1998) p160ROCK mediates RhoA activation of Na-H exchange. EMBO J 17:4712–4722

    CAS  PubMed  PubMed Central  Google Scholar 

  • Vexler ZS, Symons M, Barber DL (1996) Activation of Na+-H+ exchange is necessary for RhoA-induced stress fiber formation. J Biol Chem 271:22281–22284

    CAS  PubMed  Google Scholar 

  • Wakabayashi S, Shigekawa M, Pouyssegur J (1997) Molecular physiology of vertebrate Na+/H+ exchangers. Physiol Rev 77:51–74

    CAS  PubMed  Google Scholar 

  • Wakabayashi S, Morihara H, Yokoe S, Nakagawa T, Moriwaki K, Tomoda K, Asahi M (2019) Overexpression of Na(+)/H(+) exchanger 1 specifically induces cell death in human iPS cells via sustained activation of the rho kinase ROCK. J Biol Chem 294:19577–19588. https://doi.org/10.1074/jbc.RA119.010329

    Article  CAS  PubMed  Google Scholar 

  • Woo S, Gomez TM (2006) Rac1 and RhoA promote neurite outgrowth through formation and stabilization of growth cone point contacts. J Neurosci 26:1418–1428

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wu KL, Khan S, Lakhe-Reddy S, Jarad G, Mukherjee A, Obejero-Paz CA, Konieczkowski M, Sedor JR, Schelling JR (2004) The NHE1 Na+/H+ exchanger recruits ezrin/radixin/moesin proteins to regulate Akt-dependent cell survival. J Biol Chem 279:26280–26286

    CAS  PubMed  Google Scholar 

  • Yamaguchi Y, Katoh H, Yasui H, Mori K, Negishi M (2001) RhoA inhibits the nerve growth factor-induced Rac1 activation through rho-associated kinase-dependent pathway. J Biol Chem 276:18977–18983

    CAS  PubMed  Google Scholar 

  • Yoshizaki H et al (2003) Activity of rho-family GTPases during cell division as visualized with FRET-based probes. J Cell Biol 162:223–232

    CAS  PubMed  PubMed Central  Google Scholar 

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  1. Department of Cellular and Physiological Sciences, Life Sciences Institute, The University of British Columbia, Vancouver, BC, Canada

    Wun Chey Sin, Nicola Tam, David Moniz, Connie Lee & John Church

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  1. Wun Chey Sin
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Sin, W.C., Tam, N., Moniz, D. et al. Na/H exchanger NHE1 acts upstream of rho GTPases to promote neurite outgrowth. J. Cell Commun. Signal. 14, 325–333 (2020). https://doi.org/10.1007/s12079-020-00556-5

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