Neogenin-RGMa Signaling at the Growth Cone Is Bone Morphogenetic Protein-independent and Involves RhoA, ROCK, and PKC

The repulsive guidance molecule RGMa has been shown to induce outgrowth inhibition of neurites by interacting with the transmembrane receptor neogenin. Here we show that RGMa-induced growth cone collapse is mediated by activation of the small GTPase RhoA, its downstream effector Rho kinase and PKC. In contrast to DRG cultures from neogenin-/- mice, in which no RGMa-mediated growth cone collapse and activation of RhoA occurred, treatment of wild type DRG neurites with soluble RGMa led to a marked activation of RhoA within 3 min followed by collapse, but left Rac1 and Cdc42 unaffected. Furthermore, preincubation of DRG axons with the bone morphogenetic protein (BMP) antagonist noggin had no effect on RGMa-mediated growth cone collapse, implying that the role of RGM in axonal guidance is neogenin- and not BMP receptor-dependent. Pretreatment with 1) C3-transferase, a specific inhibitor of the Rho GTPase; 2) Y-27632, a specific inhibitor of Rho kinase; and 3) Gö6976, the general PKC inhibitor, strongly inhibited the collapse rate of PC12 neurites. Growth cone collapse induced by RGMa was abolished by the expression of dominant negative RhoA, but not by dominant negative Rac1. In contrast to RGMa, netrin-1 induced no growth cone retraction but instead reduced RGMa-mediated growth cone collapse. These results suggest that activation of RhoA, Rho kinase, and PKC are physiologically relevant and important elements of the RGMa-mediated neogenin signal transduction pathway involved in axonal guidance. The repulsive guidance molecule RGMa has been shown to induce outgrowth inhibition of neurites by interacting with the transmembrane receptor neogenin. Here we show that RGMa-induced growth cone collapse is mediated by activation of the small GTPase RhoA, its downstream effector Rho kinase and PKC. In contrast to DRG cultures from neogenin-/- mice, in which no RGMa-mediated growth cone collapse and activation of RhoA occurred, treatment of wild type DRG neurites with soluble RGMa led to a marked activation of RhoA within 3 min followed by collapse, but left Rac1 and Cdc42 unaffected. Furthermore, preincubation of DRG axons with the bone morphogenetic protein (BMP) antagonist noggin had no effect on RGMa-mediated growth cone collapse, implying that the role of RGM in axonal guidance is neogenin- and not BMP receptor-dependent. Pretreatment with 1) C3-transferase, a specific inhibitor of the Rho GTPase; 2) Y-27632, a specific inhibitor of Rho kinase; and 3) Gö6976, the general PKC inhibitor, strongly inhibited the collapse rate of PC12 neurites. Growth cone collapse induced by RGMa was abolished by the expression of dominant negative RhoA, but not by dominant negative Rac1. In contrast to RGMa, netrin-1 induced no growth cone retraction but instead reduced RGMa-mediated growth cone collapse. These results suggest that activation of RhoA, Rho kinase, and PKC are physiologically relevant and important elements of the RGMa-mediated neogenin signal transduction pathway involved in axonal guidance. Neogenin-RGMa signaling at the growth cone is bone morphogenetic protein-independent and involves RhoA, ROCK, and PKC.Journal of Biological ChemistryVol. 285Issue 53PreviewPAGE 16430: Full-Text PDF Open Access During the development of the central and peripheral nervous system, target-derived axon extension is guided by attractive and repulsive diffusible or membrane-bound factors acting over short and long distances (1.Tessier-Lavigne M. Goodman C.S. Science. 1996; 274: 1123-1133Crossref PubMed Scopus (2706) Google Scholar). During axonal chemorepulsion, repulsive guidance cues hinder neurite outgrowth, turn growth cones away from a guidance cue, and in the case of growth cone collapse induce a dramatic retraction of the growth cone. Although the repulsive guidance molecules (RGM) 2The abbreviations used are: RGM, repulsive guidance molecule; DCC, deleted in colorectal carcinoma; RasGAP, Ras GTPase-activating protein; DRG, dorsal root ganglia; PKC, protein kinase C; NGF, nerve growth factor; BMP, bone morphogenetic protein; GST, glutathione S-transferase; GFP, green fluorescent protein. and their receptor neogenin have been identified as guidance factors (2.Monnier P.P. Sierra A. Macchi P. Deitinghoff L. Andersen J.S. Mann M. Flad M. Hornberger M.R. Stahl B. Bonhoeffer F. Mueller B.K. Nature. 2002; 419: 392-395Crossref PubMed Scopus (257) Google Scholar, 3.Brinks H. Conrad S. Vogt J. Oldekamp J. Sierra A. Deitinghoff L. Bechmann I. Varez-Bolado G. Heimrich B. Monnier P.P. Mueller B.K. Skutella T. J. Neurosci. 2004; 24: 3862-3869Crossref PubMed Scopus (80) Google Scholar, 4.Rajagopalan S. Deitinghoff L. Davis D. Conrad S. Skutella T. Chedotal A. Mueller B.K. Strittmatter S.M. Nat. Cell Biol. 2004; 6: 756-762Crossref PubMed Scopus (227) Google Scholar, 5.Wilson N.H. Key B. Dev. Biol. 2006; 296: 485-498Crossref PubMed Scopus (97) Google Scholar, 6.Matsunaga E. Chedotal A. Dev. Growth Differ. 2004; 46: 481-486Crossref PubMed Scopus (57) Google Scholar), the cytoplasmic signaling mechanisms responsible for triggering neogenin-mediated and -directed growth cone collapse remain to be clarified. Recently Hata et al. (7.Hata K. Fujitani M. Yasuda Y. Doya H. Saito T. Yamagishi S. Mueller B.K. Yamashita T. J. Cell Biol. 2006; 173: 47-58Crossref PubMed Scopus (236) Google Scholar) demonstrated that neurite extension of cerebellar granule neurons was inhibited when these cells were plated on a monolayer of RGMa overexpressing cells and that this effect can be blocked with RhoA and Rho kinase inhibitors. Furthermore, increased amounts of GTP-RhoA were observed in these primary neurons after the addition of soluble RGMa. Neogenin, which is expressed by growing nerve cells in the developing vertebrate brain, consists of four immunoglobulin-like domains followed by six fibronectin type III domains, a transmembrane domain, and an intracellular domain (4.Rajagopalan S. Deitinghoff L. Davis D. Conrad S. Skutella T. Chedotal A. Mueller B.K. Strittmatter S.M. Nat. Cell Biol. 2004; 6: 756-762Crossref PubMed Scopus (227) Google Scholar, 8.Fitzgerald D.P. Seaman C. Cooper H.M. Dev. Dyn. 2006; 235: 1720-1725Crossref PubMed Scopus (26) Google Scholar, 9.Vielmetter J. Kayyem J.F. Roman J.M. Dreyer W.J. J. 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Growth Differ. 2004; 46: 481-486Crossref PubMed Scopus (57) Google Scholar, 12.Mehlen P. Cell Death Differ. 2005; 121003Crossref PubMed Scopus (8) Google Scholar, 13.Matsunaga E. Tauszig-Delamasure S. Monnier P.P. Mueller B.K. Strittmatter S.M. Mehlen P. Chedotal A. Nat. Cell Biol. 2004; 6: 749-755Crossref PubMed Scopus (223) Google Scholar, 14.Matsunaga E. Nakamura H. Chedotal A. J. Neurosci. 2006; 26: 6082-6088Crossref PubMed Scopus Google Scholar). Recently et al. K. T. K. K. 2006; PubMed Scopus Google Scholar) no effect of netrin-1 on axon or of dorsal root but a general of axon outgrowth from DRG by netrin-1 after in In addition to the of RGMa and neogenin it has been shown that RGMa, and as BMP A. Y. E. J. Neurosci. 2004; 24: PubMed Scopus Google Scholar, Y. Y. J. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Y. Nat. 2006; PubMed Scopus Google Scholar). Although no functional were it was that RGM the of cells in which are expressed to BMP these cells to or to levels of BMP Y. Y. Nat. 2006; PubMed Scopus Google Scholar). be involved in the RGMa role in axonal guidance and growth cone collapse. guidance molecule has to a of intracellular that results in a the guidance of axon extension or A. Science. PubMed Scopus Google Scholar, J.F. Neurosci. 26: PubMed Scopus Google Scholar). is a of that axon guidance signaling involves the of to the Rho of small of Rho and by and These are to as the guidance signal to and the of marked in the and of axons have been observed after of the Rho in vitro and in B.K. Neurosci. PubMed Scopus Google Scholar, B.K. H. Nat. 2005; PubMed Scopus Google Scholar). In these suggest that and Cdc42 are that neurite Rho is a negative that the inhibition or collapse of growth guidance receptor and neogenin have been shown to intracellular Rho GTPase (4.Rajagopalan S. Deitinghoff L. Davis D. Conrad S. Skutella T. Chedotal A. Mueller B.K. Strittmatter S.M. Nat. Cell Biol. 2004; 6: 756-762Crossref PubMed Scopus (227) Google Scholar, K. Fujitani M. Yasuda Y. Doya H. Saito T. Yamagishi S. Mueller B.K. Yamashita T. J. Cell Biol. 2006; 173: 47-58Crossref PubMed Scopus (236) Google Scholar, B.K. H. Nat. 2005; PubMed Scopus Google Scholar). that Rho have in the RGMa guidance of growth cone collapse. the signal transduction in primary neurons and PC12 cells collapse, we on PKC, the small GTPase Rho and its downstream and Rho In as PC12 and primary activation of Rho to growth cone collapse, neurite or neurite growth These are by a specific inhibitor of Rho and the RhoA, and not Cdc42 and at these K. I. 2005; Google Scholar, A. M. S. J. Biol. Full Text PDF PubMed Google Scholar). of the downstream of Rho is the kinase PubMed Scopus Google Scholar), which is inhibited by the M. T. H. T. T. T. H. K. J. M. S. Nature. 1997; PubMed Scopus Google Scholar). In this we the signal transduction of RGMa-mediated growth cone collapse. neogenin we that the of RGMa and neogenin is specific and important in axon BMP signaling in this used primary neurons and PC12 cells to the of of Rho and Rac1 in growth cone collapse induced by RGMa. that RhoA chemorepulsion, but not Rac1 or that PKC is involved in addition to the RhoA pathway and that of of these can repulsive mediated by the neogenin with netrin-1 that the repulsive effect of RGMa be by preincubation with Neogenin for neogenin were by M. Tessier-Lavigne In in were over and in was with to of RGMa and neogenin J. Varez-Bolado G. Skutella T. 2004; PubMed Scopus Google Scholar, J. D. 2004; PubMed Scopus Google Scholar), on and PC12 Cell and neurite outgrowth a of dorsal root cells was from wild type or neogenin-/- and for on in with and RGMa-induced growth cone collapse in detail, we used PC12 cells in with and in of with and for at in of was to the and the cells were for an of DRG, PC12 Cell and were used to RGMa, and RGMa, of RGMa and the of in or in was with amounts of or were with These were at for an of an and was to and the were at for min at were and with and the were at for The were at for and the were with and with the were with and for min at were to and to with to and RGM protein. with the PC12 cells were with specific for RhoA and a for neogenin from negative we used a specific for the neogenin for RhoA the primary was in the was used as a and and as PC12 cells were with The In Cell Death and were used for the of was in a by the RGMa J. Varez-Bolado G. Skutella T. 2004; PubMed Scopus Google Scholar, J. D. 2004; PubMed Scopus Google Scholar) the signal or and by with the and was and the with the protein were for collapse The were with an to induce a of the RGMa protein. of of protein were used for the collapse protein as the In a was to the of the or PC12 or was min after the of the collapse and was observed and for min with a Cell at a of The and growth cones were in and the were with are expressed as the cells were with and for of the collapse after addition of of of was to the PC12 cultures collapse by Rho kinase was inhibited by Rho kinase inhibitor to the cultures min the collapse inhibition of protein kinase the cells were for with of to PKC inhibitor activation was blocked by the BMP antagonist noggin at for netrin-1 was for min or in with for collapse with PC12 with the for Rac1 dominant negative Rac1 or Rac1 RhoA or Rac1 were expression the cells were plated on as followed by the collapse is on the of and to to and The were after with and to PC12 cells were plated on or was to the and the cells were with after and min of The were to the and were to amounts of and Rac1 by on RhoA, and RhoA was used for of the protein the of Rho and PKC kinase we used for Rho and PKC were inhibited with of the specific inhibitor for Rho and of the specific inhibitor for PKC. The kinase were at a of of the were by of with at 3 in In were at The for DRG and PC12 neurites were a of growth cones and retraction of the neurites. were with the Cell and RGMa Growth in in that neogenin is expressed by dorsal root at but RGMa is not Furthermore, that neogenin and RGMa directly and In a we the growth cone collapse of RGMa with primary dorsal root cells from and neogenin In the in vitro the growth cones of dorsal root cells to collapse min after the addition of to the and effect was observed after the of protein and Furthermore, no RGMa-induced growth cone collapse was observed in DRG cells from neogenin-/- The RGMa protein was with a specific the In to RGMa, which induced growth cone retraction in PC12 cells not the induced a and neogenin directly Rho GTPase we with the DRG cultures and observed a marked activation of RhoA in but not in neogenin-/- within 3 min after the of RGMa Furthermore, no activation of Rac1 and Cdc42 was observed in not in RGMa-mediated Growth of the of the BMP pathway in the RGMa-induced collapse it has been shown that RGMa, and as and A. Y. E. J. Neurosci. 2004; 24: PubMed Scopus Google Scholar, Y. Y. J. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Y. Nat. 2006; PubMed Scopus Google Scholar), we be involved in RGMa-mediated growth cone collapse. that DRG and PC12 cells but not we the BMP antagonist noggin RGMa-induced growth cone collapse. no case in which the RGMa-induced collapse be by noggin in primary DRG neurites or PC12 cells not is with the results of neogenin and that neogenin a BMP receptor is to the RGMa signal to the pathway to the collapse in the primary that the results are that neogenin a role in RGMa-induced signaling of Neogenin and Rho but in PC12 PC12 cells to growth cone collapse induced by RGMa as by these cells neogenin but not RGMa or the role of the Rho GTPase in RGMa-induced collapse of PC12 we used to RhoA and neogenin are expressed by these cells and PC12 cells growing on a were after of treatment and with neogenin- and Neogenin and RhoA were strongly in the the growth and to a lesser in the neurites of PC12 cells as and The growth cones of the PC12 cells with RGMa Growth in PC12 used PC12 cells as a to RGMa-induced growth cone collapse in During RGMa induced a collapse of PC12 cells and and retraction of the growth cones min after the addition of of RGMa to the observed no PC12 cells in after the addition of the and the of growth cone retraction was in the In the addition of the protein not induce collapse and Rho and Neogenin RGMa-induced Growth Rho are involved in RGMa-induced growth cone collapse the neogenin we the effect of an inhibitor of Rho abolished the repulsive effect of RGMa on PC12 growth The neurites not show growth cones with The of RGMa was abolished after PC12 cells were with of the or not the RGMa-induced collapse of PC12 cells not The of PC12 cells increased from to in cultures to in and PC12 cells the of RGMa. results were after inhibition of by the Rho inhibitor the addition of RGMa, PC12 cells were with of reduced the of RGMa and the of PC12 cells increased from to the functional RGMa and we the PC12 with an the of neogenin. with the functional blocked the collapse of growth cones by PKC in Neogenin a we PKC is involved in the neogenin-mediated signal transduction PKC a of protein that are in a of with high in neuronal Gö6976, a general PKC inhibitor that in their to RGMa-induced growth cone collapse of PC12 to were with not and the were with with reduced neurite growth cone collapse after the addition of RGMa of neurite retraction outgrowth that the effect of this PKC inhibitor was with that of a inhibitor that RGMa-induced neurite of RhoA, but Rac1 RGMa-induced Growth the role of Rho in the growth cone collapse induced by RGMa, we expressed of and dominant negative or Rho RhoA, and Rac1 green fluorescent protein In cultures of PC12 a for cells The of dominant and RhoA and Rac1 on neurite retraction were in cultures after The expression of dominant negative RhoA a and in the of neurites and the of growth expression of RhoA not the RGMa-induced neurite retraction In expression of dominant negative Rac1 no in the of neurite retraction These are with the that RhoA neogenin-mediated growth cone collapse. of RhoA by neogenin directly Rho GTPase we on PC12 observed a marked activation of RhoA within 3 min after the of RGMa Cdc42 Rac1 was with were to activation of RhoA, and Rac1 in PC12 this PC12 cells were with RGMa or protein. were and the of RhoA was a downstream of which binds to RGMa induced an in RhoA, no RhoA activation was in cultures treatment this effect In contrast to RGMa-induced activation of RhoA, of or Cdc42 with no of and Cdc42 in cultures with the cultures These results suggest that treatment of PC12 cells with RGMa results in activation of but not or In PC12 no were observed in the levels of RhoA, and Rho and PKC kinase were increased in PC12 cells 3 min after with RGMa in to and kinase inhibition and The in Rho kinase was observed after to after PKC kinase was min after and after of RhoA in with of RhoA and PKC were to collapse of these inhibitors. Rho kinase and PKC blocked RGMa-induced growth cone collapse of these that RhoA and PKC be involved to in the signaling pathway induced by RGMa (7.Hata K. Fujitani M. Yasuda Y. Doya H. Saito T. Yamagishi S. Mueller B.K. Yamashita T. J. Cell Biol. 2006; 173: 47-58Crossref PubMed Scopus (236) Google Scholar, B.K. H. Nat. 2005; PubMed Scopus Google Scholar). RGMa with netrin-1 to netrin-1 growth cone collapse in PC12 cells and to the activation of the addition of netrin-1 to the no growth cones be observed over a of min Furthermore, no activation of RhoA was observed not is with et al. K. T. K. K. 2006; PubMed Scopus Google Scholar), not a specific effect of netrin-1 on axon or of DRG but a general of axon outgrowth from DRG by netrin-1 after in In addition we that preincubation of PC12 neurites with netrin-1 the RGMa-mediated collapse Neogenin and RGMa in Cell Death of and PC12 we the of RGMa on the of and PC12 In no in was observed after addition of or RGMa. and no after the addition of RGMa not The and not a in vitro and in neogenin and its RGMa have been in mechanisms axonal guidance (4.Rajagopalan S. Deitinghoff L. Davis D. Conrad S. Skutella T. Chedotal A. Mueller B.K. Strittmatter S.M. Nat. Cell Biol. 2004; 6: 756-762Crossref PubMed Scopus (227) Google Scholar). In axonal growth cones are away from a guidance and in the case of growth cone collapse a dramatic retraction of the growth cone the signal transduction the repulsive guidance of RGMa by its receptor we growth cone collapse with primary DRG neurites and with PC12 cells as a for and for a role of neogenin in RGMa-induced signaling we closely the of the RGMa with its receptor neogenin in from and neogenin DRG neurites from the not to RGMa and no activation of RhoA after with RGMa. These with neogenin that RGMa not induce growth cone collapse in primary dorsal root cells its receptor neogenin. the growth cones of DRG neurites by growth cone retraction was not observed in the DRG neurites of the neogenin Furthermore, activation of the RhoA signal transduction pathway was not induced in the DRG cells after the addition of RGMa to the the of the BMP pathway in the RGMa-induced collapse of the with the receptor neogenin. are in that axon guidance in the neuronal RGMa and be in and repulsive axon guidance. Recently it was shown that RGMa, and as BMP A. Y. E. J. Neurosci. 2004; 24: PubMed Scopus Google Scholar, Y. Y. J. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Y. Nat. 2006; PubMed Scopus Google Scholar). we be involved in RGMa-mediated growth cone collapse. The with the neogenin-/- that not a BMP is to the RGMa signal to the pathway to the collapse in the primary In addition we the BMP antagonist noggin RGMa-induced growth cone collapse. we no in which the RGMa-induced collapse be by the BMP antagonist In the of axonal guidance the BMP to a role in axonal guidance is which was shown to induce the collapse of axon growth that it can directly on growth cones to a in A. A. S. J. S. 24: Full Text Full Text PDF PubMed Scopus Google Scholar, F. Tessier-Lavigne M. 2005; PubMed Scopus Google Scholar). The mechanisms the effect of on growth cones is not Although signaling type and it is not these the of axons of the Recently et al. Y. Y. Nat. 2006; PubMed Scopus Google Scholar) demonstrated that RGMa is a and but not functional was that RGM the of cells in which are expressed to BMP these cells to or to levels of BMP the collapse of DRG growth cones induced by the addition of RGMa to the not the neogenin and or not induce growth cone collapse in we that the and is not to the growth cone collapse of DRG neurites. with the BMP antagonist which not RGMa-induced growth cone collapse in it that a role in axonal guidance as of PC12 cells neogenin but not RGMa or the in the developing nervous of Rho and reduced the RGMa of PC12 growth of collapse with the of of the RhoA protein. RGMa-induced neurite retraction was blocked by of the PC12 cells with RhoA with that activation of Rho and an signal to the as or kinase S. H. C. P. Nature. PubMed Scopus Google Scholar, M. T. S. A. A. T. K. K. S. Science. PubMed Scopus Google Scholar). Furthermore, that Rac1 Cdc42 is involved in the collapse of growth cones after with RGMa. In these results that RGMa can the activation of RhoA in PC12 is by the results of Hata et al. (7.Hata K. Fujitani M. Yasuda Y. Doya H. Saito T. Yamagishi S. Mueller B.K. Yamashita T. J. Cell Biol. 2006; 173: 47-58Crossref PubMed Scopus (236) Google Scholar), that outgrowth inhibition of cerebellar granule neurons induced by RGMa overexpressing cells can be blocked with RhoA and Rho kinase inhibitors. demonstrated that PKC is involved to RhoA in neogenin-mediated growth cone collapse et al. J. Kayyem J.F. Roman J.M. Dreyer W.J. J. 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Cell Biol. 2006; 173: 47-58Crossref PubMed Scopus (236) Google Scholar) demonstrated the of RGMa signaling in and the of this that PKC and RhoA are involved in neogenin-mediated growth cone collapse a the inhibition of axonal in the central nervous