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Nerve Growth Factor Signals through TrkA, Phosphatidylinositol 3-Kinase, and Rac1 to Inactivate RhoA during the Initiation of Neuronal Differentiation of PC12 Cells*

Open AccessPublished:July 19, 2002DOI:https://doi.org/10.1074/jbc.M203617200
      In PC12 rat pheochromocytoma cells, nerve growth factor (NGF)-induced neuronal differentiation is blocked by constitutively active dominant mutants of RhoA but augmented by negative ones, suggesting a not yet elucidated inhibitory signaling link between NGF receptors and RhoA. Here we show that NGF treatment rapidly translocates RhoA from the plasma membrane to the cytosol and simultaneously decreases RhoA affinity to its target Rho-associated kinase (ROK), a key mediator of neurite outgrowth. This effect was transient, because after 2 days of NGF treatment, RhoA relocated from the cytosol to the plasma membrane, and its GTP loading returned to a level found in undifferentiated cells. Inhibition of RhoA is mediated by activation of the TrkA receptor, because NGF failed to induce RhoA translocation and inhibition of ROK binding in nnr5 cells that lack TrkA, whereas the inhibition was reconstituted in receptor add-back B5 cells. In MM17-26 cells, which due to expression of dominant negative Ras do not differentiate, NGF-stimulated transient RhoA inhibition was unaffected. The inhibitory pathway from TrkA to RhoA involves phosphatidylinositol-3-kinase (PI3K), because the inhibitors LY294002 or wortmannin prevented NGF-induced RhoA translocation and increased RhoA association with ROK. Furthermore, inhibition of PI3K significantly reduced NGF- mediated Rac1 activation, whereas dominant negative Rac1 abolished the inhibitory signaling to RhoA. Taken together, these data indicate that NGF-mediated activation of TrkA receptor stimulates PI3K, which in turn increases Rac1 activity to induce transient RhoA inactivation during the initial phase of neurite outgrowth.
      GEF
      guanine nucleotide exchange factor
      GAP
      GTPase-activating protein
      GDI
      guanine nucleotide dissociation inhibitor
      NGF
      nerve growth factor
      C3 exoenzyme
      Clostridium botulinum Rho-ADP-ribosyltransferase
      TBST
      Tris-buffered saline with Tween 20
      GST
      glutathioneS-transferase
      HMF
      heavy membrane fraction
      TrkA
      NGF receptor
      GTPγS
      guanosine 5′-O- thiotriphosphate
      The small GTPases RhoA and Rac1 are members of the Rho subfamily within the Ras superfamily of GTPases. RhoA plays an important role in the organization of the actin cytoskeleton, gene transcription, cell cycle progression, cell transformation, and membrane trafficking (for reviews, see Refs.
      • Hall A.
      ,
      • Kjoller L.
      • Hall A.
      ,
      • Welsh C.F.
      • Assoian R.K.
      ). RhoA cycles between the GDP-bound inactive and active forms. Three classes of molecules are known to interact and regulate GDP/GTP cycling of RhoA: guanine nucleotide exchange factors (GEFs)1 catalyze the exchange of GDP for GTP, GTPase-activating proteins (GAPs) stimulate the intrinsic GTPase activity of RhoA, and guanine nucleotide dissociation inhibitors (GDIs) inhibit the exchange of GDP for GTP and also stabilize cytosolic RhoA form binding GTP (
      • Kjoller L.
      • Hall A.
      ). For RhoA to transmit signals, two criteria must be satisfied: first, it must in a GTP-loaded form; second, RhoA, which is geranylgeranylated, must be in the right signaling compartment attached to the plasma membrane, where it can interact with its regulators and targets. Thus, RhoA in its GTP-bound active form associates with the inner surface of the plasma membrane (
      • Noguchi Y.
      • Nakamura S.
      • Yasuda T.
      • Kitagawa M.
      • Kohn L.D.
      • Saito Y.
      • Hirai A.
      ), whereas GDI-mediated inactivation of RhoA translocates it to the cytoplasm and prevents it from binding to GTP.
      The role of RhoA during neuronal differentiation was first suggested by studies using the Rho-specific ADP-ribosyltransferase C3 toxin (Clostridium botulinum C3 Rho-ADP-ribosylating exoenzyme), which induces neurite outgrowth in naive (not treated with NGF) PC12 cells (
      • Nishiki T.
      • Narumiya S.
      • Morii N.
      • Yamamoto M.
      • Fujiwara M.
      • Kamata Y.
      • Sakaguchi G.
      • Kozaki S.
      ). Furthermore, RhoA has been shown to be involved in the regulation of neurite outgrowth (
      • Tigyi G.
      • Miledi R.
      ,
      • Jalink K.
      • van Corven E.J.
      • Hengeveld T.
      • Morii N.
      • Narumiya S.
      • Moolenaar W.H.
      ,
      • Kozma R.
      • Sarner S.
      • Ahmed S.
      • Lim L.
      ,
      • Zipkin I.D.
      • Kindt R.M.
      • Kenyon C.J.
      ,
      • Lehmann M.
      • Fournier A.
      • Selles-Navarro I.
      • Dergham P.
      • Sebok A.
      • Tigyi G.
      • McKerracher L.
      ). In our earlier studies (
      • Sebok A.
      • Nusser N.
      • Debreceni B.
      • Guo Z.
      • Santos M.F.
      • Szeberenyi J.
      • Tigyi G.
      ), we found that expression of activated V14RhoA mutant prevented NGF-induced neurite outgrowth. In contrast, dominant negative RhoA (N19RhoA) expression led to an increase in neurite initiation and branching. Furthermore, RhoA was shown to have a dual role during neuronal differentiation. Inactivation of RhoA appears necessary for the initiation of neuronal differentiation, although during later stages of neurite elongation, introduction of N19RhoA causes the formation of short neurites (
      • Sebok A.
      • Nusser N.
      • Debreceni B.
      • Guo Z.
      • Santos M.F.
      • Szeberenyi J.
      • Tigyi G.
      ). Although these findings suggest an important role of RhoA in NGF-induced neuronal morphogenesis, little is known about the signal transduction pathways that couple NGF signaling to RhoA.
      In their active forms, RhoA proteins interact with and modulate the activity of effector proteins that include the serine/threonine protein kinase N (p120, PKN), p160 RhoA-associated kinase ROKα (ROK or ROCK-II), p150 RhoA-binding kinase ROKβ (ROCK-I), citron-K, and nonkinases rhophilin, rhotekin, citron-N, and p140mDia (
      • Sahai E.
      • Alberts A.S.
      • Treisman R.
      ,
      • Bishop A.L.
      • Hall A.
      ). Among these, ROK has been shown to mediate the formation of stress fibers, focal adhesions, regulation of myosin phosphorylation, and c-fos expression (for review, see Ref.
      • Amano M.
      • Fukata Y.
      • Kaibuchi K.
      ). Moreover, activation of ROK is sufficient to induce neurite retraction in the NGF-differentiated PC12 cells (
      • Katoh H.
      • Aoki J.
      • Ichikawa A.
      • Negishi M.
      ).
      Rac1 is an essential mediator of axonal growth, guidance, and branching (
      • Kozma R.
      • Sarner S.
      • Ahmed S.
      • Lim L.
      ,
      • Zipkin I.D.
      • Kindt R.M.
      • Kenyon C.J.
      ,
      • Luo L.
      • Liao Y.J.
      • Jan L.Y.
      • Jan Y.N.
      ,
      • Lundquist E.A.
      • Reddien P.W.
      • Hartwieg E.
      • Horvitz H.R.
      • Bargmann C.I.
      ,
      • Ng J.
      • Nardine T.
      • Harms M.
      • Tzu J.
      • Goldstein A.
      • Sun Y.
      • Dietzl G.
      • Dickson B.J.
      ). In fibroblasts, a coupled activation of Rho by Rac and Cdc42 has been described (
      • Ridley A.
      • Paterson H.F.
      • Johnston C.L.
      • Diekman D.
      • Hall A.
      ), linking these GTPases into a system that regulates almost all aspects of cytoskeletal organization. However, this activation cascade appears to be a cell type-specific phenomenon; and in neuronal cells, Rac1 and RhoA, although both activable by Cdc42, have opposing effects on neurite outgrowth (
      • Kozma R.
      • Sarner S.
      • Ahmed S.
      • Lim L.
      ).
      In the present study, we investigated the effect of NGF treatment on the GTP loading and membrane association of RhoA during the initiation and elongation phase of neuronal differentiation. We show that NGF through its TrkA receptor transiently inhibits RhoA signaling, as evidenced by its cytoplasmic translocation and diminished ability to interact with ROKα. However, during the elongation phase of neuronal differentiation, 2 days after NGF treatment, RhoA was relocated to the plasma membrane and its activation state returned to the control level. We provide evidence that TrkA, independently from Ras-mediated signaling, regulates PI3K, which through the small GTPase Rac1 mediates RhoA inactivation, which is a permissive signal for the initiation of neurite outgrowth. The present results establish an inhibitory coupling between the TrkA and RhoA through a novel negative regulatory mechanism connecting Rac1 to RhoA in PC12 cells.

      DISCUSSION

      In the present report, we sought to establish the mechanism underlying our earlier observations using constitutively active and dominant negative RhoA mutants (
      • Sebok A.
      • Nusser N.
      • Debreceni B.
      • Guo Z.
      • Santos M.F.
      • Szeberenyi J.
      • Tigyi G.
      ), which provided circumstantial evidence for a dual role of RhoA in NGF-induced differentiation of PC12 cells. Activated RhoA prevented the withdrawal of the cell from the cell cycle and abolished neurite outgrowth, whereas dominant negative RhoA enhanced differentiation. This observation implied that NGF signaling should negatively couple to RhoA to promote the early events of differentiation, which include the cessation of cell proliferation and the initiation of neurite outgrowth. In contrast to naive PC12 cells, in differentiated cells, we found that the expression of activated RhoA accelerated the rate of neurite elongation, whereas dominant negative RhoA reduced it. In the present study, we first investigated the effect of NGF signaling on RhoA by measuring its translocation and GTP-loading as measured by the affinity to its target, ROK.
      Here we provide evidence that a brief treatment with NGF leads to RhoA inactivation, as shown by its rapid translocation from the membrane to the cytosol and a decrease in its ability to associate with its downstream target, ROK (Fig. 1). These results provide a mechanism for our earlier phenomenological observation that NGF somehow inactivated RhoA-ROK signaling during the initiation phase of neuronal differentiation. We extended our study to determine not only the localization but also the activation state of RhoA during neurite elongation. Again, in agreement with the previous study using RhoA mutants, we found that a prolonged 2-day NGF treatment increased RhoA expression and its association with the plasma membrane during the neurite elongation phase. Although prolonged NGF treatment caused no elevation in the GTP-bound state of RhoA because the amount of ROK-bound RhoA returned to the a level seen in naive cells, this change represents a 70% increase as compared with the initiation phase (compare Fig. 1 and Fig. 2). Taken together, these results support our dual-role model proposed earlier: NGF treatment rapidly inactivates RhoA during the initiation phase, but it returns to the plasma membrane compartment with a basal level of GTP loading during the elongation phase of neuronal differentiation.
      Which NGF receptor mediates the inhibitory effect to RhoA? Using nnr5 cells deficient in functional TrkA receptor and a receptor add-back clone the B5 cells, we found evidence that TrkA is necessary for the inhibitory coupling to RhoA.
      We next turned our focus to TrkA-linked signaling pathways, specifically the Ras pathway because it has a central role in mediating NGF-induced neuronal differentiation. Settleman et al. (
      • Settleman J.
      • Narasimhan V.
      • Foster L.C.
      • Weinberg R.A.
      ) reported a cross-talk between Ras and Rho by showing that p190Rho-GAP was tyrosine-phosphorylated and formed a complex with the SH2 domain of p120Ras-GAP, suggesting that the latter may act as a Ras effector negatively regulating the activity of RhoA. Booden et al.(
      • Booden M.A.
      • Sakaguchi D.S.
      • Buss J.E.
      ) found that expression of constitutively activated Ras elicited neurite outgrowth that was prevented by the co-expression of inhibitory Rho only in the presence of increased amounts of inhibitory Rac1. These results suggest that RhoA and Rac1 were coupled to Ras; however, these authors did not elicit differentiation using NGF, but rather with an activated Ras mutant. We reported that activation of RhoA during neurite retraction caused by lysophosphatidic acid was independent of Ras (
      • Tigyi G.
      • Fischer D.J.
      • Sebok A.
      • Yang C.
      • Dyer D.L.
      • Miledi R.
      ), suggesting the possibility of Ras-independent signaling from NGF toward RhoA. Recently, Boglari and Szeberenyi (
      • Boglari G.
      • Szeberenyi J.
      ) showed that a TrkA-independent Ras pathway exists that is sufficient to mediate neurite outgrowth. Using a PC12 subclone, the M-M17-26 line that expresses a dominant negative N17Ras (
      • Szeberenyi J.
      • Cai H.
      • Cooper G.M.
      ), we found no significant difference between the effect of NGF on the inactivation of RhoA in wild-type and N17Ras-expressing cells. The M-M17-26 cell line has been well characterized, and there is no evidence for NGF-induced ERK1/2 activation in these cells (data not shown), whereas NGF activates PI3K and consequently Akt (
      • Yao R.
      • Cooper G.M.
      ). Taken together, our results provide further evidence for a dichotic signaling originating from TrkA: a Ras-dependent branch that activates the MAPK cascade and a Ras-independent one that mediates the inactivation of RhoA and is linked to the PI3K-Akt pathway.
      The lipid kinase PI3K can mediate signals from Ras to Rac1 required for the oncogenic transformation of Rat-1 cells (
      • Tang Y., Yu, J.
      • Field J.
      ). In vitrodata suggest that Rac1 specifically interacts with phosphatidylinositol 3,4,5-trisphosphate, a product of PI3K (
      • Missy K.
      • Van Poucke V.
      • Raynal P.
      • Viala C.
      • Mauco G.
      • Plantavid M.
      • Chap H.
      • Payrastre B.
      ). RhoA also displays significant, although much weaker, binding to this lipid (
      • Missy K.
      • Van Poucke V.
      • Raynal P.
      • Viala C.
      • Mauco G.
      • Plantavid M.
      • Chap H.
      • Payrastre B.
      ). Expression of an activated Ras mutant caused increased Rac1 GTP binding and PI3K activity in membranes of PC12 cells (
      • Sarner S.
      • Kozma R.
      • Ahmed S.
      • Lim L.
      ).
      A hierarchical cascade linking CDC42, Rac1, and RhoA was first described in fibroblasts (
      • Ridley A.
      • Paterson H.F.
      • Johnston C.L.
      • Diekman D.
      • Hall A.
      ). According to this hypothesis based on studies conducted in fibroblasts, activation of Cdc42 induces activation of Rac1, which in turn leads to activation of Rho (for review, see Refs.
      • Kjoller L.
      • Hall A.
      and
      • Scita G.
      • Tenca P.
      • Frittoli E.
      • Tocchetti A.
      • Innocenti M.
      • Giardina G.
      • Di Fiore P.P.
      ). To extend this model, functional studies established PI3K as an effector of Ras and an upstream modulator of Rac1 activity (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Khwaja A.
      • Marte B.M.
      • Pappin D.
      • Das P.
      • Waterfield M.D.
      • Ridley A.
      • Downward J.
      ). However, in Swiss 3T3 fibroblast cells, wortmannin does not interfere with Ras-mediated actin remodeling (
      • Nobes C.D.
      • Hall A.
      ). Hence, a secondary pathway is likely to exist (receptor tyrosine kinase → PI3K → Rac1) that is Ras-independent and turned on by the activation of receptor tyrosine kinases, including TrkA. The Ras-independent pathway for Rac1 regulation is supported by the phosphorylation-dependent direct association of PI3K with the Tyr751 residue of TrkA (
      • Yamashita H.
      • Avraham S.
      • Jiang S.
      • Dikic I.
      • Avraham H.
      ). PI3K has been shown to regulate at least two of the Rac1-GEFs, Vav and Sos-1 (
      • Han J.
      • Luby-Phelps K.
      • Das B.
      • Shu X.
      • Xia Y.
      • Mosteller R.D.
      • Krishna U.M.
      • Falck J.R.
      • White M.A.
      • Broek D.
      ,
      • Nimnual A.S.
      • Yatsula B.A.
      • Bar-Sagi D.
      ). We found no change in the amount of RhoA in the HMF after NGF treatment of cells pretreated with either LY294002 or wortmannin (Fig. 5). These results suggest that PI3K activation is required for the NGF-induced regulation of RhoA. In cells expressing dominant negative Rac1, NGF did not induced translocation of RhoA from the membrane to the cytosol (Fig. 4). Furthermore, the amount of ROK-associated RhoA also showed no detectable decrease in response to NGF treatment in N17Rac1-expressing cells (Fig. 4, B and C). Finally, LY294002 or wortmannin substantially reduced NGF-induced activation of Rac1 in PC12 cells (Fig. 6 A). In contrast, NGF caused a rapid increase in phosphorylation of Akt in both vector- and N17Rac1-transfected PC12 cells (Fig. 6 B). Hence, our data suggest that PI3K acts upstream from Rac1 in the NGF-activated signal transduction pathway inhibiting RhoA (Fig. 7).
      Figure thumbnail gr7
      Figure 7A scheme of the signaling pathways linking TrkA to the inhibition of RhoA. TrkA, independently of Ras, activates PI3K. PI3K activates Rac1, and active Rac1 induces the translocation of RhoA to the cytoplasm, where it may complex with Rho-GDIs and also inhibits its ability to bind to ROK.
      However, the relationship between Rac1 and Rho is more complex and likely depends on the cell type. In contrast to the Cdc42 → Rac1 → Rho hierarchical cascade described in fibroblasts, no evidence for such a relationship has been found in neuronal cells. For example, in N1E-115 cells, PAK5 mediates signals from Cdc42 and Rac1 leading to the inactivation of RhoA (
      • Dan C.
      • Nath N.
      • Liberto M.
      • Minden A.
      ). Sander et al. (
      • Sander E.E.
      • ten Klooster J.P.
      • van Delft S.
      • van der Kammen R.A.
      • Collard J.G.
      ) showed in NIH3T3 cells that activation of Rac1 leads to the inhibition of RhoA, suggesting that Rac1 is upstream of RhoA. Yamaguchi et al.(
      • Yamaguchi Y.
      • Katoh H.
      • Yasui H.
      • Mori K.
      • Negishi M.
      ) reported recently that RhoA suppressed NGF-induced Rac1 activation through the Rho-associated kinase pathway in PC12 cells, suggesting that Rho acts upstream to Rac1. In their experiments, however, NGF treatment was performed following an 18-h serum starvation and growth factor deprivation of PC12 cells, suggesting that this effect is more pertinent to an anti-apoptotic rescue effect of NGF rather than that of inducing neuronal differentiation. Under the conditions used in our assays, we found no inhibiton of NGF-induced Rac1 activation in response to NGF, suggesting that the prolonged serum starvation used by those authors may have affected the outcome of the assay. In our hands an 18-h-long serum withdrawal has adverse effect of the adherence of the cells, and cells are beginning to undergo apoptosis (data not shown).
      Taken together, our results in naive PC12 cells support a Ras-independent signal transduction pathway linking TrkA to Rac1 through PI3K, which mediates the inhibition of RhoA during the early stages of neuronal differentiation (Fig. 7). The signaling complexes involved in coupling these molecules is subject of ongoing studies.

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