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DCC-dependent Phospholipase C Signaling in Netrin-1-induced Neurite Elongation

2005; Elsevier BV; Volume: 281; Issue: 5 Linguagem: Inglês

10.1074/jbc.m512767200

1083-351X

Yi Xie, Hong Yan, Xiao-Yue Ma, Xiu-Rong Ren, Susan L. Ackerman, Lin Mei, Wen‐Cheng Xiong,

Hippo pathway signaling and YAP/TAZ

Netrins, a family of secreted molecules, play important roles in axon pathfinding during nervous system development. Although phosphatidylinositol signaling has been implicated in this event, how netrin-1 regulates phosphatidylinositol signaling remains poorly understood. Here we provide evidence that netrin-1 stimulates phosphatidylinositol bisphosphate hydrolysis in cortical neurons. This event appears to be mediated by DCC (deleted in colorectal cancer), but not neogenin or Unc5h2. Netrin-1 induces phospholipase Cγ (PLCγ) tyrosine phosphorylation. Inhibition of PLC activity attenuates netrin-1-induced cortical neurite outgrowth. These results suggest that netrin-1 regulates phosphatidylinositol turnover and demonstrate a crucial role of PLC signaling in netrin-1-induced neurite elongation. Netrins, a family of secreted molecules, play important roles in axon pathfinding during nervous system development. Although phosphatidylinositol signaling has been implicated in this event, how netrin-1 regulates phosphatidylinositol signaling remains poorly understood. Here we provide evidence that netrin-1 stimulates phosphatidylinositol bisphosphate hydrolysis in cortical neurons. This event appears to be mediated by DCC (deleted in colorectal cancer), but not neogenin or Unc5h2. Netrin-1 induces phospholipase Cγ (PLCγ) tyrosine phosphorylation. Inhibition of PLC activity attenuates netrin-1-induced cortical neurite outgrowth. These results suggest that netrin-1 regulates phosphatidylinositol turnover and demonstrate a crucial role of PLC signaling in netrin-1-induced neurite elongation. Proper wiring in developing brains requires neurite outgrowth and growth cone navigation. Netrins, a family of secreted factors, promote axon outgrowth (1Livesey F.J. Cell. Mol. Life Sci. 1999; 56: 62-68Crossref PubMed Scopus (61) Google Scholar, 2Colavita A. Culotti J.G. Dev. 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Cell. 1994; 78: 409-424Abstract Full Text PDF PubMed Scopus (1136) Google Scholar). Netrins act through two classes of receptors: DCC and Unc5. The DCC family includes DCC and neogenin in vertebrates (5Keino-Masu K. Masu M. Hinck L. Leonardo E.D. Chan S.S. Culotti J.G. Tessier-Lavigne M. Cell. 1996; 87: 175-185Abstract Full Text Full Text PDF PubMed Scopus (863) Google Scholar), Unc40 in Caenorhabditis elegans (6Chan S.S. Zheng H. Su M.W. Wilk R. Killeen M.T. Hedgecock E.M. Culotti J.G. Cell. 1996; 87: 187-195Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar), and Frazzled in Drosophila (7Kolodziej P.A. Timpe L.C. Mitchell K.J. Fried S.R. Goodman C.S. Jan L.Y. Jan Y.N. Cell. 1996; 87: 197-204Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, 8Hiramoto M. Hiromi Y. Giniger E. Hotta Y. Nature. 2000; 406: 886-889Crossref PubMed Scopus (100) Google Scholar). DCC is required for the attractive response (9Culotti J.G. Merz D.C. Curr. Opin. Cell Biol. 1998; 10: 609-613Crossref PubMed Scopus (118) Google Scholar). Unc5 in C. elegans and Unc5A, -5B, and -5C in vertebrates belong to the Unc5 family, which appears to mediate the repulsive response (10Hedgecock E.M. Culotti J.G. Hall D.H. Neuron. 1990; 4: 61-85Abstract Full Text PDF PubMed Scopus (714) Google Scholar, 11Hong K. Hinck L. Nishiyama M. Poo M.M. Tessier-Lavigne M. Stein E. Cell. 1999; 97: 927-941Abstract Full Text Full Text PDF PubMed Scopus (567) Google Scholar, 12Keleman K. Dickson B.J. Neuron. 2001; 32: 605-617Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 13Ackerman S.L. Kozak L.P. Przyborski S.A. Rund L.A. Boyer B.B. Knowles B.B. Nature. 1997; 386: 838-842Crossref PubMed Scopus (311) Google Scholar, 14Leonardo E.D. Hinck L. Masu M. Keino-Masu K. Ackerman S.L. Tessier-Lavigne M. Nature. 1997; 386: 833-838Crossref PubMed Scopus (420) Google Scholar). DCC and Unc5 proteins are transmembrane proteins without any obvious catalytic activity, and thus it remains unknown exactly how they initiate downstream signaling to mediate or regulate axonal outgrowth and guidance. Nevertheless, perturbation of Rho family GTPases inhibits netrin-induced neurite outgrowth (15Li X. Saint-Cyr-Proulx E. Aktories K. Lamarche-Vane N. J. Biol. Chem. 2002; 277: 15207-15214Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Pharmacological inhibition of extracellular signal-regulated kinase (ERK) attenuates netrin-1-induced neurite outgrowth and growth cone turning (16Forcet C. Stein E. Pays L. Corset V. Llambi F. Tessier-Lavigne M. Mehlen P. Nature. 2002; 417: 443-447Crossref PubMed Scopus (203) Google Scholar, 17Ming G.L. Wong S.T. Henley J. Yuan X.B. Song H.J. Spitzer N.C. Poo M.M. Nature. 2002; 417: 411-418Crossref PubMed Scopus (348) Google Scholar). Inhibition of focal adhesion kinase (FAK), 3The abbreviations used are: FAK, focal adhesion kinase; PIP2, phosphatidylinositol bisphosphate; IP3, inositol 1,4,5-trisphosphate; BDNF, brain-derived neurotrophic factor; PLC, phospholipase C; E, embryonic day; NFAT, nuclear factor of activated T cells; SAM, sterile α motif. 3The abbreviations used are: FAK, focal adhesion kinase; PIP2, phosphatidylinositol bisphosphate; IP3, inositol 1,4,5-trisphosphate; BDNF, brain-derived neurotrophic factor; PLC, phospholipase C; E, embryonic day; NFAT, nuclear factor of activated T cells; SAM, sterile α motif. a major tyrosine kinase localized in focal adhesions and implicated in cell spreading and migration, blocks netrin-1-induced neurite elongation and growth cone guidance (18Ren X.R. Ming G.L. Xie Y. Hong Y. Sun D.M. Zhao Z.Q. Feng Z. Wang Q. Shim S. Chen Z.F. Song H.J. Mei L. Xiong W.C. Nat. Neurosci. 2004; 7: 1204-1212Crossref PubMed Scopus (188) Google Scholar, 19Li W. Lee J. Vikis H.G. Lee S.H. Liu G. Aurandt J. Shen T.L. Fearon E.R. Guan J.L. Han M. Rao Y. Hong K. Guan K.L. Nat. Neurosci. 2004; 7: 1213-1221Crossref PubMed Scopus (185) Google Scholar, 20Liu G. Beggs H. Jurgensen C. Park H.T. Tang H. Gorski J. Jones K.R. Reichardt L.F. Wu J. Rao Y. Nat. Neurosci. 2004; 7: 1222-1232Crossref PubMed Scopus (215) Google Scholar). Treatment of wortmannin, an inhibitor of phosphatidylinositol 3-kinase, attenuates netrin-1-induced growth cone turning in Xenopus spinal neurons (27Ming G. Song H. Berninger B. Inagaki N. Tessier-Lavigne M. Poo M. Neuron. 1999; 23: 139-148Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Phosphoinositides are quantitatively minor phospholipids of cell membranes, but their metabolism is highly active and tightly regulated. They (e.g. PIP2) function either as precursors of second messengers such as inositol 1,4,5-trisphosphate (IP3) and diacylglycerol or by directly interacting with both actin-binding and pleckstrin homology domain-containing proteins to regulate their spatiotemporal distribution and/or activity. In addition, PIP2 functions as a cofactor for small GTP-binding proteins (e.g. Arf) and phospholipase D (21Hilpela P. Vartiainen M.K. Lappalainen P. Curr. Top. Microbiol. Immunol. 2004; 282: 117-163PubMed Google Scholar, 22Yin H.L. Janmey P.A. Annu. Rev. Physiol. 2003; 65: 761-789Crossref PubMed Scopus (560) Google Scholar). In the nervous system, PIP2 plays an important role in membrane trafficking at the synapse. Synaptic vesicle exocytosis and endocytosis require phosphatidylinositol 4,5-bisphosphate (23De Camilli P. Takei K. Neuron. 1996; 16: 481-486Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 24Wenk M.R. De Camilli P. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 8262-8269Crossref PubMed Scopus (254) Google Scholar, 25Cremona O. De Camilli P. J. Cell Sci. 2001; 114: 1041-1052Crossref PubMed Google Scholar, 26Paolo G.D. Moskowitz H.S. Gipson K. Wenk M.R. Voronov S. Obayashi M. Flavell R. Fitzsimonds R.M. Ryan T.A. Camilli P.D. Nature. 2004; 431: 415-422Crossref PubMed Scopus (296) Google Scholar). Using Xenopus growth cone-turning assays, Poo and colleagues (27Ming G. Song H. Berninger B. Inagaki N. Tessier-Lavigne M. Poo M. Neuron. 1999; 23: 139-148Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar) have demonstrated that perturbation of phospholipase Cγ (PLCγ) activity by pretreatment with brain-derived neurotrophic factor (BDNF) attenuates netrin-1-induced neurite outgrowth. Although PLCγ is implicated in netrin-1-induced growth cone guidance, exactly how netrin-1 regulates this signaling event and its role in netrin-1-induced neurite outgrowth remain largely unknown. In this paper, we have characterized the potential role of PIP2 hydrolysis in netrin-1 signaling. We show that netrin-1 stimulates PIP2 hydrolysis in cultured cortical neurons. This event appears to be mediated by DCC. In addition, netrin-1 increases tyrosine phosphorylation of PLCγ, a time course similar to that of PIP2 hydrolysis induced by netrin-1. Furthermore, inhibition of PLC activity by U73122 attenuated netrin-1-induced neurite elongation. These results demonstrate a crucial role of PLC signaling in netrin-1 induced neurite outgrowth. Reagents and Animals—Monoclonal antibodies were purchased form the following sources: anti-FLAG from Sigma, anti-Myc (9e10) from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-PLCβ-1 and anti-PLCγ from Upstate Biotechnology Inc. (Lake Placid, NY). Goat polyclonal antibodies (anti-DCC and anti-PLCγ-PY783) were purchased from Santa Cruz. Rabbit polyclonal anti-PY861 and anti-PY397 antibodies were from Biosource International, Inc. Rabbit polyclonal anti-neogenin was generated using glutathione S-transferase-neogenin (amino acids 1158–1527) as an antigen. Stable HEK293 cells expressing human netrin-1 and Slit-2 were provided by J. Y. Wu and Y. Rao (Washington University, St. Louis, MO) (28Li H.S. Chen J.H. Wu W. Fagaly T. Zhou L. Yuan W. Dupuis S. Jiang Z.H. Nash W. Gick C. Ornitz D.M. Wu J.Y. Rao Y. Cell. 1999; 96: 807-818Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). Unless otherwise indicated, ∼200 ng/ml human netrin-1 was used for stimulation. U73122 was obtained from Biomol Research Laboratories, Inc. (Plymouth, PA). DCC null mutant mice were maintained in B6 background, and DCCkanga mice were maintained in C. AKR background. Genotyping of both DCCkanga and DCC null mutant mice was performed by PCR as described previously (18Ren X.R. Ming G.L. Xie Y. Hong Y. Sun D.M. Zhao Z.Q. Feng Z. Wang Q. Shim S. Chen Z.F. Song H.J. Mei L. Xiong W.C. Nat. Neurosci. 2004; 7: 1204-1212Crossref PubMed Scopus (188) Google Scholar). Expression Vectors—The cDNAs encoding neogenin, DCC, or DCC mutants were amplified by PCR and subcloned into mammalian expression vectors downstream of a signal peptide and a FLAG epitope tag (MDYKDDDDKGP) or Myc epitope tag under the control of the cytomegalovirus promoter (18Ren X.R. Ming G.L. Xie Y. Hong Y. Sun D.M. Zhao Z.Q. Feng Z. Wang Q. Shim S. Chen Z.F. Song H.J. Mei L. Xiong W.C. Nat. Neurosci. 2004; 7: 1204-1212Crossref PubMed Scopus (188) Google Scholar). Deletion mutations in DCC were generated using the QuikChange kit (Strategene). DCC/SAM is a chimeric protein of DCC with the SAM domain of the EphB1 receptor substitution of the DCC-P3 domain, which was constructed as described (29Stein E. Zou Y. Poo M. Tessier-Lavigne M. Science. 2001; 291: 1976-1982Crossref PubMed Scopus (198) Google Scholar). The authenticity of all constructs was verified by DNA sequencing. HEK293 Cell Culture and Transfection—HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 100 units/ml penicillin G and streptomycin (Invitrogen). Cells were plated at a density of 106 cells/10-cm culture dish and allowed to grow for 12 h before transfection using the calcium phosphate precipitation method (30Xiong W. Parsons J.T. J. Cell Biol. 1997; 139: 529-539Crossref PubMed Scopus (150) Google Scholar). Thirty-six hours after transfection, cells were lysed in modified radioimmune precipitation buffer (50 mm Tris-HCl, pH 7.4, 150 mm sodium chloride, 1% Nonidet P-40, 0.25% sodium deoxycholate, and proteinase inhibitors) (31Ren X.R. Du Q.S. Huang Y.Z. Ao S.Z. Mei L. Xiong W.C. J. Cell Biol. 2001; 152: 971-984Crossref PubMed Scopus (99) Google Scholar). Lysates were subjected to immunoprecipitation or immunoblotting. Cortical Neuronal Culture—Primary cortical neurons were cultured as described previously (18Ren X.R. Ming G.L. Xie Y. Hong Y. Sun D.M. Zhao Z.Q. Feng Z. Wang Q. Shim S. Chen Z.F. Song H.J. Mei L. Xiong W.C. Nat. Neurosci. 2004; 7: 1204-1212Crossref PubMed Scopus (188) Google Scholar). Briefly, embryos (E17) were removed from anesthetized pregnant Sprague-Dawley rats or mice. Cerebral cortexes were dissected out and chopped into small pieces after meninges were completely removed. After incubation in phosphate-buffered saline solution containing 0.125% (w/v) trypsin (Sigma) for 20 min at 37 °C, digested tissues were mechanically triturated by repeated passages through a Pasteur pipette in phosphate-buffered saline solution containing 0.05% (w/v) DNase (Sigma). Dissociated cells were suspended in the neurobasal medium with B-27 supplement (Invitrogen) and 100 units/ml penicillin/streptomycin and were plated on poly-d-lysine-coated dishes (Corning). After a 2-day incubation at 37 °C in a 5% CO2 atmosphere, 10 μm cytosine arabinoside was added to inhibit the proliferation of glial cells. PIP2 Hydrolysis Assay—HEK293 cells, neurons, or mouse embryonic fibroblasts were incubated in inositol-free Dulbecco's modified Eagle's medium containing 5 μCi/ml myo-2-[3H]inositol (1 μCi/μl, Amersham Biosciences), and 10% dialyzed fetal calf serum for 20 h. Cells were washed with phosphate-buffered saline and incubated for an additional hour in the same medium lacking isotope. 20 mm LiCl was added to the culture, and incubation continued for another 25 min. [3H]inositol-incorporated cells were stimulated with netrin-1 for different times. Inositol phosphates were extracted in 10 mm formic acid and purified by ion-exchange chromatography using AG1-X8 resin as described previously (32Hara S. Swigart P. Jones D. Cockcroft S. J. Biol. Chem. 1997; 272: 14908-14913Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Each experiment was repeated at least three times. Explant Cultures—Explant assays were carried out as described previously (33Metin C. Deleglise D. Serafini T. Kennedy T.E. Tessier-Lavigne M. Development. 1997; 124: 5063-5074PubMed Google Scholar, 34Richards L.J. Koester S.E. Tuttle R. O'Leary D.D. J. Neurosci. 1997; 17: 2445-2458Crossref PubMed Google Scholar, 35Finger J.H. Bronson R.T. Harris B. Johnson K. Przyborski S.A. Ackerman S.L. J. Neurosci. 2002; 22: 10346-10356Crossref PubMed Google Scholar). Embryos were dissected from the uterine horn of anesthetized pregnant mice. For cortical explants, telencephalic vesicles of E15 embryos were dissected out in L-15 medium (Invitrogen), and the pia mater was removed. The dorsolateral cortex was cut with thin tungsten needles into ∼200 × 200-μm pieces that spanned the full thickness of the cortical wall. Explants were embedded in a three-dimensional collagen (Roche Diagnostics) matrix with the ventricular side up. After polymerization, gels were incubated with Ham's F-12 medium supplemented with 5% heat-inactivated horse serum (Invitrogen) and 100 units/ml penicillin/streptomycin at 37 °C in a 5% CO2 atmosphere. Neurite outgrowth was analyzed after 19–43 h in culture. Total neurite length for each explant was obtained by adding the lengths of all neurites from each explant (regardless of bundle thickness). Each experiment was repeated three times, and six explants for each treatment were analyzed (n = 2 × 3 = 6). Statistical Analysis—Student's t test was used to determine significance of effects. Data are presented as the mean of all independent replicates ± S.E. Netrin-1 Stimulation of PLC Signaling in a Time-dependent Manner—To address whether netrin-1 regulates PLC signaling, we measured PLC-mediated PIP2 hydrolysis in cultured rat cortical neurons in response to netrin-1. Rat cortical neurons (E17, 3 days in vitro) were labeled with [3H]inositol, stimulated with or without netrin-1, and assayed for the production of inositol phosphates, an indicator of PIP2 hydrolysis. BDNF, a growth factor that stimulates PLC signaling, was used as a positive control. As shown in Fig. 1A, netrin-1 as well as BDNF stimulated PIP2 hydrolysis, supporting the notion of conserved signaling pathways of netrin-1 and BDNF (27Ming G. Song H. Berninger B. Inagaki N. Tessier-Lavigne M. Poo M. Neuron. 1999; 23: 139-148Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, 36Graef I.A. Wang F. Charron F. Chen L. Neilson J. Tessier-Lavigne M. Crabtree G.R. Cell. 2003; 113: 657-670Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). Interestingly, Slit-2, a repulsive cue that negatively regulates neurite outgrowth (37Brose K. Tessier-Lavigne M. Curr. Opin. Neurobiol. 2000; 10: 95-102Crossref PubMed Scopus (302) Google Scholar), failed to stimulate this activity (Fig. 1A). In addition, netrin-1 induction of PIP2 hydrolysis appeared to be time-dependent, with a maximal activity observed within 15 min of netrin-1 stimulation (Fig. 1B). These results indicate that netrin-1 stimulates PLC signaling in neurons in a time-dependent manner. Dependence of Netrin-1 Stimulation of PLC Signaling on DCC—We next investigated the role of netrin-1 receptors in netrin-1 stimulation of PIP2 hydrolysis. To this end, we reconstituted netrin-1 regulation of PIP2 hydrolysis in HEK293 cells, which do not express endogenous DCC (data not shown). Netrin-1 stimulation did not elicit PIP2 hydrolysis in control HEK293 cells (Fig. 2A, lanes 1 and 2). However, transfection with DCC rendered HEK293 cells able to respond to netrin-1 with an increase in PIP2 hydrolysis (Fig. 2A, lanes 3 and 4), implying dependence on the DCC protein. Remarkably, netrin-1 stimulation did not induce, or elicited very weakly if at all, PIP2 hydrolysis in HEK293 cells transfected with Unc5h2 or with neogenin, a DCC-related receptor (Fig. 2A, lanes 5–8). These results suggest that DCC, but not neogenin or Unc5h2, may mediate netrin-1-induced PLC signaling. To determine the role of DCC in vivo in netrin-1-induced PLC signaling, we turned to neurons isolated from DCC knock-out mice (Fig. 2B). Used as control were neurons from wild type or heterozygote mutant mice (Fig. 2B). As observed with rat cortical neurons, cortical neurons from DCC wild type or heterozygote mice responded to netrin-1 with an increase in PIP2 hydrolysis (Fig. 2B, lanes 1–4). However, the induction of PIP2 hydrolysis was completely eliminated in neurons from DCC homozygote mice (Fig. 2B, lanes 5 and 6), demonstrating a necessity for DCC in netrin-1 activation of PLC signaling in neurons. Note that neogenin was expressed at a similar level in neurons from DCC-/- mice as compared with wild type neurons (Fig. 2B), consistent with the notion that DCC, but not neogenin, mediates netrin-1 induction of PIP2 hydrolysis in neurons. Requirement of the DCC-P3 Domain for Netrin-1-induced PLC Signaling—The cytoplasmic region of DCC contains no catalytic domain but has three conserved regions: P1, P2, and P3. DCC interacts via the P domains with other receptors to form homo- or heterodimers important for netrin-1 function (9Culotti J.G. Merz D.C. Curr. Opin. Cell Biol. 1998; 10: 609-613Crossref PubMed Scopus (118) Google Scholar, 29Stein E. Zou Y. Poo M. Tessier-Lavigne M. Science. 2001; 291: 1976-1982Crossref PubMed Scopus (198) Google Scholar, 38Kennedy T.E. Biochem. Cell Biol. 2000; 78: 569-575Crossref PubMed Scopus (78) Google Scholar, 39Stein E. Tessier-Lavigne M. Science. 2001; 291: 1928-1938Crossref PubMed Scopus (476) Google Scholar). To identify which domain is necessary for netrin-1-induced PIP2 hydrolysis, we compared the effects of DCC with DCCΔP3, a P3 deletion mutant, because P3 domain is required for netrin-1-stimulated neurite outgrowth and growth cone turning in Xenopus spinal neurons (29Stein E. Zou Y. Poo M. Tessier-Lavigne M. Science. 2001; 291: 1976-1982Crossref PubMed Scopus (198) Google Scholar, 39Stein E. Tessier-Lavigne M. Science. 2001; 291: 1928-1938Crossref PubMed Scopus (476) Google Scholar). Interestingly, expression of this mutant in HEK293 cells blocked netrin-1 stimulation of PLC signaling, whereas expression of wild type DCC showed a significant response to netrin-1 (Fig. 3A, lanes 3–6). These results suggest a role of the DCC-P3 domain in netrin-1-induced PLC signaling. We next determined the role of DCC-P3 domain in vivo in netrin-1 induction of PIP2 hydrolysis by taking advantage of DCCkanga mice. These mice have a spontaneous deletion of exon 29 that encodes the DCC-P3 domain and show abnormal projection of corticospinal tract fibers to the spinal cord (35Finger J.H. Bronson R.T. Harris B. Johnson K. Przyborski S.A. Ackerman S.L. J. Neurosci. 2002; 22: 10346-10356Crossref PubMed Google Scholar). Unlike mice homozygous for the targeted allele of DCC, which die perinatally (40Fazeli A. Dickinson S.L. Hermiston M.L. Tighe R.V. Steen R.G. Small C.G. Stoeckli E.T. Keino-Masu K. Masu M. Rayburn H. Simons J. Bronson R.T. Gordon J.I. Tessier-Lavigne M. Weinberg R.A. Nature. 1997; 386: 796-804Crossref PubMed Scopus (663) Google Scholar), DCCkanga homozygotes have a relatively normal life span (35Finger J.H. Bronson R.T. Harris B. Johnson K. Przyborski S.A. Ackerman S.L. J. Neurosci. 2002; 22: 10346-10356Crossref PubMed Google Scholar). We reasoned that netrin-1-induced PIP2 hydrolysis may be impaired in DCCkanga mice if the DCC-P3 domain plays an important role in this event. Neurons were isolated from DCCkanga mutant and wild type embryos. Wild type mouse cortical neurons responded to netrin-1 with an increase in PIP2 hydrolysis, as did DCCkanga heterozygous neurons (Fig. 3B, lanes 1–4). In contrast, the induction of PIP2 hydrolysis was significantly attenuated in neurons from homozygous embryos (Fig. 3B, lanes 5 and 6). These results suggest a crucial role of the DCC-P3 domain in PIP2 hydrolysis by netrin-1. Independence of DCC-P3 Homodimerization or FAK for Netrin-1-induced PLC Signaling—DCC-P3 domain not only binds to itself to mediate DCC homodimerization but also interacts with FAK (18Ren X.R. Ming G.L. Xie Y. Hong Y. Sun D.M. Zhao Z.Q. Feng Z. Wang Q. Shim S. Chen Z.F. Song H.J. Mei L. Xiong W.C. Nat. Neurosci. 2004; 7: 1204-1212Crossref PubMed Scopus (188) Google Scholar, 29Stein E. Zou Y. Poo M. Tessier-Lavigne M. Science. 2001; 291: 1976-1982Crossref PubMed Scopus (198) Google Scholar). Both events are required for netrin-1 functions (18Ren X.R. Ming G.L. Xie Y. Hong Y. Sun D.M. Zhao Z.Q. Feng Z. Wang Q. Shim S. Chen Z.F. Song H.J. Mei L. Xiong W.C. Nat. Neurosci. 2004; 7: 1204-1212Crossref PubMed Scopus (188) Google Scholar, 29Stein E. Zou Y. Poo M. Tessier-Lavigne M. Science. 2001; 291: 1976-1982Crossref PubMed Scopus (198) Google Scholar). We thus examined the effects of DCC dimerization and FAK on netrin-1-induced PIP2 hydrolysis. To test whether self-association of P3 contributes to netrin-1-induced PIP2 hydrolysis, we constructed DCC/SAM, a chimeric protein of DCC with the SAM domain of the EphB1 receptor substituted for the P3 domain. This SAM domain has been demonstrated to mediate self-association of the DCC cytoplasmic domain when it replaces the P3 domain and can substitute fully for P3 in mediating the ligand-regulated chemoattractant function (29Stein E. Zou Y. Poo M. Tessier-Lavigne M. Science. 2001; 291: 1976-1982Crossref PubMed Scopus (198) Google Scholar). Note that transfection of DCC/SAM blocked netrin-1 response in PIP2 hydrolysis (Fig. 3A, lanes 7 and 8, and Fig. 3C), suggesting a slight role of DCC-P3 domain-mediated dimerization in this event. We next examined the role of FAK in netrin-1 induction of PIP2 hydrolysis, as FAK is a P3 binding partner (18Ren X.R. Ming G.L. Xie Y. Hong Y. Sun D.M. Zhao Z.Q. Feng Z. Wang Q. Shim S. Chen Z.F. Song H.J. Mei L. Xiong W.C. Nat. Neurosci. 2004; 7: 1204-1212Crossref PubMed Scopus (188) Google Scholar). To this end, FAK-/- fibroblasts transfected with DCC were used. As shown in Fig. 3D, netrin-1 was able to elicit PIP2 hydrolysis in both fak+/+ and fak+/- fibroblasts expressing DCC, suggesting a slight role of FAK in this event. In aggregate, our results suggest that neither P3 domain-mediated dimerization nor P3-FAK binding appeared to be required for netrin-1 induction of PLC signaling, thus implicating an involvement of other P3-dependent-binding proteins in this event. DCC has been found to be phosphorylated at tyrosine 1420 (Tyr1420), a site close to the P3 region and implicated in netrin-1 signaling (19Li W. Lee J. Vikis H.G. Lee S.H. Liu G. Aurandt J. Shen T.L. Fearon E.R. Guan J.L. Han M. Rao Y. Hong K. Guan K.L. Nat. Neurosci. 2004; 7: 1213-1221Crossref PubMed Scopus (185) Google Scholar, 41Meriane M. Tcherkezian J. Webber C.A. Danek E.I. Triki I. McFarlane S. Bloch-Gallego E. Lamarche-Vane N. J. Cell Biol. 2004; 167: 687-698Crossref PubMed Scopus (93) Google Scholar). We thus examined whether DCC-Tyr1420 phosphorylation contributes to netrin-1-induced PIP2 hydrolysis. To this end, the DCC-Y1420F mutant was expressed into HEK293 cells, and netrin-1-induced PIP2 hydrolysis was examined. The netrin-1 response in PIP2 hydrolysis was reduced, but not abolished, in cells expressing DCC-Y1420F mutant (Fig. 3A, lanes 9 and 10). Note that the basal activity appeared to be increased in cells expressing the Y1420F mutant (Fig. 3A, lanes 9). These results suggest a potential role of DCC Tyr1420 phosphorylation in netrin-1-induced PIP2 hydrolysis. Netrin-1 Induction of Tyrosine Phosphorylation of PLCγ in Rat Cortical Neurons—PLC, a large family of proteins including PLCβ and PLCγ, is a major enzyme that catalyzes the hydrolysis of PIP2 into IP3 and diacylglycerol. To understand further how netrin-1 induces PIP2 hydrolysis, we examined whether netrin-1 activates PLC. Both PLCγ and -β are highly expressed in embryonic rat brain (Fig. 4A). We examined whether netrin-1 stimulates PLCγ by increasing its tyrosine phosphorylation. As shown in Fig. 4B, netrin-1 rapidly induced tyrosine phosphorylation of PLCγ within 5 min in rat cortical neurons, peaking at 30 min and gradually returning to basal levels after 60 min, a transient time course similar to that of netrin-1-induced PIP2 hydrolysis. Note that this time course appeared to be slightly different from that of netrin-1-induced FAK tyrosine phosphorylation (Fig. 4B), which peaked earlier, at 15 min, and lasted longer (18Ren X.R. Ming G.L. Xie Y. Hong Y. Sun D.M. Zhao Z.Q. Feng Z. Wang Q. Shim S. Chen Z.F. Song H.J. Mei L. Xiong W.C. Nat. Neurosci. 2004; 7: 1204-1212Crossref PubMed Scopus (188) Google Scholar). In addition, netrin-1 also increased the phosphorylation of Akt (Fig. 4B), a downstream kinase of the phosphatidylinositol 3-kinase pathway that is implicated in netrin-1-induced growth cone turning (27Ming G. Song H. Berninger B. Inagaki N. Tessier-Lavigne M. Poo M. Neuron. 1999; 23: 139-148Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar), with a different time course than that of netrin-1 induction of PLCγ phosphorylation. These results suggest that tyrosine phosphorylation of PLCγ by netrin-1 may not be FAK-dependent, which is in line with the observation that FAK is not required for netrin-1-induced PIP2 hydrolysis. Requirement of PLC Signaling for Netrin-1-induced Cortical Neurite Elongation—To explore the involvement of PLC signal transduction in mediating netrin-1 functions, we tested whether inhibition of PLC affects netrin-1-stimulated neurite outgrowth in rat embryonic cortical explants. In the absence of netrin-1, neurites are few and short (Fig. 5A). A significant increase in neurite outgrowth (both in number and length) was observed when explants were stimulated with netrin-1 (Fig. 5, B and I) (33Metin C. Deleglise D. Serafini T. Kennedy T.E. Tessier-Lavigne M. Development. 1997; 124: 5063-5074PubMed Google Scholar, 34Richards L.J. Koester S.E. Tuttle R. O'Leary D.D. J. Neurosci. 1997; 17: 2445-2458Crossref PubMed Google Scholar). Importantly, this event was inhibited by a PLC-specific inhibitor, U73122, but not by STI571, an inhibitor for Abl tyrosine kinase (Fig. 5, C–H). Of note is that the inhibitory effect by U73122 appeared to be dose-dependent. Whereas a weak effect was observed at a concentration of 10 nm, treatment with 500 nm U73122 showed significant inhibition (Fig. 5, C–F and I). In addition, this inhibitory effect appeared to be specific and was not associated with necrosis of the explants (Fig. 5, A, C, and E). Furthermore, U73122 had no effect on netrin-1-independent neurite outgrowth (data not shown), suggesting that the observed inhibitory effect by U73122 does not result from a general inhibition of neurite outgrowth but rather from specific inhibition of netrin-1-induced neurite elongation. Taken together, these results support the notion that PIP2 hydrolysis may be involved in netrin-1-stimulated cortical neurite outgrowth. Netrin-1 induced PIP2 hydrolysis appears to be an important mechanism for netrin-1-induced neurite elongation. This notion is supported by the following observations. First, PIP2 hydrolysis is increased in neurons challenged with netrin-1. Netrin-1 induced PIP2 hydrolysis occurs rapidly after stimulation, apparently prior to changes in neurite outgrowth. Second, netrin-1 stimulates PLCγ in a time-dependent manner. Third, treatment of neurons with U73122, an inhibitor of PLCs, attenuates netrin-1-stimulated neurite outgrowth. Fourth, PLCγ signaling has been implicated in nerve growth factor- and netrin-1-induced growth cone guidance of Xenopus spinal neurons (27Ming G. Song H. Berninger B. Inagaki N. Tessier-Lavigne M. Poo M. Neuron. 1999; 23: 139-148Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Finally, it has been demonstrated that IP3 receptor-mediated calcium release from intracellular stores plays an important role in the axonal extension of chick dorsal root ganglia (42Takei K. Shin R.M. Inoue T. Kato K. Mikoshiba K. Science. 1998; 282: 1705-1708Crossref PubMed Scopus (159) Google Scholar). Netrin-1 acts through two families of receptors: DCCs and UNC-5s. The DCC family includes DCC and neogenin in vertebrates (5Keino-Masu K. Masu M. Hinck L. Leonardo E.D. Chan S.S. Culotti J.G. Tessier-Lavigne M. Cell. 1996; 87: 175-185Abstract Full Text Full Text PDF PubMed Scopus (863) Google Scholar). Although DCC is required for growth cone attraction by netrins (9Culotti J.G. Merz D.C. Curr. Opin. Cell Biol. 1998; 10: 609-613Crossref PubMed Scopus (118) Google Scholar), the role of neogenin in netrin-1 signaling remains unclear. Interestingly, recent observations indicate that neogenin, but not DCC, appears to be a receptor for RGM (repulsive guidance molecule), which repels retinal axons (43Rajagopalan 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 (218) Google Scholar). Our data have indicated a role of DCC, but not neogenin or Unc5h2, in netrin-1-induced PIP2 hydrolysis, supporting the notion that different functions could be mediated by DCC and neogenin. DCC, an Ig family receptor, contains a cytoplasmic region with three conserved domains, namely P1, P2, and P3 (7Kolodziej P.A. Timpe L.C. Mitchell K.J. Fried S.R. Goodman C.S. Jan L.Y. Jan Y.N. Cell. 1996; 87: 197-204Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). The P domains are important for DCC functions. For example, P3 is both necessary and sufficient for binding to another molecule of DCC to form a homodimer (29Stein E. Zou Y. Poo M. Tessier-Lavigne M. Science. 2001; 291: 1976-1982Crossref PubMed Scopus (198) Google Scholar, 39Stein E. Tessier-Lavigne M. Science. 2001; 291: 1928-1938Crossref PubMed Scopus (476) Google Scholar). The homodimerization appears to be necessary for neurite outgrowth and attractive turning in Xenopus spinal neurons (29Stein E. Zou Y. Poo M. Tessier-Lavigne M. Science. 2001; 291: 1976-1982Crossref PubMed Scopus (198) Google Scholar, 39Stein E. Tessier-Lavigne M. Science. 2001; 291: 1928-1938Crossref PubMed Scopus (476) Google Scholar). In addition, P3 interacts with Robo1, a receptor of the repulsive cue, Slit (39Stein E. Tessier-Lavigne M. Science. 2001; 291: 1928-1938Crossref PubMed Scopus (476) Google Scholar). This interaction is believed to be critical for Slit-2-induced silencing of netrin-1 attraction (39Stein E. Tessier-Lavigne M. Science. 2001; 291: 1928-1938Crossref PubMed Scopus (476) Google Scholar). Furthermore, P3 interacts with FAK and phosphatidylinositol transfer protein α, which are required for netrin-1-induced neurite outgrowth (18Ren X.R. Ming G.L. Xie Y. Hong Y. Sun D.M. Zhao Z.Q. Feng Z. Wang Q. Shim S. Chen Z.F. Song H.J. Mei L. Xiong W.C. Nat. Neurosci. 2004; 7: 1204-1212Crossref PubMed Scopus (188) Google Scholar, 19Li W. Lee J. Vikis H.G. Lee S.H. Liu G. Aurandt J. Shen T.L. Fearon E.R. Guan J.L. Han M. Rao Y. Hong K. Guan K.L. Nat. Neurosci. 2004; 7: 1213-1221Crossref PubMed Scopus (185) Google Scholar, 20Liu G. Beggs H. Jurgensen C. Park H.T. Tang H. Gorski J. Jones K.R. Reichardt L.F. Wu J. Rao Y. Nat. Neurosci. 2004; 7: 1222-1232Crossref PubMed Scopus (215) Google Scholar, 44Xie Y. Ding Y.Q. Hong Y. Feng Z. Navarre S. Xi C.X. Zhu X.J. Wang C.L. Ackerman S.L. Kozlowski D. Mei L. Xiong W.C. Nat. Cell Biol. 2005; 7: 1124-1132Crossref PubMed Scopus (79) Google Scholar). In this paper, we provide evidence for an important role of P3 in netrin-1-induced PLC signaling. This event appears to be independent of P3-mediated self-association or FAK interaction, consistent with our recent observation of an important role of PITPα interaction in this event (44Xie Y. Ding Y.Q. Hong Y. Feng Z. Navarre S. Xi C.X. Zhu X.J. Wang C.L. Ackerman S.L. Kozlowski D. Mei L. Xiong W.C. Nat. Cell Biol. 2005; 7: 1124-1132Crossref PubMed Scopus (79) Google Scholar). However, the data presented here support a role of DCC and PLCγ tyrosine phosphorylation in netrin-1-induced PIP2 hydrolysis and regulation of neurite outgrowth. This is suggested by a similar time course of induction of both DCC and PLCγ tyrosine phosphorylation and PIP2 hydrolysis upon netrin-1 stimulation, an attenuation of netrin-1-induced PIP2 hydrolysis in DCC-Y1420F-expressing cells, and an inhibition of neurite outgrowth by a PLC inhibitor. These observations are consistent with previous studies showing that DCC tyrosine phosphorylation contributes to netrin-1-induced neurite outgrowth (41Meriane M. Tcherkezian J. Webber C.A. Danek E.I. Triki I. McFarlane S. Bloch-Gallego E. Lamarche-Vane N. J. Cell Biol. 2004; 167: 687-698Crossref PubMed Scopus (93) Google Scholar), that PLCγ signaling is required in nerve growth factor- and netrin-1-induced growth cone guidance of Xenopus spinal neurons (27Ming G. Song H. Berninger B. Inagaki N. Tessier-Lavigne M. Poo M. Neuron. 1999; 23: 139-148Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar), and that IP3 receptor-mediated calcium release from intracellular stores plays an important role in axonal extension of chick dorsal root ganglia (42Takei K. Shin R.M. Inoue T. Kato K. Mikoshiba K. Science. 1998; 282: 1705-1708Crossref PubMed Scopus (159) Google Scholar). Our results that both netrin-1 and BDNF activate PLCγ and PIP2 hydrolysis provide additional evidence for conserved signaling pathways between netrins and neurotrophins. This notion is in line with the observation that perturbation of PLC signaling by pretreatment of BDNF blocks netrin-1-induced neurite turning in Xenopus spinal neurons (27Ming G. Song H. Berninger B. Inagaki N. Tessier-Lavigne M. Poo M. Neuron. 1999; 23: 139-148Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). In addition, this notion is supported by a recent demonstration that both neurotrophins and netrins stimulate calcineurin-dependent nuclear localization of NFATc4 and activation of NFAT-mediated gene transcription in cultured primary neurons and also that calcineurin-NFAT signaling is required for both neurotrophin- and netrin-1-induced neurite outgrowth (36Graef I.A. Wang F. Charron F. Chen L. Neilson J. Tessier-Lavigne M. Crabtree G.R. Cell. 2003; 113: 657-670Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). In light of these observations, we speculate that netrin-1, via DCC-PLCγ, regulates PIP2 hydrolysis, enhances IP3 production, elevates the intracellular calcium concentration, and stimulates calcineurin-NFAT signaling, which is important for netrin-1-induced neurite elongation. In addition, it is possible that PIP2-bound actin-binding and pleckstrin homology domain-containing signaling proteins may be regulated by netrin-1-induced PIP2 hydrolysis. This regulation may be in a time- and space-dependent manner that is crucial for neurite outgrowth and growth cone turning. However, whether netrin-1 stimulates neurite outgrowth through such a mechanism remains to be studied further.