Received: 6 November 2013 /Accepted: 8 December 2013# Springer Science+Business Media New York 2014
Abstract Resident progenitor cells expressing nerve/glial an-tigen 2 (NG2) such as oligodendrocyte precursor cells (OPC)and pericytes persist in the adult brain. The transmembraneproteoglycan NG2 regulates migration of both these cell typesin response to growth factors or specific components of theextracellular matrix. This role of NG2 is linked to the control ofcell polarity. The polarization of OPC toward an acute lesion inthe brain is impaired in NG2-deficient mice, supporting thisconcept. A review of the signaling pathways impinged on byNG2 reveals key proteins of cell polarity: phosphatidylinositol3-kinase, focal adhesion kinase, Rho GTPases, and polaritycomplex proteins. In the scope of cell migration, I discuss herehow the interplay of NG2 with signaling transmitted by extra-cellular cues can control the establishment of cell polarity, and Ipropose a model to integrate the apparent opposite effects ofNG2 on cellular dynamics.
The chondroitin sulfate proteoglycan (CSPG) CSPG4/AN2/NG2/MCSP, hence called NG2, is expressed in various pro-genitor cells: mesenchymal stem cells, perivascular cells suchas pericytes and smooth muscle cells (SMC), skeletal myo-blasts, chondroblasts, and oligodendrocyte precursor cells(OPC) [1β4]. Although difficult to observe during develop-ment [5], the consequences of NG2 deletion appear amplifiedin the adult brain in pathological conditions and are related to
the progenitor role of NG2-expressing cells. Absence of NG2in perivascular cells inhibits de novo angiogenesis and in-creases tumor vessel leakiness [6, 7]. OPC migrating to in-jured sites have a disturbed polarity toward a stab woundapplied in the cortex of NG2-deficient mice [8]. Indeed,NG2 is known to modulate OPC migration [9, 10] and itsexpression is also associated with invasiveness in melanomaand glioma tumors [11].
The proteoglycan NG2 is a transmembrane protein com-posed of a very large 2,225-amino acid extracellular domainand a short 76-amino acid cytoplasmic domain (in mouse, rat,and human) [12] (Fig. 1). The extracellular domain containing2 laminin G-type motifs and 15 potential CSPG motifsallowing anchorage of glycosaminoglycan (GAG) chains,among which only the site at serine 999 was shown to beutilized [13], plays a role in adhesion and growth factorsignaling as a coreceptor [14, 15]. Although dispensable forthe coreceptor function of NG2 [6], the cytoplasmic domain ofNG2 is required for its role in adhesion and migration, indi-cating that NG2 triggers directly intracellular signaling [16].In the cytoplasmic domain, distinct threonines have beenshown to be phosphorylated by different stimuli such asprotein kinase C Ξ± (PKCΞ±) and extracellular signal-regulated kinase (ERK) [17, 18] and the C-terminal PDZ-binding motif was found associated with the PDZ proteinsGRIP, syntenin, and MUPP1 [19, 20]. Seminal works havelinked NG2 to the control of the RhoGTPases Cdc42 and Rac[21, 22] which are important regulators of cell polarity andmigration. Moreover, it was reported that NG2 expressioncould induce polarization of a glioma cell line [13]. Thisimplication in polarity seems conserved through species sincethe NG2 homologue in Drosophila is required for directingmigration toward specific targets [23].
My recent work has increased the number of RhoGTPasesregulated by NG2 with the discovery of the constitutive stim-ulation of RhoA by NG2 in OPC and the deciphering of the
molecular actors of this signaling pathway [8]. By consideringits regulation of RhoGTPases, its role in adhesion, and itsinteraction with growth factors, NG2 appears as a centralregulator of cell polarity in the response to extracellular cues.Within the scope of migration, I will present here mechanismsregulated by NG2 and discuss how they can affect polarity ofNG2-expressing cells.
Migration, a Polarized Process
Cell migration can be summarized as a biphasic process. First,actin polymerization produces a protrusive force resulting inmembrane ruffling and cellular process outgrowth. Second,actin contraction retracts the cell body, resulting in the move-ment of the cell [24, 25]. Nevertheless, several prerequisitesare needed to obtain efficient migration in response to thesetwo cytoskeletal changes [26]. The interaction with the sub-stratum orientates the movement. The adhesion of the newlyformed cell process provides a new anchorage point that thecell will follow, but this is provided by the loss of anchorageon the opposite side of the cell during the contraction phase. Itfollows that the cell can move forward only if the front and therear of the cell respond differently and coordinately.
The front-rear polarity is regulated by proteins specificallyacting at one pole of the cell (Fig. 2). Phosphatidylinositol 3-kinase (PI3K) and its product, phosphatidylinositol triphos-phate (PIP3), are preferentially localized in the nascent protru-sion, while the phosphatase tensin homologue (PTEN) and itsproduct, phosphatidylinositol diphosphate (PIP2), are found atthe rear of the cell [27]. At the cytoplasmic surface of theplasma membrane, PIP3 recruits the Arp2/3/WASP complexregulating actin cytoskeleton branching and activates guanine-nucleotide exchange factors (GEF) that in turn activate RhoGTPases [24]. The Rho GTPases Rac and Cdc42 stimulateactin polymerization at the leading front, and behind RhoAstimulates actin contraction through Rho-associated kinases(ROCK) to pull the cell body forward. Among the important
Fig. 1 Structure of NG2. The N-terminal globular domain containing twolaminin G-type motifs is stabilized by disulfide bonds. The central rodlikeextracellular domain containing chondroitin sulfate proteoglycan (CSPG)repeats binds to collagens V and VI. The juxtamembrane globular domainpresenting linked oligosaccharides can be proteolytically cleaved resulting inectodomain release. The intracellular domain containing threonines phosphor-ylated by distinct stimuli presents a C-terminal PDZ-binding motif
Fig. 2 Directed migration. (a) Gradients of ECM components or growthfactors (GF) orientate migration toward higher concentrations of extra-cellular cues by activating PI3K at one pole of the cell. The cell becomespolarized with PI3K and its product PIP3 localized at the front pole whilePTEN and its product PIP2 are found at the rear of the cell. Structuralcomponents of the cell arrange along the front-rear axis: microtubuleorganizing center (MTOC) and Golgi apparatus localize between thenucleus and the front of the cell. (b) The Rho GTPase Rac stimulatingactin polymerization is activated at the leading front downstream of ECM
receptors and growth factor receptors (GFR). The polarity complex PARregulates directional migration by activating Rac at the front pole throughthe GEF Tiam1. (c) Localized RhoA activity at the edge of the extensionregulates initial events of membrane protrusion. RhoA regulates alsoactin contraction through ROCK and focal adhesion (FA) maturationthrough ROCK and Dia1. (d) Proteoglycans such as HSPG contributeto growth factor signaling and adhesion-dependent FAK activation andserve as docking sites for metalloproteases
Mol Neurobiol
regulators of migration, polarity complex proteins which arerestricted to specific cellular domains in static cells have beenshown to localize at the leading front during migration [28].
Different kinds of extracellular cues induce polarized migra-tion. Cells can sense andmigrate toward higher concentrations ofadhesive cues such as the glycoproteins of the matrix or towardsoluble cues such as chemokines and growth factors. Chemokinereceptors and growth factor receptors collaborate with integrinsto locally activate PI3K. Among the first substances to redistrib-ute to one side of the cell, PI3K generates PIP3, thus defining theleading front of the cell and initiating movement [24, 29].
The Role of NG2 in Adhesion
Integrins and Proteoglycans in Cell Adhesion
Cells need to attach to a substratum to generate the forcesnecessary for movement. Inside tissues, they are in contactwith other cells and a complex network of macromolecules,the extracellular matrix (ECM). Attachment to the surround-ing ECM is provided not only by membrane receptors, mainlyintegrins, but also by cell surface proteoglycans [30]. Integrinsare heterodimeric receptors with a large extracellular domainbinding the ECM which they link to the cytoskeleton througha short cytoplasmic domain. Their specificity is defined by theassociation of Ξ± and Ξ² subunits [31]. Proteoglycans are mac-romolecules composed of a core protein linked covalently toGAG chains such as chondroitin sulfate or heparan sulfate:these are found with NG2 and syndecan, respectively, twoproteins involved in cell migration [12, 32].
Binding of integrins to extracellular substrates inducesintegrin clustering and formation of adhesion complexes inwhich adaptator proteins couple the integrins to actin cytoskele-ton [33]. The nascent adhesions formed at the edge of the cellmature into focal complexes which evolve to focal adhesionsunder the tension resulting from mechanical forces coming fromoutside and inside the cell [34, 35]. Substrate stiffness is at theorigin of the external mechanical forces whereas actomyosincontraction under the control of ROCK generates internal forces[36]. Focal adhesion kinase (FAK) is recruited to focal adhesionsduring this process andmediates the activation of PI3K, Rac, andCdc42, thus promoting actin polymerization [37]. Concomitant-ly, integrins recruit cell surfacemetalloproteinases which degradethe ECM, clearing the space necessary for the moving of the cellbody in three-dimensional environments [38]. Importantly, po-larized migration requires the disassembly of integrin-mediatedadhesion at the rear of the cell to allow a concerted movement.Detachment of the cell rear involves integrin severing and cyto-skeleton contraction driven by ROCK1 [39, 40].
Cell surface proteoglycans regulate many processes togeth-er with the integrins. The case of syndecan-4 especially wellillustrates this convergence of signals. Indeed, syndecan-4
regulates focal adhesion assembly in cells that adhere tofibronectin via Ξ±5Ξ²1 integrin, but not Ξ±4Ξ²1 integrin [41].By recruiting and activating PKCΞ± and FAK to focal adhe-sions, syndecan-4 regulates adhesion and migration in syner-gy with integrins [42β44]. In this case, the activation of PKCΞ±could require the PDZ domain protein syntenin [45]. Similarlyto integrins, cell surface proteoglycans also recruit matrixmetalloproteinases (MMP). The pro-invasive effect of theproteoglycan CD44 is thus achieved by binding MMP9 [46].
NG2 Works in Synergy with Integrins
The implication of the proteoglycan NG2 in adhesion hasbeen shown in mammals and in Drosophila. During embry-onic development of Drosophila, the homologue of NG2,expressed in a subset of myotubes, is enriched in myotubecell protrusions where it stabilizes the attachment to tendoncells [23, 47]. The loss of function of this gene results inrounded unattached muscles. This effect is mediated throughGRIP and could also involve interaction with integrins via theextracellular domain of NG2 [47]. NG2 seems conserved inmammals and diverse organisms such as chicken, zebrafish,and Drosophila (score of 43 % between Drosophila andhuman) with the maintenance of domains such as laminin G-type motifs, CSPG repeats, and PDZ-binding motif [48].However, the knowledge of its role in lower organisms is stilllimited to findings in Drosophila myotubes. NG2 binds toseveral extracellular matrix components including collagen II,collagen V, tenascin, and laminin, and its best known interac-tion was characterized with collagen VI [14, 49]. The centralnon-globular domain of NG2 is responsible for collagen VIbinding and regulates in vitro migration of glioma towardgradients of soluble collagen VI [50, 51]. As a consequenceof the affinity of NG2 for collagen VI, NG2-expressing cellscan retain this glycoprotein at the cell surface and couldtherefore modulate the assembly of the pericellular matrix[49]. NG2 regulates adhesion also by collaborating with spe-cific integrins such as Ξ±3Ξ²1 and Ξ±4Ξ²1 [52, 53]. For instance,concomitant stimulation of NG2 and Ξ±4Ξ²1 integrin enhancedintegrin-mediated cell spreading [54, 55]. This was observedin a melanoma cell line where spreading and FAK phosphor-ylation were greatly enhanced on a fibronectin fragmentallowing Ξ±4Ξ²1 integrin binding combined with anti-NG2antibody as a substrate compared to spreading on either fibro-nectin fragment or anti-NG2 antibody alone [54]. This prop-erty of NG2 in Ξ±4Ξ²1-dependent adhesion on fibronectinreflects the role of syndecan-4 in Ξ±5Ξ²1-dependent adhesion.Indeed, both stimulate FAK phosphorylation and interact withsyntenin [45, 56], and it was proposed that NG2 interacts withthe heparin-binding domain of fibronectin like syndecan-4[54, 57]. It would be interesting to know if NG2 exerts thiseffect via PKCΞ±, as is the case with syndecan-4. It remains toidentify the extracellular components binding NG2 and
Mol Neurobiol
triggering its activation, since the effect on focal adhesionswas only seen by stimulation with anti-NG2 antibodies [54,55]. Otherwise, NG2 was implied in the proteolysis of colla-gen I by binding the metalloproteinase MT3-MMP via itschondroitin sulfate glycosaminoglycan chain, allowing inva-sion through collagen I [58].
Haptotaxis, defined as the migration toward a gradient ofcellular adhesion sites, depends on signaling pathways regulatedby integrins and cell surface proteoglycans [59, 60]. Thus,persistence of the migration of fibroblasts toward higher concen-trations of fibronectin is dependent on FAK [61]. In addition tothe regulation of FAK, the ability of NG2 to stimulate migrationtoward gradients of collagen VI demonstrates its ability to con-vert the binding to ECM into directional signals for the cell [51].NG2 thus appears as a potential transducer of haptotaxis. Thereported localization of NG2 in thin cellular extensions related tofilopodia [23, 55], which are involved in the exploration of thecell's immediate environment [62], corroborates this idea. How-ever, the reported shedding of the extracellular domain of NG2could impede the intracellular signaling of NG2. ANG2 cleavedectodomain of 290 kDa was isolated from the central nervoussystem, and the cleavage was augmented in stab wound lesions[63, 64]. The cleaved ectodomain could contribute to the ECMin a similar fashion to secreted proteoglycans such as hyalectanand small leucin-rich proteoglycans, which organize the matrixthrough their interactions with their multiple partners [32]. Theclose interaction at their external surfaces of the manifold myelinmembranes wrapping axons is allowed by the loss of glycocalyxsurrounding oligodendrocytes during differentiation [65]. Down-regulation of NG2 during oligodendrocyte differentiation couldalso contribute to this process by reducing steric repulsion andelectrostatic forces generated by its linked oligosaccharides.Deposition of CSPG is a common feature of the glial scarsformed after brain lesions, and the GAG chains are usuallydetrimental for the repair process [66]. Similarly, the GAG chainof NG2 was shown to inhibit axonal growth in vitro [67, 68].However, whether NG2 is beneficial or deleterious in vivo forneuronal plasticity after lesion remains a matter of debate. Thepresent review does not aim to discuss trans-signaling of NG2 tocells which are NG2-negative, but many publications dealingwith this subject can be found in the literature [69β72].
NG2, a Coreceptor for Growth Factors
Growth Factors and HSPG as Coreceptors
Platelet-derived growth factors (PDGF) and fibroblast growthfactors (FGF) regulate proliferation, survival, differentiation,and migration, in development as well as during woundhealing [73, 74]. Two isoforms of PDGF (PDGF-A, PDGF-B) and 23 isoforms of FGF have been reported. Binding totheir specific receptors induces the dimerization and the
subsequent activation of receptor tyrosine kinases by auto-phosphorylation. An early common intracellular signalingevent induced by these growth factors consists in the activa-tion of PI3K. Indeed, phosphorylated FGF receptors recruitthe key adaptator protein FRS2, recruiting subsequently acomplex involving GRB2, GAB1, and PI3K. Phosphorylationof the PDGF receptor creates docking sites for SH2 domainproteins such as PI3K [73]. Thus, a PDGF gradient inducesPI3K recruitment and activation toward high concentrationsof the growth factor which in turn results in a polarization ofthe cell [75]. Centrosomes and PDGF receptor- and Src-bearing endosomes are thus oriented toward the gradient.The polarized activation of PI3K is crucial for chemotaxissince its aberrant recruitment evenly distributed over the plas-ma membrane, via over-stimulation by a v-Src kinase con-struct, results in loss of polarity toward the PDGF gradient[75].
PDGF have been reported to bind to various collagens [76]and fibronectin [77]. PDGF and FGF bind also to glycopro-teins of the ECM such as the thrombospondin [78, 79] andSPARC [80, 81]. However, a class of low-affinity receptors,the heparan sulfate proteoglycans (HSPG), is the major com-ponent of the matrix involved in FGF and PDGF binding [82,83]. Thus, the extracellular matrix proteoglycan perlecanbinds basic fibroblast growth factor (bFGF) and promotesbFGF signaling mediating skin wound healing in the mouse[84]. In Xenopus laevis, heparin was shown to enhance thedirect association between the dimer PDGF-AA and fibronec-tin; treatment with heparinase III, which degrades heparansulfate chains of HSPG, impaired the PDGF-AA-guidedmesendoderm movement across a fibronectin-rich ECM[77]. The binding of FGF and PDGF to HSPG involves anelectrostatic interaction between basic amino acid residues ingrowth factors and negatively charged groups found in hepa-rin sulfate includingN-sulfated saccharide domains containing2-O-sulfate groups [85, 86]. Effective binding of FGF to itsreceptor requires the presence of cell surface HSPG whichstabilize the FGF ligand-receptor interaction [87]. Among cellsurface proteoglycans, glypicans and syndecans are generallyconsidered as coreceptors for heparin sulfate-binding growthfactors [88].
NG2 as a Coreceptor for PDGF-AA and bFGF
NG2 as a proteoglycan shares binding partners with HSPG.PDGF-AA and bFGF bind NG2 with high affinity whereasPDGF-BB, TGF-Ξ²1, VEGF, and EGF exhibit little or nobinding to NG2 [15]. This characteristic of NG2 seems crucialfor responsiveness of SMC to growth factors. It was shownthat anti-NG2 antibodies inhibited chemotactic responses ofrat SMC to PDGF-AA in transwell assays, but not to PDGF-BB [89]. This result was confirmed using SMC from NG2knockout mice, which were attracted by PDGF-BB but
Mol Neurobiol
unresponsive to PDGF-AA, whereas SMC from wild-typemice were attracted by both factors [90]. Indeed, autophos-phorylation of PDGFΞ± receptor was not observed in NG2-deficient SMC, indicating a defect in signal transduction at thelevel of receptor activation. The description of the coreceptorfunction of NG2 for bFGF was more extensive [6]. In aheparan sulfate-deficient cell line, expression of NG2 wassufficient to restore the bFGF mitogenic response, conferringto NG2 the same cofactor role as HSPG. Contrary to HSPG,NG2 acts surprisingly in a GAG-independent manner. On onehand, NG2 is able to retain bFGF to the cell surface. On theother hand, NG2 associates with FGFR1 and FGFR3 even inthe absence of bFGF. All these functions are mediated by theextracellular domain of NG2. Angiogenesis and the associatedcell proliferation induced by ectopic bFGF implantation incorneal neovascularization assays are strongly impaired inNG2 null mice, illustrating the importance of NG2 for thebFGF response of pericytes and SMC [6, 91].
In the brain, OPC identified by NG2 staining expressPDGFΞ± receptor and FGF receptors [92, 93]. Indeed, OPCare responsive to both PDGF-AA and bFGF [94]. In agree-ment with the role of NG2 in SMC, I observed that thedownregulation of NG2 by RNAi abrogates the chemotacticmigration of the Oli-neu OPC cell line toward PDGF-AA(Fig. 3). The delayed myelination found in the postnatalcerebellum of NG2 null mice would be then supported bythe defect of PDGF-AA signaling and more precisely the lossof mitogenic effect of PDGF-AA. Indeed, a delayed prolifer-ation of OPC was found concurring with this phenotype [5].However, in contrast to SMC, our results do not support therequirement of NG2 for bFGF signaling in OPC [8]. Indeed,the process outgrowth promoting effect of bFGF is retained inOPC with NG2 downregulation. Whereas NG2 appears to bean essential coreceptor for PDGF signaling in OPC, it is likelythat OPC express another coreceptor triggering bFGFsignaling.
NG2, a Core Regulator of RhoGTPases and PolarityComplex Proteins
Polarity Complexes and RhoGTPases
Cell polarity is established by an asymmetrical distribution ofsignaling, adhesion molecules, and the cytoskeleton, as wellas a distinct orientation of membrane trafficking pathways[95]. Cell polarity can be observed during asymmetric divi-sion, in apical-basal polarity, or in the front-rear polarityspecific to migration. Core polarity regulators form threepolarity complexes, namely PAR (Cdc42/Par3/Par6/aPKC),CRB (Crumbs/Pals1/PATJ), and SCRIB (Scribble/Dlg/LgL)complexes. These complexes are composed of ubiquitousproteins conserved throughout evolution and were first dis-covered in Drosophila and Caenorhabditis elegans [96]. Inepithelial cells, the PAR complex regulates tight junctionassembly [97] and segregates the CRB complex in the apicaldomain and the SCRIB complex in the basolateral domain[28]. In migrating cells, proteins from the different polaritycomplexes relocalize to the leading front to promote the front-rear polarity [28, 98]. Most of the processes regulated by thePAR complex are triggered through the localized recruitmentof the GEF Tiam1which specifically stimulates Rac activities:tight junction biogenesis [97], process outgrowth fromneurites and axons [99, 100], and persistent migration bystabilizing front-rear polarity [101].
Rho GTPases control polarity by the regulation and thecoordination of the cytoskeleton remodeling. GTP bindinginduces a conformational change which promotes interactionwith downstream effectors. Direct effectors of Rac are in-volved in cytoskeleton rearrangement such as PAK [102] orIRSp53 which activates the actin branching protein Arp2/3through WAVE2 [21, 103]. Among the direct effectors ofRhoA, ROCK proteins stimulate actomyosin contraction byinhibiting the myosin light chain phosphatase, ROCK1
Fig. 3 Transfilter chemotaxis and chemokinesis of Oli-neu OPC stablyexpressing either a control or a NG2-directed shRNA in response toPDGF-AA. Cells that had migrated to the lower side of the filter werecounted after 6 h of migration. In chemotaxis assays, only the medium inthe bottom well contains PDGF whereas in chemokinesis assays, PDGFis present on both sides of the filter. Results are expressed as a percentageof basal migration, i.e., migration without chemoattractant. Data represent
the meanΒ±SEM from at least three independent experiments. Controlcells display chemotaxis but no significant chemokinesis in response toPDGF. Downregulation of NG2 abolishes PDGF-dependent migration.The loss of directed migration (chemotaxis) without enhancement ofrandom migration (chemokinesis) indicates that migration is not dis-turbed by a loss of polarity but probably rather by a loss of sensitivityto PDGF-AA. * P<0,05 analyzed by t test, n.s = non-significant
Mol Neurobiol
regulating cellular tail retraction, whereas ROCK2 seemsimportant in limiting Rac-induced process outgrowth [40,104]. Conversely, the RhoA effector Dia1 regulates mem-brane protrusion [105, 106]. The cycle of Rho GTPases be-tween an inactive GDP-bound and an active GTP-bound stateis highly regulated. GEF catalyze the exchange of GDP forGTP whereas GTPase-activating proteins (GAP) enhance thelow intrinsic GTP hydrolysis activity of Rho GTPases [107].Polarity complexes control RhoGTPase activity through theseregulators: the association of Par3 with the GEF Tiam1 stim-ulates Rac1 and stabilizes front-rear polarity [101]; the com-plex Par6/aPKC inactivates RhoA through p190 RhoGAP[108]. Respectively, Rho GTPases regulate polarity com-plexes, RhoA inducing Par3 phosphorylation by ROCK,thereby impairing Par3-mediated Rac activation [109].
NG2 Regulates RhoGTPases and Recruits Polarity ComplexProteins
Several laboratories have demonstrated that in various celltypes NG2 influences the activity of RhoGTPases. Gliomacell lines transfected with NG2 and allowed to spread onsurfaces coated with different antibodies directed againstNG2 exhibited specific membrane protrusions. Spreading ofthe cells on the antibody D120 recognizing the region respon-sible for type VI collagen binding induced cellular extensionsrelated to filopodia, whereas the antibody N143 recognizingan extracellular membrane proximal domain of NG2 promot-ed lamellipodia protrusion [16]. NG2 was already observed infilopodia in glioma cells spread on poly-L-lysine, laminin,tenascin, and type VI collagen [110]. In melanoma cellsspreading on a combination of Ξ±4Ξ²1 integrin-binding fibro-nectin fragment and anti-NG2 antibody, microspike formationwas induced and NG2 colocalized with Ξ±4 [55]. Interestingly,clustering of NG2 in melanoma recruits the RhoGTPaseCdc42 and induces its activation [21], which could explainthe contribution of NG2 in filopodia extensions as thefilopodia formation is controlled by Cdc42 [111β113]. Theinduction of lamellipodia by the antibody N143 was shown tobe mediated via stimulation of Rac GTPase activity [22]. Newperspectives came from studies demonstrating that NG2 couldbe phosphorylated by PKCΞ± on threonine 2256 in the cyto-plasmic tail [17, 18]. Interestingly, phorbol ester stimulation ofPKC induced lamellipodia where NG2 was localized. More-over, an NG2 mutant bearing a substitution of threonine 2256by a glutamate to mimic the phosphorylation by PKCΞ± in-duced also leading edge lamellipodia formation where it lo-calized with activated Ξ²1 integrin and stimulated migrationwhen expressed in astrocytoma [17, 18]. Using a similarmutant, we demonstrated that the phosphorylation of threo-nine 2256 on NG2 increases Rac activity and stimulatesprocess outgrowth and migration of OPC [8]. In addition,
phosphorylation of threonine 2314 by ERK was shown tostimulate proliferation [18].
The regulation of RhoGTPases byNG2was observed via theapplication of external constraints on NG2: application of differ-ent anti-NG2 antibodies to stimulate either Cdc42 or Rac. How-ever, no report studied the constitutive regulation ofRhoGTPases by NG2. We demonstrated that NG2 downregula-tion induces a dramatic decrease of RhoA activity in OPC [8].We showed that NG2, via the multiple PDZ protein MUPP1which binds to its C-terminus PDZ-binding motif [19], recruitsthe RhoA GEF Syx1 and maintains a ring of activated RhoA atthe cell periphery. At first, this primary signaling of NG2 canexplain the in vivo behavior of OPC which was revealed in astudy by two-photon excitation time-lapse imaging [114]. Cel-lular processes of OPC retracted when they contacted a processof the same or adjacent OPC. In mouse brains, this mutualrepulsion appears to keep apart OPC from each other. Thisbehavior is exemplified by the contact inhibition of locomotion(CIL) observed notably in neural crest cells, a cell type-specificphenomenon inducing the change of migration direction whentwo cells contact each other [115]. The change of directioninduced by CIL is mediated by the RhoA/ROCK pathway. Inour own experiments, in vitro clustering of OPCwas inhibited inthe presence of NG2, and ROCK inhibition had the same effectas NG2 downregulation, releasing the mutual repulsion charac-teristic of OPC [8]. The specific mode of migration induced byNG2 could partly explain its prevalence in progenitor cells. Theother consequence of this constitutive activation of RhoA isclearly its interference with growth factor signaling. Growthfactors trigger process outgrowth and migration stimulationthrough Rac activation. For example, bFGF stimulates neuriteoutgrowth via the Rac GEF Ξ²-PIX [8, 116]. However, RhoAactivity at themembrane inhibits process outgrowth [117]. RhoAperforms this effect by inhibiting the recruitment and the activa-tion of Rac in protrusions through a ROCK2-dependent pathway[118]. In agreement with these observations, we have shown thatthe stimulation of RhoA by NG2 reduces sensitivity to bFGFand favors the bipolar shape and persistent movement of OPCtoward a bFGF gradient. This restraining effect of NG2 towardbFGF could explain our in vivo observations. Three days afterthe application of a stab wound in the sensory motor cortex ofmice, we observed a high production of bFGF next to thewound. In WT mice, OPC polarized toward the wound in theperilesional region, whereas OPC in NG2β/β mice were unableto polarize toward the wound, possibly because of an over-stimulation producing concurrent leading fronts on the same cell.Indeed, the decrease of RhoA activity in OPC with altered NG2expression increases the basal Rac activity and potentiates Racactivation by bFGF, leading to a multiprocess morphology [8].
Whereas Ξ²-PIX downregulation inhibited bFGF-dependentoutgrowth in OPC, it did not affect outgrowth induced byphosphorylation on Thr2256 of NG2 which was mediated byTiam1 [8]. Tiam1 is known to be recruited by the PAR complex
Mol Neurobiol
proteins Par3 and Par6 during neurite outgrowth and axonspecification [100]. In addition, PATJ was shown to recruitPar3 at the leading edge of epithelial cells during directionalmigration [98]. In accordance with these results, we found thatphosphorylation of NG2 on Thr2256 stimulates Rac activityand induces process outgrowth through Tiam1, PAR complexproteins, and CRB complex proteins such as PATJ [8]. Here, thecomplex interactions of RhoGTPases appear in this pathway asa functional NG2/RhoA signaling pathway is required to in-duce process outgrowth in OPC. The study of the spatiotem-poral dynamics of RhoGTPases in migrating cells has shownthat initial events of membrane protrusion are orchestrated bylocalized RhoA activity at the edge of the cell extension [119,120]. This role of RhoA might be mediated by the recruitmentof its effector Dia1 at the membrane [121]. Indeed, a strongactivation of RhoA by SDF-1 induces neurite retractationthrough ROCK whereas a moderate activation of RhoA facil-itates axon elongation via Dia1 [122]. We can also postulatethat the recruitment of polarity complex proteins by NG2 seen
during migration and process outgrowth may be involved in theregulation of the asymmetric segregation of the EGF receptorby NG2 in OPC [123] via the documented role of the PARcomplex in asymmetric division [124].
Integration of the Multimodal Role of NG2 in Migration:a Place for PKCΞ±?
The function of NG2 is clearly ambivalent (Fig. 4a). On onehand, NG2 is a selective facilitator of migration. First, itenhances signals inducing the front pole specification suchas PIP3production by PI3K. Via its coreceptor function, NG2allows PDGF-AA and bFGF signaling in cells devoid of otherproper coreceptors. Thus, NG2 facilitates the growth factor-dependent activation of PI3K (Fig. 4a(b)). On fibronectinsubstrates, NG2 binding amplifies integrin signaling notablythrough FAK stimulation [55] which is important in polarityestablishment [95]. FAK recruitment to focal adhesions
Fig. 4 aMultiple roles of NG2 in OPC migration. From right to left: (a)NG2 serves as a docking site for the protease MT3-MMP which cleavescollagen I. (b) NG2 allows PDGF-AA signaling through its associationwith the growth factors (GF) and the growth factor receptors (GFR).Growth factors (PDGF-AA and bFGF) induce PI3K activation andPKCΞ± activation through PLC. (c) Phosphorylation of NG2 onThr2256 by PKCΞ± induces CRB complex (PATJ/Pals1/Crb2) recruit-ment. The CRB complex interacts with the PAR complex (aPKC/Par3/Par6) which stimulates Rac activity via the GEF Tiam1. (d) Extracellularbinding of NG2 enhances focal adhesion formation and FAK activation.FAK stimulates Rac activity through p130cas. (e) NG2 interacts withMUPP1 via its PDZ-binding motif and recruits the GEF Syx1 whichstimulates at the membrane RhoA activity and the effector ROCK. (aβe)Rac and PI3K stimulate actin polymerization at the leading front whereasROCK controls actin contraction inside the rear pole and favors focaladhesion (FA) maturation. MTOC microtubule organizing center. b Pro-posed model to explain the differential effect of NG2 on OPC migration:
NG2 enhances the migration rate of OPC toward a gradient of growthfactor but not inside a homogenous concentration [8]. 1.NG2maintains ahigh activity of RhoA at the membrane. 2.Growth factors stimulate Racactivity through PI3K and GEF (Ξ²-PIX for bFGF). However, the growthfactor activation of Rac is limited by the high RhoA/ROCK activityinduced by NG2 which prevents process outgrowth. 3. Activation ofPKCΞ± by growth factors induces the phosphorylation of NG2 on threo-nine 2256 and converts RhoA activation initially mediated by NG2 intoRac activation. 4. The recruitment of polarity complex proteins by NG2induces leading front specification. Mutual inhibition between RhoA andthe PAR complex segregates Rac activity at the front and RhoA activity inthe rear: Par6/aPKC inactivates RhoA through p190 RhoGAP; ROCKphosphorylates Par3 and impairs Tiam1-mediated Rac activation. Thisconfiguration stabilizes the front-rear axis and limits additional processoutgrowth in the rear pole, favoring therefore rather directional migrationthan random migration
Mol Neurobiol
should lead to the stimulation of PI3K via Ras signaling [125].RhoA is a central regulator of the mechanical forces respon-sible for focal adhesion maturation. ROCK controls the intra-cellular tension driven by actomyosin contraction, whereas theother RhoA effector Dia1 regulates the response of focaladhesions to the forces coming from inside as well as outsidethe cell [126, 127]. Therefore, it can be postulated that mem-brane stimulation of RhoA by NG2 contributes to the matu-ration of focal adhesion. Second, NG2 stimulates the RhoGTPases Cdc42 and Rac, important for actin polymerizationat the leading front. The clustering of NG2with antibodies canstimulate Cdc42 [21], a well-known regulator of cell polarity,notably via the regulation of centrosome orientation [128].The characterization of the cognate ligand of NG2 responsiblefor such an effect would yield information about another cueinducing polarization of NG2 cells. Moreover, in NG2-enhanced spreading, FAK activation is associated with therecruitment of p130Cas [21, 22, 129] and probably PI3K toadhesion sites, both activators of Rac (Fig. 4a(d)). Third, wehave shown that NG2 phosphorylated on threonine 2256 isable to recruit the polarity complex proteins specific to thefront pole of migrating cells and so to potentially initiatemigration [8] (Fig. 4a(c)). On the other hand, NG2 filtersextracellular signals by the constitutive activation of RhoA atthe cell periphery (Fig. 4a(e)). RhoA inhibits Rac throughROCK and therefore lowers the sensitivity to growth factorsas we described in the case of bFGF [8].
Phosphorylation of NG2 on threonine 2256 by PKCΞ±could reconcile the apparent opposite effects of NG2: PKCΞ±may indeed represent an important molecular switch drivingNG2-dependent directed migration (Fig. 4b). PKCΞ± is aserine/threonine protein kinase recruited notably to focal ad-hesions and activated by ECM receptors such asΞ²1 integrin orsyndecan-4 [41, 130β132] and also activated by growth fac-tors. Indeed, growth factor receptors activate phospholipase C,hydrolysing PIP2 to generate diacylglycerol (DAG) and ino-sitol triphosphate and thus triggering intracellular calciumrelease which, together with DAG, activates the PKCΞ±[133]. In certain conditions such as growth factor stimulation,PKCΞ± may phosphorylate NG2 and thus induce the recruit-ment of polarity complex proteins, acting to reverse the stim-ulation of RhoA and promoting stimulation of Rac. Thislocalized event in response to a gradient would reinforce theestablishment of a front pole with actin polymerization activ-ity, whereas maintenance of the NG2/RhoA pathway in therear pole would inhibit further process outgrowth. Such amechanism could explain the preponderance and aggressivebehavior of tumor cells expressing NG2. Considering thatactivity-dependent intracellular calcium rise can enhancePKCΞ± activity, calcium influx through glutamate receptorscould also control cell polarization via NG2. For instance,NMDA stimulation promotes OPC chemotaxis and the GEFTiam1, a downstream effector of phosphorylated NG2, was
shown to be a likely mediator of the promigratory effect ofglutamate [134].
Conflict of Interest The author declares that he has no conflict ofinterest.
References
1. Levine JM, Nishiyama A (1996) The NG2 chondroitin sulfateproteoglycan: a multifunctional proteoglycan associated with im-mature cells. Perspect Dev Neurobiol 3:245β259
2. Ozerdem U, Grako KA, Dahlin-Huppe K, Monosov E, StallcupWB (2001) NG2 proteoglycan is expressed exclusively by muralcells during vascular morphogenesis. Dev Dyn: Off Publ AmAssocAnatomists 222:218β227
3. Trotter J, Karram K, Nishiyama A (2010) NG2 cells: properties,progeny and origin. Brain Res Rev 63:72β82
4. Birnbaum T, Hildebrandt J, Nuebling G, Sostak P, Straube A (2011)Glioblastoma-dependent differentiation and angiogenic potential ofhuman mesenchymal stem cells in vitro. J Neuro-Oncol 105:57β65
5. Kucharova K, Stallcup WB (2010) The NG2 proteoglycan pro-motes oligodendrocyte progenitor proliferation and developmentalmyelination. Neuroscience 166:185β194
6. Cattaruzza S, Ozerdem U, Denzel M, Ranscht B, Bulian P,Cavallaro U, Zanocco D, Colombatti A, Stallcup WB, Perris R(2013) Multivalent proteoglycan modulation of FGF mitogenicresponses in perivascular cells. Angiogenesis 16:309β327
7. Gibby K, YouWK, Kadoya K, Helgadottir H, Young LJ, Ellies LG,Chang Y, Cardiff RD, Stallcup WB (2012) Early vascular deficitsare correlated with delayed mammary tumorigenesis in the MMTV-PyMT transgenic mouse following genetic ablation of the NG2proteoglycan. Breast Cancer Res: BCR 14:R67
9. Niehaus A, Stegmuller J, Diers-Fenger M, Trotter J (1999) Cell-surface glycoprotein of oligodendrocyte progenitors involved inmigration. J Neurosci 19:4948β4961
10. StegmΓΌller J, Schneider S, Hellwig A, Garwood J, Trotter J (2002)AN2, the mouse homologue of NG2, is a surface antigen on glialprecursor cells implicated in control of cell migration. J Neurocytol31:497β505
11. Campoli M, Ferrone S, Wang X (2010) Functional and clinicalrelevance of chondroitin sulfate proteoglycan 4. Adv Cancer Res109:73β121
12. Stallcup WB, Huang FJ (2008) A role for the NG2 proteoglycan inglioma progression. Cell Adh Migr 2:192β201
13. Stallcup WB, Dahlin-Huppe K (2001) Chondroitin sulfate andcytoplasmic domain-dependent membrane targeting of the NG2proteoglycan promotes retraction fiber formation and cell polariza-tion. J Cell Sci 114:2315β2325
14. Burg MA, Tillet E, Timpl R, Stallcup WB (1996) Binding of theNG2 proteoglycan to typeVI collagen and other extracellular matrixmolecules. J Biol Chem 271:26110β26116
15. Goretzki L, Burg MA, Grako KA, Stallcup WB (1999) High-affinity binding of basic fibroblast growth factor and platelet-
Mol Neurobiol
derived growth factor-AA to the core protein of the NG2 proteo-glycan. J Biol Chem 274:16831β16837
16. Fang X, Burg MA, Barritt D, Dahlin-Huppe K, Nishiyama A,Stallcup WB (1999) Cytoskeletal reorganization induced by en-gagement of the NG2 proteoglycan leads to cell spreading andmigration. Mol Biol Cell 10:3373β3387
18. Makagiansar IT, Williams S, Mustelin T, Stallcup WB (2007)Differential phosphorylation of NG2 proteoglycan by ERK andPKCalpha helps balance cell proliferation and migration. J CellBiol 178:155β165
19. Barritt DS, Pearn MT, Zisch AH, Lee SS, Javier RT, Pasquale EB,Stallcup WB (2000) The multi-PDZ domain protein MUPP1 is acytoplasmic ligand for the membrane-spanning proteoglycan NG2.J Cell Biochem 79:213β224
20. StegmΓΌller J, Werner H, Nave KA, Trotter J (2003) The proteogly-can NG2 is complexed with alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors by the PDZ glutamatereceptor interaction protein (GRIP) in glial progenitor cells.Implications for glial-neuronal signaling. J Biol Chem 278:3590β3598
22. Majumdar M, Vuori K, Stallcup WB (2003) Engagement of theNG2 proteoglycan triggers cell spreading via rac and p130cas. CellSignal 15:79β84
23. Schnorrer F, Kalchhauser I, Dickson BJ (2007) The transmembraneprotein Kon-tiki couples to Dgrip to mediate myotube targeting inDrosophila. Dev Cell 12:751β766
24. Friedl P, Wolf K (2003) Tumour-cell invasion and migration: diver-sity and escape mechanisms. Nat Rev Cancer 3:362β374
26. Petrie RJ, Doyle AD, Yamada KM (2009) Random versusdirectionally persistent cell migration. Nat Rev Mol Cell Biol 10:538β549
27. Maeda YT, Inose J, Matsuo MY, Iwaya S, Sano M (2008) Orderedpatterns of cell shape and orientational correlation during spontane-ous cell migration. PLoS One 3:e3734
28. Etienne-Manneville S (2008) Polarity proteins in migration andinvasion. Oncogene 27:6970β6980
29. Dawes AT, Edelstein-Keshet L (2007) Phosphoinositides and Rhoproteins spatially regulate actin polymerization to initiate and main-tain directed movement in a one-dimensional model of a motile cell.Biophys J 92:744β768
30. Hood JD, Cheresh DA (2002) Role of integrins in cell invasion andmigration. Nat Rev Cancer 2:91β100
32. Theocharis AD, Skandalis SS, Tzanakakis GN, Karamanos NK(2010) Proteoglycans in health and disease: novel roles for proteo-glycans in malignancy and their pharmacological targeting. FEBS J277:3904β3923
33. Huttenlocher A, Horwitz AR (2011) Integrins in cell migration.Cold Spring Harb Perspect Biol 3:a005074
34. Balaban NQ, Schwarz US, Riveline D, Goichberg P, Tzur G,Sabanay I, Mahalu D, Safran S, Bershadsky A, Addadi L et al(2001) Force and focal adhesion assembly: a close relationshipstudied using elastic micropatterned substrates. Nat Cell Biol 3:466β472
35. Galbraith CG, Yamada KM, Sheetz MP (2002) The relationshipbetween force and focal complex development. J Cell Biol 159:695β705
36. Lavelin I, Wolfenson H, Patla I, Henis YI, Medalia O, Volberg T,Livne A, Kam Z, Geiger B (2013) Differential effect of actomyosinrelaxation on the dynamic properties of focal adhesion proteins.PLoS One 8:e73549
37. Scales TM, Parsons M (2011) Spatial and temporal regulation ofintegrin signalling during cell migration. Curr Opin Cell Biol 23:562β568
38. Mueller SC, Ghersi G, Akiyama SK, Sang QX, Howard L, Pineiro-SanchezM,Nakahara H, YehY, ChenWT (1999) A novel protease-docking function of integrin at invadopodia. J Biol Chem 274:24947β24952
39. Regen CM, Horwitz AF (1992) Dynamics of beta 1 integrin-mediated adhesive contacts in motile fibroblasts. J Cell Biol 119:1347β1359
40. Vega FM, Fruhwirth G, Ng T, Ridley AJ (2011) RhoA and RhoChave distinct roles in migration and invasion by acting throughdifferent targets. J Cell Biol 193:655β665
42. Lim ST, Longley RL, Couchman JR, Woods A (2003) Directbinding of syndecan-4 cytoplasmic domain to the catalytic domainof protein kinase C alpha (PKC alpha) increases focal adhesionlocalization of PKC alpha. J Biol Chem 278:13795β13802
43. Keum E, Kim Y, Kim J, Kwon S, Lim Y, Han I, Oh ES (2004)Syndecan-4 regulates localization, activity and stability of proteinkinase C-alpha. Biochem J 378:1007β1014
44. Morgan MR, Humphries MJ, Bass MD (2007) Synergistic controlof cell adhesion by integrins and syndecans. Nat Rev Mol Cell Biol8:957β969
45. Hwangbo C, Kim J, Lee JJ, Lee JH (2010) Activation of the integrineffector kinase focal adhesion kinase in cancer cells is regulated bycrosstalk between protein kinase Calpha and the PDZ adapterprotein mda-9/Syntenin. Cancer Res 70:1645β1655
46. Yu Q, Stamenkovic I (1999) Localization of matrix metalloprotein-ase 9 to the cell surface provides a mechanism for CD44-mediatedtumor invasion. Gene Dev 13:35β48
47. Estrada B, Gisselbrecht SS, Michelson AM (2007) The transmem-brane protein Perdido interacts with Grip and integrins to mediatemyotube projection and attachment in the Drosophila embryo.Development 134:4469β4478
48. Staub E, Hinzmann B, Rosenthal A (2002) A novel repeat in themelanoma-associated chondroitin sulfate proteoglycan defines anew protein family. FEBS Lett 527:114β118
49. NishiyamaA, StallcupWB (1993) Expression of NG2 proteoglycancauses retention of type VI collagen on the cell surface. Mol BiolCell 4:1097β1108
50. Tillet E, Ruggiero F, Nishiyama A, Stallcup WB (1997) Themembrane-spanning proteoglycan NG2 binds to collagens V andVI through the central nonglobular domain of its core protein. J BiolChem 272:10769β10776
51. Burg MA, Nishiyama A, Stallcup WB (1997) A central segment ofthe NG2 proteoglycan is critical for the ability of glioma cells to bindand migrate toward type VI collagen. Exp Cell Res 235:254β264
52. Fukushi J, Makagiansar IT, Stallcup WB (2004) NG2 proteoglycanpromotes endothelial cell motility and angiogenesis via engagementof galectin-3 and alpha3beta1 integrin.Mol Biol Cell 15:3580β3590
53. Chekenya M, Krakstad C, Svendsen A, Netland IA, Staalesen V,Tysnes BB, Selheim F, Wang J, Sakariassen PO, Sandal T et al(2008) The progenitor cell marker NG2/MPG promoteschemoresistance by activation of integrin-dependent PI3K/Akt sig-naling. Oncogene 27:5182β5194
Mol Neurobiol
54. Iida J, Meijne AM, Spiro RC, Roos E, Furcht LT, McCarthy JB(1995) Spreading and focal contact formation of human melanomacells in response to the stimulation of both melanoma-associatedproteoglycan (NG2) and alpha 4 beta 1 integrin. Cancer Res 55:2177β2185
55. Yang J, PriceMA,Neudauer CL,Wilson C, Ferrone S, XiaH, Iida J,Simpson MA, McCarthy JB (2004) Melanoma chondroitin sulfateproteoglycan enhances FAK and ERK activation by distinct mech-anisms. J Cell Biol 165:881β891
56. Chatterjee N, Stegmuller J, Schatzle P, KarramK, Koroll M,WernerHB, Nave KA, Trotter J (2008) Interaction of syntenin-1 and theNG2 proteoglycan in migratory oligodendrocyte precursor cells. JBiol Chem 283:8310β8317
57. Mahalingam Y, Gallagher JT, Couchman JR (2007) Cellular adhe-sion responses to the heparin-binding (HepII) domain of fibronectinrequire heparan sulfate with specific properties. J Biol Chem 282:3221β3230
58. Iida J, Pei D, Kang T, Simpson MA, Herlyn M, Furcht LT,McCarthy JB (2001) Melanoma chondroitin sulfate proteoglycanregulates matrix metalloproteinase-dependent human melanomainvasion into type I collagen. J Biol Chem 276:18786β18794
59. Dertinger SK, Jiang X, Li Z, Murthy VN, Whitesides GM (2002)Gradients of substrate-bound laminin orient axonal specification ofneurons. Proc Natl Acad Sci U S A 99:12542β12547
60. Gunawan RC, Silvestre J, Gaskins HR, Kenis PJ, Leckband DE (2006)Cell migration and polarity onmicrofabricated gradients of extracellularmatrix proteins. Langmuir: ACS J Surf Colloids 22:4250β4258
61. Rhoads DS, Guan JL (2007) Analysis of directional cell migrationon defined FN gradients: role of intracellular signaling molecules.Exp Cell Res 313:3859β3867
62. Etienne-Manneville S, Hall A (2002) Rho GTPases in cell biology.Nature 420:629β635
63. Nishiyama A, Lin XH, StallcupWB (1995) Generation of truncatedforms of the NG2 proteoglycan by cell surface proteolysis. Mol BiolCell 6:1819β1832
64. Asher RA, Morgenstern DA, Properzi F, Nishiyama A, Levine JM,Fawcett JW (2005) Two separate metalloproteinase activities areresponsible for the shedding and processing of the NG2 proteogly-can in vitro. Mol Cell Neurosci 29:82β96
65. Bakhti M, Snaidero N, Schneider D, Aggarwal S, Mobius W,Janshoff A, Eckhardt M, Nave KA, Simons M (2013) Loss ofelectrostatic cell-surface repulsion mediates myelin membrane ad-hesion and compaction in the central nervous system. Proc NatlAcad Sci U S A 110:3143β3148
66. Bartus K, James ND, Bosch KD, Bradbury EJ (2012) Chondroitinsulphate proteoglycans: key modulators of spinal cord and brainplasticity. Exp Neurol 235:5β17
67. Ughrin YM, Chen ZJ, Levine JM (2003) Multiple regions of theNG2 proteoglycan inhibit neurite growth and induce growth conecollapse. J Neurosci 23:175β186
68. Lee SI, ZhangW,RaviM,WeschenfelderM,BastmeyerM, Levine JM(2013) Atypical protein kinase C and Par3 are required forproteoglycan-induced axon growth inhibition. J Neurosci 33:2541β2554
69. de Castro R Jr, Tajrishi R, Claros J, StallcupWB (2005) Differentialresponses of spinal axons to transection: influence of the NG2proteoglycan. Exp Neurol 192:299β309
70. Yang Z, Suzuki R, Daniels SB, Brunquell CB, Sala CJ, NishiyamaA (2006) NG2 glial cells provide a favorable substrate for growingaxons. J Neurosci 26:3829β3839
71. Hossain-Ibrahim MK, Rezajooi K, Stallcup WB, Lieberman AR,Anderson PN (2007) Analysis of axonal regeneration in the centraland peripheral nervous systems of the NG2-deficient mouse. BMCNeurosci 8:80
72. Massey JM, Amps J, Viapiano MS, Matthews RT, Wagoner MR,Whitaker CM, Alilain W, Yonkof AL, Khalyfa A, Cooper NG et al
(2008) Increased chondroitin sulfate proteoglycan expression indenervated brainstem targets following spinal cord injury creates abarrier to axonal regeneration overcome by chondroitinase ABCand neurotrophin-3. Exp Neurol 209:426β445
73. Heldin CH, Westermark B (1999) Mechanism of action and in vivorole of platelet-derived growth factor. Physiol Rev 79:1283β1316
74. Turner N, Grose R (2010) Fibroblast growth factor signalling: fromdevelopment to cancer. Nat Rev Cancer 10:116β129
75. Platek A, Vassilev VS, de Diesbach P, Tyteca D, Mettlen M,Courtoy PJ (2007) Constitutive diffuse activation ofphosphoinositide 3-kinase at the plasma membrane by v-Src sup-presses the chemotactic response to PDGF by abrogating the polar-ity of PDGF receptor signalling. Exp Cell Res 313:1090β1105
76. Somasundaram R, Schuppan D (1996) Type I, II, III, IV, V, and VIcollagens serve as extracellular ligands for the isoforms of platelet-derived growth factor (AA, BB, and AB). J Biol Chem 271:26884β26891
77. Smith EM, Mitsi M, Nugent MA, Symes K (2009) PDGF-A inter-actions with fibronectin reveal a critical role for heparan sulfate indirected cell migration during Xenopusgastrulation. Proc Natl AcadSci U S A 106:21683β21688
79. Margosio B, Marchetti D, Vergani V, Giavazzi R, Rusnati M, PrestaM, Taraboletti G (2003) Thrombospondin 1 as a scavenger formatrix-associated fibroblast growth factor 2. Blood 102:4399β4406
80. Raines EW, Lane TF, Iruela-Arispe ML, Ross R, Sage EH (1992)The extracellular glycoprotein SPARC interacts with platelet-derived growth factor (PDGF)-AB and -BB and inhibits the bindingof PDGF to its receptors. Proc Natl Acad Sci U S A 89:1281β1285
81. Brekken RA, Sage EH (2000) SPARC, a matricellular protein: at thecrossroads of cell-matrix. Matrix Biol: J Int Soc Matrix Biol 19:569β580
82. Lustig F, Hoebeke J, Ostergren-Lunden G, Velge-Roussel F,Bondjers G, Olsson U, Ruetschi U, Fager G (1996) Alternativesplicing determines the binding of platelet-derived growth factor(PDGF-AA) to glycosaminoglycans. Biochemistry 35:12077β12085
83. Harmer NJ, Ilag LL,Mulloy B, Pellegrini L, Robinson CV, BlundellTL (2004) Towards a resolution of the stoichiometry of the fibro-blast growth factor (FGF)-FGF receptor-heparin complex. J MolBiol 339:821β834
84. Zhou Z, Wang J, Cao R, Morita H, Soininen R, Chan KM, Liu B,Cao Y, Tryggvason K (2004) Impaired angiogenesis, delayedwound healing and retarded tumor growth in perlecan heparansulfate-deficient mice. Cancer Res 64:4699β4702
85. Turnbull JE, Fernig DG, Ke Y, WilkinsonMC, Gallagher JT (1992)Identification of the basic fibroblast growth factor binding sequencein fibroblast heparan sulfate. J Biol Chem 267:10337β10341
86. Naimy H, Buczek-Thomas JA, Nugent MA, Leymarie N, Zaia J(2011) Highly sulfated nonreducing end-derived heparan sulfatedomains bind fibroblast growth factor-2 with high affinity and areenriched in biologically active fractions. J Biol Chem 286:19311β19319
87. Yayon A, Klagsbrun M, Esko JD, Leder P, Ornitz DM (1991) Cellsurface, heparin-like molecules are required for binding of basicfibroblast growth factor to its high affinity receptor. Cell 64:841β848
88. Schaefer L, Schaefer RM (2010) Proteoglycans: from structuralcompounds to signaling molecules. Cell Tissue Res 339:237β246
89. Grako KA, Stallcup WB (1995) Participation of the NG2 proteo-glycan in rat aortic smooth muscle cell responses to platelet-derivedgrowth factor. Exp Cell Res 221:231β240
90. Grako KA, Ochiya T, Barritt D, Nishiyama A, Stallcup WB (1999)PDGF (alpha)-receptor is unresponsive to PDGF-AA in aortic
Mol Neurobiol
smooth muscle cells from the NG2 knockout mouse. J Cell Sci112(Pt 6):905β915
91. Ozerdem U, Stallcup WB (2004) Pathological angiogenesis is re-duced by targeting pericytes via the NG2 proteoglycan.Angiogenesis 7:269β276
92. Redwine JM, Blinder KL, Armstrong RC (1997) In situ expressionof fibroblast growth factor receptors by oligodendrocyte progenitorsand oligodendrocytes in adult mouse central nervous system. JNeurosci Res 50:229β237
93. He Y, Cai W,Wang L, Chen P (2009) A developmental study on theexpression of PDGFalphaR immunoreactive cells in the brain ofpostnatal rats. Neurosci Res 65:272β279
94. Zhang H, Vutskits L, Calaora V, Durbec P, Kiss JZ (2004) A role forthe polysialic acid-neural cell adhesion molecule in PDGF-inducedchemotaxis of oligodendrocyte precursor cells. J Cell Sci 117:93β103
95. Fukata M, Nakagawa M, Kaibuchi K (2003) Roles of Rho-familyGTPases in cell polarisation and directional migration. Curr OpinCell Biol 15:590β597
97. Mertens AE, Rygiel TP, Olivo C, van der Kammen R, Collard JG(2005) The Rac activator Tiam1 controls tight junction biogenesis inkeratinocytes through binding to and activation of the Par polaritycomplex. J Cell Biol 170:1029β1037
98. Shin K, Wang Q, Margolis B (2007) PATJ regulates directionalmigration of mammalian epithelial cells. EMBO Rep 8:158β164
99. Kunda P, Paglini G, Quiroga S, Kosik K, Caceres A (2001)Evidence for the involvement of Tiam1 in axon formation. JNeurosci 21:2361β2372
100. Nishimura T, Yamaguchi T, Kato K, Yoshizawa M, Nabeshima Y,Ohno S, Hoshino M, Kaibuchi K (2005) PAR-6βPAR-3 mediatesCdc42-induced Rac activation through the Rac GEFs STEF/Tiam1.Nat Cell Biol 7:270β277
101. Pegtel DM, Ellenbroek SI, Mertens AE, van der Kammen RA, deRooij J, Collard JG (2007) The Par-Tiam1 complex controls persis-tent migration by stabilizing microtubule-dependent front-rear po-larity. Curr Biol 17:1623β1634
102. Manser E, Leung T, Salihuddin H, Zhao ZS, Lim L (1994) A brainserine/threonine protein kinase activated by Cdc42 and Rac1.Nature 367:40β46
103. Derivery E, Gautreau A (2010) Generation of branched actin net-works: assembly and regulation of the N-WASP and WAVE molec-ular machines. BioEssays: News Rev Mol Cel Dev Biol 32:119β131
104. Fujisawa K, Fujita A, Ishizaki T, Saito Y, Narumiya S (1996)Identification of the Rho-binding domain of p160ROCK, a Rho-associated coiled-coil containing protein kinase. J Biol Chem 271:23022β23028
105. Maiti S, Michelot A, Gould C, Blanchoin L, Sokolova O, Goode BL(2012) Structure and activity of full-length formin mDia1.Cytoskeleton 69:393β405
106. Goh WI, Lim KB, Sudhaharan T, Sem KP, Bu W, Chou AM,Ahmed S (2012) mDia1 and WAVE2 proteins interact directly withIRSp53 in filopodia and are involved in filopodium formation. JBiol Chem 287:4702β4714
107. Rossman KL, Der CJ, Sondek J (2005) GEF means go: turning onRHO GTPases with guanine nucleotide-exchange factors. Nat RevMol Cell Biol 6:167β180
108. Zhang H, Macara IG (2008) The PAR-6 polarity protein regulatesdendritic spine morphogenesis through p190 RhoGAP and the RhoGTPase. Dev Cell 14:216β226
109. Nakayama M, Goto TM, Sugimoto M, Nishimura T, Shinagawa T,Ohno S, Amano M, Kaibuchi K (2008) Rho-kinase phosphorylatesPAR-3 and disrupts PAR complex formation. Dev Cell 14:205β215
110. Lin XH, Grako KA, Burg MA, Stallcup WB (1996) NG2 proteo-glycan and the actin-binding protein fascin define separate popula-tions of actin-containing filopodia and lamellipodia during cellspreading and migration. Mol Biol Cell 7:1977β1993
111. Kozma R, Ahmed S, Best A, Lim L (1995) The Ras-related proteinCdc42Hs and bradykinin promote formation of peripheral actinmicrospikes and filopodia in Swiss 3T3 fibroblasts. Mol Cell Biol15:1942β1952
112. Nobes CD, Hall A (1995) Rho, rac, and cdc42 GTPases regulate theassembly of multimolecular focal complexes associated with actinstress fibers, lamellipodia, and filopodia. Cell 81:53β62
113. Krugmann S, Jordens I, Gevaert K, Driessens M, VandekerckhoveJ, Hall A (2001) Cdc42 induces filopodia by promoting the forma-tion of an IRSp53:Mena complex. Curr Biol 11:1645β1655
114. Hughes EG, Kang SH, Fukaya M, Bergles DE (2013)Oligodendrocyte progenitors balance growth with self-repulsionto achieve homeostasis in the adult brain. Nat Neurosci 16:668β676
115. Carmona-Fontaine C, Matthews HK, Kuriyama S, Moreno M,Dunn GA, Parsons M, Stern CD, Mayor R (2008) Contact inhibi-tion of locomotion in vivo controls neural crest directional migra-tion. Nature 456:957β961
116. Shin EY, Shin KS, Lee CS, Woo KN, Quan SH, Soung NK, KimYG, Cha CI, Kim SR, Park D et al (2002) Phosphorylation of p85beta PIX, a Rac/Cdc42-specific guanine nucleotide exchange factor,via the Ras/ERK/PAK2 pathway is required for basic fibroblastgrowth factor-induced neurite outgrowth. J Biol Chem 277:44417β44430
117. Kranenburg O, Poland M, Gebbink M, Oomen L, Moolenaar WH(1997) Dissociation of LPA-induced cytoskeletal contraction fromstress fiber formation by differential localization of RhoA. J Cell Sci110(Pt 19):2417β2427
118. Yamaguchi Y, Katoh H, Yasui H, Mori K, Negishi M (2001) RhoAinhibits the nerve growth factor-induced Rac1 activation through Rho-associated kinase-dependent pathway. J Biol Chem 276:18977β18983
119. Pertz O, Hodgson L, Klemke RL, Hahn KM (2006) Spatiotemporaldynamics of RhoA activity in migrating cells. Nature 440:1069β1072
120. Machacek M, Hodgson L, Welch C, Elliott H, Pertz O, Nalbant P,Abell A, Johnson GL, Hahn KM, Danuser G (2009) Coordinationof Rho GTPase activities during cell protrusion. Nature 461:99β103
121. Kurokawa K, Matsuda M (2005) Localized RhoA activation as arequirement for the induction of membrane ruffling. Mol Biol Cell16:4294β4303
122. Arakawa Y, Bito H, Furuyashiki T, Tsuji T, Takemoto-Kimura S,Kimura K, Nozaki K, Hashimoto N, Narumiya S (2003) Control ofaxon elongation via an SDF-1alpha/Rho/mDia pathway in culturedcerebellar granule neurons. J Cell Biol 161:381β391
123. Sugiarto S, Persson AI, Munoz EG, Waldhuber M, Lamagna C,Andor N, Hanecker P, Ayers-Ringler J, Phillips J, Siu J et al (2011)Asymmetry-defective oligodendrocyte progenitors are glioma pre-cursors. Cancer Cell 20:328β340
124. Goldstein B, Macara IG (2007) The PAR proteins: fundamentalplayers in animal cell polarization. Dev Cell 13:609β622
125. Cote JF, Vuori K (2007) GEF what? Dock180 and related proteinshelp Rac to polarize cells in newways. Trends Cell Biol 17:383β393
126. Riveline D, Zamir E, Balaban NQ, Schwarz US, Ishizaki T,Narumiya S, Kam Z, Geiger B, Bershadsky AD (2001) Focalcontacts as mechanosensors: externally applied local mechanicalforce induces growth of focal contacts by an mDia1-dependent andROCK-independent mechanism. J Cell Biol 153:1175β1186
127. BershadskyAD, BallestremC, Carramusa L, ZilbermanY,Gilquin B,Khochbin S, Alexandrova AY, Verkhovsky AB, Shemesh T, KozlovMM (2006) Assembly and mechanosensory function of focal adhe-sions: experiments and models. Eur J Cell Biol 85:165β173
128. Etienne-Manneville S, Hall A (2001) Integrin-mediated activationof Cdc42 controls cell polarity in migrating astrocytes throughPKCzeta. Cell 106:489β498
Mol Neurobiol
129. Raftopoulou M, Hall A (2004) Cell migration: Rho GTPases leadthe way. Dev Biol 265:23β32
130. NgT, ShimaD, Squire A, Bastiaens PI, Gschmeissner S, HumphriesMJ, Parker PJ (1999) PKCalpha regulates beta1 integrin-dependentcell motility through association and control of integrin traffic.EMBO J 18:3909β3923
131. Han J, Lim CJ, Watanabe N, Soriani A, Ratnikov B,Calderwood DA, Puzon-McLaughlin W, Lafuente EM,Boussiotis VA, Shattil SJ et al (2006) Reconstructing and
deconstructing agonist-induced activation of integrinalphaIIbbeta3. Curr Biol 16:1796β1806
132. Jaken S, Parker PJ (2000) Protein kinase C binding partners.BioEssays: News Rev Mol Cel Dev Biol 22:245β254
133. Denning MF (2012) Specifying protein kinase C functions in mel-anoma. Pigment Cell Melanoma Res 25:466β476
134. Xiao L, Hu C, YangW, Guo D, Li C, Shen W, Liu X, Aijun H, DanW, He C (2013) NMDA receptor couples Rac1-GEFTiam1 to directoligodendrocyte precursor cell migration. Glia 61:2078β2099
