Journal of Neurocytology 31, 423–435 (2002)

The NG2 proteoglycan: Past insightsand future prospectsWILLIAM B. S TALLCUP

The Burnham Institute, Cancer Research Center, 10901 North Torrey Pines Road, La Jolla, CA 92037, USAstallcup@burnham.org

Received 25 January 2001; revised 24 September 2002; accepted 1 October 2002

Abstract

The NG2 chondroitin sulfate proteoglycan is a valuable marker for several types of incompletely-differentiated precursorcells, including oligodendrocyte progenitors in the central nervous system, developing mesenchymal cells in cartilage, muscle,and bone, and pericytes/smooth muscle cells in developing vasculature. In addition to extending our knowledge about thedevelopmental roles of these cell types, current studies on NG2 are also providing information about the molecular mechanismsthrough which the proteoglycan itself influences progenitor development. This research suggests that interaction of NG2 withextracellular and intracellular ligands regulates signaling events that are important for both cell proliferation and cell migration.

In the 20 years since its discovery, the NG2 proteogly-can has become a valuable marker for identification ofoligodendrocyte progenitor cells in the central nervoussystem. Recent work demonstrates that NG2 is also aneffective marker for other immature cell types outsidethe CNS, especially mural cells in developing vascu-lature. Immunomapping studies on the proteoglycantherefore provide a means of elucidating the detailsof cellular development in a variety of tissues. Someprogress is also being made in defining the molecularmechanisms that underlie NG2-mediated signaling inresponse to a variety of stimuli. Hopefully, the comingyears will see an increase in the pace of our acquisitionof knowledge about the functional role played by NG2in the development of both normal and pathologicaltissues.

This article will begin with a review of the founda-tion of work that has been done on expression of theproteoglycan in the central nervous system. It will thensummarize some of the more recent progress made inusing NG2 to study the development of other tissues,stressing common features exhibited by NG2-positivecell types. The discussion will conclude with a descrip-tion of NG2 participation in transmembrane signalingevents, focusing on what has been learned about inter-actions of the proteoglycan with both extracellular andintracellular ligands.

Structure of NG2

Our first reports concerning NG2 identified themolecule only as a serological epitope present on a sub-

population of neural cell lines. Prior to the introduc-tion of monoclonal antibodies, the epitope was initiallycharacterized by a sequentially-absorbed rabbit anti-serum (Wilson et al., 1981; Stallcup, 1981). We were thenable to prepare monoclonal antibodies that appeared torecognize this same epitope (Stallcup et al., 1981). Iden-tification of NG2 as a chondroitin sulfate proteoglycanfollowed two years later as a result of immunoprecipita-tion experiments which showed that NG2 migrated onSDS-PAGE as a pair of components: a well-defined 300kDa band and a much more amorphous high molec-ular weight smear (Stallcup et al., 1983). The proteo-glycan nature of NG2 was revealed by the ability ofthe high molecular weight component to incorporate35SO4 and by the ability of chondroitinase ABC to con-vert this smear quantitatively into the 300 kDa coreprotein. Pulse-chase experiments with 35S-methionineshowed that the core protein (deglycosylated molec-ular weight 260 kDa) was initially synthesized as a275 kDa entity that contained immature, high mannoseoligosaccharide chains. Within 60 minutes of synthesis,the oligosaccharide chains of this NG2 precursor weremodified in the Golgi to yield the mature 300 kDa coreglycoprotein. Appearance of the chondroitin sulfate-containing proteoglycan species was also noticeable af-ter this 60 minute lag period (Stallcup et al., 1983). Basedon the fact that the use of detergent was required for ex-traction of NG2 from cell lines, we also concluded thatthe proteoglycan was likely to be an integral membranecomponent.

Independent of our own studies, experiments inother laboratories had identified a serological epitope

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on human melanoma cells which came to be known asthe high molecular weight melanoma-associated anti-gen or HMW-MAA (Houghton et al., 1982). This entitywas identified as a cell surface chondroitin sulfate pro-teoglycan in the laboratory of Ralph Reisfeld (Bumol& Reisfeld, 1982; Bumol et al., 1984). As we noted atthe time (Stallcup et al., 1983), the similarities betweenNG2 and the melanoma proteoglycan were too obvi-ous to ignore. However, it was not until our first aminoacid sequences became available that the homology be-tween the two molecules was firmly established. Basedon a personal communication from Bob Spiro, we real-ized that the amino-terminal 18 residues of NG2 wereidentical to those of the human melanoma proteogly-can. Thanks almost entirely to the cloning efforts ofAkiko Nishiyama, we published the complete cDNAand primary amino acid sequences for NG2 in 1991(Nishiyama et al., 1991a). At the time the 2325 aminoacid NG2 core protein sequence was one of the largestto be established by molecular cloning. Subsequently,the published sequence of the human melanoma pro-teoglycan firmly established it as the human homologof NG2 (Pluschke et al., 1996). One apparent differencebetween the two molecules in the juxtamembrane por-tion of the extracellular domain was reconciled by ourfinding of three nucleotides that were missing from theoriginal NG2 sequence (Nishiyama et al., 1999a). Themouse NG2 homolog, designated AN2, has also beenidentified (Niehaus et al., 1999; Schneider et al., 2001).Throughout the remainder of this article I will refer tothese molecules collectively as NG2.

The amino acid sequence of NG2 confirmed our orig-inal classification of the proteoglycan as a membrane-spanning molecule. A single 25-residue transmem-brane domain divides the core protein into a relativelyshort 76 amino acid cytoplasmic tail and an extensive2225-residue extracellular domain. We further dividedthis ectodomain into three subdomains, an arrange-ment that initially seemed somewhat arbitrary, but laterproved to have some structural merit. Predictions ofsecondary structure suggested that the NG2 ectomainshould contain two globular domains connected bya more extended central segment (Burg et al., 1997).Electron micrographs of rotary shadowed NG2 prepa-rations reveal just such “dumbbell-shaped’’ structures(Tillet et al., 1997). In our model, domain 1, compris-ing the amino-terminal one-third of the ectodomain, isproposed to be stabilized in a globular conformation byintramolecular disulfide bonding. The central domain2 was proposed to contain the site or sites for additionof chondroitin sulfate chains. Subsequently, we haveshown that NG2 contains only a single chondroitinsulfate substitution at serine-999 (Stallcup & Dahlin-Huppe, 2001). The alpha-helical, amino-terminal por-tion of domain 2 also contains the site for binding col-lagen V and VI (Burg et al., 1997; Tillet et al., 1997). Theglobular juxtamembrane one-third of the ectodomain

was designated domain 3. This segment has been foundto contain sites for proteolysis of NG2 that lead to itsrelease from the cell surface (Nishiyama et al., 1995).Figure 1 presents a schematic representation of our cur-rent ideas about NG2 structure.

Although four repeats resembling the Ca++-binding motif of cadherins are scattered through theectodomain, little similarity was found between NG2and other molecules. In light of the existence of multi-member families of other proteoglycans such as syn-decans, glypicans, aggrecans, etc. (Yamaguchi, 2000,2001), our inability so far to identify other NG2-likemolecules is somewhat surprising. Even in the cyto-plasmic domain, NG2 does not have strong resem-blance to other transmembrane proteins. There are,however, some recognizable motifs that may prove tobe functionally important. At the extreme C-terminus,a QYWV sequence fits the pattern for a PDZ-bindingmotif (Songyang et al., 1997) and may be respon-sible for interaction of NG2 with MUPP1, a multi-PDZ domain-containing cytoplasmic scaffolding pro-tein (Barritt et al., 2000). The NG2 cytoplasmic domainalso contains phosphothreonine residues. Four threo-nine residues appear to be possible candidates for thisphosphorylation (Nishiyama et al., 1991a), but the iden-tity of the phosphorylated residue(s) has not been estab-lished. Finally, although a classical PXXP SH3-bindingmotif is not present (Yu et al., 1994), the C-terminal halfof the NG2 cytoplasmic tail is very rich in prolines. Thesignificance of this remains to be demonstrated.

Expression of NG2 in the central nervous system

The expression pattern of NG2 has always been oneof its most interesting features. Long before we hadany data concerning function, the ability to use NG2as a marker for unique cell types was a strong motiva-tion for our work. Since we originally identified NG2as a surface component of a subset of neural cell lines,we initially tried to understand NG2 distribution bycomparing its expression with the phenotype of thesecells. The phenotype was defined largely by ion flux as-says to detect the presence of voltage-dependent Na+

and K+ channels (Stallcup & Cohn, 1976a, b; Arner &Stallcup, 1981; Wilson et al., 1981). These studies indi-cated that NG2 was never expressed on cell lines thatlacked voltage-dependent ion channels. Instead it wasexpressed by a few cell lines with K+ channels and bya few cell lines with both Na+ and K+ channels. How-ever, cells that were found to be capable of generatinga full-fledged action potential were negative for NG2.Based on these results, we postulated that NG2 expres-sion might be characteristic of immature neural cellscapable of differentiating into either glia or neurons;hence the designation of the molecule as “nerve/glialantigen 2’’ (NG1 did not stand the test of time!). This hy-pothesis seemed plausible in light of the fact that most

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Fig. 1. Structure of NG2. This diagram illustrates the domainstructure of the NG2 proteoglycan. The N-terminal globulardomain 1 is stabilized by intramolecular disulfide bonds (theactual number of disulfides is not known). The central domain2 contains both the type VI collagen-binding site (bold seg-ment) and the single chondroitin sulfate chain (irregular line).The membrane-proximal globular domain 3 contains at leasttwo sites for proteolytic processing of NG2 (arrowheads). Thecytoplasmic tail contains a PDZ-binding motif (filled circle),a proline-rich segment (grid-like motif), and several potentialsites for threonine phosphorylation (open circle).

of our cell lines were derived from tumors that wereinduced in rat embryos by administration of ethylni-trosourea near the end of the second week of gestation(Schubert et al., 1974), a time point at which many im-mature progenitor cells are expected to exist.

Examination of primary cultures prepared from em-bryonic rat forebrain and early postnatal rat cerebel-lum only served to strengthen the impression that NG2-positive cells were incompletely-differentiated, multi-potent progenitors. NG2 was expressed by a populationof small stellate cells that were clearly distinct fromthe bulk of well-defined neurons and glia in the cul-tures (Stallcup, 1981). A percentage of these cells werepositive for the astrocyte-specific marker GFAP, al-though the majority of well-developed GFAP-positiveastrocytes were NG2-negative. Similarly, a portion ofNG2-positive cells could be labeled with tetanus toxin,which binds to ganglioside receptors on neurons. Onceagain, though, the majority of tetanus toxin-positiveneurons were negative for NG2. Thus, NG2-positivecells seemed to exhibit some properties of both neu-rons and glia. However, since NG2 was not expressedby clear-cut neuronal or glial cells, we still felt that NG2might be expressed by immature progenitor cells priorto their differentiation to either of the mature cell types(Stallcup, 1981).

These puzzles were resolved to a large extent by thework of Raff and co-workers (Raff et al., 1983; Raff,1989; Richardson et al., 1990) who identified a popu-lation of progenitor cells in the rat optic nerve that arecapable of giving rise in culture to either oligodendro-cytes or to type II astrocytes (hence the term O2A cells).Re-examining our cell culture data in this context al-lowed us to show that the NG2-positive stellate cells inour cultures were, in fact, O2A cells. When grown inserum, these cells differentiate into GFAP-positive as-trocytes. In serum-free, defined medium they becomegalactocerebroside-positive oligodendrocytes (Stallcup& Beasley, 1987; Levine & Stallcup, 1987). NG2 expres-sion is lost during these differentiation processes, sothat mature astrocytes and oligodendrocytes are nega-tive for NG2. Our initial data concerning NG2 expres-sion by cells with a neuronal phenotype was also ex-plained by the reports from the Raff group. In additionto expressing gangliosides recognized by the A2B5 anti-body, another marker for O2A cells, NG2-positive pro-genitors also express the gangliosides that are respon-sible for tetanus toxin binding. In light of our initialreport that NG2-positive neural cell lines are charac-terized by the presence of voltage-dependent ion chan-nels, it is of interest to note that O2A glial cells are alsoequipped with components characteristic of electricallyactive membranes (Sontheimer et al., 1989; Barres et al.,1990; Steinhauser & Gallo, 1996).

Subsequent work by a number of groups has estab-lished NG2 as an excellent marker for glial progeni-tor cells in vivo (Levine et al., 1993; Nishiyama et al.,

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1996a, 1999b; Trapp et al., 1997; Reynolds & Hardy,1997; Keirstead et al., 1998). What has not been com-pletely resolved is the question of whether the O2Alineage operates in vivo in the same manner seen incell cultures. While it seems clear that NG2-positiveprogenitors give rise to oligodendrocytes in vivo, evi-dence for the derivation of astrocytes from this samelineage is less abundant (Luskin et al., 1993; Grove et al.,1993; Levison & Goldman, 1993; Levine et al., 1993;Nishiyama et al., 1996a; Trapp et al., 1997; Reynolds &Hardy, 1997; Levison et al., 1999). The current fashionis therefore to refer to these cells as oligodendrocyteprogenitors. As outlined below, it remains to be seenwhether this is an oversimplification.

Our results have shown that NG2 is not expressedby the undifferentiated neural progenitor cells in ei-ther the primary or secondary germinal zones of thedeveloping rat central nervous system. Instead, NG2is first expressed by cells that have moved out of thegerminal zones and made an initial commitment to theoligodendrocyte lineage (Stallcup et al., 1983). NG2 ex-pression in these cells is preceded by expression of thePDGF α-receptor, which is critical for progenitor de-velopment and provides another valuable marker forthis cell population (Richardson et al., 1988; Raff et al.,1988; Ellison & deVellis, 1994). Oligodendrocyte pro-genitors expressing both NG2 and PDGF α-receptor canbe seen in the rat CNS as early as embryonic day 16–17(Nishiyama et al., 1996a). From their initial appearancein the ventral spinal cord, the distribution of these pro-genitors expands with time so that by the end of the firstweek postnatally they are located throughout the CNS,in both gray and white matter. The density of the pro-genitors decreases thereafter, as many of them differen-tiate into mature oligodendrocytes and down-regulateboth NG2 and PDGF α-receptor expression just as seenin vitro (Nishiyama et al., 1996a, b).

Large numbers of progenitors are still present in theadult CNS, however. They are clearly distinguishedfrom mature oligodendrocytes and astrocytes by theirfailure to express markers characteristic of either celltype and by their continued expression of both NG2and PDGF α-receptor. The quantity and widespreaddistribution of these cells has led to much speculationas to whether they still function exclusively as oligo-dendrocyte progenitors at this stage of development, orwhether they actually represent a novel, differentiatedglial cell type with a specialized function (Nishiyamaet al., 1999b). In favor of a progenitor role for these cellsis the finding that at least some of them are still mitoticand can still give rise to oligodendrocytes in the adultCNS (Levison et al., 1999). In the adult spinal cord, upto 50% of the mitotically-active cells identified by BrdUincorporation are positive for NG2 (Horner et al., 2000).Moreover, localized populations of NG2-positive cellsincrease in number following physical CNS injury ordemyelinating lesions, once again illustrating their abil-

ity to proliferate (Keirstead et al., 1998; Wu et al., 2000;Zhang et al., 2001; McTigue et al., 2001; Jones et al., 2002).

On the other hand, it has been established for sometime that “adult’’ and “perinatal’’ progenitors have dis-tinct properties in cell culture (Wolswijk & Noble, 1989;Wolswijk et al., 1991; Chan et al., 1990), suggestingthat they may not serve equivalent functions in vivo.NG2-positive cells in adult gray matter have a distinc-tive multi-polar astrocyte-like morphology which hasled some workers to classify them as smooth proto-plasmic astrocytes with few glial filaments (Levine &Card, 1987). These cells are often seen in close prox-imity to neurons, where they may play some type ofsupporting role (Ellsion & deVellis, 1994; Nishiyamaet al., 1996a). In this respect it is significant that NG2immunoreactivity has been localized to synapses inthe hippocampus (Ong & Levine, 1999). Indeed, NG2-positive hippocampal progenitors are even reported toreceive glutamatergic synaptic inputs from pyramidalneurons (Bergles et al., 2000). It is not presently knownwhether this synaptic stimulation plays a role in thedevelopment of the NG2-positive cells or whether thecells perform some type of differentiated neuromod-ulatory function in response to the synaptic input. Inwhite matter an extremely close spatial relationship ex-ists between NG2-positive cells and myelinated axons.Processes from these morphologically-complex NG2-positive cells have been shown to contribute in a spe-cific manner to the cellular make-up of nodes of Ran-vier, perhaps indicative of a differentiated function incontrolling the ultrastructural or ionic environment ofthe node (Butt et al., 1999). Taken together, this informa-tion suggests that NG2-positive cells in the adult CNSmay not be a homogeneous population of progenitorsand that our understanding of their role in the nervoussystem is incomplete.

Expression of NG2 outside the centralnervous system

Far from being restricted to oligodendrocyte progeni-tors in the CNS, NG2 has a widespread distribution inmany other tissues. In the peripheral nervous system,for example, the mouse NG2 homolog AN2 is reportedto be present on Schwann cells (Schneider et al., 2001).This does not agree with other published work (Martinet al., 2001) or with our own unpublished studies onrat sciatic nerve which suggests instead that NG2 is ex-pressed by perineurial cells. Preliminary evidence sug-gests that NG2 is substantially up-regulated followingsciatic nerve injury, in parallel to what has been seen inthe CNS (Zhang et al., 2001).

Outside the nervous system, NG2 is expressed by avariety of mesenchymal cell types. In the developingrat limb, we have shown that NG2 expression is notseen on undifferentiated cells of the embryonic day14 limb bud, but is up-regulated in condensations of

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mesenchymal cells that represent immature chondrob-lasts (Nishiyama et al., 1991b). These NG2-positive car-tilagenous condensations appear in a proximal to distalgradient along the developing limb, beginning with thehumerus or femur and culminating in the appearanceof NG2 in the digits by embryonic day 17. As chon-droblasts differentiate into mature chondrocytes, NG2expression is largely down-regulated. This pattern pro-vides a clear parallel to the situation we have describedfor oligodendrocyte progenitor cells in the CNS. In bothcases NG2 is not expressed by undifferentiated pro-genitors, but is expressed by a more developmentallyrestricted population of partially-differentiated pro-genitor cells that have made an initial commitment to aparticular lineage. These restricted progenitors are stillmitotic and are likely to retain a degree of develop-mental plasticity. When these cells undergo terminaldifferentiation and become quiescent, NG2 expres-sion is down-regulated. Recent work with othermesenchymal cell types suggests that NG2 is alsoexpressed in developing skeletal muscle and bone(Petrini et al., 2003; Fukushi et al., 2003). Clearly, muchremains to be done here to determine whether NG2expression in these tissues exhibits the same type ofdevelopmental regulation seen in chondroblasts andoligodendrocyte progenitors.

Much of our recent work has focused on NG2 ex-pression in developing vasculature. NG2 promises toprovide a tool for the study of cardiovascular develop-ment that will be even more useful than it has been forthe study of oligodendrocyte development in the CNS.Although some controversy has existed in the litera-ture over the cellular specificity of NG2 expression inthe vasculature, our most recent studies demonstrateconvincingly that NG2 is not present on vascular en-dothelial cells, but instead is restricted to the surfaces ofvascular mesenchymal/mural cells. This is true of vas-culature formed either by the process of vasculogenesisor by the process of angiogenesis. Thus, in the develop-ing heart NG2 is expressed by cardiomyocytes, in largevessels it is expressed by smooth muscle cells, and inmicrovasculature it is present on pericytes (Ozerdemet al., 2001). NG2 expression by vascular pericytes oc-curs both in normally developing microvessels and inmicrovessels that develop during pathological angio-genesis (Schlingemann et al., 1990; Burg et al., 1999;Ozerdem et al., 2002).

Just as oligodendrocyte progenitors are charac-terized by their co-expression of NG2 and PDGFα-receptor, microvascular pericytes are characterizedby their co-expression of NG2 and PDGF β-receptor.PDGF β-receptor is just as important to the devel-opment of pericytes (Lindahl et al., 1997; Hellstromet al., 1999) as PDGF α-receptor is to the developmentof oligodendrocyte progenitors. The ability to recog-nize and study pericytes via use of these dual markersshould allow a tremendous expansion of our knowl-

edge concerning these poorly understood cells. Theyare notorious for the apparent heterogeneity of their de-velopmental origins and for their developmental plas-ticity (Sims et al., 2000; Schor & Canfield, 1998; Allt& Lawrenson, 2001). Lessons learned from studies ofpericyte biology may be applicable to understandingthe possible heterogeneity, plasticity, and specializationof oligodendrocyte progenitors in the CNS, and viceversa. For example, capillaries in the CNS are denselyinvested by pericytes in a well-regulated manner, sothat in looking at developing CNS tissue one encoun-ters numerous NG2-positive pericytes in addition toNG2-positive oligodendrocyte progenitors (Nishiyamaet al., 1996a). As the brain matures and CNS capillar-ies become stable and quiescent, pericyte expressionof NG2 decreases (Miller et al., 1995), as we have seenfor other NG2-positive cell types. In contrast, tumorneovasculature exhibits heterogeneous and poorly-regulated investment of endothelial tubes by pericytes.Figure 2 shows examples of capillaries in a mouse mam-mary tumor that are incompletely invested by peri-cytes, as judged by double staining for the endothelialcell marker CD31 (b, d, f) and the pericyte markers NG2(a, c, g) and PDGF β-receptor (e, h). The lack of preciseoverlap between pericytes and endothelial cells can beseen in panels c, d (NG2 and CD31) and e, f (PDGFβ-receptor and CD31). Pericyte co-expression of bothNG2 and PDGF β-receptor is illustrated in panels g andh. It will be of considerable interest to determine the ex-tent to which heterogeneous pericyte investment is re-sponsible for the unregulated growth of tumor vessels.

Functions of NG2

Very early in our work, the expression pattern of NG2on immature progenitor cells suggested to us thatthe proteoglycan might contribute to processes suchas cell proliferation and motility which are critical toprogenitor biology. This impression was strengthenedby the finding that NG2 is often re-expressed by tu-mor cells, which are usually characterized by increasedproliferation and migration. Indeed, we have shownthat expression of NG2 increases the tumorigenic andmetastatic properties of mouse melanoma cells (Burget al., 1998). In addition to its wide expression onmelanomas (Real et al., 1985), NG2 is also found onglioblastomas (Schrappe et al., 1989; Chekenya et al.,1999), chondrosarcomas (Leger et al., 1994), and lym-phoid leukemias (Smith et al., 1996). Subsequent studiesin our lab and others have tried to identify mechanismsby which NG2 might influence aspects of cell behav-ior such as proliferation and migration. A large part ofthis research has been concerned with defining the in-teractions of NG2 with extracellular and intracellularbinding partners in an attempt to establish the molecu-lar basis for an interface between NG2 and cytoplasmicsignaling processes.

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Fig. 2. NG2 expression in mammary tumor vasculature. NG2 localization in vasculature was studied in sections of mammarytumors taken from 90 day-old female MMTV/PyMT transgenic mice. Sections were double-stained for NG2 and the endothelialmarker CD31 (a, b and c, d), for PDGF β-receptor and CD31 (e, f), and for NG2 and PDGF β-receptor (g, h). At low magnification(a, b. bar = 20 µm) NG2 and CD31 can both be seen in the tumor vasculature. At higher magnification, however (c, d. bar =10 µm), the lack of precise co-localization for the two markers is evident. NG2-positive pericytes can be found in vessels thatappear to be deficient in CD31-positive endothelial cells. This same pattern is seen in panels e and f, where PDGF β-receptorpositive pericytes and CD31-positive endothelial cells are often distributed in a non-overlapping fashion. In contrast, excellentco-localization is seen for the two pericyte markers (g and h).

EXTRACELLULAR LIGANDS FOR NG2

We have shown that NG2 is capable of binding withfairly high affinity to the two growth factors, bFGFand PDGF-AA (Goretzki et al., 1999). This growth fac-tor binding activity of NG2 does not appear to de-pend on the presence of the chondroitin sulfate chain,since chondroitin sulfate-free NG2 core protein inter-acts normally with both factors. This is in contrast tothe behavior of heparan sulfate proteoglycans, whichinteract with bFGF via their glycosaminoglycan chains(Rapraeger et al., 1995). It is of considerable interest tous that bFGF and PDGF-AA are critical mitogens foroligodendrocyte progenitors (Bogler et al., 1990; Baronet al., 2000), and we have speculated that NG2 expres-sion is important for the ability of progenitors to re-spond to these growth factors. Evidence for this hypoth-esis comes from experiments showing that progenitorstreated in vitro with blocking antibodies against NG2fail to proliferate normally in response to growth fac-tors (Nishiyama et al., 1996b). Additional support hasrecently been obtained from preliminary cell culture ex-periments comparing oligodendrocyte progenitors de-rived from wild-type and NG2 knockout mice (Fig. 3).Treatment of wild-type progenitors with bFGF and

PDGF-AA maintains their undifferentiated A2B5+O4−

phenotype over the course of several days. Over thissame time period, NG2-null progenitors appear to beinsensitive to the growth factors and progress to theA2B5−O4+ phenotype characteristic of initial differen-tiation along the oligodendrocyte pathway.

Additional studies with aortic smooth muscle cellsalso reflect the importance of NG2 in potentiating cellu-lar responses to PDGF-AA. In contrast to microvascularpericytes which express PDGF β-receptor, macrovascu-lar smooth muscle cells express PDGF α-receptor anddepend on signaling via this receptor for proper devel-opment (Schatteman et al., 1995). Blocking antibodiesagainst NG2 inhibit both mitosis and migration of ratsmooth muscle cells in response to PDGF-AA (Grako& Stallcup, 1995). Comparisons of smooth muscle cellsfrom wild-type and NG2 knockout mice also show thiseffect. The defect in the NG2-null cells appears to oc-cur at the level of activation of the PDGF α-receptorby PDGF-AA, since receptor autophosphorylation inresponse to the growth factor is not readily observedin these cells (Grako et al., 1999). Based on the avail-able data our working hypothesis is that, like a numberof other proteoglycans, NG2 may potentiate the effect

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Fig. 3. Differentiation of oligodendrocyte progenitors from wild-type and NG2-null mice. Oligodendrocyte progenitors derivedfrom the cerebral cortex of 1 day-old wild-type (wt) and NG2-null mice (ko) were cultured in N2 medium, either unsupplemented(control) or supplemented with 20 ng/ml PDGF-AA (+AA), 20 ng/ml bFGF (+FGF), or a combination of both growth factors(+AA+FGF). After 3 days, immunofluoresence staining was used to assess the percentage of cells in each culture that expressedthe markers A2B5, O4, and GC (galactocerebroside). (Percentages do not always add up 100% since individual cells sometimesexpress more than one marker.) In the absence of added growth factors both sets of cultures were characterized by largepercentages of O4+ cells, indicative of initial differentiation to pre-oligodendrocytes. In the presence of both growth factors, themajority of wild-type cells were still positive for the A2B5 marker, indicating that they had largely retained the phenotype ofundifferentiated progenitors. In contrast, the presence of both growth factors had no obvious effect on the NG2-null population.These cultures were still dominated by cells with the partially-differentiated O4+ phenotype. These results suggest a role forNG2 in the responsiveness of oligodendrocyte progenitors to PDGF-AA and bFGF.

of growth factors by sequestering them at the cell sur-face and presenting them to their respective signalingreceptors.

Matrix metalloproteinases (MMPs) appear to rep-resent another type of cell surface ligand for NG2.Membrane-type 3 matrix metalloproteinase (MT3-MMP) has been shown to form a complex with NG2that is critical for the ability of melanoma cells to de-grade and invade a type I collagen-containing matrix(Iida et al., 2001). Following this same theme, we havedemonstrated the ability of the NG2 ectodomain tointeract with the kringle domains of proteins such asplasminogen (Goretzki et al., 2000). The importance ofthe NG2/plasminogen interaction is illustrated by theenhanced ability of plasminogen activator to convertplasminogen to active plasmin when the zymogen iscomplexed with NG2. This activation to plasmin is acritical event for the motility of both normal and neo-plastic cells. The binding of NG2 to kringle domains

has an additional implication in the case of plasmino-gen. The anti-angiogenic protein angiostatin is a prote-olytic fragment of plasminogen that retains four of thekringle domains (O’Reilly et al., 1994; Cao et al., 1996),and therefore also exhibits strong binding to NG2. Bind-ing to NG2 blocks the ability of angiostatin to inhibitendothelial cell proliferation (Goretzki et al., 2000), pro-viding another mechanism by which NG2 may be ableto regulate angiogenesis.

In each of the above examples, NG2 is not thoughtto serve as a primary signaling molecule, but insteadfunctions in a role that is auxilliary to the actions ofother receptors or proteases. In other instances NG2itself may act as the signal transducing molecule, asappears to be the case in interactions of the proteogly-can with certain extracellular matrix components (Burget al., 1996). By far the best characterized of these inter-actions is that of NG2 with type VI collagen. Indicativeof a viable interaction between the two molecules, NG2

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is co-localized with collagen VI in a number of develop-ing tissues, including vasculature (Stallcup et al., 1990;Rand et al., 1993). We have shown that NG2 functionsvery effectively as a cell surface receptor for anchor-ing type VI collagen to the cell surface (Stallcup et al.,1990; Nishiyama & Stallcup, 1993). The functional sig-nificance of this NG2/collagen VI interaction is sug-gested by an increased ability of NG2-transfected cellsto migrate in response to collagen VI (Burg et al., 1997).Experiments with NG2 fragments and with NG2 dele-tion mutants both indicate that the binding site for col-lagen VI lies in the N-terminal half of the extended cen-tral portion (domain 2) of the NG2 ectodomain (Burget al., 1997; Tillet et al., 1997). In rotary shadowed prepa-rations, type VI collagen can be seen tightly alignedwith this central domain, spanning the space betweenthe two extracellular globular domains of the proteo-glycan (Tillet et al., 1997).

Even though the single NG2 chondroitin sulfatechain at ser-999 is located within the designated col-lagen VI binding domain (Stallcup & Dahlin-Huppe,2001), this substitution appears to have little effecton NG2/collagen VI binding. Chondroitin sulfate-freeNG2 core protein interacts normally with type VI col-lagen in solid phase binding assays (Burg et al., 1996;Tillet et al., 1997), and anchorage of collagen VI occursnormally on cells expressing a chondroitin sulfate-freemutant of NG2 (unpublished data). The interaction ofNG2 with collagen VI also appears to trigger cellularresponses that do not require a contribution from β1integrins, which are also known to serve as collagenVI receptors. In GD25 cells which are deficient in theβ1 subunit and therefore lack the ability to express β1-containing heterodimers on the cell surface, the pres-ence of NG2 still promotes cell spreading on type VIcollagen-coated surfaces (Tillet et al., 2002).

NG2-MEDIATED TRANSMEMBRANE SIGNALING

The apparent ability of NG2 to promote cell spreadingand cell motility in response to collagen VI indicatesthat engagement of NG2 must trigger transmembranesignaling events that lead to dynamic rearrangementsof the actin cytoskeleton. Such a role for the pro-teoglycan had been suggested early on by experi-ments showing that treatment with anti-NG2 anti-bodies could inhibit the attachment, spreading, andgrowth of melanoma cells (Bumol et al., 1984; Harper& Reisfeld, 1987). Subsequent work suggested an in-teraction of NG2 with the cytoskeleton (Lin et al.,1996a, b) and demonstrated that engagement of NG2by specific antibodies activated signaling events in-volved in promoting melanoma cell spreading (Iidaet al., 1995). Using monoclonal antibody-coated sur-faces as model substrata, we were able to show thatNG2-mediated spreading and migration requires thepresence of the NG2 cytoplasmic domain (Fang et al.,

1999). This seemed consistent with the idea that, as amembrane-spanning protein, NG2 itself might be ca-pable of interacting with the cytoskeleton and/or withcytoplasmic signaling machinery. This lack of depen-dence on an auxilliary signaling molecule is also evi-dent in the aforementioned ability of NG2 to respondto collagen VI even in the absence of β1 integrins. Al-though it remains for us to define motifs in the NG2 cy-toplasmic tail that are responsible for interacting withsignaling pathways, we found that cell migration wasabrogated by elimination of the carboxy-terminal halfof the cytoplasmic domain. This segment contains thePDZ-binding motif, the proline-rich segment, and twoof the threonines that are candidates for phosphoryla-tion. More focused mutations will be required to evalu-ate the respective roles of these individual elements inNG2-mediated signaling.

We also noted that, depending on the extracellularepitope recognized by the substratum, engagement ofNG2 could promote the extension of either lamellipo-dia or filopodia (Fang et al., 1999). These two types ofmorphological behavior are highly suggestive of theinvolvement of the small GTPases rac and cdc42, re-spectively, in the spreading process (Ridley et al., 1992;Nobes & Hall, 1995). The activation of cdc42 has nowbeen directly demonstrated following the engagementof NG2 on human melanoma cells (Eisenmann et al.,1999). Using human astrocytoma cells, we have recentlyshown that rac is activated to its GTP-bound form dur-ing cell spreading on surfaces coated with a specificNG2 monoclonal antibody. In addition, we employedtransfection with the dominant negative form of racto confirm the involvement of this small GTPase inNG2-mediated cell spreading (Majumdar et al., 2002).The activation of cdc42 and rac can in some respectsbe regarded as parallel processes, since both are ableto interact with p21-activated kinases (PAKs) to initi-ate additional downstream signaling events associatedwith changes in cell motility and morphology (Manseret al., 1994; Sells et al., 1999). The aforementioned stud-ies of Eisenmann et al. (1999) and Majumdar et al. (2002)also implicate p130cas as an essential component ofboth the cdc42 and rac-mediated signaling cascades.Since p130cas serves a key role in coupling transmem-brane signaling to cell motility (Klemke et al., 1998; Caryet al., 1998), it will be important to establish its posi-tion relative to the small GTPases in the NG2-mediatedpathway.

It seems certain that additional adaptors will be re-quired to link NG2 with these signaling pathways. TheMUPP1 cytoplasmic scaffolding protein is one candi-date for such a role (Barritt et al., 2000). The C-terminalPDZ-binding motif of NG2 binds to the first of 13 PDZdomains in MUPP1. Interaction of other proteins (mem-brane spanning components such as the serotonin re-ceptor or cytoplasmic components such as the APCtumor suppressor) with the remaining PDZ domains

The NG2 proteoglycan 431

of the MUPP1 scaffold may be critical for clusteringNG2 together with signaling components at appropri-ate subcellular locations. In this respect it is very inter-esting that NG2 is highly localized to retraction fibersthat form at the trailing edges of motile cells (Lin et al.,1996b). We have speculated that NG2 in retraction fibersmight activate signaling events that facilitate release ofthe trailing edge from the substratum, thus increasingcell motility. Recently, we have shown that expression ofNG2 greatly enhances the tendency of cells to form re-traction fibers and to assume the polarized morphologycharacteristic of motile cells: i.e. actin-rich lamellipodiaat one pole and retraction fibers at the other (Stallcup &Dahlin-Huppe, 2001). NG2 is thus part of the molec-ular segregation of specific components that distin-guishes the two poles of the cell (Gomez-Mouton et al.,2001).

In addition to a requirement for the cytoplasmic do-main for targeting of NG2 to retraction fibers, we alsofound the chondroitin sulfate chain to be necessaryfor correct targeting to this membrane microdomain(Stallcup & Dahlin-Huppe, 2001). Although initiallysurprising to us in light of the lack of effect of the chon-droitin sulfate chain on ligand binding, this findingnevertheless is consistent with a number of recent re-ports on the importance of glycosyl groups in generaland glycosaminoglycans in particular for controllingthe sorting of proteins to specific subcellular locations(Scheiffele et al., 1995; Mertens et al., 1996; Gut et al.,1998; Prydz & Dalen, 2000). The role of the chondroitinsulfate chain in targeting NG2 to the membrane, andthe subsequent ability of NG2 to trigger the formationof retraction fibers and the acquisition of cell polarity,will be focal points for the further understanding ofsignaling events mediated by this proteoglycan.

Acknowledgments

We are very grateful for support over the years by NIHgrants RO1 NS21990, RO1 NS32767, RO1 AR44400, RO1CA95287, and PO1 HD25938.

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