Neurochemical Research, Vol. 23, No. 3, 1998, pp. 291-303

The Role of Globo-Series Glycolipids in Neuronal Cell Differentiation—A Review* Toshio Ariga1 and Robert K. Yu1-2 (Accepted February 6, 1997)

Alterations in glycolipid composition as well as glycosyltransferase activities during cellular differentiation and growth have been well documented. However, the underlying mechanisms for the regulation of glycolipid expression remain obscure. One of the major obstacles has been the lack of a well defined model system for studying these phenomena. We have chosen PC 12 pheochromocytoma cells as a model because (a) the properties of these cells have been well characterized, and (b) they respond to nerve growth factor (NGF) by differentiating into sympathetic-like neurons and are amenable to well-controlled experimentation. Thus, PC12 cells represent a suitable model for studying changes in glycolipid metabolism in relation to cellular differentiation. We have previously shown that subcloned PC12 cells accumulate a unique series of globo-series neutral glycolipids which are not expressed in parental PC12 cells. This unusual change in glycolipid distribution is accompanied by changes in the activities of specific glycosyltransferases involved in their synthesis and is correlated with neuritogenesis and/or cellular differentiation in this cell line. We have further demonstrated that changes in the glycosyltransferase activities may be modulated by the phosphorylation states of the cells via protein kinase systems. We conclude that these unique globo-series glycolipids may play a functional role in the initiation and/or maintenance of neurite outgrowth in PC12 cells.

KEY WORDS: Cellular differentiation; globo-series glycolipids; gangliosides; PC12 pheochromocytoma cells; protein kinases; glycolipid antibodies; signal transduction.

transformation (for reviews: see 7, 9-11). These changes correlate well with the putative functions of glycolipids, such as their role in regulation of cell-cell interactions, cellular differentiation and proliferation, as well as oncogenesis (7,12-16). Thus, glycolipids have gained much attention for their functions in normal as well as malignant cells. Although the mechanisms underlying control of cellular growth and differentiation are poorly understood, there are numerous reports indicating that exog-

INTRODUCTION Glycolipids are important constituents of the plasma membranes in virtually all vertebrate tissues (3-6). They constitute part of the glycoconjugate network extending from the membrane surface and are important in governing the properties and functions of cells (7,8). During the past two decades, considerable evidence has accumulated indicating that the composition of glycolipids can undergo remarkable changes during cellular growth, differentiation, and oncogenic

Abbreviations: PC12 cells, PC12 pheochromocytoma cells; NGF, nerve growth factor; FRK, forskolin; GalGb3, Galctl-SGalal4Galpl-4Glc|31-rCer. The nomenclature used for glycolipids is based on that recommended by the IUPAC-IUB Commission on Biochemical Nomenclature [1] except gangliosides, which are abbreviated according to Svennerholm F21.

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Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298-0614. 2 Address reprint requests to Dr. Robert K. Yu. * Special issue dedicated to Dr. William T. Norton.

291 0364-3190/98/0300-0291S15.00/0 © 1998 Plenum Publishing Corporation

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Fig. 1. Two possible modes of action of NGF on neurite outgrowth in PC 12 pheochromocytoma cells. PC12D cells closely resemble "NGF-primed" PC12 cells because they respond to differentiation agents such as NGF much faster than the parental PC 12 cells. This is likely due to the fact that PC12D cells are more differentiated than the parental PC12 cells. (Reproduced from 58).

Fig. 2. Time course of neurite outgrowth in PC12 cells. PC12D cells respond to differentiation agents such as NGF much faster than the parental PC12 cells.

enously administered glycolipids or their antibodies in culture can affect cellular growth and differentiation. Exogenously administrated glycolipids have been shown to have profound effects on cellular events in a variety of cells, including neurons (5,17-20) and glia (21,22). These studies have promoted an intense interest in investigating the neurotrophic and neuritogenic properties of glycolipids in a variety of in vitro and in vivo conditions (for a review, see 23). For example, exogenously administered GM1 is known to stimulate the neurite outgrowth of neuroblastoma cells (24-27) and GM3 induces human promyelocytic leukemia HL-60 cells to monocytic differentiation (28,29). Yim et al. (22) re-

Ariga and Yu ported an effect of GM3 on the outgrowth of cellular processes and induction of glycoproteins in cultured oligodendrocytes. Recently we found that exogenously administered GM3 promoted protein kinase A (PKA) activity and inhibited protein kinase C (PKC) activity in cultured brain microvascular endothelial cells (30); this observation may present a potential mechanism for regulation of endothelial cell functions. GM3 monoclonal antibody can trigger differentiation of Neuro-2a cells in culture (31). These observations implicate glycolipids in diverse cellular functions, which may be triggered by modulation of transmembrane signal transduction leading to regulation of cellular proliferation and differentiation (8,11,32). In this review, we describe some of the critical changes in glycolipid composition and their biosynthesis in an attempt to gain a better understanding of the molecular and biochemical bases underlying these changes in a neuronal cell line, the PC12 cells. 1. Glycolipids in the Pheochromocytoma Cell Line, PCI2. PC12 pheochromocytoma cells are a clonal cell line derived from rat adrenal medullary pheochromocytomas (33) which share some properties of normal adrenal chromaffin cells originating from sympathetic nerve ganglia. They have the capacity to store and secrete catecholamines in response to cholinergic agonists and increasing extracellular K+ ion concentrations (34). In addition, these cells display characteristic responses to nerve growth factor (NGF) such as neurite outgrowth, increased cellular adhesion, and increased levels of choline acetyltransferase, acetylcholine esterases, and norepinephrine uptake sites (35,36). Therefore, PC 12 cells have been subjected to intensive investigations as a model for studying neuronal function and differentiation (35). In addition to the parental cell line, several subclones of PC 12 cells have been described. The subcloned PC12h cells established by Dr. Hatanaka (37) are capable of short neurite formation even in the absence of nerve growth factor (NGF) and exhibit NGF-stimulated tyrosine hydroxylase activation. PC12D cells are another subline of PC12 cells; however, they differ from the parental PC12 cells in that they extend neurites very quickly (within 24 h rather than several days) in response to not only NGF, but also to cyclic AMP-enhancing agents. Hence, they are similar to "NGF-primed cells" (38) (Figs. 1 and 2). These subclones are considered to be more differentiated than the parental cells in morphology because of their enhanced ability for neurite outgrowth even in the basal condition. The structures of complex glycolipids in PC 12 cells have been studied. Early studies by us (39) and others (40,41) revealed that PC 12 cells contain many ganglio-

The Role of Globo-Series Glycolipids in Neuronal Cell Differentiation—A Review

Fig. 3. Structures of fucosylated gangliosides in PC 12 cells.

Fig. 4. Structures of unique globo-series glycolipids accumulated in subcloned PC 12 cells

Fig. 5. Phase-contrast micrographs of PC12 cells, a, parental PC12 cells; b, subcloned PC12D cells; c, subcloned PC12h cells. Bar indicates 50 (j.m.

sides having unusual chromatographic mobilities as compared to brain-type gangliosides, and these gangliosides might contain fucose. Moreover, NGF treatment produced an almost 3-fold increase in the incorporation of labeled [3H]-fucose into the ganglioside fractions (42,43) and induced the increased labeling of polysialogangliosides (44). These fucosylated gangliosides were characterized by us as fucosylated GM1 (Fuc-GMl), fucosyiated GDlb (Fuc-GDlb), and the corresponding fucosylated gangliosides with blood group B determinant (BGM1 and BGDlb, respectively) (9,39) (Fig. 3). These

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observations prompted us to suggest that their up-regulation may be induced by activation of a-fucosyltransferase (FucT) and/or a-galactosyltransferase (39). This view was shared by Schwaiting et al. (42) who further indicated that the increased levels of fucosylated gangliosides in response to NGF might reflect a molecular event underlying PC 12 cell differentiation. In addition to the above ganglioside species, globotetraosylceramide (globoside) is also a major neutral glycolipid in the parental PC12 cells maintained in culture (9,42). After prolonged treatment of the cells with NGF, the levels of complex neutral glycolipids were elevated (42), but not those of phospholipids (9,43). Moreover, we recently found that the glycolipid composition changed dramatically during subcloning and that the subcloned PC12h cells and PC12D cells accumulated an unusual neutral glycolipid: Gala 1-3 Gala l-4Gal (314Glcpl-l'Cer (Galal-3Gb3), which was not expressed in the parental PC 12 cells in appreciable amounts (45). In addition, the subcloned PC 12 cells accumulated other unusual neutral glycolipids, some of which carried repetitive terminal Gala 1-3 residues (globo-series glycolipids) (46): Galal-3Galal-3Gb3Cer, Galal3Galal-3Galal-3Gb3Cer, Fucal-2Galal-3Gb3Cer, and GalNAc(M-3Galal-3Gb3Cer (Fig. 4). Since the subcloned PC12h and PC12D cells are more differentiated than the parental PC 12 cells (Fig. 5), these globoseries glycolipids may play a critical role in the initiation/maintenance of neurite outgrowth, at least in these cell systems and their up-regulation during cellular differentiation is most likely a result of the activation of several related glycosyltransferases (9,46). 2. Glycosyltransferases in Cellular Differentiation and Neuritogenesis. Alteration in the biosynthesis of the sugar moieties of glycoconjugates is a hallmark of transformed cells (7). Over 100 glycosyltransferases are required for the synthesis of carbohydrate structures on glycoproteins and glycolipids. Many reports have documented that the glycosyltransferases, which are present primarily in the Golgi apparatus (47,48) and occasionally on the cell surface (49) may be activated during various stages of cellular differentiation which may in turn affect cellular recognition and other cellular behavior (7). The activities of sialyltransferase (ST), galactosyltransferase (GalT), and fucosyltransferase (FucT) have been found to be elevated in the sera of patients with malignant tumors (12), suggesting that these glycosyltransferases may serve as valuable markers for malignant rumors. Furthermore, the expression of glycolipids may be controlled by differential expression of specific glycosyltransferase genes or post-translational regulation of these activities (50).

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Fig. 6. Immunostaining of PC12D cells with a polyclonal anti-GalGb3 antibody, a and b, Phase-contrast micrograph and the corresponding fluorescence micrograph, respectively, of untreated cells. PC12D cells were 100% immunoreactive against this antibody, c and d, Phasecontrast micrograph and the corresponding fluorescence micrograph, respectively, of PC12D cells after 24 h of NGF treatment. Most of the cells have long varicose processes (c). Strong immunoreactivity can be seen along these processes including growth cones (arrowheads in d). Fine cell processes are also positively stained (arrow). Bar indicates 50 |im. (Reproduced from 58).

Fig. 7. Immunostaining of parental PC 12 cells with a polyclonal anti-GalGb3 antibody, a and b, Phase-contrast micrograph and the corresponding fluorescence micrograph, respectively, of untreated cells. No definitely immunoreactive cells are observed in b; c and d, Phase-contrast micrograph and the corresponding fluorescence migrograph, respectively, of PC 12 cells after 96 h of NGF treatment. A substantial number of PC12 cells are immunoreactive against this antibody after NGF exposure, but some nonreactive cells (asterisks) are still recognized. Both immunoreactive (arrowheads) and nonimmunoreactive (arrows) cellular processes can be seen. None of the intense immunoreactivity at the growth cone (arrowheads) is observed. Bar indicates 50 |0.m. (Reproduced from 58).

To investigate the metabolic basis for the synthesis of Galod-3Gb3, a major neutral glycolipid that accumulates in the differentiated subcloned PC12h and

Ariga and Yu PC12D cells, we assayed the UDP-galactose: globotriaosylceramide cd-3-galactosyltransferase (GalGb3-synthase) activity using cell free homogenates as the enzyme source and Gb3 and [14C]-UDP-galactose as the substrates. Upon isolation of the neutral glycolipids from the reaction mixture, a radioactive band corresponding to authentic Galcd-3Gb3, was separated by thin-layer chromatography (TLC) (51). We found that the enzyme activities in PC12h and PC12D cells were increased twofold as compared to that in the less differentiated parental PC 12 cells (51). This may explain the accumulation of large amounts of globo-series glycolipids in these subcloned cell lines (46). The increase of globo-series glycolipids and changes in the glycosyltransferase activity may underlie differentiation of PC 12 cells (51). 2.1. Effects of Inducers of Differentiation on Glycosyltransferase Activities. The glycolipid composition of cells is known to change dramatically during cellular differentiation (for reviews see 8, 11, 52). NGF and forskolin (FRK) are known to induce cellular differentiation, including process formation in PC 12 cells. At the stage of cellular differentiation, neuronal cells require the addition of newly formed membrane materials, including proteins and lipids which are synthesized at the perikarya and transported to the growing tips of neuronal processes, namely, the growth cones. Thus, substances localized and concentrated at the growth cones, i.e., GAP-43 (53,54), |3-galactosyltransferase (55), SNAP-25 (56), and pp60c-src/pp60c-src-N (57), are considered to be closely related to neuronal development, regeneration, and/or differentiation. After PC12D cells were treated with NGF or FRK for 24 hrs, we found that the whole cell processes including varicoses and growth cones became strongly immunoreactive with a rabbit polyclonal antibody against the globo-series neutral glycolipid, Galod-3Gb3 (58) (Fig. 6). Since in the neurites Galcd3Gb3 is expressed at the growth cones, it suggests that this unique glycolipid may be critical to neuronal growth, at least in PC12D cells. Similarly, when the parental PC 12 cells, which are considered to be less differentiated that the PC12D cells, were induced to differentiate by treatment with NGF/FRK for 4 days, the percentage of anti-GalGb3 positive cells increased from less than 5% to 20% (Fig. 7). In addition, the activity of GalGb3-synthase increased significantly in PC12D cells during NGF- and FRK-induced differentiation and the maximal activity occurred at 8 and 12 hr post-treatment, respectively (Fig. 8). Hence, the increase of globoseries glycolipids, especially Galcd-3Gb3, may be a general response to membrane synthesis accompanying cellular differentiation in PC 12 cells. The unique distribution of the globo-series glycolipids in the extending

The Role of Globo-Series Glycolipids in Neuronal Cell Differentiation—A Review

Fig. 8. Time course for induction of GalGbS-synthase activity. The enzymes activity was determined at 4, 8, 12, and 24h using NGF or FRK. in PC12D cells. Filled bars represent percentage of activities after NGF treatment, hatched bars represent percentage of activities after FRK. treatment, and empty bars represent control (100%). The values are expressed as mean percentages of normal control (untreated PC12D cells; n = 5 individual samples). *p < 0.01; **p < 0.05.

growth cones of the neurites may be one of the essential factors in the initiation and elongation of neurites. This concept is supported by the fact that a concentration gradient exists in neutral glycolipids of growth cones, but not of mixed membranes, isolated from 16- to 18-dayold fetal rat brains (59,60). This implies that a functional specification of the neutral glycolipids in axonal growth guidance. 2.2. Expression of Unique Globo-Series Glycolipid Antigens in Rat Tissues. We reported that cultured PC12h cells contained a unique GalGbS-synthase which catalyzes the synthesis of Galcd-3Gb3 from Gb3. In order to examine the generality of this finding, we investigated other tissues and cells which also arise from a neural crest origin. For this purpose, we examined GalGbS-synthase activity in rat tissues including dorsal root ganglia (DRG) (61). Among the E-16 embryonic tissues examined, DRG had the highest enzyme activity (226 ± 14 pmol/h/mg protein) compared with other tissues such as brain (37.4 ± 2.2), spinal cord (36.6 ± 6.6), and liver (17.7 ± 0.7). The enzyme activities in one-day postnatal rat tissues are as follows: cerebrum (8.4 pmol/h/mg protein), cerebellum (10.6), liver (5.3), kidney (119), and spleen (29.9). These results suggest that this enzyme activity is expressed in a tissue-specific manner and is especially enriched in embryonic DRG.

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To further define the spatial and temporal expression of these globo-series glycolipids in DRG, we examined cultured DRG from El6 rat embryos employing a polyclonal antibody which reacted with Galcd-3Gb3. DRG cells were examined after 5, 8, 12 and 15 days in culture. Immunostaining was confined primarily to the neuronal cell bodies while Schwann cells and fibroblasts were negative. The staining was very weak on day 5, began to intensify by day 8, peaked at day 12 and remained constant at day 15 (Fig. 9). This expression pattern coincides with the intense neurite outgrowth in DRG cultures and strongly suggests the involvement of Galod-3Gb3 in neuritogenesis. Since glycolipids are implicated in cellular adhesion and adhesion molecules are known to influence the process of differentiation (6,62,63), these unique globo-series glycolipids are likely involved in neurite initiation/elongation and cellular differentiation in cultured PC 12 cells as well as DRG. Furthermore, their synthesis may be regulated by the modulation of the corresponding glycosyltransferases during these processes. These results also suggest that globo-series glycolipids may be a differentiation marker for subcloned PC12 cells. 3. Glycolipids as Second Messenger of Protein Kinase Systems and Signal Transduction. It is widely accepted that glycolipids play a crucial role in many dynamic cellular processes, including the regulation of differentiation and proliferation (4,7,64) and that addition of exogenous gangliosides to several cell lines grown in tissue culture medium causes growth inhibition by extending the length of the Gl phase of the cell cycle, and blocks cellular proliferation in the presence of growth factors (51,65). Although the exact mechanisms by which glycolipids may influence these diverse systems have largely remained obscure, evidence is accumulating suggesting that glycolipids are modulators of transmembrane signaling by direct or indirect action on several protein kinases, including protein kinase C (PKC), cAMP-dependent protein kinase (PKA), Ca2Vcalmodulin-dependent protein kinase II (CaM-kinase II), GQlb-sensitive ecto-kinase, and receptor-mediated kinase systems (8,17,64,66-76). Thus, glycolipids in the plasma membrane or exogenously added glycolipids may participate in signal transduction processes by serving as receptors for various ligands such as calcium, growth factors, hormones, and mitogenic factors (17,52). The physiological role of PKC stimulation by gangliosides is not clear. Higashi et al. (77,78) isolated calmodulin from a soluble cytosol fraction of mouse brains and suggested that gangliosides may modulate the CaM-dependent enzyme by binding to CaM and to the enzyme itself. The above studies have

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Fig. 9. Temporal staining of rat embryonic dorsal root ganglion (DRG) in culture. A, B, C, and D are phase-contrast micrographs of DRG at 5, 8, 12, and 15 days in culture. E, F, G, and H are micrographs of immunostaining obtained with an antibody against microtuble-associated protein tau during the same period. I, J, K, and L are micrographs of immunostaining using a polyclonal anti-GalGb3 antibody during the same period. (Reproduced from 61).

prompted increasing attention on the role of glycolipids as modulators of membrane-associated protein kinases and trans-membrane signal transduetion events. 3. /. Differential Effects of Glycolipids on Protein Kinase C Activity in PC12D Pheochromocytoma Cells. Many extracellular agents regulate cellular differentiation and function through their binding to specific receptors at the cell surface. Phosphorylation-dephosphorylation of proteins is known to be a major regulatory mechanism in the subsequent intracellular transduetion of these signals (79). A large family of kinases have been shown to play a key role in the regulation of cellular growth, differentiation, and function (11,52). Constitutive activation of PKC leads to growth abnormalities in vitro and tumor promotion in vivo; stimulation of PKC in cultured astrocytes results in biochemical and morphological alterations associated with the transformed phenotype (80). It is known that glycolipids can be inserted into the cell membrane and modulate membrane-associated cellular functions through interactions with functional membrane bound proteins such as

growth factor receptors (8,52). Several recent studies have indicated that PKC is an important component of the NGF-sensitive phosphorylation system in PC 12 cells (81,82). Hilbush and Levine (83) reported that GM1 modulated NGF signal transduetion in PC12 cells. Recently Mutoh et al. (84) have reported that GM1 ganglioside binds to Trk, the high-affinity tyrosine kinase-type receptor for NGF and enhances the action of NGF in PC 12 cells. Hall et al. (85) reported that PKC plays a role in mediating the neuritogenic effects of NGF by virtue of a sphingosine-sensitive pathway. In our own studies comparing PKC activities in PC 12 cells and the two ubclones PC12D and PC12h, we found that the activity of this enzyme was significantly elevated in the subclones with the highest activity in the PC12D cells. Since it is known that certain glycolipids may function as modulators of PKC activity (86), we examined the structure-activity relationship of a variety of sphingolipids, including 17 gangliosides, 10 neutral glycolipids, as well as sulfatide, psychosine and ceramide, on PKC activity in PC12D cells. Using an in vitro assay system,

The Role of Globo-Series Glycolipids in Neuronal Cell Differentiation—A Review

Fig. 10. Effects of the number of sialic acid residues on PK.C activity at the ganglioside concentrations of 25 u,M. The values are expressed as mean percentages of normal control (untreated PC12D cells; n = 5 individual samples). *p < 0.05; **p, 0.01, ***p < 0.001 (forasialoganglioside). (Reproduced from 86).

Fig. 11. Effects of the terminal sugar residues on PKC activity at the neutral glycolipid concentrations. The values are expressed as mean percentages of normal control (untreated PC12D cells; n = 5 individual samples.)

we found that all but one (GDlb) of the gangliosides inhibited the PKC activity at concentrations between 25100 |aM, and the potency was proportional to the number of sialic acid residues; the rank order for inhibitory potency was trisialo > disialo > monosialogangliosides (Fig. 10). However, at lower concentrations several gangliosides, including GM1 and LM1, behaved as mild activators for PKC activity. In contrast to Tsuji et al. (68) who reported that GQlb at nanomolar concentration stimulated cellular proliferation and neurite outgrowth,

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perhaps by stimulating a novel ecto-protein kinase system in certain neuroblastoma cells, we found that GQlb had no effect within the range of 0.1-10 (j,M, but acted as a mild activator at the concentration of 25 (J.M for the PKC activity. It is known that glycolipids, even in minute amounts, may have profound effects on protein kinase systems. Thus, the above observation is consistent with the finding that de-N-acetyl GM3, GQlb, and polysialogangliosides, which are present at negligible or undetectable levels in human carcinoma A431 cells (72), human neuroblastoma cells (68), and rat brain (69,70), are stimulators of certain kinases. On the other hand, fucosylated GM1 and the GM1 containing blood group B determinant, which are abundant in PC 12 cells, were potent inhibitors of PKC activity. The neutral glycolipids LacCer, Gb3, GalGb3, and GA1, all of which have a terminal galactose residue, were found to be ineffective or acted as mild activators for the PKC activity. In contrast, GA2, Gb4 and Gb5 which have terminal N-acetylgalactosamine residue, were potent inhibitors of the PKC activity. Thus, the terminal sugar residue may play a pivotal role in determining the effect of glycolipids in modulating PKC activity (Fig. 11). In addition, we also found that GalCer containing non-hydroxylated fatty acids acted as a potent activator for the PKC activity. Ceramide and GlcCer appeared to be ineffective for modulating PKC activity, whereas psychosine and sulfatides appeared to be inhibitory. The overall data indicate that the terminal N-acetylgalactosamine residue as well as negatively charged functional groups, such as the sialic acid or sulfate residue, may be structurally important for suppressing PKC activity. Additionally, the terminal N-acetylgalactosamine residue may act as inhibitory structural elements. There have been several reports indicating that modification or substitution of the functional groups of glycolipids as well as their fatty acid chains may modify their effects on protein kinase systems. For example, deN-acetyl GM3 stimulated tyrosine phosphokinase activity associated with the EOF or insulin receptor and enhanced proliferation of various cell lines in culture; this is in striking contrast to GM3 and lyso-GM3 which exhibited an inhibitory effect on the receptor-associated tyrosine kinase and on cellular growth (64,72). Thus, the carbohydrate head groups and the hydrophobic groups of gangliosides and neutral glycolipids may modulate the PKC system in their unique manner, which may in turn affect various biological processes in the cell (11). Such variations in structural requirement for the modulatory roles for glycolipids have been shown for several growth factor receptors (8). An understanding of the structural requirement for the modulatory effect of gly-

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Fig. 12. Time course of PK.C activity using various differentiating agents (A) and GalGb3-synthase activity using various differentiating agents in addition to GalCer (B). The values are expressed as mean percentages of normal control (untreated PC12D cells; n = 6 individual samples).

colipids should help elucidate their role in signal transduction pathways and their biological functions. 3.2. Possible Roles of Protein Kinase Systems in Regulating Glycosyltransfera.se Activities. A number of phosphorylation systems are altered by treatment of cells with differentiating agents, e.g., nerve growth factor (NGF), forskolin (FRK), staurosporine (STP), and K252a. The activities of PKA and PKC have also been reported to increase when cells are treated with NGF (81,87). Extrapolation from these studies permits the generalization that the biochemical mechanism by which NGF acts on the cell is through a sequence of phosphorylation reactions starting at the membrane. A number of reports indicate that inducers of differentiation such as 12-O-tetradecanoylphorbol-13-acetate (TPA), dimethylsulfoxide (DMSO), butyrate, and retinoic acid (RA) have profound effects on glycosyltransferase activities (17,18,70,88-91). We have reported that these differentiating agents are capable of inducing changes in the glycolipid pattern in human squamous SQCC/Y1 cells (14) and HL-60 cells (18). Since these agents modulate the phosphorylation state of the cell, there is a possibility that glycosyltransferase activities may be under the control of protein kinase systems. For example, GM3-synthase (90,91) and GM2-synthase (91,92) have been shown to be regulated by this mechanism. Scheideler and Dawson (93) demonstrated that treatment of mouse neuroblastoma cells with reagents known to regulate PKA had an inhibitory effect on GM2-synthase activity. Xia et al. (18) also reported a correlation between the activities of PKC and GM3-synthase. However, direct evidence that the activities of glycosyltransferases are under phosphorylation-dephosphorylation control is still

unavailable. The major obstacle in proving this is a lack of pure enzymes and specific antibodies directed against them. In PC 12 cells, specific glycosyltransferases for the synthesis of the globo-series glycolipids may be regulated by the system of phosphorylation-dephosphorylation induced by differentiating agents (19). Recently, we measured the activities of UDP-galactose: globotriaosylceramide al-3galactosyltransferase (a-GalT, GalGb3synthase) and protein kinase C (PKC) in PC12D cells induced to differentiate by nerve growth factor (NGF), forskolin (FRK), staurosporine (STP), retinoic acid (RA), 2-chloroadenosine (ClAd), or galactosylceramide (GalCer). NGF, STP, FRK, and RA stimulated, whereas ClAd inhibited the PKC activity (19) (Fig. 12). At the concentration of 25 (4.M, GalCer having normal fatty acids was found to be a stimulator, whereas GalCer having hydroxylated fatty acids was ineffective in modulating the PKC activity. Interestingly, all stimulators of PKC activities, including GalCer having normal fatty acids also activated the GalGb3-synthase. On the other hand, GalCer having ct-hydroxylated fatty acids had no effect and ClAd was found to be a potent inhibitor for the GalGb3-synthase activity (19). These data suggest that the increase in GalGb3-synthase activity in GalCertreated cells is likely mediated directly or indirectly through a PKC-dependent process. Roth et al. (88) recently reported an increased glycoprotein (3-GalT activity in PC 12 cells induced by either FRK or ClAd and that the induction of activity is dependent on protein kinase A. In our own study, ClAd appeared to be a potent inhibitor for the glycolipid a-GalT activity. This discrepancy might reflect differences in the source of

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Fig. 13. Effects of NGF and antibody on GalGb3-synthase (A) and PKC (B) activities. The values are expressed as mean percentages of normal control (untreated PC12D cells; n = 5 individual samples).

GalT, the kinase systems, and/or the cell lines. In initial experiments using a soluble rat brain sialyltransferase preparation (94), we found that addition of 100 (iM of ATP resulted in nearly a 50% reduction in the activities of sialyltransferase-I (ST-I), -II, -III, and -IV, presumably due to their phosphorylation by endogenous protein kinases (11,95). Inhibition of enzyme activity could also be demonstrated using a highly purified rat brain ST-IV preparation (11,95). This PKC-dependent inhibition of ST-IV activity was reversed by treatment of the phosphorylated enzyme with a rat brain membrane bound phosphatase. The significance of this finding was further underscored by our recent discovery that ST-IV was tightly associated with a subtype of the 14-3-3 protein, which is a family of acidic proteins with PKC inhibitor activity (96). It is postulated that this protein may play an indirect role in the post-translational regulation of ST-IV activity by modulating the activity of PKC. Analysis of the phosphoamino acids generated from the phosphorylated ST-IV revealed that serine residues were phosphorylated (11,95). Thus, our results demonstrate a role for PKC, which may be activated by external signals, in the regulation of sialyltransferases for ganglioside synthesis through the mechanism of phosphorylation and dephosphorylation (11).

4. The Effects of Glycolipid Antibodies on Cellular Differentiation. The mechanisms underlying the control of cellular growth and differentiation are poorly understood. There are several reports documenting that antibodies may affect cellular growth and differentiation (31,97). Anti-GMl antibodies can inhibit dendritic spouting induced in dorsal root ganglia by NGF (20), elevation of ornithine decarboxylase activity induced by NGF in cell culture (97), and the growth of glioma cells (98). Anti-GM3 and anti-GMl antibodies also inhibit Balb/3T3 and NIL hamster fibroblast differentiation (99). Morphological changes in retinal cells from chicken embryos and rat basophilic leukemia cells in the presence of antiganglioside antibodies in culture have been demonstrated (100,101). Wu et al. (25) recently reported that anti-GMl antibody, but not anti-GM2 antibody, and the B-subunit of cholera toxin inhibited neurite outgrowth in neuroblastoma Neuro-2a cells. On the other hand, neuraminidase and GM1 stimulate neurite outgrowth in neuroblastoma cells (24) Chatterjee et al. (31) have reported that a monoclonal antibody against GM3 triggers differentiation of Neuro-2a cells in culture and that the GM3-antibody-mediated differentiated cells contain more complex gangliosides in addition to GM3 and GM2 gangliosides. None of these changes associ-

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ated with antibody binding were seen for other anti-cell surface antibodies (101). These results point to a pivotal role for cell surface GM1 and GM3 in cellular differentiation induced by many neuritogenic agents. By using specific antibodies directed against glycolipids, it is possible to test the above hypotheses regarding the regulation of enzyme activities and the subsequent differential expression of glycolipids during cellular differentiation. To further investigate this phenomenon in PC12D cells, we added anti-GalGb3 monoclonal antibody (102) to the culture medium together with or without NGF and anti-NGF polyclonal antibody, and measured the activities of GalGbS-synthase and PKC. We found that both anti-GalGb3 monoclonal antibody and anti-NGF polyclonal antibody effectively down-regulated the activities of GalGbS-synthase and PKC (Figs. 13A and 13B). In addition, these antibodies caused the cells to dedifferentiate into a round morphology. These changes were not observed by addition of other monoclonal anti-glycolipid antibodies. Our observation is in accord with the recent findings by Gill et al. (103) who reported that neurite outgrowth in PC 12 cells induced by NGF was abolished by exposure of these cells to anti-NGF and the shape of the cells became round. To assess the possible "signaling effect" of GalGb3, we examined the effect of exogenously added GalGb3. In addition, we used a liposome system containing GalGb3 (5 to 25 joM) because GalGb3 was not readily soluble in the culture medium. We found that GalGb3 had no effect on the GalGb3-synthase nor PKC activity. In addition, we previously reported that GalGb3 had no effect on PKC in vitro (11). Thus, GalGb3 itself does not exert any signaling effect. Our data suggest that changes in the activity of GalGb3-synthase may be closely related to events underlying cellular differentiation and the enzyme activity may be under the control of protein kinase systems via the modulation of phosphorylation states of the cell. Moreover, Benzil et al. (80) have reported that GM3-antibody-induced differentiation can be inhibited by protein kinase A (PKA) inhibitor and that GM3-antibody mediates its effect via PKC. Neuro-2a neuroblastoma cells, when induced to differentiate via a cAMP-dependent pathway by treatment with anti-GM3 monoclonal antibody, accumulated a high level of pp60c-src protein and pp60c-src kinase activity just before the onset of neurite formation (104). Oliver et al. (101) have also reported that a striking morphological change can be effected in rat basophilic leukemia cells by treatment with a monoclonal antibody against gangliosides and this effect appears to be accompanied by activation of PKC. Taken together, these reports suggest a direct or indirect involvement of protein kinases such as PKC in cel-

lular processes related to growth cessation, differentiation, and function of the cell lines (8,17).

CONCLUSION The experiments described here indicate that alterations in glycolipid composition and glycosyltransferase activities correlate with neuritogenesis and/or cellular differentiation in the neuronal PC12 cells. We have demonstrated that the unique globo-series glycolipids, expressed exclusively in subcloned PC12 cells, participate in initiation and/or maintenance of neurite outgrowth by these cells. We have also shown that the glycolipid changes correlate with glycosyltransferase (GalGb3-synthase) activities which may be modulated by protein kinase systems. Thus, the enzyme GalGb3-synthase may play a key role in the regulation of globo-series glycolipid synthase during differentiation. To obtain a better understanding of the molecular mechanisms underlying the regulatory events in glycolipid synthesis during PC 12 cellular differentiation and/or neurite outgrowth, it is necessary to isolate the gene for this enzyme. Future studies will focus on the use of molecular biological probes to address the regulation and differential expression of GalGb3-synthase activities in neural cell function and differentiation.

ACKNOWLEDGMENTS This work was supported by a USPHS grant NS11853. The authors gratefully acknowledge Dr. Ritsuko Katoh-Semba, Aichi Prefecture Colony, for providing the PC12D cells, and Dr. Tadashi Tai, The Tokyo Metropolitan Institute of Medical Science, for providing the monoclonal antibodies against glycolipids. The authors would also like to thank Drs. Takashi Kanda, Shubro Pal, Guichao Zeng, Xinbin Gu, John Bigbee and other members of the laboratory for participating in parts of the experimental work this paper is based on. The authors also acknowledge Professors Thomas Seyfried, Cara-Lynne Schengrund, and Tadashi Miyatake for their valuable suggestions for this work.

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