Characterization of the bone morphogenetic protein-2 as a neurotrophic factor. Induction of neuronal differentiation of PC12 cells in the absence of mitogen-activated protein kinase activation.

Rat pheochromocytoma PC12 cells are shown to express a single class of high affinity binding sites for bone morphogenetic protein (BMP)-2 (1,300 receptors/cell, Kd = 31.3 pM). Affinity cross-linking using radiolabeled BMP-2 demonstrated the presence of six components with apparent molecular masses of 170, 155, 105, 90, 80, and 70 kDa. BMP-2 induced morphological changes in PC12 cells with the concomitant expression of three neurofilament proteins. Thus, BMP-2 would appear to be another neurotrophic factor that, like nerve growth factor or basic fibroblast growth factor, stimulates the neuronal differentiation of PC12 cells. Unlike nerve growth factor and basic fibroblast growth factor, however, BMP-2 failed to induce the activation of either 41- and 43-kDa mitogen-activated protein (MAP) kinases or the MAP kinase/extracellular signal-regulated kinase kinase (MEK). Also, BMP-2 did not induce the expression of the c-fos gene in PC12 cells. Activin A was also capable of inducing the neuronal differentiation of PC12 cells without activating MAP kinases and MEK. These findings show a clear dissociation between the requirement for the activation of the MAP kinase cascade and the ability of BMP-2 and activin A to induce PC12 cell neuronal differentiation. In addition, these results suggest that the activation of MAP kinases and MEK is not an absolute requirement for PC12 cell differentiation.

Bone morphogenetic proteins (BMPs) 1 are a family of proteins, which were originally identified and characterized on the basis of their novel ability to induce cartilage and bone formation in ectopic extraskeletal sites in vivo (Refs. 1 and 2 and reviewed in Ref. 3). Recently, several members of this protein family have been cloned, expressed in mammalian cells, and characterized; these include BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7 (also referred to as osteogenic protein (OP)-1) and OP-2 (4 -8).
All the BMPs contain the characteristic seven highly conserved cysteines in their carboxyl-terminal portions and thus belong to the TGF-␤ superfamily, which includes TGF-␤s, activins, inhibins, and Mü llerian inhibiting substance (reviewed in Ref. 9). The members of the TGF-␤ superfamily are multifunctional, e.g. TGF-␤s are known to be implicated in the regulation of a wide range of biological phenomena such as cell proliferation, cell differentiation, angiogenesis, immunosuppression, inflammation, tissue repair, and embryogenesis. In this respect, BMP gene expression has been observed in various tissues including kidney, brain, and skin (10 -12). Increasing evidence suggests a regulatory role for BMPs in embryonic development (13)(14)(15)(16), indicating that the BMP family of proteins may have much broader biological effects unrelated to bone formation. Despite this, the precise physiological function and the mechanism of action of BMPs remain largely unknown.
As a first step toward the elucidation of the biological action of BMPs, we recently examined the distribution of specific cellular binding proteins (receptors) for BMP-2 on a wide variety of cell types and tissues. We have shown that high affinity receptors for BMP-2 are present not only on osteoblastic cells but are also distributed ubiquitously on a wide variety of other types of cells; these include fibroblasts, keratinocytes, astrocytes, kidney epithelial cells, and tumor cells of lung, liver, kidney, stomach, and neuronal tissue. On the other hand, cells of hematopoietic origin and vascular endothelial cells do not express specific binding sites for BMP-2. These results suggest that a majority of cell types with the exception of hematopoietic cells and vascular endothelial cells may be potential targets for BMP-2 action (17).
The presence of specific cellular binding proteins for BMP-2 on neuronal cells seems particularly interesting as several of the TGF-␤ superfamily members have recently been shown to possess neurotrophic activity. For example, activin has been demonstrated to be a nerve cell survival molecule (18); TGF-␤ has been shown to be neurotrophic for the dorsal root ganglia neurons of neonatal rat (19); glial cell line-derived neurotrophic factor, a distantly related member of the TGF-␤ superfamily, has been shown to promote the differentiation and survival of embryonic midbrain dopaminergic neurons (20); BMP-7 has been shown to induce the expression of the neural cell adhesion molecule in neural cells (21).
In the present study, we investigate the possibility that BMP-2 acts as a neurotrophic factor. We show that BMP-2 induces the neuronal differentiation of rat pheochromocytoma PC12 cells through a signaling pathway, which is distinct from * This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan and by grants from the Research Foundation for Pharmaceutical Sciences of Japan and CIBA-GEIGY Foundation (Japan) for the Promotion of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Contributed equally to this work. ¶ Recipient of a fellowship from the Japan Society for the Promotion of Science for Japanese Junior Scientists.

EXPERIMENTAL PROCEDURES
Materials-Recombinant human BMP-2, expressed in silkworm larvae, was purified to homogeneity as described previously (22). Recombinant human TGF-␤1 was purchased from Wako Chemical Co. (Osaka, Japan). ␤-NGF purified from mouse submaxillary glands and recombinant human bFGF were from Toyobo Co. (Osaka, Japan). Purified human activin A and purified bovine inhibin A were kindly provided by Dr. K. Miyamoto (National Cardiovascular Center, Research Institute, Osaka, Japan). Other chemicals and reagents were of the purest grade available.
Cell Culture-Rat pheochromocytoma cells (PC12) were obtained through the Japanese Cancer Research Resources Bank. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% horse serum and 5% fetal bovine serum. For neurite outgrowth assays, PC12 cells were seeded in the growth medium at 5 ϫ 10 3 cells/well in collagen (type I)-coated 12-well culture plates and allowed to grow for 24 h. The cells were then incubated in serum-free medium (Dulbecco's modified Eagle's medium containing 2 mg/ml bovine serum albumin (Boehringer Mannheim GmbH, Germany), 1 g/ml insulin, 2 g/ml transferrin, 30 nM Na 2 SeO 3 , 20 nM progesterone, and 10 mM Hepes, pH 7.4) for 24 h. BMP-2 (30 ng/ml), TGF-␤1 (10 ng/ml), activin A (30 ng/ml), bFGF (10 ng/ml), or NGF (20 ng/ml) was added, and neurite outgrowth was measured after 4 days. For quantification of neurite outgrowth, five random photographs were made per well, and cells bearing processes longer than twice the length of cell body were considered positive. A total of over 500 cells were counted,and each data point corresponded to the counts obtained from two independent wells.
Iodination of BMP-2 and Binding Assay-Recombinant human BMP-2 was radioiodinated by using the Bolton and Hunter reagent (Amersham), and the binding of 125 I-labeled BMP-2 to PC12 cells was analyzed as described previously (17). Briefly, PC12 cells were seeded at 6 ϫ 10 5 cells/well in collagen (type I)-coated 24-well culture plates and allowed to grow for 24 h. The monolayers were then washed twice and incubated in binding buffer (Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 25 mM Hepes, pH 7.4) for 1 h at 37°C. After washing, cells were incubated in 0.3 ml binding buffer containing 1ϳ500 pM 125 I-labeled BMP-2 for 2 h at room temperature on a rocker platform. At the end of the incubation, the cells were washed five times with binding buffer and then solubilized with 750 l of solubilization buffer (1% Triton X-100, 10% glycerol, 20 mM Hepes, pH 7.5, 0.05% bovine serum albumin) for 20 min at 37°C. The radioactivity in aliquots of 600 l was measured by a Packard gamma counter. Nonspecific binding was determined by using a 100-fold excess of unlabeled BMP-2. The number of cells in replicate wells was determined by counting in a hematocytometer. Each data point was determined in triplicate,and binding data were analyzed according to the method of Scatchard (23).
Cross-linking of 125 I-Labeled BMP-2 to Cell Surface Receptors-Binding of radiolabeled BMP-2 to PC12 cells was carried out as described above, except that cells were in 35-mm culture dishes, 250 pM of 125 Ilabeled BMP-2 was used, and binding incubation was for 4 h at 4°C. After washing the cell monolayer, bound 125 I-labeled BMP-2 was affinity cross-linked with 0.5 mM disuccinimidyl suberate (Pierce) as described previously (17). Samples were then run on SDS-PAGE gels, and the gels were analyzed using a Fujix Bioimaging analyzer BAS 1500 (Fuji Photo Film Co., Tokyo).
MAP Kinase Activity Assay-Cell lysates prepared as described above were pretreated with 0.1% SDS for 20 min at 4°C (24,25), diluted 1:10 in cell lysis buffer, and then immunoprecipitated by incubating for 3 h at 4°C with the anti-MAP kinase antibody preadsorbed to protein-A Sepharose (Pharmacia Biotech Inc.). The immunoprecipitates were washed three times with cell lysis buffer and twice with kinase buffer (50 mM Tris-HCl, pH 8.0, 25 mM MgCl 2 , 1 mM dithiothreitol, 0.5 mM EGTA, 10% glycerol). Kinase assays were performed by incubating the immunoprecipitates in 30 l of kinase buffer containing 20 M ATP, 1 Ci [␥-32 P]ATP (3000 Ci/mmol) (Amersham), and 0.5 mg/ml myelin basic protein (MBP) (Sigma). After 30 min at 30°C, the reaction was stopped by adding 10 l of 0.6% HCl containing 1 mM ATP and 1% bovine serum albumin. Samples (30 l) were then spotted onto 1.5 ϫ 1.5-cm squares of phosphocellulose papers (P81, Whatman), washed five times in 180 mM phosphoric acid, and the radioactivity incorporated into MBP was determined by liquid scintillation spectrometry. In some experiments, the phosphorylation reaction was terminated by adding 10 l of 4 ϫ SDS sample buffer, and the incorporation of 32 P into MBP was examined by SDS-PAGE followed by autoradiography. Both assays always gave identical results. Our anti-MAP kinase antibody recognized both the 41-and 43-kDa MAP kinases, and thus all the MBP phosphorylation activity of each of the immunoprecipitates shown in this report is the sum of the activities of these two species of MAP kinase. The synchronous activation of both the 41-and 43-kDa MAP kinases in mitogen-stimulated cells was confirmed by doing the kinase detection assay within a polyacrylamide gel containing MBP (data not shown; see Ref. 24).
MEK Activity Assay-Cell lysates prepared as described above were immunoprecipitated by incubating for 3 h at 4°C with the anti-MEK1 antibody (Transduction Laboratories) preadsorbed to protein-A Sepharose. The immunoprecipitates were washed three times with cell lysis buffer and twice with kinase buffer (50 mM Tris-HCl, pH 8.0, 25 mM MgCl 2 , 1 mM dithiothreitol, 0.5 mM EGTA, 10% glycerol). Kinase assays were performed by incubating the immunoprecipitates in 30 l of kinase buffer containing 20 M ATP, 1 Ci [␥-32 P]ATP (3000 Ci/mmol) (Amersham), and 1 g of a catalytically inactive 41-kDa MAP kinaseglutathione S-transferase fusion protein (GST-ERK2 K52R ). After a 60min incubation at 30°C, the reaction was terminated by the addition of 10 l of 4 ϫ SDS sample buffer, and the incorporation of 32 P into GST-ERK2 K52R was examined by SDS-PAGE followed by autoradiography or subjected to quantitative analysis using a Fujix Bioimaging analyzer BAS 1500. The cDNA for ERK2 K52R was kindly provided by Dr. M. J. Weber (University of Virginia Health Sciences Center) and expressed in Escherichia coli as a GST-fusion protein. GST-ERK2 K52R was purified by affinity chromatography using glutathione-Sepharose beads (Pharmacia) as described (28).
RNA Isolation and Northern Blot Analysis-Total RNA was isolated from growth factor-stimulated PC12 cells according to the method of Chomczynski and Sacchi (29). 20 g of total RNA was fractionated on 1.2% agarose gels containing 2.2 M formaldehyde and then transferred to a Zeta-probe membrane (Bio-Rad). The filter-bound RNA was hybridized with a randomly primed, 32 P-labeled v-fos cDNA probe (pfos-1 obtained through Japanese Cancer Research Resources Bank) (30) for 16 h at 42°C. The filters were then washed under stringent conditions according to the instruction manual (Bio-Rad).

RESULTS
Specific Binding of BMP-2 to PC12 Cells and Affinity Crosslinking of 125 I-Labeled BMP-2-Radiolabeled BMP-2 specifically bound to rat pheochromocytoma PC12 cells in a dose-dependent manner; a typical set of binding curves and corresponding Scatchard analysis are shown in Fig. 1A. These gave a linear plot characteristic of a single high affinity binding site with 1,300 receptors/cell and a dissociation constant (K d ) of 31.3 pM. The specific binding proteins for BMP-2 in PC12 cells were further characterized by an affinity cross-linking technique using the homobifunctional cross-linking agent, disuccinimidyl suberate. Analysis by SDS-PAGE repeatedly revealed six cross-linked macromolecular components (Fig. 1B). The apparent molecular masses of these species were 170, 155, 105, 90, 80, and 70 kDa, all of which have been commonly detected in several human and mouse fibroblast cell lines as described previously (17). The cross-linking to all these macromolecules was inhibited by a 100-fold excess of unlabeled BMP-2.
BMP-2 Induces Neurite Outgrowth in PC12 Cells-We examined the effects of BMP-2 on PC12 cells. Upon treatment with BMP-2, there was a change in the morphology of PC12 cells. The most prominent change was the formation of neurite-like processes similar to those observed when PC12 cells are treated with NGF (Fig. 2). The process-inducing activity of BMP-2 was dose dependent and was maximal at a concentration of 30 ng/ml (1 nM) (Fig. 3). Although the majority (more than 85%) of PC12 cells responded to BMP-2-stimulation and started to extend processes within the initial 1ϳ2 days, branching and intermingling of the processes in BMP-2-treated PC12 cells was slightly less conspicuous compared with those observed in NGF-treated cells (Fig. 2). In PC12 cells, bFGF and activin A were found to induce, respectively, moderate and low process formation; however, this was not the case for TGF-␤1 and inhibin A up to a concentration of 50 ng/ml ( Fig. 2 and data not shown).
To confirm that the processes observed in BMP-2-treated cells are indeed neurites, we examined for the expression of NF proteins. NF is a member of the intermediate filament subclass and is expressed specifically in neurons. Mammalian NF is composed of three subunits consisting of ϳ200-kDa (H), ϳ160-kDa (M), and ϳ70 kDa (L) proteins (31), and NGF-stimulation of PC12 cells has previously been reported to increase the level of all of these NF proteins (26,27). As shown in Fig. 4, the expression of NF-H, NF-M, and NF-L proteins was markedly increased in BMP-2-stimulated PC12 cells with a time course similar to that observed in NGF-treated cells. These results demonstrate that the processes induced in BMP-2-treated PC12 cells are actually neurites, clearly indicating that PC12 cells undergo neuronal differentiation upon stimulation with BMP-2. Basic FGF and activin A were also found to, respectively, increase the marked and moderate expression of these three NF proteins in PC12 cells.
BMP-2 Does Not Induce the Tyrosine Phosphorylation and Activation of 41-and 43-kDa MAP Kinases in PC12 Cells-It has recently been suggested that the activation of MAP kinases is necessary and sufficient for PC12 cell differentiation (32). For this reason, we examined for the activation of 41-and 43-kDa MAP kinases in BMP-2-stimulated PC12 cells. The activation of these MAP kinases was examined by three different assay procedures: by measuring the appearance of their active forms, which show reduced mobility in SDS-PAGE due to phosphorylation of the specific threonine and tyrosine residues (24,33), by measurement of their tyrosine phosphorylation, and by a direct in vitro kinase assay of the immunoprecipitates using MBP as the substrate. These analyses always gave essentially an identical time-course profile for MAP kinase activation. As shown in Figs. 5 and 6, NGF induced a rapid, marked, and sustained activation of 41-and 43-kDa MAP kinases in PC12 cells. MAP kinase activation reached the maximal level within 10 min and then declined gradually. However, a considerable level of activation (ϳ25% of maximum) could still be detected even 4 h after stimulation of the cells. Such a prolonged activation of MAP kinases in NGFtreated PC12 cells has been previously reported (34,35). Basic FGF also induced a rapid but transient activation of MAP kinases in PC12 cells. In contrast, BMP-2 treatment of PC12 cells stimulated neither the tyrosine phosphorylation nor the activation of the 41-and 43-kDa MAP kinases significantly at any time point up to 12 h after stimulation. This was also the case for activin A.
BMP-2 Does Not Induce the Activation of MEK in PC12 Cells-Activation of the 41-and 43-kDa MAP kinases is achieved by combined tyrosine and threonine phosphorylation catalyzed by a dual specificity kinase, MEK (36,37). It has been reported, however, that in Rat 1a cells active Raf-1 leads to the activation of MEK with no subsequent activation of MAP kinases (38). Thus, we next examined the state of activation of MEK in BMP-2-treated PC12 cells. As shown in Fig. 7, NGF induced a rapid, marked, and sustained activation of MEK, and bFGF induced a rapid but transient MEK activation in PC12 cells. The time-course profile of MEK activation was very similar to that of MAP kinase activation irrespective of the factor that was used to treat the cells. In contrast, BMP-2-treatment of PC12 cells did not stimulate the activation of MEK at any time point analyzed up to 6 h. This was also the case for activin A (data not shown). The good correlation between the kinetics of activation of 41-/43-kDa MAP kinases and that of MEK suggests that MEK and MAP kinases constitute a linear pathway in this signaling process.
BMP-2 Does Not Induce the Expression of Protooncogene c-fos in PC12 Cells-It has recently been reported that the ternary complex factor (p62 TCF /Elk-1) is phosphorylated by MAP kinases. This phosphorylation results in enhanced ternary complex formation and activation of c-fos expression (39,40). We therefore wished to see if BMP-2 could stimulate the expression of c-fos in PC12 cells. Treatment of PC12 cells with NGF induced the rapid and transient expression of c-fos gene (Fig.   8). In contrast, BMP-2 did not stimulate c-fos gene expression in these cells at any time point analyzed up to 5 h. DISCUSSION PC12 cells express a single class of high affinity binding sites for BMP-2. Affinity cross-linking experiments have revealed six macromolecular components that bind specifically to BMP-2. The apparent molecular masses of these affinity-labeled complexes are 170, 155, 105, 90, 80, and 70 kDa, and all have been detected in several human and mouse fibroblast cell lines (17). As discussed previously, 125 I-labeled BMP-2 might have bound to cellular proteins with molecular masses of ϳ140, ϳ75 (this value corresponds to that of type II receptor for TGF-␤), and ϳ52 kDa (this value corresponds to that of type I receptor for TGF-␤); cross-linking of the ligand monomer (16 kDa) or dimer (30 kDa) to each of these proteins would then generate the observed 170-/155-, 105-/90-, and 80-/70-kDa species, respectively (17).
BMP-2 induces the neuronal differentiation of PC12 cells. This was confirmed not only by morphological analysis of the cells but also by the observed expression of three neurofilament proteins (NF-H, NF-M, and NF-L; Fig. 4). Among the other members of the TGF-␤ superfamily, activin A was found to weakly induce the neuronal differentiation of PC12 cells. In contrast, both TGF-␤ and inhibin A were negative. BMP-2 also shows neurotrophic effects on several neurons obtained from chick and mouse brain, e.g. BMP-2 induces neurite outgrowth in sensory neurons obtained from 10-day-old chick embryos, and it supports the survival of striatum neurons obtained from 14-day-old mouse embryos. 2 The induction of neuronal differentiation of PC12 cells by BMP-4 has recently been demon-strated, but only in the presence of serum (41). BMP-2 induces neurite outgrowth in PC12 cells both in the presence (data not shown) and absence of serum (Fig. 2).
We have investigated whether or not the MAP kinase cascade is involved in the signaling pathway of BMP-2 that induces the neuronal differentiation of PC12 cells. The MAP kinase cascade is the major cytoplasmic kinase pathway that is commonly activated in a wide variety of cells by diverse extracellular stimuli. Activation of the cascade is known to elicit a wide array of physiological responses such as cell division, differentiation, and secretion (42)(43)(44)(45)(46)(47), and it has recently been shown that the activation of MEK (32) and MAP kinases (48) is necessary and sufficient for PC12 cell differentiation.
NGF induces the rapid, marked and sustained activation of MEK (Fig. 7) and 41-/43-kDa MAP kinases (Figs. 5 and 6) and the rapid and transient expression of c-fos gene (Fig. 8). Basic FGF also induces the rapid activation of MEK and MAP kinases, but the activated state is transient. In contrast, BMP-2 induces neither the activation of MEK and MAP kinases nor the expression of c-fos gene. Activin A also induces the differentiation of PC12 cells without activating MEK nor MAP kinases. All of these findings show a clear dissociation between FIG. 6. Kinetics of MAP kinase activation induced by NGF, bFGF, or BMP-2 in PC12 cells. PC12 cells were treated with 20 ng/ml NGF, 10 ng/ml bFGF, or 30 ng/ml BMP-2 for the indicated periods of time. MAP kinase assay was performed by incubating cell lysates (10 g of protein) with anti-MAP kinase antibody followed by the kinase reaction; radioactivity incorporated into MBP was determined as described under "Experimental Procedures." Each value represents the mean Ϯ S.E. of triplicate determinations of a representative experiment. Similar results were obtained in five independent experiments. FIG. 7. Kinetics of MEK activation induced by NGF, bFGF, or BMP-2 in PC12 cells. PC12 cells were treated with 20 ng/ml NGF, 10 ng/ml bFGF, or 30 ng/ml BMP-2 for the indicated periods of time. MEK assay was performed by incubating cell lysates (20 g of protein) with anti-MEK1 antibody followed by the kinase reaction. Radioactivity incorporated into GST-ERK2 K52R was quantified with a Fujix Bioimaging analyzer BAS 1500. Each value represents the mean Ϯ S.E. of duplicate determinations of a representative experiment. 32 P incorporation into GST-ERK2 K52R is also shown by autoradiography (inset). Similar results were obtained in three independent experiments.

FIG. 5. Growth factor-induced activation of 41-and 43-kDa MAP kinases in PC12 cells.
PC12 cells were treated with 20 ng/ml NGF, 10 ng/ml bFGF, 30 ng/ml BMP-2, or 30 ng/ml activin A for the indicated periods of time. Cell were then lysed, and cell lysates (10 g of protein) were resolved by SDS-PAGE, blotted, and probed with anti-MAP kinase antibody or anti-phosphotyrosine antibody, followed by ECL detection. Closed arrowheads indicate positions of the phosphorylated (activated) forms of 41-and 43-kDa MAP kinases (pp41, pp43), while open arrowheads indicate positions of the unphosphorylated forms of these MAP kinases (p41, p43). Data shown are representative of three to five separate experiments that gave essentially the identical results.
the requirement for the activation of MAP kinases and MEK and the ability of BMP-2 and activin A to induce PC12 cell neuronal differentiation. In other words, the cytoplasmic signaling pathways of BMP-2 and activin A appear to be totally independent of the MAP kinase cascade.
As far as we know, BMP-2 stimulates the growth of Swiss 3T3 cells without inducing the activation of Ras, MEK, MAP kinases, or other recently identified cytoplasmic signaling pathways such as the Janus kinase (JAK)/signal transducers and activators of transcription (STAT) pathway and the c-Jun NH 2 -terminal kinase (JNK) pathway. 3 Activation of the JAK/ STAT pathway has been commonly observed in cells stimulated with a variety of cytokines (interferons, interleukins, erythropoietin, etc.) and with EGF and platelet-derived growth factor (reviewed in Ref. 49), while the JNK pathway has been reported to be activated by treatment of cells with UV radiation, pro-inflammatory cytokines, and environmental stress (reviewed in Refs. 46,47,50). Thus, the signaling pathway of BMP-2, which originates at the cell surface receptors that have been suggested to possess serine/threonine kinase activity (51)(52)(53)(54), seems quite unique and apparently independent of those well characterized cytoplasmic kinase cascades. The precise cytoplasmic signaling pathway of BMP-2 through which it elicits its effects on the regulation of cell proliferation/differentiation remains to be determined.
In conclusion, we have demonstrated in this report that BMP-2 is able to induce the neuronal differentiation of PC12 cells by a signaling pathway that is totally independent of the MAP kinase cascade. Our results also indicate that the activation of MEK and MAP kinases is not an absolute requirement for PC12 cell differentiation.