Nerve Growth Factor-induced Growth Arrest and Induction of p21 in NIH-3T3 Cells Expressing TrkA

Treatment of NIH-3T3 cells expressing human TrkA with nerve growth factor (NGF) resulted in a rapid cessation of growth. Cells stopped dividing within 24 h of NGF treatment and failed to divide as long as NGF was present, accumulating in the G1 stage of the cell cycle. NGF caused a prolonged activation of mitogen-activated protein kinase relative to EGF. NGF treatment of cells greatly increased levels of the p21 protein, an inhibitor of cyclin-dependent kinases, without affecting levels of p27 or p16. Levels of p21remained elevated for at least 48 h following NGF addition. EGF had little effect on p21 expression in the same parental cells expressing the human EGF receptor. NGF treatment of cells completely inhibited the activity of the cyclin-dependent protein kinases CDK2 and CDK4. Inhibition correlated with a 10-20-fold increase in the amount of p21 complexed with CDK2 and CDK4. Levels of CDK2 and CDK4 were decreased following NGF treatment of cells; however, levels of cyclin E and cyclin D were increased. These data indicate that NGF can induce cell cycle arrest of NIH-3T3, perhaps through modulation of p21 levels. The data also show that distinct signals are generated by TrkA versus the EGF receptor in NIH-3T3 cells.

Treatment of NIH-3T3 cells expressing human TrkA with nerve growth factor (NGF) resulted in a rapid cessation of growth. Cells stopped dividing within 24 h of NGF treatment and failed to divide as long as NGF was present, accumulating in the G 1 stage of the cell cycle. NGF caused a prolonged activation of mitogen-activated protein kinase relative to EGF. NGF treatment of cells greatly increased levels of the p21 Cip1/WAF1 protein, an inhibitor of cyclin-dependent kinases, without affecting levels of p27 KIP1 or p16 INK4 . Levels of p21 Cip1/WAF1 remained elevated for at least 48 h following NGF addition. EGF had little effect on p21 Cip1/WAF1 expression in the same parental cells expressing the human EGF receptor. NGF treatment of cells completely inhibited the activity of the cyclin-dependent protein kinases CDK2 and CDK4. Inhibition correlated with a 10 -20-fold increase in the amount of p21 Cip1/WAF1 complexed with CDK2 and CDK4. Levels of CDK2 and CDK4 were decreased following NGF treatment of cells; however, levels of cyclin E and cyclin D were increased. These data indicate that NGF can induce cell cycle arrest of NIH-3T3, perhaps through modulation of p21 Cip1/WAF1 levels. The data also show that distinct signals are generated by TrkA versus the EGF receptor in NIH-3T3 cells.
Nerve growth factor (NGF) 1 is a member of the family of neurotrophic factors and plays a significant role in growth, differentiation, and survival of various types of neurons (1,2). NGF can also induce the differentiation of PC-12 pheochromocytoma cells into neuron-like cells (3). These effects are thought to be mediated primarily through binding of NGF to the high affinity NGF receptor TrkA and subsequent signal generation by TrkA (4,5). A second receptor, p75, a member of the tumor necrosis factor-␣ family, also binds NGF (as well as other neurotrophic factors) and may be involved in some aspects of NGF signaling (6).
NGF binding to TrkA has been shown to activate the Ras/ Raf/MAP kinase pathway (7), perhaps through tyrosine phosphorylation of Shc proteins by TrkA (8). Phospholipase C-␥1 (9, 10), and phosphatidylinositol 3-kinase (11) have also been shown to be involved in signaling by TrkA. Activation of TrkA also results in tyrosine phosphorylation of the SNT protein in neuronal cells (12). Many of these pathways are also activated by mitogenic factors such as EGF and platelet-derived growth factor (13), raising the question of whether signaling specificity resides in the receptors themselves or with the cellular background in which the receptors are expressed. In fact, it has been reported that when expressed in NIH-3T3 cells, TrkA functions much like the EGF receptor, stimulating cell division and cellular transformation (14). Conversely, in some neuroblastoma cell lines (15) and in PC-12 cells (3), TrkA mediates NGF-dependent growth arrest and differentiation. In this report we find that NGF treatment of NIH-3T3 cells expressing TrkA leads to growth inhibition that may be mediated through activation of the MAP kinase pathway and through induction of synthesis of the cyclin-dependent kinase inhibitor p21 Cip1/WAF1 . Cell Culture and TrkA Expression-A strain of NIH-3T3 cells expressing no detectable TrkA (17) was transfected with human TrkA cDNA (a kind gift from G. Parada, National Institutes of Health), which had been inserted into the REP4 mammalian expression vector (Invitrogen) using the calcium phosphate procedure. Cells were selected in 50 units/ml hygromycin and resistant clones screened for 125 I-NGF binding. Twelve clones were isolated, which expressed approximately 25,000 -125,000 high affinity TrkA receptors. NIH-3T3 cells expressing human EGF receptor have been described previously (18). All cells were grown in Dulbecco's modified Eagle's medium with 5% calf serum.

Materials-
Assay of MAP Kinase Activity-Immune complex assay of MAP kinase activity was performed essentially according to Boulton et al. (19). Briefly, cells grown on 60-mm dishes were serum-starved for 6 h prior to additions. Cells were lysed in 1 ml of MIPA buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 0.5% desoxycholate, 1% Nonidet P-40, 50 mM NaF, 1 mM para-nitrophenyl phosphate, 1 mM NaVO 3 , 10 g/ml aprotinin, 10 g/ml leupeptin, and 1 mM benzamidine). Lysates were cleared by centrifugation and immune-precipitated with antibodies against the p42 and p44 forms of MAP kinase for 60 min at 4°C. Immune complexes were bound to protein A-Sepharose beads and washed three times with lysis buffer. 50 l of 25 mM HEPES, 10 mM MgCl 2 , 1 mM dithiothreitol, 0.3 mg/ml myelin basic protein solution were added to the beads, prior to the addition of 50 M [␥-32 P]ATP for 15 min at room temperature. Reactions were stopped with SDS sample buffer and 32 P-labeled myelin basic protein resolved on SDS gels. Immunoblotting of p21 Cip1/WAF1 , p27 KIP1 , p16 INK4 , CDK2, CDK4, Cyclin E, and Cyclin D-Cells expressing TrkA or, in some cases, EGF receptor were plated at 2 ϫ 10 4 cells/ml in 35-mm dishes and allowed to adhere for 4 h. NGF or EGF was then added at the indicated concentrations for indicated periods of time. Cells were washed twice with 2 ml of phosphate-buffered saline and lysed in 100 l of SDS sample buffer. 20 g of protein from each whole cell lysate was applied to SDS gels, protein transferred to nitrocellulose, and membranes probed with the appropriate antibodies. For determination of the amount of p21 Cip1/WAF1 bound to CDK2 or CDK4, cells were lysed in MIPA buffer, lysates immune-precipitated with antibody to CDK2 or CDK4, immu-* 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. noprecipitated proteins resolved on SDS gels, proteins transferred to nitrocellulose, and membranes were immunoblotted with antibody against p21 Cip1/WAF1 .
Immune Complex Assays of Cyclin-dependent Kinase Activity-CDK2 activity in immune complexes from control or NGF-treated cells using histone H1 as substrate was assayed according to Ewen et al. (20). CDK4 activity in immune complexes was assayed using glutathione S-transferase-Rb protein as substrate according to Matsushime et al. (21). Measurements of [ 3 H]thymidine incorporation and anchorage-independent growth were performed as described previously (18).

RESULTS
The TrkA high affinity receptor for NGF was expressed in NIH-3T3 cells to compare transmembrane signaling between TrkA and the EGF receptor in a non-transformed cell background. A representative clone expressing about 100,000 TrkA receptors/cell (TRK1 cells) was used throughout this work; in some cases, results using this clone were compared with those using the same parental cells expressing a similar number of EGF receptors (EGFR1 cells) (18).
We first examined the effects NGF and EGF, respectively, on the growth of TRK1 and EGFR1 cells cultured in normal medium with 5% calf serum. As shown in Fig. 1A, EGF had little additional effect on the serum-dependent growth rate of EGFR1 cells; however, NGF completely arrested growth of TRK1 cells. This effect was evident within 24 h of NGF addition. NGF also inhibited [ 3 H]thymidine incorporation when added to TRK1 cells growing in normal medium (Fig. 1B). In addition, NGF treatment of TRK1 cells had a dramatic effect on cellular morphology (Fig. 1C). In the presence of NGF for 4 days, many cells had become multinucleate, assuming a round, flattened shape with long processes. Cell cycle analysis of normally growing TRK1 cells and cells cultured in the presence of NGF for 4 days indicated that NGF caused a build up of cells in the G 1 stage of the cell cycle (not shown). NGF-treated cells did not appear to be undergoing apoptosis (not shown).
Since NGF had previously been shown to stimulate anchorage-independent growth of another strain of NIH-3T3 cells expressing TrkA (14), we cultured TRK1 cells in soft agar in the presence and absence of NGF (Table I). Surprisingly, NGF did not induce colony formation of TRK1 cells in agar. When examined microscopically, TRK1 cells in the presence of NGF appeared to divide once or twice, forming clusters of 2-4 large cells. As expected, EGF-stimulated EGFR1 cells formed large colonies in agar.
Studies using PC-12 cells have shown that NGF treatment results in a more prolonged activation of MAP kinase than does treatment with EGF, suggesting a role for MAP kinase in the distinct actions of these growth factors (22,23). We found similar results when the effects of NGF and EGF on MAP kinase activation were compared in TRK1 and EGFR1 cells ( Fig. 2A). NGF and EGF produced equivalent stimulation of MAP kinase activity at the 30-min and 2-h time points; however, at the 8-and 24-h time points, little stimulation by EGF was apparent, while the NGF stimulation was still 4 -6 times above background.
The frank inhibition of growth of TRK1 cells by NGF and build-up of cells in G 1 suggested that NGF could be causing inhibition of CDKs. p27 KIP1 , p21 Cip1/WAF1 , and p16 INK4 are NGF Induction of p21 Cip1/WAF1 30842 recently described inhibitors of these kinases, which have been shown to be regulated during differentiation and growth arrest (24 -28). While treatment of TRK1 cells with NGF had little effect on protein levels of p27 KIP1 or p16 INK4 (data not shown), NGF caused a rapid and dramatic increase in levels of p21 Cip1/WAF1 protein (Fig. 3A). Increased levels of p21 Cip1/WAF1 were seen as early as 2 h following NGF addition and were maximal by 6 h (Fig. 3B). NGF at 1 nM was sufficient to achieve maximal response (Fig. 3C). EGF treatment of EGFR1 cells had little effect on levels of p21 Cip1/WAF1 . NGF has been reported to induce synthesis of transforming growth factor-␤ in PC-12 cells (29), which may increase p21 Cip1/WAF1 levels in some cells (30); however, transforming growth factor-␤ had no effect on levels of p21 Cip1/WAF1 in TRK1 cells (data not shown).
To further examine the relationship between NGF-dependent inhibition of growth and induction of p21 Cip1/WAF1 , the effects of NGF on the activities of CDK2 and CDK4, cyclin-dependent kinases known to be involved in G 1 /S transition and known to bind p21 Cip1/WAF1 , were determined (Fig. 4A). NGF treatment of TRK1 cells completely inhibited the activities of CDK2 and CDK4 as judged by in vitro kinase assays. As shown in Fig. 4B, NGF treatment reduced the detectable levels of CDK2 protein by about 80% and reduced the level of CDK4 protein about 50%. The amount of p21 Cip1/WAF1 co-precipitating with CDK2 and CDK4 antibodies increased 10 -20-fold following NGF treatment (Fig. 4C). Somewhat surprisingly, NGF treatment of TRK1 cells resulted in increases in the levels of cyclin E and cyclin D (Fig. 4D). DISCUSSION This is the first report in which NGF has been shown to increase cellular levels of p21 Cip1/WAF1 and the first instance in which levels of p21 Cip1/WAF1 are regulated through activation of a tyrosine kinase receptor. Increased expression of p21 Cip1/WAF1 occurs following exposure of cells to ␥-irradiation (31), in cells undergoing senescence (32), during differentiation of myoblasts and keratinocytes (33)(34)(35)(36), and in response to treatment with transforming growth factor-␤ (30). In most cases, up-regulation of p21 Cip1/WAF1 levels correlates with cell cycle arrest, seemingly dependent on the relative stoichiometric amounts of p21 Cip1/WAF1 present in complexes with CDK kinases (37). Expression of p21 Cip1/WAF1 may also directly inhibit DNA synthesis by blocking proliferating cell nuclear antigen from activating DNA polymerase (38 -40), although recent data suggest that the proliferating cell nuclear antigen binding and inhibition of growth may be disassociated (41,42).
Here, we find a clear NGF-induced increase in the level of p21 Cip1/WAF1 protein and a dramatic increase in the amount of a Single cell suspensions (2.5 ϫ 10 3 cells/35-mm dish) were plated in 0.3% agar containing vehicle or NGF or EGF. Cells were incubated for 10 days, at which time colonies were counted microscopically. Colonies larger than 0.1 mm were scored as positive. Each value represents the mean and standard error from triplicate cultures. NGF Induction of p21 Cip1/WAF1 30843 p21 Cip1/WAF1 complexed with CDK2 and CDK4. These changes correlate with a complete inhibition of measurable CDK2 and CDK4 activity and may form the basis for the observed NGFinduced growth arrest in TRK1 cells. Similar inhibition of G 1 cyclin-dependent kinases has been found in terminally differentiating cells (34) and in cells undergoing senescence (43,44). NGF has been shown to induce growth arrest in parallel with differentiation in PC-12 cells (3) and a number of neuroblastoma cell lines (15), suggesting a possible role for NGF-mediated suppression of growth in neurons. It will be of interest to determine whether NGF induces expression of p21 Cip1/WAF1 in these cell types. NGF treatment of TRK1 cells down-regulated protein levels of CDK2 and CDK4 (CDK2 was particularity affected), while levels of cyclin D and cyclin E increased. Induction of macrophage-like differentiation of HL-60 cells by 12-O-tetradecanoylphorbol-13-acetate results in a similar decrease in expression of CDK2 and an up-regulation of cyclin D1 (45). Several reports (34,46) have also shown a large increase in expression of cyclin D3 following differentiation of L6 cells or skeletal myoblasts into myotubes. Expression of cyclin D1 is enhanced in PC12 h cells undergoing neuronal differentiation after treatment with NGF (47) and in senescent human fibroblasts relative to cells that have undergone fewer divisions (43,44). Decreases in CDK levels seem a logical response to stimuli that induce growth arrest; however, increases in levels of cyclin D are surprising in light of the ability of cyclin D1 to function as an oncogene (48) and to shorten G 1 when overexpressed (49). At present, it is not clear whether increased expression of G 1 cyclins during growth arrest is a result of cells arresting at a point the cell cycle where these cyclins are normally expressed at high levels, or is due to loss of a feedback loop for inhibition of expression, or whether cyclins play an active role in growth arrest. At high intracellular concentrations cyclin D could interact with proteins other than CDK4 and Rb.
In contrast to our findings, it has been reported previously that TrkA functions as a typical mitogenic growth factor receptor when expressed in NIH-3T3 cells (14). Since activation of TrkA can induce growth arrest and differentiation in neuronal derived cell lines, these data led to proposals that the ability of TrkA to stimulate differentiation and survival was probably a consequence of the cellular background in which it is expressed rather than due to any intrinsic signaling properties (14,50). Our data support the opposite contention, that signals from a mitogenic receptor such as the EGF receptor and those from TrkA are not equivalent. It may be that TrkA has an inherent capacity to generate signals that negatively influence cell cycle progression through induction of synthesis of inhibitors such as p21 Cip1/WAF1 . This would represent a critical point of divergence from signaling by mitogenic receptors. We can only speculate that the differences in growth responses to NGF between TRK1 cells and the cells used by Cordo-Cardo et al. (14) are due to strain differences or differences in the expression levels of TrkA.
Further study of TrkA signaling using NIH-3T3 cells should more precisely delineate pathways involved in NGF-dependent induction of p21 Cip1/WAF1 . TRK1 cells represent an easily manipulated, non-transformed cell in vitro system and may prove useful for studies of other aspects of NGF action.