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The Ras Suppressor, RSU-1, Enhances Nerve Growth Factor-Induced Differentiation of PC12 Cells and Induces P21cip Expression1

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The Ras Suppressor, RSU-1, Enhances Nerve Growth Factor-Induced Differentiation of PC12 Cells and Induces P21cip Expression1 Vol. 10, 555–564, August 1999 Cell Growth & Differentiation 555 The Ras Suppressor, RSU-1, Enhances Nerve Growth Factor-induced Differentiation of PC12 Cells and Induces p21CIP Expression1 L. Masuelli, S. Ettenberg, F. Vasaturo, Introduction 2 3 K. Vestergaard-Sykes, and M. L. Cutler Rsu-1, which was isolated based on its ability to suppress Department of Pathology, Uniformed Services University of the Health Ras transformation, encodes a Mr 33,000 protein that con- Sciences, Bethesda, Maryland 20814 [L. M., S. E., F. V., M. L. C.]; tains a series of leucine-rich amphipathic repeats homolo- Department of Experimental Medicine, First University of Rome, Rome 00161, Italy [L. M.]; and United States Department of Agriculture gous to the leucine repeats found in yeast adenylyl cyclase Laboratories, Beltsville, Maryland 20705 [K. V-S.] (1–3). These repeats are required for the activation of adeny- lyl cyclase by Ras in Saccharomyces cerevisiae (4, 5), and similar repeats are required for a Ras-induced differentiation Abstract pathway in Caenorhabditis elegans (3). Rsu-1 binds to Raf-1 The Rsu-1 Ras suppressor gene was isolated based on in in vitro binding assays, suggesting that Rsu-1 may stabi- its ability to inhibit v-Ras transformation. Using Rsu-1 lize Ras-Raf association and/or inhibit the association of Ras transfectants of the pheochromocytoma cell line PC12, with other effectors. Rsu-1 expression inhibited RasGAP we demonstrated previously that Rsu-1 expression activity, resulting in an increase in Ras-GTP, and inhibited the inhibited Jun kinase activation but enhanced Erk2 activation of Jun kinase by EGF4 (6). Activation of the Erk activation in response to epidermal growth factor. In kinase pathway was not inhibited by Rsu-1 expression; in the present study, the Rsu-1 PC12 transfectants were contrast, Rsu-1 expression resulted in increased stimulation used to investigate the role of Rsu-1 in nerve growth of Erk in response to EGF (6). factor (NGF)- and v-Ki-ras-mediated neuronal The infection of PC12 pheochromocytoma cells with Ki- differentiation. NGF-induced neurite extension was MSV or Ha-MSV and microinjection of activated Ras p21 enhanced, not inhibited, by the expression of Rsu-1 in results in the induction of a program of differentiation phe- PC12 cells. The activation of Erk kinase activity in notypically demonstrated by neurite outgrowth (7, 8). This response to NGF was sustained longer in the Rsu-1 program of differentiation is biochemically characterized by transfectants compared with the vector control cells. induction of a specific gene expression program (9, 10). The During NGF-mediated differentiation, an increase in the stimulation of PC12 cells with NGF results in the activation of expression of specific mRNAs for the early response a program of neuronal differentiation (11). NGF activation of genes Fos, cJun, and NGF1a was detected in both the the tyrosine kinase activity of trk, as part of a high affinity vector control and Rsu-1 transfectants. The expression NGF receptor, results in tyrosine phosphorylation of trk and of the differentiation-specific genes VGF8 and SCG10 activation of downstream effectors (12–14). This activation was similar in Rsu-1 transfectants compared with the requires Ras (15, 16) and depends upon sustained activation vector control cells. The induction of Rsu-1 expression of the Erk pathway (17, 18). In the present study, we tested in these cell lines did not inhibit v-Ki-ras-induced the ability of Rsu-1 expression, which can inhibit Ras-in- differentiation, as measured by neurite extension. duced transformation and Jun kinase activation, to inhibit These data suggest that although Rsu-1 blocked some v-Ki-Ras-induced and NGF-induced differentiation of PC12 Ras-dependent response(s), these responses were not cells. Previous studies have demonstrated that increased required for differentiation. Moreover, the induction of expression of RasGAP, resulting in a decrease in Ras-GTP Rsu-1 expression in the PC12 clones resulted in levels, inhibited NGF induced-differentiation (19) and that WAF/CIP growth inhibition and p21 expression. Hence, Jun kinase activation was not necessary for differentiation in Rsu-1 expression enhances NGF-induced response to NGF (20). Hence, overexpression of Rsu-1 dur- differentiation while inhibiting the growth of cells. ing NGF stimulation of PC12 cells will assess the importance of elevated and sustained activation of Erk kinase as well as the requirement for activation of other pathways downstream Received 2/24/99; revised 4/14/99; accepted 7/1/99. of Ras for the initiation of the differentiation program. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Previous studies of NGF-induced differentiation of PC12 advertisement in accordance with 18 U.S.C. Section 1734 solely to indi- cells indicated there are numerous changes brought about cate this fact. by cell cycle regulatory proteins that can promote growth 1 This work was supported in part by a grant from the Childhood Brain Tumor Foundation (to M. L. C.) and by DOD Grant DAMD 17-97-1-7089 arrest in G1. NGF-stimulated PC12 cells exhibit an increase (to M. L. C.). 2 Present address: Department of Surgical Oncology, Medical College of Virginia, Richmond, VA. 3 To whom requests for reprints should be addressed, at B3122, Uni- 4 The abbreviations used are: EGF, epidermal growth factor; NGF, nerve formed Services University of Health Sciences, 4301 Jones Bridge Road, growth factor; cdk, cyclin-dependent kinase; GPDH, glyceraldehyde-3- Bethesda, MD 20814. Phone: (301) 295-3453; Fax: (301) 295-1640; E- phosphate dehydrogenase; MMTV, mouse mammary tumor virus; MuLV, mail: [email protected]. murine leukemia virus. 556 Rsu-1 Enhances PC12 Differentiation in p21WAF/CIP when propagated in serum-containing media (21). However, stimulation of cells maintained in low levels of serum did not require induction of p21WAF/CIP for differenti- ation (22). Recent studies on the role of p21CIP in cell cycle regulation indicated that activation of Raf can result in in- duction of p21CIP expression (23, 24). Therefore, in addition to the activation of the Ras-Raf pathway in response to NGF, differentiation relies on induction of p21WAF/CIP or serum deprivation to inhibit cell cycle progression. In this study, Rsu-1 transfectants of PC12 cells, which contain Rsu-1 under the control of a dexamethasone-induc- ible MMTV promoter, were induced to differentiate by the addition of NGF or by infection with Ki-MSV. The activation of early-response genes as well as differentiation-specific markers was determined by Northern blotting after both in- fection and NGF treatment. The activation of Erk and Jun kinase as well as the extent of neurite outgrowth was meas- ured in response to NGF exposure and Ki-MSV infection. The results demonstrate that the effect of increased Rsu-1 ex- pression is not an inhibition of Ras-induced differentiation but rather an enhancement of the process. Moreover, the result of NGF-induced stimulation of the Rsu-1 transfectants is the induction of a rapid differentiation process. Results The Ras suppressor, Rsu-1, was introduced into PC12 cells to test its effect on Ras-dependent processes of growth factor-induced mitogenesis and differentiation. Our previous work described the construction of a vector carrying the Rsu-1 cDNA under the control of a dexamethasone-induc- Fig. 1. Thymidine incorporation after stimulation of Rsu-1 transfectant ible promotor and its introduction into PC12 cells (6). Two cell line with mitogens. PC12-a, a vector control transfectant cell line, and clones expressing Rsu-1 in response to dexamethasone, PC12-20, an Rsu-1 transfectant cell line, were treated with dexametha- sone for 24 h in reduced serum medium. EGF (100 ng/ml) or insulin (400 PC12-20 and PC12-26, were used in the studies described ng/ml) was added to the cells, and [3H]thymidine pulse labeling was here, along with a pMAM vector control clone, PC12-a (6). performed at 0, 24, 48, and 72 h after the addition of mitogen. Triplicate samples were used to calculate the mean for each time point; bars, SD. The cells were stimulated with NGF or infected with Ki-MSV SDs are shown and were ,10% of the mean at all time points. to initiate differentiation; the response of the cells to mito- genic compounds was tested by stimulation with insulin and EGF. Growth of PC12 Rsu-1 Transfectants in Response to regulating signal transduction leading to neuronal differenti- Mitogens. To investigate the effects of mitogenic factors ation, two Rsu-1 transfectant PC12 cell clones and a vector signaling through Ras on the growth of PC12 cells overex- control PC12 cell line were exposed to NGF. The phenotypic pressing Rsu-1, the effect of two growth factors, epidermal appearance of the cells in the form of neurite outgrowth and growth factor and insulin, was tested. The PC12 vector con- the expression of differentiation-specific RNA were investi- trol and Rsu-1 transfectant cells were plated in serum con- gated over a period of 4 days. taining medium. Rsu-1 expression was induced for 24 h with The extent of neurite outgrowth after NGF addition to the 0.5 mM dexamethasone before the addition of EGF or insulin. cells was analyzed in cultures of PC12-a vector control cell The cells were harvested at different time points after [3H]thy- line and in the PC12-20 Rsu-1 transfectant clones. PC12 cell midine pulse labeling, as indicated in Fig. 1. [3H]Thymidine clones growing on dishes were stimulated with two different incorporation studies demonstrated that the vector control concentrations of NGF. The degree of neurite extension was cell line PC12-a proliferated rapidly in response to EGF or determined by microscopically counting cells at 24 and 72 h insulin, whereas proliferation of the Rsu-1 transfectant after NGF addition and quantitating the number of cells ex- PC12-20 was nearly completely blocked.
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