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Cell Growth & Differentiation Vol. 10, 555-564, August 1999
© 1999 American Association for Cancer Research

The Ras Suppressor, RSU-1, Enhances Nerve Growth Factor-induced Differentiation of PC12 Cells and Induces p21CIP Expression1

L. Masuelli, S. Ettenberg, F. Vasaturo, K. Vestergaard-Sykes2 and M. L. Cutler3

Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814 [L. M., S. E., F. V., M. L. C.]; Department of Experimental Medicine, First University of Rome, Rome 00161, Italy [L. M.]; and United States Department of Agriculture Laboratories, Beltsville, Maryland 20705 [K. V-S.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
The Rsu-1 Ras suppressor gene was isolated based on its ability to inhibit v-Ras transformation. Using Rsu-1 transfectants of the pheochromocytoma cell line PC12, we demonstrated previously that Rsu-1 expression inhibited Jun kinase activation but enhanced Erk2 activation in response to epidermal growth factor. In the present study, the Rsu-1 PC12 transfectants were used to investigate the role of Rsu-1 in nerve growth factor (NGF)- and v-Ki-ras-mediated neuronal differentiation. NGF-induced neurite extension was enhanced, not inhibited, by the expression of Rsu-1 in PC12 cells. The activation of Erk kinase activity in response to NGF was sustained longer in the Rsu-1 transfectants compared with the vector control cells. During NGF-mediated differentiation, an increase in the expression of specific mRNAs for the early response genes Fos, cJun, and NGF1a was detected in both the vector control and Rsu-1 transfectants. The expression of the differentiation-specific genes VGF8 and SCG10 was similar in Rsu-1 transfectants compared with the vector control cells. The induction of Rsu-1 expression in these cell lines did not inhibit v-Ki-ras-induced differentiation, as measured by neurite extension. These data suggest that although Rsu-1 blocked some Ras-dependent response(s), these responses were not required for differentiation. Moreover, the induction of Rsu-1 expression in the PC12 clones resulted in growth inhibition and p21WAF/CIP expression. Hence, Rsu-1 expression enhances NGF-induced differentiation while inhibiting the growth of cells.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Rsu-1, which was isolated based on its ability to suppress Ras transformation, encodes a Mr 33,000 protein that contains a series of leucine-rich amphipathic repeats homologous to the leucine repeats found in yeast adenylyl cyclase (1, 2, 3) . These repeats are required for the activation of adenylyl cyclase by Ras in Saccharomyces cerevisiae (4 , 5) , and similar repeats are required for a Ras-induced differentiation pathway in Caenorhabditis elegans (3) . Rsu-1 binds to Raf-1 in in vitro binding assays, suggesting that Rsu-1 may stabilize Ras-Raf association and/or inhibit the association of Ras with other effectors. Rsu-1 expression inhibited RasGAP activity, resulting in an increase in Ras-GTP, and inhibited the activation of Jun kinase by EGF4 (6) . Activation of the Erk kinase pathway was not inhibited by Rsu-1 expression; in contrast, Rsu-1 expression resulted in increased stimulation of Erk in response to EGF (6) .

The infection of PC12 pheochromocytoma cells with Ki-MSV or Ha-MSV and microinjection of activated Ras p21 results in the induction of a program of differentiation phenotypically demonstrated by neurite outgrowth (7 , 8) . This program of differentiation is biochemically characterized by induction of a specific gene expression program (9 , 10) . The stimulation of PC12 cells with NGF results in the activation of a program of neuronal differentiation (11) . NGF activation of the tyrosine kinase activity of trk, as part of a high affinity NGF receptor, results in tyrosine phosphorylation of trk and activation of downstream effectors (12, 13, 14) . This activation requires Ras (15 , 16) and depends upon sustained activation of the Erk pathway (17 , 18) . In the present study, we tested the ability of Rsu-1 expression, which can inhibit Ras-induced transformation and Jun kinase activation, to inhibit v-Ki-Ras-induced and NGF-induced differentiation of PC12 cells. Previous studies have demonstrated that increased expression of RasGAP, resulting in a decrease in Ras-GTP levels, inhibited NGF induced-differentiation (19) and that Jun kinase activation was not necessary for differentiation in response to NGF (20) . Hence, overexpression of Rsu-1 during 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 of Ras for the initiation of the differentiation program.

Previous studies of NGF-induced differentiation of PC12 cells indicated there are numerous changes brought about by cell cycle regulatory proteins that can promote growth arrest in G1. NGF-stimulated PC12 cells exhibit an increase 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 differentiation (22) . Recent studies on the role of p21CIP in cell cycle regulation indicated that activation of Raf can result in induction 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-inducible 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 infection and NGF treatment. The activation of Erk and Jun kinase as well as the extent of neurite outgrowth was measured in response to NGF exposure and Ki-MSV infection. The results demonstrate that the effect of increased Rsu-1 expression 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
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
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-inducible promotor and its introduction into PC12 cells (6) . Two clones expressing Rsu-1 in response to dexamethasone, PC12-20 and PC12-26, were used in the studies described here, along with a pMAM vector control clone, PC12-a (6) . The cells were stimulated with NGF or infected with Ki-MSV to initiate differentiation; the response of the cells to mitogenic compounds was tested by stimulation with insulin and EGF.

Growth of PC12 Rsu-1 Transfectants in Response to Mitogens.
To investigate the effects of mitogenic factors signaling through Ras on the growth of PC12 cells overexpressing Rsu-1, the effect of two growth factors, epidermal growth factor and insulin, was tested. The PC12 vector control and Rsu-1 transfectant cells were plated in serum containing medium. Rsu-1 expression was induced for 24 h with 0.5 µM dexamethasone before the addition of EGF or insulin. The cells were harvested at different time points after [3H]thymidine pulse labeling, as indicated in Fig. 1Citation . [3H]Thymidine incorporation studies demonstrated that the vector control cell line PC12-a proliferated rapidly in response to EGF or insulin, whereas proliferation of the Rsu-1 transfectant PC12-20 was nearly completely blocked. These data suggest that overexpression of Rsu-1 can inhibit proliferation of these cells in response to mitogens.



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Fig. 1. Thymidine incorporation after stimulation of Rsu-1 transfectant cell line with mitogens. PC12-a, a vector control transfectant cell line, and PC12-20, an Rsu-1 transfectant cell line, were treated with dexamethasone for 24 h in reduced serum medium. EGF (100 ng/ml) or insulin (400 ng/ml) was added to the cells, and [3H]thymidine pulse labeling was 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. SDs are shown and were <10% of the mean at all time points.

 
Analysis of NGF-mediated Differentiation in PC12-Rsu-1 Transfectant Cell Lines.
Stimulation of PC12 cells with NGF can induce neuronal differentiation, characterized by neurite outgrowth and expression of specific differentiation markers (25, 26, 27) . To analyze a possible role for Rsu-1 in regulating signal transduction leading to neuronal differentiation, two Rsu-1 transfectant PC12 cell clones and a vector control PC12 cell line were exposed to NGF. The phenotypic appearance of the cells in the form of neurite outgrowth and the expression of differentiation-specific RNA were investigated over a period of 4 days.

The extent of neurite outgrowth after NGF addition to the cells was analyzed in cultures of PC12-a vector control cell line and in the PC12-20 Rsu-1 transfectant clones. PC12 cell clones growing on dishes were stimulated with two different concentrations of NGF. The degree of neurite extension was determined by microscopically counting cells at 24 and 72 h after NGF addition and quantitating the number of cells extending short or long neurites. As shown in Table 1Citation , both cell lines differentiated when exposed to NGF. The Rsu-1-overexpressing cell lines, PC12-20, differentiated earlier and faster than the vector control cell line PC12-a. The overall level of differentiation was approximately 3-fold greater for the Rsu-1 transfectants than the vector control cells. More striking were the appearance of long neurites in the Rsu-1 transfectants (Table 1)Citation . The photograph in Fig. 2Citation Citation illustrates the appearance of the vector control and the Rsu-1 transfectant clones maintained on plastic dishes in complete medium in the presence and absence of NGF.


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Table 1 Differentiation of Rsu-1 transfectant in response to NGF

 


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Fig. 2. Differentiation of Rsu-1 transfectant cell lines in response to NGF. PC12 Rsu-1 transfectant and vector control transfectant cells were seeded in plastic tissue cultures dishes in complete medium, induced with 0.5 µM dexamethasone for 48 h, followed by maintenance in the presence or absence of NGF (50 ng/ml) for 24 h. Cells were photographed by confocal microscopy using a x40 objective (A) and by phase contrast microscopy at x400 (B). Panels a and b, clone a; panels c and d, clone 20; panels e and f, clone 26. Panel a, c, and e, cells exposed to only dexamethasone; panels b, d, and f, cells exposed to dexamethasone and NGF.

 


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Fig. 2A. Continued.

 
The expression of RNA for early response genes (Fos, c-Jun, and NGF1A/EGR1) and for differentiation-specific genes (VGF8 and SCG10/strathmin) in response to NGF was analyzed. Initially, RNA was extracted from dexamethasone-induced PC12-a control and PC12-20 Rsu-1 transfectant cells after a time course of NGF treatment (0–60 min); the level of specific RNA was determined by Northern blotting and hybridization to specific probes. As shown in Fig. 3ACitation , there was an increase in the expression of c-fos, c-Jun, and NGF1A by 30 min after NGF addition in both the Rsu-1 and vector control cell lines. Quantitation revealed that the magnitude of the Fos response was 2-fold greater in the Rsu-1-overexpressing cell line, PC12-20, compared with the vector control cell line, PC12-a (Fig. 3B)Citation . Fos induction by NGF has been reproducibly elevated 2–2.5-fold over the control cell line at 45 and 60 min. Expression of late-response genes specific for neuronal differentiation, VGF8 and SCG10, were also analyzed over a longer time course (0–72 h) in both Rsu-1 transfectants and the vector control clone. There were no significant differences in SCG10 expression between the Rsu-1 transfectant cell lines and the control. However, VGF8 expression was sustained longer in response to NGF in the Rsu-1 transfectants (Fig. 4)Citation . Again, the greater intensity of Fos induction was evident in the Rsu-1 transfectants compared to the control clone. The somewhat elevated neurite extension and induction of VGF8 RNA, which is specific to the induction of the differentiation program by NGF, in the Rsu-1 transfectants demonstrates that this Ras-dependent process is not inhibited by Rsu-1 expression. In fact, it is somewhat enhanced compared with the control cell line.



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Fig. 3. Expression of early-response genes and differentiation-specific genes after NGF stimulation of Rsu-1 transfectant cell lines. A, Northern blot of RNA from NGF-stimulated PC12-a and PC12-20 transfectant cell lines. Northern blots were prepared from 10 µg of total RNA extracted from cells exposed to NGF (200 ng/ml) for 0, 30, 45, or 60 min. The blots were hybridized sequentially to the indicated specific probes. B, the hybridization signals from the blot in Fig. 2ACitation were quantitated and normalized to the GPDH hybridization signal. The results are shown graphically.

 


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Fig. 4. Expression of early-response genes and differentiation-specific markers after 72 h of NGF stimulation of Rsu-1 transfectant cell lines. Northern blots were prepared from total RNA isolated from PC12-a, PC12-20, and PC12-26 transfectant cell lines exposed to NGF (200 ng/ml) for 0, 1, 6, 24, 48, or 72 h. The blots were hybridized to probes for the differentiation-specific markers SCG10, VGF8, as well as the early-response genes Fos and NGF1A. Rsu-1 and GPDH (GAPDH) hybridization are shown as controls. Arrows, position of specific induced bands.

 
Infection of PC12 Rsu-1 Transfectants with Ki-Ras.
The introduction of viral Ras into PC12 cells by infection with Ki-MSV, microinjection of v-Ras p21, or transfection of a vector carrying v-Ras under the control of an inducible promoter resulted in initiation of differentiation in PC12 cells (7 , 8 , 10) . Because Rsu-1 expression was able to inhibit Ras-induced transformation of fibroblasts, the ability of Rsu-1 to alter v-Ras-induced differentiation of PC12 cells was tested. Ki-MSV was used to infect PC12 vector control cells and Rsu-1 transfectants. The cells were examined for phenotypic differentiation, and RNA was isolated to check for the induction of Ras-induced transcriptional response. As a control for changes occuring in response to v-Ras, a PC12-derived cell line containing activated v-Ras under the control of a dexamethasone-inducible MMTV promoter (GSRas-1) was used (10) . In addition, MuLV infection of the PC12 clones served as a negative control.

The effect of v-Ras infection on phenotypic differentiation of PC12 vector control and Rsu-1 transfectants was examined. The percentage of differentiated cells in the cultures was determined microscopically at 48 and 96 h after infection. As seen in Table 2Citation , the percentage of differentiated cells was similar in control and Rsu-1 transfectants. Again, these results indicate that the expression of Rsu-1 does not inhibit v-Ras-induced differentiation.


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Table 2 Differentiation of Rsu-1 transfectant in response to Ki-MSV

 
Alteration of expression of early-response genes in the GSRas-1 cells was checked 24 h after dexamethasone induction of v-Ras expression. The results shown in Fig. 5aCitation indicate that Fos but not Jun expression increased in GSRas-1 cells. RNAs isolated from Ki-MSV-infected control and Rsu-1 transfectant PC12 clones at 72 h after infection were analyzed for expression of early-response genes as well as for expression of v-ras- and Rsu-1-specific messages by Northern blotting (Fig. 5B)Citation . An increase in Fos expression was present in the Rsu-1 transfectant cell line PC12-20 after v-Ras infection compared with the vector control cell line PC12-a. The expression of cJun after Ki-MSV infection was less in the Rsu-1 transfectants relative to the control cell line. Equivalent expression of Ki-MSV-specific RNA was present in both cell lines (Ras, Fig. 5Citation ). These data indicate that the introduction of v-Ras into the Rsu-1 transfectants resulted in v-Ras expression and induction of specific Ras-induced genes.



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Fig. 5. Induction of Fos and cJun RNA expression by Ras infection of Rsu-1 transfectant cell lines. A, GS-Ras1 cells, which contain v-Ras under the control of an MMTV promoter, and PC12 clone a vector control cells were treated with dexamethasone for 24 h. RNA was extracted from the cells, and 10 µg of total RNA were analyzed by Northern blotting for levels of Fos and c-Jun RNA. The amounts of specific hybridization were quantitated and are represented graphically. B, PC12-clone a and PC12-clone 20 cells were treated with dexamethasone and infected with Ki-MSV. Forty-eight h after infection, the dexamethasone was removed from the culture, and 24 h later (72 h after infection), the cells were lysed for isolation of RNA. RNA was assayed for Rsu-1, Ras, Fos, and c-Jun transcripts as in Fig. 4ACitation .

 
Erk and RafB Kinase Activation in Rsu-1 Transfectants.
The activation of Erk kinase and Jun kinase pathways after exposure to NGF was analyzed in PC12 Rsu-1 transfected and vector control cell lines. Two Rsu-1 transfectant cell clones, PC12-20 and PC12-26, were used for these experiments. The cells were plated, induced for 24 h with 0.5 µM dexamethasone, and treated with NGF (100 ng/ml) for 0, 1, 4, 6, or 24 h before lysis. Cell lysates were used to analyze Erk kinase by immunoprecipitation and phosphorylation of myelin basic protein substrate. As shown on Fig. 6ACitation , the Erk kinase pathway was activated in response to NGF in both the PC12-20 and PC12-26 cell lines as well as the vector control cell line PC12-a. The results of three experiments were used to quantitate the level of Erk activity and revealed a sustained increase in Erk activity in the Rsu-1 transfectants exposed to NGF compared with identically treated control cells. These differences in Erk activation are similar to those obtained after EGF stimulation of the Rsu-1 transfectants maintained in the same way (6) . Analysis of Jun kinase activation revealed little or no increase of c-Jun phosphorylation after NGF treatment in the control cells in agreement with the published results of others (data not shown; Ref. 20 ). These data support the conclusions of others that although the Erk mitogen-activated protein kinase pathway is important for NGF-induced differentiation, the Jun kinase pathway is not significantly activated by NGF and is not required for the induction of the differentiation process. The ability of Rsu-1 to enhance differentiation in response to NGF is most likely due to the sustained activation of Erk kinase in these cells.



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Fig. 6. Induction of Erk 1 and RafB after NGF stimulation. A, dexamethasone-induced PC12 clone a, clone 20, and clone 26 were stimulated with NGF (100 ng/ml). At 0, 1, 4, 6, and 24 h after NGF addition, the cells were lysed, and the amount of ERK-1 and ERK-2 activity in the lysates was determined using an immune kinase reaction. The results are the means of three experiments and are shown as fold-increase in phosphorylation of myelin basic protein substrate. Bars, SD. B, dexamethasone-induced PC12 clone a, clone 20, and clone 26 were stimulated with NGF (100 ng/ml). At 0, 1, and 4 h after NGF addition, the cells were lysed, and the amount of RafB activity in the lysates was determined using an immune kinase reaction. The autoradiogram of labeled substrate, Mek-1, is shown.

 
The likely mechanism for the activation of Erk in response to NGF is via the activation of RafB and Mek. Therefore, the level of RafB activation was determined using an immune kinase assay with purified Mek1 as a substrate. The results in Fig. 6BCitation demonstrate that the level of RafB activation was enhanced in the Rsu-1 transfectants relative to the vector control cells. Analysis of Raf-1 activation using the same cell lysates revealed a much lower level of Mek phosphorylation in all clones (data not shown). These results support the conclusion that there is an enhanced RafB-Mek-Erk stimulation by NGF in the Rsu-1 transfectants.

Induction of p21CIP Expression after Induction of Rsu-1 Expression in PC12 Cells.
The addition of NGF to PC12 cells maintained in serum containing medium results in expression of p21CIP. Several studies have demonstrated this increase, as well as a decrease in cdk-4- and cdk-6-associated kinase activity for Rb, indicating that p21CIP induction may play an important role in G1 growth arrest necessary for the initiation of the differentiation program (21 , 22) . To assess the contribution of Rsu-1 to p21CIP induction and because Rsu-1 expression inhibits growth in these cells (Fig. 1)Citation , the level of p21CIP was determined at time points after the induction of Rsu-1 expression. The results of Western blotting of Rsu-1 transfectants and control lysates are shown in Fig. 7ACitation . Increased p21CIP expression is evident 48 h after the induction of Rsu-1 expression in the Rsu-1 transfectant cell lines, but identical treatment of the vector control cell line did not induce p21CIP. As a control, the level of another cell cycle inhibitor, p27KIP, was checked on the same cell lysates. The results in Fig. 7ACitation show that p27KIP, was induced equally in the control and Rsu-1 transfectants. Hence, the expression of Rsu-1 in these cells results in specific p21CIP induction. We also examined NGF-stimulated transfectant and control cells for p21CIP expression. We noted that after dexamethasone and NGF stimulation of PC12 Rsu-1 transfectants, p21CIP expression was maximal by 48 h; however, a large increase in p21CIP was not detected in the vector control cell line until 72 h of NGF treatment. Again, the level of p27KIP did not increase in response to NGF in either the Rsu-1 transfectants or the control cells. Although the earlier increase in p21CIP expression in the Rsu-1 transfectants stimulated with NGF supports a role for p21CIP in NGF-induced differentiation, our results indicate that increases in p21CIP alone are clearly not sufficient to initiate differentiation.



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Fig. 7. Induction of p21CIP and p27KIP in Rsu-1 PC12 transfectants. A, at various times after dexamethasone addition, the vector control clone a and the Rsu-1 transfectant clones 20 and 26 were lysed, and the levels of p21CIP and p27KIP were determined by Western blotting of 50 µg of cell lysate. The results for vector control and two Rsu-1 transfectants are shown. B, at various times after dexamethasone and NGF (100 ng/ml) addition, the vector control clone a and the Rsu-1 transfectant clones 20 and 26 were lysed, and the levels of p21CIP and p27KIP were determined by Western blotting of 50 µg of cell lysate.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Activation of a program of differentiation in the PC12 cell line by NGF requires sustained activation of the Erk kinase pathway (17) . This is achieved by a Ras-dependent mechanism (28) . Induction of differentiation by v-Ras also results in an increase in Erk kinase activation via the Raf-Mek-Erk pathway (10 , 28) . In this study we demonstrate that PC12 transfectant cell lines expressing the suppressor of Ras transformation, Rsu-1, are capable of differentiating in response to NGF. The induction of the differentiation process is accompanied in both control and Rsu-1 transfectant cell lines by activation of the Erk kinases and early-response gene transcription. The activation of Jun kinase, a step blocked by Rsu-1 in PC12 cells (6) , occurs at a low level in the control cells as a result of NGF stimulation. However, the inhibition of Jun kinase activation may prevent proliferation of Rsu-1 transfectants in response to insulin and EGF. The Rsu-1 transfectants differentiate more rapidly than the control cells in response to NGF, indicating that, as shown by others, this pathway does not require a high level of Jun kinase activation. However, the Rsu-1 transfectants exhibit increased RafB and Erk activation as well as an increase in the level of Fos transcriptional activation compared with the control cells.

After infection of the PC12 transfectant cell lines with Ki-MSV and expression of v-Ki-Ras, little activation of Jun kinase was detected in control cell lines or the Rsu-1 transfectants. The Rsu-1 transfectants exhibited the same or higher levels of Erk kinase activation and phenotypic differentiation as the control transfectants. These data indicate that the pathway(s) downstream of Ras that are inhibited by Rsu-1 in the process leading to transformation in fibroblasts, i.e., the activation of Jun kinase, may be only selectively required for the induction of a stable differentiation-specific response to v-Ras in PC12 cells.

Activation of Ras results in the activation of Raf kinase as well as other Ras effectors. Previous studies suggest two mechanisms by which Rsu-1 expression may result in an increase in Erk activation. Rsu-1 binds Raf1 in in vitro binding assays. Although a similar in vivo association has not been detected, there may be stabilization of a complex involving Raf and Rsu-1 that promotes signaling by RafB. Also, Rsu-1 expression inhibits the activity of RasGAP (6) . RasGAP, the Ras GTPase-activating protein, has Ras effector function in addition to its regulatory function. Expression of RasGAP inhibits NGF-induced differentiation of PC12 cells, presumably due to inhibition of Erk activation (19 , 29) . In Rsu-1 PC12 transfectants, the increase in RafB and Erk activation in response to NGF may be a direct result of decreased RasGAP activity and an increase in GTP-Ras. RasGAP effector function has been associated with activation of the small GTPase, Rho (30 , 31) . Rho regulates actin cytoskeletal changes (32 , 33) , and Rho can function as a negative regulator of PC12 differentiation (34, 35, 36, 37, 38) . Hence, the inhibition of RasGAP activity in response to Rsu-1 expression might be expected to inhibit Rho activity and to sustain neurite outgrowth.

Another potential role for Rho in PC12 differentiation is in regulating levels of p21WAF/CIP, the cdk inhibitor (39) . In PC12 cells, the activation of p21CIP apparently contributes to the growth arrest required or associated with the induction of the differentiation program by NGF (21 , 22) . In PC12 cells that are maintained in very low serum during the NGF-induced differentiation process, there is no induction of p21CIP, presumably because G1 growth arrest is accomplished by removal of mitogenic factors (21 , 22) . However, high level activation of the Raf-Mek-Erk kinase pathway induced expression of p21CIP (23 , 24) . Olson et al. (39) have demonstrated that this growth-inhibitory event, i.e., high levels of p21CIP, can be down-regulated by activated Rho. In these instances, the control of the level of p21CIP by Rho allows cells to avoid G1 arrest. Analysis of the Rsu-1 PC12 cell transfectants found a higher level of p21CIP in these cells than the control cells, even without the addition of NGF. This result suggests that Rsu-1 inhibits the activation of a specific Ras and Raf-dependent pathway by inhibiting a Rho-dependent event.

In conclusion, expression of Rsu-1 can enhance Ras-regulated differentiation of PC12 cells. Because another leucine repeat protein has been identified as important in a Ras-dependent differentiation process (3) , this may be a common function for a small group of leucine repeat proteins. The dual Rsu-1 activities of enhancing Ras-dependent differentiation, but inhibiting Ras-dependent transformation, may result from the requirement for activation of specific Ras effectors for each function. Elucidation of the mechanism by which Rsu-1 inhibits RasGAP function should provide additional understanding of the control of these pathways.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Culture.
PC12 transfectants were grown in DMEM with 10% charcoal-stripped horse serum and 4% fetal bovine serum "complete medium" and induced with dexamethasone as described previously (6) . Low-serum medium contained 3% charcoal-stripped horse serum and 0.5% fetal bovine serum.

Antibodies.
Monoclonal antibodies against ERK-2 were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY) or Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Polyclonal antibodies against ERK-1 and ERK-2 and RafB were obtained from Santa Cruz Biotechnology, Inc. Monoclonal Jun kinase antibody was from PharMingen, and monoclonal antibody recognizing phosphorylated Jun kinase was obtained from Santa Cruz Biotechnology. Antibodies to p21WAF/CIP and cyclin D1 were obtained from Santa Cruz Biotechnology.

Western Blotting.
Seventy-five µg of lysate were separated by SDS-PAGE, transferred to nitrocellulose (Schleicher & Schuell, Keene, NH), and blocked overnight in 5% nonfat milk in Tris-buffered saline (TBS). The filters were reacted with primary antibodies at the concentration of 1 µg/ml, washed in TBS/0.1% Tween 20 (T-TBS), and incubated with the specific horseradish peroxidase-conjugated secondary antibody (Amersham Life Science, Inc., Arlington Heights, IL). The filters were developed by chemiluminescence using the ECL chemiluminescence system (Amersham Life Science, Inc.) and Kodak X-OMAT films.

Northern Blotting.
Total RNA was isolated from cell cultures induced with 0.5 µM dexamethasone for 48 h and treated with 100 ng/ml of NGF 2.5S (Boehringer Mannheim) for different periods of time, by lysis with guanidine thiocyanate and purification on cesium chloride gradient or using Tripure reagent (Boehringer Mannheim) according to the manufacturer’s protocol. Ten µg of total RNA were separated on a 1% agarose/4-morpholinepropanesulfonic acid/formaldehyde gel and transferred to nylon membrane. The filters were cross-linked and hybridized with the following probes: a 237-bp fragment corresponding to amino acids 101–179 from the cDNA sequence of SCG10 (27) ; a 299-bp fragment corresponding to bases 481–780 of cDNA sequence of VGF8 (25) ; and a 307-bp fragment corresponding to bases 1051–1358 of the cDNA sequence of NGF1A (26) . These probes were obtained by reverse transcription and PCR amplification. Other probes included the entire open reading frame of human c-jun, a 2-kb fragment of human c-fos, and the probe for GDPH were obtained from American Tissue Culture Collection. Hybridizations were performed as described previously (6) . The filters were quantitated using a Packard Beta Scan system. The filters were stripped by boiling for 5 min in washing buffer and were reused for hybridization.

Kinase Assays.
PC12 transfectants were maintained in regular medium before stimulation with 100 ng/ml NGF 2.5S (Boehringer Mannheim). For Erk-2 phosphorylation gel shift assays, 50 µg of NP40/DOC lysates from stimulated or unstimulated cells were resolved by SDS-PAGE on a 10% Tris-glycine gel, transferred on nitrocellulose, reacted by Western blotting analysis with a specific anti-ERK-2 monoclonal antibody, and detected by chemiluminescence.

For myelin basic protein phosphorylation assay, 100 µg of lysates (buffer: 0.5% Triton X-100 10% glycerol, 100 mM NaCl, and 20 mM HEPES) from stimulated or unstimulated cells were immunoprecipitated using polyclonal antibody that recognize both ERK-1 and ERK-2. The immunoprecipitates were washed twice with lysis buffer and twice with a kinase buffer containing 30 mM HEPES (pH 7.4), 10 mM MgCl2, and 1 mM DTT. The immunoprecipitates were resuspended in 30 µl of kinase buffer containing 5 µg of myelin basic protein and 10 µCi of {gamma}-ATP (32P; DuPont-NEN, Boston, MA) and incubated 30 min at 30°C. The reactions were stopped by the addition of 2x sample buffer and resolved by SDS-PAGE. After transfer on polyvinylidene difluoride (Millipore Corp., Bedford, MA) membrane, the filters were subjected to autoradiography and quantitated using a Beta Scan system. For Mek-1 phosphorylation assay, 400 µg of lysates were precipitated with anti-RafB as described above. The reactions were carried out and quantitated as described above using kinase buffer [25 mM Tris (pH 7.5), 2 mM MnCl2, 1 mM EDTA, 10 mM MgCl2, and 1 mM DTT] and 2 µg of Mek-1 (Santa Cruz Biotechnology, Inc.).

Viral Infection.
Cells (106) were plated on poly-D-lysine-coated, 100-mm dishes in complete medium containing 0.5 µM dexamethasone for 48 h. Prior to the infection, the cells were incubated with 2.5 µg/ml of Polybrene in complete medium for 4 h in the incubator. The infection was carried out by incubation of cells with the Ki-MSV (MuLV) at a multiplicity of infection of 1–5 in 2 ml of medium containing 2.5 µg/ml of Polybrene for 4 h. The inoculum was then diluted to 10 ml and left on the cells overnight. After replacing the virus with dexamethasone containing medium, the cells were incubated for up to 5 days, and differentiation was analyzed by counting the number of neurite-bearing cells microscopically. For the RNA isolation experiments, the cells were incubated for 2 days after infection in dexamethasone containing medium and then for 2 additional days in regular medium lacking dexamethasone. RNA and proteins were extracted from these cultures and analyzed by Northern blotting and Western blotting as described.

[3H]Thymidine Incorporation.
Cells (4 x 103) were plated on 96-well plates in 5% horse serum containing medium; after 24 h, the cells were induced by addition of 0.5 µM dexamethasone, and triplicate wells were treated with the appropriate growth factor for 24, 48, and 72 h prior to labeling with 0.5 µCi/well [3H]thymidine (Amersham) for 6 h. The cells were then harvested using a cell harvester, transferred onto filters, and quantified by scintillation counting.


    Acknowledgments
 
We are grateful to Dr. Simon Halegoua for the GS-Ras1 cell line.


    Footnotes
 
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.

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 (to M. L. C.). Back

2 Present address: Department of Surgical Oncology, Medical College of Virginia, Richmond, VA. Back

3 To whom requests for reprints should be addressed, at B3122, Uniformed Services University of Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814. Phone: (301) 295-3453; Fax: (301) 295-1640; E-mail: mcutler{at}usuhs.mil Back

4 The abbreviations used are: EGF, epidermal growth factor; NGF, nerve growth factor; cdk, cyclin-dependent kinase; GPDH, glyceraldehyde-3-phosphate dehydrogenase; MMTV, mouse mammary tumor virus; MuLV, murine leukemia virus. Back

Received for publication 2/24/99. Revision received 4/14/99. Accepted for publication 7/ 1/99.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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