Ral-specific guanine nucleotide exchange factor activity opposes other Ras effectors in PC12 cells by inhibiting neurite outgrowth

Ras proteins can activate at least three classes of downstream target proteins: Raf kinases, phosphatidylinositol-3 phosphate (PI3) kinase, and Ral-specific guanine nucleotide exchange factors (Ral-GEFs). In NIH 3T3 cells, activated Ral-GEFs contribute to Ras-induced cell proliferation and oncogenic transformation by complementing the activities of Raf and PI3 kinases. In PC12 cells, activated Raf and PI3 kinases mediate Ras-induced cell cycle arrest and differentiation into a neuronal phenotype. Here, we show that in PC12 cells, Ral-GEF activity acts opposite to other Ras effectors. Elevation of Ral-GEF activity induced by transfection of a mutant Ras protein that preferentially activates Ral-GEFs, or by transfection of the catalytic domain of the Ral-GEF Rgr, suppressed cell cycle arrest and neurite outgrowth induced by nerve growth factor (NGF) treatment. In addition, Rgr reduced neurite outgrowth induced by a mutant Ras protein that preferentially activates Raf kinases. Furthermore, inhibition of Ral-GEF activity by expression of a dominant negative Ral mutant accelerated cell cycle arrest and enhanced neurite outgrowth in response to NGF treatment. Ral-GEF activity may function, at least in part, through inhibition of the Rho family GTPases, CDC42 and Rac. In contrast to Ras, which was activated for hours by NGF treatment, Ral was activated for only approximately 20 min. These findings suggest that one function of Ral-GEF signaling induced by NGF is to delay the onset of cell cycle arrest and neurite outgrowth induced by other Ras effectors. They also demonstrate that Ras has the potential to promote both antidifferentiation and prodifferentiation signaling pathways through activation of distinct effector proteins. Thus, in some cell types the ratio of activities among Ras effectors and their temporal regulation may be important determinants for cell fate decisions between proliferation and differentiation.     

Colostrinin-Driven Neurite Outgrowth Requires p53 Activation in PC12 Cells

Summary 1. Colostrinin (CLN) induces maturation and differentiation of murine thymocytes, promotes proliferation of peripheral blood leukocytes, induces immunomodulator cytokines, and ameliorates oxidative stress-mediated activation of c-Jun NH2-terminal kinases. 2. Here we report that upon treatment with CLN, medullary pheochromocytoma (PC12) cells ceased to proliferate and extend neurites. 3. The arrest of CLN-treated PC12 cells in the G1 phase of the cell cycle was due to an increase in the phosphorylation of p53 at serine¹⁵ (p53^ser15) and expression of p21^WAF1. PC12 cells treated with inhibitory oligonucleotides to p53 lacked p53^ser15 and p21^WAF1 expression, and did not show morphological changes after CLN exposure. Transfection with inhibitory oligonucleotides to p21^WAF1 had no effect on p53 activation; however, cells failed to arrest or extend neurites. An oligonucleotide inhibiting luciferase expression had no effect on CLN-mediated p53 activation, p21^WAF1 expression, growth arrest, or neurite outgrowth. 4. We conclude that CLN induces delicate cassettes of signaling pathways common to cell proliferation and differentiation, and mediates activities that are similar to those of hormones and neurotrophins, leading to neurite outgrowth.     

Histone deacetylase inhibitors differentially stabilize acetylated p53 and induce cell cycle arrest or apoptosis in prostate cancer cells

In LNCaP prostate cancer cells CG-1521, a new inhibitor of histone deacetylases, alters the acetylation of p53 in a site-specific manner. While p53 is constitutively acetylated at Lys320 in LNCaP cells, treatment with CG-1521, stabilizes the acetylation of p53 at Lys373, elevating p21 (and inducing cell cycle arrest). Treatment with CG-1521 also promotes Bax translocation to the mitochondria and cleavage, and apoptosis. TSA stabilizes the acetylation of p53 at Lys382, elevating p21 levels and inducing cell cycle arrest, but does not induce Bax translocation or apoptosis. In LNCaP cells CG-1521, but not TSA, promotes the rapid degradation of HDAC2. These data suggest that the acetylation of p53 at Lys373 is required for the p53-mediated induction of cell cycle arrest and apoptosis, while acetylation of p53 at Lys382 induces only cell cycle arrest. In p53(-/-) PC3 cells both compounds induce p21 and cell cycle arrest, but not Bax translocation or apoptosis, suggesting that both compounds can also induce p21 through a p53-independent mechanism.     

Cellular senescence induced by p53-ras cooperation is independent of p21waf1 in murine embryo fibroblasts

Oncogenic activation in primary murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the p53 tumor suppressor pathway. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras with p53 produced an irreversible cell cycle arrest that displayed features of cellular senescence. Introduction of a conditional murine p53 allele (p53val135) into double p53/p21-null mouse embryonic fibroblasts showed that p21waf1 was not required for this effect, since p53-/-;p21-/- double-null cells undergo terminal growth arrest with features of senescence following coexpression of oncogenic Ras and p53. Our results indicate that oncogenic activation of the Ras pathway in murine fibroblasts converts p53 into a senescence inducer through a p21waf1-independent mechanism.     

Concerted Regulation of Wild-Type p53 Nuclear Accumulation and Activation by S100B and Calcium-Dependent Protein Kinase C

The calcium ionophore ionomycin cooperates with the S100B protein to rescue a p53-dependent G(1) checkpoint control in S100B-expressing mouse embryo fibroblasts and rat embryo fibroblasts (REF cells) which express the temperature-sensitive p53Val135 mutant (C. Scotto, J. C. Deloulme, D. Rousseau, E. Chambaz, and J. Baudier, Mol. Cell. Biol. 18:4272-4281, 1998). We investigated in this study the contributions of S100B and calcium-dependent PKC (cPKC) signalling pathways to the activation of wild-type p53. We first confirmed that S100B expression in mouse embryo fibroblasts enhanced specific nuclear accumulation of wild-type p53. We next demonstrated that wild-type p53 nuclear translocation and accumulation is dependent on cPKC activity. Mutation of the five putative cPKC phosphorylation sites on murine p53 into alanine or aspartic residues had no significant effect on p53 nuclear localization, suggesting that the cPKC effect on p53 nuclear translocation is indirect. A concerted regulation by S100B and cPKC of wild-type p53 nuclear translocation and activation was confirmed with REF cells expressing S100B (S100B-REF cells) overexpressing the temperature-sensitive p53Val135 mutant. Stimulation of S100B-REF cells with the PKC activator phorbol ester phorbol myristate acetate (PMA) promoted specific nuclear translocation of the wild-type p53Val135 species in cells positioned in early G(1) phase of the cell cycle. PMA also substituted for ionomycin in the mediating of p53-dependent G(1) arrest at the nonpermissive temperature (37.5 degrees C). PMA-dependent growth arrest was linked to the cell apoptosis response to UV irradiation. In contrast, growth arrest mediated by a temperature shift to 32 degrees C protected S100B-REF cells from apoptosis. Our results suggest a model in which calcium signalling, linked with cPKC activation, cooperates with S100B to promote wild-type p53 nuclear translocation in early G(1) phase and activation of a p53-dependent G(1) checkpoint control.     

The involvement of MAPK signaling pathways in determining the cellular response to p53 activation: cell cycle arrest or apoptosis

The effect of ERK, p38, and JNK signaling on p53-dependent apoptosis and cell cycle arrest was investigated using a Friend murine erythroleukemia virus (FVP)-transformed cell line that expresses a temperature-sensitive p53 allele, DP16.1/p53ts. In response to p53 activation at 32 degrees C, DP16.1/p53ts cells undergo p53-dependent G(1) cell cycle arrest and apoptosis. As a result of viral transformation, these cells express the spleen focus forming env-related glycoprotein gp55, which can bind to the erythropoietin receptor (EPO-R) and mimics many aspects of EPO-induced EPO-R signaling. We demonstrate that ERK, p38 and JNK mitogen-activated protein kinases (MAPKs) are constitutively active in DP16.1/p53ts cells. Constitutive MEK activity contributes to p53-dependent apoptosis and phosphorylation of p53 on serine residue 15. The pro-apoptotic effect of this MAPK kinase signal likely reflects an aberrant Ras proliferative signal arising from FVP-induced viral transformation. Inhibition of MEK alters the p53-dependent cellular response of DP16.1/p53ts from apoptosis to G(1) cell cycle arrest, with a concomitant increase in p21(WAF1), suggesting that the Ras/MEK pathway may influence the cellular response to p53 activation. p38 and JNK activity in DP16.1/p53ts cells is anti-apoptotic and capable of limiting p53-dependent apoptosis at 32 degrees C. Moreover, JNK facilitates p53 protein turnover, which could account for the enhanced apoptotic effects of inhibiting this MAPK pathway in DP16.1/p53ts cells. Overall, these data show that intrinsic MAPK signaling pathways, active in transformed cells, can both positively and negatively influence p53-dependent apoptosis, and illustrate their potential to affect cancer therapies aimed at reconstituting or activating p53 function.     

p53‐mediated regulation of cell cycle progression: Pronounced impact of cellular microenvironment

Data on the biological effects of some overexpressed oncogenes and their cooperation with cellular factors are, at least partially, contradictory. There are reports on the strong pro‐apoptotic action of temperature‐sensitive (ts) p53^135val in transformed cells at permissive temperature. However, in our experience very high levels of p53^135val induce in transformed rat cells at permissive temperature cell cycle arrest but not apoptosis. Comparison of the experimental protocols reveals that cells used for transfection strongly differ. Therefore, we decided to explore the impact of primary cells used for generation of cell clones on the biological effects evoked by p53 and c‐Ha‐Ras. In the present study, we used primary rat cells (RECs) isolated from rat embryos of different age: at 13.5 gd (y) and 15.5 gd (o). We immortalized rat cells using ts p53^135val mutant and additionally generated transformed cells after co‐transfection with oncogenic Ras. The RECs were transfected with a constitutively activated Ha‐Ras protein, a mutation that is found in a wide variety of human tumors. The ts p53^135Val mutant, switching between wild‐type (wt) and mutant conformation, offers the possibility to study the escape from p53‐mediated cell cycle control in a model of malignant transformation in cells with the same genetic background. Surprisingly, the kinetics of cell proliferation at non‐permissive temperature and that of cell cycle arrest at 32°C strongly differed between cell clones established from yRECs and oRECs, thereby indicating that overexpression of genes such as ts p53^135Val mutant and oncogenic‐Ha‐Ras does not fully override the intrinsic cellular program. J. Cell. Physiol. 219: 459–469, 2009. © 2009 Wiley‐Liss, Inc.     

Signal transduction by nerve growth factor and fibroblast growth factor in PC12 cells requires a sequence of src and ras actions

We have investigated the roles of pp60c-src and p21c-ras proteins in transducing the nerve growth factor (NGF) and fibroblast growth factor (FGF) signals which promote the sympathetic neuronlike phenotype in PC12 cells. Neutralizing antibodies directed against either Src or Ras proteins were microinjected into fused PC12 cells. Each antibody both prevented and reversed NGF- or FGF-induced neurite growth, a prominent morphological marker for the neuronal phenotype. These data demonstrate the involvement of both pp60c-src and p21c-ras proteins in NGF and FGF actions in PC12 cells, and establish a physiological role for the pp60c-src tyrosine kinase in signal transduction pathways initiated by receptor tyrosine kinases in these cells. Additional microinjection experiments, using PC12 transfectants containing inducible v-src or ras oncogene activities, demonstrated a specific sequence of Src and Ras actions. Microinjection of anti-Ras antibody blocked v-src-induced neurite growth, but microinjection of anti-Src antibodies had no effect on ras oncogene-induced neurite growth. We propose that a cascade of Src and Ras actions, with Src acting first, is a significant feature of the signal transduction pathways for NGF and FGF. The Src-Ras cascade may define a functional cassette in the signal transduction pathways used by growth factors and other ligands whose receptors have diverse structures and whose range of actions on various cell types include mitogenesis and differentiation.     

Oncogenic ras and p53 cooperate to induce cellular senescence

Oncogenic activation of the mitogen-activated protein (MAP) kinase cascade in murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the ARF/p53 tumor suppressor pathway. To investigate whether p53 is sufficient to induce senescence, we introduced a conditional murine p53 allele (p53(val135)) into p53-null mouse embryonic fibroblasts and examined cell proliferation and senescence in cells expressing p53, oncogenic Ras, or both gene products. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras or activated mek1 with p53 enhanced both p53 levels and activity relative to that observed for p53 alone and produced an irreversible cell cycle arrest that displayed features of cellular senescence. p19(ARF) was required for this effect, since p53(-/-) ARF(-/-) double-null cells were unable to undergo senescence following coexpression of oncogenic Ras and p53. Although the levels of exogenous p53 achieved in ARF-null cells were relatively low, the stabilizing effects of p19(ARF) on p53 could not explain the cooperation between oncogenic Ras and p53 in promoting senescence. Hence, enforced p53 expression without oncogenic ras in p53(-/-) mdm2(-/-) double-null cells produced extremely high p53 levels but did not induce senescence. Taken together, our results indicate that oncogenic activation of the MAP kinase pathway in murine fibroblasts converts p53 into a senescence inducer through both quantitative and qualitative mechanisms.     

p53 can inhibit cell proliferation through caspase-mediated cleavage of ERK2/MAPK

Stimulation of the Ras/MAPK cascade can either activate p53 and promote replicative senescence and apoptosis, or degrade p53 and promote cell survival. Here we show that p53 can directly counteract the Ras/MAPK signaling by inactivating ERK2/MAPK. This inactivation is due to a caspase cleavage of the ERK2 protein and contributes to p53-mediated growth arrest. We found that in Ras-transformed cells, growth arrest induced by p53, but not p21(Waf1), is associated with a strong reduction in ERK2 activity, phosphorylation, and protein half-life, and with the appearance of caspase activity. Likewise, DNA damage-induced cell cycle arrest correlates with p53-dependent ERK2 downregulation and caspase activation. Furthermore, caspase inhibitors or expression of a caspase-resistant ERK2 mutant interfere with ERK2 cleavage and restore proliferation in the presence of p53 activation, indicating that caspase-mediated ERK2 degradation contributes to p53-induced growth arrest. These findings strongly point to ERK2 as a novel p53 target in growth suppression.     

Cytokines inhibit p53-mediated apoptosis but not p53-mediated G1 arrest.

Murine erythroleukemia cells that lack endogenous p53 expression were transfected with a temperature-sensitive p53 allele. The temperature-sensitive p53 protein behaves as a mutant polypeptide at 37 degrees C and as a wild-type polypeptide at 32 degrees C. Three independent clones expressing the temperature-sensitive p53 protein were characterized with respect to p53-mediated G1 cell cycle arrest, apoptosis, and differentiation. Clone ts5.203 responded to p53 activation at 32 degrees C by undergoing G1 arrest, apoptosis, and differentiation. Apoptosis was seen in cells representative of all phases of the cell cycle and was not restricted to cells arrested in G1. The addition of a cytokine (erythropoietin, c-kit ligand, or interleukin-3) to the culture medium of ts5.203 cells blocked p53-mediated apoptosis and differentiation but not p53-mediated G1 arrest. These observations indicate that apoptosis and G1 arrest can be effectively uncoupled through the action of cytokines acting as survival factors and are consistent with the idea that apoptosis and G1 arrest represent separate functions of p53. Clones ts15.15 and tsCB3.4 responded to p53 activation at 32 degrees C by undergoing G1 arrest but not apoptosis. We demonstrate that tsCB3.4 secretes a factor with erythropoietin-like activity and that ts15.15 secretes a factor with interleukin-3 activity and suggest that autocrine secretion of these cytokines blocks p53-mediated apoptosis. These data provide a framework in which to understand the variable responses of cells to p53 overexpression.     

Rit contributes to nerve growth factor-induced neuronal differentiation via activation of B-Raf-extracellular signal-regulated kinase and p38 mitogen-activated protein kinase cascades

Rit is one of the original members of a novel Ras GTPase subfamily that uses distinct effector pathways to transform NIH 3T3 cells and induce pheochromocytoma cell (PC6) differentiation. In this study, we find that stimulation of PC6 cells by growth factors, including nerve growth factor (NGF), results in rapid and prolonged Rit activation. Ectopic expression of active Rit promotes PC6 neurite outgrowth that is morphologically distinct from that promoted by oncogenic Ras (evidenced by increased neurite branching) and stimulates activation of both the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase signaling pathways. Furthermore, Rit-induced differentiation is dependent upon both MAP kinase cascades, since MEK inhibition blocked Rit-induced neurite outgrowth, while p38 blockade inhibited neurite elongation and branching but not neurite initiation. Surprisingly, while Rit was unable to stimulate ERK activity in NIH 3T3 cells, it potently activated ERK in PC6 cells. This cell type specificity is explained by the finding that Rit was unable to activate C-Raf, while it bound and stimulated the neuronal Raf isoform, B-Raf. Importantly, selective down-regulation of Rit gene expression in PC6 cells significantly altered NGF-dependent MAP kinase cascade responses, inhibiting both p38 and ERK kinase activation. Moreover, the ability of NGF to promote neuronal differentiation was attenuated by Rit knockdown. Thus, Rit is implicated in a novel pathway of neuronal development and regeneration by coupling specific trophic factor signals to sustained activation of the B-Raf/ERK and p38 MAP kinase cascades.     

Nerve growth factor withdrawal-mediated apoptosis in naive and differentiated PC12 cells through p53/caspase-3-dependent and -independent pathways

Programmed cell death is regulated in response to a variety of stimuli, including the tumor suppressor protein p53, that can mediate cell cycle arrest through p21/Waf1 and apoptosis through the Bcl-2/Bax equilibrium and caspases. Neuronal cell apoptosis has been reported to require p53, whereas other data suggest that neuronal cell death may be independent of p53. Comparison of wild type PC12 to a temperature-sensitive PC12 cell line that depresses the normal function of p53 has permitted investigation of the importance of p53 in a variety of cell functions. This study examined the role of p53 in trophic factor withdrawal-mediated apoptosis in both na?ve and differentiated PC12 cells. Our data show that as PC12 cells differentiate they are more poised to undergo apoptosis than their undifferentiated counterparts. Survival assays with XTT (sodium 3'-1-(phenylaminocarbonyl)-3,4-tetrazolium-bis(4-methoxy-6-nitro)benzene sulfonic acid) and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) demonstrated that lack of p53 is initially protective against apoptosis. The window of protection is about 20 h for na?ve and 36 h for differentiated cells. Apoptosis involved caspases 3, 6, and 9. However, caspase 3 activation was absent in cells lacking p53, concomitant with the delayed apoptosis. When the expression of caspase 3 was silenced with interference RNA, wild type PC12 cells revealed a morphology and biochemistry similar to PC12[p53ts] cells, indicating that caspase 3 accounts for the observed delay in apoptosis in p53 dysfunction. These results suggest that p53 is important, but not essential, in factor withdrawal-mediated apoptosis. Parallel pathways of caspase-mediated apoptosis are activated later in the absence of functional p53.     

Proliferative inhibition by dominant-negative Ras rescues naive and neuronally differentiated PC12 cells from apoptotic death.

We have used the nerve growth factor (NGF)-responsive PC12 cell line as a model to examine the role of cell cycle progression in apoptotic neuronal cell death triggered by withdrawal of trophic support. Because p21 Ras plays a key role in mitogenic signaling, we tested whether interference with the activity of this protein would affect cell cycle progression and thereby apoptotic death after trophic factor deprivation. For this purpose, we exploited PC12 cells transfected with an inducible form of dominant-inhibitory Ras. In contrast to non-transfected and uninduced cells, which continue to synthesize DNA when deprived of trophic support, PC12 cells induced to express dominant-inhibitory Ras showed little thymidine incorporation. When non-transfected and uninduced cells were deprived of trophic support, these underwent rapid apoptotic death that could be prevented by NGF. However, cells in which dominant-inhibitory Ras was induced and which were consequently quiescent did not die upon withdrawal of trophic support and showed long-term survival in the absence of NGF or other trophic factors. Moreover, induction of dominant-inhibitory Ras also rescued non-dividing, neuronally differentiated PC12 cells from death caused by NGF withdrawal. These findings suggest a relationship between proliferative capacity and neuronal apoptosis and raise the hypothesis that following withdrawal of trophic support, neurons undergo an unsuccessful and fatal attempt to re-enter the cell cycle.     

Domains of E1A that bind p105Rb, p130, and p300 are required to block nerve growth factor-induced neurite growth in PC12 cells.

Nerve growth factor (NGF) causes PC12 cells to cease division and undergo sympathetic neuron-like differentiation, including neurite outgrowth. We have tested whether differentiation and division share overlapping control mechanisms in these cells. To do this, we have perturbed the activity of proteins known to participate in cell-cycle regulation by introducing the E1A oncogene or its mutant forms via microinjection into PC12 cells. The E1A protein binds to several putative cell cycle control proteins, including p105Rb (the product of the retinoblastoma susceptibility gene), as well as others of unknown function such as p130, p107, and p300. Similar to previous results, we find that wild-type E1A abrogates NGF-induced neurite extension. However, NGF does cause neurite outgrowth in the presence of E1A mutants known to have greatly reduced binding to either p105Rb and p130 or p300. Our experiments suggest that p105Rb, p130, and p300 may participate either in E1A-mediated inhibition of differentiation or in the NGF signal transduction pathway. We also report here that NGF affects phosphorylation of p105Rb, suggesting that Rb mediates at least some of NGF's effects. Our results raise the possibility that putative cell-cycle control proteins may participate not only in NGF-induced cessation of division but also in differentiation.