Two closely related human members of chitinase-like family, CHI3L1 and CHI3L2, activate ERK1/2 in 293 and U373 cells but have the different influence on cell proliferation

The activation of extracellular signal-regulated kinases (ERK1/2) has been associated with specific outcomes. Sustained activation of ERK1/2 by nerve growth factor (NGF) is associated with translocation of ERKs to the nucleus of PC12 cells and precedes their differentiation into sympathetic-like neurons whereas transient activation by epidermal growth factor (EGF) leads to cell proliferation. It was demonstrated that different growth factors initiating the same cellular signaling pathways may lead to the different cell destiny, either to proliferation or to the inhibition of mitogenesis and apoptosis. Thus, further investigation on kinetic differences in activation of certain signal cascades in different cell types by biologically different agents are necessary for understanding the mechanisms as to how cells make a choice between proliferation and differentiation.It was reported that chitinase 3-like 1 (CHI3L1) protein promotes the growth of human synovial cells as well as skin and fetal lung fibroblasts similarly to insulin-like growth factor 1 (IGF1). Both are involved in mediating the mitogenic response through the signal-regulated kinases ERK1/2. In addition, CHI3L1 which is highly expressed in different tumors including glioblastomas possesses oncogenic properties. As we found earlier, chitinase 3-like 2 (CHI3L2) most closely related to human CHI3L1 also showed increased expression in glial tumors at both the RNA and protein levels and stimulated the activation of the MAPK pathway through phosphorylation of ERK1/2 in 293 and U87 MG cells. The work described here demonstrates the influence of CHI3L2 and CHI3L1 on the duration of MAPK cellular signaling and phosphorylated ERK1/2 translocation to the nucleus. In contrast to the activation of ERK1/2 phosphorylation by CHI3L1 that leads to a proliferative signal (similar to the EGF effect in PC12 cells), activation of ERK1/2 phosphorylation by CHI3L2 (similar to NGF) inhibits cell mitogenesis and proliferation.     

Borna disease virus persistent infection activates mitogen-activated protein kinase and blocks neuronal differentiation of PC12 cells

Persistence of Borna disease virus (BDV) in the central nervous system causes damage to specific neuronal populations. BDV is noncytopathic, and the mechanisms underlying neuronal pathology are not well understood. One hypothesis is that infection affects the response of neurons to factors that are crucial for their proliferation, differentiation, or survival. To test this hypothesis, we analyzed the response of PC12 cells persistently infected with BDV to the neurotrophin nerve growth factor (NGF). PC12 is a neural crest-derived cell line that exhibits features of neuronal differentiation in response to NGF. We report that persistence of BDV led to a progressive change of phenotype of PC12 cells and blocked neurite outgrowth in response to NGF. Infection down-regulated the expression of synaptophysin and growth-associated protein-43, two molecules involved in neuronal plasticity, as well as the expression of the chromaffin-specific gene tyrosine hydroxylase. We showed that the block in response to NGF was due in part to the down-regulation of NGF receptors. Moreover, although BDV caused constitutive activation of the ERK1/2 pathway, activated ERKs were not translocated to the nucleus efficiently. These observations may account for the absence of neuronal differentiation of persistently infected PC12 cells treated with NGF.     

Role of phosphoinositide 3-kinase and endocytosis in nerve growth factor-induced extracellular signal-regulated kinase activation via Ras and Rap1

Neurotrophins promote multiple actions on neuronal cells including cell survival and differentiation. The best-studied neurotrophin, nerve growth factor (NGF), is a major survival factor in sympathetic and sensory neurons and promotes differentiation in a well-studied model system, PC12 cells. To mediate these actions, NGF binds to the TrkA receptor to trigger intracellular signaling cascades. Two kinases whose activities mediate these processes include the mitogen-activated protein (MAP) kinase (or extracellular signal-regulated kinase [ERK]) and phosphoinositide 3-kinase (PI3-K). To examine potential interactions between the ERK and PI3-K pathways, we studied the requirement of PI3-K for NGF activation of the ERK signaling cascade in dorsal root ganglion cells and PC12 cells. We show that PI3-K is required for TrkA internalization and participates in NGF signaling to ERKs via distinct actions on the small G proteins Ras and Rap1. In PC12 cells, NGF activates Ras and Rap1 to elicit the rapid and sustained activation of ERKs respectively. We show here that Rap1 activation requires both TrkA internalization and PI3-K, whereas Ras activation requires neither TrkA internalization nor PI3-K. Both inhibitors of PI3-K and inhibitors of endocytosis prevent GTP loading of Rap1 and block sustained ERK activation by NGF. PI3-K and endocytosis may also regulate ERK signaling at a second site downstream of Ras, since both rapid ERK activation and the Ras-dependent activation of the MAP kinase kinase kinase B-Raf are blocked by inhibition of either PI3-K or endocytosis. The results of this study suggest that PI3-K may be required for the signals initiated by TrkA internalization and demonstrate that specific endocytic events may distinguish ERK signaling via Rap1 and Ras.     

SB203580 promotes EGF-stimulated early morphological differentiation in PC12 cell through activating ERK pathway.

MAP kinases have important role in PC12 cell differentiation, since the activities of both extracellular regulated protein kinase (ERK) and p38 have been indicated as necessary signal for PC12 cell differentiation. Epidermal growth factor (EGF) and NGF both activate ERK and p38 in PC12 cells, but only NGF trigger differentiation. It has been proposed that the duration of ERK activation determines the switch from proliferation to differentiation, since EGF causes more transient activation of ERK than NGF in PC12 cells. Here we report that treatment of PC12 cells with EGF in the presence of SB203580, a widely used p38 inhibitor, caused differentiation. The pro-differentiation effect of SB203580 in EGF-treated PC12 cells was found to be independent of its function of p38 inhibition but was through an effect on the ERK pathway that has been recently reported (Kalmes et al. [1999] FEBS Lett. 444: 71-74; Hall-Jackson et al. [1999] Onc. 18: 2047-2054). We found that SB203580 by itself did not affect the activity of ERK1/2 but significantly extended EGF-induced ERK activation in PC12 cells, which resulted in early morphological differentiation. Our data indicated that although both ERK and p38 are required for PC12 cell differentiation, activation of p38 is not required when ERK is superactivated. Our data provided further evidence for the threshold theory that differentiation is determined by the duration of ERK activation.     

Inactivation of dual-specificity phosphatases is involved in the regulation of extracellular signal-regulated kinases by heat shock and hsp72

Extracellular signal-regulated kinase 1 (ERK1) and ERK2 (ERK1/2) dramatically enhance survival of cells exposed to heat shock. Using Cos-7 cells and primary human fibroblasts (IMR90 cells), we demonstrated that heat shock activates ERKs via two distinct mechanisms: stimulation of the ERK-activating kinases, MEK1/2, and inhibition of ERK dephosphorylation. Under milder heat shock conditions, activation of ERKs proceeded mainly through stimulation of MEK1/2, whereas under more severe heat shock MEK1/2 could no longer be activated and the inhibition of ERK phosphatases became critical. In Cos-7 cells, nontoxic heat shock caused rapid inactivation of the major ERK phosphatase, MKP-3, by promoting its aggregation, so that in cells exposed to 45 degrees C for 20 min, 90% of MKP-3 became insoluble. MKP-3 aggregation was reversible and, 1 h after heat shock, MKP-3 partially resolubilized. The redistribution of MKP-3 correlated with an increased rate of ERK dephosphorylation. Similar heat-induced aggregation, followed by partial resolubilization, was found with a distinct dual-specificity phosphatase MKP-1 but not with MKP-2. Therefore, MKP-3 and MKP-1 appeared to be critical heat-labile phosphatases involved in the activation of ERKs by heat shock. Expression of the major heat shock protein Hsp72 inhibited activation of MEK1/2 and prevented inactivation of MKP-3 and MKP-1. Hsp72DeltaEEVD mutant lacking a chaperone activity was unable to protect MKP-3 from heat inactivation but interfered with MEK1/2 activation similar to normal Hsp72. Hence, Hsp72 suppressed ERK activation by both protecting dual-specificity phosphatases, which was dependent on the chaperone activity, and suppressing MEK1/2, which was independent of the chaperone activity.     

ERK1/2-driven and MKP-mediated inhibition of EGF-induced ERK5 signaling in human proximal tubular cells

The MEK1-ERK1/2 signaling pathway has been implicated in the regulation of renal epithelial cell proliferation, epithelial-to-mesenchymal transition and the induction of an invasive cell phenotype. Much less information is available about the MEK5-ERK5 module and its role in renal epithelial cell proliferation and differentiation. In the present study we have investigated the regulation of these two families of extracellular signal-regulated kinases in epidermal growth factor (EGF)-stimulated human kidney-2 (HK-2) cells and a possible interaction between ERK1/2 and ERK5. Here we report that 5 ng/ml EGF led to a strong stimulation of HK-2 cell proliferation, which was largely U0126-sensitive. Both synthetic MEK1/2 inhibitors U0126 and Cl-1040, when used at 10 and 1 microM, respectively, inhibited basal and EGF-induced ERK1/2 phosphorylation but not ERK5 phosphorylation. Long-term inhibition of MEK1/2-ERK1/2 signaling and/or vanadate-sensitive protein phosphatases enhanced and prolonged EGF-induced ERK5 phosphorylation, while transient expression of an adenoviral constitutively active MEK1 (Ad-caMEK1) construct completely blocked EGF-induced ERK5 phosphorylation. Expression of Ad-caMEK1 in HK-2 cells resulted in the upregulation of the dual-specificity phosphatases MKP-3/DUSP6, MKP-1/DUSP1, and DUSP5. The EGF-mediated time-dependent induction of MKP-3, MKP-1 and DUSP5 mRNA levels was U0126-sensitive at a concentration, which blocked EGF-mediated ERK1/2 phosphorylation but not ERK5 phosphorylation. Furthermore, U0126 inhibited EGF-induced MKP-3 and MKP-1 protein expression. Both MKP-3 and MKP-1 co-immunoprecipitated with ERK5 in unstimulated as well as in EGF-stimulated HK-2 cells. These results suggest the existence of an ERK1/2-driven negative feed-back regulation of ERK5 signaling in EGF-stimulated HK-2 cells, which is mediated by MKP-3, DUSP5 and/or MKP-1.     

Extracellular signal-regulated kinases 2 autophosphorylates on a subset of peptides phosphorylated in intact cells in response to insulin and nerve growth factor: analysis by peptide mapping.

The phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2) in response to insulin in Rat 1 HIRc B cells and in response to nerve growth factor (NGF) in PC12 cells has been examined. ERK1 and ERK2 are phosphorylated on serine in the absence of the stimuli and additionally on tyrosine and threonine residues after exposure to NGF and insulin. NGF stimulates tyrosine phosphorylation of ERK1 more rapidly than threonine phosphorylation. Two-dimensional phosphopeptide maps of both ERK1 and ERK2 phosphorylated in intact cells treated with NGF or with insulin display the same three predominant phosphopeptides that comigrate when digests of ERK1 and ERK2 are mixed. As many as five additional phosphopeptides are detected under certain conditions. Autophosphorylated recombinant ERK2 also contains the three tryptic phosphopeptides found in ERKs labeled in intact cells. These experiments demonstrate that ERK1 and ERK2 are phosphorylated on related sites in response to two distinct extracellular signals. The data also support the possibility that autophosphorylation may be involved in the activation of the ERKs.     

Nerve growth factor stimulates the concentration of TrkA within lipid rafts and extracellular signal-regulated kinase activation through c-Cbl-associated protein

Nerve growth factor (NGF) acts through its receptor, TrkA, to elicit the neuronal differentiation of PC12 cells through the action of extracellular signal-regulated kinase 1 (ERK1) and ERK2. Upon NGF binding, TrkA translocates and concentrates in cholesterol-rich membrane microdomains or lipid rafts, facilitating formation of receptor-associated signaling complexes, activation of downstream signaling pathways, and internalization into endosomes. We have investigated the mechanisms responsible for the localization of TrkA within lipid rafts and its ability to activate ERK1 and ERK2. We report that NGF treatment results in the translocation of activated forms of TrkA to lipid rafts, and this localization is important for efficient activation of the ERKs. TrkA is recruited and retained within lipid rafts through its association with flotillin, an intrinsic constituent of these membrane microdomains, via the adapter protein, c-Cbl associated protein (CAP). Mutant forms of CAP that lack protein interaction domains block TrkA localization to lipid rafts and attenuate ERK activation. Importantly, suppression of endogenous CAP expression inhibited NGF-stimulated neurite outgrowth from primary dorsal root ganglion neurons. These data provide a mechanism for the lipid raft localization of TrkA and establish the importance of the CAP adaptor protein for NGF activation of the ERKs and neuronal differentiation.     

Mechanisms regulating the constitutive activation of the extracellular signal-regulated kinase (ERK) signaling pathway in ovarian cancer and the effect of ribonucleic acid interference for ERK1/2 on cancer cell proliferation

The ERK1/2 MAPK pathway is a critical signaling system that mediates ligand-stimulated signals for the induction of cell proliferation, differentiation, and cell survival. Studies have shown that this pathway is constitutively active in several human malignancies and may be involved in the pathogenesis of these tumors. In the present study we examined the ERK1/2 pathway in cell lines derived from epithelial and granulosa cell tumors, two distinct forms of ovarian cancer. We show that ERK1 and ERK2 are constitutively active and that this activation results from both MAPK kinase-dependent and independent mechanisms and is correlated with elevated BRAF expression. MAPK phosphatase 1 (MKP-1) expression, which is involved in ERK1/2 deactivation, is down-regulated in the cancer cells, thus further contributing to ERK hyperactivity in these cells. Treatment of these cancer cell lines with the proteasome inhibitor ZLLF-CHO increased MKP-1 but not MKP-2 expression and decreased ERK1/2 phosphorylation. More importantly, silencing of ERK1/2 protein expression using RNA interference led to the complete suppression of tumor cell proliferation. These results provide evidence that the ERK pathway plays a major role in ovarian cancer pathogenesis and that down-regulation of this master signaling pathway is highly effective for the inhibition of ovarian tumor growth.     

Multiple signaling conduits regulate global differentiation-specific gene expression in PC12 cells

PC12 cells serve as a model for exploring nerve growth factor (NGF)-stimulated signal pathways that mediate neural differentiation. We previously demonstrated that neurofilament light chain (NFLC) gene induction by NGF requires collaborative extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling. Herein, we investigate the broader requirement for integrated ERK and JNK signaling in NGF-stimulated gene expression. NGF stimulates differentiation as well as maintenance of cell viability while insulin-like growth factor-1 (IGF-1) stimulates only trophic actions in PC12 cells. Affymetrix Genechips were used to identify genes whose expression specifically increased in response to NGF, but not IGF-1. From the set of NGF-specific genes, the induction by NGF of ten genes with diverse predicted cellular functions was tested for ERK and JNK pathway requirements using the protein kinase inhibitors, PD98059 and SP600125, respectively. Like NFLC, induction of urokinase plasminogen activator (uPAR), transin/matrix metalloproteinase 3 (MMP3), Fra-1 and transforming growth factor beta 1 (TGF beta 1) required collaborative ERK and JNK signaling while the increased expression of cortexin, rat collapsin response mediator protein 4 (rCRMP4), rat growth and transformation-dependent protein (RGT), and synapsin II required neither mitogen-activated protein kinase (MAPK) pathway. NGF-induction of the bradykinin B2 receptor and c-Ret mRNAs was partially inhibited by SP600125, but not PD98059. Reporter constructs containing the promoters for ERK/JNK-dependent genes (NFLC, transin, uPAR) as well as an ERK/JNK-independent gene (synapsin II) revealed that both sets of genes required functional Ras signaling for activation by NGF. Integrated signaling through the ERK and JNK MAPKs, therefore, represents a general conduit for NGF-dependent gene expression, but additional Ras-dependent signaling pathways distinct from the ERKs and JNKs must contribute as well. Thus, multiple signaling conduits control global differentiation-specific gene expression in PC12 cells.     

GD3 synthase gene expression in PC12 cells results in the continuous activation of TrkA and ERK1/2 and enhanced proliferation

A rat pheochromocytoma cell line (PC12) transfected with ganglioside GD3 synthase gene showed a marked change in the ganglioside profile and enhanced proliferation and no response of neurite extension to nerve growth factor (NGF) stimulation. In these transfectant cells, a continuous phosphorylation of TrkA and the activation of ERK1/2 without NGF treatment were observed. Proliferation inhibition experiments with kinase inhibitors such as herbimycin A, K-252a, and PD98059 revealed that the enhanced proliferation was actually due to the activation of the Ras/MEK/ERK pathway. A TrkA dimer was detected in the GD3 synthase transfectant cells regardless of NGF treatment by cross-linking and immunoblotting. The increased expression of GD1b and GT1b in these transfectant cells might induce the conformational change of TrkA to form a dimer and to be activated continuously. These results may indicate regulatory roles of gangliosides in cell proliferation under physiological and malignant processes.