A protein kinase inhibitor, staurosporine, mimics nerve growth factor induction of neurotensin/neuromedin N gene expression.

Nerve growth factor (NGF) cooperates with glucocorticoids, activators of adenylate cyclase, and lithium to induce the expression of teh gene encoding the neuropeptides neurotensin and neuromedin N (NT/N gene) in PC 12 pheochromocytoma cells. High level expression requires simultaneous treatment with three or all four inducers. To examine the mechanism underlying this complex synergism, we have examined the effects of protein kinase inhibitors and other agents which influence intracellular signal transduction on NT/N gene expression. Two structurally similar bacterial alkaloids, staurosporine and K-252a, inhibit several protein kinases in vitro, including protein kinase C and cyclic nucleotide-dependent kinases. K-252a has been reported to specifically inhibit the effects of NGF on PC12 pheochromocytoma cells. Surprisingly, staurosporine in combination with other inducers markedly potentiated NT/N gene expression. In contrast, K-252a had no effect on NT/N gene expression when added simultaneously with other inducers. Expression of the NT/N gene was also potentiated by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which directly activates protein kinase C, and by bradykinin, which stimulates phosphatidylinositol turnover in PC12 cells, and these effects were not blocked by staurosporine. Staurosporine was generally more effective in stimulating NT/N gene expression when used in inducer combinations that did not include NGF. These results, taken together with recent evidence that staurosporine is also able to induce neurite outgrowth from PC12 cells, suggest that the effects of staurosporine and NGF may converge, in part, on a common intracellular target.     

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.     

NGF induction of the gene encoding the protease transin accompanies neuronal differentiation in PC12 cells

Various proteases have been found to be released by the growth cones of developing neurons in culture and have been hypothesized to play a role in the process of axon elongation. We report here that nerve growth factor (NGF) induced the gene encoding the metalloprotease transin in PC12 cells with a time course coincident with the initial appearance of neurites by these cells. Acidic and basic fibroblast growth factors also stimulated transin mRNA expression and neurite outgrowth, whereas various other agents had no effects on either of these phenomena. In contrast, dexamethasone was found to inhibit the induction of transin mRNA when added with, or following, NGF treatment. Finally, we show that sequences contained within 750 bp of the 5' untranscribed region of the transin gene confer responsiveness to NGF and dexamethasone.     

A branched signaling pathway for nerve growth factor is revealed by Src-, Ras-, and Raf-mediated gene inductions.

A myriad of gene induction events underlie nerve growth factor (NGF)-induced differentiation of PC12 cells. To dissect the signal transduction pathways which lead to NGF actions, we have assessed the relative roles of NGF receptor, Src, Ras, and Raf activities in mediating specific gene inductions. We have used the PC12 cell line as well as sublines which inducibly express activated forms of either Src, Ras, or Raf or a dominant inhibitory form of Ras (p21N17 Ras) to study the expression of multiple NGF-inducible mRNAs. The NGF induction of NGFI-A, transin, and VGF mRNAs was mimicked by activated forms of Src, Ras, or Raf and was blocked by p21N17 Ras. The NGF induction of SCG10 mRNA was mimicked only by activated Src and Ras and was blocked by p21N17 Ras, while the induction of Thy-1 mRNA was mimicked only by activated Src and was not blocked by p21N17 Ras. The NGF induction of mRNAs for two sodium channel types was neither mimicked by any activated oncoprotein nor blocked by p21N17 Ras. From these and previous results, we suggest a model in which a linear order of NGF receptor, Src, Ras, and Raf activities is used by NGF to elicit gene inductions. These signaling components define branchpoints in the pathway to specific gene induction events, providing a mechanism for generating a host of diverse NGF actions.     

Modulation of nerve growth factor-induced activation of MAP kinase in PC12 cells by inhibitors of nitric oxide synthase

Nerve growth factor (NGF) increases expression of nitric oxide synthase (NOS) isozymes leading to enhanced production of nitric oxide (NO). NOS inhibitors attenuate NGF-mediated increases in cholinergic gene expression and neurite outgrowth. Mechanisms underlying this are unknown, but the mitogen-activated protein (MAP) kinase pathway plays an important role in NGF signaling. Like NGF, NO donors activate Ras leading to phosphorylation of MAP kinase. The present study investigated the role of NO in NGF-mediated activation of MAP kinase in PC12 cells. Cells were treated with 50 ng/mL NGF to establish the temporal pattern for rapid and sustained activation phases of MAP kinase kinase (MEK)-1/2 and p42/p44-MAP kinase. Subsequently, cells were pretreated with NOS inhibitors Nomega-nitro-L-arginine methylester and s-methylisothiourea and exposed to NGF for up to 24 h. NGF-induced activation of MEK-1/2 and p42/p44-MAP kinase was not dependent on NO, but sustained phosphorylation of MAP kinase was modulated by NO. This modulation did not occur at the level of Ras-Raf-MEK signaling or require activation of cGMP/PKG pathway. NOS inhibitors did not affect NGF-mediated phosphorylation of MEK. Expression of constitutively active-MEKK1 in cells led to phosphorylation of p42/p44-MAP kinase and robust neurite outgrowth; constitutively active-MKK1 also caused differentiation with neurite extension. NOS inhibitor treatment of cells expressing constitutively active kinases did not affect MAP kinase activation, but neurite outgrowth was attenuated. NOS inhibitors did not alter NGF-mediated nuclear translocation of phospho-MAP kinase, but phosphorylated kinases disappeared more rapidly from NOS inhibitor-treated cells suggesting greater phosphatase activity and termination of sustained activation of MAP kinase.     

Up-regulation of tissue inhibitor of metalloproteinases-3 gene expression by TGF-β in articular chondrocytes is mediated by serine/threonine and tyrosine kinases

The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulates extracellular matrix turn-over in normal animal development, cancer cell metastasis, atherosclerotic plaque rupture and erosion of arthritic cartilage. Transforming growth factor beta (TGF-β), an inducer of matrix synthesis, potently enhances mRNA and protein of a recently characterized MMP inhibitor, TIMP-3, in bovine articular chondrocytes. We examined the implication of protein kinases in the TGF-β-mediated induction of TIMP-3 expression by utilizing activators and inhibitors of these enzymes. Protein kinase A activators, dibutyryl cyclic AMP, or forskolin had little or no effect, respectively, while phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased TIMP-3 gene expression. H7, a serine/threonine protein kinase inhibitor, markedly reduced the response of TIMP-3 gene to TGF-β. Furthermore, two protein tyrosine kinase inhibitors, genistein and herbimycin A, inhibited TGF-β induction of TIMP-3. H7 and genistein also suppressed TGF-β-induced TIMP-3 protein expression. These results suggest that TGF-β signaling for TIMP-3 gene induction involves H7-sensitive serine/threonine kinase as well as herbimycin A- and genistein-sensitive protein tyrosine kinases. J. Cell. Biochem. 70:517–527, 1998. © 1998 Wiley-Liss, Inc.     

Mimicry and Inhibition of Nerve Growth Factor Effects: Interactions of Staurosporine, Forskolin, and K252a in PC12 Cells and Normal Rat Chromaffin Cells In Vitro

The structurally similar compounds staurosporine and K252a are potent inhibitors of protein kinases. K252a has previously been reported to inhibit most or all of the effects of nerve growth factor (NGF) on PC12 pheochromocytoma cells, and staurosporine has been reported both to inhibit and to mimic NGF-induced neurite outgrowth from a PC12 cell subclone in a dose-dependent manner. We have studied the interactions of these agents with each other, with NGF, and with forskolin, an activator of adenylate cyclase, on the parent PC12 cell line and on normal neonatal and adult rat chromaffin cells. Staurosporine alone or in conjunction with forskolin induces outgrowth of short neurites from PC12 cells but does not substitute for NGF in promoting cell survival. It does not abolish NGF-induced neurite outgrowth but does reverse the effects of NGF on catecholamine synthesis. K252a abolishes NGF-induced neurite outgrowth but only partially decreases outgrowth induced by NGF plus forskolin. It does not inhibit neurite outgrowth produced by staurosporine or staurosporine plus forskolin. These findings with PC12 cells suggest that staurosporine might act downstream from K252a and NGF on components of one or more signal transduction pathways by which NGF selectively affects the expression of certain traits. Both neonatal and adult rat chromaffin cells show dramatic flattening and extension of filopodia in response to staurosporine, an observation suggesting that some of the same pathways might remain active in cells that do not exhibit a typical NGF response. Only a small amount of neurite outgrowth is observed, however, and only in neonatal cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Ras-ERK pathway is required for the induction of neuronal nitric oxide synthase in differentiating PC12 cells

We have studied the role of MAP kinase pathways in neuronal nitric oxide synthase (nNOS) induction during the differentiation of PC12 cells. In nerve growth factor (NGF)-treated PC12 cells, we find nNOS induced at RNA and protein levels, resulting in increased NOS activity. We note that neither nNOS mRNA, nNOS protein nor NOS activity is induced by NGF treatment in cells that have been infected with a dominant negative Ras adenovirus. We have also used drugs that block MAP kinase pathways and assessed their ability to inhibit nNOS induction. Even though U0126 and PD98059 are both MEK inhibitors, we find that U0126, but not PD98059, blocks induction of nNOS protein and NOS activity in NGF-treated PC12 cells. Also, the p38 kinase inhibitor, SB203580, does not block nNOS induction in our clone of PC12 cells. Since the JNK pathway is not activated in NGF-treated PC12 cells, we conclude that the Ras-ERK pathway and not the p38 or JNK pathway is required for nNOS induction in NGF-treated PC12 cells. We find that U0126 is much more effective than PD98059 in blocking the Ras-ERK pathway, thereby explaining the discrepancy in nNOS inhibition. We conclude that the Ras-ERK pathway is required for nNOS induction.     

Blockage of Nerve Growth Factor Action in PC12h Cells by Staurosporine, a Potent Protein Kinase Inhibitor

Staurosporine, which has a structure similar to that of K-252a, a potent protein kinase inhibitor that blocks nerve growth factor (NGF) action in PC12 and PC12h cells, is also known as a potent inhibitor of several protein kinases. This study shows that in PC12h cells staurosporine has a dual action: at lower concentrations than that required by K-252a, it is an inhibitor of NGF induction of neurite formation and of changes in the phosphorylation of specific proteins, whereas at concentrations higher than that required to inhibit NGF-induced neurite outgrowth, it rapidly enhances outgrowth by itself.     

Protein Kinase Inhibitor H-7 Differentially Affects Early and Delayed Nerve Growth Factor Responses in PC12 Cells

Abstract: The effects of the protein kinase inhibitor H-7 on early and delayed responses to nerve growth factor (NGF) were investigated in PC12 cells. H-7 reduced the NGF-induced expression of c-Fos in a dose-dependent manner without affecting the time course of c-Fos appearance. Conversely, H-7 potentiated delayed NGF effects, i.e., neurite outgrowth and Ca²⁺/phospholipid-dependent protein kinase (PKC) induction, but not choline acetyltransferase induction. Long-term treatment with NGF resulted in an increase of at least four tyrosine-phosphorylated protein bands with molecular masses between 39 and 48 kDa, which was also potentiated by H-7. In the absence of NGF, H-7 had no significant effect on c-Fos expression, tyrosine phosphorylation of the 45 kDa protein, or choline acetyltransferase activity. However, 4 days of exposure to H-7 alone induced PKC activity and tyrosine phosphorylation of the 39-kDa protein. The action of H-7 derivatives on neurite outgrowth did not correlate with their inhibition profile of cyclic nucleotide-dependent protein kinases. Down-regulation of PKC activity by prolonged exposure to phorbol ester did not completely abolish the effects of NGF and H-7 on induction of c-Fos, choline acetyltransferase activity, and neurite outgrowth, indicating that PKC-independent pathways contribute to these actions. These results suggest that additional pathway(s) sensitive to H-7 may exist, which induce immediate early gene expression and suppress neuronal differentiation of PC12 cells.     

Nerve growth factor-induced stimulation of p38 mitogen-activated protein kinase in PC12 cells is partially mediated via G(i/o) proteins

Differentiation of PC12 cells by nerve growth factor (NGF) requires the activation of various mitogen-activated protein kinases (MAPKs) including p38 MAPK. Accumulating evidence has suggested cross-talk regulation of NGF-induced responses by G protein-coupled receptors, thus we examined whether NGF utilizes G(i/o) proteins to regulate p38 MAPK in PC12 cells. Induction of p38 MAPK phosphorylation by NGF occurred in a time- and dose-dependent manner and was partially inhibited by pertussis toxin (PTX). NGF-dependent p38 MAPK phosphorylation became insensitive to PTX treatment upon transient expressions of Galpha(z) or the PTX-resistant mutants of Galpha(i2) and Galpha(oA). Moreover, Galpha(i2) was co-immunoprecipitated with the TrkA receptor from PC12 cell lysates. To discern the participation of various signaling intermediates, PC12 cells were treated with a panel of specific inhibitors prior to the NGF challenge. NGF-induced p38 MAPK phosphorylation was abolished by inhibitors of Src (PP1, PP2, and SU6656) and MEK1/2 (U0126). Inhibition of the p38 MAPK pathway also suppressed NGF-induced PC12 cell differentiation. In contrast, inhibitors of JAK2, phospholipase C, protein kinase C and Ca(2+)/calmodulin-dependent kinase II did not affect the ability of NGF to activate p38 MAPK. Collectively, these studies indicate that NGF-dependent p38 MAPK activity may be mediated via G(i2) protein, Src, and the MEK/ERK cascade.