Eva Cano

Parallel signal processing among mammalian MAPKs.

The intracellular signalling field is dominated by the mitogen-activated protein kinase (MAPK) cascade and its control, which involves the small GTPase Ras and sequential kinase activation. Until recently, ERK1 and ERK2 were the only cloned and well-characterized mammalian MAPKs; diverse ligand-stimulated, proline-directed protein phosphorylation events were attributed to these kinases. The recent discovery of two other MAPK subtypes, the JNK/SAPK subfamily and p38/RK (mammalian equivalents of HOG1 in yeast), reveals extreme complexity within the family and, most intriguingly, the existence in mammalian cells of parallel MAPK cascades that can be activated simultaneously.

Anisomycin-activated protein kinases p45 and p55 but not mitogen-activated protein kinases ERK-1 and -2 are implicated in the induction of c-fos and c-jun.

Independent of its ability to block translation, anisomycin intrinsically initiates intracellular signals and immediate-early gene induction [L. C. Mahadevan and D. R. Edwards, Nature (London) 349:747-749, 1991]. Here, we characterize further its action as a potent, selective signalling agonist. In-gel kinase assays show that epidermal growth factor (EGF) transiently activates five kinases: the mitogen-activated protein (MAP) kinases ERK-1 and -2, and three others, p45, p55, and p80. Anisomycin, at inhibitory and subinhibitory concentrations, does not activate ERK-1 and -2 but elicits strong sustained activation of p45 and p55, which are unique in being serine kinases whose detection is enhanced with poly-Glu/Tyr or poly-Glu/Phe copolymerized in these gels. Translational arrest using emetine or puromycin does not activate p45 and p55 but does prolong EGF-stimulated ERK-1 and -2 activation. Rapamycin, which blocks anisomycin-stimulated p70/85S6k activation without affecting nuclear responses, has no effect on p45 or p55 kinase. p45 and p55 are activable by okadaic acid or UV irradiation, and both kinases phosphorylate the c-Jun NH2-terminal peptide 1-79, putatively placing them within c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) subfamily of MAP kinases. Thus, the EGF- and anisomycin-activated kinases p45 and p55 are strongly implicated in signalling to c-fos and c-jun, whereas the MAP kinases ERK-1 and -2 are not essential for this process.

p38/RK is essential for stress-induced nuclear responses: JNK/SAPKs and c-Jun/ATF-2 phosphorylation are insufficient

The ERK, JNK/SAPK and p38/RK MAP kinase subtypes (reviewed in [1]) are differentially activated in mammalian cells by various stimuli, which elicit induction of immediate-early (IE) genes, such as c-fos and c-jun (reviewed in [1-3]), as well as phosphorylation of histone H3 [4] and HMG-14 [5]. Anisomycin and UV radiation have been suggested to induce c-fos and c-jun transcription via JNK/SAPK-mediated phosphorylation of TCF (ternary complex factor), for c-fos induction [6-8], and c-Jun and/or ATF-2 for c-jun induction [9-11] [12,13]. We report here that anisomycin and ultraviolet radiation (UV) activate MAP kinase kinase-6 (MKK6) [14,15], p38/RK [16] [17,18] and MAPKAP kinase-2 (MAPKAP K-2) [17-19]. By using the p38/RK inhibitor SB 203580 [20,21], we show that activation of p38/RK and/or its downstream effectors are essential for anisomycin- and UV-stimulated c-fos/c-jun induction and histone H3/HMG-14 phosphorylation, whereas JNK/SAPK activation and phosphorylation of c-Jun and ATF-2 are insufficient for these responses.

Anisomycin Selectively Desensitizes Signalling Components Involved in Stress Kinase Activation and fos and jun Induction

ABSTRACT Anisomycin, a translational inhibitor secreted byStreptomyces spp., strongly activates the stress-activated mitogen-activated protein (MAP) kinases JNK/SAPK (c-Jun NH2-terminal kinase/stress-activated protein kinase) and p38/RK in mammalian cells, resulting in rapid induction of immediate-early (IE) genes in the nucleus. Here, we have characterized this response further with respect to homologous and heterologous desensitization of IE gene induction and stress kinase activation. We show that anisomycin acts exactly like a signalling agonist in eliciting highly specific and virtually complete homologous desensitization. Anisomycin desensitization of a panel of IE genes (c-fos, fosB, c-jun,junB, and junD), using epidermal growth factor (EGF), basic fibroblast growth factor, (bFGF), tumor necrosis factor alpha (TNF-α), anisomycin, tetradecanoyl phorbol acetate (TPA), and UV radiation as secondary stimuli, was found to be extremely specific both with respect to the secondary stimuli and at the level of individual genes. Further, we show that anisomycin-induced homologous desensitization is caused by the fact that anisomycin no longer activates the JNK/SAPK and p38/RK MAP kinase cascades in desensitized cells. In anisomycin-desensitized cells, activation of JNK/SAPKs by UV radiation and hyperosmolarity is almost completely lost, and that of the p38/RK cascade is reduced to about 50% of the normal response. However, all other stimuli produced normal or augmented activation of these two kinase cascades in anisomycin-desensitized cells. These data show that anisomycin behaves like a true signalling agonist and suggest that the anisomycin-desensitized signalling component(s) is not involved in JNK/SAPK or p38/RK activation by EGF, bFGF, TNF-α, or TPA but may play a significant role in UV- and hyperosmolarity-stimulated responses.

ERK activation induces phosphorylation of Elk-1 at multiple S/T-P motifs to high stoichiometry

Elk-1, a member of the TCF family of Ets domain proteins, contains a C-terminal transcriptional activation domain with multiple copies of the MAPK core consensus sequence S/T-P. This region is phosphorylated by MAP kinases in vitro and in vivo, but the extent and kinetics of phosphorylation at the different sites have not been investigated in detail. We prepared antisera against the phosphorylated forms of residues T353, T363, T368, S383, S389 and T417. The antisera specifically recognize the phosphorylated Elk-1 C terminus and are specific for their cognate sites, as assessed by peptide competition and mutagenesis experiments. Analysis of cells stably expressing Elk-1 in vivo shows that following serum or TPA stimulation, residues T353, T363, T368, S383, S389 and T417 become phosphorylated with similar kinetics. Mutation of any one site does not prevent phosphorylation of the others. Mutation to alanine of S383, F378 or W379, which virtually abolishes transcriptional activation by Elk-1, does not affect phosphorylation of any sites tested. Analysis of Elk-1 using two-dimensional gel electrophoresis shows that following ERK activation Elk-1 receives at least six phosphates in addition to those present prior to stimulation. We propose that the Elk-1 C-terminal regulatory domain becomes stoichiometrically phosphorylated following growth factor stimulation.

Effects of the inhibition of p38/RK MAP kinase on induction of five fos and jun genes by diverse stimuli.

The ERK, JNK/SAPK and p38/RK MAP kinase subtypes are differentially activated by physiological, pharmacological and stress stimuli; all three subtypes are implicated in immediate-early (IE) gene induction by these agents. Here, we have asked whether inhibition of a single MAP kinase subtype under these conditions would generally alter induction of several IE genes in a similar way or whether this would differentially up- and down-regulate particular IE genes, an issue which bears on the question of whether individual MAP kinases are strictly targeted to specific IE genes, or whether they might catalyse phosphorylation events that affect several IE genes in the same way. SB 203580, an inhibitor of p38/RK, has been used to analyse the role of this kinase in the induction of five IE genes (c-fos, fosB, c-jun, junB and junD) under diverse conditions of stimulation. In C3H 10T½ cells, p38/RK and its downstream kinase MAPKAP K-2 are activated by all stimuli used with the exception of TPA. The specificity of SB 203580 as a p38/RK inhibitor in these cells is demonstrated; it does not affect ERKs or JNK/SAPKs but does result in a small increase in the activity of the upstream kinase MKK6, the principal p38/RK activator in these cells. We find that inhibition of p38/RK under these conditions produces general effects on all five IE genes as a group in three ways. First, induction of all five genes in response to okadaic acid or tumour necrosis factor-α (TNF-α) is not significantly altered by SB 203580. Second, in cells stimulated with anisomycin or U.V. radiation, SB 203580 potently inhibits all of the induced IE genes. Finally, SB 203580 enhances induction of all five IE genes in EGF-treated cells; these enhanced mRNA levels are not due to stabilisation of labile mRNA transcripts. The significance of these results to current thinking on the relationship between distinct MAP kinase subtypes and specific IE genes is discussed.

Depolarization of Neural Cells Induces Transcription of the Down Syndrome Critical Region 1 Isoform 4 via a Calcineurin/Nuclear Factor of Activated T Cells-dependent Pathway

In this study we showed that the transcriptional regulation of Down syndrome critical region isoform 4 (DSCR1.4) is mediated by the calcineurin/nuclear factor of activated T cells (NFAT) pathway in neural cells. Stimuli that elicit an increase in the intracellular concentrations of calcium, such as membrane depolarization, induced de novo transcription of DSCR1.4, with mRNA expression peaking after 4 h and then declining. Action via the physiologically relevant L-type calcium channel was confirmed by blockade with nifedipine and verapamil. This calcium-dependent transcription of DSCR1.4 was inhibited by the calcineurin inhibitors cyclosporin A and FK506. Deletional analysis showed that the calcium- and calcineurin-dependent activation is mediated by the promoter region between nucleotides –350 and –166, a region that contains putative NFAT-binding motifs. Exogenous NFATc2 potently augmented the DSCR1.4 promoter transcriptional activity, and the involvement of endogenous NFAT signaling pathway in DSCR1.4 transcription was confirmed by the suppression of depolarization-inducible promoter activity with the NFAT inhibitor peptide VIVIT. Exogenous overexpression of DSCR1 protein (calcipressin 1) resulted in the inhibition of the transcription of DSCR1.4 and NFAT-dependent signaling. These findings suggest that calcineurin-dependent induction of DSCR1.4 product may represent an important auto-regulatory mechanism for the homeostatic control of NFAT signaling in neural cells.

Calcium/calcineurin signaling in primary cortical astrocyte cultures: Rcan1-4 and cyclooxygenase-2 as NFAT target genes.

The calcineurin/nuclear factor of activated T cells (NFAT) signaling pathway mediates important cell responses to calcium, but its activity and function in astrocytes have remained unclear. We show that primary cortical astrocyte cultures express the regulatory and catalytic subunits of the phosphatase calcineurin as well as the calcium‐regulated NFAT family members (NFATc1, c2, c3, and c4). NFATs are activated by calcium‐mobilizing agents in astrocytes, and this activation is blocked by the calcineurin inhibitor cyclosporine A. Microarray screening identified cyclooxygenase‐2 (Cox‐2), which is implicated in brain injury, and Rcan 1‐4, an endogenous calcineurin inhibitor, as genes up‐regulated by calcineurin‐dependent calcium signals in astrocytes. Mobilization of intracellular calcium with ionophore potently augments the promoter activity and mRNA and protein expression of Rcan 1‐4 and Cox‐2 induced by combined treatment with phorbol esters. Moreover, Rcan 1‐4 expression is efficiently induced by calcium mobilization alone. For both the genes, the calcium signal component is dependent on calcineurin and is replicated by exogenous expression of a constitutively active NFAT, strongly suggesting that the calcium‐induced gene activation is mediated by NFATs. Finally, we report that calcineurin‐dependent expression of Cox‐2 and Rcan 1‐4 is induced by physiological calcium mobilizing agents, such as thrombin, agonists of purinergic and glutamate receptors, and L‐type voltage‐gated calcium channels. These findings provide insights into calcium‐initiated gene transcription in astrocytes, and have implications for the regulation of calcium responses in astrocytes.

Tumor Necrosis Factor-α (TNF-α), Interferon-γ, and Interleukin-6 but Not TNF-β Induce Differentiation of Neuroblastoma Cells: The Role of Nitric Oxide

Abstract: Tumor necrosis factor‐a (TNF‐α), interferon‐γ (IFN‐7), and interleukin‐6 (IL‐6), but not TNF‐β, can induce the in vitro differentiation of the neuroblastoma cell line N103 in a dose‐dependent manner. Differentiation of N103 was accompanied by the arrest of cell growth and neurite formation. The induction of neuroblastoma cell differentiation by TNF‐α and IFN‐γ can be specifically inhibited by a nitric oxide (NO) synthase inhibitor, l‐NG‐monomethylarginine. In contrast, the differentiation of N103 cells by IL‐6 was not affected by l‐NG‐monomethylarginine. These results indicate that TNF‐α and IFN‐γ, but not IL‐6, induce the differentiation of neuroblastoma cells via NO. This is confirmed by the finding that the culture super‐ natants of N103 cells induced by TNF‐α and IFN‐γ, but not that by IL‐6, contained high levels of NO2−, the production of which was inhibited by l‐NG‐monomethylarginine. Furthermore, the differentiation of N103 cells can be induced directly in a dose‐dependent manner by the addition of nitroprusside, a generator of NO, into the culture medium. These data therefore indicate that NO may be an important mediator in the induction of neuronal cell differentiation by certain cytokines such as TNF‐α and IFN‐γ and that neuronal cells, in addition to the macrophagelike brain cells, can be induced by immunological stimuli to produce large quantities of NO.

Regulator of calcineurin 1 (Rcan1) has a protective role in brain ischemia/reperfusion injury

BackgroundAn increase in intracellular calcium concentration [Ca2+]i is one of the first events to take place after brain ischemia. A key [Ca2+]i-regulated signaling molecule is the phosphatase calcineurin (CN), which plays important roles in the modulation of inflammatory cascades. Here, we have analyzed the role of endogenous regulator of CN 1 (Rcan1) in response to experimental ischemic stroke induced by middle cerebral artery occlusion.MethodsAnimals were subjected to focal cerebral ischemia with reperfusion. To assess the role of Rcan1 after stroke, we measured infarct volume after 48 h of reperfusion in Rcan1 knockout (KO) and wild-type (WT) mice. In vitro studies were performed in astrocyte-enriched cortical primary cultures subjected to 3% oxygen (hypoxia) and glucose deprivation (HGD). Adenoviral vectors were used to analyze the effect of overexpression of Rcan1-4 protein. Protein expression was examined by immunohistochemistry and immunoblotting and expression of mRNA by quantitative real-time Reverse-Transcription Polymerase Chain Reaction (real time qRT-PCR).ResultsBrain ischemia/reperfusion (I/R) injury in vivo increased mRNA and protein expression of the calcium-inducible Rcan1 isoform (Rcan1-4). I/R-inducible expression of Rcan1 protein occurred mainly in astroglial cells, and in an in vitro model of ischemia, HGD treatment of primary murine astrocyte cultures induced Rcan1-4 mRNA and protein expression. Exogenous Rcan1-4 overexpression inhibited production of the inflammatory marker cyclo-oxygenase 2. Mice lacking Rcan1 had higher expression of inflammation associated genes, resulting in larger infarct volumes.ConclusionsOur results support a protective role for Rcan1 during the inflammatory response to stroke, and underline the importance of the glial compartment in the inflammatory reaction that takes place after ischemia. Improved understanding of non-neuronal mechanisms in ischemic injury promises novel approaches to the treatment of acute ischemic stroke.