Biomedical subjects

Matthias Gaestel

Publications and source records attributed to Matthias Gaestel.

32 records · Page 2Linked to original sources

PubMed2004

IFN-gamma regulation in anti-CD4 antibody-induced T cell unresponsiveness.

The anti-rat CD4 mAb RIB5/2 is very potent in inducing allospecific tolerance in vivo. It is interesting that the unresponsiveness is breakable by exogenous IL-2 applied during the induction phase of tolerance. The molecular mechanisms underlying anti-CD4 antibody-mediated inhibition of allospecific T cell activation and how this is antagonized by exogenous IL-2 were investigated. Anti-CD4 treatment, in vivo and in vitro, completely abrogated IL-2 production by alloreactive T cells. In contrast, anti-CD4-treated alloactivated T cells showed similar IFN-gamma mRNA expression as untreated alloactivated T cells but did not secrete any protein. Thus, the anti-CD4 antibody cannot prevent IFN-gamma mRNA expression but is interfering with posttranscriptional mechanisms that control IFN-gamma production during alloactivation of T cells. Addition of IL-2 but not IL-15 to anti-CD4-treated alloactivated T cells restored IFN-gamma protein production without leading to enhanced IFN-gamma mRNA expression. Further investigations revealed a diminished activation of translation initiation factor eIF2alpha in anti-CD4-treated T cells, which was restored by exogenous IL-2. As activated eIF2alpha is essential for the translation of IFN-gamma mRNA, the results may explain the reversibility of anti-CD4-induced unresponsiveness by exogenous IL-2. Furthermore, these results not only shed further light onto the molecular mechanisms of tolerance induction but also reveal the possible weaknesses of anti-CD4 antibody-induced unresponsiveness.

Animals↗

PubMed2003

Role of p38alpha Map kinase in Type I interferon signaling.

Multiple signaling pathways are activated during engagement of the Type I interferon (IFN) receptor to mediate biological responses, including the Jak-Stat and Rac1/p38 Map kinase signaling cascades. In the present study we sought to determine the functional relevance of the p38alpha isoform in IFN signaling, using cells from mouse embryos with targeted disruption of the p38alpha gene. Our data demonstrate that p38alpha activation is essential for Type I IFN-dependent transcriptional regulation via ISRE or GAS elements. On the other hand, the function of p38alpha is not required for IFN-dependent Ser727 or Tyr701 phosphorylation of Stat1 and does not impact on the formation of ISGF3 or SIF nuclear binding complexes. In efforts to identify downstream effectors of p38 that may mediate IFN-dependent transcriptional responses, we found that IFNalpha activates the kinase Msk1, a known regulator of histone phosphorylation and chromatin remodeling. In other studies, we demonstrate that Type I IFN-dependent activation of the kinases MapKapK-2 and MapKapK-3 is defective in the absence of p38alpha, while Type I IFN-dependent antiviral properties are decreased in cells with targeted disruption of the MapKapK-2 gene. Altogether, our data establish that the p38alpha Map kinase pathway regulates activation of downstream effectors that participate in the induction of IFN-dependent gene transcription, to mediate IFN-responses.

Animals↗

PubMed2003

Analysis of the interaction of small heat shock proteins with unfolding proteins.

The ubiquitous small heat shock proteins (sHsps) are efficient molecular chaperones that interact with nonnative proteins, prevent their aggregation, and support subsequent refolding. No obvious substrate specificity has been detected so far. A striking feature of sHsps is that they form large complexes with nonnative proteins. Here, we used several well established model chaperone substrates, including citrate synthase, alpha-glucosidase, rhodanese, and insulin, and analyzed their interaction with murine Hsp25 and yeast Hsp26 upon thermal unfolding. The two sHsps differ in their modes of activation. In contrast to Hsp25, Hsp26 undergoes a temperature-dependent dissociation that is required for efficient substrate binding. Our analysis shows that Hsp25 and Hsp26 reacted in a similar manner with the nonnative proteins. For all substrates investigated, complexes of defined size and shape were formed. Interestingly, several different nonnative proteins could be incorporated into defined sHsp-substrate complexes. The first substrate protein bound seems to determine the complex morphology. Thus, despite the differences in quaternary structure and mode of activation, the formation of large uniform sHsp-substrate complexes seems to be a general feature of sHsps, and this unique chaperone mechanism is conserved from yeast to mammals.

Animals↗

PubMed2003

Affinity purification of ARE-binding proteins identifies polyA-binding protein 1 as a potential substrate in MK2-induced mRNA stabilization.

An important determinant for the expression level of cytokines and proto-oncogenes is the rate of degradation of their mRNAs. AU-rich sequence elements (AREs) in the 3(') untranslated regions have been found to impose rapid decay of these mRNAs. ARE-containing mRNAs can be stabilized in response to external signals which activate the p38 MAP kinase cascade including the p38 MAP kinase substrate MAPKAP kinase 2 (MK2). In an attempt to identify components downstream of MK2 in this pathway we analyzed several proteins which selectively interact with the ARE of GM-CSF mRNA. One of them, the cytoplasmic poly(A)-binding protein PABP1, co-migrated with a protein that showed prominent phosphorylation by recombinant MK2. Phosphorylation by MK2 was confirmed using PABP1 purified by affinity chromatography on poly(A) RNA. The selective interaction with an ARE-containing RNA and the phosphorylation by MK2 suggest that PABP1 plays a regulatory role in ARE-dependent mRNA decay and its modulation by the p38 MAP kinase cascade.

3' Untranslated Regions↗

PubMed2003

Elimination of protein kinase MK5/PRAK activity by targeted homologous recombination.

MK5 (mitogen-activated protein kinase [MAPK]-activated protein kinase 5), also designated PRAK (p38-regulated and -activated kinase), was deleted from mice by homologous recombination. Although no MK5 full-length protein and kinase activity was detected in the MK5 knockout mice, the animals were viable and fertile and did not display abnormalities in tissue morphology or behavior. In addition, these mice did not show increased resistance to endotoxic shock or decreased lipopolysaccharide-induced cytokine production. Hence, MK5 deletion resulted in a phenotype very different from the complex inflammation-impaired phenotype of mice deficient in MK2, although MK2 and MK5 exhibit evolutional, structural, and apparent extensive functional similarities. To explain this discrepancy, we used wild-type cells and embryonic fibroblasts from both MK2 and MK5 knockout mice as controls to reexamine the mechanism of activation, the interaction with endogenous p38 MAPK, and the substrate specificity of both enzymes. In contrast to MK2, which shows interaction with and chaperoning properties for p38 MAPK and which is activated by extracellular stresses such as arsenite or sorbitol treatment, endogenous MK5 did not show these properties. Furthermore, endogenous MK5 is not able to phosphorylate Hsp27 in vitro and in vivo. We conclude that the differences between the phenotypes of MK5- and MK2-deficient mice result from clearly different functional properties of both enzymes.

Animals↗

PubMed2002

Genetic dissection of the cellular pathways and signaling mechanisms in modeled tumor necrosis factor-induced Crohn's-like inflammatory bowel disease.

Recent clinical evidence demonstrated the importance of tumor necrosis factor (TNF) in the development of Crohn's disease. A mouse model for this pathology has previously been established by engineering defects in the translational control of TNF mRNA (Tnf(Delta)(ARE) mouse). Here, we show that development of intestinal pathology in this model depends on Th1-like cytokines such as interleukin 12 and interferon gamma and requires the function of CD8(+) T lymphocytes. Tissue-specific activation of the mutant TNF allele by Cre/loxP-mediated recombination indicated that either myeloid- or T cell-derived TNF can exhibit full pathogenic capacity. Moreover, reciprocal bone marrow transplantation experiments using TNF receptor-deficient mice revealed that TNF signals are equally pathogenic when directed independently to either bone marrow-derived or tissue stroma cell targets. Interestingly, TNF-mediated intestinal pathology was exacerbated in the absence of MAPKAP kinase 2, yet strongly attenuated in a Cot/Tpl2 or JNK2 kinase-deficient genetic background. Our data establish the existence of redundant cellular pathways operating downstream of TNF in inflammatory bowel disease, and demonstrate the therapeutic potential of selective kinase blockade in TNF-mediated intestinal pathology.

Animals↗

PubMed2002

Inhibition of SAPK2a/p38 prevents hnRNP A0 phosphorylation by MAPKAP-K2 and its interaction with cytokine mRNAs.

Lipopolysaccharide (LPS) stimulates production of inflammatory mediators, partly by stabilizing [interleukin-6 (IL-6), cyclooxygenase 2 (COX-2)] and/or stimulating translation [tumour necrosis factor-alpha (TNF-alpha)] of their mRNAs. Such regulation depends on AU-rich elements (AREs) within the 3'-untranslated regions and is partially suppressed by SB 203580 (which inhibits SAPK2a/p38). The LPS-induced production of TNF-alpha and IL-6 is suppressed in MAPKAP-K2-deficient mice (a kinase activated by SAPK2a/p38). Here, we identify 18 macrophage proteins that bind to AREs and show that hnRNP A0 is a major substrate for MAPKAP-K2 in this fraction. MAPKAP-K2 phosphorylated hnRNP A0 at Ser84 in vitro and this residue became phosphorylated in LPS-stimulated cells. Phosphorylation was prevented by SB 203580 and suppressed in macrophages derived from MAPKAP-K2-deficient mice. The mRNAs encoding TNF-alpha, COX-2 and macrophage inflammatory protein-2 (MIP-2) bound to hnRNP A0 in LPS-stimulated macrophages, an interaction prevented by SB 203580. The LPS-induced stabilization of MIP-2 mRNA and production of MIP-2 protein were abolished when macrophages were incubated with SB 203580 plus PD 184352 (which inhibits the classical MAP kinase cascade). Our data suggest that LPS-induced binding of hnRNP A0 to AREs may contribute to the post-transcriptional regulation of specific mRNAs.

Animals↗

PubMed2002

Mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 deficiency protects brain from ischemic injury in mice.

Mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MK2) is one of several kinases directly regulated by p38 MAP kinase. A role of p38 MAP kinase in ischemic brain injury has been previously suggested by pharmacological means. In the present study, we provide evidence for a role of MK2 in cerebral ischemic injury using MK2-deficient (MK2(-/-)) mice. MK2(-/-) mice subjected to focal ischemia markedly reduced infarct size by 64 and 76% after transient and permanent ischemia, respectively, compared with wild-type mice. Furthermore, MK2(-/-) mice had significant reduction in neurological deficits. Real-time PCR analysis identified a significantly lower expression in interleukin-1beta mRNA (53% reduction) but not in tumor necrosis factor-alpha mRNA in MK2(-/-) mice over wild-type animals after ischemic injury. The significant reduction in interleukin-1beta was also confirmed in MK2(-/-) mice by enzyme-linked immunosorbent assay. The marked neuroprotection from ischemic brain injury in MK2(-/-) mice was not associated with the alteration of hemodynamic or systemic variables, activation of caspase-3, or apoptosis. Our data provide new evidence for the involvement of MAP kinase pathway in focal ischemic brain injury and suggest that this effect might be associated with the expression of interleukin-1beta in the ischemic brain tissue.

Animals↗

PubMed2002

Cytoskeletal changes in hypoxic pulmonary endothelial cells are dependent on MAPK-activated protein kinase MK2.

Exposure to hypoxia causes structural changes in the endothelial cell layer that alter its permeability and its interaction with leukocytes and platelets. One of the well characterized cytoskeletal changes in response to stress involves the reorganization of the actin cytoskeleton and the formation of stress fibers. This report describes cytoskeletal changes in pulmonary microvascular endothelial cells in response to hypoxia and potential mechanisms involved in this process. The hypoxia-induced actin redistribution appears to be mediated by components downstream of MAPK p38, which is activated in pulmonary endothelial cells in response to hypoxia. Our results indicate that kinase MK2, which is a substrate of p38, becomes activated by hypoxia, leading to the phosphorylation of one of its substrates, HSP27. Because HSP27 phosphorylation is known to alter actin distribution in response to other stimuli, we postulate that it also causes the actin redistribution observed in hypoxia. This notion is supported by the observations that similar actin redistribution occurs in cells overexpressing constitutively active MK2 or phosphomimicking HSP27 mutant. Overexpressing dominant negative MK2 blocks the effects of hypoxia on the actin cytoskeleton. Taken together these results indicate that hypoxia stimulates the p38-MK2-HSP27 pathway leading to significant alteration in the actin cytoskeleton.

Animals↗

PubMed2002

Mitogen-activated protein kinase-activated protein kinase 2-deficient mice show increased susceptibility to Listeria monocytogenes infection.

Mitogen-activated protein kinase-activated protein kinase 2 (MK2) is one of several kinases activated through direct phosphorylation by p38 mitogen-activated protein kinase. MK2 regulates LPS-induced TNF mRNA translation, and targeted mutation of the MK2 gene renders mice more resistant to D-galactosamine plus LPS-induced liver damage. In the present study, we investigated the role of MK2 in immune defense against Listeria monocytogenes infection. MK2-deficient mice displayed diminished resistance to L. monocytogenes due to impaired control of bacterial growth. The increase in bacterial load in MK2(-/-) mice was associated with normal levels of IL-1 beta, IL-6, and IFN-gamma, whereas TNF production was strongly attenuated. In line, MK2-deficient bone marrow-derived macrophages showed impaired release of TNF, but not of IL-1 beta, in response to various bacterial stimuli in addition to decreased phagocytosis of fluorescence-labeled bacteria. Furthermore, spleen cells from MK2(-/-) mice displayed diminished IFN-gamma synthesis after stimulation with L. monocytogenes. In contrast, MK2 deficiency had no effect on macrophage generation of NO or on oxidative burst activity in response to L. moocytogenes. These results indicate an essential role of MK2 in host defense against intracellular bacteria probably via regulation of TNF and IFN-gamma production required for activation of antibacterial effector mechanisms.

Animals↗

PubMed2002

Identification of a phosphorylation site on skeletal muscle myosin light chain kinase that becomes phosphorylated during muscle contraction.

A protein phosphorylated efficiently in vitro by MAP kinase-activated protein kinase-2 (MAPKAP-K2) was purified from skeletal muscle extracts and identified as the calcium/calmodulin-dependent myosin light chain kinase (MLCK). The phosphorylation site was mapped to Ser(161), a residue shown previously to be autophosphorylated by MLCK. The residue equivalent to Ser(161) became phosphorylated in vivo when rat hindlimbs were stimulated electrically. However, phosphorylation was triggered within seconds, whereas activation of MAPKAP-K2 required several minutes. Moreover, contraction-induced Ser(161) phosphorylation was similar in wild-type or MAPKAP-K2-/- mice. These results indicate that contraction-induced phosphorylation is probably catalyzed by MLCK and not MAPKAP-K2. Ser(161) phosphorylation induced the binding of MLCK to 14-3-3 proteins, but did not detectably affect the kinetic properties of MLCK. The sequence surrounding Ser(161) is unusual in that residue 158 is histidine. Previously, an arginine located three residues N-terminal to the site of phosphorylation was thought to be critical for the specificity of MAPKAP-K2.

14-3-3 Proteins↗

PubMed2002

Distinct cellular functions of MK2.

Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2) is activated upon stress by p38 MAPK alpha and -beta, which bind to a basic docking motif in the C terminus of MK2 and which subsequently phosphorylate its regulatory sites. As a result of activation MK2 is exported from the nucleus to the cytoplasm and cotransports active p38 MAPK to this compartment. Here we show that the amount of p38 MAPK is significantly reduced in cells and tissues lacking MK2, indicating a stabilizing effect of MK2 for p38. Using a murine knockout model, we have previously shown that elimination of MK2 leads to a dramatic reduction of tumor necrosis factor (TNF) production in response to lipopolysaccharide. To further elucidate the role of MK2 in p38 MAPK stabilization and in TNF biosynthesis, we analyzed the ability of two MK2 isoforms and several MK2 mutants to restore both p38 MAPK protein levels and TNF biosynthesis in macrophages. We show that MK2 stabilizes p38 MAPK through its C terminus and that MK2 catalytic activity does not contribute to this stabilization. Importantly, we demonstrate that stabilizing p38 MAPK does not restore TNF biosynthesis. TNF biosynthesis is only restored with MK2 catalytic activity. We further show that, in MK2-deficient macrophages, formation of filopodia in response to extracellular stimuli is reduced. In addition, migration of MK2-deficient mouse embryonic fibroblasts (MEFs) and smooth muscle cells on fibronectin is dramatically reduced. Interestingly, reintroducing catalytic MK2 activity into MEFs alone is not sufficient to revert the migratory phenotype of these cells. In addition to catalytic activity, the proline-rich N-terminal region is necessary for rescuing the migratory phenotype. These data indicate that catalytic activity of MK2 is required for both cytokine production and cell migration. However, the proline-rich MK2 N terminus provides a distinct role restricted to cell migration.

Adenoviridae↗

PubMed2002

In the cellular garden of forking paths: how p38 MAPKs signal for downstream assistance.

Mitogen-activated protein kinases (MAPKs) are evolutionarily conserved enzymes which connect cell-surface receptors to regulatory targets within cells and convert receptor signals into various outputs. In mammalian cells, four distinct MAPKs have been identified: the extracellular signal-related kinases (ERK)-1/2, the c-jun N-terminal kinases or stress-activated protein kinases 1 (JNK1/2/3, or SAPK1s), the p38 MAPKs (p38 alpha/beta/gamma/delta, or SAPK2s), and the ERK5 or big MAP kinase 1 (BMK1). The p38 MAPK cascade is activated by stress or cytokines and leads to phosphorylation of its central elements, the p38 MAPKs. Downstream of p38 MAPKs there is a diversification and extensive branching of signalling pathways. For that reason, we will focus in this review on the different signalling events that are triggered by p38 activity, and analyse how these events contribute to specific gene expression and cellular responses.

Amino Acid Sequence↗

PubMed2001

MK2 targets AU-rich elements and regulates biosynthesis of tumor necrosis factor and interleukin-6 independently at different post-transcriptional levels.

We demonstrate that lipopolysaccharide-induced tumor necrosis factor (TNF) biosynthesis becomes independent of MAPKAP kinase 2 (MK2) when the AU-rich element (ARE) of the TNF gene is deleted. In spleen cells and macrophages where TNF biosynthesis is restored as a result of this deletion, interleukin (IL)-6 biosynthesis is still dependent on MK2. In MK2-deficient macrophages the half-life of IL-6 mRNA is reduced more than 10-fold, whereas the half-life of TNF mRNA is only weakly decreased. It is shown that the stability of a reporter mRNA carrying the AU-rich 3'-untranslated region (3'-UTR) of IL-6 is increased by MK2. The data provide in vivo evidence that the AU-rich 3'-UTRs of TNF and IL-6 are downstream to MK2 signaling and make MK2 an essential component of mechanisms that regulate biosynthesis of IL-6 at the levels of mRNA stability, and of TNF mainly through TNF-ARE-dependent translational control.

3' Untranslated Regions↗