P38alpha Activation Research Articles

Disruption of TAB1/p38α Interaction Using a Cell-permeable Peptide Limits Myocardial Ischemia/Reperfusion Injury

Targeting the adaptor protein (transforming growth factor-β (TGF-β)-activated protein kinase 1 (TAK1)-binding protein 1) (TAB1)-mediated non-canonical activation of p38α to limit ischemia/reperfusion (I/R) injury after an acute myocardial infarction seems to be attractive since TAB1/p38α interaction occurs specifically in very limited circumstances and possesses unique structural basis. However, so far no TAB1/p38α interaction inhibitor has been reported due to the limited knowledge about the interfaces. In this study, we sought to identify key amino acids essential for the unique mode of interaction with computer-guided molecular simulations and molecular docking. After validation of the predicted three-dimensional (3-D) structure of TAB1/p38α complex, we designed several peptides and evaluated whether they could block TAB1/p38α interaction with selectivity. We found that a cell-permeable peptide worked as a selective TAB1/p38α interaction inhibitor and decreased myocardial I/R injury. To our knowledge, this is the first TAB1/p38α interaction inhibitor.

The kinase p38α serves cell type–specific inflammatory functions in skin injury and coordinates pro- and anti-inflammatory gene expression

The mitogen-activated protein kinase p38 mediates cellular responses to injurious stress and immune signaling. Among the many p38 isoforms, p38 alpha is the most widely expressed in adult tissues and can be targeted by various pharmacological inhibitors. Here we investigated how p38 alpha activation is linked to cell type-specific outputs in mouse models of cutaneous inflammation. We found that both myeloid and epithelial p38 elicit inflammatory responses, yet p38 alpha signaling in each cell type served distinct inflammatory functions and varied depending on the mode of skin irritation. In addition, myeloid p38 alpha limited acute inflammation via activation of anti-inflammatory gene expression dependent on mitogen- and stress-activated kinases. Our results suggest a dual function for p38 alpha in the regulation of inflammation and show mixed potential for its inhibition as a therapeutic strategy.

Regulation of Ncx1 Gene Expression in the Normal and Hypertrophic Heart

The Na+/Ca2+ exchanger (NCX1) is crucial in the regulation of [Ca2+]i in the cardiac myocyte. The exchanger is upregulated in cardiac hypertrophy, ischemia, and failure. This upregulation can have an effect on Ca2+ transients and possibly contribute to diastolic dysfunction and an increased risk of arrhythmias. Studies from both in vivo and in vitro model systems have provided an initial skeleton of the potential signaling pathways that regulate the exchanger during development, growth, and hypertrophy. The Ncx1 gene is upregulated in response to alpha-adrenergic stimulation. We have shown that this is via p38alpha activation of transcription factors binding to the Ncx1 promotor at the -80 CArG element. Interestingly, most of the elements, including the CArG element, which we have demonstrated to be important for regulation of Ncx1 expression are in the proximal 184 bp of the promotor. Using a transgenic mouse, we have shown that the proximal 184 bp is sufficient for expression of reporter genes in adult cardiomyocytes and for the correct spatiotemporal pattern of Ncx1 expression in development but not for upregulation in response to pressure overload.

Multiple Activation Mechanisms of p38α Mitogen-activated Protein Kinase

The p38alpha MAPK participates in a variety of biological processes. Activation of p38alpha is mediated by phosphorylation on specific regulatory tyrosine and threonine sites, and the three dual kinases, MAPK kinase 3 (MKK3), MKK4, and MKK6, are known to be the upstream activators of p38alpha. In addition to activation by upstream kinases, p38alpha can autoactivate when interacting with transforming growth factor-beta-activated protein kinase 1-binding protein 1 (TAB1). Here we used MKK3 and MKK6 double knock-out (MKK3/6 DKO) and MKK4/7 DKO mouse embryonic fibroblast (MEF) cells to examine activation mechanisms of p38alpha. We confirmed that the MKK3/6 pathway is a primary mechanism for p38alpha phosphorylation in MEF cells, and we also showed the presence of other p38alpha activation pathways. We show that TAB1-mediated p38alpha phosphorylation in MEF cells did not need MKK3/4/6, and it accounted for a small portion of the total p38alpha phosphorylation that was induced by hyperosmolarity and anisomycin. We observed that a portion of peroxynitrite-induced phospho-p38alpha is associated with an approximately 85-kDa disulfide complex in wild-type MEF cells. Peroxynitrite-induced phosphorylation of p38alpha in the approximately 85-kDa complex is independent from MKK3/6 because only phospho-p38alpha not associated with the disulfide complex was diminished in MKK3/6 DKO cells. In addition, our data suggest interference among different pathways because TAB1 had an inhibitory effect on p38alpha phosphorylation in the peroxynitrite-induced approximately 85-kDa complex. Mutagenesis analysis of the cysteines in p38alpha revealed that no disulfide bond forms between p38alpha and other proteins in the approximately 85-kDa complex, suggesting it is a p38alpha binding partner(s) that forms disulfide bonds, which enable it to bind to p38alpha. Therefore, multiple mechanisms of p38alpha activation exist that can influence each other, be simultaneously activated by a given stimulus, and/or be selectively used by different stimuli in a cell type-specific manner.

Regulation of the maintenance of peripheral T-cell anergy by TAB1-mediated p38 alpha activation.

In anergic T cells, T-cell receptor (TCR)-mediated responses are functionally inactivated by negative regulatory signals whose mechanisms are poorly understood. Here, we show that CD4(+) T cells anergized in vivo by superantigen Mls-1(a) express a scaffolding protein, transforming growth factor beta-activated protein kinase 1-binding protein 1 (TAB1), that negatively regulates TCR signaling through the activation of mitogen-activated protein kinase p38 alpha. TAB1 was not expressed in naive and activated CD4(+) T cells. Inhibition of p38 activity in anergic T cells by a chemical inhibitor resulted in the recovery of interleukin 2 (IL-2) and the inhibition of IL-10 secretion. T-cell hybridoma 2B4 cells transduced with TAB1-containing retrovirus (TAB1-2B4 cells) showed activated p38 alpha, inhibited extracellular signal-regulated kinase (ERK) activity, culminating in reduced IL-2 levels and increased IL-10 production. The use of a p38 inhibitor or cotransfection of a dominant-negative form of p38 in TAB1-2B4 cells resulted in the recovery of ERK activity and IL-2 production. These results imply that TAB1-mediated activation of p38 alpha in anergic T cells regulates the maintenance of T-cell unresponsiveness both by inhibiting IL-2 production and by promoting IL-10 production.

Role of p38α 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.

A p38 inhibitor allows to dissociate differentiation and apoptotic processes triggered upon LIF withdrawal in mouse embryonic stem cells.

Mouse embryonic stem cells remain pluripotent when maintained in the presence of leukemia inhibitory factor (LIF). Upon LIF withdrawal, most cells differentiate into various lineages, while some die by apoptosis within 3 days. We have analyzed the activation pattern of the mitogen-activated protein kinase (MAPK) families and characterized the expression profile of selected genes modulated during differentiation or apoptosis. We show that p38 MAPKs are activated first, during the apoptotic crisis, while extracellular-regulated kinases and c-Jun N-terminal kinases are induced after the apoptotic crisis in differentiated cells. However, by using both p38 kinase inhibitors (PD169316 and SB203580) and a p38alpha(-/-) cell line, we demonstrate that p38alpha activation is rather a consequence than a cause of apoptosis. We thus reveal novel properties of PD169316, which induces cell survival without impairing cell differentiation, and identify PD169316-sensitive targets like the fibroblast growth factor-5, Brachyury and bcl-2 genes. Finally, we demonstrate that overexpression of the PD169316 - regulated bcl-2 gene prevents LIF withdrawal - induced cell death.

Indole-based heterocyclic inhibitors of p38alpha MAP kinase: designing a conformationally restricted analogue.

p38alpha Mitogen Activated Protein Kinase (MAP kinase) is an intracellular soluble serine threonine kinase. p38alpha kinase is activated in response to cellular stresses, growth factors and cytokines such as interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha). The central role of p38alpha activation in settings of both chronic and acute inflammation has led efforts to find inhibitors of this enzyme as possible therapies for diseases such as rheumatoid arthritis, where p38alpha activation is thought to play a causal role. Herein, we report structure-activity relationship studies on a series of indole-based heterocyclic inhibitors that led to the design and identification of a new class of p38alpha inhibitors.

P38 mitogen-activated protein kinase is activated and linked to TNF-alpha signaling in inflammatory bowel disease.

Inflammatory bowel diseases (IBD)--Crohn's disease and ulcerative colitis--are relapsing chronic inflammatory disorders which involve genetic, immunological, and environmental factors. The regulation of TNF-alpha, a key mediator in the inflammatory process in IBD, is interconnected with mitogen-activated protein kinase pathways. The aim of this study was to characterize the activity and expression of the four p38 subtypes (p38alpha-delta), c-Jun N-terminal kinases (JNKs), and the extracellular signal-regulated kinases (ERK)1/2 in the inflamed intestinal mucosa. Western blot analysis revealed that p38alpha, JNKs, and ERK1/2 were significantly activated in IBD, with p38alpha showing the most pronounced increase in kinase activity. Protein expression of p38 and JNK was only moderately altered in IBD patients compared with normal controls, whereas ERK1/2 protein was significantly down-regulated. Immunohistochemical analysis of inflamed mucosal biopsies localized the main expression of p38alpha to lamina propria macrophages and neutrophils. ELISA screening of the supernatants of Crohn's disease mucosal biopsy cultures showed that incubation with the p38 inhibitor SB 203580 significantly reduced secretion of TNF-alpha. In vivo inhibition of TNF-alpha by a single infusion of anti-TNF-alpha Ab (infliximab) resulted in a highly significant transient increase of p38alpha activity during the first 48 h after infusion. A significant infliximab-dependent p38alpha activation was also observed in THP-1 myelomonocytic cells. In human monocytes, infliximab enhanced TNF-alpha gene expression, which could be inhibited by SB 203580. In conclusion, p38alpha signaling is involved in the pathophysiology of IBD.

The p38-MAPK/SAPK Pathway is Required for Human Keratinocyte Migration on Dermal Collagen

Human keratinocyte motility plays an important role in the re-epithelialization of human skin wounds. The wound bed over which human keratinocytes migrate is rich in extracellular matrices, such as fibrin, fibronectin, and collagen, and serum factors, such as platelet-derived growth factor and transforming growth factor beta 1. Extracellular matrices and the serum factors bind to cell surface receptors and initiate a cascade of intracellular signaling events that regulate cell migration. In this study, we identified an intracellular signaling pathway that mediates collagen- driven motility of human keratinocytes. Pharmaco logic inhibition of the activation of p38-alpha and p38-beta mitogen-activated protein kinase activation potently blocked collagen-driven human keratinocyte migration. Transfection of the same keratinocytes with the kinase-negative mutants of p38-alpha or p38-beta mitogen-activated protein kinase markedly inhibited keratinocyte migration on collagen. Attachment of keratinocytes to collagen activated p38 mitogen- activated protein kinase, as well as p44/p42 ERKs. Interestingly, activation of the p38 mitogen-activated protein kinase cascade by overexpressing the constitutively active MKK3 and MKK6, MKK3b(E) and MKK6b(E), could neither initiate migration in the absence of collagen nor enhance collagen-driven migration. This study provides evidence that the p38-MAPK/SAPK pathway is necessary, but insufficient, for mediating human keratinocyte migration on collagen.

The role of differential activation of p38-mitogen-activated protein kinase in preconditioned ventricular myocytes.

Activation of protein kinase C (PKC) and more recently mitogen-activated protein kinases (MAPKs) have been associated with the cardioprotective effect of ischemic preconditioning. We examined the interplay between these kinases in a characterized model of ischemic preconditioning in cultured rat neonatal ventricular cardiocytes where ectopic expression of active PKC-delta results in protection. Two members of the MAPK family, p38 and p42/44, were activated transiently during preconditioning by brief simulated ischemia/reoxygenation. Overexpression of active PKC-delta, rather than augmenting, completely abolished this activation. We therefore determined whether a similar process occurred during lethal prolonged simulated ischemia. In contrast to ischemia, brief, lethal-simulated ischemia activated only p38 (2.8+/-0.45 vs. basal, P<0.01), which was attenuated by expression of active PKC-delta or by preconditioning (0.48+/-0.1 vs. ischemia, P<0.01). To determine whether reduced p38 activation was the cause or an effect of protection, we used SB203580, a p38 inhibitor. SB203580 reduced ischemic injury (CK release 38.0+/-3.1%, LDH release 77.3+/-4.0%, and MTT bioreduction 127.1+/-4.8% of control, n=20, P<0.05). To determine whether p38 activation was isoform selective, myocytes were infected with adenoviruses encoding wild-type p38alpha or p38beta. Transfected p38alpha and beta show differential activation (P<0.001) during sustained simulated ischemia, with p38alpha remaining activated (1.48+/-0.36 vs. basal) but p38beta deactivated (0.36+/-0.1 vs. basal, P<0.01). Prior preconditioning prevented the activation of p38alpha (0.65+/-0.11 vs. ischemia, P<0.05). Moreover, cells expressing a dominant negative p38alpha, which prevented ischemic p38 activation, were resistant to lethal simulated ischemia (CK release 82.9+/-3.9% and MTT bioreduction 130.2+/-6.5% of control, n=8, P<0.05). Thus, inhibition of p38alpha activation during ischemia reduces injury and may contribute to preconditioning-induced cardioprotection in this model.