Role Of P38α Research Articles

Uncoupling p38α nuclear and cytoplasmic functions and identification of two p38α phosphorylation sites on β-catenin: implications for the Wnt signaling pathway in CRC models

BackgroundActivation of the Wnt pathway has been linked to colorectal cancer (CRC). Previous reports suggest that Wnt3a can activate p38. Besides, p38α feeds into the canonical Wnt/β-catenin pathway by inhibiting GSK3β through phosphorylation. Recently, we identified p38α as a new druggable member of β-catenin chromatin-associated kinase complexes in CRC.MethodsThe functional relationship between p38α and β-catenin was characterized in CRC cells, patient-derived CRC stem cells, patient-derived tumor intestinal organoids, and in vivo models (C57BL/6-APCMin/+ mice). The role of p38α in β-catenin transcriptional activity was assessed by pharmacological inhibition with ralimetinib.ResultsWe used the GSK3β inhibitor TWS-119, which promotes the activation of Wnt signaling, to uncouple p38α nuclear/cytoplasmatic functions in the Wnt pathway. Upon GSK3β inhibition, nuclear p38α phosphorylates β-catenin at residues S111 and T112, allowing its binding to promoter regions of Wnt target genes and the activation of a transcriptional program implicated in cancer progression. If p38α is pharmacologically inhibited in addition to GSK3β, β-catenin is prevented from promoting target gene transcription, which is expected to impair carcinogenesis.Conclusionsp38α seems to play a dual role as a member of the β-catenin destruction complex and as a β-catenin chromatin-associated kinase in CRC. This finding may help elucidate mechanisms contributing to human colon tumor pathogenesis and devise new strategies for personalized CRC treatment.

Characterization of p38α Signaling Networks in Cancer Cells Using Quantitative Proteomics and Phosphoproteomics

p38α (encoded by MAPK14) is a protein kinase that regulates cellular responses to almost all types of environmental and intracellular stresses. Upon activation, p38α phosphorylates many substrates both in the cytoplasm and nucleus, allowing this pathway to regulate a wide variety of cellular processes. While the role of p38α in the stress response has been widely investigated, its implication in cell homeostasis is less understood. To investigate the signaling networks regulated by p38α in proliferating cancer cells, we performed quantitative proteomic and phosphoproteomic analyses in breast cancer cells in which this pathway had been either genetically targeted or chemically inhibited. Our study identified with high confidence 35 proteins and 82 phosphoproteins (114 phosphosites) that are modulated by p38α and highlighted the implication of various protein kinases, including MK2 and mTOR, in the p38α-regulated signaling networks. Moreover, functional analyses revealed an important contribution of p38α to the regulation of cell adhesion, DNA replication, and RNA metabolism. Indeed, we provide experimental evidence supporting that p38α facilitates cancer cell adhesion and showed that this p38α function is likely mediated by the modulation of the adaptor protein ArgBP2. Collectively, our results illustrate the complexity of the p38α-regulated signaling networks, provide valuable information on p38α-dependent phosphorylation events in cancer cells, and document a mechanism by which p38α can regulate cell adhesion.

Roles of p38α and p38β mitogen‑activated protein kinase isoforms in human malignant melanoma A375 cells.

Skin cancer is one of the most common cancers worldwide. Melanoma accounts for ~5% of skin cancers but causes the large majority of skin cancer-related deaths. Recent discoveries have shown that the mitogen-activated protein kinase (MAPK) signaling pathway is critical for melanoma development and progression. Many oncogenic pathways that cause melanoma tumorigenesis have been identified, most of which are due to RAF/MEK/ERK (MAPK) pathway activation. However, the precise role of p38 remains unclear. Using specific short hairpin (sh) RNA to silence p38α and p38β, the present findings demonstrated that p38α was a crucial factor in regulating cell migration in the A375 melanoma cell line. Silencing p38α downregulated the expression of epithelial-mesenchymal transition markers, such as matrix metallopeptidase (MMP) 2, MMP9, twist family bHLH transcription factor 1, snail family transcriptional repressor 1 and vimentin, while mesenchymal-epithelial transition markers, such as E-cadherin, were upregulated. Of note, the results also demonstrated that p38α silencing impaired vascular endothelial growth factor expression, which regulates tumor angiogenesis. Furthermore, p38α knockdown inhibited cell proliferation in melanoma cells. In addition, silencing p38α induced senescence-like features, but not cell cycle arrest. Expression of the senescence markers p16, p21, p53 and β-galactosidase was upregulated, and an increase in the number of senescence-associated β-galactosidase-positive cells was observed in a p38α knockdown stable clone. However, no significant difference was found between control and p38β stable knockdown cells. Taken together, the present results suggested that p38α knockdown impaired migration and proliferation, and increased senescence, in A375 melanoma cells. However, p38β may not be involved in melanoma tumorigenesis. Therefore, targeting p38α may be a valuable approach towards inhibiting tumor growth and metastasis in patients with melanoma.

Abstract 22: Apoptosis Signal-Regulating Kinase 1 (ASK1) is a Novel Master Molecular Switch Controlling Microglia/Macrophage Reactions That Impact Short- and Long-Term Stroke Outcomes

Introduction: Recent studies suggest that the functional status of microglia/macrophages (Mi/MΦ) profoundly impacts stroke outcome. However, the molecular mechanisms regulating Mi/MΦ reactions after brain ischemia/reperfusion (I/R) are poorly understood. Hypothesis: Activation of ASK1, a key MAPKKK upstream of multiple inflammation-regulating pathways, critically contributes to I/R brain injury via priming Mi/MΦ towards a neurotoxic phenotype. Methods: We generated mice with cell-specific, tamoxifen-induced knockout of ASK1 in neurons (nKO) or Mi/MΦ (mKO). Brain injury, neuroinflammation, and various behavioral deficits were assessed after 1 h MCAO and reperfusion. Results: MCAO induced transient ASK1-MKK4-JNK3 activation (6-24 h) in neurons and persistent ASK1-MKK3-p38/STAT1 activation (3-5 d) in Mi/MΦ in WT, but not in ASK1 nKO or mKO mice, respectively. ASK1 nKO reduced infarct sizes by 28.5% at 2 d after MCAO (p<0.01 vs WT, n=8-10/group), but failed to prevent the release of HMGB1 from necrotic cells and Mi/MΦ activation, or exert any effect on long term (28 d) stroke outcomes. In contrast to neuronal ASK1 KO, ASK1 mKO markedly improved both short and long term sensorimotor/cognitive functions after I/R (p<0.01 vs WT, n=14-16/group). ASK1 mKO attenuated the activation of p38α and STAT1 (immunostaining), inhibited the expression of 12 proinflammatory markers in Mi/MΦ (FACS-sorting followed by qPCR; p<0.05, n=4/group), and mitigated brain inflammation (ELISA array; 19 out of 80 markers, p<0.05, n=5/group) 3 d after I/R. In consistent with the role of p38α and STAT1 as downstream mediators of ASK1 signaling in Mi/MΦ, either p38α or STAT1 mKO alleviated neuroinflammation (3 d) and improved long term (28 d) stroke outcomes (n=12/group), partially mimicking the effects of ASK1 mKO. Conclusions: ASK1-dependent activation of p38α and STAT1 promotes inflammation, I/R brain injury, and maladaptive behavior by priming Mi/MΦ towards a neurotoxic phenotype.

Genetic and Pharmacological Inhibition of p38α Improves Locomotor Recovery after Spinal Cord Injury

One of the mitogen-activated protein kinases, p38α plays a crucial role in various inflammatory diseases and apoptosis of various types of cells. In this study, we investigated the pathophysiological roles of p38α in spinal cord injury (SCI), using a mouse model. Lateral hemisection at T9 of the SC was performed in wild type (WT) and p38α+/- mice (p38α-/- showed embryonic lethality). p38α+/- mice showed a better functional recovery from SCI-associated paralyzed hindlimbs compared to WT mice at 7 days post-injury (dpi), which remained until 28 dpi (an end time point of monitoring the behavior). In histopathological analysis at 28 dpi, there was more axonal regeneration with remyelination on the caudal side of the lesion epicenter in p38α+/- mice than in WT mice. At 7 dpi, infiltration of inflammatory cells into the lesion and expression of cytokines in the lesion were reduced in p38α+/- mice compared with WT mice. At the same time point, the number of apoptotic oligodendrocytes in the white matter at the caudal boarder of the lesion of p38α+/- mice was lower than that of WT mice. At 14 dpi, more neural and oligodendrocyte precursor cells in the gray matter and white matter, respectively, were observed around the lesion epicenter of p38α+/- mice compared with the case of WT mice. At the same time point, astrocytic scar formation was less apparent in p38α+/- than in WT mice, while compaction of inflammatory immune cells associated with the wound contraction was more apparent in p38α+/- than in WT mice. Furthermore, we verified the effectiveness of oral administration of SB239063, a p38α inhibitor on the hindlimb locomotor recovery after SCI. These results suggest that p38α deeply contributes to the pathogenesis of SCI and that inhibition of p38α is a beneficial strategy to recovery from SCI.

P38α has an important role in antigen cross-presentation by dendritic cells.

The role of the p38 signaling pathway in the innate and adaptive immune responses has been well documented, especially in inflammatory cytokine production by dendritic cells (DCs). However, whether the p38 signaling pathway affects the important antigen (Ag) presentation function of DCs remains largely unknown. In this study, we reported that the deletion of p38α resulted in an impaired cross-presentation ability of CD8+ conventional DCs (cDCs) and a reduction in the direct presentation ability of CD8- cDCs ex vivo. Further study revealed that p38α had a crucial role in Ag processing by CD8+ cDCs but did not affect the Ag uptake or co-stimulation of T cells. Moreover, p38α deficiency led to reduced cross-priming of T cells in vivo. The production of the IL-12p40 and IL-12p70 cytokines by p38α-deficient cDCs was also significantly reduced. Our study identified a new role for p38α in modulating the important antigen cross-presentation function of DCs.

3D-QSAR studies on disubstituted dibenzosuberone derivatives as p38α MAP kinase inhibitors using CoMFA and COMSIA

The p38 mitogen-activated protein kinases belong to the mitogen-activated protein kinase superfamily. They are strongly activated by different stress signals and inflammatory cytokines. p38α mitogen-activated protein kinase is broadly expressed in most cell types and plays an important role in the coordination of the cellular responses such as proliferation, differentiation & survival. The role of p38α in various cancers and cellular functions makes it an interesting kinase drug target. In the present study, a series of recently published disubstituted dibenzosuberones derivatives were taken as p38α inhibitors and three-dimensional quantitative structure-activity relationship study was performed. A reasonable Comparative Molecular Field Analysis (q2 = 0.689, NOC = 6, r2 = 0.942) and Comparative Molecular Similarity Indices Analysis (q2 = 0.645, NOC = 6, r2 = 0.921) models were developed. All the developed models were validated using external test set, bootstrapping, progressive sampling and rm2 metric calculations. The validation of models showed acceptable statistical values and proved to be predictive and robust. The contour map analysis revealed the important insight on favorable substituents to increase the potency of the compounds. Negative substitution near the oxygen atom of R1 position and hydrophobic & hydrogen bond acceptor groups near the phenyl ring of R1 position could enhance the activity. Likewise, positive hydrophobic substitution near the phenyl ring at R3 position favors the increase in the inhibitory activity. Our finding could be helpful to design a more potent derivative of p38α kinase inhibitors in future.Open image in new window

Roles of p38α mitogen-activated protein kinase in mouse models of inflammatory diseases and cancer.

The p38α mitogen‐activated protein kinase pathway not only regulates the production of inflammatory mediators, but also controls processes related to tissue homeostasis, such as cell proliferation, differentiation and survival, which are often disrupted during malignant transformation. The versatility of this signaling pathway allows for the regulation of many specific functions depending on the cell type and context. Here, we discuss mouse models that have been used to identify in vivo functions of p38α signaling in the pathogenesis of inflammatory diseases and cancer. Experiments using genetically modified mice and pharmacological inhibitors support that targeting the p38α pathway could be therapeutically useful for some inflammatory diseases and tumor types.

ELUCIDATING THE ROLE OF P38γ IN PATHOLOGICAL CARDIAC HYPERTROPHY

P38-mitogen activated protein kinases (of which there are 4 isoforms; α, β, γ and δ) are serine/threonine kinases that are activated in response to stress stimuli. Classically studies have only examined the role of p38α in the myocardium. However, p38γ is also highly and preferentially expressed in the myocardium. The aim of this study is to investigate the role of p38γ in the progression of pathological hypertrophy following abdominal aortic banding. Comparisons of cardiac function and structure of wild type (WT) and p38γ knock out (KO) mice in response to abdominal aortic banding found that KO mice develop less ventricular hypertrophy than their corresponding WT controls, and have preserved cardiac function. We observed that basal p38γ myocardial staining is primarily localised at the membranes and throughout the cytoplasm. Following aortic constriction nuclear staining of p38γ incre ases but no accumulation of p38α is observed. This suggests that the two isoforms play distinct roles in the heart. To elucidate its signaling pathway and identify endogenous substrates of p38γ we have generated an analogue sensitive p38γ, which is mutated at a gatekeeper residue, to specifically track endogenous substrates in the myocardium. The mutation allows only the mutant kinase, but not WT kinases, to utilise analogues of ATP that are expanded at the N-6 position and contain a visible tag on the γ-phosphate. Transfer of this tag to substrates allows subsequent isolation and identification. Using this technique we have been able to identify, amongst other substrates, Cypher and Calpastatin as novel targets of p38γ.

Muscle‐specific deletion of p38α/β MAPK improves glucose tolerance and reduces body fat but impairs exercise capacity

The p38 MAPK belong to the stress‐activated kinase family, and p38γ MAPK is vital to exercise‐induced PGC‐1α expression, mitochondrial biogenesis and angiogenesis in skeletal muscle. However, the roles of p38α and p38β in skeletal muscle remain unclear likely due to redundanct function. To determine the function of these isoforms in skeletal muscle, we generated muscle‐specific p38α/β double knockout mice (DMKO). We then subjected these mice to 12 weeks of 45% high‐fat diet (HFD). DMKO mice showed improved glucose tolerance compared with wild‐type (WT) littermates on normal chow and HFD. DMKO mice showed reductions in total body weight, and muscle and epididymal fat mass compared with WT mice, accompanied by increased energy expenditure at equivalent caloric consumption. DMKO mice showed reduced exercise capacity as assessed by treadmill running. These findings demonstrate protective effects of p38α/β MAPK deletion in skeletal muscle regarding the development of insulin resistance and gains in body weight and fat mass, associated with increased energy expenditure. However, p38α/β MAPK deletion impairs exercise capacity suggesting a critical role in muscle function that requires further examination.

P38 mitogen‐activated protein kinase signaling, ERCC1 expression, and viability of lung cancer cells from never or light smoker patients

Expression of DNA-repair proteins and activated mitogen-activated protein kinases (MAPKs) may differ according to smoking status. The authors investigated whether p38 MAPK activity contributed to the viability of cisplatin in lung cancer cell lines from never or light smokers and to ERCC1 mRNA expression. Activated p38 MAPK was tested as a predictor for ERCC1 levels in 117 lung adenocarcinomas. Cell viabilities of NCI-H1975, NCI-H1793, NCI-H1650, and NCI-H1651 cell lines, derived from never or light smokers, were measured after treatment with the p38 MAPK inhibitor SB202190 and cisplatin. The role of p38α (MAPK14) and p38β (MAPK11) isoforms and ERCC1 was evaluated using RNA interference. ERCC1 protein-level expression was predicted by activated p38 MAPK in lung adenocarcinoma tissues. The p38-specific inhibitor SB202190 strongly decreased cell viability (43%-63%). SB202190 plus cisplatin significantly decreased cell viability in every cell line, including cisplatin-resistant NCI-H1793. Genetic inhibition, targeting both MAPK11 and MAPK14, reduced the viability of the different cell lines: down-regulation of p38β accounted for most of this effect. Cisplatin's effect was greater after MAPK11 down-regulation for NCI-H1651, and MAPK14 down-regulation for NCI-H1650. In addition, both SB202190 and MAPK11 inhibition reduced excision repair cross-complementing 1 mRNA levels. Lung cancer cells from never or light smokers rely on p38 MAPK signaling for survival. MAPK11 is involved in that pathway and might contribute to ERCC1 expression. Sensitization to cisplatin can be achieved by pharmacological inhibition of p38 MAPK signaling.

Role of p38α in apoptosis: implication in cancer development and therapy

This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 2.0 France Licence.

Abstract 1015: P38 mitogen-activated protein kinase signaling and viability of lung cancer cells from never or light smoker patients

Abstract Purpose: Expression levels of DNA repair proteins and activated mitogen-activated protein kinases (MAPKs) may differ according to smoking status. We investigated whether P38 MAPK activity contributed to the viability and sensitivity to cisplatin of lung cancer cells lines from never or light smokers and to ERCC1 mRNA expression. Methods: Activated P38 MAPK was tested by means of immunohistochemistry as a predictor of ERCC1 levels in 117 lung adenocarcinomas. Cell viability of NCI-H1975, NCI-H1793, NCI-H1650 and NCI-H1651 was measured after treatment with the P38 MAPK inhibitor SB202190 and cisplatin. The role of P38α (MAPK14), P38β (MAPK11) isoforms and ERCC1 was evaluated using ARN interference. ERCC1 mRNA was measured by quantitative PCR. Results: ERCC1 protein expression was predicted by activated P38 MAPK in lung adenocarcinoma tissues. The P38 specific inhibitor SB202190 induced a strong decrease (P<0.01) in cell viability of NCI-H1975 (43%) and NCI-H1650 (63%) compared to control. When SB202190 was combined with cisplatin, cell viability decreased (P<0.01) in every cell line including cisplatin resistant NCI-H1793. The combination of drugs reduced cell viability more than cisplatin (P<0.01) for NCI-H1793, NCI-H1650 and NCI-H1651, and it was synergistic for NCI-H1793 and NCI-H1651. Genetic inhibition targeting both MAPK11 and MAPK14 reduced the viability of every cell line (P<0.01) with a dose-response for three of them. The decrease in cell viability with genetic inhibition at 25 nM was higher (P<0.01) than with cisplatin for NCI-H1793 (67% vs. 35%) and NCI-H1650 (55% vs. 38%). In separate experiments, MAPK11 downregulation reduced viability of every cell line (P<0.01), while MAPK14 downregulation reduced viability of NCI-H1651. The effect of cisplatin was higher after MAPK11 downregulation for NCI-H1651 and after MAPK14 downregulation for NCI-H1650. In addition, both SB202190 and MAPK11 inhibition reduced excision repair cross-complementing 1 mRNA levels. Conclusion: lung cancer cells from never or light smokers rely on P38 MAPK signaling for their survival whether or not they harbor EGFR mutations. MAPK11 is involved in that pathway and might contribute to ERCC1 expression. Sensitization to cisplatin can be achieved by pharmacological inhibition of P38 MAPK signaling in some lung cancer models from never smokers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1015. doi:10.1158/1538-7445.AM2011-1015

Cell Density-Dependent Inhibition of Epidermal Growth Factor Receptor Signaling by p38α Mitogen-Activated Protein Kinase via Sprouty2 Downregulation

Contact inhibition is a fundamental process in multicellular organisms aimed at inhibiting proliferation at high cellular densities through poorly characterized intracellular signals, despite availability of growth factors. We have previously identified the protein kinase p38alpha as a novel regulator of contact inhibition, as p38alpha is activated upon cell-cell contacts and p38alpha-deficient cells are impaired in both confluence-induced proliferation arrest and p27(Kip1) accumulation. Here, we establish that p27(Kip1) plays a key role downstream of p38alpha to arrest proliferation at high cellular densities. Surprisingly, p38alpha does not directly regulate p27(Kip1) expression levels but leads indirectly to confluent upregulation of p27(Kip1) and cell cycle arrest via the inhibition of mitogenic signals originating from the epidermal growth factor receptor (EGFR). Hence, confluent activation of p38alpha uncouples cell proliferation from mitogenic stimulation by inducing EGFR degradation through downregulation of the EGFR-stabilizing protein Sprouty2 (Spry2). Accordingly, confluent p38alpha-deficient cells fail to downregulate Spry2, providing them in turn with sustained EGFR signaling that facilitates cell overgrowth and oncogenic transformation. Our results provide novel mechanistic insight into the role of p38alpha as a sensor of cell density, which induces confluent cell cycle arrest via the Spry2-EGFR-p27(Kip1) network.

Adenosine induces endothelial apoptosis by activating protein tyrosine phosphatase: a possible role of p38alpha.

Endothelial cell (EC) apoptosis is important in vascular injury, repair, and angiogenesis. Homocysteine and/or adenosine exposure of ECs causes apoptosis. Elevated homocysteine or adenosine occurs in disease states such as homocysteinuria and tissue necrosis, respectively. We examined the intracellular signaling mechanisms involved in this pathway of EC apoptosis. Inhibition of protein tyrosine phosphatase (PTPase) attenuated homocysteine- and/or adenosine-induced apoptosis and completely blocked apoptosis induced by the inhibition of S-adenosylhomocysteine hydrolase with MDL-28842. Consistent with this finding, the tyrosine kinase inhibitor genistein enhanced apoptosis in adenosine-treated ECs. Adenosine significantly elevated the PTPase activity in the ECs. Mitogen-activated protein kinase activities were examined to identify possible downstream targets for the upregulated PTPase(s). Extracellular signal-regulated kinase (ERK) 1 activity was slightly elevated in adenosine-treated ECs, whereas ERK2, c-Jun NH(2)-terminal kinase-1, or p38beta activities differed little. The mitogen-activated protein kinase-1 inhibitor PD-98059 enhanced DNA fragmentation, suggesting that increased ERK1 activity is a result but not a cause of apoptosis in adenosine-treated ECs. Adenosine-treated ECs had diminished p38alpha activity compared with control cells; this effect was blunted on PTPase inhibition. These results indicate that PTPase(s) plays an integral role in the induction of EC apoptosis upon exposure to homocysteine and/or adenosine, possibly by the attenuation of p38alpha activity.

Requirement for p38α in Erythropoietin Expression: A Role for Stress Kinases in Erythropoiesis

Activity of the p38α MAP kinase is stimulated by various stresses and hematopoietic growth factors. A role for p38α in mouse development and physiology was investigated by targeted disruption of the p38α locus. Whereas some p38α −/− embryos die between embryonic days 11.5 and 12.5, those that develop past this stage have normal morphology but are anemic owing to failed definitive erythropoiesis, caused by diminished erythropoietin (Epo) gene expression. As p38α-deficient hematopoietic stem cells reconstitute lethally irradiated hosts, p38α function is not required downstream of Epo receptor. Inhibition of p38 activity also interferes with stabilization of Epo mRNA in human hepatoma cells undergoing hypoxic stress. The p38α MAP kinase plays a critical role linking developmental and stress-induced erythropoiesis through regulation of Epo expression.