P38 MAPK-dependent Phosphorylation Research Articles

P38 MAPK Signaling in the Retina: Effects of Aging and Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is the leading cause of irreversible visual impairment worldwide. Age is the greatest risk factor for AMD but the underlying mechanism remains unascertained, resulting in a lack of effective therapies. Growing evidence shows that dysregulation of the p38 MAPK signaling pathway (SP) contributes to aging and neurodegenerative diseases; however, information about its alteration in the retina with age and during AMD development is limited. To assess the contribution of alterations in p38 MAPK signaling to AMD, we compared age-associated changes in p38 MAPK SP activity in the retina between Wistar rats (control) and OXYS rats, which develop AMD-like retinopathy spontaneously. We analyzed changes in the mRNA levels of genes of this SP in the retina (data of RNA-seq) and evaluated the phosphorylation/activation of key kinases using Western blotting at different stages of AMD-like pathology including the preclinical stage. p38 MAPK SP activity increased in the retinas of healthy Wistar rats with age. The manifestation and dramatic progression of AMD-like pathology in OXYS rats was accompanied by hyperphosphorylation of p38 MAPK and MK2 as key p38 MAPK SP kinases. Retinopathy progression co-occurred with the enhancement of p38 MAPK-dependent phosphorylation of CryaB at Ser59 in the retina.

P38 MAPK-dependent phosphorylation of TFEB promotes monocyte-to-macrophage differentiation.

The transcription factor EB (TFEB) regulates energy homeostasis and cellular response to a wide variety of stress conditions, including nutrient deprivation, oxidative stress, organelle damage, and pathogens. Here we identify S401 as a novel phosphorylation site within the TFEB proline-rich domain. Phosphorylation of S401 increases significantly in response to oxidative stress, UVC light, growth factors, and LPS, whereas this increase is prevented by p38 MAPK inhibition or depletion, revealing a new role for p38 MAPK in TFEB regulation. Mutation of S401 in THP1 cells demonstrates that the p38 MAPK/TFEB pathway plays a particularly relevant role during monocyte differentiation into macrophages. TFEB-S401A monocytes fail to upregulate the expression of multiple immune genes in response to PMA-induced differentiation, including critical cytokines, chemokines, and growth factors. Polarization of M0 macrophages into M1 inflammatory macrophages is also aberrant in TFEB-S401A cells. These results indicate that TFEB-S401 phosphorylation links differentiation signals to the transcriptional control of monocyte differentiation.

P38 MAPK-dependent phosphorylation of transcription factor SOX2 promotes an adaptive response to BRAF inhibitors in melanoma cells

Despite recent advances in the development of BRAF kinase inhibitors (BRAFi) for BRAF-mutant melanomas, development of resistance remains a major clinical problem. In addition to genetic alterations associated with intrinsic resistance, several adaptive response mechanisms are known to be rapidly activated to allow cell survival in response to treatment, limiting efficacy. A better understanding of the mechanisms driving resistance is urgently needed to improve the success of BRAF-targeted therapies and to make therapeutic intervention more durable. In this study, we identify the mitogen-activated protein kinase (MAPK) p38 as a novel mediator of the adaptive response of melanoma cells to BRAF-targeted therapy. Our findings demonstrate that BRAFi leads to an early increase in p38 activation, which promotes phosphorylation of the transcription factor SOX2 at Ser251, enhancing SOX2 stability, nuclear localization, and transcriptional activity. Furthermore, functional studies show that SOX2 depletion increases sensitivity of melanoma cells to BRAFi, whereas overexpression of a phosphomimetic SOX2-S251E mutant is sufficient to drive resistance and desensitize melanoma cells to BRAFi in vitro and in a zebrafish xenograft model. We also found that SOX2 phosphorylation at Ser251 confers resistance to BRAFi by binding to the promoter and increasing transcriptional activation of the ATP-binding cassette drug efflux transporter ABCG2. In summary, we unveil a p38/SOX2-mediated mechanism of adaptive response to BRAFi, which provides prosurvival signals to melanoma cells against the cytotoxic effects of BRAFi prior to acquiring resistance.

Neurotrophic factor GDNF regulates intestinal barrier function in inflammatory bowel disease.

Impaired intestinal epithelial barrier (IEB) function with loss of desmosomal junctional protein desmoglein 2 (DSG2) is a hallmark in the pathogenesis of inflammatory bowel disease (IBD). While previous studies have reported that glial cell line-derived neurotrophic factor (GDNF) promotes IEB function, the mechanisms are poorly understood. We hypothesized that GDNF is involved in the loss of DSG2, resulting in impaired IEB function as seen in IBD. In the inflamed intestine of patients with IBD, there was a decrease in GDNF concentrations accompanied by a loss of DSG2, changes of the intermediate filament system, and increased phosphorylation of p38 MAPK and cytokeratins. DSG2-deficient and RET-deficient Caco2 cells revealed that GDNF specifically recruits DSG2 to the cell borders, resulting in increased DSG2-mediated intercellular adhesion via the RET receptor. Challenge of Caco2 cells and enteroids with proinflammatory cytokines as well as dextran sulfate sodium-induced (DSS-induced) colitis in C57Bl/6 mice led to impaired IEB function with reduced DSG2 mediated by p38 MAPK-dependent phosphorylation of cytokeratins. GDNF blocked all inflammation-induced changes in the IEB. GDNF attenuates inflammation-induced impairment of IEB function caused by the loss of DSG2 through p38 MAPK-dependent phosphorylation of cytokeratin. The reduced GDNF in patients with IBD indicates a disease-relevant contribution to the development of IEB dysfunction.

P38 MAPK-dependent alphaB-crystallin phosphorylation in Alzheimer's disease-like pathology in OXYS rats.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder. Development of AD is closely related to alterations of proteostasis including the deposition of amyloid beta (Aβ) and hyperphosphorylated tau in the brain. Molecular chaperones such as αB-crystallin (CryaB) can prevent aggregation of proteins and enable their correct refolding. The p38 mitogen-activated protein kinase signaling (MAPK) pathway regulates CryaB activity through phosphorylation of CryaB. Here, we examined CryaB and phospho-(p-)Ser59-CryaB protein amounts in the prefrontal cortex of the senescence-accelerated OXYS rats at different stages of the development of AD-like pathology and in Wistar rats (control). We compared this result with the changes in the expression of genes involved in the p38 MAPK signaling pathway in the prefrontal cortex of both rat strains. Manifestation and progression of AD-like pathology in OXYS rats were accompanied by an increased level of detergent-insoluble p-Ser59-CryaB in the brain cortex, while the CryaB amount did not change. p-Ser59-CryaB was absent in the detergent-soluble protein fraction of the cortex in both rat strains. Immunostaining of brain slices revealed notable colocalization of p-Ser59-CryaB with Aβ in 18-month-old OXYS rats. According to RNA sequencing data, the development of AD-like pathology in OXYS rats is accompanied in the cortex by up- and downregulation of genes involved in p38 MAPK signaling. In general, we demonstrated that the manifestation AD-like pathology in OXYS rats occurs against the background of activation of p38 MAPK-dependent CryaB phosphorylation and alterations of the p38 MAPK signaling pathway. The increased p-Ser59-CryaB amount and its colocalization with Aβ can be considered a response to the accumulation of protein aggregates and may be an important part of an endogenous mechanism of AD development.

Taxol Induces Brk-dependent Prosurvival Phenotypes in TNBC Cells through an AhR/GR/HIF-driven Signaling Axis.

The metastatic cascade is a complex process that requires cancer cells to survive despite conditions of high physiologic stress. Previously, cooperation between the glucocorticoid receptor (GR) and hypoxia-inducible factors (HIF) was reported as a point of convergence for host and cellular stress signaling. These studies indicated p38 MAPK-dependent phosphorylation of GR on Ser134 and subsequent p-GR/HIF-dependent induction of breast tumor kinase (PTK6/Brk), as a mediator of aggressive cancer phenotypes. Herein, p-Ser134 GR was quantified in human primary breast tumors (n = 281) and the levels of p-GR were increased in triple-negative breast cancer (TNBC) relative to luminal breast cancer. Brk was robustly induced following exposure of TNBC model systems to chemotherapeutic agents (Taxol or 5-fluorouracil) and growth in suspension [ultra-low attachment (ULA)]. Notably, both Taxol and ULA resulted in upregulation of the Aryl hydrocarbon receptor (AhR), a known mediator of cancer prosurvival phenotypes. Mechanistically, AhR and GR copurified and following chemotherapy and ULA, these factors assembled at the Brk promoter and induced Brk expression in an HIF-dependent manner. Furthermore, Brk expression was upregulated in Taxol-resistant breast cancer (MCF-7) models. Ultimately, Brk was critical for TNBC cell proliferation and survival during Taxol treatment and in the context of ULA as well as for basal cancer cell migration, acquired biological phenotypes that enable cancer cells to successfully complete the metastatic cascade. These studies nominate AhR as a p-GR binding partner and reveal ways to target epigenetic events such as adaptive and stress-induced acquisition of cancer skill sets required for metastatic cancer spread.Implication: Breast cancer cells enlist intracellular stress response pathways that evade chemotherapy by increasing cancer cell survival and promoting migratory phenotypes. Mol Cancer Res; 16(11); 1761-72. ©2018 AACR.

Abstract 3457: Chemotherapy enables Brk/PTK6-dependent survival of triple-negative breast cancer cells via induction of an AhR/GR/HIF signaling axis

Abstract The metastatic cascade is a complex process that requires cancer cells to survive despite exposure to conditions of high physiologic stress. We previously showed that breast tumor kinase (Brk; also known as PTK6), a mediator of aggressive breast cancer phenotypes, is induced in breast cancer cells in response to a convergence of cellular and hormonal stress signals mediated by cross talk between hypoxia-inducible factors (HIFs) and glucocorticoid receptors (GR). Specifically, p38-MAPK dependent phosphorylation of GR-Ser134 (p-GR) and p-GR/HIF transcriptional complexes mediated heightened Brk gene expression in response to multiple inputs to stress pathway activation. Following studies in mice to demonstrate that Brk is a GR target gene in the mammary gland, immunohistochemistry (IHC) was performed on human primary breast tumor tissues using total and p-GR antibodies. To model stress-induced p-GR action relevant to tumor progression, molecular markers of stress signaling were measured in triple negative breast cancer (TNBC) cell lines treated with chemotherapy (Taxol and 5-flourouricil) and following growth in suspension (ultra-low attachment (ULA)). Co-immunoprecipitation and ChIP assays were used to demonstrate association of p-GR with novel co-regulatory factors in transcription complexes at the Brk promoter. Cell viability and migration assays were performed following Brk knock-out using CRISPR/Cas9 gene editing. Systemic Dex administration in mice confirmed that Brk is a GR target gene in vivo. In human breast tumor samples, phospho-GR was significantly associated with TNBC relative to luminal cancers. Chemotherapy and ULA induced activation of p38 MAPK, phosphorylation of GR, and upregulation of HIFs as well as the Aryl hydrocarbon receptor (AhR), a known mediator of cancer cell survival under cellular stress. Moreover, AhR and GR co-purified constitutively, and following chemotherapy or ULA culture, these factors assembled at the Brk promoter in a HIF-dependent manner. Brk induction was critical for TNBC cell survival during Taxol treatment or during ULA culture and for cancer cell migration, acquired biological phenotypes that enable cancer cells to successfully complete the metastatic cascade.These studies define AhR as a novel p-GR binding partner and show that increased p-GR/AhR and Brk expression drive a migratory phenotype relevant to TNBC progression. Strategies to target p-GR signaling may ameliorate stress-induced acquisition of aggressive cancer phenotypes required for metastatic cancer spread that are linked to high breast cancer patient mortality. Citation Format: Tarah M. Regan Anderson, Shihong Ma, Carlos J. Perez Kerkvliet, Taylor M. Helle, Raisa Krutilina, Ganesh V. Raj, John A. Cidlowski, Kathryn L. Schwertfeger, Tiffany N. Seagroves, Carol A. Lange. Chemotherapy enables Brk/PTK6-dependent survival of triple-negative breast cancer cells via induction of an AhR/GR/HIF signaling axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3457.

Loss of Drosha underlies dopaminergic neuron toxicity in models of Parkinson\u2019s disease

MiRNAs, a group of powerful modulator of gene expression, participate in multiple cellular processes under physiological and pathological conditions. Emerging evidence shows that Drosha, which controls the initial step in canonical miRNA biogenesis, is involved in modulating cell survival and death in models of several diseases. However, the role of Drosha in Parkinson’s disease (PD) has not been well established. Here, we show that the level of Drosha decreases in 6-OHDA-induced cellular and animal models of PD. 6-OHDA induced a p38 MAPK-dependent phosphorylation of Drosha. This triggered Drosha degradation. Enhancing the level of Drosha protected the dopaminergic (DA) neurons from 6-OHDA-induced toxicity in both in vitro and in vivo models of PD and alleviated the motor deficits of PD mice. These findings reveal that Drosha plays a critical role in the survival of DA neurons and suggest that stress-induced destabilization of Drosha may be part of the pathological process in PD.

Do neuroinflammatory pathways contribute to serotoninergic alterations in autism? p38alpha MAPK signaling drives perturbed serotonin clearance, receptor hypersensitivity, and social behavior deficits in the SERT Ala56 mouse

Autism Spectrum Disorder (ASD) is a prevalent neurodevelopmental disorder with no FDA approved medications that alleviate core symptoms. Aberrant proinflammatory cytokine signaling involving the interleukin-1 receptor and p38 MAPK have been reported in ASD, a disorder where 25% of subjects exhibit elevated serotonin (5-HT) blood levels (hyperserotonemia). In ASD subjects, we identified functional coding variation in the 5-HT transporter (SERT), mutations that elevate SERT activity and perturb p38 MAPK-dependent transporter regulation. Mice that express the most common of these variants, SERT Ala56, exhibit hyperserotonemia, elevated, p38 MAPK-dependent SERT phosphorylation, increased hippocampal 5-HT clearance, 5-HT receptor hypersensitivity, increased repetitive behavior and disrupted social interactions. Treatment of SERT Ala56 mice with the novel, selective, CNS penetrant, p38alpha MAPK inhibitor, MW150, or conditional elimination of p38alpha MAPK in 5-HT neurons, normalized 5-HT receptor hypersensitivity, elevated 5-HT clearance and social deficits. As p38alpha MAPK signaling supports the IL-1beta and TNF-alpha-induced activation of SERT, our findings suggest that proinflammatory, p38alpha MAPK activation may contribute to ASD risk via a perturbation of 5-HT signaling. Our studies also indicate that p38alpha MAPK may be a relevant target for the pharmacologic treatment of ASD. Funding: This work was supported by NIH grants MH078028, MH094527, NS007491 and U01AG043415. Additional support was provided by the Simons Foundation Autism Research Initiative and the PhRMA Foundation.

Mechanisms of corticosteroid insensitivity in COPD alveolar macrophages exposed to NTHi

BackgroundNon-typeable Haemophilus influenza (NTHi) infection is common in COPD. Corticosteroids can have limited therapeutic effects in COPD patients. NTHi causes corticosteroid insensitive cytokine production from COPD alveolar macrophages. We investigated the mechanisms by which NTHi causes corticosteroid insensitive inflammatory responses, and the effects of NTHi exposure on COPD macrophage polarisation.MethodAlveolar macrophages from COPD patients and controls were exposed to NTHi in conjunction with the corticosteroid dexamethasone and/or the p38 MAPK inhibitor BIRB-796. Cytokine release, GR phosphorylation and modulation and macrophage phenotype were analysed.ResultsDexamethasone significantly inhibited NTHi induced TNF-α, IL-6 and IL-10 from COPD macrophages but, CXCL8 was not suppressed.BIRB-796 combined with dexamethasone caused significantly greater inhibition of all cytokines than either drug alone (p < 0.05 all comparisons). NTHi caused phosphorylation of GR S226 reducing GR nuclear localisation, an effect regulated by p38 MAPK. NTHi altered macrophage polarisation by increasing IL-10 and decreasing CD36, CD206, CD163 and HLA-DR.ConclusionNTHi exposure causes p38 MAPK dependent GR phosphorylation associated with decreased GR function in COPD alveolar macrophages. Combining a p38 MAPK inhibitor with corticosteroids can enhance anti-inflammatory effects during NTHi exposure of COPD alveolar macrophages. NTHi causes macrophage polarisation that favours bacterial persistence.

OCT-4: a novel estrogen receptor-α collaborator that promotes tamoxifen resistance in breast cancer cells

Tamoxifen has shown great success in the treatment of breast cancer; however, long-term treatment can lead to acquired tamoxifen (TOT) resistance and relapse. TOT classically antagonizes estradiol (E2) -dependent breast cancer cell growth, but exerts partial agonist/antagonist behavior on gene expression. Although both E2 and TOT treatment of breast cancer cells results in recruitment of the estrogen receptor (ER) to common and distinct genomic sites, the mechanisms and proteins underlying TOT preferential recruitment of the ER remains poorly defined. To this end, we performed in silico motif-enrichment analyses within the ER-binding peaks in response to E2 or TOT, to identify factors that would specifically recruit ER to genomic binding sites in the presence of TOT as compared to E2. Intriguingly, we found Nkx3-1 and Oct-transcription factor homodimer motifs to be enriched in TOT preferential binding sites and confirmed the critical role of Oct-3/4 (aka Oct-4) in directing ER recruitment to TOT preferential genomic binding sites, by chromatin immunoprecipitation (ChIP) analyses. Further investigation revealed Oct-4 expression to be basally repressed by Nkx3-1 in MCF-7 cells and TOT treatment appeared to elevate Nkx3-1 degradation through a p38MAPK-dependent phosphorylation of the E3 ligase, Skp2 at serine-64 residue, as observed by quantitative mass-spectrometry analyses. Consistently, Oct-4 upon induction by phospho-Ser64-Skp2-mediated proteasomal degradation of Nkx3-1, participated in ER transcriptional complexes along with p38MAPK and Skp2 in a tamoxifen-dependent manner leading to TOT-dependent gene activation and cell proliferation of the TOT-resistant MCF-7-tamr breast cancer cells. Notably, Oct-4 levels were highly elevated in MCF-7-tamr cells, and appeared critical for their TOT sensitivity in cell proliferation assays. Furthermore, overexpression of Oct-4 enhanced tumor growth in the presence of tamoxifen in mice in vivo. Collectively, our work presents a novel mechanism for tamoxifen-specific gene activation by ER, secondary to its TOT preferential recruitment to genomic sites by specific activation of Oct-4, a phenomenon that appears to underlie tamoxifen resistance in breast cancer cells and in xenograft tumor models, and could be useful in designing therapeutic interventions to improve treatment outcome.

Modulation of radiation injury response in retinal endothelial cells by quinic acid derivative KZ-41 involves p38 MAPK.

Radiation-induced damage to the retina triggers leukostasis, retinal endothelial cell (REC) death, and subsequent hypoxia. Resultant ischemia leads to visual loss and compensatory retinal neovascularization (RNV). Using human RECs, we demonstrated that radiation induced leukocyte adhesion through mechanisms involving p38MAPK, p53, and ICAM-1 activation. Additional phenotypic changes included p38MAPK-dependent tyrosine phosphorylation of the focal adhesion scaffolding protein, paxillin (Tyr118). The quinic acid derivative KZ-41 lessened leukocyte adhesion and paxillin-dependent proliferation via inhibition of p38MAPK-p53-ICAM-1 signaling. Using the murine oxygen-induced retinopathy (OIR) model, we examined the effect of KZ-41 on pathologic RNV. Daily ocular application of a KZ-41-loaded nanoemulsion significantly reduced both the avascular and neovascular areas in harvested retinal flat mounts when compared to the contralateral eye receiving vehicle alone. Our data highlight the potential benefit of KZ-41 in reducing both the retinal ischemia and neovascularization provoked by genotoxic insults. Further research into how quinic acid derivatives target and mitigate inflammation is needed to fully appreciate their therapeutic potential for the treatment of inflammatory retinal vasculopathies.

Basal p38 Mitogen-Activated Protein Kinase Regulates Unliganded Glucocorticoid Receptor Function in Airway Smooth Muscle Cells

Like many steroid receptors, the glucocorticoid (GC) receptor (GR) is a phosphoprotein. Although there are multiple phosphorylation sites critical for GR transcriptional activity (i.e., serine [S]203, S211, and S226), their respective role in driving GR functions is highly cell specific. We have recently identified protein phosphatase 5 as an essential Ser/Thr phosphatase responsible for impairing GR function via S211 dephosphorylation in airway smooth muscle (ASM) cells. Because p38 mitogen-activated protein kinase (MAPK) directly phosphorylates GR in different cell types in a stimulus- and cell-dependent manner, we investigated the role of p38 MAPK on GR phosphorylation and function in ASM cells. Cells were transfected with 100 nM p38 MAPK small interfering RNA or 2 μg MAPK kinase 3 expression vector (a specific kinase that directly activates p38 MAPK) in the presence or absence of fluticasone (100 nM) and/or p38 MAPK pharmacological inhibitor SB203580. We found that p38 MAPK blockade positively regulates GR nuclear translocation and GR-dependent induction of the steroid-target gene GC-induced leucine zipper in a hormone-independent manner. We also found that p38 MAPK-dependent regulation of GR functions was associated with a differential action on GR phosphorylation at S203 and S211 residues. This study demonstrated that the inactive state of GR in resting conditions is not only ensured by the absence of the GC ligand but also by p38 MAPK-dependent phosphorylation of unliganded GR at specific residues, which appears to be important in determining the overall GC responsiveness of ASM cells.

86 : IFN-γ, ER-stress, autophagy and antimicrobial defense: A novel ATF6/C/EBP-β centric signaling pathway

The IFN family of cytokines operates a frontline defense against pathogens and neoplastic cells in vivo by controlling the expression of several genes. The Death-Associated Protein Kinase 1 (DAPK1), an IFN-g induced enzyme, controls cell cycle, apoptosis, autophagy, and tumor metastasis; and its expression is frequently down regulated in a number of human tumors. Previously, we have shown that transcription factor C/EBP-b is required for the basal and IFN-g-induced expression of DAPK1. Here, we show that ATF6, an ER stress-induced transcription factor, interacts with C/EBP-b in an IFN-stimulated manner and is obligatory for Dapk1 expression. IFN-stimulated proteolytic processing and p38 MAPK dependent phosphorylation of ATF6; and ERK1/2-mediated phosphorylation of C/EBP-b are necessary for these interactions. The activation of this pathway is independent from the IFN-induced JAK-STAT pathways. More importantly, IFN-g-failed to activate autophagy in cells lacking either ATF6 or C/EBP-b. Consistent with these observations, the Atf6-/- mice were highly susceptible to lethal bacterial infections compared to the wildtype mice. These studies, for the first time, not only unravel a novel IFN signaling pathway that controls cell growth, autophagy and antibacterial defense but also expand the role of ATF6 beyond ER-stress.

Skepinone-L, a Novel Potent and Highly Selective Inhibitor of p38 MAP Kinase, Effectively Impairs Platelet Activation and Thrombus Formation

Background/Aims: Platelets are critically important for primary haemostasis and the major players in thrombotic vascular occlusion. Platelets are activated by agonists, such as thrombin and collagen-related peptide as well as second-wave mediators including thromboxane A₂ via different intracellular signaling pathways resulting in degranulation, aggregation and thrombus formation. Platelet activation is paralleled by phosphorylation and activation of p38 MAPK. The limited specificity of hitherto known p38 MAPK inhibitors precluded safe conclusions on the precise role of p38 MAPK in the regulation of platelet function. The present study examined the impact of Skepinone-L, a novel and highly selective inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), on platelet activation and thrombus formation. Methods: Experiments were performed in freshly isolated human platelets. Protein phosphorylation was quantified by Western blotting, thromboxane B₂ synthesis by enzyme immunoassay, ATP release by ChronoLume luciferin assay, cytosolic Ca²⁺ concentration by Fura-2 fluorescence-measurements, platelet aggregation by a light transmissions measurement and in vitro thrombus formation by a flow chamber. Results: Skepinone-L (1 μM) virtually abrogated the phosphorylation of platelet p38 MAPK substrate Hsp27 following stimulation with CRP (1 μg/ml), thrombin (5 mU/ml) or thromboxane A₂ analogue U-46619 (1 μM). Furthermore, Skepinone-L significantly blunted activation-dependent platelet secretion and aggregation following threshold concentrations of CRP, thrombin and thromboxane A₂ analogue U-46619. Skepinone-L did not impair platelet Ca²⁺ signaling but prevented agonist-induced thromboxane A₂ synthesis through abrogation of p38 MAPK-dependent phosphorylation of platelet cytosolic phospholipase A₂ (cPLA₂). Skepinone-L further markedly blunted thrombus formation under low (500-s) and high (1700-s) arterial shear rates. Conclusions: The present study discloses a powerful inhibiting effect of p38 MAPK-blocker Skepinone-L on platelet activation and thrombus formation.

Autism gene variant causes hyperserotonemia, serotonin receptor hypersensitivity, social impairment and repetitive behavior

Fifty years ago, increased whole-blood serotonin levels, or hyperserotonemia, first linked disrupted 5-HT homeostasis to Autism Spectrum Disorders (ASDs). The 5-HT transporter (SERT) gene (SLC6A4) has been associated with whole blood 5-HT levels and ASD susceptibility. Previously, we identified multiple gain-of-function SERT coding variants in children with ASD. Here we establish that transgenic mice expressing the most common of these variants, SERT Ala56, exhibit elevated, p38 MAPK-dependent transporter phosphorylation, enhanced 5-HT clearance rates and hyperserotonemia. These effects are accompanied by altered basal firing of raphe 5-HT neurons, as well as 5HT(1A) and 5HT(2A) receptor hypersensitivity. Strikingly, SERT Ala56 mice display alterations in social function, communication, and repetitive behavior. Our efforts provide strong support for the hypothesis that altered 5-HT homeostasis can impact risk for ASD traits and provide a model with construct and face validity that can support further analysis of ASD mechanisms and potentially novel treatments.

Osmotic Stress Inhibits Proteasome by p38 MAPK-dependent Phosphorylation

Osmotic stress causes profound perturbations of cell functions. Although the adaptive responses required for cell survival upon osmotic stress are being unraveled, little is known about the effects of osmotic stress on ubiquitin-dependent proteolysis. We now report that hyperosmotic stress inhibits proteasome activity by activating p38 MAPK. Osmotic stress increased the level of polyubiquitinated proteins in the cell. The selective p38 inhibitor SB202190 decreased osmotic stress-associated accumulation of polyubiquitinated proteins, indicating that p38 MAPK plays an inhibitory role in the ubiquitin proteasome system. Activated p38 MAPK stabilized various substrates of the proteasome and increased polyubiquitinated proteins. Proteasome preparations purified from cells expressing activated p38 MAPK had substantially lower peptidase activities than control proteasome samples. Proteasome phosphorylation sites dependent on p38 were identified by measuring changes in the extent of proteasome phosphorylation in response to p38 MAPK activation. The residue Thr-273 of Rpn2 is the major phosphorylation site affected by p38 MAPK. The mutation T273A in Rpn2 blocked the proteasome inhibition that is mediated by p38 MAPK. These results suggest that p38 MAPK negatively regulates the proteasome activity by phosphorylating Thr-273 of Rpn2.

Post-transcriptional regulation of TNF-induced expression of ICAM-1 and IL-8 in human lung microvascular endothelial cells: An obligatory role for the p38 MAPK-MK2 pathway dissociated with HSP27

The tumor necrosis factor-alpha (TNF)-induced inflammatory response in human lung microvascular endothelial cells (MVECs) is an early event in acute lung injury. Studies have shown that p38 mitogen-activated protein kinase (MAPK), MAPK-activated protein kinase 2 (MK2) and heat shock protein 27 (HSP27) are involved in the expression of pro-inflammatory mediators in other cell types. However, their role in the TNF-induced inflammatory response in lung MVECs has not been determined. We evaluated the role of p38 MAPK, MK2 and HSP27 in regulating the TNF-induced expression of ICAM-1 and IL-8 in human lung MVECs. Inhibition of p38 MAPK reduced ICAM-1 and IL-8 expression without influencing NF-κB activation or ICAM-1 and IL-8 mRNA levels. TNF stimulation induced p38 MAPK-dependent phosphorylation of MK2 and HSP27. MK2 silencing reduced ICAM-1 and IL-8 expression without influencing NF-κB activation or ICAM-1 and IL-8 mRNA levels. HSP27 silencing reduced cellular HSP27 levels and HSP27 phosphorylation following TNF stimulation but had no effect on ICAM-1 and IL-8 expression. Our study demonstrates for the first time that MK2 mediates post-transcriptional regulation by p38 MAPK of the TNF-induced expression of ICAM-1 and IL-8 in human lung MVECs, and that this regulation by the p38 MAPK/MK2 pathway is dissociated from HSP27 phosphorylation.

Rap1 Signal Controls B Cell Receptor Repertoire and Generation of Self-Reactive B1a Cells

We previously reported that the mice deficient for SPA-1, a Rap1 GTPase-activating protein, developed hematopoietic stem cell disorders. Here, we demonstrate that SPA-1(-/-) mice show an age-dependent increase in B220(high) B1a cells producing anti-dsDNA antibody and lupus-like nephritis. SPA-1(-/-) peritoneal B1 cells revealed the altered Vkappa gene repertoire, including skewed Vkappa4 usage and the significant Igkappa/Iglambda isotype inclusion indicative of extensive receptor editing. Rap1GTP induced OcaB gene activation via p38MAPK-dependent Creb phosphorylation, and consistently, SPA-1(-/-) immature BM B cells showing high Rap1GTP exhibited the augmented expression of OcaB and Vkappa4 genes. SPA-1(-/-) BM cells could transfer the autoimmunity in association with the generation of peritoneal B220(high) B1a cells in Rag-2(-/-) recipients. Finally, a portion of SPA-1(-/-) mice developed B1 cell leukemia with hemolytic autoantibody. Present results suggest that the regulated Rap1 signal in the immature B cells plays a role in modifying the B cell receptor repertoire and in maintaining the self-tolerance.

P38MAPK-dependent phosphorylation and degradation of SRC-3/AIB1 and RARα-mediated transcription

Nuclear retinoic acid (RA) receptors (RARs) activate gene expression through dynamic interactions with coregulators in coordination with the ligand and phosphorylation processes. Here we show that during RA-dependent activation of the RARalpha isotype, the p160 coactivator pCIP/ACTR/AIB-1/RAC-3/TRAM-1/SRC-3 is phosphorylated by p38MAPK. SRC-3 phosphorylation has been correlated to an initial facilitation of RARalpha-target genes activation, via the control of the dynamics of the interactions of the coactivator with RARalpha. Then, phosphorylation inhibits transcription via promoting the degradation of SRC-3. In line with this, inhibition of p38MAPK markedly enhances RARalpha-mediated transcription and RA-dependent induction of cell differentiation. SRC-3 phosphorylation and degradation occur only within the context of RARalpha complexes, suggesting that the RAR isotype defines a phosphorylation code through dictating the accessibility of the coactivator to p38MAPK. We propose a model in which RARalpha transcriptional activity is regulated by SRC-3 through coordinated events that are fine-tuned by RA and p38MAPK.