Ubiquitin plays an atypical role in GPCR-induced p38 MAP kinase activation on endosomes.

Protease-activated receptor 1 (PAR1) is a G protein-coupled receptor (GPCR) for thrombin and promotes inflammatory responses through multiple pathways including p38 mitogen-activated protein kinase signaling. The mechanisms that govern PAR1-induced p38 activation remain unclear. Here, we define an atypical ubiquitin-dependent pathway for p38 activation used by PAR1 that regulates endothelial barrier permeability. Activated PAR1 K63-linked ubiquitination is mediated by the NEDD4-2 E3 ubiquitin ligase and initiated recruitment of transforming growth factor-β-activated protein kinase-1 binding protein-2 (TAB2). The ubiquitin-binding domain of TAB2 was essential for recruitment to PAR1-containing endosomes. TAB2 associated with TAB1, which induced p38 activation independent of MKK3 and MKK6. The P2Y1 purinergic GPCR also stimulated p38 activation via NEDD4-2-mediated ubiquitination and TAB1-TAB2. TAB1-TAB2-dependent p38 activation was critical for PAR1-promoted endothelial barrier permeability in vitro, and p38 signaling was required for PAR1-induced vascular leakage in vivo. These studies define an atypical ubiquitin-mediated signaling pathway used by a subset of GPCRs that regulates endosomal p38 signaling and endothelial barrier disruption

Quantification of p38/synaptophysin in highly purified adrenal medullary chromaffin vesicles

p38/synaptophysin is a membrane protein present in clear (synaptic) vesicles of neurons and endocrine ceHs [1-4]. From the amino acid sequence deduced from cDNAs encoding p38/synaptophysin, a model with several membrane spanning polypeptide segments and a carboxy-terminal protein domain exposed to the cytoplasmic surface has been constructed [5-7]. The function of p38/synaptophysin is not known. It has been suggested to form a transmembrane channel for ions, or to interact with cytoplasmic factors via its cytoplasmic domain [7]. Since synaptophysin binds Ca2 +, it may also play a role in the release of neurotransmitters stored in clear (synaptic) vesicles [3]. Recently it has been reported [8] that p38/synaptophysin also occurs in hormone containing large dense core vesicles. This would imply that p38/synaptophysin could fulfill similar functions as described above in chromaffin and other secretory ceHs containing large dense core vesicles. In dear (synaptic) vesicles p38/synaptophysin constitutes 7.51Jfo of the vesicle membrane proteins [I]. The amount of p38/synaptophysin in large dense core vesides is not known. Here we report on the quantification of p38/synaptophysin in highly purified chromaffin secretory veside

Heat Shock Protein 70 Protects the Heart from Ischemia/Reperfusion Injury through Inhibition of p38 MAPK Signaling.

BackgroundHeat shock protein 70 (Hsp70) has been shown to exert cardioprotection. Intracellular calcium ([Ca2+]i) overload induced by p38 mitogen-activated protein kinase (p38 MAPK) activation contributes to cardiac ischemia/reperfusion (I/R) injury. However, whether Hsp70 interacts with p38 MAPK signaling is unclear. Therefore, this study investigated the regulation of p38 MAPK by Hsp70 in I/R-induced cardiac injury.MethodsNeonatal rat cardiomyocytes were subjected to oxygen-glucose deprivation for 6 h followed by 2 h reoxygenation (OGD/R), and rats underwent left anterior artery ligation for 30 min followed by 30 min of reperfusion. The p38 MAPK inhibitor (SB203580), Hsp70 inhibitor (Quercetin), and Hsp70 short hairpin RNA (shRNA) were used prior to OGD/R or I/R. Cell viability, lactate dehydrogenase (LDH) release, serum cardiac troponin I (cTnI), [Ca2+]i levels, cell apoptosis, myocardial infarct size, mRNA level of IL-1β and IL-6, and protein expression of Hsp70, phosphorylated p38 MAPK (p-p38 MAPK), sarcoplasmic/endoplasmic reticulum Ca2+-ATPase2 (SERCA2), phosphorylated signal transducer and activator of transcription3 (p-STAT3), and cleaved caspase3 were assessed.ResultsPretreatment with a p38 MAPK inhibitor, SB203580, significantly attenuated OGD/R-induced cell injury or I/R-induced myocardial injury, as evidenced by improved cell viability and lower LDH release, resulted in lower serum cTnI and myocardial infarct size, alleviation of [Ca2+]i overload and cell apoptosis, inhibition of IL-1β and IL-6, and modulation of protein expressions of p-p38 MAPK, SERCA2, p-STAT3, and cleaved-caspase3. Knockdown of Hsp70 by shRNA exacerbated OGD/R-induced cell injury, which was effectively abolished by SB203580. Moreover, inhibition of Hsp70 by quercetin enhanced I/R-induced myocardial injury, while SB203580 pretreatment reversed the harmful effects caused by quercetin.ConclusionsInhibition of Hsp70 aggravates [Ca2+]i overload, inflammation, and apoptosis through regulating p38 MAPK signaling during cardiac I/R injury, which may help provide novel insight into cardioprotective strategies

CXCL12-induced neurotoxicity critically depends on NMDA receptor-gated and L-type Ca2+ channels upstream of p38 MAPK.

BackgroundThe chemokine receptor CXCR4 (CD184) and its natural ligand CXCL12 contribute to many physiological processes, including decisions about cell death and survival in the central nervous system. In addition, CXCR4 is a co-receptor for human immunodeficiency virus (HIV)-1 and mediates the neurotoxicity of the viral envelope protein gp120. However, we previously observed that CXCL12 also causes toxicity in cerebrocortical neurons but the cellular mechanism remained incompletely defined.MethodsPrimary neuronal-glial cerebrocortical cell cultures from rat were exposed to a neurotoxicity-inducing CXCL12 concentration for different times and the activity of the stress-associated mitogen-activated protein kinase p38 (p38 MAPK) was assessed using an in vitro kinase assay. Neurotoxicity of CXCL12 and cellular localization of p38 MAPK was analyzed by immunofluorescence microscopy. Pharmacological inhibition of NMDA-type glutamate receptor-gated ion channels (NMDAR) of L-type Ca2+ channels was employed during 12- and 24-h exposure to neurotoxic amounts of CXCL12 to study the effects on active p38 MAPK and neuronal survival by Western blotting and microscopy, respectively. Neurotoxicity of CXCL12 was also assessed during pharmacological inhibition of p38 MAPK.ResultsHere, we show that a neurotoxic amount of CXCL12 triggers a significant increase of endogenous p38 MAPK activity in cerebrocortical cells. Immunofluorescence and Western blotting experiments with mixed neuronal-glial and neuron-depleted glial cerebrocortical cells revealed that the majority of active/phosphorylated p38 MAPK was located in neurons. Blockade of NMDAR-gated ion channels or L-type Ca2+ channels both abrogated an increase of active p38 MAPK and toxicity of CXCL12 in cerebrocortical neurons. Inhibition of L-type Ca2+ channels with nimodipine kept the active kinase at levels not significantly different from baseline while blocking NMDAR with MK-801 strongly reduced phosphorylated p38 MAPK below baseline. Finally, we confirmed that directly blocking p38 MAPK also abrogated neurotoxicity of CXCL12.ConclusionsOur findings link CXCL12-induced neuronal death to the regulation of NMDAR-gated ion channels and L-type Ca2+ channels upstream of p38 MAPK activation

The role of p38 MAPK and its substrates in neuronal plasticity and neurodegenerative disease

A significant amount of evidence suggests that the p38-mitogen-activated protein kinase (MAPK) signalling cascade plays a crucial role in synaptic plasticity and in neurodegenerative diseases. In this review we will discuss the cellular localisation and activation of p38 MAPK and the recent advances on the molecular and cellular mechanisms of its substrates: MAPKAPK 2 (MK2) and tau protein. In particular we will focus our attention on the understanding of the p38 MAPK-MK2 and p38 MAPK-tau activation axis in controlling neuroinflammation, actin remodelling and tau hyperphosphorylation, processes that are thought to be involved in normal ageing as well as in neurodegenerative diseases. We will also give some insight into how elucidating the precise role of p38 MAPK-MK2 and p38 MAPK-tau signalling cascades may help to identify novel therapeutic targets to slow down the symptoms observed in neurodegenerative diseases such as Alzheimer's and Parkinson's disease

Role of p38 MAPK inhibitor in conditioned fear

p38 mitogen activated protein kinase (p38) is a kinase that has been implicated in cellular plasticity, stress, and psychiatric disorders and recently in the process of DNA repair. Recently, we have shown that p38 is responsible for inhibiting Glycogen Synthase Kinase 3β (GSK3β), which has also been shown to be involved in the same processes and recently in the process of DNA repair. We have also shown that GSK3β is regulated by stress and that its inhibition produces exaggerated conditioned fear. The goal of this study is to examine whether inhibiting p38 will result in a similar exaggeration of conditioned fear. To this end, mice were injected systemically with the potent and selective inhibitor of p38, SB203580 or vehicle prior to tone and foots­hock fear conditioning and tested for freezing to the tone one day later. Mice injected with SB203580 showed greater tone freezing than mice injected with vehicle. In contrast to tone freezing, SB203580 injections did not affect freezing to the context. Injections of SB203580 prior to 24 hours after fear conditioning, but before fear testing, also did not affect freezing. These data suggest that p38 plays a role in regulating the strength of conditioned fear. The fact that p38 regulates GSK3β and that inhibition of GSK3β also produces exaggerated conditioned fear raises the possibility that a p38 to GSK3β pathway may be regulating the strength of conditioned fear

p38 Mitogen-activated Protein Kinase Regulates Oscillation of Chick Pineal Circadian Clock

Extracellular signal-regulated kinase (ERK) and p38 are members of the mitogen-activated protein kinase (MAPK) family, and in some cases these kinases serve for closely related cellular functions within a cell. In a wide range of animal clock structures, ERK plays an important role in the circadian time-keeping mechanism. Here we found that immunoreactivity to p38 protein was uniformly distributed among cells in the chick pineal gland. On the other hand, a constant level of activated p38 was detected over the day, predominantly in the follicular and parafollicular pinealocytes that are potential circadian clock-containing cells. Chronic application of SB203580, a selective and reversible inhibitor of p38, to the cultured chick pineal cells markedly lengthened the period of the circadian rhythm of the melatonin release (up to 28.7 h). Noticeably, despite no significant temporal change of activated p38 level, a 4-h pulse treatment with SB203580 delayed the phase of the rhythm only when delivered during the subjective day. These results indicate a time-of-day-specific role of continuously activated p38 in the period length regulation of the chick pineal clock and suggest temporally separated regulation of the clock by two MAPKs, nighttime-activated ERK and daytime-working p38

Differential regulation of anti-inflammatory genes by p38 MAP kinase and MAP kinase kinase 6.

BackgroundConventional p38α inhibitors have limited efficacy in rheumatoid arthritis, possibly because p38 blockade suppresses the counter-regulatory mechanisms that limit inflammation. In contrast, targeting the upstream MAP kinase kinases, MKK3 and MKK6, partially maintains p38-mediated anti-inflammatory responses in bone marrow-derived macrophages (BMDM). In this study, we explored the mechanisms that preserve anti-inflammatory gene expression by evaluating differential regulation of IL-10 and p38-dependent anti-inflammatory genes in MKK3-/-, MKK6-/-, and p38 inhibitor-treated wildtype cells.MethodsBMDM from wild type (WT), MKK3-/-, and MKK6-/- mice were pre-treated with p38 inhibitor SB203580 (SB), JNK inhibitor SP600125 (SP), and/or ERK inhibitor PD98059 (PD) and stimulated with LPS. Supernatant protein levels were measured by multiplex bead immunoassay. mRNA expression was determined by qPCR and protein expression by Western blot analysis. De novo IL-10 mRNA synthesis was quantified in cells treated with ethynyl-uridine and LPS followed by reverse transcription and qPCR. mRNA half-life was measured in LPS-treated cells that were then incubated with actinomycin D ± SB203580.ResultsPre-treatment of WT BMDM with p38 inhibitor significantly reduced IL-10 production in the three groups, while ERK and JNK inhibitors had minimal effects. IL-10 production was significantly decreased in MKK3-/- BMDM compared with either WT or MKK6-/- cells. IL-10 mRNA expression was modestly reduced in MKK3-/- BMDM but was preserved in MKK6-/- cells compared with WT. De novo IL-10 mRNA synthesis was inhibited in MKK3-/- and p38 inhibitor pre-treated cells, but not MKK6-/- cells compared with WT. IL-10 mRNA half-life was markedly reduced in p38 inhibitor-treated WT cells while MKK-deficiency had minimal effect. DUSP1 mRNA levels were preserved in MKK-deficient cells but not in p38 inhibitor-treated WT cells. Tristetraprolin mRNA and protein levels were reduced in p38 inhibitor-treated WT cells compared with MKK6-/- cells.ConclusionUnlike p38-inhibition, the absence of MKK6 mostly preserves IL-10 and TTP protein expression in BMDM. MKK6-deficiency also spares DUSP1 and IL-1RA, which are key negative regulators of the inflammatory response. Together, these data suggest that MKK6 is a potential therapeutic target in RA

Regulation of peripheral inflammation by spinal p38 MAP kinase in rats.

BackgroundSomatic afferent input to the spinal cord from a peripheral inflammatory site can modulate the peripheral response. However, the intracellular signaling mechanisms in the spinal cord that regulate this linkage have not been defined. Previous studies suggest spinal cord p38 mitogen-activated protein (MAP) kinase and cytokines participate in nociceptive behavior. We therefore determined whether these pathways also regulate peripheral inflammation in rat adjuvant arthritis, which is a model of rheumatoid arthritis.Methods and findingsSelective blockade of spinal cord p38 MAP kinase by administering the p38 inhibitor SB203580 via intrathecal (IT) catheters in rats with adjuvant arthritis markedly suppressed paw swelling, inhibited synovial inflammation, and decreased radiographic evidence of joint destruction. The same dose of SB203580 delivered systemically had no effect, indicating that the effect was mediated by local concentrations in the neural compartment. Evaluation of articular gene expression by quantitative real-time PCR showed that spinal p38 inhibition markedly decreased synovial interleukin-1 and -6 and matrix metalloproteinase (MMP3) gene expression. Activation of p38 required tumor necrosis factor alpha (TNFalpha) in the nervous system because IT etanercept (a TNF inhibitor) given during adjuvant arthritis blocked spinal p38 phosphorylation and reduced clinical signs of adjuvant arthritis.ConclusionsThese data suggest that peripheral inflammation is sensed by the central nervous system (CNS), which subsequently activates stress-induced kinases in the spinal cord via a TNFalpha-dependent mechanism. Intracellular p38 MAP kinase signaling processes this information and profoundly modulates somatic inflammatory responses. Characterization of this mechanism could have clinical and basic research implications by supporting development of new treatments for arthritis and clarifying how the CNS regulates peripheral immune responses

DENDRITIC CELL DIFFERENTIATION BLOCKED BY PRIMARY EFFUSION LYMPHOMA-RELEASED FACTORS IS PARTIALLY RESTORED BY INHIBITION OF P38 MAPK

To better understand the molecular mechanisms underlying the dendritic cell (DC) defects in cancer, we analyzed which signaling pathway is implicated in the abnormal monocyte differentiation into DC determined by the presence of Primary effusion lymphoma (PEL) released factors. Our results indicate that the DC, obtained in this condition, together with phenotypic abnormalities and reduced allostimulatory function, showed hyperphosphorylation of signal transducer and activator of transcription 3 (STAT3) and p38 mitogen-activated protein kinase (MAPK) molecules, in comparison to the DC differentiated in the absence of PEL-released factors. The inhibition of p38 MAPK but not of STAT3 phosphorylation, with specific inhibitors, was able to revert the effect of the PEL-released factors on the DC phenotype. This study suggests that p38 MAPK signaling pathway is an important contributor to the abnormal differentiation of DC in PEL