Attenuated P38 Mitogen-activated Protein Kinase Research Articles

ITGAM sustains MAPK signaling and serves as an adverse prognostic factor and therapeutic target in acute myeloid leukemia.

Acute myeloid leukemia (AML) is the second most frequently occurring type of leukemia in adults. Despite breakthroughs in genetics, the prognosis of AML patients remains dismal. The aim of this study is to find new therapeutic targets and diagnostic markers for AML and to explore their mechanisms of action. The expression patterns of integrin subunit alpha M (ITGAM) were investigated across different cell types using the Human Protein Atlas (HPA) database. The ITGAM levels across cancer types were analyzed using the Gene Expression Profiling Interactive Analysis (GEPIA) database. Prognostic correlations in AML individuals were evaluated using The Cancer Genome Atlas (TGCA) database. ITGAM-associated functions were evaluated by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The AML cells were transfected with short-hairpin RNA targeting ITGAM or a control, and subsequently subjected to analysis in order to ascertain the impact of ITGAM on proliferation and apoptosis. The expression of ITGAM was significantly higher in the AML patient samples compared to the control samples. High ITGAM expression was significantly associated with poor overall survival (OS). The knockdown of ITGAM in the AML cells resulted in a decrease in proliferation and an increase in apoptosis. This was accompanied by cell cycle arrest at the G1 phase and a downregulation of protein production for cyclin D1, cyclin E1, cyclin-dependent kinase 2 (CDK2), and cyclin-dependent kinase 4 (CDK4). A pathway analysis and a western blot analysis revealed that ITGAM positively regulated mitogen-activated protein kinase (MAPK) signaling by silencing attenuated p38 MAPK (P38), c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) phosphorylation, while the total protein levels remained unchanged. ITGAM can serve as a potential prognostic biomarker and therapeutic target for AML. ITGAM production was elevated in AML and indicated poor survival. Silencing ITGAM suppressed AML cell viability and induced apoptosis by blocking cell cycle progression, likely by impeding the activation of the MAPK pathway. Further investigations that directly target the ITGAM-MAPK axis may offer novel strategies for mitigating AML pathogenesis and overcoming chemotherapy resistance.

Pulsed ultrasound prevents lipopolysaccharide-induced muscle atrophy through inhibiting p38 MAPK phosphorylation in C2C12 myotubes

ObjectiveInflammation contributes to skeletal muscle atrophy via protein degradation induced by p38 mitogen-activated protein kinase (MAPK) phosphorylation. Meanwhile, pulsed ultrasound irradiation provides the mechanical stimulation to the target tissue, and has been reported to show anti-inflammatory effects. This study investigated the preventive effects of pulsed ultrasound irradiation on muscle atrophy induced by lipopolysaccharide (LPS) in C2C12 myotubes. MethodsC2C12 myotubes were used in this research. The pulsed ultrasound (a frequency of 3 MHz, duty cycle of 20%, intensity of 0.5 W/cm2) was irradiated to myotube before LPS administration. ResultsThe LPS increased phosphorylation of p38 MAPK and decreased the myofibril and myosin heavy chain protein (P < 0.05), followed by atrophy in C2C12 myotubes. The pulsed ultrasound irradiation attenuated p38 MAPK phosphorylation and myotube atrophy induced by LPS (P < 0.05). ConclusionsPulsed ultrasound irradiation has the preventive effects on inflammation-induced muscle atrophy through inhibiting phosphorylation of p38 MAPK.

Fibroblast Growth Factor Type 1 Ameliorates High-Glucose-Induced Oxidative Stress and Neuroinflammation in Retinal Pigment Epithelial Cells and a Streptozotocin-Induced Diabetic Rat Model.

Diabetic retinopathy (DR) is a common complication of diabetes that causes severe visual impairment globally. The pathogenesis of DR is related to oxidative stress and chronic inflammation. The fibroblast growth factor type 1 (FGF-1) mitogen plays crucial roles in cell function, development, and metabolism. FGF-1 is involved in blood sugar regulation and exerts beneficial antioxidative and anti-inflammatory effects on various organ systems. This study investigated the antioxidative and anti-inflammatory neuroprotective effects of FGF-1 on high-glucose-induced retinal damage. The results revealed that FGF-1 treatment significantly reversed the harmful effects of oxidative stress and inflammatory mediators in retinal tissue in a streptozotocin-induced diabetic rat model. These protective effects were also observed in the in vitro model of retinal ARPE-19 cells exposed to a high-glucose condition. We demonstrated that FGF-1 attenuated p38 mitogen-activated protein kinase and nuclear factor-κB pathway activation under the high-glucose condition. Our results indicated that FGF-1 could effectively prevent retinal injury in diabetes. The findings of this study could be used to develop novel treatments for DR that aim to reduce the cascade of oxidative stress and inflammatory signals in neuroretinal tissue.

Protective role of c-Jun NH2-terminal kinase-associated leucine zipper protein (JLP) in curcumin-induced cancer cell death

Previous studies have established the antitumor activity of curcumin, a major component of turmeric. Increasing evidence indicates that curcumin induces autophagy, the activation of mitogen-activated protein kinase (MAPK) intracellular signaling pathways, and reactive oxygen species (ROS)-mediated cell death. The c-Jun NH2-terminal kinase (JNK)-associated leucine zipper protein (JLP), a scaffold protein for MAPK signaling pathways, has been identified as a candidate biomarker for cancer. In this study, we explored the role of JLP in curcumin-induced cancer cell death. We found that JLP knockdown (KD) increases cell death and intracellular ROS levels. Furthermore, JLP KD impaired lysosomal accumulation around perinuclear regions, which led to the inhibition of autophagosome–lysosome fusion, and attenuated p38 MAPK activation in curcumin-treated cells. The decreases in cell viability and p38 MAPK activation were reversed by expressing wild-type JLP but not a JLP mutant lacking the p38 MAPK-binding domain. In addition, the inactivation of a key gene involved in autophagy increased sensitivity to curcumin-induced cell death. Together, these results suggest that JLP mediates the induction of autophagy by regulating lysosome positioning and p38 MAPK signaling, indicating an overall protective role in curcumin-induced ROS-mediated cancer cell death.

The cardioprotective effects of secretory leukocyte protease inhibitor against myocardial ischemia/reperfusion injury.

Protease enzymes generated from injured cells and leukocytes are the primary cause of myocardial cell damage following ischemia/reperfusion (I/R). The inhibition of protease enzyme activity via the administration of particular drugs may reduce injury and potentially save patients' lives. The aim of the current study was to investigate the cardioprotective effects of treatment with recombinant human secretory leukocyte protease inhibitor (rhSLPI) on in vitro and ex vivo models of myocardial I/R injury. rhSLPI was applied to isolated adult rat ventricular myocytes (ARVMs) subjected to simulated I/R and to ex vivo murine hearts prior to I/R injury. Cellular injury, cell viability, reactive oxygen species (ROS) levels, and levels of associated proteins were assessed. The results demonstrated that administration of rhSLPI prior to or during sI/R significantly reduced the death and injury of ARVMs and significantly reduced intracellular ROS levels in ARVMs during H2O2 stimulation. In addition, treatment of ARVMs with rhSLPI significantly attenuated p38 mitogen-activated protein kinase (MAPK) activation and increased the activation of Akt. Furthermore, pretreatment of ex vivo murine hearts with rhSLPI prior to I/R significantly decreased infarct size, attenuated p38 MAPK activation and increased Akt phosphorylation. The results of the current study demonstrated that treatment with rhSLPI induced a cardioprotective effect and reduced ARVM injury and death, intracellular ROS levels and infarct size. rhSLPI also attenuated p38 MAPK phosphorylation and activated Akt phosphorylation. These results suggest that rhSLPI may be developed as a novel therapeutic strategy of treating ischemic heart disease.

Protective Effect of Polydeoxyribonucleotide Against Renal Ischemia-Reperfusion Injury in Mice

BackgroundPolydeoxyribonucleotide (PDRN) is an A2A receptor agonist that induces vascular endothelial growth factor (VEGF) production during the pathological condition of low tissue perfusion. Ischemia-reperfusion injury (IRI) is a major problem after renal transplantation. In the present study, we investigated whether PDRN exhibits reno-protective effects against ischemia-reperfusion–induced acute kidney injury in mice. MethodsRenal ischemia-reperfusion injury was induced in male C57BL/6 mice by bilateral renal pedicle occlusion for 30 minutes, followed by reperfusion for 48 hours. PDRN (8 mg/kg body weight intraperitoneally) was administered 30 minutes before IRI. ResultsTreatment with PDRN significantly decreased neutrophil gelatinase-associated lipocalin levels in the urine, blood urea nitrogen level, and serum creatinine levels as well as kidney tubular injury. Western blotting showed that PDRN significantly increased the levels of vascular endothelial growth factor and B-cell lymphoma protein and attenuated p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, inducible nitric oxide synthase, and Bcl-2–associated X protein levels 48 hours after IRI. ConclusionsOur findings suggest that PDRN is a potential therapeutic agent for acute ischemia-induced renal damage.

Inhibition of Na+/H+ exchanger 1 by cariporide reduces burn-induced intestinal barrier breakdown

Severe burns initiate an inflammatory cascade within the gut, which leads to intestinal mucosal injury. Although Na+/H+ exchanger 1 (NHE1) is recognised as a pivotal player in several inflammatory processes, its role in burn-induced intestinal injury is relatively unknown. We hypothesised that NHE1 might be involved in the increased intestinal permeability and barrier breakdown after severe burns. Thus, we here investigate whether the inhibition of NHE1 has a protective effect on burn-induced intestinal injury. Mice were subjected to a 30% total body surface area (TBSA) full-thickness steam burn. Cariporide was used to assess the function of NHE1 in mice with burn-induced intestinal injury by fluorescence spectrophotometry, Western blotting and enzyme linked immunosorbent assay (ELISA). We found that severe burn increased intestinal permeability, associated with the up-regulation of NHE1 and raised inflammatory cytokine levels. Mice treated with the NHE1 inhibitor cariporide had significantly attenuated burn-induced intestinal permeability and a reduced inflammatory response. NHE1 inhibition also reduced nuclear factor-κB (NF-κB) activation and attenuated p38 mitogen-activated protein kinase (MAPK) phosphorylation. Our study suggests that NHE1 plays an important role in burn-induced intestinal permeability through the regulation of the inflammatory response. Inhibition of NHE1 may be adopted as a potential therapeutic strategy for attenuating intestinal barrier breakdown.

P38 Mitogen-activated protein kinase and extracellular signal-regulated kinase signaling pathways are not essential regulators of formyl peptide-stimulated p47phox activation in neutrophils

Three structurally unrelated p38 mitogen-activated protein kinase (MAPK) inhibitors, (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole (SB203580), 1-5-tert-butyl-2-p-tolyl-2H-pyrazol-3-yl)-3-[4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl] urea (BIRB 796) and 5-(2,6-dichlorophenyl)-2-[2,4-difluorophenyl]thio]-6H-pyrimido[1,6-b]pyridazin-6-one (VX 745) showed approximately 40% inhibition of formyl-Met-Leu-Phe (fMLP)-stimulated neutrophil superoxide anion (O2•−) generation at concentrations that greatly diminished p38 MAPK activity. However, a significant inhibition of p47phox activation occurred at concentrations much higher than the corresponding IC50 values of these inhibitors in blocking p38 MAPK activity. 4-Ethyl-2(p-methoxyphenyl)-5-(4′-pyridyl)-IH-imidazole (SB202474), an inactive analogue of SB203580, at a concentration (30μM) which significantly attenuated p38 MAPK activity, had no effect on p47phox activation, whereas it inhibited O2•− generation with an IC50 value of approximately 16μM. Moreover, both SB203580 and BIRB 796 had no effect on protein kinase B (PKB)/Akt Ser473 phosphorylation and S100A9 protein membrane translocation at concentrations that effectively blocked p38 MAPK activity. Pretreatment of cells with two structurally unrelated MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitors, 2-(2-amino-3-methoxy-phenyl)-chromen-4-one (PD 98059) and 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), at concentrations that effectively blocked MEK activity, attenuated p47phox phosphorylation but did not affect the recruitment of p47phox to p22phox or O2•− generation. Both p47phox activation and O2•− generation were attenuated by a protein kinase C (PKC) inhibitor 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF 109203X) in the concentration range that effectively blocked PKC activity. Taken together, these results suggest that the ERK-mediated Ser phosphorylation of p47phox is not implicated in the assembly of NADPH oxidase or O2•− generation, and that O2•− generation is partly attributable to p38 MAPK signaling through mechanisms other than p47phox activation, Akt activation and S100A9 membrane recruitment in fMLP-stimulated neutrophils.

Lidocaine Attenuates Proinflammatory Cytokine Production Induced by Extracellular Adenosine Triphosphate in Cultured Rat Microglia

Our previous studies demonstrated that intrathecal lidocaine treatment could produce prolonged reversal of established hyperalgesia or allodynia, both induced by chronic constriction injury. Indeed, intrathecal lidocaine treatment remarkably suppressed the activation of p38 mitogen-activated protein kinase (MAPK) in hyperactive microglia. In the present study we suggest that lidocaine may act directly on the microglia and attenuate the release of cytokines. We assessed the influence of lidocaine on the levels of phospho-p38 MAPK, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, and intracellular calcium triggered by extracellular adenosine triphosphate (ATP) in cultured rat microglia. Our experimental methods included Western blot, real-time reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and calcium imaging. We found that lidocaine (in a dose-dependent manner) significantly attenuated p38 MAPK activation triggered by 1 mM ATP, by inhibiting the transcription of 3 cytokine messenger RNAs and causing a decrease in their respective protein concentrations (TNF-alpha, IL-1beta, and IL-6, P < 0.05, vs. the ATP group). SB203580, an antagonist of P38, attenuated ATP-activated elevation in protein levels of TNF-alpha, IL-1beta, and IL-6 in the microglia. The high level of intracellular calcium ([Ca(2+)]i) that is induced by ATP was decreased by the addition of 10 mM lidocaine (P < 0.05 vs. the ATP group). These findings indicate that lidocaine can directly act on microglia. Lidocaine, by inhibiting the increase of intracellular calcium, also inhibited p38 MAPK activation and attenuated the production of proinflammatory cytokines (including TNF-alpha, IL-1beta, and IL-6), which were triggered by extracellular ATP in cultured rat microglia.

Inhibitory effects of Bulnesia sarmienti aqueous extract on agonist-induced platelet activation and thrombus formation involves mitogen-activated protein kinases

Ethnopharmacological relevance B. sarmienti has long been recognized in folk medicine as a medicinal plant with various medicinal uses. Traditionally, it has been appreciated for the skin-healing properties of its essence. The bark has also been employed to treat stomach and cardiovascular disorders and reported to have antitumor, antioxidant and anti-inflammatory activities. However, information on its antiplatelet activity is limited. Aim of the study To examined the effects of B. sarmienti aqueous extract (BSAE) in platelet physiology. Materials and methods The anti-platelet activity of BSAE was studied using rat platelets for in vitro determination of the extract effect on agonist-induced platelet aggregation, ATP secretion, [Ca 2+] i mobilization and MAP kinase phosphorylation. The extract in vivo effects was also examined in arterio-venous shunt thrombus formation in rats, and tail bleeding time in mice. Result HPLC chromatographic analysis revealed that B. sarmienti extract contained (+)-catechin (C), (−)-epigallocatechin (EGC), (−)-epicatechin (EC), and (−)-epicatechin gallate (ECG). BSAE, significantly and dose dependently, inhibited collagen, thrombin, or ADP-induced platelet aggregation. The 50 percent inhibitory concentrations (IC 50) of the extract for collagen, thrombin and ADP-induced platelet aggregation were 45.3 ± 2.6, 100 ± 5.6 and 110 ± 4.6 μg/ml, respectively. Collagen activated ATP release and thrombin-induced intracellular Ca 2+ concentration were reduced in BSAE-treated platelets. In addition, the extract in vivo activity showed that BSAE at 100 mg/kg significantly attenuated thrombus formation in rat extracorporeal shunt model while mice tail bleeding time was not affected. Moreover, BSAE attenuated p38 mitogen-activated protein kinase (p38 MAPK), c-Jun N-terminal kinase 1 (JNK1) and extracellular-signal-regulated protein kinase 2 (ERK2) phosphorylations. Conclusion BSAE inhibits platelet activation, granule secretion, aggregation, and thrombus formation without affecting bleeding time, and that this effect is mediated by inhibition of P38, JNK1 and ERK2 phosphorylations. The ability of BSAE to inhibit platelet function might be relevant in cases involving aberrant platelet activation where the plant extract could be considered as a candidate to anti-platelet and antithrombotic agent.

Angiotensin II-Induced Migration of Vascular Smooth Muscle Cells Is Mediated by p38 Mitogen-Activated Protein Kinase-Activated c-Src through Spleen Tyrosine Kinase and Epidermal Growth Factor Receptor Transactivation

Angiotensin II (Ang II) stimulates protein synthesis by activating spleen tyrosine kinase (Syk) and DNA synthesis through epidermal growth factor receptor (EGFR) transactivation in vascular smooth muscle cells (VSMCs). This study was conducted to determine whether Syk mediates Ang II-induced migration of aortic VSMCs using a scratch wound approach. Treatment with Ang II (200 nM) for 24 h increased VSMC migration by 1.56 +/- 0.14-fold. Ang II-induced VSMC migration and Syk phosphorylation as determined by Western blot analysis were minimized by the Syk inhibitor piceatannol (10 microM) and by transfecting VSMCs with dominant-negative but not wild-type Syk plasmid. Ang II-induced VSMC migration and Syk phosphorylation were attenuated by inhibitors of c-Src [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2)], p38 mitogen-activated protein kinase (MAPK) [4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole (SB202190)], and extracellular signal-regulated kinase (ERK) 1/2 [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio) butadiene (U0126)]. SB202190 attenuated p38 MAPK and c-Src but not ERK1/2 phosphorylation, indicating that p38 MAPK acts upstream of c-Src and Syk. The c-Src inhibitor PP2 attenuated Syk and ERK1/2 phosphorylation, suggesting that c-Src acts upstream of Syk and ERK1/2. Ang II- and epidermal growth factor (EGF)-induced VSMC migration and EGFR phosphorylation were inhibited by the EGFR blocker 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) (2 microM). Neither the Syk inhibitor piceatannol nor the dominant-negative Syk mutant altered EGF-induced cell migration or Ang II- and EGF-induced EGFR phosphorylation. The c-Src inhibitor PP2 diminished EGF-induced VSMC migration and EGFR, ERK1/2, and p38 MAPK phosphorylation. The ERK1/2 inhibitor U0126 (10 microM) attenuated EGF-induced cell migration and ERK1/2 but not EGFR phosphorylation. These data suggest that Ang II stimulates VSMC migration via p38 MAPK-activated c-Src through Syk and via EGFR transactivation through ERK1/2 and partly through p38 MAPK.

Mitogen-activated protein kinase (MAPK) phosphatase-1 and -4 attenuate p38 MAPK during dexamethasone-induced insulin resistance in 3T3-L1 adipocytes.

Prolonged use of glucocorticoids induces pronounced insulin resistance in vivo. In vitro, treatment of 3T3-L1 adipocytes with dexamethasone for 48 h reduces the maximal level of insulin- and stress (arsenite)-induced glucose uptake by approximately 50%. Although phosphatidylinositol 3-kinase signaling was slightly attenuated, phosphorylation of its downstream effectors such as protein kinase B and protein kinase C-lambda remained intact. Nor was any effect of dexamethasone treatment observed on insulin- or arsenite-induced translocation of glucose transporter 4 (GLUT4) toward the plasma membrane. However, for a maximal response to either arsenite- or insulin-induced glucose uptake in these cells, functional p38 MAPK signaling is required. Dexamethasone treatment markedly attenuated p38 MAPK phosphorylation coincident with an up-regulation of the MAPK phosphatases MKP-1 and MKP-4. Employing lentivirus-mediated ectopic expression in fully differentiated 3T3-L1 adipocytes demonstrated a differential effect of these phosphatases: whereas MKP-1 was a more potent inhibitor of insulin-induced glucose uptake, MKP-4 more efficiently inhibited arsenite-induced glucose uptake. This coincided with the effects of these phosphatases on p38 MAPK phosphorylation, i.e. MKP-1 and MKP-4 attenuated p38 MAPK phosphorylation by insulin and arsenite, respectively. Taken together, these data provide evidence that in 3T3-L1 adipocytes dexamethasone inhibits the activation of the GLUT4 in the plasma membrane by a p38 MAPK-dependent process, rather than in a defect in GLUT4 translocation per se.

Activation of p38 MAPK Induced by a Multi-cycle Ischaemic Preconditioning Protocol is Associated with Attenuated p38 MAPK Activity During Sustained Ischaemia and Reperfusion

The role of p38 mitogen-activated protein kinase (MAPK) in ischaemic preconditioning remains controversial. Since most previous studies focussed on events only during sustained ischaemia, the aim of this study was to establish the activation pattern of p38 MAPK during a multicycle preconditioning protocol, sustained ischaemia as well as reperfusion and to correlate these events with functional recovery of the isolated perfused rat heart. Isolated perfused rat hearts were preconditioned by 3×5 min global ischaemia followed by 25 min global ischaemia and 30 min reperfusion. Non-preconditioned hearts were subjected to 25 min global ischaemia and 30 min reperfusion. Hearts were freeze-clamped and p38 MAPK activation in tissue lysates was assessed by standard Western blotting techniques, using a dual phospho-p38 MAPK antibody as well as a non-radioactive IP-kinase assay. The results showed that transient dual phosphorylation and activation of p38 MAPK occurs during a 3×5 min preconditioning protocol: the activation was maximal during the first episode, becoming progressively lower during the second and third episodes. p38 MAPK activation was significantly less during both sustained ischaemia and reperfusion in preconditioned hearts, when compared with non-preconditioned hearts. Attenuation of p38 MAPK activity during sustained ischaemia and reperfusion was associated with improved functional recovery. The effect of inhibition of p38 MAPK activation on cardioprotection was further evaluated in adult, isolated cardiomyocytes. Administration of SB 203580 (1–10μM ) before and during the preconditioning protocol, had no effect on cell morphology and viability after 2 h hypoxia, compared to untreated preconditioned cardiomyocytes. When administered to non-preconditioned cells before the onset of 2 h hypoxia, it caused a significant improvement in both morphology and viability. In summary, the results suggest that attenuation of the kinase activity during sustained ischaemia and reperfusion may be an essential element of the preconditioning process.