Mechanisms regulating the nuclear translocation of p38 MAP kinase

p38 mitogen‐activated protein kinase (MAPK) is of fundamental importance in a cell's response to environmental stresses, cytokines and DNA damage. p38 resides in the cytoplasm of resting cells, and translocates into the nucleus upon activation, yet the exact mechanisms remain largely unclear. We show here that the phosphorylation‐dependent nuclear translocation of p38 is a common phenomenon when cells are stimulated with various stresses. On the other hand, the nuclear export of p38 requires its dephosphorylation, and it is exported both in a MK2‐dependent and a nuclear export signal (NES)‐independent manner. Although different p38‐regulated/activated protein kinase (PRAK) mutants all dictate the intracellular localization of p38, results from a PRAK‐deficient cell line indicate that it plays no role in this process. Microtubule depolymerizing reagent nocodazole and dynein inhibitor EHNA both block the nuclear translocation of p38, demonstrating roles for microtubules and dynein in p38 transport. Taken together, stress‐induced nuclear accumulation of p38 is a phosphorylation‐dependent, microtubule‐ and dynein‐associated process. J. Cell. Biochem. 110: 1420–1429, 2010. © 2010 Wiley‐Liss, Inc.     

Oxidative stress-induced intestinal epithelial cell apoptosis is mediated by p38 MAPK

Free oxygen radicals are involved in the pathogenesis of necrotizing enterocolitis (NEC) in premature infants. The stress-activated p38 mitogen-activated protein kinase (MAPK) has been implicated in gut injury. Here, we found that phosphorylated p38 was detected primarily in the villus tips of normal intestine, whereas it was expressed in the entire mucosa in NEC. H(2)O(2) treatment resulted in a rapid phosphorylation of p38 MAPK and subsequent apoptosis of rat intestinal epithelial (RIE)-1 cells; this induction was attenuated by treatment with SB203580, a selective p38 MAPK inhibitor, or transfection with p38alpha siRNA. Moreover, SB203580 also blocked H(2)O(2)-induced PKC activation. In contrast, the PKC inhibitor (GF109203x) did not affect p38 activation, indicating that p38 MAPK activation occurs upstream of PKC activation in H(2)O(2)-induced apoptosis. H(2)O(2) treatment also decreased mitochondrial membrane potential; pretreatment with SB203580 attenuated this response. Our study demonstrates that the p38 MAPK/PKC pathway plays an important role as a pro-apoptotic cellular signaling during oxidative stress-induced intestinal epithelial cell injury.     

Chondrogenesis induced by actin cytoskeleton disruption is regulated via protein kinase C-dependent p38 mitogen-activated protein kinase signaling

Disruption of the actin cytoskeleton in subconfluent mesenchymal cells induces chondrogenic differentiation via protein kinase C (PKC) alpha signaling. In this study, we investigated the role of p38 mitogen-activated protein (MAP) kinase in the chondrogenic differentiation of mesenchymal cells that is induced by depolymerization of the actin cytoskeleton. Treatment of mesenchymal cells derived from chick embryonic limb buds with cytochalasin D (CD) disrupted the actin cytoskeleton with concomitant chondrogenic differentiation. The chondrogenesis was accompanied by an increase in p38 MAP kinase activity and inhibition of p38 MAP kinase with SB203580 blocked chondrogenesis. Together these results suggest an essential role for p38 MAP kinase in chondrogenesis. In addition, inhibition of p38 MAP kinase did not alter CD-induced increased expression and activity of PKC alpha, whereas down-regulation of PKC by prolonged exposure of cells to phorbol ester inhibited CD-induced p38 MAP kinase activation. Our results therefore suggest that PKC is involved in the regulation of chondrogenesis induced by disruption of the actin cytoskeleton via a p38 MAP kinase signaling pathway.     

Soy sauce increased the oxidative stress tolerance of nematode via p38 MAPK pathway

Soy sauce – a fermented food made from soybeans and wheat – is considered a healthy seasoning, but little scientific evidence is available to support this. In this study, physiological effects of soy sauce were analyzed using Caenorhabditis elegans. When soy sauce was fed to C. elegans together with Escherichia coli OP50, fat accumulation decreased, and resistance to oxidative stress by H₂O₂ was greatly increased in the nematodes. qRT-PCR revealed that mRNA expression of oxidative stress tolerance genes, including sod, ctl, and gpx, was markedly increased in soy sauce-fed nematodes. Worms ingesting soy sauce showed high mitochondrial membrane potential and reactive oxygen species (ROS) and low intracellular ROS, suggesting that soy sauce induced mitohormesis and decreased cytoplasmic ROS. Therefore, soy sauce ingestion affects the mitochondria and may alter the fat metabolism in C. elegans. Furthermore, the increase in oxidative stress tolerance is mediated through p38 MAPK pathway.     

The p38 Pathway Regulates Oxidative Stress Tolerance by Phosphorylation of Mitochondrial Protein IscU

The p38 pathway is an evolutionarily conserved signaling pathway that responds to a variety of stresses. However, the underlying mechanisms are largely unknown. In the present study, we demonstrate that p38b is a major p38 MAPK involved in the regulation of oxidative stress tolerance in addition to p38a and p38c in Drosophila. We further show the importance of MK2 as a p38-activated downstream kinase in resistance to oxidative stresses. Furthermore, we identified the iron-sulfur cluster scaffold protein IscU as a new substrate of MK2 both in Drosophila cells and in mammalian cells. These results imply a new mechanistic connection between the p38 pathway and mitochondria iron-sulfur clusters.     

Eclalbasaponin I causes mitophagy to repress oxidative stress-induced apoptosis via activation of p38 and ERK in SH-SY5Y cells

Oxidative stress accompanying excessive accumulation of reactive oxygen species (ROS) and mitochondrial dysfunction leads to the occurrence of neurodegenerative diseases. Our previous study showed that Eclalbasaponin I (EcI), a triterpene saponin isolated from Aralia elata (Miq.) Seem. (A. elata), repressed oxidative stress in human neuroblastoma SH-SY5Y cells. However, the detailed mechanism remains unclear. In this study, pretreatment with EcI in SH-SY5Y cells significantly activated the p38-mitogenactivated protein kinase (p38), the extracellular regulated protein kinase (ERK), whereas it did not affect the c-jun NH2 terminal kinases (JNK). In accordance with the initial findings, EcI-induced neuroprotective effect was attenuated by SB203580 (SB, a p38 inhibitor) or FR180204 (FR, an ERK inhibitor), being further confirmed by specific small interfering RNA (siRNA). Inhibition of either p38 or ERK up-regulated the apoptosis induction in EcI- and H₂O₂-cotreated cells. Furthermore, p38 or ERK suppression enhanced intracellular and mitochondrial ROS generation, decreased the activities of endogenous antioxidant defences as well as the mitochondrial membrane potential (MMP), resulting in dysfunction of mitochondria. In addition, EcI-induced autophagy and mitophagy were obviously down-regulated when p38 or ERK activation was blocked. Cumulatively, these findings supported that EcI-caused mitophagy contributed to the neuroprotective effect through p38 or ERK activation. Mitophagy induction might be an effective therapeutic intervention in neurodegenerative diseases.     

A protective effect of curcumin on cardiovascular oxidative stress indicators in systemic inflammation induced by lipopolysaccharide in rats

ObjectiveInflammation has been considered as an important factor in cardiovascular diseases (CVD). Curcumin has been well known for its anti-inflammatory effects. In current research, protective effect of curcumin on cardiovascular oxidative stress indicators in systemic inflammation induced by lipopolysaccharide (LPS) was investigated in rats.Material and methodsThe animals were divided into five groups and received the treatments during two weeks [1]: Control in which vehicle was administered instead of curcumin and saline was injected instead of LPS [2], LPS group in which vehicle of curcumin plus LPS (1 mg/kg) was administered [3-5], curcumin groups in them three doses of curcumin (5, 10 and 15 mg/kg) before LPS were administered.ResultsAdministration of LPS was followed by an inflammation status presented by an increased level of white blood cells (WBC) (p < 0.001). An oxidative stress status was also occurred after LPS injection which was presented by an increased level of malondialdehyde (MDA) while, a decrease in thiols, superoxide dismutase (SOD) and catalase(CAT) in all heart, aorta and serum (p < 0.001). The results also showed that curcumin decreased WBC (doses: 10 and 15 mg/kg) (p < 0.001) accompanying with a decrease in MDA (P < 0.01 and P < 0.001). Curcumin also improved the thiols and the activities of SOD and catalase (P < 0.05, P < 0.01 and P < 0.001).ConclusionBased on our findings, curcumin can ameliorates oxidative stress and inflammation induced by LPS in rats to protect the cardiovascular system.     

大鼠脑内远位触液神经元p38丝裂原活化蛋白激酶的分布及在噪声应激时的表达

目的：观察脑内远位触液神经元内p-p38丝裂原活化蛋白激酶（MAPK）的分布及其在噪声应激时的表达。方法：用霍乱毒素亚单位B与辣根过氧化物酶复合物（CB-HRP）标记和免疫组织化学相结合的双重标记技术．观察SD大鼠脑实质内远位触液神经元中p-p38MAPK的分布：进一步制作噪声应激动物模型,观察噪声应激后该类神经元中p-p38MAPK的表达变化。结果：在脑干的特定部位恒定出现被CB-HRP标记的两组神经细胞簇,其他脑区未见CB-HRP标记神经细胞簇。不予应激刺激,该细胞簇内仅有个别神经元见有CB-HRP／p—p38MAPK;噪声应激刺激1d时,上述特定部位细胞簇的CB-HRP／p-p38MAPK双重标记神经元数目没有明显变化;噪音应激刺激5d时,CB-HRP／p—p38MAPK双重标记神经元数目较对照组显著增多（P〈0．05）;噪音应激刺激10d时CB-HRP／p—p38MAPK双重标记神经元数目较对照组显著增多（P〈0．05）;噪音应激刺激20d时,CB-HRP／p—p38MAPK双重标记神经元数目较对照组显著增多（P〈0．01）：结论：在脑干特定部位恒定存在的两组被CBHRP标记的细胞团为远位触液神经元,其中少数触液神经元有p-p38MAPK表达,且当给予动物噪声应激刺激时,p-p38MAPK免疫阳性神经元和CB-HRP／p—p38MAPK双重标记神经元数量显著增加,提示脑实质内的这种远位触液神经元中的P—p38MAPK可能参与了机体对噪声应激的信息传递或调控,其作用随应激天数增加而日趋增强．     

Induction of apoptosis in breast cancer cells by apomine is mediated by caspase and p38 mitogen activated protein kinase activation

The 1,1-bisphosphonate ester family member apomine (SR-45023A) is known to have anti-tumour activity in various cancer cell types. The aims of this study were to determine the effect of apomine on the growth of two breast cancer cell lines, MCF-7 and MDA-MB-231, to ascertain whether any growth inhibitory effects found were due to induction of apoptosis, and to investigate the mechanism of action of apomine. Apomine caused significant growth inhibition of both cell lines after 72h of treatment. Apomine-induced growth inhibition was associated with caspase and p38 MAPK activation and DNA fragmentation. Apomine had no effect on Ras localisation, nor did addition of mevalonate to treatment media prevent apomine-induced apoptosis. We conclude that apomine induces apoptosis in breast cancer cells, an effect that is independent of oestrogen receptor status and is not via inhibition of the mevalonate pathway. Our study suggests apomine is a potential anti-neoplastic drug in breast cancer treatment.     

Streptozotocin directly impairs cardiac contractile function in isolated ventricular myocytes via a p38 map kinase-dependent oxidative stress mechanism

Streptozotocin (STZ) has long been used to induce experimental diabetes mellitus to study diabetic complications such as diabetic cardiomyopathy. However, direct impact of STZ on cardiac function is unknown. This study was designed to evaluate the cardiac contractile effect of STZ in isolated adult rat ventricular myocytes. Contractile properties were assessed with an IonOptix MyoCam system including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90), and maximal velocities of shortening/relengthening (+/-dL/dt). Intracellular Ca2+ handling was evaluated with the fluorescent dye fura-2. Acute exposure of STZ (10(-9)-10(-5) M) depressed PS, prolonged TR90, and decreased electrically stimulated intracellular Ca2+ rise in a concentration-dependent manner. TPS,+/-dL/dt, resting intracellular Ca2+ level, and intracellular Ca2+ clearing rate were unaffected. The STZ-induced mechanical alterations were alleviated by the antioxidant vitamin C (100 microM) and the p38 MAP kinase inhibitor SB203580 (1 microM). 2', 7'-Dichlorofluorescein diacetate staining revealed enhanced production of reactive oxygen species following STZ treatment, which was prevented by either vitamin C or SB203580. Collectively, our data provided convincing evidence that the tool drug for experimental diabetes STZ may itself cause deleterious cardiac contractile dysfunction via an oxidative stress and p38 MAP kinase-dependent mechanism. Thus, caution should be taken when assessing diabetic heart complications using STZ-induced diabetic models.     

The lipid peroxidation end-product 4-HNE induces COX-2 expression through p38MAPK activation in 3T3-L1 adipose cell

Oxidative stress and low grade chronic inflammation are increased in accumulating fat. Our objective was to test whether 4-hydroxynonenal (4-HNE), an end-product of lipid peroxidation, affects cyclooxygenases in 3T3-L1 adipose cells. 4-HNE increased COX-2 mRNA and protein expression and p38MAP-kinase phosphorylation in a dose-dependent manner. Pretreatment of 3T3-L1 cells by a selective inhibitor of p38MAPK (PD 169316) abolished 4-HNE and glucose oxidase induced COX-2 expression. Our results show that oxidative stress induces COX-2 expression through the production of 4-HNE which activates p38MAPKinase, suggesting that 4-HNE links oxidative stress and chronic inflammation through the activation of cyclooxygenase.     

P38 mitogen activated protein kinase expression and regulation by interleukin-4 in human B cell non-Hodgkin lymphomas

The prevalence and regulation of p38 mitogen activated protein kinase (MAPK) expression in human lymphomas have not been extensively studied. In order to elucidate the role of p38 MAPK in lymphomagenesis, we examined the expression of native and phosphorylated p38 (p-p38) MAPK in cell lines derived from human hematopoietic neoplasms including B cell lymphoma-derived cell lines using Western blot analysis. The p-p38 MAPK protein was also analyzed in 30 B cell non-Hodgkin lymphoma (NHL) tissue biopsies by immunohistochemistry. Our results show that the expression of p38 MAPK was up-regulated in most of the cell lines as compared with peripheral blood lymphocytes, while the expression of p-p38 MAPK was more variable. A subset of B cell NHL biopsies showed increased expression of p-p38 MAPK relative to reactive germinal center cells. Interleukin-4 (IL-4) induced a dose-dependent increase in the expression of p-p38 MAPK (1.6- to 2.8-fold) in cell lines derived from activated B cell-like diffuse large B cell lymphoma (DLBCL) but not those from germinal center-like DLBCL. No change was seen in native p38 MAPK. The in vitro kinase activity of p38 MAPK, however, was induced (1.6- to 3.2-fold) in all five cell lines by IL-4. Quantitative fluorescent RT-PCR demonstrated that all four isoforms of p38 MAPK gene were expressed in the lymphoma cell lines, with p38γ and p38β isoforms being predominant. IL-4 stimulation increased the expression of β, γ, and δ isoforms but not α isoform in two cell lines. In conclusion, there is constitutive expression and activation of p38 MAPK in a large number of B-lymphoma-derived cell lines and primary lymphoma tissues, supportive of its role in lymphomagenesis. The differential IL-4 regulation of p38 MAPK expression in cell lines derived from DLBCL may relate to the cellular origin of these neoplasms.

Electronic supplementary material

The online version of this article (doi:10.1007/s12308-009-0049-5) contains supplementary material, which is available to authorized users.     

FABP4 inhibitor BMS309403 decreases saturated-fatty-acid-induced endoplasmic reticulum stress-associated inflammation in skeletal muscle by reducing p38 MAPK activation

Aims

Fatty acid binding protein 4 (FABP4) inhibitors have been proposed as potential therapeutic approaches against insulin resistance-related inflammation and type 2 diabetes mellitus. However, the underlying molecular mechanisms by which these molecules drive these effects in skeletal muscle remain unknown. Here, we assessed whether the FABP4 inhibitor BMS309403 prevented lipid-induced endoplasmic reticulum (ER) stress-associated inflammation in skeletal muscle.

Influence of crocetin on high-cholesterol diet induced atherosclerosis in rats via anti-oxidant activity together with inhibition of inflammatory response and p38 MAPK signaling pathway

The present study aimed to investigate the effect and possible mechanism of action of crocetin on the high cholesterol diet (HCD) induced atherosclerosis rat. The Wistar rats were used in the current investigation. The rats were divided into following group, Group I: control, Group II: HCD induced AS, Group III: AS + crocetin (25 mg/kg), Group IV: AS + crocetin (50 mg/kg) and Group V: AS + Simvastatin, respectively. AS was induced in the rats using the vitamin D₃ and HCD. The rats received the pre-determined treatment for the 10 weeks. After the study period, the level of lipid profile, malonaldehyde (MDA) and superoxide dismutase (SOD) were also estimated. The proinflammatory cytokines viz., tumor necrosis factor (TNF)-α and interleukin (IL)-6 were scrutinized using the ELISA kits. We also estimated the expression of phosphorylated p38 (p-p38) MAPK using the Western blot techniques. The results revealed that the AS was successfully induced in the rats. The AS control group rats showed the modulated level of lipid profile, and decreased the level of the SOD and boost the level of the MDA as compared with the normal control. However, crocetin thrived in enhancing the lipid profile toward the standard value in the normal control group rats. The crocetin and simvastatin group rats significantly inhibited the expression of the p-p38 MAPK as compared to the AS group rats. In conclusion, the current investigation revealed that the crocetin reduced the HCD induced dyslipidemia in the Wistar rats, the possible mechanism of action may be connected to the antioxidative, down regulating of p-p38 MAPK and antiinflammatory effect by crocetin.     

p38 mitogen-activated protein kinase (MAPK) promotes cholesterol ester accumulation in macrophages through inhibition of macroautophagy

p38 MAPK has been strongly implicated in the development of atherosclerosis, but its role in cholesterol ester accumulation in macrophages and formation of foam cells, an early step in the development of atherosclerosis, has not been investigated. We addressed this issue and made some brand new observations. First, elevated intracellular cholesterol level induced by the exposure to LDL-activated p38 MAPK and activation of p38 MAPK with anisomycin increased the ratio of cholesterol esters over free cholesterol, whereas inhibition of p38 MAPK with SB203580 or siRNA reduced the LDL loading-induced intracellular accumulation of free cholesterol and cholesterol esters in macrophages. Second, exposure to LDL cholesterol inhibited autophagy in macrophages, and inhibition of autophagy with 3-methyladenine increased intracellular accumulation of cholesterol (free cholesterol and cholesterol esters), whereas activation of autophagy with rapamycin decreased intracellular accumulation of free cholesterol and cholesterol esters induced by the exposure to LDL cholesterol. Third, LDL cholesterol loading-induced inhibition of autophagy was prevented by blockade of p38 MAPK with SB203580 or siRNA. Neutral cholesterol ester hydrolase was co-localized with autophagosomes. Finally, LDL cholesterol loading and p38 activation suppressed expression of the key autophagy gene, ulk1, in macrophages. Together, our results provide brand new insight about cholesterol ester accumulation in macrophages and foam cell formation.