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.     

F-Box Only Protein 31 (FBXO31) Negatively Regulates p38 Mitogen-activated Protein Kinase (MAPK) Signaling by Mediating Lysine 48-linked Ubiquitination and Degradation of Mitogen-activated Protein Kinase Kinase 6 (MKK6)

The p38 MAPK signal transduction pathway plays an important role in inflammatory and stress responses. MAPKK6 (MKK6), a dual specificity protein kinase, is a p38 activator. Activation of the MKK6-p38 pathway is kept in check by multiple layers of regulations, including autoinhibition, dimerization, scaffold proteins, and Lys-63-linked polyubiquitination. However, the mechanisms underlying deactivation of MKK6-p38, which is crucial for maintaining the magnitude and duration of signal transduction, are not well understood. Lys-48-linked ubiquitination, which marks substrates for proteasomal degradation, is an important negative posttranslational regulatory machinery for signal pathway transduction. Here we report that the accumulation of F-box only protein 31 (FBXO31), a component of Skp1·Cul1·F-box protein E3 ligase, negatively regulated p38 activation in cancer cells upon genotoxic stresses. Our results show that FBXO31 binds to MKK6 and mediates its Lys-48-linked polyubiquitination and degradation, thereby functioning as a negative regulator of MKK6-p38 signaling and protecting cells from stress-induced cell apoptosis. Taken together, our findings uncover a new mechanism of deactivation of MKK6-p38 and substantiate a novel regulatory role of FBXO31 in stress response.     

p38 MAPK信号转导途径在关节软骨细胞中的激活和作用

p38信号转导途径是MAPK途径的一种,软骨细胞是关节软骨中唯一的细胞成分。软骨细胞中的p38 MAPK可以被多种细胞因子、机械因素等所激活,它与软骨细胞表型的保持和分化、软骨细胞的肥大化和钙化、凋亡、软骨基质金属蛋白酶的合成、软骨炎性细胞因子的产生等有密切关系,可能在关节炎的发生发展中发挥了重要作用。     

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.     

p38 MAPK通路在脂多糖致呼吸道上皮损伤中的作用

脂多糖（Lipopolysaccharide,LPS）是革兰阴性杆菌细胞壁的主要组成成分,也是一种很强的炎症反应和氧化应激诱导剂。呼吸道上皮是机体防御外界细菌、病毒、香烟烟雾等生物和化学因素损伤的天然屏障,在维持呼吸道局部微环境稳态中可发挥重要作用,也是吸入性药物治疗的主要靶细胞。呼吸道上皮结构完整性缺陷或功能紊乱还参与了哮喘、慢性阻塞性肺疾病等多种肺部疾病的发生和发展。LPS可引起呼吸道上皮损伤,但其具体的分子机制目前尚不清楚。p38丝裂原活化蛋白激酶（P38mitogen-activated protein kinase,p38 MAPK）作为MAPK家族四个亚家族成员之一,包含四个成员：p38α、p38β、p38γ和p38δ,可通过经典和非经典的p38 MAPK信号通路激活方式及通过激酶活性无关的功能参与调控炎症反应、细胞生长、细胞分化和细胞死亡等多种病理生理过程。本文就p38 MAPK信号通路在LPS致呼吸道上皮损伤中的作用做一综述。     

p38 MAPK通路在脂多糖致呼吸道上皮损伤中的作用

脂多糖(Lipopolysaccharide,LPS)是革兰阴性杆菌细胞壁的主要组成成分,也是一种很强的炎症反应和氧化应激诱导剂。呼吸道上皮是机体防御外界细菌、病毒、香烟烟雾等生物和化学因素损伤的天然屏障,在维持呼吸道局部微环境稳态中可发挥重要作用,也是吸入性药物治疗的主要靶细胞。呼吸道上皮结构完整性缺陷或功能紊乱还参与了哮喘、慢性阻塞性肺疾病等多种肺部疾病的发生和发展。LPS可引起呼吸道上皮损伤,但其具体的分子机制目前尚不清楚。p38丝裂原活化蛋白激酶(P38mitogen-activated protein kinase,p38 MAPK)作为MAPK家族四个亚家族成员之一,包含四个成员:p38α、p38β、p38γ和p38δ,可通过经典和非经典的p38 MAPK信号通路激活方式及通过激酶活性无关的功能参与调控炎症反应、细胞生长、细胞分化和细胞死亡等多种病理生理过程。本文就p38 MAPK信号通路在LPS致呼吸道上皮损伤中的作用做一综述。     

Facilitation of Mitochondrial Outer and Inner Membrane Permeabilization and Cell Death in Oxidative Stress by a Novel Bcl-2 Homology 3 Domain Protein

We identified a sequence homologous to the Bcl-2 homology 3 (BH3) domain of Bcl-2 proteins in SOUL. Tissues expressed the protein to different extents. It was predominantly located in the cytoplasm, although a fraction of SOUL was associated with the mitochondria that increased upon oxidative stress. Recombinant SOUL protein facilitated mitochondrial permeability transition and collapse of mitochondrial membrane potential (MMP) and facilitated the release of proapoptotic mitochondrial intermembrane proteins (PMIP) at low calcium and phosphate concentrations in a cyclosporine A-dependent manner in vitro in isolated mitochondria. Suppression of endogenous SOUL by diced small interfering RNA in HeLa cells increased their viability in oxidative stress. Overexpression of SOUL in NIH3T3 cells promoted hydrogen peroxide-induced cell death and stimulated the release of PMIP but did not enhance caspase-3 activation. Despite the release of PMIP, SOUL facilitated predominantly necrotic cell death, as revealed by annexin V and propidium iodide staining. This necrotic death could be the result of SOUL-facilitated collapse of MMP demonstrated by JC-1 fluorescence. Deletion of the putative BH3 domain sequence prevented all of these effects of SOUL. Suppression of cyclophilin D prevented these effects too, indicating that SOUL facilitated mitochondrial permeability transition in vivo. Overexpression of Bcl-2 and Bcl-x_L, which can counteract the mitochondria-permeabilizing effect of BH3 domain proteins, also prevented SOUL-facilitated collapse of MMP and cell death. These data indicate that SOUL can be a novel member of the BH3 domain-only proteins that cannot induce cell death alone but can facilitate both outer and inner mitochondrial membrane permeabilization and predominantly necrotic cell death in oxidative stress.     

MAPK磷酸酯酶-1通过抑制JNK和p38的磷酸化调节SH-SY5Y神经母细胞瘤的凋亡

目的研究MKP-1在SH-SY5Y神经母细胞瘤中的抗凋亡。方法建立稳定表达MKP-1的SH-SY5Y细胞,用H2O2诱导细胞凋亡,并通过Western blotting比较分析MKP-1的表达对JNK和p38磷酸化的调节。结果①H2O2诱导SH-SY5Y细胞表达MKP-1,同时导致JNK和p38的去磷酸化;②在稳定表达MKP-1的SH-SY5Y细胞中,MKP-1可以抑制JNK和p38的磷酸化。③稳定表达MKP-1的SH-SY5Y细胞抵抗H2O2诱导细胞凋亡的能力比对照细胞提高了1倍左右。结论MKP-1对神经细胞的凋亡具有重要的调节作用,提示MKP-1作为调节ERK、JNK和p38蛋白激酶信号途径的重要分子,可能对退行性神经系统疾病的发病机制和治疗有重要的作用。     

盐敏感性高血压大鼠纹蛋白水平变化对醛固酮及p38MAPK通路的影响

摘要 目的：探讨盐敏感性高血压大鼠纹蛋白水平变化对醛固酮及p38MAPK通路的影响。方法：通过向新生大鼠皮下注射辣椒素和高盐饮食构建盐敏感性高血压大鼠模型,分别使用醛固酮（ALDO）和醛固酮、依普利酮联合给药,实验分组为：1）正常对照组;2）模型组;3）假手术组;4）ALDO组;5）ALDO+依普利酮组。通过ELISA检测大鼠血浆中ALDO含量和外周血肾素含量,通过qPCR和WB检测大鼠心脏、肾脏、血管平滑肌中striatin和p38 MAPK的mRNA和蛋白的表达变化。结果：造模后在给药前大鼠血压有不同程度的升高,给药后ALDO组和ALDO+ Eplerenone组血压均有一定程度的下降;给药后ALDO+Eplerenone组大鼠血浆中ALDO和外周血中肾素含量升高;在肾脏与Control组和Model组相比,ALDO+Eplerenone组的striatin和p38 MAPK蛋白表达水平显著升高,p38 MAPK的 mRNA表达水平显著升高;在心脏中与Model组相比,ALDO+Eplerenone组的p38 MAPK蛋白表达水平显著升高;在主动脉中与Control组和Model组相比,ALDO+Eplerenone组的striatin和p38 MAPK蛋白表达水平显著降低。结论：纹蛋白水平的改变与盐敏感高血压具有相关性,其可通过调节ALDO/MR→p38MAPK相关通路影响盐敏感性高血压。     

Phosphorylation of Raptor by p38beta participates in arsenite-induced mammalian target of rapamycin complex 1 (mTORC1) activation

Cell growth is influenced by environmental stress. Mammalian target of rapamycin (mTOR), the central regulator of cell growth, can be positively or negatively regulated by various stresses through different mechanisms. The p38 MAP kinase pathway is essential in cellular stress responses. Activation of MK2, a downstream kinase of p38α, enhances mTOR complex 1 (mTORC1) activity by preventing TSC2 from inhibiting mTOR activation. The p38β-PRAK cascade targets Rheb to inhibit mTORC1 activity upon glucose depletion. Here we show the activation of p38β participates in activation of mTOR complex 1 (mTORC1) induced by arsenite but not insulin, nutrients, anisomycin, or H(2)O(2). Arsenite treatment of cells activates p38β and induces interaction between p38β and Raptor, a regulatory component of mTORC1, resulting in phosphorylation of Raptor on Ser(863) and Ser(771). The phosphorylation of Raptor on these sites enhances mTORC1 activity, and contributes largely to arsenite-induced mTORC1 activation. Our results shown here and in previous work demonstrate that the p38 pathway can regulate different components of the mTORC1 pathway, and that p38β can target different substrates to either positively or negatively regulate mTORC1 activation when a cell encounters different environmental stresses.     

Glial Fibrillary Acidic Protein is Greatly Modified by Oxidative Stress in Aceruloplasminemia Brain

Aceruloplasminemia is an autosomal recessive disorder of iron metabolism caused by mutations in the ceruloplasmin (Cp) gene. The neuropathological hallmark of this disease is intracellular iron overload, which is thought to lead to neuronal cell death through increased oxidative stress. We evaluated and characterized protein oxidation in the brain of a patient with this disease. The protein carbonyl content in the cerebral cortex of the patient was elevated compared to controls. Furthermore, peptide mass fingerprinting and partial amino acid sequencing identified glial fibrillary acidic protein (GFAP) as the major carbonylated protein in the cerebral cortex of the patient. In conjunction with the facts that Cp mainly localizes to astrocytes in the central nervous system and that astrocytes are loaded with much more iron than neurons in the cerebral cortex, our findings indicate that Cp deficiency may primarily damage astrocytes. We speculate that the dysfunction of astrocytes may be causatively related to neuronal cell loss in aceruloplasminemia.     

Mitochondrial Glutathione Transport Is a Key Determinant of Neuronal Susceptibility to Oxidative and Nitrosative Stress

Mitochondrial oxidative stress significantly contributes to the underlying pathology of several devastating neurodegenerative disorders. Mitochondria are highly sensitive to the damaging effects of reactive oxygen and nitrogen species; therefore, these organelles are equipped with a number of free radical scavenging systems. In particular, the mitochondrial glutathione (GSH) pool is a critical antioxidant reserve that is derived entirely from the larger cytosolic pool via facilitated transport. The mechanism of mitochondrial GSH transport has not been extensively studied in the brain. However, the dicarboxylate (DIC) and 2-oxoglutarate (OGC) carriers localized to the inner mitochondrial membrane have been established as GSH transporters in liver and kidney. Here, we investigated the role of these carriers in protecting neurons from oxidative and nitrosative stress. Immunoblot analysis of DIC and OGC in primary cultures of rat cerebellar granule neurons (CGNs) and cerebellar astrocytes showed differential expression of these carriers, with CGNs expressing only DIC and astrocytes expressing both DIC and OGC. Consistent with these findings, butylmalonate specifically reduced mitochondrial GSH in CGNs, whereas both butylmalonate and phenylsuccinate diminished mitochondrial GSH in astrocytes. Moreover, preincubation with butylmalonate but not phenylsuccinate significantly enhanced susceptibility of CGNs to oxidative and nitrosative stressors. This increased vulnerability was largely prevented by incubation with cell-permeable GSH monoethylester but not malate. Finally, knockdown of DIC with adenoviral siRNA also rendered CGNs more susceptible to oxidative stress. These findings demonstrate that maintenance of the mitochondrial GSH pool via sustained mitochondrial GSH transport is essential to protect neurons from oxidative and nitrosative stress.