p53蛋白在周期调节蛋白A1变异引起的雄性小鼠生殖细胞凋亡中的作用

为研究p5 3蛋白在周期调节蛋白A1(cyclinA1)变异引起的雄性小鼠生殖细胞凋亡中的作用 ,以p5 3基因敲除的小鼠和周期调节蛋白A1基因敲除的小鼠杂交 ,获取同胎生单基因变异和双基因同时变异的雄性后代共 4组 12只 .比较它们的性腺和生殖细胞发育 ,并用TUNEL染色法观察和比较生殖细胞的凋亡情况 .在睾丸最大横切面上观察到 :周期调节蛋白A1变异组凋亡细胞最多 (348± 10 4个 ) ,明显高于p5 3 周期调节蛋白A1双基因变异组 (12 1± 38个 ) ,t=3 2 5 79,P =0 0 4 72 .p5 3变异组凋亡细胞最少 (45± 2 4个 ) ,配对t检验显示有非常显著性差异 ,t=8 4 0 13,P =0 0 0 35 .这一研究结果提示 ,p5 3基因可能在雄性生殖细胞的发育中起监视作用 ,并在周期调节蛋白A1变异引起发育异常时启动p5 3途径造成异常细胞的凋亡 .     

Rotenone Directly Induces BV2 Cell Activation via the p38 MAPK Pathway

Parkinson’s disease (PD) is the second most common neurodegenerative disease. Although its pathogenesis is still unclear, increasing evidence suggests that mitochondrial dysfunction induced by environmental toxins, such as mitochondrial complex I inhibitors, plays a significant role in the disease process. The microglia in PD brains are highly activated, and inflammation is also an essential element in PD pathogenesis. However, the means by which these toxins activate microglia is still unclear. In the present study, we found that rotenone, a mitochondrial complex I inhibitor, could directly activate microglia via the nuclear factor kappa B (NF-κB) signaling pathway, thereby inducing significantly increased expression of inflammatory cytokines. We further observed that rotenone induced caspase-1 activation and mature IL-1β release, both of which are strictly dependent on p38 mitogen-activated protein kinase (MAPK). The activation of p38 is associated with the presence of reactive oxygen species (ROS) produced by rotenone. Removal of these ROS abrogated the activation of the microglia. Therefore, our data suggest that the environmental toxin rotenone can directly activate microglia through the p38 MAPK pathway.     

Hypertonicity-induced p38MAPK Activation Elicits Recovery of Corneal Epithelial Cell Volume and Layer Integrity

In hypertonicity-stressed (i.e., 600 mOsm) SV40-immortalized rabbit and human corneal epithelial cell layers (RCEC and HCEC, respectively), we characterized the relationship between time-dependent changes in translayer resistance, relative cell volume and modulation of MAPK superfamily activities. Sulforhodamine B permeability initially increased by 1.4- and 2-fold in RCEC and HCEC, respectively. Subsequently, recovery to its isotonic level only occurred in RCEC. Light scattering revealed that in RCEC 1) regulatory volume increase (RVI) extent was 20% greater; 2) RVI half-time was 2.5-fold shorter. However, inhibition of Na-K-2Cl cotransporter and Na/K-ATPase activity suppressed the RVI response more in HCEC. MAPK activity changes were as follows: 1) p38 was wave-like and faster as well as larger in RCEC than in HCEC (90- and 18-fold, respectively); 2) increases in SAPK/JNK activity were negligible in comparison to those of p38; 3) Erk1/2 activity declined to 30-40% of their basal values. SB203580, a specific p38 inhibitor, dose dependently suppressed the RVI responses in both cell lines. However, neither U0126, which inhibits MEK, the kinase upstream of Erk, nor SP600125, inhibitor of SAPK/JNK, had any effect on this response. Taken together, sufficient activation of the p38 limb of the MAPK superfamily during a hypertonic challenge is essential for maintaining epithelial cell volume and translayer resistance. On the other hand, Erk1/2 activity restoration seems to be dependent on cell volume recovery.     

Activation of p38MAPK contributes to expanded polyglutamine-induced cytotoxicity

Background

The signaling pathways that may modulate the pathogenesis of diseases induced by expanded polyglutamine proteins are not well understood.

Methodologies/Principal Findings

Herein we demonstrate that expanded polyglutamine protein cytotoxicity is mediated primarily through activation of p38MAPK and that the atypical PKC iota (PKCι) enzyme antagonizes polyglutamine-induced cell death through induction of the ERK signaling pathway. We show that pharmacological blockade of p38MAPK rescues cells from polyglutamine-induced cell death whereas inhibition of ERK recapitulates the sensitivity observed in cells depleted of PKCι by RNA interference. We provide evidence that two unrelated proteins with expanded polyglutamine repeats induce p38MAPK in cultured cells, and demonstrate induction of p38MAPK in an in vivo model of neurodegeneration (spinocerebellar ataxia 1, or SCA-1).

Conclusions/Significance

Taken together, our data implicate activated p38MAPK in disease progression and suggest that its inhibition may represent a rational strategy for therapeutic intervention in the polyglutamine disorders.     

Activation by 2-arachidonoylglycerol of platelet p38MAPK/cPLA2 pathway

The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) is described as a platelet agonist able to induce aggregation and to increase intracellular calcium. In the present report we have confirmed these data and demonstrated that the inhibitor of p38MAPK SB203580 and the inhibitor of cPLA(2) metabolism ETYA affect both these parameters. Thus, we aimed to define the role of p38MAPK/cytosolic phospholipase A(2) (cPLA(2)) pathway in 2-AG-induced human platelet activation. p38MAPK activation was assayed by phosphorylation. cPLA(2) activation was assayed by phosphorylation and as arachidonic acid release and thromboxane B(2) formation. It was shown that 2-AG in a dose- and time-dependent manner activates p38MAPK peaking at 10 μM after 1 min of incubation. The 2-AG effect on p38MAPK was not impaired by apyrase, indomethacin or RGDS peptide but it was significantly reduced by SR141716, specific inhibitor of type-1 cannabinoid receptor and unaffected by the specific inhibitor of type-2 cannabinoid receptor SR144528. Moreover, the incubation of platelets with 2-AG led to the phosphorylation of cPLA(2) and its activation. Platelet pretreatment with SB203580, inhibitor of p38MAPK, abolished both cPLA(2) phosphorylation and activation. In addition SR141716 strongly impaired cPLA(2) phosphorylation, arachidonic acid release and thromboxane B(2) formation, whereas SR144528 did not change these parameters. Finally platelet stimulation with 2-AG led to an increase in free oxygen radical species. In conclusion, data provide insight into the mechanisms involved in platelet activation by 2-AG, indicating that p38MAPK/cPLA(2) pathway could play a relevant role in this complicated process.     

Endothelium-Derived 5-Methoxytryptophan Protects Endothelial Barrier Function by Blocking p38 MAPK Activation

The endothelial junction is tightly controlled to restrict the passage of blood cells and solutes. Disruption of endothelial barrier function by bacterial endotoxins, cytokines or growth factors results in inflammation and vascular damage leading to vascular diseases. We have identified 5-methoxytryptophan (5-MTP) as an anti-inflammatory factor by metabolomic analysis of conditioned medium of human fibroblasts. Here we postulated that endothelial cells release 5-MTP to protect the barrier function. Conditioned medium of human umbilical vein endothelial cells (HUVECs) prevented endothelial hyperpermeability and VE-cadherin downregulation induced by VEGF, LPS and cytokines. We analyzed the metabolomic profile of HUVEC conditioned medium and detected 5-MTP but not melatonin, serotonin or their catabolites, which was confirmed by enzyme-linked immunosorbent assay. Addition of synthetic pure 5-MTP preserved VE-cadherin and maintained barrier function despite challenge with pro-inflammatory mediators. Tryptophan hydroxylase-1, an enzyme required for 5-MTP biosynthesis, was downregulated in HUVECs by pro-inflammatory mediators and it was accompanied by reduction of 5-MTP. 5-MTP protected VE-cadherin and prevented endothelial hyperpermeability by blocking p38 MAPK activation. A chemical inhibitor of p38 MAPK, SB202190, exhibited a similar protective effect as 5-MTP. To determine whether 5-MTP prevents vascular hyperpermeability in vivo, we evaluated the effect of 5-MTP administration on LPS-induced murine microvascular permeability with Evans blue. 5-MTP significantly prevented Evans blue dye leakage. Our findings indicate that 5-MTP is a new class of endothelium-derived molecules which protects endothelial barrier function by blocking p38 MAPK.     

Activation of p38 MAPK by oxidative stress underlying epirubicin-induced vascular endothelial cell injury

Epirubicin, an anthracycline antitumor drug, often causes vascular injury such as vascular pain, phlebitis, and necrotizing vasculitis. However, an effective prevention for the epirubicin-induced vascular injury has not been established. The purpose of this study is to identify the mechanisms of cell injury induced by epirubicin in porcine aorta endothelial cells (PAECs). PAECs were exposed to epirubicin for 10 min followed by further incubation without epirubicin. The exposure to epirubicin (3-30 μM) decreased the cell viability concentration and time dependently. Epirubicin increased the activity of caspase-3/7, apoptotic cells, and intracellular lipid peroxide levels, and also induced depolarization of mitochondrial membranes. These intracellular events were reversed by glutathione (GSH) and N-acetylcysteine (NAC), while epirubicin rather increased intracellular GSH slightly and L-buthionine-(S,R)-sulfoximine, a specific inhibitor of GSH synthesis, had no effect on the epirubicin-induced cell injury. The epirubicin-induced cell injury and increase of caspase-3/7 activity were also attenuated by p38 mitogen-activated protein kinase (MAPK) inhibitors, SB203580 and PD169316. Moreover, epirubicin significantly enhanced the phosphorylation of p38 MAPK, and these effects were attenuated by GSH and NAC. In contrast, a c-Jun N-terminal kinase inhibitor SP600125, an extracellular signal-regulated kinase inhibitor PD98059, and a p53 inhibitor pifithrin α did not affect the epirubicin-induced cell injury and increase of caspase-3/7 activity. These results indicate that an activation of p38 MAPK by oxidative stress is involved in the epirubicin-induced endothelial cell injury.     

Thrombin Enhances NGF-Mediated Neurite Extension via Increased and Sustained Activation of p44/42 MAPK and p38 MAPK

Rapid neurite remodeling is fundamental to nervous system development and plasticity. It involves neurite extension that is regulated by NGF through PI3K/AKT, p44/42 MAPK and p38 MAPK. It also involves neurite retraction that is regulated by the serine protease, thrombin. However, the intracellular signaling pathway by which thrombin causes neurite retraction is unknown. Using the PC12 neuronal cell model, we demonstrate that thrombin utilizes the PI3K/AKT pathway for neurite retraction in NGF-differentiated cells. Interestingly, however, we found that thrombin enhances NGF-induced neurite extension in differentiating cells. This is achieved through increased and sustained activation of p44/42 MAPK and p38 MAPK. Thus, thrombin elicits opposing effects in differentiated and differentiating cells through activation of distinct signaling pathways: neurite retraction in differentiated cells via PI3K/AKT, and neurite extension in differentiating cells via p44/42 MAPK and p38 MAPK. These findings, which also point to a novel cooperative role between thrombin and NGF, have significant implications in the development of the nervous system and the disease processes that afflicts it as well as in the potential of combined thrombin and NGF therapy for impaired learning and memory, and spinal cord injury which all require neurite extension and remodeling.     

Biphasic Activation of p38MAPK Suggests That Apoptosis Is a Downstream Event in Pemphigus Acantholysis

In pemphigus vulgaris and pemphigus foliaceus (PF), autoantibodies against desmoglein-3 and desmoglein-1 induce epidermal cell detachment (acantholysis) and blistering. Activation of keratinocyte intracellular signaling pathways is emerging as an important component of pemphigus IgG-mediated acantholysis. We previously reported activation of p38 mitogen-activated protein kinase (MAPK) in response to pathogenic pemphigus vulgaris and PF IgG. Inhibition of p38MAPK blocked pemphigus IgG-induced cytoskeletal reorganization in tissue culture and blistering in pemphigus mouse models. We now extend these observations by demonstrating two peaks of p38MAPK activation in pemphigus tissue culture and mouse models. Administration of the p38MAPK inhibitor SB202190 before PF IgG injection blocked both peaks of p38MAPK phosphorylation and blister formation, consistent with our previous findings; however, administration of the inhibitor 4 h after PF IgG injection blocked only the later peak of p38MAPK activation but failed to block blistering. Examination of the temporal relationship of p38MAPK phosphorylation and apoptosis showed that apoptosis occurs at or after the second peak of p38MAPK activation. The time course of p38MAPK activation and apoptotic markers, as well as the ability of inhibitors of p38MAPK to block activation of the proapoptotic proteinase caspase-3, suggest that activation of apoptosis is downstream to, and a consequence of, p38MAPK activation in pemphigus acantholysis. Furthermore, these observations suggest that the earlier peak of p38MAPK activation is part of the mechanism leading to acantholysis, whereas the later peak of p38MAPK and apoptosis may not be essential for acantholysis.Pemphigus is a group of related autoimmune diseases characterized by blistering in the skin. The histologic hallmark of these disorders is termed acantholysis, which describes the loss of adhesion between adjacent epithelial cells. The two major variants are pemphigus foliaceus (PF)² and pemphigus vulgaris (PV). In PF, acantholysis is observed beneath the stratum corneum and within the granular layer of epidermal epithelia, whereas in PV, blister formation occurs above the basal layer of epidermal epithelia and mucosal epithelium. Passive transfer of IgG purified from both PV and PF patient sera reproduces the clinical, histological, and immunologic features of the human diseases, demonstrating that these autoantibodies are pathogenic (1, 2). In PF, autoantibodies target the desmosomal cadherin desmoglein (dsg) 1, whereas in PV, autoantibodies initially target dsg3 (3, 4) in mucosal PV and then subsequently target both dsg1 and dsg3 in mucocutaneous PV (5-7).The mechanism by which pemphigus autoantibodies induce blistering has been under investigation. Work from a number of laboratories has suggested that activation of intracellular events is induced by binding of PF or PV IgG to dsg1 and dsg3, respectively (8-14). Previously, we have reported that PV IgG activate p38MAPK and heat shock protein (HSP) 27 in human keratinocyte tissue cultures (15). Significantly, p38MAPK inhibitors blocked PV IgG-induced keratin filament retraction and actin reorganization in human keratinocyte tissue cultures. Furthermore, we have demonstrated that both PV and PF IgG induce phosphorylation of p38MAPK and HSP25, the murine HSP27 homologue, in mouse models and that inhibitors of p38MAPK block blistering in both the PV (16) and the PF (17) passive transfer mouse models. Additionally, in human skin biopsies from both PV and PF patients, phosphorylation of p38MAPK and HSP27 has been observed (18). Collectively, these observations suggest that activation of p38MAPK within the target keratinocyte contributes directly to loss of cell-cell adhesion induced by pemphigus autoantibodies.Both p38MAPK and HSP27 have been implicated in the regulation of the intermediate filament and actin cytoskeletons (19-25); the ability of p38MAPK inhibitors to block both pemphigus IgG-activated cytoskeletal reorganization and pemphigus IgG-activated blistering suggests that p38MAPK may be acting upstream of the cytoskeleton in the mechanism of acantholysis; however, p38MAPK signaling has been implicated in other cellular responses (reviewed in Ref. 26). For example, there is abundant evidence for p38MAPK involvement in apoptosis (27-29); however, the role of p38MAPK in apoptosis seems to be cell type- and stimulus-dependent. Although p38MAPK signaling promotes cell death in some cell lines, it also functions to enhance survival, growth, and differentiation in other cell lines (30). Several reports describe increased apoptosis of keratinocytes in pemphigus (31-35); however, the relationship between PV IgG-mediated p38MAPK signaling, the induction of apoptosis, and the relationship of apoptosis to blistering has not been defined. This study was undertaken to investigate the relationship between p38MAPK activation, apoptosis, and acantholysis.     

Regulation of autophagy by p38α MAPK

Autophagy is induced in mammalian cells by nutrient deprivation, which acts through repression of the protein kinase mammalian target of rapamycin (mTOR) and may involve other unknown mechanisms. Mitogen-activated protein kinases (MAPKs), and in particular p38 MAPK, are implicated in amino acid signalling. Furthermore, the extracellular signal-regulated kinase (ERK) and p38 regulate autophagy in response to various stimuli. However, the molecular mechanisms of p38 regulation of autophagy are still widely unknown. Our recent data suggest that p38α MAPK negatively regulates the interaction of mAtg9 and a novel mAtg9 binding partner, p38IP, to control the levels of autophagy induced in response to starvation.     

亚砷酸盐诱导HSP27的细胞内移位依赖于p38 MAPK介导的磷酸化

为研究HSP27的磷酸化与其细胞内定位之间的关系,利用定点突变和DNA重组技术构建EGFP融合的HSP27野生型和第82位丝氨酸突变体的真核表达载体并转染NIH 3T3细胞,观察两者在静息状态和亚砷酸盐刺激下的细胞内定位情况.利用p38 MAPK特异性抑制剂SB203580预处理细胞后,观察对HSP27磷酸化和细胞内定位的影响.结果发现,野生型HSP27受到NaAsO₂刺激后移位入核,而其突变体HSP27(S82A)不能入核.同时,SB203580的预处理使HSP27的磷酸化和NaAsO₂诱导的移位入核都被阻断.这些结果表明,p38介导的HSP27磷酸化在其细胞内定位中具有重要作用     

Cell size homeostasis is maintained by CDK4-dependent activation of p38 MAPK

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p38MAPK介导的胶质细胞iNOS的转录激活机制

丝裂原激活蛋白激酶(MAPK)酶级联反应系统参与胶质细胞中iNOS的合成.通过瞬时转染p38MAPK途径中上游激酶,MAPK激酶3(MKK3)和MAPK激酶6 (MKK6 )表达质粒,进一步了解p38MAPK级联传导信号系统调节iNOS基因在胶质细胞中的转录激活机制.MKK3或MKK6表达质粒与接有荧光素酶(luciferase ,Luc)的大鼠iNOS启动基因质粒(iNOS Luc)联合转染C6星形胶质细胞株引起iNOS Luc的激活,并且使细胞因子诱导的iNOSmRNA的表达增强.这两种效应都能够被p38MAPK抑制剂SB2 0 35 80所抑制.MKK3 6也可以诱导核因子κB(NFκB Luc)依赖的转录活性.这些分子水平的研究结果为p38MAPK信号级联传导途径在调节大鼠胶质细胞中iNOS基因转录激活中的重要作用,包括转录因子NFκB的作用提供了证据.通过阻断iNOS表达或NO的生成,抑制细胞炎症发生,为防治神经细胞炎症反应性疾病提供实验依据.