Inhibition of nucleoside transport by p38 MAPK inhibitors

While investigating the ability of p38 MAPK to regulate cytarabine (Ara C)-dependent differentiation of erythroleukemia K562 cells, we observed effects that indicated that the imidazoline class of p38 MAPK inhibitors prevented nucleoside transport. Incubation of K562 cells with SB203580, SB203580-iodo, or SB202474, an analogue of SB203580 that does not inhibit p38 MAPK activity, inhibited the uptake of [3H]Ara C or [3H]uridine and the differentiation of K562 cells. Consistent with the effects of these compounds on the nitrobenzylthioinosine (NBMPR)-sensitive equilibrative nucleoside transporter (ENT1), incubation with SB203580 or SB203580-iodo eliminated the binding of [3H]NBMPR to K562 cells or membranes isolated from human erythrocytes. Furthermore, using a uridine-dependent cell type (G9c), we observed that SB203580 or SB203580-iodo efficiently inhibited the salvage synthesis of pyrimidine nucleotides in vivo. Thus these studies demonstrate that the NBMPR-sensitive equilibrative nucleoside transporters are novel and unexpected targets for the p38 MAPK inhibitors at concentrations typically used to inhibit protein kinases.     

高碳酸血症对大鼠机械通气肺损伤时炎症因子和p38MAPK 表达的影响

目的:探讨高碳酸血症对大鼠机械通气性肺损伤(VILI)时炎症因子和p38MAPK表达的影响。方法:健康雄性Wistar大鼠30只,体重220～280g,采用随机数字表法,将大鼠随机分3组(n=10):对照组(C组)、机械通气肺损伤组(V组)和高碳酸血症组(H组)。C组保留自主呼吸,V组和H组行机械通气4 h。采用高气道压机械通气模式制备机械通气性肺损伤模型。H组通过调整吸入的CO2浓度来维持动脉血PaCO2分别为80～100mmHg。机械通气结束时,测定支气管肺泡灌洗液(BALF)中总蛋白、TNF-α和巨噬细胞炎症蛋白-2(MIP-2)的浓度;取肺组织,测定湿干重比(W/D比)、细胞间粘附分子(ICAM-1)和p38MAPK蛋白的表达水平以及p38MAPK的活性,并观察病理学结果,进行肺损伤评分。结果:与C组比较,V组肺损伤评分、W/D比、ICAM-1表达水平、BALF中总蛋白浓度、TNF-α和MIP-2浓度和肺组织p38MAPK活性升高,PaO2降低(P<0.05);与V组比较,H组肺损伤评分、W/D比、ICAM-1表达水平、BALF中总蛋白浓度、TNF-α和MIP-2浓度和肺组织p38MAPK活性降低,PaO2升高(P<0.05)。结论:高碳酸血症通过调节p38MAPK的表达,从而抑制炎症反应减轻大鼠机械通气肺损伤。     

One-way cross-talk between p38(MAPK) and p42/44(MAPK). Inhibition of p38(MAPK) induces low density lipoprotein receptor expression through activation of the p42/44(MAPK) cascade.

In this paper, we report that SB202190 alone, a specific inhibitor of p38(MAPK), induces low density lipoprotein (LDL) receptor expression (6-8-fold) in a sterol-sensitive manner in HepG2 cells. Consistent with this finding, selective activation of the p38(MAPK) signaling pathway by expression of MKK6b(E), a constitutive activator of p38(MAPK), significantly reduced LDL receptor promoter activity. Expression of the p38(MAPK) alpha-isoform had a similar effect, whereas expression of the p38(MAPK) betaII-isoform had no significant effect on LDL receptor promoter activity. SB202190-dependent increase in LDL receptor expression was accompanied by induction of p42/44(MAPK), and inhibition of this pathway completely prevented SB202190-induced LDL receptor expression, suggesting that p38(MAPK) negatively regulates the p42/44(MAPK) cascade and the responses mediated by this kinase. Cross-talk between these kinases appears to be one-way because modulation of p42/44(MAPK) activity did not affect p38(MAPK) activation by a variety of stress inducers. Taken together, these findings reveal a hitherto unrecognized one-way communication that exists between p38(MAPK) and p42/44(MAPK) and provide the first evidence that through the p42/44(MAPK) signaling cascade, the p38(MAPK) alpha-isoform negatively regulates LDL receptor expression, thus representing a novel mechanism of fine tuning cellular levels of cholesterol in response to a diverse set of environmental cues.     

Inhibition of p38 MAPK facilitates ex vivo expansion of skin epithelial progenitor cells

Ex vivo expansion of skin epithelial stem cells has long attracted great interest because of the potential utilization in transplantation and gene therapy. The use of cultured stem or progenitor cells was limited by the lack of applicable culturing system with both satisfactory expansion efficacy and well suppressed differentiation ex vivo. The p38 mitogen-activated protein kinase (MAPK) pathways are responsible for cell growth and differentiation process. We investigated the function of p38 inhibitor SB203580 in the ex vivo expansion of skin epithelial progenitor cells by comparing media with or without addition of this inhibitor. Our results showed that the culturing medium with murine 3T3 feeder layers added with 10 μM SB203580 was more effective in promoting clonal growth of human skin epithelial progenitors or stem cells than the conventional medium without SB203580. The clone initial day in cells treated with 10 μM SB203580 came 2 d earlier with higher colony formation efficiency. The skin epithelial progenitor cells treated with 10 μM SB203580 formed clones that were uniformly smaller in size, longer in sustained proliferation, shorter in clone doubling time, higher in S-phase cells percentage, and lower in levels of differentiation markers such as K10 along with higher levels of stem-cell-associated markers such as p63, K15, and ABCG2 than those cultured in the conventional medium. Collectively, these results indicate that the p38 MAPK pathways inhibitor SB203580 can be used as a culture medium additive that helps to achieve more effective ex vivo expansion of skin epithelial progenitor cells.     

Desmosome signaling. Inhibition of p38MAPK prevents pemphigus vulgaris IgG-induced cytoskeleton reorganization

In the human autoimmune blistering disease pemphigus vulgaris (PV) pathogenic antibodies bind the desmosomal cadherin desmoglein-3 (dsg3), causing epidermal cell-cell detachment (acantholysis). Pathogenic PV dsg3 autoantibodies were used to initiate desmosome signaling in human keratinocyte cell cultures. Heat shock protein 27 (HSP27) and p38MAPK were identified as proteins rapidly phosphorylated in response to PV IgG. Inhibition of p38MAPK activity prevented PV IgG-induced HSP27 phosphorylation, keratin filament retraction, and actin reorganization. These observations suggest that PV IgG binding to dsg3 activates desmosomal signal transduction cascades leading to (i) p38MAPK and HSP27 phosphorylation and (ii) cytoskeletal reorganization, supporting a mechanistic role for signaling in PV IgG-induced acantholysis. Targeting desmosome signaling via inhibition of p38MAPK and HSP27 phosphorylation may provide novel treatments for PV and other desmosome-associated blistering diseases.     

The regulation of Th1 responses by the p38 MAPK

IL-12 is a dimeric cytokine that is produced primarily by APCs. In this study we examined the role that the p38 MAPKs (MAPK/p38) play in regulating IL-12 production. We show that inhibition of p38 dramatically increased IL-12 production upon stimulation, while decreasing TNF-α. This reciprocal effect on these two cytokines following MAPK/p38 inhibition occurred in many different APCs, following a variety of different stimuli. IL-12 production was also increased in macrophages treated with small interfering RNA to limit p38α expression, and in macrophages deficient in MKK3, a kinase upstream of p38. The increase in IL-12 production following MAPK/p38 inhibition appears to be due to enhanced IL-12 (p40) mRNA stability. We show that MAPK/p38 inhibition can promote Th1 immune responses and thereby enhance vaccine efficacy against leishmaniasis. In a mouse model of Leishmania major infection, vaccination with heat-killed L. major plus CpG and SB203580 elicited complete protection against infection compared with heat-killed L. major plus CpG without SB203580. Thus, this work suggests that MAPK/p38 inhibitors may be applied as adjuvants to bias immune responses and improve vaccinations against intracellular pathogens.     

The suppressive role of p38 MAPK in cellular vacuole formation

Vacuolization of the cytoplasm is one of the dramatic and frequently observed phenomena in various cell types. Cellular vacuoles occur spontaneously or via a wide range of inductive stimuli, but the molecular mechanism involved in this process remains largely unknown. In this study, we investigated the role of the p38 and JNK pathways in the formation of cytoplasmic vacuoles. We found that p38 and JNK agonist anisomycin abolishes spontaneous cytoplasmic vacuolization of HepG2 cells through p38 activation, but not through JNK activation. Importantly, blocking the activity of p38 or suppression the expression of p38 elicits cytoplasmic vacuoles formation in various cancer cells. Furthermore, cytoplasmic vacuoles induced by p38 blocking are derived from the perinuclear region. These observations provide direct evidence for a role of p38 signaling in regulating the formation of cytoplasmic vacuoles. J. Cell. Biochem. 114: 1789–1799, 2013. © 2013 Wiley Periodicals, Inc.     

Inhibition of cross-bridge formation has no effect on contraction-associated phosphorylation of p38 MAPK in mouse skeletal muscle

Mitogen-activated protein kinases (MAPKs), in particular p38 MAPK, are phosphorylated in response to contractile activity, yet the mechanism for this is not understood. We tested the hypothesis that the force of contraction is responsible for p38 MAPK phosphorylation in skeletal muscle. Extensor digitorum longus (EDL) muscles isolated from adult male Swiss Webster mice were stimulated at fixed length at 10 Hz for 15 min and then subjected to Western blot analysis for the phosphorylation of p38 MAPK and ERK1/2. Contralateral muscles were fixed at resting length and were not stimulated. Stimulated muscles showed a 2.5-fold increase in phosphorylated p38 MAPK relative to nonstimulated contralateral controls, and there was no change in the phosphorylation of ERK1/2. When contractile activity was inhibited with N-benzyl-p-toluene sulfonamide (BTS), a specific inhibitor of actomyosin ATPase, force production decreased in both a time- and concentration-dependent manner. Preincubation with 25, 75, and 150 µM BTS caused 78 ± 4%, 97 ± 0.2%, and 99 ± 0.2% inhibition in contractile force, respectively, and was stable after 30 min of treatment. Fluorescence measurements demonstrated that Ca²⁺ cycling was minimally affected by BTS treatment. Surprisingly, BTS did not suppress the level of p38 MAPK phosphorylation in stimulated muscles. These data do not support the view that force generation per se activates p38 MAPK and suggest that other events associated with contraction must be responsible. calcium; energetics; mechanical stress; metabolism     

Dancing with p53: The role of p38MAPK in mitosis of p53-deficient tetraploid cells

Comment on: Vitale I, et al. Cell Cycle 2010; 9:2823-9.     

抑制p38MAPK信号通路对Bpiv3复制的影响

由牛副流感病毒3型(Bovine parainfluenza virus type 3,Bpiv3)感染引起的牛副流感病已成为各国牛场最重要的传染病之一,每年都会给世界养牛业造成巨大的经济损失,但关于该病致病的分子机制研究较少。本研究通过观察Bpiv3感染对MDBK细胞中丝裂原活化蛋白激酶(MKK3)及其下游分子p38丝裂酶原活化的蛋白激酶(p38MAPK)的表达的影响,探讨相关的信号转导机制,对p38 MAPK通路在Bpiv3感染过程中的作用进行了初步研究。Bpiv3感染细胞后,采用Western Blot检测MKK3,p38 MAPK在蛋白水平的表达变化,并采用ELISA法检测细胞上清中IL-6,IL-8,IL-13和TNF-α的水平变化,采用SPSS 12软件进行统计学分析。结果表明,Bpiv3在感染后能够诱导MKK3的激活以及p38的磷酸化,激活了p38 MAPK信号通路。而且p38 MAPK信号通路参与了Bpiv3的复制过程。ELISA检测Bpiv3感染后以及使用抑制剂SB202190处理后的细胞上清中IL-6、IL-8、IL-13和TNF-α的水平发现,p38 MAPK信号通路参与了Bpiv3诱导的炎症反应。研究证实Bpiv3感染能够激活p38 MAPK通路,显著上调MKK3的表达并诱导p38发生磷酸化,进一步激活下游分子发挥生物学活性,促进Bpiv3的复制及诱导促炎细胞因子的产生。p38 MAPK信号通路的激活可能是Bpiv3感染诱发炎症反应的机制之一。     

低剂量顺铂通过p38MAPK介导的DNMT1下调降低p21与p16启动子甲基化上调p21与p16表达

低剂量顺铂可通过诱导p21与p16表达而诱导肿瘤细胞早衰,但其机制不明。本研究探讨了低剂量顺铂诱导的HeLa细胞衰老过程中p21与p16的上调机制。低剂量顺铂（4 μmol/L）处理HeLa细胞后,DNA甲基转移酶DNMT1蛋白水平降低;p21与p16启动子甲基化水平降低,二者mRNA及蛋白质水平升高;顺铂对DNMT1蛋白水平的降低作用与其激活p38MAPK有关,用SB203580抑制p38MAPK可部分逆转顺铂对DNMT1蛋白水平以及p21与p16启动子甲基化的降低作用,从而部分逆转顺铂对p21与p16表达的诱导;抑制p38MAPK 也可部分逆转低剂量顺铂诱导的HeLa细胞早衰。上述结果表明,低剂量顺铂可通过p38MAPK信号通路下调p21与p16启动子甲基化水平,进而上调二者的表达。这些结果为解析低剂量顺铂诱导肿瘤细胞早衰的信号转导机制提供了实验依据。     

Inhibition of p38 MAPK Signaling Augments Skin Tumorigenesis via NOX2 Driven ROS Generation

p38 mitogen-activated protein kinases (MAPKs) respond to a wide range of extracellular stimuli. While the inhibition of p38 signaling is implicated in the impaired capacity to repair ultraviolet (UV)-induced DNA damage—a primary risk factor for human skin cancers—its mechanism of action in skin carcinogenesis remains unclear, as both anti-proliferative and survival functions have been previously described. In this study, we utilized cultured keratinocytes, murine tumorigenesis models, and human cutaneous squamous cell carcinoma (SCC) specimens to assess the effect of p38 in this regard. UV irradiation of normal human keratinocytes increased the expression of all four p38 isoforms (α/β/γ/δ); whereas irradiation of p53-deficient A431 keratinocytes derived from a human SCC selectively decreased p38α, without affecting other isoforms. p38α levels are decreased in the majority of human cutaneous SCCs assessed by tissue microarray, suggesting a tumor-suppressive effect of p38α in SCC pathogenesis. Genetic and pharmacological inhibition of p38α and in A431 cells increased cell proliferation, which was in turn associated with increases in NAPDH oxidase (NOX2) activity as well as intracellular reactive oxygen species (ROS). These changes led to enhanced invasiveness of A431 cells as assessed by the matrigel invasion assay. Chronic treatment of p53^-/-/SKH-1 mice with the p38 inhibitor SB203580 accelerated UV-induced SCC carcinogenesis and increased the expression of NOX2. NOX2 knockdown suppressed the augmented growth of A431 xenografts treated with SB203580. These findings indicate that in the absence of p53, p38α deficiency drives SCC growth and progression that is associated with enhanced NOX2 expression and ROS formation.     

Renoprotective Effects of Alpha Lipoic Acid on Iron Overload-Induced Kidney Injury in Rats by Suppressing NADPH Oxidase 4 and p38 MAPK Signaling

Biological Trace Element Research - We aimed to investigate the protective effect of alpha lipoic acid (ALA), a powerful antioxidant, against oxidative kidney damage induced by iron overload in...     

Cholesterol Perturbation in Mice Results in p53 Degradation and Axonal Pathology through p38 MAPK and Mdm2 Activation

Perturbation of lipid metabolism, especially of cholesterol homeostasis, can be catastrophic to mammalian brain, as it has the highest level of cholesterol in the body. This notion is best illustrated by the severe progressive neurodegeneration in Niemann-Pick Type C (NPC) disease, one of the lysosomal storage diseases, caused by mutations in the NPC1 or NPC2 gene. In this study, we found that growth cone collapse induced by genetic or pharmacological disruption of cholesterol egress from late endosomes/lysosomes was directly related to a decrease in axonal and growth cone levels of the phosphorylated form of the tumor suppressor factor p53. Cholesterol perturbation-induced growth cone collapse and decrease in phosphorylated p53 were reduced by inhibition of p38 mitogen-activated protein kinase (MAPK) and murine double minute (Mdm2) E3 ligase. Growth cone collapse induced by genetic (npc1−/−) or pharmacological modification of cholesterol metabolism was Rho kinase (ROCK)-dependent and associated with increased RhoA protein synthesis; both processes were significantly reduced by P38 MAPK or Mdm2 inhibition. Finally, in vivo ROCK inhibition significantly increased phosphorylated p53 levels and neurofilaments in axons, and axonal bundle size in npc1−/− mice. These results indicate that NPC-related and cholesterol perturbation-induced axonal pathology is associated with an abnormal signaling pathway consisting in p38 MAPK activation leading to Mdm2-mediated p53 degradation, followed by ROCK activation. These results also suggest new targets for pharmacological treatment of NPC disease and other diseases associated with disruption of cholesterol metabolism.     

低强度激光照射对老龄小鼠的抗氧化系统的影响

目的：研究低强度激光照射对机体抗氧化系统的影响。方法：采用不同剂量的低强度He-Ne激光照射老龄小鼠肝区,照射后观察T－SOD,Cn-SOD,Mn-SOD,T-AO,GSH-PX,GSH,MDA等指标。结果：能量密度分别为2．48J／cm^2,12．48J／cm^2,18．72J／cm^2的低强度He-Ne激光可提高,Cu,Zn-SOD,Mn-SOD,总SOD,及GSH－PX活性,可提高GSH含量和总抗氧化能力,降低MDA含量,其中以能量密度为18．72J／cm^2最为显著,使老龄小鼠以上各项指标恢复接近正常水平。结论：低强度激光照射可以提高机体抗氧化酶的活性和抗氧化剂的含量,增强机体抗氧化能力。     

The role of RIP2 in p38 MAPK activation in the stressed heart

The activation of p38 MAPK by dual phosphorylation aggravates myocardial ischemic injury and depresses cardiac contractile function. SB203580, an ATP-competitive inhibitor of p38 MAPK and other kinases, prevents this dual phosphorylation during ischemia. Studies in non-cardiac tissue have shown receptor-interacting protein 2 (RIP2) lies upstream of p38 MAPK, is SB203580-sensitive and ischemia-responsive, and aggravates ischemic injury. We therefore examined the RIP2-p38 MAPK signaling axis in the heart. Adenovirus-driven expression of wild-type RIP2 in adult rat ventricular myocytes caused robust, SB203580-sensitive dual phosphorylation of p38 MAPK associated with activation of p38 MAPK kinases MKK3, MKK4, and MKK6. The effect of SB203580 was recapitulated by unrelated inhibitors of RIP2 or the downstream MAPK kinase kinase, TAK1. However, overexpression of wild-type, kinase-dead, caspase recruitment domain-deleted, or kinase-dead and caspase recruitment domain-deleted forms of RIP2 had no effect on the activating dual phosphorylation of p38 MAPK during simulated ischemia. Similarly, p38 MAPK activation and myocardial infarction size in response to true ischemia did not differ between hearts from wild-type and RIP2 null mice. However, both p38 MAPK activation and the contractile depression caused by the endotoxin component muramyl dipeptide were attenuated by SB203580 and in RIP2 null hearts. Although RIP2 can cause myocardial p38 MAPK dual phosphorylation in the heart under some circumstances, it is not responsible for the SB203580-sensitive pattern of activation during ischemia.     

Inhibition of actin polymerization decreases osteogeneic differentiation of mesenchymal stem cells through p38 MAPK pathway

Background

Mesenchymal Stem Cells (MSC) are important candidates for therapeutic applications due to their ex vivo proliferation and differentiation capacity. MSC differentiation is controlled by both intrinsic and extrinsic factors and actin cytoskeleton plays a major role in the event. In the current study, we tried to understand the initial molecular mechanisms and pathways that regulate the differentiation of MSC into osteocytes or adipocytes.

Results

We observed that actin modification was important during differentiation and differentially regulated during adipogenesis and osteogenesis. Initial disruption of actin polymerization reduced further differentiation of MSC into osteocytes and osteogenic differentiation was accompanied by increase in ERK1/2 and p38 MAPK phosphorylation. However, only p38 MAPK phosphorylation was down regulated upon inhibition of actin polymerization which as accompanied by decreased CD49E expression.

Conclusion

Taken together, our results show that actin modification is a pre-requisite for MSC differentiation into osteocytes and adipocytes and osteogenic differentiation is regulated through p38 MAPK phosphorylation. Thus by modifying their cytoskeleton the differentiation potential of MSC could be controlled which might have important implications for tissue repair and regeneration.