C-reactive protein induces high-mobility group box-1 protein release through activation of p38MAPK in macrophage RAW264.7 cells

BACKGROUND: C-reactive protein (CRP) is widely used as a sensitive biomarker for inflammation. Increasing evidence suggests that CRP plays a role in inflammation. High-mobility group box-1 (HMGB1), a primarily nuclear protein, is passively released into the extracellular milieu by necrotic or damaged cells and is actively secreted by monocytes/macrophages. Extracellular HMGB1 as a potent inflammatory mediator has stimulated immense curiosity in the field of inflammation research. However, the molecular dialogue implicated between CRP and HMGB1 in delayed inflammatory processes remains to be explored. METHODS AND RESULTS: The levels of HMGB1 in culture supernatants were determined by Western blot analysis and enzyme-linked immunosorbent assay in macrophage RAW264.7 cells. Purified CRP induced the release of HMGB1 in a dose- and time-dependent fashion. Immunofluorescence analysis revealed nuclear translocation of HMGB1 in response to CRP. The binding of CRP to the Fc gamma receptor in RAW264.7 cells was confirmed by fluorescence-activated cell sorter analysis. Pretreatment of cells with IgG-Fc fragment, but not IgG-Fab fragment, efficiently blocked this binding. CRP triggered the activation of p38MAPK and ERK1/2, but not Jun N-terminal kinase. Moreover, both p38MAPK inhibitor SB203580 and small interfering RNA significantly suppressed the release of HMGB1, but not the MEK1/2 inhibitor U-0126. CONCLUSION: We demonstrated for the first time that CRP, a prominent risk marker for inflammation including atherosclerosis, could induce the active release of HMGB1 by RAW264.7 cells through Fc gamma receptor/p38MAPK signaling pathways, thus implying that CRP plays a crucial role in the induction, amplification, and prolongation of inflammatory processes, including atherosclerotic lesions.     

Expression and clinical significance of the nin one binding protein and p38 MAPK in prostate carcinoma

Background: Prostate carcinoma is a major cause of morbidity and mortality. The MAPK Signaling Pathway plays an important role in multiple tumors, including prostate carcinoma. MAPK signaling is mediated by ERK1/2, JNK and p38 MAPK, which are important in the control of cell proliferation, differentiation and apoptosis. However, relatively little is known about the regulatory mechanism of p38 MAPK in prostate cancers. NOB1 is among the most novel topic in MAPK studies currently. Recent studies found its vital role in tumor metastasis in glioblastoma proliferation, however, its expression profile and its prognostic value in prostate carcinoma have not been investigated. Methods: To determine the relationship between NOB1 and p38 MAPK expressions, a population-based study was conducted for immunohistochemical staining analysis of tumor tissues, in matched malignant and nonmalignant prostatectomy samples from 132 PCa patients. Moreover, Western blot analysis and NOB1 interference studies of prostate cancer cell lines. To evaluate the diagnostic and prognostic between NOB1 and p38 MAPK in prostate cancer (PCa) tissue after radical prostatectomy, the hypothesis that prostate cancers with NOB1 expression have distinct clinical, prognostic and molecular attributes was tested. Results: Among 132 prostate cancers, NOB1 expression was detected in 117 (88.7%) tumors by immunohistochemistry. NOB1 and p38 MAPK expression had significant positive correlation with carcinogenesis, tumor progression and patient survival. Immunohistochemically, NOB1 expression in prostate cancer was independently associated with p38 MAPK activation (P=0.0002). Furthermore, p38 MAPK expression was completely suppressed by NOB1 interference in the prostate cancer cell lines DU-145 and PC-3. Conclusions: NOB1 expression status was closely correlated with important histopathologic characteristics and the recurrence and metastasis of prostate carcinomas. These data support a potential link between NOB1 and p38 MAPK, and suggest that NOB1 may identify a subset of prostate cancer patients with a poor prognosis. This study proved that NOB1 in PCa tissue can be used, in combination with traditional clinicopathological factors, as promising diagnostic and prognostic tools.     

p38MAPK在小鼠早胚中的表达及其对胚泡发育的作用

目的观察p38丝裂原活化蛋白激酶(p38MAPK)在小鼠早胚中的表达及其作用,探讨p38MAPK与胚泡植入的相关关系。方法取小鼠胚胎的2细胞,4细胞,8细胞,桑葚胚,胚泡等不同发育阶段的早胚,用免疫组化及免疫荧光法测定早胚中p38MAPK的表达及变化情况。用体外胚胎培养技术:将2细胞期小鼠早胚随机分组,接种到添加不同浓度p38MAPK特异性抑制剂SB220025的培养液内进行培养,台盼蓝染色观察p38MAPK特异性抑制剂对小鼠早胚发育的影响。结果 p38MAPK在小鼠胚胎发育各细胞期均呈阳性表达,随着妊娠进展,其表达逐渐加强。体外实验显示:抑制剂组早胚不能发育到胚泡阶段,抑制剂恢复实验发现抑制剂组胚卵仍具存活能力。结论 p38MAPK在小鼠胚胎发育过程中起作用,p38MAPK特异性抑制剂可抑制小鼠早胚的发育。     

α-Naphthoflavone inhibits 3T3-L1 pre-adipocytes differentiation via modulating p38MAPK signaling

α-Naphthoflavone (α-NF) is a synthetic flavonone derivative and is well known as a potent inhibitor of aromatase in a variety of systems. However, its role in lipid metabolism remains far from understood. The aim of current study was to investigate the effects of α-NF on 3T3-L1 pre-adipocytes differentiation and the mechanism through which it acts. Treatment of 3T3-L1 cells with α-NF in conjunction with a hormone cocktail resulted in α-NF mediated suppression of adipocyte differentiation in a dose dependent manner. At the molecular level, our findings demonstrated that α-NF inhibited the mid and late phase, but not the early phase of adipogenic markers expression during 3T3-L1 adipogenesis. The phosphorylation of p38 was activated upon adipogenic stimulation, yet was substantially suppressed by α-NF treatment. α-NF also synergistically inhibited expression of the adipogenic marker peroxisome proliferator-activated receptor gamma (PPARγ) expression together with p38 selective inhibitor, SB203580. Our study demonstrated for the first time that α-NF is capable of suppressing 3T3-L1 adipocyte differentiation and that this effect likely occurs through repression of the p38MAPK signaling pathway.     

反义p38 MAPK寡核苷酸对AT1R介导的血管收缩的影响及其机制

目的研究反义p38 MAPK寡核苷酸在血管紧张素Ⅱ1型受体（AT1R）介导的血管平滑肌收缩反应中的作用及其机制。方法采用反义寡核苷酸（ASODN）基因封闭技术来特异性调控p38 MAPK的表达,观察p38 MAPK在AT1R介导的大鼠主动脉血管平滑肌收缩中的作用及其与血管平滑肌肌球蛋白轻链激酶（MLCK）的关系。结果 AngⅡ诱导大鼠主动脉产生浓度依赖性的收缩,AT1R拮抗剂losartan和p38 MAPK-ASODN处理均可拮抗AngⅡ诱导的血管收缩反应。同时,AngⅡ处理可明显升高血管MLCK的活性,AT1R拮抗剂和p38 MAPK-ASODN也明显抑制了AngⅡ的这一作用。结论 p38 MAPK参与了AT1R介导的血管收缩反应的调节,其机制与MLCK调节途径有关。     

p38MAPK通过上调组蛋白乙酰化水平影响APP表达

 摘要：目的 研究p38MAPK信号通路的激活对淀粉前体蛋白（β-Amyloid precursor protein, APP）表达的影响及其相关表观遗传学机制。方法 采用体外培养神经母细胞瘤细胞（SH-SY5Y）,Western blot方法检测p38MAPK信号通路激活后APP蛋白的表达及组蛋白乙酰化酶(Histone Acetyltranferase, HAT)和组蛋白去乙酰化酶(Histone Deacetyltranferase, HDAC)的表达情况;光密度值法检测组蛋白H3和H4整体乙酰化水平。结果 p38MAPK信号通路经特异性激动剂激活72小时后,APP表达明显增高至对照组1.5倍;同时组蛋白H3整体乙酰化水平增高,但H4乙酰化水平无明显改变;Western blot结果显示,组蛋白乙酰化酶 CBP表达增高至对照组2.5倍,而组蛋白去乙酰化酶HDAC3表达下降至对照组40%。结论 以上结果提示,p38MAPK信号通路可能通过对组蛋白乙酰化水平的调节影响APP蛋白的表达。     

Cochlear protection from acoustic injury by inhibitors of p38 mitogen-activated protein kinase and sequestosome 1 stress protein

This study evaluated the protective role of p38 mitogen-activated protein kinase (p38 MAPK) inhibitors and sequestosome 1 (Sqstm1/A170/p62), a stress-induced signal modulator, in acoustic injury of the cochlea in mice. Two weeks after the exposure of mice to acoustic stress, threshold shifts of the auditory brainstem response (ABR) from the pre-exposure level and hair cell loss were evaluated. The activation of p38 MAPK was observed in cochlea by immunostaining 4 h after acoustic stress. To examine the role of p38 MAPK in tissue injury, its inhibitors were i.p. injected into male wild-type C57BL mice before the acoustic overexposure. The inhibitors SB202190 and SB203580 but not the inactive analogue SB202474 dose-dependently decreased the auditory threshold shift and outer hair cell loss induced by acoustic overexposure, suggesting the involvement of p38 MAPK in ototoxicity. We found that acoustic overexposure induced the up-regulation of Sqstm1 mRNA expression in the cochlea of wild-type mice and that SQSTM1-deficient mice exhibited an enhanced ABR threshold shift and hair cell loss, suggesting a role of SQSTM1 in the protection of tissue from acoustic stress.     

The kinase p38α functions in dendritic cells to regulate Th2-cell differentiation and allergic inflammation

Dendritic cells (DCs) play a critical role in controlling T helper 2 (Th2) cell-dependent diseases, but the signaling mechanism that triggers this function is not fully understood. We showed that p38α activity in DCs was decreased upon HDM stimulation and dynamically regulated by both extrinsic signals and Th2-instructive cytokines. p38α-specific deletion in cDC1s but not in cDC2s or macrophages promoted Th2 responses under HDM stimulation. Further study showed that p38α in cDC1s regulated Th2-cell differentiation by modulating the MK2−c-FOS−IL-12 axis. Importantly, crosstalk between p38α-dependent DCs and Th2 cells occurred during the sensitization phase, not the effector phase, and was conserved between mice and humans. Our results identify p38α signaling as a central pathway in DCs that integrates allergic and parasitic instructive signals with Th2-instructive cytokines from the microenvironment to regulate Th2-cell differentiation and function, and this finding may offer a novel strategy for the treatment of allergic diseases and parasitic infection.     

Rap1a activation by CalDAG-GEFI and p38 MAPK is involved in E-selectin-dependent slow leukocyte rolling

Rolling leukocytes are exposed to different adhesion molecules and chemokines. Neutrophils rolling on E-selectin induce integrin αLβ2-mediated slow rolling on ICAM-1 by activating a phospholipase C (PLC)γ2-dependent and a separate PI3Kγ-dependent pathway. E-selectin-signaling cooperates with chemokine signaling to recruit neutrophils into inflamed tissues. However, the distal signaling pathway linking PLCγ2 (Plcg2) to αLβ2-activation is unknown. To identify this pathway, we used different Tat-fusion-mutants and gene-deficient mice in intravital microscopy, autoperfused flow chamber, peritonitis, and biochemical studies. We found that the small GTPase Rap1 is activated following E-selectin engagement and that blocking Rap1a in Pik3cg-/- mice by a dominant-negative Tat-fusion mutant completely abolished E-selectin-mediated slow rolling. We identified CalDAG-GEFI (Rasgrp2) and p38 MAPK as key signaling intermediates between PLCγ2 and Rap1a. Gαi-independent leukocyte adhesion to and transmigration through endothelial cells in inflamed postcapillary venules of the cremaster muscle were completely abolished in Rasgrp2-/- mice. The physiological importance of CalDAG-GEFI in E-selectin-dependent integrin activation is shown by complete inhibition of neutrophil recruitment into the inflamed peritoneal cavity of Rasgrp2-/- leukocytes treated with pertussis toxin to block Gαi-signaling. Our data demonstrate that Rap1a activation by p38 MAPK and CalDAG-GEFI is involved in E-selectin-dependent slow rolling and leukocyte recruitment.     

抑制Rac1通过降低磷酸化p38MAPK提高1型糖尿病小鼠的心脏功能

目的:探讨抑制Rac1对1型糖尿病小鼠心肌细胞肥大、心脏功能的影响及其作用机制。方法:50只8周龄雄性C57小鼠随机分为对照组(control,n=10)、Rac1抑制剂NSC23766对照组(NSC,n=10)、1型糖尿病组(STZ,n=15)及NSC治疗组(STZ+NSC,n=15)。小鼠腹腔注射链脲佐菌素(STZ)建立1型糖尿病动物模型,血糖升高后给予小鼠腹腔注射NSC23766,实验于8周末结束,记录实验小鼠生存率、测量小鼠体重及左室重量并计算左室重量指数,运用超声心动图检测小鼠心脏功能,心肌组织进行HE染色结合图像分析软件定量心肌细胞大小,运用实时定量RT-PCR技术检测心肌组织心房利钠肽(ANP)、脑利尿肽(BNP)、β-肌球蛋白重链(β-MHC)mRNA表达,以Westernblotting定量心肌组织磷酸化p38丝裂素活化蛋白激酶(pho-p38MAPK)表达。结果:抑制Rac1后:(1)糖尿病小鼠生存率提高、糖尿病小鼠左室重量指数降低(P0.01)、心脏左室射血分数(EF)增加、左室短轴缩短率增加(FS)(P0.01);(2)心肌细胞大小明显降低(P0.01);(3)心肌肥大相关基因ANP、BNP、β-MHC表达明显降低(P0.01);(4)心肌组织pho-p38MAPK表达明显降低(P0.01)。结论:抑制Rac1活性显著改善1型糖尿病小鼠心脏功能、降低心肌细胞肥大,其机制可能与明显降低心肌组织磷酸化p38MAPK密切相关。     

TPX2通过p38 MAPK信号通路诱导直肠癌HR-8348细胞凋亡

目的:研究下调非洲爪蟾驱动蛋白样蛋白2靶蛋白(TPX2)对直肠癌细胞凋亡的影响及机制。方法:用TPX2小干扰RNA(si RNA)转染直肠癌HR-8348细胞,记为TPX2 si RNA组;以不做转染的细胞作为正常对照(control)组;以转染si RNA阴性对照(si RNA-NC)的细胞作为si RNA-NC组;用p38 MAPK抑制剂处理敲减TPX2表达后的直肠癌HR-8348细胞记为TPX2 si RNA+SB203580组。RT-qPCR和Western blot测定TPX2的表达水平,MTT法测定细胞存活率,流式细胞术测定细胞凋亡,Western blot测定细胞中p38 MAPK、p-p38 MAPK、cleaved caspase-3和Bcl-2的蛋白水平。结果:TPX2 si RNA转染后HR-8348细胞中TPX2的m RNA和蛋白表达水平显著下降(P 0. 05),而转染si RNA-NC对HR-8348细胞中TPX2的m RNA和蛋白水平没有影响。敲减TPX2表达后的直肠癌HR-8348细胞存活率降低,凋亡率升高,细胞中的cleaved caspase-3、p-p38 MAPK/p38 MAPK蛋白水平明显升高,Bcl-2水平水平降低,与control组比较,差异有统计学意义(P 0. 05)。与TPX2 si RNA组相比,TPX2 si RNA+SB203580组的HR-8348细胞凋亡率、cleaved caspase-3水平和p-p38 MAPK/p38 MAPK蛋白水平明显降低,存活率明显升高(P 0. 05)。结论:TPX2表达下调可以通过激活p38 MAPK促进直肠癌HR-8348细胞凋亡。     

Ketamine reduces inducible superoxide generation in human neutrophils in vitro by modulating the p38 mitogen-activated protein kinase (MAPK)-mediated pathway

Many cellular stresses and inflammatory stimuli can activate p38 mitogen-activated protein kinase (MAPK), a serine/threonine kinase in the MAPK family. The different stimuli act via different receptors or signalling pathways to induce phosphorylation of the cytosolic protein p47^phox, one subunit of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Formyl–methionyl–leucyl–phenylalanine (fMLP) has been shown to induce the p38 MAPK phosphorylation during the respiratory burst in human neutrophils. Here, we show that treatment with S(+)-ketamine or R(-)-ketamine at different concentrations (50, 100, 200, 400 µM) reduced fMLP-induced superoxide anion generation and p47^phox phosphorylation in neutrophils in a concentration-dependent manner (y = −0·093x + 93·35 for S(+)-ketamine and y = −0·0982x + 95·603 for R(-)-ketamine, respectively). While treatment with 50 µM ketamine inhibited fMLP-induced superoxide generation by 10%, treatment with 400 µM S(+)-ketamine and R(-)-ketamine reduced fMLP-induced superoxide generation to 60·5 ± 8·3% and 60·0 ± 8·5%, respectively, compared with that in neutrophils treated with fMLP alone. Furthermore, treatment with ketamine down-regulated both fMLP-induced p47^phox and isoproterenol-induced p38 MAPK phosphorylation and superoxide production. Interestingly, treatment with SB203580, the p38 MAPK inhibitor, also mitigated fMLP-induced superoxide anion generation and p38 MAPK and p47^phox phosphorylation as well as apoptosis in a concentration-dependent fashion in neutrophils. Therefore, ketamine racemes inhibited fMLP-induced superoxide anion generation and p47^phox phosphorylation by modulating fMLP-mediated p38 MAPK activation in neutrophils.     

Suppression of the clinical and cytokine response to endotoxin by RWJ-67657, a p38 mitogen-activated protein-kinase inhibitor, in healthy human volunteers

Sepsis resulting in multiorgan failure and death is still a major problem in intensive care medicine, despite extensive attempts to interfere in the supposed underlying mechanism of a deranged immune system. This is not only due to the persistent lacunae in knowledge about the immune system in sepsis but also due to the lack of sufficient instruments for intervention. Inhibitors of the p38 mitogen-activated protein kinase (p38MAPK) have been used to study the signalling pathway of the immune response. In vitro and animal studies have demonstrated that blocking p38MAPK could mitigate the pro-inflammatory response and improve survival after endotoxaemia. Using an endotoxaemia model in healthy human volunteers we evaluated the attenuation of clinical and cytokine response to endotoxin after inhibition of p38MAPK by an oral dose of RWJ-67657, a pyrindinyl imidazole. We measured the clinical parameters temperature, blood pressure and heart rate. The proinflammatory cytokines tumour necrosis factor-alpha, interleukin-6 and interleukin-8 were measured by ELISA at various points during a 24-h period. Drug toxicity was evaluated by routine clinical and laboratory examinations. After a single dose dose of RWJ-67657 the temperature and blood pressure response remained at the basal level. The inhibition of TNF-alpha, IL-6 and IL-8 response was a dose dependent. With the maximum dosage, reduction in peak serum levels of the proinflammatory cytokines was greater than 90%. There was no drug-related toxicity. Interpretation: We conclude that inhibition of p38MAPK by RWJ-67657 might be a tool to intervene in the deranged immune response in sepsis and other inflammatory diseases.     

Cisplatin causes cell death via TAB1 regulation of p53/MDM2/MDMX circuitry

The interdependence of p53 and MDM2 is critical for proper cell survival and cell death and, when altered, can lead to tumorigenesis. Mitogen-activated protein kinase (MAPK) signaling pathways function in a wide variety of cellular processes, including cell growth, migration, differentiation, and death. Here we discovered that transforming growth factor β-activated kinase 1 (TAK1)-binding protein 1 (TAB1), an activator of TAK1 and of p38α, associates with and inhibits the E3 ligase activity of MDM2 toward p53 and its homolog, MDMX. Depletion of TAB1 inhibits MDM2 siRNA-mediated p53 accumulation and p21 induction, partially rescuing cell cycle arrest induced by MDM2 ablation. Interestingly, of several agents commonly used as DNA-damaging therapeutics, only cell death caused by cisplatin is mitigated by knockdown of TAB1. Two mechanisms are required for TAB1 to regulate apoptosis in cisplatin-treated cells. First, p38α is activated by TAB1 to phosphorylate p53 N-terminal sites, leading to selective induction of p53 targets such as NOXA. Second, MDMX is stabilized in a TAB1-dependent manner and is required for cell death after cisplatin treatment. Interestingly TAB1 levels are relatively low in cisplatin-resistant clones of ovarian cells and in ovarian patient''s tumors compared with normal ovarian tissue. Together, our results indicate that TAB1 is a potential tumor suppressor that serves as a functional link between p53–MDM2 circuitry and a key MAPK signaling pathway.     

Activation of p38 MAPK by feline infectious peritonitis virus regulates pro-inflammatory cytokine production in primary blood-derived feline mononuclear cells

Feline infectious peritonitis (FIP) is an invariably fatal disease of cats caused by systemic infection with a feline coronavirus (FCoV) termed feline infectious peritonitis virus (FIPV). The lethal pathology associated with FIP (granulomatous inflammation and T-cell lymphopenia) is thought to be mediated by aberrant modulation of the immune system due to infection of cells such as monocytes and macrophages. Overproduction of pro-inflammatory cytokines occurs in cats with FIP, and has been suggested to play a significant role in the disease process. However, the mechanism underlying this process remains unknown. Here we show that infection of primary blood-derived feline mononuclear cells by FIPV WSU 79-1146 and FIPV-DF2 leads to rapid activation of the p38 MAPK pathway and that this activation regulates production of the pro-inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta). FIPV-induced p38 MAPK activation and pro-inflammatory cytokine production was inhibited by the pyridinyl imidazole inhibitors SB 203580 and SC 409 in a dose-dependent manner. FIPV-induced p38 MAPK activation was observed in primary feline blood-derived mononuclear cells individually purified from multiple SPF cats, as was the inhibition of TNF-alpha production by pyridinyl imidazole inhibitors.     

三七皂苷单体R1 抑制低氧高二氧化碳诱导的肺动脉平滑肌细胞p38 MAPK信号通路的活化

目的: 探讨三七皂苷单体R₁(R₁)减轻低氧高二氧化碳(CO₂)性肺动脉收缩的作用及其与p38 MAPK信号通路的关系。方法: 原代培养雄性SD大鼠肺动脉平滑肌细胞(PASMCs),取第2至5代对数生长期细胞至低氧高CO₂(1% O₂, 6% CO₂)条件下继续培养,并分别用8、40、100 mg/L R₁ 孵育24 h后收集细胞,采用免疫印迹法测定p38 MAPK磷酸化蛋白表达,半定量RT-PCR检测p38 MAPK mRNA的表达。结果: Western blotting和RT-PCR结果显示,低氧高CO₂组p-p38 MAPK蛋白和p38 MAPK mRNA表达明显高于对照组(N)组(P<0.01)。与低氧高CO₂组相比,R₁(8、40、100 mg/L)不同程度抑制了p-p38 MAPK蛋白和p38 MAPK mRNA的表达(P<0.01),并呈剂量依赖关系。结论: 低氧高CO₂诱导PASMCs p38 MAPK活化,三七皂苷单体R₁可能通过抑制p38 MAPK通路减轻低氧高CO₂性肺动脉收缩。