碱性成纤维细胞生长因子对人涎腺腺样囊性癌ACC-2细胞株增殖及MEK/ERK、MKP-1通路的影响

目的:观察碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)对人涎腺腺样囊性癌ACC-2细胞株增殖及MEK1/2、ERK1/2及MKP-1表达的影响。方法:培养人涎腺腺样囊性癌细胞株(ACC-2),MTT比色法测定不同浓度bFGF对细胞增殖的影响;免疫沉淀法纯化蛋白并ERK试剂盒测定ERK活性;免疫印迹法测定p-MEK1/2、p-ERK1/2及MKP-1表达。结果:MTT实验显示bFGF明显增强ACC-2细胞增殖,免疫沉淀法显示bFGF上调ERK活性,免疫印迹法显示bFGF明显增强p-MEK1/2、p-ERK1/2表达及抑制MKP-1表达。结论:bFGF可促进人涎腺腺样囊性癌ACC-2细胞株增殖,其途径与上调ERK活性、激活MEK/ERK通路、抑制MKP-1表达有关。     

辛伐他汀通过MEK/ERK信号转导通路上调突触素表达

目的研究辛伐他汀(SV)对体外培养大鼠大脑皮层神经元突触素(SYP)的影响及信号转导机制。方法从新生Sprague-Dawley大鼠大脑皮层分离和培养神经元,4 d后分为对照组、SV处理组(2、4、8μmol/L作用48 h)、PD98059处理组和SV+PD98059处理组(先加入10μmol/L阻断剂PD98059作用30 min,再加入10μmol/L SV作用48 h)。应用免疫荧光检测法检测SYP表达,Western blot检测磷酸化丝裂原细胞外激酶(p-MEK)、磷酸化细胞外调节蛋白激酶1(p-ERK1)和磷酸化细胞外调节蛋白激酶2(p-ERK2)水平。结果免疫荧光检测显示,4μmol/L SV可明显增强SYP免疫反应性。Western blot检测显示,8μmol/L SV可显著增高SYP、p-MEK、p-ERK1和p-ERK2水平。应用PD98059可显著降低SV引起的SYP、p-MEK、p-ERK1和p-ERK2水平上调。结论SV可通过激活MEK/ERK信号转导通路上调SYP表达。     

Knockdown of mitogen‐activated protein kinase (MAPK) signalling in the midgut of Anopheles stephensi mosquitoes using antisense morpholinos

Arthropod‐borne infectious diseases are responsible for nearly 1.5 million deaths annually across the globe, with malaria responsible for >50% of these deaths. Recent efforts to enhance malaria control have focused on developing genetically modified Anopheles mosquitoes that are resistant to malaria parasite infection by manipulating proteins that are essential to the immune response. Although this approach has shown promise, the lack of efficient genetic tools in the mosquito makes it difficult to investigate innate immunity using reverse genetics. Current gene knockdown strategies based on small interfering RNA are typically labourious, inefficient, and require extensive training. In the present study, we describe the use of morpholino antisense oligomers to knockdown MEK‐ERK signalling in the midgut of Anopheles stephensi through a simple feeding protocol. Anti‐MEK morpholino provided in a saline meal was readily ingested by female mosquitoes with minimal toxicity and resulted in knockdown of total MEK protein levels 3–4 days after morpholino feeding. Further, anti‐MEK morpholino feeding attenuated inducible phosphorylation of the downstream kinase ERK and, as predicted by previous work, reduced parasite burden in mosquitoes infected with Plasmodium falciparum. To our knowledge, this is the first example of morpholino use for target protein knockdown via feeding in an insect vector. Our results suggest this method is not only efficient for studies of individual proteins, but also for studies of phenotypic control by complex cell signalling networks. As such, our protocol is an effective alternative to current methods for gene knockdown in arthropods.     

Involvement of RSK phosphorylation in PTTH-stimulated ecdysone secretion in prothoracic glands of the silkworm,Bombyx mori

It is well known that phosphorylation of extracellular signal-regulated kinase (ERK) is involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in insect prothoracic glands (PGs). In the present study, we further investigated the downstream signalling pathways. Our results showed that PTTH stimulated p90 ribosomal S6 kinase (RSK) phosphorylation at Thr573 in Bombyx mori PGs both in vitro and in vivo. The in vitro PTTH stimulation was stage- and dose-dependent. The absence of Ca²⁺ reduced PTTH-stimulated RSK phosphorylation. Stimulation of RSK phosphorylation was also observed after treatment with either A23187 or thapsigargin. A phospholipase C (PLC) inhibitor, U73122, blocked PTTH-stimulated RSK phosphorylation. These results indicate the involvement of Ca²⁺ and PLC. Treatment with diphenylene iodonium (DPI), a mitochondrial oxidative phosphorylation inhibitor, blocked PTTH-regulated RSK phosphorylation, indicating its redox regulation. A mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor, U0126, but not a phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, decreased PTTH-stimulated RSK phosphorylation, indicating that ERK is an upstream signalling. A protein kinase C (PKC) inhibitor, chelerythrine C, inhibited PTTH-stimulated RSK phosphorylation, and a PKC activator, phorbol 12-myristate acetate (PMA) stimulated RSK phosphorylation, indicating the involvement of PKC. BI-D1870, a specific RSK inhibitor, partly prevented PTTH-stimulated RSK phosphorylation and significantly inhibited PTTH-stimulated ecdysteroid secretion, indicating that PTTH-stimulated RSK phosphorylation is involved in ecdysteroidogenesis. Taken together, these data indicate that PTTH activates RSK phosphorylation which plays important roles in PTTH-stimulated ecdysteroidogenesis.     

模拟失重条件下骨形态发生蛋白2诱导成骨细胞蛋白激酶MEK 1的活性变化

背景:模拟失重条件下,骨形态发生蛋白2诱导的大鼠骨肉瘤成骨样细胞(ROS17/2.8)的Ⅰ型胶原α1链的基因表达下降,而丝裂原激活的蛋白激酶信号传导通路中的蛋白激酶MEK1参与了骨形态发生蛋白2对成骨细胞Ⅰ型胶原α1链mRNA表达的调节过程,但是在模拟失重条件下MEK1的活性如何变化尚不清楚。目的:观察模拟失重条件下由骨形态发生蛋白2诱导的ROS17/2.8细胞中MEK1激酶活性的变化。设计:不完全随机对照的实验。单位:解放军第四军医大学航空航天生物动力学教研室。材料:大鼠骨肉瘤成骨样细胞。方法:实验于2002-08/2003-01在北京航天医学工程研究所航天细胞与分子生物学实验室完成。在地面1G和回转器模拟失重条件下培养ROS17/2.8细胞,培养时间分别为24,48和72h。分为7组:第1组,空白对照组,即1G条件下不加骨形态发生蛋白2培养24h;第2组,1G培养24h;第3组,失重培养24h;第4组,1G培养48h;第5组,失重培养48h;第6组,1G培养72h;第7组,失重培养72h;第2组至第7组均加入骨形态发生蛋白2。培养结束前1h加入骨形态发生蛋白2(500mg/L),1h后提取细胞蛋白,应用WesternBlotting法检测MEK1的活性。主要观察指标:观察细胞中的总ERK1/2和磷酸化ERK1/2(p-ERK1/2)的含量。结果:①模拟失重条件下骨形态发生蛋白2诱导后ROS17/2.8细胞的总ERK1/2的表达:各实验组细胞的总ERK1/2蛋白表达量无明显差异。②模拟失重条件下骨形态发生蛋白2诱导后ROS17/2.8细胞的磷酸化ERK1/2的表达:第1组,细胞培养液中不含骨形态发生蛋白2在1G重力条件下培养24h,p-ERK1/2呈低水平表达,第2组,即培养液中加入骨形态发生蛋白2在1G条件下培养24h,p-ERK1/2的表达量明显高于第1组(P<0.01)。各模拟失重组p-ERK1/2表达量均低于相同时间点1G重力对照组,即第3,5,7组的表达分别低于第2,4,6组(P<0.01),且随着失重时间延长,第3,5,7组的p-ERK1/2表达量呈逐渐降低的趋势(P<0.01)。结论:模拟失重条件下骨形态发生蛋白2诱导的成骨细胞丝裂原激活的蛋白激酶信号通路中蛋白激酶MEK1的活性下降。     

KRAS突变对肺损伤大鼠蛋白激酶信号途径的影响

目的 研究鼠类肉瘤病毒癌(KRAS)基因突变对肺损伤大鼠蛋白激酶信号途径的影响。方法 建立大鼠急性肺损伤模型。根据组织芯片KRAS蛋白表达分组,KRAS阴性组:KRAS蛋白表达H-score计分<3分),KRAS阳性组(KRAS蛋白表达H-score计分> 3分)。KRAS突变型组:突变型KRAS蛋白的表达H-score计分<3分,KRAS野生型组:突变型KRAS蛋白的表达H-score计分> 3分。采用免疫组化法检测10种肺损伤相关蛋白[磷脂酰肌醇-3-激酶(PI3K),KRAS,KRAS突变蛋白,NRAS,BRAF,丝裂原活化的细胞外信号调节激酶(MEK),细胞外信号调节激酶(ERK),磷酸化MEK1(p MEK1),磷酸化MEK2(p MEK2)和磷酸化ERK1/2(p ERK1/2)]的阳性表达,采用组织化学评分法(H-score)对蛋白表达情况进行定量并分组,比较各组KRAS下游蛋白及MEK2/ERK通路蛋白的表达情况。结果①与KRAS阴性组相比,KRAS阳性组该基因下游蛋白中BRAF、MEK、ERK表达显著上升(P <0. 05),而NRAS无显著差异(P> 0. 05);②与KRAS野生型相比,KRAS突变型基因下游蛋白中BRAF、MEK、ERK表达显著上升(P <0. 05),而NRAS无显著差异(P> 0. 05);③与KRAS阴性组相比,KRAS阳性组pMEK2,pERK1/2蛋白表达显著上升(P <0. 05),而p MEK1无显著差异(P> 0. 05)。结论 KRAS蛋白阳性大鼠肺损伤组织中,该蛋白下游蛋白BRAF、MEK和ERK(RAS/RAF/MEK/ERK信号通路的下游蛋白)在KRAS基因突变的肺损伤组织中的表达量显著增加,且MEK2/ERK通路蛋白MEK、p MEK2和ERK表达率也显著增加,提示KRAS蛋白通过调控其下游蛋白表达调控肺组织损伤,该过程可能与MEK2/ERK通路相关。     

Identification of ecdysteroidogenic enzyme genes and their expression during pupal diapause in the cabbage armyworm,Mamestra brassicae

In this study, we identified ecdysteroidogenic enzymes in the cabbage armyworm, Mamestra brassicae, and demonstrated reduced expression of these genes during diapause. Some insects employ a temporary developmental arrest, diapause, to survive in severe environments. The titres of the moulting hormone ecdysteroid were reduced in diapause pupae of M. brassicae; therefore, ecdysteroidogenesis might be suppressed by a diapause‐specific mechanism. To clarify expression changes of ecdysteroidogenic enzyme genes during diapause in M. brassicae, we first identified the genes for seven ecdysteroidogenic enzymes: Neverland, Non‐molting glossy (Nm‐g), CYP307A1 (Spook), CYP306A1 (Phantom), CYP302A1 (Disembodied), CYP315A1 (Shadow) and CYP314A1 (Shade). Enzymatic assays using heterologous expression in Drosophila Schneider 2 (S2) cells and analysis of mRNA distribution indicated that the identified genes were ecdysteroidogenic enzymes of M. brassicae. Expression levels of these ecdysteroidogenic enzyme genes were compared between prothoracic glands in different pupal stages throughout diapause. Immediately after pupation, diapause‐destined pupae showed similar expression levels of ecdysteroidogenic enzyme genes to those of nondiapause pupae. All of these genes showed reduced gene expression after diapause initiation. Expression was immediately increased in diapause‐destined pupae at the postdiapause quiescence phase. These results indicate that reduced expression of ecdysteroidogenic enzyme genes suppresses ecdysteroidogenesis and maintains developmental arrest during diapause.     

Urolithin C increases glucose-induced ERK activation which contributes to insulin secretion

Polyphenols exert pharmacological actions through protein-mediated mechanisms and by modulating intracellular signalling pathways. We recently showed that a gut-microbial metabolite of ellagic acid named urolithin C is a glucose-dependent activator of insulin secretion acting by facilitating L-type Ca²⁺ channel opening and Ca²⁺ influx into pancreatic β-cells. However, it is still unknown whether urolithin C regulates key intracellular signalling proteins in β-cells. Here, we report that urolithin C enhanced glucose-induced extracellular signal-regulated kinases 1/2 (ERK1/2) activation as shown by higher phosphorylation levels in INS-1 β-cells. Interestingly, inhibition of ERK1/2 with two structurally distinct inhibitors led to a reduction in urolithin C effect on insulin secretion. Finally, we provide data to suggest that urolithin C-mediated ERK1/2 phosphorylation involved insulin signalling in INS-1 cells. Together, these data indicate that the pharmacological action of urolithin C on insulin secretion relies, in part, on its capacity to enhance glucose-induced ERK1/2 activation. Therefore, our study extends our understanding of the pharmacological action of urolithin C in β-cells. More generally, our findings revealed that urolithin C modulated the activation of key multifunctional intracellular signalling kinases which participate in the regulation of numerous biological processes.     

Block of C/EBP alpha function by phosphorylation in acute myeloid leukemia with FLT3 activating mutations

Mutations constitutively activating FLT3 kinase are detected in approximately 30% of acute myelogenous leukemia (AML) patients and affect downstream pathways such as extracellular signal-regulated kinase (ERK)1/2. We found that activation of FLT3 in human AML inhibits CCAAT/enhancer binding protein alpha (C/EBPalpha) function by ERK1/2-mediated phosphorylation, which may explain the differentiation block of leukemic blasts. In MV4;11 cells, pharmacological inhibition of either FLT3 or MEK1 leads to granulocytic differentiation. Differentiation of MV4;11 cells was also observed when C/EBPalpha mutated at serine 21 to alanine (S21A) was stably expressed. In contrast, there was no effect when serine 21 was mutated to aspartate (S21D), which mimics phosphorylation of C/EBPalpha. Thus, our results suggest that therapies targeting the MEK/ERK cascade or development of protein therapies based on transduction of constitutively active C/EBPalpha may prove effective in treatment of FLT3 mutant leukemias resistant to the FLT3 inhibitor therapies.     

LncRNA UCA1介导Raf/MEK/ERK信号通路调控乳腺癌细胞生物学作用的机制研究

目的 探讨长链非编码RNA(long non-coding RNA, LncRNA)尿路上皮癌胚抗原1(urothelial carcinoma antigen1,UCA1)在乳腺癌细胞中的表达及其对乳腺癌细胞生物学作用的机制研究。方法 实时定量聚合酶链式反应(quantitative real-time PCR, qRT-PCR)法检测LncRNA UCA1在正常人乳腺上皮细胞MCF-10A与乳腺癌细胞BT-474,MCF-7,SKBR-3和MDA-MB453中的表达;选择BT-474和MCF-7细胞随机分为三组,分别转染UCA1-siR,NC-siR及Control,再通过qRT-PCR法验证转染后BT-474和MCF-7细胞中LncRNA UCA1表达;通过CCK-8法、Transwell实验和流式细胞仪检测BT-474和MCF-7细胞增殖、侵袭、凋亡能力;利用Western blot检测两种细胞中RAF/MEK/ERK通路相关蛋白的表达。结果 与MCF-10A相比,LncRNA UCA1在BT-474,MCF-7, SKBR-3和MDA-MB453细胞中表达水平分别上调了133...     

Homeodomain‐interacting protein kinase (Hipk) phosphorylates the small SPOC family protein Spenito

The Drosophila homeodomain‐interacting protein kinase (Hipk) is a versatile regulator involved in a variety of pathways, such as Notch and Wingless signalling, thereby acting in processes including the promotion of eye development or control of cell numbers in the nervous system. In vertebrates, extensive studies have related its homologue HIPK2 to important roles in the control of p53‐mediated apoptosis and tumour suppression. Spenito (Nito) belongs to the group of small SPOC family proteins and has a role, amongst others, as a regulator of Wingless signalling downstream of Armadillo. In the present study, we show that both proteins have an enzyme–substrate relationship, adding a new interesting component to the broad range of Hipk interactions, and we map several phosphorylation sites of Nito. Furthermore, we were able to define a preliminary consensus motif for Hipk target sites, which will simplify the identification of new substrates of this kinase.     

Activation of the spinal ERK signaling pathway contributes naloxone-precipitated withdrawal in morphine-dependent rats

Extracellular signal-regulated kinase (ERK), a mitogen-activated protein kinases (MAPK), transduces a broad range of extracellular stimuli into diverse intracellular responses. Recent studies have showed that ERK activation in the supraspinal level involved in the development of drug dependence, especially in psychological dependence. In this study, we reported that the spinal ERK signaling pathway was activated by chronic morphine injection. There was a further increase in ERK activation after naloxone-precipitated withdrawal. Furthermore, attenuation of the spinal ERK phosphorylation by intrathecal a MAPK kinase (MEK) inhibitor U0126 or knockdown of the spinal ERK by antisense oligonucleotides not only decreased the scores of morphine withdrawal, but also attenuated withdrawal-induced allodynia, which were accompanied by decreased ERK phosphorylation in the spinal cord. The spinal ERK inhibition or knockdown also reduced morphine withdrawal-induced phosphorylation of cAMP response element binding protein (CREB), which is one of the important downstream substrates of ERK pathway, and Fos expression. The involvement of the spinal ERK in morphine withdrawal was supported by our finding that intrathecal N-methyl-D-aspartate receptor antagonist MK-801 or protein kinase C inhibitor chelerythrine chloride suppressed withdrawal-induced ERK activation in the spinal cord and attenuated morphine withdrawal symptoms. These findings suggest activation of the spinal ERK signaling pathway contributes naloxone-precipitated withdrawal in morphine-dependent rats.     

HOXC10 promotes proliferation and attenuates lipid accumulation of sheep bone marrow mesenchymal stem cells

Homeodomain-containing gene C10 (HOXC10), known to regulate cell differentiation and proliferation, is a key negative regulator in the browning of white adipose tissue in mice. Sheep is an important farm animal that provides meat for human consumption, with fat content being an important meat quality determinant; however, there is no report about the role of HOXC10 in sheep adipocytes or adipogenesis. In this study, we investigated the effect of HOXC10 on proliferation and adipogenic differentiation in sheep bone marrow mesenchymal stem cells (sBMSCs). In sBMSCs, HOXC10 overexpression promoted cell proliferation and upregulated the expression of p-PI3K, p-AKT, p-p70S6K, p-MEK, and p-ERK, whereas HOXC10 knockdown was associated with the opposite effects. These results suggested that HOXC10 may promote cell proliferation by activating the MEK/ERK and PI3K/AKT/mTOR/p70S6K signaling pathways. In addition, we found that HOXC10 expression was negatively associated with lipid accumulation in adipogenic-differentiated sBMSCs. HOXC10 overexpression in sBMSCs significantly decreased lipid droplet accumulation and suppressed the expression of adipogenic-specific genes, including ACC, LPL, PPARG, and FABP4, while HOXC10 knockdown was associated with the opposite effects. Furthermore, our study suggested a new regulatory mechanism of the effect of HOXC10 on lipid accumulation and metabolism; HOXC10 may negatively regulate lipid accumulation in adipogenic-differentiated sBMSCs, at least in part, by suppressing LPL expression. Overall, our research not only contributes to a better understanding of the mechanism of lipid accumulation and metabolism in sheep, but also shed light on meat quality control in the future.     

ERK信号转导通路在癫痫大鼠脑部作用的研究

目的:研究ERK信号转导通路在癫痫发病机制中的作用。方法:将54只雄性Wistar大鼠分为正常组6只,假模型组和癫痫组各24只,后2组按大鼠存活时间各分为30 min组1、.5 h组5、h组和8 h组各6只。癫痫组腹腔注射戊四氮制作癫痫大鼠模型,假模型组以生理盐水代替戊四氮腹腔注射。应用免疫组化染色、组织病理学染色及Western blot检测,分别检测各组大鼠大脑皮质ERK1、ERK2和p-ERK1/2的表达及神经元的损伤情况。结果:免疫组化染色结果显示正常组和假模型组大鼠脑内有少量的ERK1和ERK2阳性神经元,无p-ERK1/2阳性神经元;癫痫组致痫30 min后脑内p-ERK1/2阳性细胞开始出现,同时ERK1和ERK2阳性细胞较正常水平也有增多,1.5 h后3种阳性细胞均明显增多(P<0.05),5 h后3者开始减少,8 h后仅见少量阳性神经元。组织病理学染色示癫痫组皮质出现散在异常神经元。Western blot检测显示痫性发作各时点大鼠皮质内ERK1、ERK2和p-ERK1/2蛋白表达较正常水平显著增多(P<0.05),1.5 h时点组的蛋白表达水平高于其他各时点组(P<0.05)。结论:皮质ERK信号途径在戊四氮致痫模型中被激活,参与癫痫发作的病理生理过程。     

Activation of Akt1 by human 5-hydroxytryptamine (serotonin)1B receptors is sensitive to inhibitors of MEK.

Akt1/protein kinase B and the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase 1 (ERK1) and ERK2 have been shown to promote cell survival in a cell-specific manner. Since many receptors activate both pathways, inhibitors are commonly used to study the relative role of each pathway. In the present study, we examined the effects of PD098059 and U0126, two structurally dissimilar inhibitors of MAP kinase kinase (MEK1/2), on the activation of ERK and Akt stimulated by human 5-hydroxytryptamine(1B) (serotonin) (5-HT1B) receptors. Surprisingly, pathways for activation of both ERK and Akt were found to be sensitive to the two MEK inhibitors at concentrations commonly used to selectively inhibit the activation of ERK. Both compounds caused complete inhibition of phosphorylation of ERK and a maximal 60% inhibition of 5-HT1B receptor-mediated phosphorylation of Akt. Inhibition of Akt activation required almost complete inhibition of ERK. Transfection with cDNA for activated forms of MEK1/2 caused increased phosphorylation of ERK but not of Akt, demonstrating that independent activation of MEK/ERK was insufficient for activation of Akt. Therefore, it is not clear whether inhibition of activation of Akt resulted from selective inhibition of MEK or from additional actions on other unidentified common pathways. Nevertheless, our findings that PD098059 and U0126 inhibit activation of Akt at commonly used concentrations demonstrate that in at least some systems, these compounds inhibit activation of both ERK and Akt, and cannot be used to discern the relative roles of each pathway in mediating cellular responses.     

丙泊酚对大鼠海马ERK1/2磷酸化和c-fos mRNA表达水平的影响

目的探讨丙泊酚对大鼠海马ERK1/ERK2磷酸化和c-fos mRNA表达水平的影响。方法成年雄性SD大鼠64只,体重250-280 g,随机分为2组(n=32),丙泊酚组(P组)于训练前15 min腹腔注射丙泊酚9 mg/kg,容量2 ml/kg;生理盐水组(S组)注射等容量生理盐水。采用避暗箱实验测试大鼠认知功能。首先对大鼠进行训练,记录100 s内大鼠不再钻入暗室所需的训练次数。于训练结束后1、3、24 h(T1-3)时点记录大鼠在明室停留的时间即记忆潜伏期。各组于给药后15 min(T0)和T1-3记忆能力测试结束后,各断头处死8只大鼠,分离海马,测定ERK1/ERK2、磷酸化ERK1/ERK2(p-ERK1/ERK2)、c-fos mRNA的表达水平。结果与S组比较,P组大鼠不再钻入暗室所需的训练次数增加,T2和T3时记忆潜伏期缩短,T2,3时ERK1磷酸化水平降低,T0-3时ERK2磷酸化水平降低(P0.01),T0,3时海马c-fos mRNA表达水平无明显变化(P0.05),各时点海马ERK1、ERK2的表达差异均无统计学意义(P0.05)。结论丙泊酚可抑制大鼠海马ERK1/ERK2的磷酸化水平,对c-fos mRNA的表达尚不确定。     

Intensity-dependent activation of extracellular signal-regulated protein kinase 5 in sensory neurons contributes to pain hypersensitivity

Alterations in the intracellular signal transduction pathway in primary afferents may contribute to pain hypersensitivity. Recently, we have reported that the phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) occurs in primary afferent neurons in response to noxious stimulation of the peripheral tissue, i.e., activity-dependent activation of ERK1/2 and p38 MAPK in dorsal root ganglion (DRG) neurons. In the present study, we investigated the phosphorylation of ERK5, also known as big MAPK1, in the DRG by noxious stimulation using immunohistochemistry. Capsaicin injection induced phosphorylated ERK5 (p-ERK5) in small-to-medium diameter sensory neurons with a peak at 2 min after capsaicin injection. Furthermore, we examined the p-ERK5 labeling in the DRG after noxious heat and cold stimuli and found a stimulus intensity-dependent increase in the number of activated neurons. Most of these p-ERK5-immunoreactive neurons were small- and medium-sized neurons, which coexpressed transient receptor potential (TRP) ion channel TRPV1 and TRPA1 after noxious heat and cold stimuli, respectively. In contrast, there was no change in ERK5 phosphorylation in the spinal dorsal horn. The i.t. administration of ERK5 antisense oligodeoxynucleotide reversed heat hyperalgesia, but not mechanical allodynia, produced by capsaicin injection. Taken together, these findings suggest that the in vivo activation of the ERK5 signaling pathway in sensory neurons by noxious stimulation may be, at least in part, correlated with functional activity and, further, involved in the development of pain hypersensitivity.     

ERK activation is required for hydrostatic pressure‐induced tensile changes in engineered articular cartilage

The objective of this study was to identify ERK 1/2 involvement in the changes in compressive and tensile mechanical properties associated with hydrostatic pressure treatment of self‐assembled cartilage constructs. In study 1, ERK 1/2 phosphorylation was detected by immunoblot, following application of hydrostatic pressure (1 h of static 10 MPa) applied at days 10–14 of self‐assembly culture. In study 2, ERK 1/2 activation was blocked during hydrostatic pressure application on days 10–14. With pharmacological inhibition of the ERK pathway by the MEK1/ERK inhibitor U0126 during hydrostatic pressure application on days 10–14, the increase in Young's modulus induced by hydrostatic pressure was blocked. Furthermore, this reduction in Young's modulus with U0126 treatment during hydrostatic pressure application corresponded to a decrease in total collagen expression. However, U0126 did not inhibit the increase in aggregate modulus or GAG induced by hydrostatic pressure. These findings demonstrate a link between hydrostatic pressure application, ERK signalling and changes in the biomechanical properties of a tissue‐engineered construct. Copyright © 2012 John Wiley & Sons, Ltd.