Glucose-Regulated Protein 94 Mediates the Proliferation and Metastasis through the Regulation of ETV1 and MAPK Pathway in Colorectal Cancer

Colorectal cancer (CRC) is a worldwide health problem. Glucose-regulated protein 94 (GRP94) is known as an important endoplasmic reticulum-stress response protein that shows correlation with aggressive cancer behavior. However, the role of GRP94 in CRC is still unclear. Our results showed that silencing GRP94 (GRP94-KD) reduced cell proliferation, invasion and migration of CRC cells and suppressed tumorigenesis in the xenograft mouse model. Rescue assay showed that ETV1 overexpression reversed the effect of GRP94 on cell proliferation and migration. In the molecular mechanism, we found that knockdown of GRP94 inhibited the level of MAPK pathway, including ERK/p-ERK, JNK/p-JNK, and p38/p-p38 signals. Cyclooxygenase-2 and epithelial-mesenchymal transformation biomarkers, such as N-cadherin, vimentin, and β-catenin were suppressed in GRP94 knockdown cells. Treatment of specific inhibitors of MAPK pathway showed that ERK/p-ERK, and p38/p-p38 inhibitors significantly influenced ETV1 expression as compared to JNK/p-JNK inhibitor. Our results indicated that silencing GRP94 repressed the ability of EMT process, cancer cell proliferation, metastasis, and CRC tumorigenesis. Therefore, GRP94 may play an important role in CRC by regulating ETV1 and MAPK pathway.     

磷酸化的丝裂原活化蛋白激酶在大鼠睾丸的表达

目的研究丝裂原活化蛋白激酶(MAPKs)的3个亚家族成员细胞外信号调节激酶(ERK)、c-Jun N-端激酶(JNK)和p38 MAPK的活化形式在睾丸中的定位,了解MAPKs在生精过程中所起的作用。方法免疫组织化学方法检测正常大鼠睾丸中磷酸化的p-ERKJ、NK、p38 MAPK的表达情况。结果正常大鼠睾丸中p-ERK主要分布于精原细胞、细线前期到粗线期的初级精母细胞以及9~12期长形精子细胞的细胞核,p-JNK则主要位于支持细胞与支持细胞、支持细胞与生精细胞(尤其是19期精子细胞)之间,而p-p38 MAPK除了在生精小管的部分细胞胞质中有分布外,其表达最明显的部位是在间质细胞的细胞质。结论ERKJ、NK和p-38 MAPK分别定位于正常大鼠睾丸内的不同部位,提示MAPKs不同的亚家族成员分别在精子发生的不同环节中发挥主要作用。ERK可能参与生精细胞增殖、分化的信号转导,JNK则可能通过调节细胞的黏附而最终影响生精细胞的迁移与精子释放过程,而p38 MAPK除了可能与JNK一起参与精子释放的调节外,最主要的作用可能是睾酮合成分泌的调节。     

H2S通过MAPK信号通路影响缺血再灌注损伤大鼠回肠上皮细胞凋亡

 目的: 建立大鼠肠缺血再灌注(I/R)损伤模型,研究H₂S对I/R损伤大鼠回肠上皮细胞凋亡、MAPK信号通路表达的影响。方法: 30只雄性Wistar大鼠随机分为假手术(sham)组、I/R组、I/R+NaHS组。建立大鼠肠I/R损伤模型。再灌注前10 min时经大鼠尾静脉注入100μmol/kg NaHS,随后按1 mg·kg^-1·h^-1输注直到再灌注2 h。RT-PCR检测回肠组织ERK、JNK和p38MAPK的mRNA表达,Western blot检测回肠组织p-ERK、p-JNK、p-p38MAPK、p-NF-κB P65的蛋白水平。TUNEL染色检测回肠上皮细胞凋亡。敏感硫电极法测定血、回肠组织匀浆中的H₂S浓度。结果: I/R组的H₂S浓度、ERK、mRNA、p-ERK均低于sham组和I/R+NaHS组,JNK mRNA、p38MAPK mRNA、p-JNK、p-p38MAPK、p-NF-κB P65和凋亡指数高于sham组和I/R+NaHS组。结论: H₂S通过下调ERK的mRNA表达及磷酸化,上调JNK、p38MAPK mRNA的表达,促进JNK、p38MAPK、NF-κB磷酸化,从而减轻I/R损伤大鼠的回肠上皮细胞凋亡。     

活性氧与丝裂原激活蛋白激酶通路的相互作用介导高糖引起的心肌细胞损伤

目的探讨活性氧(ROS)与丝裂原激活蛋白激酶(MAPK)通路的相互作用在高糖损伤H9c2心肌细胞中的作用。方法应用细胞计数盒(CCK-8)检测细胞存活率;Hoechst 33258核染色检测凋亡细胞形态及数量的改变;双氯荧光素(DCFH-DA)染色荧光显微镜照相检测细胞内ROS水平;Western blot测定蛋白质表达水平。结果高糖(35 mmol/L葡萄糖)处理H9c2心肌细胞24 h可引起明显的损伤,表现为细胞存活率下降,凋亡细胞数量及ROS水平明显升高。另方面,高糖可明显地上调磷酸化(p)p38MAPK、细胞外信号调节蛋白激酶1/2(ERK1/2)及c-Jun N端激酶(JNK)(为MAPK家族的3个成员)的表达水平。N-乙酰半胱氨酸(NAC,为ROS清除剂)能抑制高糖引起的心肌细胞毒性和细胞凋亡,也能阻断高糖对p-p38MAPK、p-ERK1/2及p-JNK表达的上调作用。此外,p38MAPK、ERK1/2和JNK的选择性抑制剂均能抑制高糖引起的心肌损伤,并能抑制ROS生成增多。结论在高糖损伤H9c2心肌细胞中,存在ROS与MAPK通路的正相互作用,这种相互作用可能在高糖引起的心肌细胞损伤中起着重要的作用。     

盐酸石蒜碱对TPC-1细胞增殖和周期的影响

目的:探讨盐酸石蒜碱(LH)对甲状腺乳头状癌TPC-1细胞增殖和周期的影响及其机制.方法:体外培养TPC-1细胞,用不同浓度(1.25、2.5、5、10、20、40、60、80和100μmol/L)的LH处理TPC-1细胞48 h,CCK-8法检测LH作用TPC-1细胞的IC50.参照IC50,将对数生长期TPC-1细...     

Expression of the GLUT1 glucose transporter, p63 and p53 in thyroid carcinomas

Only few studies have evaluated the usefulness of the GLUT1 and p63 status of thyroid carcinomas in revealing tumorigenesis. We studied GLUT1, p53, and p63 immunoexpression in a total of 86 cases of various thyroid carcinoma types to determine the biological significance of GLUT1 and p63 expression in thyroid carcinomas. GLUT1 was detected in six cases of anaplastic carcinoma and in one case of poorly differentiated carcinoma with membranous staining. p63 was detected in five cases of anaplastic carcinoma, in one case of poorly differentiated carcinoma, and in five cases of papillary carcinoma with nuclear positivity. p53 was detected in six cases of anaplastic carcinoma, in one case of poorly differentiated carcinoma, and in one case of follicular carcinoma with nuclear positivity. Five of seven cases of anaplastic carcinoma expressed all three of these markers. The results suggest that GLUT1, p63, and p53 are not expressed in well-differentiated thyroid carcinomas, and that they are usually expressed late in the course of thyroid tumor progression. These data strongly suggest that in anaplastic carcinomas, impairment of p53-mediated repression results in increased GLUT1 and p63 expression, and that this probably reflects the differential regulation of hypoxia-responsive pathways and basal/stem cell regulatory pathways.     

盐酸吡格列酮对糖尿病大鼠肾组织TOLL 受体4 / MAPKs 信号通路的影响

目的:探讨吡格列酮对糖尿病肾病大鼠肾组织中TOLL 受体4/ MAPKs 信号通路的影响。方法:将雄性SD 随机分为对照组(n =8)和试验组(n =32)。试验组SD 鼠造模成功后随机分为3 组进行干预,应用Western blot 检测对照组和干预组大鼠肾组织中丝裂原活化蛋白激酶ERK1/2 及p38MAPK 磷酸化水平。结果:与对照组比较,各组大鼠肾组织中TOLL 受体4 表达增强,其中ERK1/2 与JNK 磷酸化水平明显升高(P<0郾05),但p38MAPK 水平变化不明显(P>0郾05);与模型组比较,吡咯列酮干预组大鼠肾组织中ERK1/2 与p38MAPK 磷酸化水平降低(P<0郾05)。结论:吡格列酮通过TOLL 受体4/ MAPKs/ERK1/2 与TOLL 受体4/ MAPKs/ JNK 信号通路来完成,延缓糖尿病肾病的发生与发展。     

MiR-126 通过MAPK 信号通路抑制oxLDL 处理的血管平滑肌细胞增殖和迁移

目的:探索miR-126 对oxLDL 处理的血管平滑肌细胞(VSMC)生物学功能的影响及其分子机制。方法:采用oxLDL 处理人主动脉血管平滑肌细胞建立模型。实时定量PCR(qRT-PCR)检测miR-126 表达。CCK-8 实验检测细胞增殖。Transwell 实验分析细胞迁移。免疫印迹分析增殖标记蛋白细胞增殖核抗原-67(Ki-67)、增殖细胞核抗原(PCNA)、迁移标记蛋白基质金属蛋白酶9(MMP-9)、血管内皮细胞生长因子(VEGF)促分裂原活化蛋白激酶(MAPK)信号通路相关蛋白ERK1/2、p-ERK1/2、p38、p-p38、JNK 和p-JNK 的表达。结果:模型组的miR-126 表达显著低于对照组(P<0.01)。与模型组相比,miR-126 mimic 组miR-126 表达极显著升高(P<0.001)。模型组和mimic control 组细胞增殖倍数明显高于对照组(P<0.05)。与模型组相比,miR-126 mimic 组细胞增殖倍数明显降低(P<0.05)。与对照组相比,模型组和mimic control 组细胞迁移能力大大提高(P<0.001)。miR-126 mimic 组细胞迁移显著低于模型组(P<0.01)。模型组和mimic control 组细胞增殖标记蛋白Ki-67和PCNA,迁移标记蛋白MMP-9 和VEFG 表达显著高于对照组(P<0.01)。与模型组相比,miR-126 mimic 组增殖标记蛋白Ki-67 和PCNA 及迁移标记蛋白MMP-9 和VEFG 表达明显减弱(P<0.05)。而且,与对照组相比,模型组和mimic control 组中p-ERK1/2/ ERK1/2、p-p38/ p38、p-JNK/ JNK 的相对蛋白表达量的比值显著升高(P<0.01)。miR-126 mimic 组p-ERK1/2/ ERK1/2、p-p38/ p38、p-JNK/ JNK 的相对蛋白表达量的比值则明显低于模型组(P<0.05)。与对照组相比,模型组Ki-67、PCNA、MMP-9、VEGF 表达明显升高(P <0.05)。与模型组相比,miR-126 mimic 组Ki-67、PCNA、MMP-9、VEGF 表达明显降低(P <0.05);Anisomycin 组Ki-67、PCNA、MMP-9、VEGF 表达明显升高(P<0.05)。miR-mimic+ Anisomycin 组Ki-67、PCNA、MMP-9 和VEGF表达与模型组无明显差异。结论:MiR-126 通过MAPK 信号通路抑制oxLDL 处理的血管平滑肌细胞增殖和迁移。     

Immunolocalization of the mitogen-activated protein kinase signaling pathway in Hassall's corpuscles of the human thymus

In the present study, we investigated the immunohistochemical localization of mitogen-activated protein kinase (MAPK) signaling pathway in the human thymus. Three members of MAPK, the extracellular signal-regulated kinase (ERK), the c-Jun N-terminal kinase (JNK) and the p38 kinase, showed differential expression patterns in the thymus medulla. The phosphorylated form of ERK (p-ERK) was abundantly present in the outer layer of Hassall's corpuscles, and the phosphorylated form of p38 kinase (p-p38 kinase) was present in the entire Hassall's corpuscles. The phosphorylated form of JNK (p-JNK) was expressed in medullary thymocytes. We also examined localization of MAPK kinases (MAPKK or MEK) which specifically activate MAPK. MEK1, an activator of ERK, was found in the outer layer of Hassall's corpuscles where p-ERK was expressed. MEK3, an activator of p38 kinase, was also expressed in the outer layer. MEK4 and MEK7, which are activators of JNK, were present in the entire Hassall's corpuscles. Thus, differential expression of MAPK in the thymus supports the concept that the MAPK signaling pathway controls the specificity of functional thymic responses to extracellular stimuli. Furthermore, the abundant expression of various elements of the pathway in Hassall's corpuscles suggests that the pathway is involved in thymic medullary epithelial maturation.     

Immunohistochemical localisation of RET and p53 mutant protein of thyroid lesions in a North-Eastern Malaysian population and its prognostic implications

AIMS: To investigate RET and p53 expression in local thyroid lesions, in order to shed light on the pathogenesis of papillary carcinoma and explain the high prevalence of this condition among the nodular hyperplasia (multi-nodular goitre) cases. METHODS: Archival thyroid tissue was retrieved from Hospital Universiti Sains Malaysia (HUSM) Pathology Department files and studied by immunohistochemistry for RET and p53 mutant protein. Normal tissues from 74 cases served as controls. RESULTS: Fifty follicular adenoma, 66 nodular hyperplasia and 53 papillary carcinoma cases were studied. RET was expressed in 5.4% of normal thyroid tissue, 18% of follicular adenomas, 22.7% of nodular hyperplasia cases and 71.7% of papillary carcinomas. Its expression in papillary carcinoma was not associated with the coexistence of nodular hyperplasia lesions. p53 was expressed by 17% of papillary carcinomas. No association was found between p53 expression of nodular hyperplasia with or without co-existing papillary carcinoma. p53, rather than RET, was an excellent predictor of tumour lymph node metastasis and capsular invasion. p53 was also a significant prognosticator of survival outcome. CONCLUSIONS: RET expression is highly prevalent in local papillary carcinoma, indicating a significant role in the pathogenesis of this tumour, with no apparent role in tumour behaviour and survival outcome. p53 on the other hand appears to be a significant factor in the latter events. The two genes appear to act in two different pathways: the former being an initiator, and the later a propagator of papillary carcinoma.     

Real-time quantitative RT-PCR identifies distinct c-RET, RET/PTC1 and RET/PTC3 expression patterns in papillary thyroid carcinoma

RET/PTC1 and RET/PTC3 are the markers for papillary thyroid carcinoma. Their reported prevalence varies broadly. Nonrearranged c-RET has also been detected in a variable proportion of papillary carcinomas. The published data suggest that a wide range in expression levels may contribute to the different frequency of c-RET and, particularly, of RET/PTC detection. However, quantitative expression analysis has never been systematically carried out. We have analyzed by real-time RT-PCR 25 papillary carcinoma and 12 normal thyroid samples for RET/PTC1, RET/PTC3 and for RET exons 10-11 and 12-13, which are adjacent to the rearrangement site. The variability in mRNA levels was marked and four carcinoma groups were identified: one lacking RET/PTC rearrangement with balanced RET exon levels similar to those of the normal samples (7/25 cases, 28%), the second (6/25 cases, 24%) with balanced RET expression and very low levels of RET/PTC1, the third with unbalanced RET exons 10-11 and 12-13 expression, high RET/PTC1 levels but no RET/PTC3 (7/25 cases, 28%), and the fourth with unbalanced RET expression, high RET/PTC1 levels and low levels of RET/PTC3 (5/25 cases, 20%). Papillary carcinomas with high RET/PTC1 expression showed an association trend for large tumor size (P=0.063). Our results indicate that the variability in c-RET and RET/PTC mRNA levels contributes to the apparent inconsistencies in their reported detection rates and should be taken into account not only for diagnostic purposes but also to better understand the role of c-RET activation in thyroid tumorigenesis.     

亚油酸乙酯抑制钛颗粒诱导的炎症反应及其作用机制

BACKGROUND: Ethyl linoleate has been proved to attenuate the inflammatory-cytokines release induced by lipopolysaccharide, but whether it can inhibit titanium-induced osteolysis and the underlying mechanism remain unclear. OBJECTIVE: To observe the effect of ethyl linoleate on the expression of inflammatory-related factors induced by titanium particles and explore its mechanism. METHODS: Forty-eight Kunming mice were randomly divided into blank control, titanium, dimethylsulfoxide (DMSO) and experimental groups. The back air pouch Inflammatory models were established in the mice of the titanium, DMSO and experimental groups, in which the 200 µL menstruum of DMSO (0.5%) and 200 µL ethyl linoleate (0.5%) were respectively administered into the pouch of the mice at 12 hours. Mice in the blank control group received no intervention. Fourteen days later, the inflammatory cell infiltration in the skin was examined through hematoxylin-eosin staining; the expression levels of inhibitor κB-α, nuclear factor-κB, inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis factor-α and interleukin-6 as well as ERK, p-ERK, JNK, p-JNK, p38 and p-p38 in MAPK signaling pathways were evaluated by western blot assay. RESULTS AND CONCLUSION: In the titanium group and DMSO group, there were numerous inflammatory cells and vacuole-like necrotic tissues in the hair follicle lacuna of dermis and loose connective tissues of hypodermis. The experiment group showed significant reduction in inflammatory cell infiltration and vacuole-like necrosis. Compared with the blank control group, the expression levels of inducible nitric oxide synthase, cyclooxygenase-2, nuclear factor-κB, tumor necrosis factor-α, interleukin-6, ERK, JNK and p38 in the DMSO and titanium groups were significantly increased, while inhibitor κB-α significantly decreased (P < 0.05). Compared with the DMSO and titanium groups, there were significantly down-regulated levels of inducible nitric oxide synthase, cyclooxygenase-2, nuclear factor-κB, tumor necrosis factor-α, interleukin-6, ERK, JNK and p38, and up-regulated inhibitor κB-α level in the experimental group (P < 0.05). In conclusion, ethyl linoleate can remarkably suppress the expressions of titanium-induced inflammatory factors associated with the inhibition of nuclear factor-κB and MAPK signaling pathway activation.     

丝裂素激活蛋白激酶在良性前列腺增生中的表达及临床意义

目的对比研究丝裂素激活蛋白激酶(MAPKs)在增生与正常前列腺组织中的表达,探讨良性前列腺增生发病的分子生物学机制。方法采用W estern B lot及免疫组织化学方法,检测增生及正常前列腺组织中ERK、JNK和p38MAPK的表达情况。结果与正常前列腺组织相比,增生组织ERK的平均灰度值(wh ite~b lack,0~255)和阳性细胞百分率明显升高,JNK和p38MAPK的检测结果则与之相反。ERK染色主要位于细胞核和细胞质内,在增生组织上皮细胞及基质细胞的染色阳性率均高于正常前列腺组织。结论ERK在良性前列腺增生组织中过表达,从而导致细胞增生指数增加,JNK和p38MAPK低表达导致凋亡减少,促进疾病的发生和发展。     

胰岛素对大鼠结肠平滑肌细胞凋亡及MAPK通路的影响

 目的 探讨胰岛素对大鼠结肠平滑肌细胞( SMCs )凋亡及丝裂原活化蛋白激酶( MAPK )信号转导通路的影响。方法 酶解法分离培养SD大鼠结肠SMCs,α-actin免疫鉴定,将大鼠结肠SMCs分为正常组,胰岛素组,胰岛素 +PD98059( ERK抑制剂)组,MTT法检测SMCs增殖,流式细胞术Annexin V-FITC/PI检测SMCs凋亡,Western blot法检测p-ERK、ERK、p-P38MAPK、P38MAPK和p-JNK、JNK表达。结果 胰岛素组较正常组细胞明显增殖,凋亡率降低,p-ERK表达增强,p-ERK/ERK 比值升高( 110.36 ± 9.5 vs 50.92 ± 6.01 ) ( P < 0.01 );p-P38MAPK、P38MAPK、p-JNK、JNK表达无差异。PD98059组较正常组细胞增殖明显下降,凋亡率升高,p-ERK表达减弱,p-ERK/ERK比值降低( 15.69 ± 2.11 vs 50.92±6.01 ) ( P < 0.01 )。结论 胰岛素可能通过激活结肠SMCs的MAPK通路中的ERK途径,促进细胞增殖,抑制凋亡,可能与P38MAPK途径和JNK途径无关。     

尼古丁抑制人牙髓干细胞增殖和分化

目的探讨尼古丁的刺激对人牙髓干细胞增殖、分化的影响。方法培养人牙髓干细胞,流式细胞计量术鉴定细胞表面抗原;用不同浓度的尼古丁(10-4、10-3和10-2 mol/L)刺激牙髓干细胞,培养0、1、2、3和4 d后,CCK8法检测细胞增殖能力;茜素红染色法检测细胞分化过程中矿化结节形成,RT-qPCR和免疫印迹(Western blot)检测牙本质涎磷蛋白(DSPP)、碱性磷酸酶(ALP)、骨桥素(OPN)及细胞外信号调节激酶(ERK)、c-Jun氨基末端激酶(JNK)、p38(p-ERK、p-JNK、p-p38)等MAPK通路相关蛋白表达。结果培养第3、4天时,与对照组相比,尼古丁刺激时A值显著降低(P<0.05);与对照组相比,尼古丁刺激时矿化结节形成数、DSPP、ALP、OPN mRNA和蛋白、p-ERK、p-JNK、p-p38蛋白表达显著降低(P<0.05)。结论尼古丁抑制人牙髓干细胞增殖、成骨分化能力。     

血清淀粉样蛋白A对骨肉瘤细胞侵袭、迁移能力以及MAPK信号通路的影响

目的:探讨沉默血清淀粉样蛋白A(SAA)的表达对骨肉瘤细胞活力、凋亡、转移能力以及丝裂原活化蛋白激酶(MAPK)信号通路的影响。方法:通过小干扰RNA(siRNA)特异性沉默SAA在骨肉瘤U2OS细胞中的表达,实验分为空白对照组、阴性对照组和实验组,阴性对照组和实验组分别转染阴性对照siRNA和SAA-siRNA,空白对照组不做任何处理。MTT法观察细胞的活力变化,Annexin V-FITC/PI双染法检测细胞的凋亡率,Transwell小室法分析细胞迁移和侵袭能力的变化,Western blot检测细胞中SAA、磷酸化p38 MAPK(p-p38 MAPK)和磷酸化c-Jun氨基末端激酶(p-JNK)的蛋白水平。结果:SAA-siRNA可显著降低SAA在骨肉瘤U2OS细胞中的表达(P 0. 05);与空白对照组相比,SAA-siRNA组细胞的活力显著降低(P 0. 05),凋亡率显著升高(P 0. 05),侵袭和迁移能力显著降低(P 0. 05)。SAA-siRNA组细胞中p-p38 MAPK和p-JNK的蛋白水平显著低于对照组(P 0. 05),而总JNK和p38蛋白表达量的差异无统计学显著性。结论:沉默SAA的表达可抑制骨肉瘤细胞活力,诱导其凋亡,降低细胞的转移能力,其作用可能与调控MAPK信号通路的活性有关。     

Expression of Jun activation domain-binding protein 1 and p27 (Kip1) in thyroid medullary carcinoma

AIMS: p27 is a prominent regulator of cell proliferation by universally inhibiting the cell cycle, while Jun activation domain-binding protein 1 (Jab1), a multifunctional cell signaling protein, contributes to carcinoma progression by degrading p27. In this study, we investigated the expression of these proteins in medullary thyroid carcinoma. METHODS: We immunohistochemically examined Jab1 and p27 expression in 64 medullary thyroid carcinomas. RESULTS: Of the 64 cases examined, decreased p27 expression was observed in 38 cases (59.4%). The p27 expression level was inversely linked to tumour size as well as plasma calcitonin level. Jab1 expression level was generally high, and 46 cases (71.9%) were classified as overexpressing Jab1. The incidence was higher than those in papillary and follicular carcinomas, which were previously reported. Jab1 expression level was inversely linked to that of p27, and all five cases with only cytoplasmic but not nuclear staining of p27 overexpressed Jab1. CONCLUSIONS: These findings suggest that (1) decrease in p27 expression may contribute to local tumour growth; (2) Jab1 expression is related to the progression of medullary carcinoma by decreasing the amount of p27 in the cell and accelerating its degradation; and (3) Jab1 may play a more vital role in the pathogenesis of medullary carcinoma than papillary and follicular carcinomas.     

Activation of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) during hypoxia in cerebral cortical nuclei of guinea pig fetus at term: role of nitric oxide

Previously we have shown that cerebral tissue hypoxia results in generation of nitric oxide (NO) free radicals as well as increased expression of mitogen-activated protein kinase like extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK). The present study tested the hypothesis that administration of l-nitro-l-arginine methyl ester (L-NAME), a NOS inhibitor, prior to hypoxia prevents the hypoxia-induced activation of p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) and in the cerebral cortex of the term guinea pig fetus. To test this hypothesis normoxic (Nx, n=6), hypoxic (Hx, n=7) and hypoxic pretreated with l-NAME (Hx+L-NAME, n=6) guinea pig fetuses at 60 days gestation were studied to determine the phosphorylated p38, ERK and JNK. Hypoxia was induced by exposing pregnant guinea pigs to FiO2 of 0.07 for 1h. l-NAME (30mg/kg i.p.) was administered to pregnant mothers 60min prior to hypoxia. Cerebral tissue hypoxia was documented biochemically by determining the tissue levels of ATP and phosphocreatine (PCr). Neuronal nuclei were isolated, purified and proteins separated using 12% SDS-PAGE, and then probed with specific phosphorylated ERK, JNK and p38 antibodies. Protein bands were detected by enhanced chemiluminescence, analyzed by imaging densitometry and expressed as absorbance (ODxmm2). The relative level of p-p38 was 51.41+/-9.80 (Nx), 173.67+/-3.63 (Hx), 58.56+/-3.40 (Hx+L-NAME), p<0.05 vs. Hx. The relative level p-ERK was 44.91+/-4.20 (Nx), 135.12+/-17.02 (Hx), 58.37+/-9.5 (Hx+L-NAME), p<0.05 vs. Hx. The relative level of p-JNK was 34.86+/-6.77 (Nx), 97.36+/-19.24 (Hx), 46.65+/-12.81 (Hx+L-NAME), p<0.05 vs. Hx. The data show that administration of l-NAME prior to hypoxia decreased the relative level of phosphorylated p38, ERK and JNK at term gestation. Since a NOS inhibitor prevented the hypoxia-induced phosphorylation of p38, ERK and JNK, we conclude that the hypoxia-induced activation of p38, ERK and JNK in the cerebral cortical nuclei of guinea pig fetus at term is NO-mediated. We speculate that NO-mediated modification of cysteine residue leading to inhibition of MAP kinase phosphatases results in increased activation of p38, ERK and JNK in the guinea pig fetus at term.     

High-level expression, activation, and subcellular localization of p38-MAP kinase in thyroid neoplasms

The p38 family of MAP kinases (p38-MAPKs) is involved in regulating the proliferation, survival, and migration of various cancer cells. The present study has investigated the expression, subcellular localization, phosphorylation, and activity of p38-MAPKs in normal and tumoural human thyroid tissues and in thyroid cell lines. The expression and nucleo-cytosolic compartmentalization of the alpha-isoform of p38-MAPKs (p38alpha-MAPK) were studied by western blotting in the WRO and B-CPAP cell lines, which are derived from human follicular and papillary thyroid carcinomas, respectively, and in the non-transformed rat thyroid cell lines FRTL-5 and PCCL3. Immunohistochemistry was used to study the expression and subcellular localization of p38alpha-MAPK, and of the phosphorylated forms of p38-MAPKs (P-p38-MAPKs) in human toxic adenomas (TAs), follicular adenomas (FAs), papillary thyroid carcinomas (PTCs), and follicular thyroid carcinomas (FTCs). The activity of p38-MAPKs in PTCs and FTCs was revealed by immunohistochemical detection of their typical phosphorylated substrate, MAPK-activated protein kinase 2/3 (MK2/3). p38alpha-MAPK was expressed in all cell lines and this expression was restricted to the cytosolic compartment. p38 MAPK activity was involved in regulating DNA synthesis in B-CPAP cells. p38alpha-MAPK and P-p38-MAPKs were strongly expressed in PTC and FTC cells, although only in the cytoplasm, whereas they were only very weakly expressed in FA cells, and absent in adjacent normal tissues. They were also expressed at a high level in TAs, but they were found in both nucleus and cytoplasm. Finally, phospho-MK2/3 immunostaining followed very similar patterns to those of p38alpha-MAPK and P-p38-MAPKs in PTCs and FTCs. Taken together, these results show for the first time that the p38-MAPK signalling cascade is functional in two types of differentiated carcinoma of the thyroid. The observation that p38-MAPK hyper-expression occurs in FTC, but not in FA, may provide an additional diagnostic tool for malignancy in some thyroid nodules.     

Inhibition of tobacco smoke-induced bladder MAPK activation and epithelial-mesenchymal transition in mice by curcumin

Tobacco smoke (TS) has been shown to cause bladder cancer. Epithelial-mesenchymal transition (EMT) is a crucial pathophysiological process in cancer development. MAPK pathways play central roles in tumorigenesis including EMT process. Curcumin is a promising chemopreventive agent for several types of cancers. In the present study we investigated the effects of TS on MAPK pathway activation and EMT alterations in the bladder of mice, and the preventive effect of curcumin was further examined. Results showed that exposure of mice to TS for 12 weeks resulted in activation of ERK1/2, JNK, p38 and ERK5 MAPK pathways as well as AP-1 proteins in bladder. TS reduced mRNA and protein expression levels of epithelial markers E-cadherin and ZO-1, while mRNA and protein expression levels of the mesenchymal markers vimentin and N-cadherin were increased. Curcumin treatment effectively attenuated TS-triggered activation of ERK1/2, JNK and p38 MAPK pathways, AP-1 proteins and EMT alterations in bladder tissue. These results suggest the protective effects of curcumin in TS-induced MAPK activation and EMT, thus providing new insights into the chemoprevention of TS-associated bladder cancer.