巨噬细胞免疫调变信号:Raf-1,MAPK p44,MAPK p42和p38 MAPK的研究

为了了解巨噬细胞免疫调变机理,我们应用LPS和PMA处理小鼠抑制性巨噬细胞,观察到Ras下游信号分子Raf-1,分裂原激活蛋白激酶MAPK p44,MAPK p42和p38 MAPK均被活化,发现forskolin能增强p38 MAPK的活性,进一步提示PKC和PKA途径增强了p38 MAPK的磷酸化效应,为我们了解LPS如何激活p38 MAPK信号通路提供了一个新的机会。     

14，15-EET通过EGFR和p44／42MAPK信号通道诱导肿瘤细胞增殖

目的探讨14,15-EET促进肿瘤细胞增殖的机制。方法Tea-8113培养于含10％小牛血清的DMEM,必要时更换无血清DMEM使其进入静止期。用14,15一EET和／或抑制剂处理后收集细胞蛋白,用Western印迹检测EGFR和p44／42MAPK（ERK1／ERK2）的磷酸化水平。结果14,15-EET以剂量依赖性方式刺激EGFR和p44／42MAPK（ERKI／ERK2）蛋白磷酸化,该效应能够被特异性的EGFR抑制剂AG1478阻断。MTT分析显示,AG1478能够完全取消14,15-EET诱导的Tea-8113细胞增殖效应。结论EGFR的活化是花生四烯酸细胞色素P450表氧化酶代谢产物14,15-EET丝裂原信号途径中的关键事件;在该途径中EGFR的活化是p44／42MAPK（ERK1／ERK2）活化的上游事件。     

神经生长因子家族成员NGF、BDNF、NT-3和NT-4在猫L6脊髓的分布

采用免疫组织化学方法观察神经生长因子家庭成员NGF,BDNF、NT3和NT4在成年猫L6脊髓的分布。结果;在L6脊髓灰质均可见四种生长因子的免疫阳性细胞,这些细胞主要是腹角及凝角深部的大神经元及背角浅层的小神经元,灰质内亦内NGF,NT3及NT4阳性的胶质细胞,但数量多少不等,其中NT3者最多,其次是NGF,NT4者最少,此外,Ⅱ板层内还可见较多BDNF及少量NT-3阳性神经膨体,本文结果表明,在成年猫脊髓存在NGF,BDNF、NT3和NT4,但其分布有差异。     

P-P42/p44在慢性肾功能不全大鼠肾组织表达特征及其作用

目的探讨慢性肾功能不全大鼠肾组织磷酸化P42/p44丝裂原活化蛋白激酶(P-P42/p44 MAPK)的表达特征及其可能的作用。方法16只Wistar大鼠随机分成实验组和对照组,每组8只。采用5/6肾切除方法构建慢性肾功能不全大鼠模型,术后120d处死大鼠,取大鼠肾组织行石蜡切片,PAS染色观察大鼠肾脏病理改变,免疫组化和Western blot法分别检测大鼠肾组织磷酸化P42/p44丝裂原活化蛋白激酶的表达特征及活性变化。结果术后120d实验组大鼠与对照组相比,出现明显的肾小球硬化和肾小管坏死等慢性肾功能不全的典型病理特征,免疫组织化学染色检测磷酸化p42/p44 MAPK黄棕色染色颗粒明显增加。Western-blot结果显示,实验组大鼠肾组织磷酸化P42/p44丝裂原活化蛋白激酶(P-P42/p44 MAPK)活性表达水平明显上调(P<0.01)。结论磷酸化P42/p44丝裂原活化蛋白激酶在慢性肾功能不全大鼠模型的肾组织中活性明显升高,可能是慢性肾功能不全时各种细胞外刺激因素介导肾脏纤维化的重要途径之一。     

Tetraspanin CD9 regulates osteoclastogenesis via regulation of p44/42 MAPK activity

Tetraspanin CD9 has been shown to regulate cell-cell fusion in sperm-egg fusion and myotube formation. However, the role of CD9 in osteoclast, another multinucleated cell type, is not still clear. Therefore, we investigated the role of CD9 in osteoclast differentiation. CD9 was expressed in osteoclast lineage cells and its expression level increased during the progression of RANKL-induced osteoclastogenesis. KMC8, a neutralizing antibody specific to CD9, significantly suppressed RANKL-induced multinucleated osteoclast formation and the mRNA expression of osteoclast differentiation marker genes. To define CD9-regulated osteoclastogenic signaling pathway, MAPK pathways were examined. KMC8 induced long-term phosphorylation of p44/42 MAPK, but not of p38 MAPK. Constitutive activation of p44/42 MAPK by overexpressing constitutive-active mutant of MEK1 almost completely blocked osteoclast differentiation. Taken together, these results suggest that CD9 expressed on osteoclast lineage cells might positively regulate osteoclastogenesis via the regulation of p44/42 MAPK activity.     

Chemokine CCL20 enhances the growth of HuH7 cells via phosphorylation of p44/42 MAPK in vitro

CCR6 is the receptor of chemokine CCL20. In the present study, we demonstrated that the surface expression of CCR6 was enhanced on the human HCC cell lines (HuH7, PLC/PRF/5, and HepG2) especially on HuH7 cells, but not on HLE or HLF cells. These HCC cell lines (HuH7, PLC/PRF/5, and HepG2) especially the HuH7 cells secreted a significant amount of CCL20 spontaneously, whereas HLE or HLF did not. Stimulation by CCL20 up-regulated the mRNA expression of CCR6 in HuH7 cells and significantly enhanced the growth of HuH7 cells. CCL20-stimulated growth of HuH7 cells was abrogated by the inhibition of downstream signal transduction pathway mediated by p44/42 MAPK, but not by p38 MAPK or SAPK/JNK. CCR6 expression in human HCC tissues was confirmed by RT-PCR. These results indicate that the growth of a proportion of human HCC cells may be mediated by CCL20-CCR6 axis, like HuH7 cells, in an autocrine or paracrine manner.     

p44/42 MAPK activation is necessary for receptor activator of nuclear factor-kappaB ligand induction by high extracellular calcium

Although extracellular calcium (Ca(2+)(o)) has been suggested to modulate bone remodeling, the exact mechanism is unclear. This study was performed to explore the signaling pathways of high Ca(2+)(o) that are responsible for controlling the expression of receptor activator of NF-kappaB ligand (RANKL) in mouse osteoblastic cells. As previously reported, high Ca(2+)(o) increased RANKL expression. However, the G protein-coupled Ca(2+)(o)-sensing receptor (CaSR) was not detected in the primary cultured mouse osteoblastic cell. The inhibition of the pertussis-sensitive G protein, phospholipase C, protein kinase C, intracellular calcium mobilization, p38 MAPK, or phosphoinositide 3-kinase did not block RANKL induction caused by high Ca(2+)(o). In contrast, the inhibition of p44/42 MAPK pathway reduced the RANKL expression induced by high Ca(2+)(o). Moreover, high Ca(2+)(o) activated p44/42 MAPK and MEK1/2. These results suggest that RANKL induction by high Ca(2+)(o) might not be mediated by CaSR and its putative downstream signaling pathways, but the pathway employing p44/42 MAPK is involved in the high Ca(2+)(o)-induced RANKL expression in mouse osteoblastic cells.     

Vitamin C down-regulates VEGF production in B16F10 murine melanoma cells via the suppression of p42/44 MAPK activation

It is known that vitamin C induces apoptosis in several kinds of tumor cells, but its effect on the regulation of the angiogenic process of tumors is not completely studied. Vascular endothelial growth factor (VEGF) is the most well-known angiogenic factor, and it has a potent function as a stimulator of endothelial survival, migration, as well as vascular permeability. Therefore, we have investigated whether vitamin C can regulate the angiogenic process through the modulation of VEGF production from B16F10 melanoma cells. VEGF mRNA expression and VEGF production at protein levels were suppressed by vitamin C. In addition, we found that vitamin C suppressed the expression of cyclooxygenase (COX)-2 and that decreased VEGF production by vitamin C was also restored by the administration of prostaglandin E2 which is a product of COX-2. These results suggest that vitamin C suppresses VEGF expression via the regulation of COX-2 expression. Mitogen-activated protein kinases are generally known as key mediators in the signaling pathway for VEGF production. In the presence of vitamin C, the activation of p42/44 MAPK was completely inhibited. Taken together, our data suggest that vitamin C can down-regulate VEGF production via the modulation of COX-2 expression and that p42/44 MAPK acts as an important signaling mediator in this process.     

Histone code of genes induced by co-treatment with a glucocorticoid hormone agonist and a p44/42 MAPK inhibitor in human small intestinal Caco-2 cells

Background

Inactivation of glucocorticoid hormones and p44/42 mitogen-activated protein kinase (MAPK) is thought to be important in small intestinal maturation and expression of genes related to intestinal differentiation and functions.

Methods

We investigated target genes induced by co-treatment for 48 h with a glucocorticoid hormone agonist, dexamethasone (Dex), and a p44/42 MAPK inhibitor, PD98059 (PD), in a small intestine-like cell line (Caco-2) using microarray analysis. We also investigated whether expression changes of the target genes induced by the co-treatment are associated with histone modifications around these genes.

Results

Co-treatment of Caco-2 cells with Dex and PD enhanced several genes related to intestinal differentiation and functions such as SCNN1A, FXYD3, LCT and LOX. Induction of the SCNN1A gene was associated with increased presence of acetylated histone H3 and H4 and di-methylated histone H3 at lysine (K) 4 around the transcribed region of the gene, and induction of the FXYD3 gene was associated with increased presence of acetylated histones H3 and H4 from the promoter/enhancer to the transcribed region of the gene. Induction of LCT and LOX genes was associated with increased presence of acetylated histone H4 on the promoter/enhancer region of the genes.

Conclusions

Histone acetylation and/or histone H3 K4 methylation around the promoter/enhancer or/and transcribed regions of target genes are associated with induction of the genes by co-treatment with Dex and PD in Caco-2 cells.

General significance

The histone code is specific to each gene with respect to induction by glucocorticoid hormone and inhibition of p44/42 MAPK in Caco-2 cells.     

Coxsackie adenovirus receptor (CAR) regulates integrin function through activation of p44/42 MAPK

The coxsackie B virus and adenovirus receptor (CAR) is an attachment receptor for Adenovirus serotype 5 (Ad5) and in many cell types forms homodimers with neighbouring cells as part of a cell adhesion complex. CAR co-operates with cell surface integrin receptors to enable efficient viral entry, but little is known about the mechanism of crosstalk between these two receptor types. Here we show that overexpression of CAR in human epithelial cells leads to increased basal activation of p44/42 MAPK and this is required for efficient Ad5 infection. We demonstrate that CAR forms homodimers in cis and that this dimerisation is enhanced in the presence of Ad5 in a phospho-p44/42-dependent manner. CAR-induced p44/42 activation also leads to increased activation of β1 and β3 integrins. Analysis of CAR mutants demonstrates that the cyto domain of CAR is required for CAR-induced p44/42 activation, integrin activation and localisation to cell junctions. This data for the first time demonstrates that signalling downstream of CAR can have a dual effect on integrins and CAR itself in order to promote efficient viral binding to cell membranes.     

猫延髓吻侧腹外侧区NPY、SOM和NT免疫反应物质的分布

生理学研究表明,延髓吻侧腹外侧区(RVL)在维持血压稳定方面起着关键的作用。本文用免疫组化ABC技术,观察了猫RVL神经肽Y(NPY)、生长抑素(SOM)和神经降压肽(NT)免疫反应(IR)细胞和纤维的分布,以便为研究此区血压调节功能的机制提供形态学资料。结果表明:NPY—、SOM—IR细胞和少量NT—IR细胞主要分布于旁巨细胞外侧核、外侧网状核吻侧部以及外侧网状核背侧紧邻的网状结构。这些细胞从RVL尾侧向吻侧逐渐减少。NPY—IR纤维分布于旁巨细胞外侧核以及外侧网状核吻侧部的腹内侧区。NT—IR纤维较多,可见两丛中等密度的NT—IR纤维:一丛位于旁巨细胞外侧核;另一丛位于面后核、疑核以及二核紧邻的区域。此外还可见少量SOM—IR纤维。