平滑肌细胞迁移的肌球蛋白轻链非磷酸化途径

为了阐明平滑肌细胞迁移存在肌球蛋白轻链非磷酸化调节途径,研究花生四烯酸(arachidonicacid,AA)对肌球蛋白轻链非磷酸化状态下平滑肌细胞迁移的影响及其相关的信号传导途径.经Boyden小室跨膜迁移实验发现,AA对培养的兔血管平滑肌SM3细胞具有明显的诱导迁移作用.然而,当预先用10μmolL肌球蛋白轻链激酶(myosinlightchainkinase,MLCK)特异性抑制剂ML7作用SM3细胞后,发现AA对SM3细胞仍然具有明显的诱导迁移作用,并呈剂量依赖性,这种诱导作用可被细胞外信号调节激酶12(ERK12)的特异性抑制剂PD98059或磷脂酶C(PLC)的特异性抑制剂U73122所拮抗.此外,Ⅱ型肌球蛋白抑制剂blebbistatin(BLB)可部分抑制“非磷酸化”状态下AA的诱导迁移作用.经Western印迹检测显示,10μmolLML7可完全抑制SM3细胞中20kD肌球蛋白轻链(MLC20)磷酸化,并且加入AA后MLC20仍为非磷酸化状态.应用免疫荧光染色法观察肌动蛋白在SM3细胞中分布的变化,发现在AA作用下肌动蛋白呈细胞边缘聚集现象,有伪足形成,细胞形态表现为迁移状态.预先用ML7作用后再加入AA,肌动蛋白的分布与上述结果相同.研究结果初步表明,在平滑肌细胞迁移的作用途径中,在MLC磷酸化调节途径受到抑制时,AA可诱导MLC非磷酸化的平滑肌细胞发生迁移,其分子机理可能与ERK12和PLC信号传导途径有关,非磷酸化的肌球蛋白直接参与了该迁移过程.     

非磷酸化肌球蛋白在血小板衍生生长因子诱导的血管平滑肌细胞迁移中的作用

为了阐明非磷酸化肌球蛋白在平滑肌细胞迁移中的作用,研究探讨了非磷酸化肌球蛋白是否介导了血小板衍生生长因子(PDGF)诱导豚鼠脑基底动脉平滑肌细胞(GbaSM-4)的迁移。研究结果显示,20ng/ml以下剂量的PDGF可诱导GbaSM-4细胞发生迁移,此时肌球蛋白轻链(MLC20)磷酸化水平无变化。该迁移作用可被肌球蛋白特异性抑制剂blebbistatin所拮抗。应用RNA干扰技术抑制肌球蛋白轻链激酶表达,经免疫印迹检测经果显示,MLC20的磷酸化水平发生了显著下降;但对PDGF诱导的迁移作用无影响;在RNA干扰后blebbistatin也可抑制其迁移作用。体外ATP酶活性测定结果显示,blebbistatin对从平滑肌中提取的非磷酸化肌球蛋白的ATP酶活性有明显的抑制作用,其主要作用位点位于肌球蛋白头的头部S1。上述结果提示,非磷酸化的肌球蛋白参与了PDGF诱导的平滑肌细胞迁移。     

柴胡提取物诱导人类白血病细胞HL-60的细胞凋亡及其机理研究

柴胡提取物诱导人类白血病细胞HL-60的细胞凋亡从而抑制其细胞生长.为了研究该过程的作用机理,我们研究了丝裂原活化蛋白激酶(MAPKs),包括胞外信号调节激酶(ERK1/2),c-jun氨基末端蛋白激酶(JNK)和p38丝裂原活化蛋白激酶(MAPK),在该过程中的磷酸化特征与动态变化.结果表明,柴胡提取物显著的增加了p38丝裂原活化蛋白激酶和胞外信号调节激酶(ERK1/2)的磷酸化作用,其增加值在测试范围内与测试剂量和作用时间成正相关,但在柴胡提取物诱导人类白血病细胞HL-60的细胞凋亡过程中,没有发现对氨基末端蛋白激酶(JNK)表现出磷酸化活性.柴胡提取物诱导白血病HL-60的细胞凋亡部分归结于对p38丝裂原活化蛋白激酶的上调节作用,这种上调节作用能够受到p38 MAPK特异性的抑制剂SB203580的部分逆转,而MEK的抑制剂U0126则对柴胡提取物诱导HL-60细胞凋亡过程中的胞外信号调节激酶(ERK1/2)的磷酸化具有显著的协同效应.这是首次报道柴胡提取物在诱导人白血病细胞HL-60细胞凋亡过程中参与p38丝裂原活化蛋白激酶的磷酸化,同时柴胡提取物作为胞外信号调节激酶(ERK1/2)抑制剂的协同作用物具有相应的药物学功能.     

JNK、ERK信号通路在NaAsO_2诱导骨髓间充质干细胞增殖中的作用

目的：研究c-jnk氨基末端激酶（JNK）、细胞外信号调节激酶（ERK）在亚砷酸钠（NaAs02）诱导骨髓间充质干细胞（BMSC）增殖中的作用。方法：体外培养骨髓间充质干细胞,四甲基偶氮唑盐比色法（MTT法）检测细胞增殖,Western-blot检测磷酸化JNK、ERK表达水平。结果：低浓度1、2μmol/LNaAs02对BMSC有明显的促进增殖作用;高浓度16、32μmol/LNaAs02则对细胞生长产生抑制作用,具有一定剂量-效应关系;2、4、8μmol/LNaAs02处理BMSC24h后,JNK磷酸化表达水平明显增加,ERK磷酸化表达水平明显降低;JNK抑制剂SP600125可明显降低高浓度16、32μmol/LNaAs02的生长抑制作用;ERK抑制剂PD98059可抑制低浓度1、2μmol/LNaAs02对BMSC的促增殖作用。结论：低浓度NaAs02激活ERK信号通路,提高细胞增殖率,可被抑制剂PD98059阻断;高浓度NaAs02激活JNK信号通路,提高细胞凋亡率,可被抑制剂SP600125阻断。NaAs02致癌机制可能与JNK、ERK信号通路作用相关。     

高转移倾向乳腺癌细胞中p-ERK促进增殖和迁移作用的信号转导途径

在建立乳腺癌细胞MCF-7高转移倾向亚克隆LM-MCF-7细胞株的基础上,为阐明LM-MCF-7细胞具有更强增殖和迁移能力的分子机制,对其相关分子及其信号转导途径进行了探讨.免疫印迹结果显示,与MCF-7细胞相比,LM-MCF-7细胞中p-ERK1/2水平显著升高.流式细胞术和“伤口愈合”实验结果表明,ERK1/2的特异性抑制剂PD98059可明显抑制LM-MCF-7细胞的高增殖和高迁移能力.免疫印迹检测发现,与MCF-7细胞相比,LM-MCF-7细胞中与增殖和迁移相关的因子,如β-catenin、细胞周期蛋白D1、磷酸化肌球蛋白轻链（p-MLC）和肌球蛋白轻链激酶（MLCK）的水平呈明显增高,PD98059对这些因子水平的增高具有抑制作用.免疫荧光染色显示,LM-MCF-7细胞中β-catenin分布在细胞核中,应用PD98059处理后,β-catenin主要分布在胞浆中.上述研究结果表明,在LM-MCF-7细胞中活化的ERK1/2水平升高,是导致该细胞增殖和迁移能力增强的重要原因之一,与ERK1/2-MLCK-p-MLC和ERK1/2-β-catenin 细胞周期蛋白D1等信号转导途径有密切的关系.     

酸性鞘磷脂水解酶和MAPK信号通路在UVA诱导细胞凋亡中的作用

为了探讨酸性鞘磷脂水解酶 (ASM)和MAPK信号通路在UVA诱导的细胞凋亡中的作用 ,用DNA梯形条带 (DNAladder)和荧光显微镜鉴定细胞凋亡 ,Western印迹分析MAPK信号通路的激活情况 .结果显示 :①经UVA照射 ,正常的淋巴母细胞JY出现严重的细胞凋亡 ,而ASM遗传性缺陷的淋巴母细胞MS1 4 1 8出现轻微凋亡 ;给予ASM特异性抑制剂NB6 ,UVA诱导的JY细胞凋亡明显减轻 ,表明UVA诱导的细胞凋亡依赖于ASM .②UVA照射后 ,磷酸化ERK含量在MS1 4 1 8细胞中明显升高 ,在JY细胞中受到抑制 ;UVA照射前给予NB6 ,JY细胞中磷酸化ERK含量上升 ,表明ASM能抑制ERK的激活 .③UVA照射后 ,磷酸化JNK含量在MS1 4 1 8细胞中几乎没有变化 ,而在JY细胞中含量升高 ;UVA照射前给予NB6 ,JY细胞中磷酸化JNK含量没有明显升高 ,表明ASM激活JNK通路 .④NB6对UVA激活的p38MAPK信号通路没有影响 ,表明p38的激活与ASM关系不大 .研究表明 ,UVA诱导的细胞凋亡是通过激活ASM、激活JNK信号通路并抑制ERK信号通路来完成的     

Rottlerin 通过抑制 PKCδ-ERK1/2 通路减轻6-羟基多巴胺所致多巴胺能细胞凋亡

近来发现新的蛋白激酶C δ亚型（PKCδ）的磷酸化激活与帕金森病（PD）中神经元的缺失有关.我们的前期研究发现, PKCδ 磷酸化参与6-羟基多巴胺引起的多巴胺能细胞的毒性作用,但其具体机制尚不清楚.本研究以TUNEL检测发现,6-羟基多巴胺引起较显著的细胞凋亡,PKC δ 抑制剂 Rottlerin 可减轻6-羟基多巴胺诱导的凋亡,总PKC抑制剂Bis、钙依赖性PKC（α和β）抑制剂Go6976对6-羟基多巴胺诱导的凋亡无影响.采用Western免疫印迹杂交实验发现,6-羟基多巴胺持续性激活 ERK 1/2 和PKC δ, Rottlerin既可抑制PKCδ的磷酸化激活,又可抑制ERK的磷酸化激活,而 MEK 抑制剂 U0126 仅能抑制 ERK 1/2 磷酸化激活,对 PKC δ 磷酸化却无显著影响.这说明 PKCδ 是6-羟基多巴胺持续性激活 ERK 1/2 的上游激酶.本研究结果提示,在凋亡过程中PKCδ仍然是ERK1/2激活的上游激酶,阻断PKCδ磷酸化可阻断ERK1/2持续激活.Rottlerin正是由于阻断PKCδ的激活,进一步阻断ERK1/2持续激活,减轻多巴胺能细胞的凋亡.因此, Rottlerin 可能对防治帕金森病患者神经元缺失有一定作用.     

大黄素对HT-29 细胞MAPKs 信号通路活化及IL-8 分泌的影响

目的:研究大黄素对IFN-和LPS刺激的人结肠癌细胞株HT-29细胞的ERK、JNK和p38 MARK和IL-8表达的影响。方法:人结肠癌细胞株HT-29细胞与40 ng/mL的IFN-共培养12 h,再加入100 ng/mL LPS刺激15 min,用大黄素预处理进行干预。ELISA检测HT-29细胞内的ERK、JNK和p38 MARK含量和细胞上清IL-8含量。结果:IFN-γ和LPS刺激后HT-29细胞的ERK、JNK和p38 MARK磷酸化水平和IL-8分泌明显升高。大黄素对p38和JNK磷酸化有明显的抑制作用,而对ERK磷酸化则没有明显抑制作用;大黄素能显著降低IFN-γ+LPS所引起的HT-29细胞IL-8的大量产生,并且呈明显的剂量依赖关系。结论:大黄素能有效抑制IFN-γ+LPS所引起的HT-29细胞p38和JNK的磷酸化,并显著降低IL-8分泌。     

丝裂原活化蛋白激酶在人冠状动脉平滑肌细胞向三维纤维蛋白胶迁移中的作用

目的:观察三维纤维蛋白(Fb)胶对体外培养的人冠状动脉平滑肌细胞(HCASMC)的趋化作用及其信号转导机制.方法:采用胶原酶消化法培养HCASMC,倒置相差显微镜观察其向三维Fb胶中迁移的能力及ERK、p38、JNK信号通路对其迁移能力的影响.Western blot检测Fb对HCASMC p-ERK、p-p38和p-JNK表达的调控.结果:向Fb胶中迁移的HCASMC呈长梭型,细胞数量增加时形成环形管腔样结构.纤维蛋白原(Fg)浓度为0.8 g/L～6.4 g/L时,HCASMC向胶中迁移的数量呈浓度依赖性增加,并随培育时间的延长逐渐增多.用Western Blot分析显示Fb以时间依赖性方式诱导ERK、p38及JNK活化,三者的选择性抑制剂PD98059 50 μmol/L、SB20358010 μmol/L及SP600125 20 μmol/L可分别抑制其活化,但对HCASMC向胶中迁移的抑制能力不尽相同.PD9805950 μmol/L对HCASMC迁移无明显影响,而SB203580 10 μmol/L和SP600125 20 μmol/L均可抑制HCASMC向Fb胶迁移,且后者抑制作用更强.结论:Fb胶通过激活细胞JNK和p38(而不是ERK信号通路)促进HCASMC向Fb胶中迁移,这种机制可能在动脉粥样硬化血栓形成及再狭窄过程中发挥重要作用.     

Signaling pathways downstream of P2 receptors in human neutrophils

Extracellular nucleotides stimulate human neutrophils by activating the purinergic P2Y₂ receptor. However, it is not completely understood which types of G proteins are activated downstream of this P2 receptor subtype. We investigated the G-protein coupling to P2Y₂ receptors and several subsequent signaling events. Treatment of neutrophils with pertussis toxin (PTX), a Gi protein inhibitor, caused only ∼75% loss of nucleotide-induced Ca²⁺ mobilization indicating that nucleotides cause Ca²⁺ mobilization both through Gi-dependent and Gi-independent pathways. However, the PLC inhibitor U73122 almost completely inhibited Ca²⁺ mobilization in both nucleotide- and fMLP-stimulated neutrophils, strongly supporting the view that both the PTX-sensitive and the PTX-insensitive mechanism of Ca²⁺ increase require activation of PLC. We investigated the dependence of ERK phosphorylation on the Gi pathway. Treatment of neutrophils with PTX caused almost complete inhibition of ERK phosphorylation in nucleotide or fMLP activated neutrophils. U73122 caused inhibition of nucleotide- or fMLP-stimulated ERK phosphorylation, suggesting that although pertussis toxin-insensitive pathways cause measurable Ca²⁺ mobilization, they are not sufficient for causing ERK phosphorylation. Since PLC activation leads to intracellular Ca²⁺ increase and PKC activation, we investigated if these intracellular events are necessary for ERK phosphorylation. Exposure of cells to the Ca²⁺ chelator BAPTA had no effect on nucleotide- or fMLP-induced ERK phosphorylation. However, the PKC inhibitor GF109203X was able to almost completely inhibit nucleotide- or fMLP-induced ERK phosphorylation. We conclude that the P2Y₂ receptor can cause Ca²⁺ mobilization through a PTX-insensitive but PLC-dependent pathway and ERK phosphorylation is highly dependent on activation of the Gi proteins.     

Inhibition of PTPs by H(2)O(2) regulates the activation of distinct MAPK pathways

It has been shown that endogenous production of reactive oxygen species (ROS) during T cell activation regulates signaling events including MAPK activation. Protein tyrosine phosphatases (PTPs) have been regarded as targets of ROS which modify the catalytic cysteine residues of the enzymes. We have analyzed the interplay between the inhibition of PTPs and the activation of MAPK by H(2)O(2). Stimulation of Jurkat T cells with H(2)O(2) induces the phosphorylation of ERK, p38, and JNK members of MAPK family. H(2)O(2) stimulation of T cells was found to inhibit the PTP activity of CD45, SHP-1, and HePTP. Transfection of cells with wtSHP-1 decreased H(2)O(2)-induced ERK and JNK phosphorylation without affecting p38 phosphorylation. Transfection with wtHePTP inhibited H(2)O(2)-induced ERK and p38 phosphorylation without inhibiting JNK phosphorylation. The Src-family kinase inhibitor, PP2, inhibited the H(2)O(2)-induced phosphorylation of ERK, p38, and JNK. The phospholipase C (PLC) inhibitor, U73122, or the protein kinase C (PKC) inhibitor, Ro-31-8425, blocked H(2)O(2)-induced ERK phosphorylation, whereas the same treatment did not inhibit p38 or JNK phosphorylation. Taken together, these results suggest that inhibition of PTPs by H(2)O(2) contributes to the induction of distinct MAPK activation profiles via differential signaling pathways.     

Intracellular signal transduction for migration and actin remodeling in vascular smooth muscle cells after sphingosylphosphorylcholine stimulation

Molecular mechanisms underlying migration of vascular smooth muscle cells (VSMCs) toward sphingosylphosphorylcholine (SPC) were analyzed in light of the hypothesis that remodeling of the actin cytoskeleton should be involved. After SPC stimulation, mitogen-activated protein kinases (MAPKs), including p38 MAPK (p38) and p42/44 MAPK (p42/44), were found to be phosphorylated. Migration of cells toward SPC was reduced in the presence of SB-203580, an inhibitor of p38, but not PD-98059, an inhibitor of p42/44. Pertussis toxin (PTX), a Gi protein inhibitor, induced an inhibitory effect on p38 phosphorylation and VSMC migration. Myosin light chain (MLC) phosphorylation occurred after SPC stimulation with or without pretreatment with SB-203580 or PTX. The MLC kinase inhibitor ML-7 and the Rho kinase inhibitor Y-27632 inhibited MLC phosphorylation but only partially inhibited SPC-directed migration. Complete inhibition was achieved with the addition of SB-203580. After SPC stimulation, the actin cytoskeleton formed thick bundles of actin filaments around the periphery of cells, and the cells were surrounded by elongated filopodia, i.e., magunapodia. The peripheral actin bundles consisted of alpha- and beta-actin, but magunapodia consisted exclusively of beta-actin. Such a remodeling of actin was reversed by addition of SB-203580 and PTX, but not ML-7 or Y-27632. Taken together, our biochemical and morphological data confirmed the regulation of actin remodeling and suggest that VSMCs migrate toward SPC, not only by an MLC phosphorylation-dependent pathway, but also by an MLC phosphorylation-independent pathway.     

Thrombin Mediates Migration of Rat Brain Astrocytes via PLC, Ca2+, CaMKII, PKCα, and AP-1-Dependent Matrix Metalloproteinase-9 Expression

Matrix metalloproteinase-9 (MMP-9) plays a crucial role in pathological processes of brain inflammation, injury, and neurodegeneration. Thrombin has been known as a regulator of MMP-9 expression and cells migration. However, the mechanisms underlying thrombin-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) remain unclear. Here, we demonstrated that thrombin induced the expression of pro-form MMP-9 and migration of RBA-1 cells, which were inhibited by pretreatment with the inhibitor of Gq-coupled receptor (GPAnt2A), Gi/o-coupled receptor (GPAnt2), PC-PLC (D609), PI-PLC (U73122), Ca²⁺-ATPase (thapsigargin, TG), calmodulin (CaMI), CaMKII (KN62), PKC (Gö6976 or GF109203X), MEK1/2 (PD98059), p38 MAPK (SB202190), JNK1/2 (SP600125), or AP-1 (Tanshinone IIA) or the intracellular calcium chelator (BAPTA/AM) and transfection with siRNA of PKCα, Erk2, JNK1, p38 MAPK, c-Jun, or c-Fos. In addition, thrombin-induced elevation of intracellular Ca²⁺ concentration was attenuated by PPACK (a thrombin inhibitor). Thrombin further induced CaMKII phosphorylation and PKCα translocation, which were inhibited by U73122, D609, KN62, TG, or BAPTA/AM. Thrombin also induced PKCα-dependent p42/p44 MAPK and JNK1/2, but not p38 MAPK activation. Finally, we showed that thrombin enhanced c-Fos expression and c-Jun phosphorylation. c-Fos mRNA levels induced by thrombin were reduced by PD98059, SP600125, and Gö6976, but not SB202190. Thrombin stimulated in vivo binding of c-Fos to the MMP-9 promoter, which was reduced by pretreatment with SP600125 or PD98059, but not SB202190. These results concluded that thrombin activated a PLC/Ca²⁺/CaMKII/PKCα/p42/p44 MAPK and JNK1/2 pathway, which in turn triggered AP-1 activation and ultimately induced MMP-9 expression in RBA-1 cells.     

Eosinophil migration induced by mast cell chymase is mediated by extracellular signal-regulated kinase pathway

Mast cell chymase is known to induce eosinophil migration in vivo and in vitro. In the present study, we investigated possible involvement of mitogen-activated protein (MAP) kinases; extracellular signal-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38, in the chymase-induced eosinophil migration. Human chymase induced a rapid phosphorylation of ERK1/2 and p38 in human eosinophilic leukemia EoL-1 cells, while no phosphorylation was detected in JNK. The chymase-induced phosphorylation of ERK and p38 was inhibited by pertussis toxin. Similar results were obtained in the experiments using mouse chymase and eosinophils. U0126 (the inhibitor for MAP/ERK kinase) suppressed chymase-induced migration of EoL-1 cells and mouse eosinophils. However, SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor) showed little effect on the migration. It is suggested therefore that chymase activates ERK and p38 probably through G-protein-coupled receptor, and that ERK but not p38 cascade may have a crucial role in chymase-induced migration of eosinophils.     

Thrombin promotes actin stress fiber formation in RPE through Rho/ROCK‐mediated MLC phosphorylation

The retinal pigment epithelium (RPE) forms the outer blood–retina barrier (BRB). Most retinal diseases involve BRB breakdown, whereupon thrombin contained in serum directly contacts the RPE. Thrombin is known to promote actin stress fiber formation, an important determinant in eye diseases involving the epithelial–mesenchymal transition (EMT) and migration of RPE cells, such as proliferative vitreoretinopathy. We analyzed thrombin effect on signaling pathways leading to myosin light chain (MLC) phosphorylation and actin stress fiber formation in primary cultures of rat RPE cells, in order to support a role for thrombin in RPE transdifferentiation. MLC phosphorylation was measured by Western blot; actin cytoskeleton was visualized using immunofluorescent phalloidin, and Rho GTPase activation was assessed by ELISA. We showed that thrombin/PAR‐1 induces the time‐ and dose‐dependent phosphorylation of MLC through the activation of Rho/ROCK and myosin light chain kinase (MLCK). ROCK increased phospho‐MLC by phosphorylating MLC and by inhibiting MLC phosphatase. Thrombin effect was abolished by the ROCK inhibitor Y‐27632, whereas MLCK inhibitor ML‐7 and PLC‐β inhibitor U73122 attenuated MLC phosphorylation by ≈50%, suggesting the activation of MLCK by PLC‐β‐mediated calcium increase. Additionally, thrombin‐induced MLC phosphorylation was blocked by the inhibitory PKCζ pseudosubstrate, wortmannin, and LY294002, indicating IP₃/PKCζ involvement in the control of MLC phosphorylation. Moreover, we demonstrated that thrombin effect on MLC induces actin stress fiber formation, since this effect was prevented by inhibiting the pathways leading to MLC phosphorylation. We conclude that thrombin stimulation of MLC phosphorylation and actin stress fiber formation may be involved in thrombin‐induced RPE cell transformation subsequent to BRB dysfunction. J. Cell. Physiol. 226: 414–423, 2011. © 2010 Wiley‐Liss, Inc.     

G(q)-dependent signalling by the lysophosphatidic acid receptor LPA(3) in gastric smooth muscle: reciprocal regulation of MYPT1 phosphorylation by Rho kinase and cAMP-independent PKA

The present study characterized the signalling pathways initiated by the bioactive lipid, LPA (lysophosphatidic acid) in smooth muscle. Expression of LPA(3) receptors, but not LPA(1) and LPA(2), receptors was demonstrated by Western blot analysis. LPA stimulated phosphoinositide hydrolysis, PKC (protein kinase C) and Rho kinase (Rho-associated kinase) activities: stimulation of all three enzymes was inhibited by expression of the G(alphaq), but not the G(alphai), minigene. Initial contraction and MLC(20) (20 kDa regulatory light chain of myosin II) phosphorylation induced by LPA were abolished by inhibitors of PLC (phospholipase C)-beta (U73122) or MLCK (myosin light-chain kinase; ML-9), but were not affected by inhibitors of PKC (bisindolylmaleimide) or Rho kinase (Y27632). In contrast, sustained contraction, and phosphorylation of MLC(20) and CPI-17 (PKC-potentiated inhibitor 17 kDa protein) induced by LPA were abolished selectively by bisindolylmaleimide. LPA-induced activation of IKK2 {IkappaB [inhibitor of NF-kappaB (nuclear factor kappaB)] kinase 2} and PKA (protein kinase A; cAMP-dependent protein kinase), and degradation of IkappaBalpha were blocked by the RhoA inhibitor (C3 exoenzyme) and in cells expressing dominant-negative mutants of IKK2(K44A) or RhoA(N19RhoA). Phosphorylation by Rho kinase of MYPT1 (myosin phosphatase targeting subunit 1) at Thr(696) was masked by phosphorylation of MYPT1 at Ser(695) by PKA derived from IkappaB degradation via RhoA, but unmasked in the presence of PKI (PKA inhibitor) or C3 exoenzyme and in cells expressing IKK2(K44A). We conclude that LPA induces initial contraction which involves activation of PLC-beta and MLCK and phosphorylation of MLC(20), and sustained contraction which involves activation of PKC and phosphorylation of CPI-17 and MLC(20). Although Rho kinase was activated, phosphorylation of MYPT1 at Thr(696) by Rho kinase was masked by phosphorylation of MYPT1 at Ser(695) via cAMP-independent PKA derived from the NF-kappaB pathway.     

Sphingosylphosphorylcholine induces proliferation of human adipose tissue-derived mesenchymal stem cells via activation of JNK

Sphingosylphosphorylcholine (SPC) has been implicated in a variety of cellular responses, including proliferation and differentiation. In this study, we demonstrate that d-erythro-SPC, but not l-threo-SPC, stereoselectively stimulated the proliferation of human adipose tissue-derived mesenchymal stem cells (hADSCs), with a maximal increase at 5 microM, and increased the intracellular concentration of Ca(2+) ([Ca(2+)](i)) in hADSCs, which do not express known SPC receptors (i.e., OGR1, GPR4, G2A, and GPR12). The SPC-induced proliferation and increase in [Ca(2+)](i) were sensitive to pertussis toxin (PTX) and the phospholipase C (PLC) inhibitor U73122, suggesting that PTX-sensitive G proteins, Gi or Go, and PLC are involved in SPC-induced proliferation. In addition, SPC treatment induced the phosphorylation of c-Jun and extracellular signal-regulated kinase, and SPC-induced proliferation was completely prevented by pretreatment with the c-Jun N-terminal kinase (JNK)-specific inhibitor SP600125 but not with the MEK-specific inhibitor U0126. Furthermore, the SPC-induced proliferation and JNK activation were completely attenuated by overexpression of a dominant negative mutant of JNK2, and the SPC-induced activation of JNK was inhibited by pretreatment with PTX or U73122. Treatment of hADSCs with lysophosphatidic acid (LPA) receptor antagonist, Ki16425, had no impact on the SPC-induced increase in [Ca(2+)](i). However, SPC-induced proliferation was partially, but significantly, attenuated by pretreatment of the cells with Ki16425.These results indicate that SPC stimulates the proliferation of hADSCs through the Gi/Go-PLC-JNK pathway and that LPA receptors may be responsible in part for the SPC-induced proliferation.     

Mechanical hyperalgesia induced by endothelin-1 in rats is mediated via phospholipase C, protein kinase C, and MAP kinases

In addition to causing overt nociception, intraplantar (ipl) endothelin (ET)-1 injection into the rat hind paw induces hyperalgesia to mechanical stimuli, mediated via local ET(B) receptors coupled to protein kinase (PK) C, but not PKA. The present study further examines the intracellular signaling mechanisms underlying this effect of ET-1. ET-1 (30 pmol) or phospate-buffered saline (PBS) was injected ipl in rats and the threshold of responsiveness to mechanical stimulation was assessed repeatedly each hour up to 8 hrs and 24 hrs, using the dynamic plantar aesthesiometer test, which detects the minimal pressure required to evoke paw withdrawal. Different groups were treated, 15 mins before ET-1 administration, with ipsilateral injection of selective inhibitors of either phospholipase (PL) A2 (1 nmol PACOCF3), PLC (30 pmol U73122), PKC (1 nmol GF109203X), p38 mitogen-activated protein kinase (MAPK; 30 nmol SB203580), extracellular signal-regulated kinase (ERK1/2; 30 nmol PD98059), c-Jun N-terminal kinase (JNK; 30 nmol SP600125), or vehicle, to assess their influence on the hyperalgesic response. The mechanical hyperalgesia caused by ET-1 started 2 hrs after injection, peaked at 5 hrs (PBS, 29 +/- 0.5 g; ET-1, 17 +/- 1.3 g) and lasted up to 8 hrs. The inhibitors of PLC, PKC, p38 MAPK, ERK1/2, and JNK caused long-lasting reductions of the mechanical hyperalgesia (inhibitions at 4 hrs of 100%, 90%, 97%, 90%, and 100%, respectively), but the PLA2 inhibitor reduced hyperalgesia only at 4 hrs (by 58%). Thus, mechanical hyperalgesia triggered by ET-1 in the rat hind paw depends importantly on signaling pathways involving PLC, PKC, p38 MAPK, ERK1/2, and JNK, whereas the contribution of PLA2 is relatively minor.