全反式维甲酸通过抑制ERK和激活p38 MAPK信号诱导KLF4基因表达

全反式维甲酸（all-trans retinoic acid, ATRA）诱导细胞分化与上调转录因子Krüppel样因子4 (KLF4)表达有关, 但目前对ATRA诱导KLF4表达的分子机制尚不清楚．为了研究ATRA在血管平滑肌细胞（VSMC）中诱导KLF4表达的分子机制,本 研究观察ATRA对视黄酸受体α (retinoic acid receptor α, RARα)和KLF4表达的影响及RARα介导ATRA诱导KLF4表达所依 赖的信号转导途径．实验结果显示,ATRA可显著诱导RARα和KLF4表达,用RARα拮抗剂Ro 41 5253阻断ATRA与受体相互作 用后,ATRA诱导的KLF4表达受到显著抑制．用p38 MAPK、ERK和Akt抑制剂阻断ATRA与RARα相互作用所激活的信号转导途径 后,发现阻断p38 MAPK信号途径显著抑制ATRA诱导的KLF4表达,抑制ERK信号途径使ATRA对KLF4表达的诱导作用明显增强, 抑制Akt信号途径不影响KLF4基因表达．表明RARα介导ATRA对KLF4表达的诱导作用,ATRA通过抑制ERK和激活p38 MAPK信号 途径发挥其对KLF4基因表达的诱导作用．     

Hyperplastic Growth of Pulmonary Artery Smooth Muscle Cells from Subjects with Pulmonary Arterial Hypertension Is Activated through JNK and p38 MAPK

Smooth muscle in the pulmonary artery of PAH subjects, both idiopathic and hereditary, is characterized by hyperplasia. Smooth muscle cells (HPASMC) isolated from subjects with or without PAH retain their in vivo phenotype as illustrated by their expression of alpha-smooth muscle actin and expression of H-caldesmon. Both non PAH and PAH HPASMC display a lengthy, approximately 94h, cell cycle. The HPASMC from both idiopathic and hereditary PAH display an abnormal proliferation characterized by continued growth under non-proliferative, non-growth stimulated conditions. This effector independent proliferation is JNK and p38 MAP kinase dependent. Blocking the activation of either abrogates the HPASMC growth. HPASMC from non PAH donors under quiescent conditions display negligible proliferation but divide upon exposure to growth factors such as PDGF-BB or FGF2 but not EGF. This growth does not involve the MAP kinases. Instead it routes via the tyrosine kinase receptor through mTOR and then 6SK. In the PAH cells PDGF-BB and FGF2 augment the dysregulated cell proliferation, also through mTOR/6SK. Additionally, blocking the activation of mTOR also modulates the MAP kinase promoted dysregulated growth. These results highlight key alterations in the growth of HPASMC from subjects with PAH which contribute to the etiology of the disease and can clearly be targeted at various regulatory points for future therapies.     

Expression of Protease-Activated Receptor 1 and 2 and Anti-Tubulogenic Activity of Protease-Activated Receptor 1 in Human Endothelial Colony-Forming Cells

Endothelial colony-forming cells (ECFCs) are obtained from the culture of human peripheral blood mononuclear cell (hPBMNC) fractions and are characterised by high proliferative and pro-vasculogenic potential, which makes them of great interest for cell therapy. Here, we describe the detection of protease-activated receptor (PAR) 1 and 2 amongst the surface proteins expressed in ECFCs. Both receptors are functionally coupled to extracellular signal-regulated kinase (ERK) 1 and 2, which become activated and phosphorylated in response to selective PAR1- or PAR2-activating peptides. Specific stimulation of PAR1, but not PAR2, significantly inhibits capillary-like tube formation by ECFCs in vitro, suggesting that tubulogenesis is negatively regulated by proteases able to stimulate PAR1 (e.g. thrombin). The activation of ERKs is not involved in the regulation of tubulogenesis in vitro, as suggested by use of the MEK inhibitor PD98059 and by the fact that PAR2 stimulation activates ERKs without affecting capillary tube formation. Both qPCR and immunoblotting showed a significant downregulation of vascular endothelial growth factor 2 (VEGFR2) in response to PAR1 stimulation. Moreover, the addition of VEGF (50–100 ng/ml) but not basic Fibroblast Growth Factor (FGF) (25–100 ng/ml) rescued tube formation by ECFCs treated with PAR1-activating peptide. Therefore, we propose that reduction of VEGF responsiveness resulting from down-regulation of VEGFR2 is underlying the anti-tubulogenic effect of PAR1 activation. Although the role of PAR2 remains elusive, this study sheds new light on the regulation of the vasculogenic activity of ECFCs and suggests a potential link between adult vasculogenesis and the coagulation cascade.     

miR-20b-5p靶向MAPK1调控脑出血过程中脑微血管内皮细胞铁死亡

摘要 目的：探讨miR-20b-5p对氧糖剥夺（OGD）/Hemin处理的脑微血管内皮细胞（BMVEC）功能的影响及机制。方法：将BMVEC分为Control组、agomir-NC组、agomir-miR-20b-5p组、antagomir-NC组和antagomir-miR-20b-5p组。使用Lipofectamine 2000试剂对细胞进行相应的转染处理。BMVEC转染后,将BMVEC再分为Control组、OGD/Hemin组（O/H组）、OGD/Hemin+agomir-NC组（O/H+agomir-NC组）、OGD/Hemin+agomir-miR-20b-5p组（O/H+agomir-miR-20b-5p组）、OGD/Hemin+antagomir-NC组（O/H+antagomir-NC组）和OGD/Hemin+antagomir-miR-20b-5p组（O/H+antagomir-miR-20b-5p组）。Control组BMVEC正常培养,其他组BMVEC进行OGD/Hemin处理。MTT法检测BMVEC增殖,TUNEL染色检测BMVEC凋亡,Transwell检测BMVEC迁移。使用试剂盒检测超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）和丙二醛（MDA）水平。使用Iron Assay试剂盒检测Fe²⁺含量。通过qRT-PCR检测miR-20b-5p和MAPK1 mRNA水平。通过Western blot检测MAPK1、Bax、Bcl-2、谷胱甘肽过氧化物酶4（GPX4）和前列腺素内过氧化物合酶2（PTGS2）蛋白表达水平。通过免疫荧光染色检测MAPK1的荧光强度水平。结果：与Control组和agomir-NC组比较,agomir-miR-20b-5p组BMVEC中的miR-20b-5p水平升高（P<0.05）。与Control组和antagomir-NC组比较,antagomir-miR-20b-5p组BMVEC中的miR-20b-5p水平降低（P<0.05）。与Control组比较,O/H组BMVEC中的miR-20b-5p水平降低,细胞活力降低,TUNEL阳性率和Bax蛋白表达水平升高,Bcl-2蛋白表达水平降低,迁移数量降低,SOD和GSH-Px活性降低,MDA含量升高,Fe²⁺含量和PTGS2的蛋白表达水平升高,GPX4的蛋白表达水平降低,MAPK1的mRNA和蛋白表达水平以及相对荧光强度升高（P<0.05）。与O/H组和O/H+agomir-NC组比较,O/H+agomir-miR-20b-5p组BMVEC中的miR-20b-5p水平升高,细胞活力升高,TUNEL阳性率和Bax蛋白表达水平降低,Bcl-2蛋白表达水平升高,迁移数量升高,SOD和GSH-Px活性升高,MDA含量降低,Fe²⁺含量和PTGS2的蛋白表达水平降低,GPX4的蛋白表达水平升高,MAPK1的mRNA和蛋白表达水平以及相对荧光强度降低（P<0.05）。与O/H组和O/H+antagomir-NC组比较,O/H+antagomir-miR-20b-5p组BMVEC中的miR-20b-5p水平降低,细胞活力降低,TUNEL阳性率和Bax蛋白表达水平升高,Bcl-2蛋白表达水平降低,迁移数量降低,SOD和GSH-Px活性降低,MDA含量升高,Fe²⁺含量和PTGS2的蛋白表达水平升高,GPX4的蛋白表达水平降低,MAPK1的mRNA和蛋白表达水平以及相对荧光强度升高（P<0.05）。结论：本研究表明上调miR-20b-5p通过抑制OGD/Hemin处理的BMVEC中MAPK1的表达从而抑制了铁死亡途径。     

Liver Kinase B1 Expression Promotes Phosphatase Activity and Abrogation of Receptor Tyrosine Kinase Phosphorylation in Human Cancer Cells

Aberrant receptor tyrosine kinase phosphorylation (pRTK) has been associated with diverse pathological conditions, including human neoplasms. In lung cancer, frequent liver kinase B1 (LKB1) mutations correlate with tumor progression, but potential links with pRTK remain unknown. Heightened and sustained receptor activation was demonstrated by LKB1-deficient A549 (lung) and HeLaS3 (cervical) cancer cell lines. Depletion (siRNA) of endogenous LKB1 expression in H1792 lung cancer cells also correlated with increased pRTK. However, ectopic LKB1 expression in A549 and HeLaS3 cell lines, as well as H1975 activating-EGF receptor mutant lung cancer cell resulted in dephosphorylation of several tumor-enhancing RTKs, including EGF receptor, ErbB2, hepatocyte growth factor receptor (c-Met), EphA2, rearranged during transfection (RET), and insulin-like growth factor I receptor. Receptor abrogation correlated with attenuation of phospho-Akt and increased apoptosis. Global phosphatase inhibition by orthovanadate or depletion of protein tyrosine phosphatases (PTPs) resulted in the recovery of receptor phosphorylation. Specifically, the activity of SHP-2, PTP-1β, and PTP-PEST was enhanced by LKB1-expressing cells. Our findings provide novel insight on how LKB1 loss of expression or function promotes aberrant RTK signaling and rapid growth of cancer cells.     

Src Tyrosine Kinase Activation by 4-Hydroxynonenal Upregulates p38, ERK/AP-1 Signaling and COX-2 Expression in YPEN-1 Cells

4-Hydroxynonenal (4-HNE), a major end product of lipid peroxidation, is highly reactive and involved in various cellular processes, such as inflammatory signaling. However, to date, the mechanistic roles of 4-HNE in inflammatory signaling related to protein tyrosine kinases have not been elucidated. In the present study, we investigated the interaction between 4-HNE and Src (a non-receptor tyrosine kinase) for its involvement in the molecular modulation of the inflammatory signaling pathway utilizing the YPEN-1 cell system. Immunoprecipitation experiments showed that 4-HNE phosphorylates (activates) Src at Tyr416 via adduct formation. In addition, LC-MS/MS and a docking simulation model revealed an addiction site at the Cys248 residue of Src, resulting in the stimulation of downstream p38, ERK/AP-1 and cyclooxygenase-2 (COX-2) signaling in YPEN-1 cells. The role of 4-HNE-activated Src in downstream inflammatory signaling was further investigated using dasatinib (a Src inhibitor) and by siRNA knockdown of Src. p38 and ERK were directly regulated by Src, as revealed by immunoblotting of the phosphorylated forms of mitogen-activated protein kinases (MAPKs), which are key elements in the signaling transduction pathway initiated by Src. The study also shows that Src modulates the HNE-enhanced activation of AP-1 and the expression of COX-2 (a target gene of AP-1). Together, the results of this study show that 4-HNE stimulates Src tyrosine kinase in activation of the inflammation process.     

MAPK磷酸酯酶-1通过抑制JNK和p38的磷酸化调节SH-SY5Y神经母细胞瘤的凋亡

目的研究MKP-1在SH-SY5Y神经母细胞瘤中的抗凋亡。方法建立稳定表达MKP-1的SH-SY5Y细胞,用H2O2诱导细胞凋亡,并通过Western blotting比较分析MKP-1的表达对JNK和p38磷酸化的调节。结果①H2O2诱导SH-SY5Y细胞表达MKP-1,同时导致JNK和p38的去磷酸化;②在稳定表达MKP-1的SH-SY5Y细胞中,MKP-1可以抑制JNK和p38的磷酸化。③稳定表达MKP-1的SH-SY5Y细胞抵抗H2O2诱导细胞凋亡的能力比对照细胞提高了1倍左右。结论MKP-1对神经细胞的凋亡具有重要的调节作用,提示MKP-1作为调节ERK、JNK和p38蛋白激酶信号途径的重要分子,可能对退行性神经系统疾病的发病机制和治疗有重要的作用。     

Expression and Activity of Cyclin-Dependent Kinase 5/p35 in Adult Rat Peripheral Nervous System

Abstract: In spite of the clarification in the temporal and spatial expression pattern of a cyclin-dependent kinase (Cdk) 5 and its neuron-specific activator, p35, in the CNS, it remains to be elucidated in the PNS. In addition, it is not known whether Cdk5 activity exists in the PNS. Therefore, we have examined their expression and activity in the PNS by immunoblot analysis, immunohistochemistry, and in vitro kinase assay. Immunoblot analysis indicated the expression of Cdk5 and p35 proteins in dorsal root ganglion (DRG) and sciatic nerve alike in the CNS. By immunohistochemistry, both proteins were shown to be present in the cell body and axon (sciatic nerve) of both DRG neurons and anterior horn cells. A co-immunoprecipitation study indicated the in vivo association between Cdk5 and p35 in both DRG and sciatic nerve. However, Cdk5 kinase activity was found only in DRG, but not in sciatic nerve. These results suggest that Cdk5 kinase activity exists and functions physiologically in the PNS and may be regulated by unknown mechanisms other than the availability of p35 as reported in developing brains.     

Interferon-γ and Nitric Oxide Down-Regulate Lipopolysaccharide-Induced Prostanoid Production in Cultured Rat Microglial Cells by Inhibiting Cyclooxygenase-2 Expression

Abstract: We have used purified microglial cultures obtained from neonatal rat brains to study some aspects of the regulation of prostanoid production in these cells. We found that nitric oxide, which is released into the culture medium along with prostanoids when the cells are exposed to lipopolysaccharide (1–100 ng/ml), can down-regulate prostanoid biosynthesis. Indeed, the abrogation of endogenous nitric oxide production, obtained by depleting the medium of the precursor l -arginine or by blocking the activity of nitric oxide synthase by the specific inhibitor N^G-monomethyl-l -arginine, led to a remarkable increase in lipopolysaccharide-induced prostanoid release. Moreover, the nitric oxide-generating compound 3-morpholinosydnonimine caused a substantial reduction of prostanoid formation, in the absence of endogenous nitric oxide, suggesting that both endogenous and exogenous nitric oxide may inhibit the induced prostanoid production. We also found that interferon-γ potentiated the effect of lipopolysaccharide on nitrite accumulation as previously described by others and depressed the lipopolysaccharide-evoked production of prostaglandin E₂, prostaglandin D₂, and thromboxane. It is interesting that the inhibitory effect of interferon-γ on prostanoid production did not appear to depend on the potentiation of NO release, as it was present also when the endogenous synthesis of nitric oxide was abrogated. Additional experiments showed that interferon-γ and nitric oxide act mainly by down-regulating the lipopolysaccharide-induced enzymatic activity and expression (analyzed by western blot) of cyclooxygenase-2. Our data indicate that microglial prostanoid biosynthesis induced by proinflammatory stimuli, such as lipopolysaccharide, is tightly regulated by nitric oxide. Interferon-γ appears to affect the balance between these local mediators by favoring nitric oxide production and inhibiting the prostanoid cascade and may thus contribute to the modulation of inflammation, local immune reactivity, and neuronal damage.     

Differential Activation of Mitogen-Activated Protein Kinases,ERK 1/2, p38MAPK and JNK p54/p46 During Postnatal Development of Rat Hippocampus

Mitogen-activated protein kinases (MAPKs) are a group of serine-threonine kinases, including p38^MAPK, ERK 1/2 and JNK p54/p46, activated by phosphorylation in response to extracellular stimuli. The early postnatal period is characterized by significant changes in brain structure as well as intracellular signaling. In the hippocampus MAPKs have been involved in the modulation of development and neural plasticity. However, the temporal profile of MAPK activation throughout the early postnatal development is incomplete. An understanding of this profile is important since slight changes in the activity of these enzymes, in response to environmental stress in specific developmental windows, might alter the course of development. The present study was undertaken to investigate the hippocampal differential activation of MAPK during postnatal period. MAPK activation and total content were evaluated by Western blotting of hippocampal tissue obtained from male Wistar rats at postnatal days (P) 1, 4, 7, 10, 14, 21, 30 and 60. The total content and phosphorylation of each MAPK was expressed as mean ± SEM and then calculates as a percentile compared to P1 (set at 100 %). The results showed: (1) phosphorylation peaks of p38^MAPK at PN4 (p = 0.036) and PN10 to PN60; (2) phosphorylation of ERK1 and ERK2 were increased with age (ERK1 p = 0.0000005 and ERK2 p = 0.003); (3) phosphorylation profile of JNK p54/p46 was not changed during the period analyzed (JNKp56 p = 0.716 and JNKp46 p = 0.192). Therefore, the activity profile of ERK 1/2 and p38^MAPK during postnatal development of rat hippocampus are differentially regulated. Our results demonstrate that ERK 1/2 and p38^MAPK are dynamically regulated during postnatal neurodevelopment, suggesting temporal correlation of MAPK activity with critical periods when programmed cell death and synaptogenesis are occurring. This suggests an important role for these MAPKs in postnatal development of rat hippocampus.

     

佛波酯诱导内皮素和FOS／JUN基因在血管内皮细胞中的表达及AP－1结合活性

佛波酯诱导内皮素和ＦＯＳ／ＪＵＮ基因在血管内皮细胞中的表达及ＡＰ－１结合活性温进坤,魏素珍（河北医学院生化教研室,石家庄０５００１７）张晨晖,姚阿卿,周爱儒,汤健（北京医科大学心血管基础研究所,北京,１０００８３）关键词内皮素基因表达;ＡＰ－１转录因...     

Notch1 Signaling Sensitizes Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Apoptosis in Human Hepatocellular Carcinoma Cells by Inhibiting Akt/Hdm2-mediated p53 Degradation and Up-regulating p53-dependent DR5 Expression

Notch signaling plays a critical role in regulating cell proliferation, differentiation, and apoptosis. Our previous study showed that overexpression of Notch1 could inhibit human hepatocellular carcinoma (HCC) cell growth by arresting the cell cycle and inducing apoptosis. HCC cells are resistant to apoptotic induction by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), so new therapeutic approaches have been explored to sensitize HCC cells to TRAIL-induced apoptosis. We are wondering whether and how Notch1 signaling can enhance the sensitivity of HCC cells to TRAIL-induced apoptosis. In this study, we found that overexpression of ICN, the constitutive activated form of Notch1, up-regulated p53 protein expression in HCC cells by inhibiting proteasome degradation. p53 up-regulation was further observed in human primary hepatocellular carcinoma cells after activation of Notch signaling. Inhibition of the Akt/Hdm2 pathway by Notch1 signaling was responsible for the suppression of p53 proteasomal degradation, thus contributing to the Notch1 signaling-mediated up-regulation of p53 expression. Accordingly, Notch1 signaling could make HCC cells more sensitive to TRAIL-induced apoptosis, whereas Notch1 signaling lost the synergistic promotion of TRAIL-induced apoptosis in p53-silenced HepG2 HCC cells and p53-defective Hep3B HCC cells. The data suggest that enhancement of TRAIL-induced apoptosis by Notch1 signaling is dependent upon p53 up-regulation. Furthermore, Notch1 signaling could enhance DR5 expression in a p53-dependent manner. Taken together, Notch1 signaling sensitizes TRAIL-induced apoptosis in HCC cells by inhibiting Akt/Hdm2-mediated p53 degradation and up-regulating p53-dependent DR5 expression. Thus, our results suggest that activation of Notch1 signaling may be a promising approach to improve the therapeutic efficacy of TRAIL-resistant HCC.Notch signaling determines cell fate and affects cell proliferation, differentiation, and apoptosis during cell development (1). As a highly conserved family, Notch coordinates a signaling cascade present in all animal species studied to date (2). Mammals have four Notch receptors that bind five different ligands, among which Notch1 signaling functions in many physiological and pathophysiological processes of numerous cell types, and its dysfunction results in a variety of developmental defects, including embryonic lethality and adult disorders. For example, the Notch1/Jagged1 signaling pathway is activated during liver regeneration and is potentially contributing to signals affecting hepatocyte growth (3, 4). Inducible inactivation of Notch1 has been shown to cause nodular regenerative hyperplasia in mouse liver (5). These studies suggest that Notch1 signaling may be involved in the liver functions and the pathogenesis of liver diseases. Our previous study demonstrated that Notch1 signaling could suppress the growth of human hepatocellular carcinoma (HCC)⁴ cells by arresting the cell cycle and inducing apoptosis (6). However, the underlying molecular mechanisms remain to be fully understood.p53, an important tumor suppressor gene, is involved in cell cycle arrest and cellular apoptosis. Its activity is mostly regulated by complex networks of post-translational modifications, including phosphorylation, ubiquitination, and proteasome degradation. One protein that is essential for determining p53 stability is Mdm2 (mouse double minute protein 2) (7). Mdm2, a nuclear phosphoprotein and an E3 ubiquitin ligase, binds to p53 and ubiquitinates p53, leading to proteosome degradation of p53 (8). Another important mechanism of p53 stability is related to its phosphorylation status, which is Mdm2-dependent or Mdm2-independent (9). As to the regulation of p53 by Notch1, there are controversial reports that Notch1 activation increased p53 expression in neural progenitor cells (10); however, suppression of p53 by Notch signaling was also well established in lymphomagenesis (11). We also reported that Notch1 signaling significantly up-regulated p53 expression in SMMC7721 HCC cells (6); however, the molecular mechanisms remained unclear and needed to be further characterized.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of a superfamily of cell death-inducing ligands, induces apoptosis in a broad range of transformed cells and tumor cells but has little or no effect on normal cells (12). Therefore, TRAIL has been regarded as a potential drug for cancer therapy (12, 13). However, several kinds of cancer, including HCC, are not sensitive to soluble TRAIL treatment (14). HCC accounts for 80–90% of liver cancers and is one of the most prevalent carcinomas throughout the world, especially in Africa and Asia. Thus, it is worthwhile to find a new strategy to overcome the resistance of HCC cells to TRAIL-induced apoptosis.Considering that Notch1 signaling up-regulates p53 and induces apoptosis of HCC cells and that there are no reports to date that address the relationship between Notch1 signaling and TRAIL-induced apoptosis, in this study, we investigated whether and how Notch1 signaling could sensitize HCC cells to TRAIL-induced apoptosis. We demonstrate that Notch1 signaling up-regulates p53 expression by inhibiting proteasome degradation via, at least in part, suppressing the phosphatidylinositol 3-kinase/Akt/Hdm2 pathway. In addition, we here report that Notch1 signaling enhances DR5 (death receptor 5) expression in a p53-dependent manner, and DR5 contributes, at least in part, to the enhancement of TRAIL-induced apoptosis by Notch1 signaling. Accordingly, Notch1 signaling sensitizes HCC cells to TRAIL-induced apoptosis.