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1.
Birukova AA Liu F Garcia JG Verin AD 《American journal of physiology. Lung cellular and molecular physiology》2004,287(1):L86-L93
Cross talk between the actin cytoskeleton and the microtubule (MT) network plays a critical role in regulation of endothelial permeability. We have previously demonstrated that MT disruption by nocodazole results in increases in MLC phosphorylation, actomyosin contraction, cell retraction, and paracellular gap formation, cardinal features of endothelial barrier dysfunction (Verin AD, Birukova A, Wang P, Liu F, Becker P, Birukov K, and Garcia JG. Am J Physiol Lung Cell Mol Physiol 281: L565-L574, 2001; Birukova AA, Smurova K, Birukov KG, Usatyuk P, Liu F, Kaibuchi K, Ricks-Cord A, Natarajan V, Alieva A, Garcia JG, and Verin AD. J Cell Physiol. In press.). Although activation of PKA opposes barrier-disrupting effects of edemagenic agents on confluent EC monolayers, information about the molecular mechanisms of PKA-mediated EC barrier protection is limited. Our results suggest that MT disassembly alters neither intracellular cAMP levels nor PKA enzymatic activity; however, elevation of cAMP levels and PKA activation by either cholera toxin or forskolin dramatically attenuates the decline in transendothelial electrical resistance induced by nocodazole in human pulmonary EC. Barrier-protective effects of PKA on EC were associated with PKA-mediated inhibition of nocodazole-induced stress fiber formation, Rho activation, phosphorylation of myosin phosphatase regulatory subunit at Thr696, and decreased MLC phosphorylation. In addition, forskolin pretreatment attenuated MT disassembly induced by nocodazole. These results suggest a critical role for PKA activity in stabilization of MT cytoskeleton and provide a novel mechanism for cAMP-mediated regulation of Rho-induced actin cytoskeletal remodeling, actomyosin contraction, and EC barrier dysfunction induced by MT disassembly. 相似文献
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Rescue of CAMDI deletion‐induced delayed radial migration and psychiatric behaviors by HDAC6 inhibitor
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Toshifumi Fukuda Shun Nagashima Takaya Abe Hiroshi Kiyonari Ryoko Inatome Shigeru Yanagi 《EMBO reports》2016,17(12):1785-1798
The DISC1‐interacting protein CAMDI has been suggested to promote radial migration through centrosome regulation. However, its physiological relevance is unclear. Here, we report the generation and characterization of CAMDI‐deficient mice. CAMDI‐deficient mice exhibit delayed radial migration with aberrant neural circuit formation and psychiatric behaviors including hyperactivity, repetitive behavior, and social abnormality typically observed in autism spectrum disorder patients. Analyses of direct targets of CAMDI identify HDAC6 whose α‐tubulin deacetylase activity is inhibited by CAMDI at the centrosome. CAMDI deficiency increases HDAC6 activity, leading to unstable centrosomes with reduced γ‐tubulin and acetylated α‐tubulin levels. Most importantly, psychiatric behaviors as well as delayed migration are significantly rescued by treatment with Tubastatin A, a specific inhibitor of HDAC6. Our findings indicate that HDAC6 hyperactivation by CAMDI deletion causes psychiatric behaviors, at least in part, through delayed radial migration due to impaired centrosomes. 相似文献
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Dhillon B Badiwala MV Maitland A Rao V Li SH Verma S 《Molecular and cellular biochemistry》2003,247(1-2):223-227
Homocysteine is an independent risk factor for atherosclerotic vascular disease. It impairs endothelial function via increasing superoxide production and quenching nitric oxide (NO) release. Tetrahydrobiopterin (BH4) is a critical cofactor that couples nitric oxide synthase and facilitates the production of nitric oxide (vs. superoxide anions). In the first study, the effects of hyperhomocysteinemia (0.1 mM, 3 h) on endothelium-dependent vasorelaxation to ACh and A23187 were examined in isolated segments of rat aortae in the presence or absence of BH4 (0.1 mM). In the second study, the effects of hyperhomocysteinemia (24 h) on nitric oxide production and superoxide release (using lucigenin chemiluminescence) were studied in human umbilical vein endothelial cells in the absence or presence of BH4 (10 M). Homocysteine incubation impaired receptor-dependent and -independent endothelial function to ACh and A23187. This effect was attenuated by BH4. Furthermore, homocysteine exposure increased superoxide production and impaired agonist-stimulated nitric oxide release. These effects were attenuated by BH4 (p < 0.05). Hyperhomocysteinemia impairs endothelial function, in part due to a diminished bioavailability of BH4 with resultant uncoupling of nitric oxide synthase. BH4 may represent an important target for strategies aimed at improving endothelial dysfunction secondary to hyperhomocysteinemia. 相似文献
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The purpose of the present study was to examine the effects of astragaloside IV, a saponin isolated from Astragalus membranaceus (Fisch) Bge, on the impairment of barrier function induced by acute high glucose in cultured human vein endothelial cells. High glucose (27.8 mM) induced a decrease in transendothelial electrical impedance and an increase in cell monolayer permeability in human umbilical vein endothelial cells. Endothelial barrier dysfunction stimulated by high glucose was accompanied by translocation and activation of protein kinase C (PKC), the redistribution of F-actin and formation of intercellular gaps, suggesting that increases in PKC activity and rearrangement of F-actin could be associated with endothelial barrier dysfunction induced by acute high glucose. Application of astragaloside IV inhibited high glucose-induced endothelial barrier dysfunction in a dose-dependent manner, which is compatible with inhibition of PKC translocation and improvement of F-actin rearrangements. Western blot analysis revealed that high glucose-induced PKC alpha and beta2 overexpression in the membrane fraction were significantly reduced by astragaloside IV. These findings indicate that astragaloside IV protected endothelial cells from high glucose-induced barrier impairment by inhibiting PKC activation, as well as improving cytoskeleton remodeling. 相似文献
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Fan Y Wu DZ Gong YQ Xu R Hu ZB 《Biochemical and biophysical research communications》2002,293(3):979-985
Metabolic responses induced by thrombin in human umbilical vein endothelial cells (HUVECs) were investigated by using the cytosensor technique. Thrombin increased the extracellular acidification rate of endothelial cells, measured as an index of metabolic activity with a cytosensor microphysiometer, in a concentration-dependent fashion with an EC(50) of 1.27+/-0.59 IU/ml, which was abolished by the MAP kinase inhibitor PD98059. When intracellular Ca(2+) was chelated or PKC was inactivated, PD98059 failed to abolish the thrombin-induced acidification rate response in HUVECs. In addition, the tyrosine kinase inhibitor genistein, PKC inhibitor calphostin C, and Na(+)/H(+)exchanger antagonist MIA also partly inhibited thrombin-induced acidification rate responses. It is suggested that thrombin stimulated rapid metabolic responses via MAP kinase in HUVECs, which are calcium- and PKC-dependent. 相似文献
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Characterisation of the interaction between circulating and in vitro cultivated endothelial progenitor cells and the endothelial barrier 总被引:2,自引:0,他引:2
Funcke F Hoyer H Brenig F Steingen C Ladage D Müller-Ehmsen J Schmidt A Brixius K Bloch W 《European journal of cell biology》2008,87(2):81-90
In vitro cultured endothelial progenitor cells (cEPC) are used for intracoronary cell therapy in cardiac regeneration. The aim of this study was to investigate whether cEPC and circulating mononuclear cells (MNC), which include a small number of in vivo circulating EPC, are able to transmigrate through the endothelial barrier into the cardiac tissue. MNC and EPC were isolated from the peripheral blood from healthy male volunteers (n = 13, 25+/-6 years) and stained with a fluorescent marker. The cells were perfused in vitro through organs with endothelial layers of different phenotypes (rat aorta, human umbilical vein, isolated mouse heart). The endothelium and the basal lamina were then stained by immunofluorescence and the cryo-sections analysed using a confocal laser scanning microscope. After perfusion through the rat aorta, an adhesion/integration of MNC was observed at the endothelial layer and the basal lamina beneath endothelial cells. However, no migration of MNC over the endothelial barrier was found. This remained true even when the cell numbers were increased (from 0.5 to 10 million cells/h), when the time of perfusion was prolonged (1.5-4 h) and when the aorta was cultivated for 24 h. In the Langendorff-perfused mouse heart with intact endothelium, no migration of MNC (1 x 10(7)) or cEPC (1 x 10(6)) was observed after 0.5 and 2 h. In conclusion, MNC and cEPC do not possess any capacity to transmigrate the endothelial barrier. In the context of stem cell therapy, these cells may therefore serve as endothelial regenerators but not as cardiomyocyte substitutes. 相似文献
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Histone deacetylases (HDACs) have been implicated in numerous biological events. However, to date, the role of HDAC6 in early embryos remains unknown. In the current study, Tubastatin A (TubA), a potent HDAC6 inhibitor, was used to block HDAC6 activity in mouse embryos. We found that TubA exposure significantly reduced the blastocyst formation of early embryos. Confocal microscopy revealed the markedly increased chromosomal congression failure in the mouse embryos treated with the HDAC6 inhibitor. Moreover, the HDAC6 inhibition resulted in the overproduction of reactive oxygen species (ROS) in embryos. In addition, we observed the accumulation of phosphorylated γH2AX in TubA-treated embryos, indicative of the increased DNA damage. In line with this, cell apoptosis of blastocysts was frequently detected in HDAC6-deficient embryos compared with their controls. Altogether, our data indicate that HDAC6 may serve as an important regulator of chromatin structure and mitochondrial function, determining the developmental potential of the early embryos of mouse. 相似文献
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旨在建立分子水平HDAC6小分子抑制剂的高通量筛选模型,用于新型HDAC6特异性小分子抑制剂的发现。建立HDAC6的昆虫表达系统,分离纯化HDAC6蛋白,利用底物Boc-Lys(Ac)-AMC对纯化的HDAC6进行测活,并对测活体系进行优化,以SAHA为阳性抑制剂,确定适合高通量筛选的酶及底物浓度,反应时间等。首先构建HDAC6昆虫真核细胞表达载体,转入昆虫细胞中表达,并利用GST亲和柱纯化获得较高纯度的GST-HDAC6融合蛋白;建立体外HDAC6分子测活方法,表明昆虫表达的GST-HDAC6融合蛋白具有去乙酰化酶活性,并通过对多种参数优化使得Z’因子达到0.60,表明分子水平的HDAC6小分子抑制剂高通量筛选体系成功建立。 相似文献
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Jae‐Hoon Kim Song Yi Park Tae‐Hwan Kim Woon‐Kyu Lee Duk‐Jae Jang Ji‐Eun Yoon Young‐Il Choi Seihwan Kim JinYeul Ma Chul‐Joong Kim Tso‐Pang Yao Jae U Jung Joo‐Yong Lee Jong‐Soo Lee 《The EMBO journal》2016,35(4):429-442
RIG‐I is a key cytosolic sensor that detects RNA viruses through its C‐terminal region and activates the production of antiviral interferons (IFNs) and proinflammatory cytokines. While posttranslational modification has been demonstrated to regulate RIG‐I signaling activity, its significance for the sensing of viral RNAs remains unclear. Here, we first show that the RIG‐I C‐terminal region undergoes deacetylation to regulate its viral RNA‐sensing activity and that the HDAC6‐mediated deacetylation of RIG‐I is critical for viral RNA detection. HDAC6 transiently bound to RIG‐I and removed the lysine 909 acetylation in the presence of viral RNAs, promoting RIG‐I sensing of viral RNAs. Depletion of HDAC6 expression led to impaired antiviral responses against RNA viruses, but not against DNA viruses. Consequently, HDAC6 knockout mice were highly susceptible to RNA virus infections compared to wild‐type mice. These findings underscore the critical role of HDAC6 in the modulation of the RIG‐I‐mediated antiviral sensing pathway. 相似文献
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Ferro T Neumann P Gertzberg N Clements R Johnson A 《American journal of physiology. Lung cellular and molecular physiology》2000,278(6):L1107-L1117
We tested the hypothesis that protein kinase C-alpha (PKC-alpha) mediates tumor necrosis factor-alpha (TNF-alpha)-induced alterations in permeability of pulmonary microvessel endothelial monolayers (PEM). The permeability of PEM was assessed by the clearance rate of Evans blue-labeled albumin. PEM lysates were analyzed for PKC-alpha mRNA (Northern cDNA blot), protein (Western immunoblot), and activity (translocation and phosphorylation of myristoylated arginine-rich C kinase substrate). Incubation of PEM with TNF-alpha (1,000 U/ml) for 4 h resulted in increases in 1) PKC-alpha protein, 2) cytoskeletal-associated PKC-alpha, 3) PKC-alpha activity, and 4) permeability to albumin. The TNF-alpha-induced increase in PKC-alpha protein, PKC-alpha activity, and permeability was prevented by a 4-h pretreatment with PKC-alpha antisense oligonucleotide but not by the scrambled nonsense oligonucleotide. The TNF-alpha-induced increase in permeability to albumin was prevented by myristoylated protein kinase C inhibitor (an inhibitor of PKC-alpha/beta, 100 microM) and calphostin (an inhibitor of the classic and novel PKC isotypes, 200 nM). The treatment with calphostin from 0.5 to 3.0 h after TNF-alpha still prevented barrier dysfunction induced by 4 h of TNF-alpha treatment. The data indicate that prolonged activation of PKC-alpha, maintained by a translation-dependent pool of PKC-alpha protein, mediates TNF-alpha-induced increases in endothelial permeability in PEM. 相似文献
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Smita Salian-Mehta Mei Xu Timothy A. McKinsey Stuart Tobet Margaret E. Wierman 《The Journal of biological chemistry》2015,290(22):14045-14056
The impact of histone deacetylases (HDACs) in the control of gonadotropin releasing hormone (GnRH) neuronal development is unknown. We identified an increase in many HDACs in GT1-7 (differentiated) compared with NLT (undifferentiated) GnRH neuronal cell lines. Increased HDAC9 mRNA and protein and specific deacetylase activity in GT1-7 cells suggested a functional role. Introduction of HDAC9 in NLT cells protected from serum withdrawal induced apoptosis and impaired basal neuronal cell movement. Conversely, silencing of endogenous HDAC9 in GT1-7 cells increased apoptosis and cell movement. Comparison of WT and mutant HDAC9 constructs demonstrated that the HDAC9 pro-survival effects required combined cytoplasmic and nuclear localization, whereas the effects on cell movement required a cytoplasmic site of action. Co-immunoprecipitation demonstrated a novel interaction of HDAC9 selectively with the Class IIb HDAC6. HDAC6 was also up-regulated at the mRNA and protein levels, and HDAC6 catalytic activity was significantly increased in GT1-7 compared with NLT cells. HDAC9 interacted with HDAC6 through its second catalytic domain. Silencing of HDAC6, HDAC9, or both, in GT1-7 cells augmented apoptosis compared with controls. HDAC6 and -9 had additive effects to promote cell survival via modulating the BAX/BCL2 pathway. Silencing of HDAC6 resulted in an activation of movement of GT1-7 cells with induction in acetylation of α-tubulin. Inhibition of HDAC6 and HDAC9 together resulted in an additive effect to increase cell movement but did not alter the acetylation of αtubulin. Together, these studies identify a novel interaction of Class IIa HDAC9 with Class IIb HDAC6 to modulate cell movement and survival in GnRH neurons. 相似文献
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Silence of HDAC6 suppressed esophageal squamous cell carcinoma proliferation and migration by disrupting chaperone function of HSP90
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Hua Tao Yuan‐Yuan Chen Zong‐Wen Sun Hua‐Lin Chen Ming Chen 《Journal of cellular biochemistry》2018,119(8):6623-6632
Esophageal carcinoma is aggressive in nature and its prognosis is largely dependent on the degree of invasion. Histone deacetylase 6 (HDAC6), as the most unique member of HDACs family, has the positive activity to promote initiation and progression of various cancers via targeting multiple non‐histone proteins in cytoplasm. In this study, we found that HDAC6 was over‐expressed in three esophageal cancer cell lines (KYSE140, KYSE170, KYSE180) when compared to non‐carcinoma esophageal epithelial cell HEEC‐1. Then two HDAC6 specific siRNAs and HDAC6 inhibitor tubastatin A greatly suppressed KYSE140 and KYSE180 cells proliferation and migration, and the inhibition of cell motility was accompanied by elevated acetylation of α‐tubulin, a target of HDAC6. Consistently, the microtubulin skeleton was stabilized after HDAC6 knockdown or inhibition. In addition, acetylation status of HSP90, another HDAC6 target, was also increased towards HDAC6 knockdown or inhibition by co‐immunoprecipitation assay. Besides, co‐treatment of HSP90 inhibitor (PU‐H71) and HDAC6 inhibitor (tubastatin A) induced a stronger cell migration inhibition compared to administration of either drug alone. Furthermore, cell proliferation of KYSE140 and KYSE180 were also compromised in response to combination of HDAC6 and HSP90 inhibitors. Additionally, co‐administration of HSP90 inhibitor and HDAC6 inhibitor strongly inhibited tumor growth in vivo. Taken together, our results indicated that HDAC6 is a promising target by inhibiting HSP90 function in ESCC. 相似文献
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Qiwen Zhu Xumeng Yu Qianqian Shen Qiumeng Zhang Mingbo Su Yubo Zhou Jia Li Yi Chen Wei Lu 《Bioorganic & medicinal chemistry》2018,26(16):4706-4715
Camptothecin plays an important role in clinical cancer treatment, and its derivatives are a favorite of pharmaceutical chemists. Herein, we have designed a series of camptothecin prodrugs that exhibit histone deacetylase (HDAC) inhibition activity based on the synergy effect between HDAC inhibitors and camptothecin derivatives. With the evaluation of stability in buffers or plasma from human or mouse model, an appropriate linker was found, so the active drug can be released efficiently and compound 21a exhibited strong antiproliferative activity in A549 and HCT-116 cell lines. These results indicated that the well-designed prodrug can be promising in cancer treatment. 相似文献
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Histone deacetylase 6 (HDAC6) catalyzes multiple reactions. We summarize the current knowledge on HDAC6, its targets and functions. Among others, HDAC6 recognizes damaged proteins and assures that these proteins are destroyed by autophagy. On the other hand, HDAC6 also modifies the tracks used by the clearance mechanism so that axonal transport becomes less efficient. We hypothesize that a disturbance in the equilibrium between the different functions of HDAC6 could play an important role in neurodegeneration. 相似文献
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Cell motility and adhesion involves dynamic microtubule (MT) acetylation/deacetylation, a process regulated by enzymes as HDAC6, a major cytoplasmic α-tubulin deacetylase. We identify G protein-coupled receptor kinase 2 (GRK2) as a key novel stimulator of HDAC6. GRK2, which levels inversely correlate with the extent of α-tubulin acetylation in epithelial cells and fibroblasts, directly associates with and phosphorylates HDAC6 to stimulate α-tubulin deacetylase activity. Remarkably, phosphorylation of GRK2 itself at S670 specifically potentiates its ability to regulate HDAC6. GRK2 and HDAC6 colocalize in the lamellipodia of migrating cells, leading to local tubulin deacetylation and enhanced motility. Consistently, cells expressing GRK2-K220R or GRK2-S670A mutants, unable to phosphorylate HDAC6, exhibit highly acetylated cortical MTs and display impaired migration and protrusive activity. Finally, we find that a balanced, GRK2/HDAC6-mediated regulation of tubulin acetylation differentially modulates the early and late stages of cellular spreading. This novel GRK2/HDAC6 functional interaction may have important implications in pathological contexts. 相似文献
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目的: 观察感染性急性肺损伤(ALI)肺血管内皮屏障功能失调小鼠肺组织中弗里德白血病病毒插入位点1(FLI-1)蛋白表达的变化,以探讨FLI-1在感染性ALI肺血管内皮屏障功能失调发生发展中的意义。方法: SPF级雄性ICR小鼠60只,腹腔注射脂多糖(LPS,7.5 mg/kg)复制ALI模型,在给予LPS 0 h、12 h、24 h、48 h后,检测小鼠肺血管内皮屏障通透性和肺湿干重比,ELISA法检测肺泡灌洗液中TNF-α和IL-6含量,Western blot法检测肺组织FLI-1和Src酪氨酸激酶(SRC)蛋白的表达。结果: 与0 h组比较,12 h组、24 h组肺血管内皮屏障通透性分别升高74.3%和162.4%,而48 h组较24 h组降低27.0%(P均<0.05);与0 h组比较,12 h组、24 h组肺湿干重比分别升高50.1%和122.9%,而48 h组较24 h组降低10.7%(P均<0.05);与0 h组比较,12 h、24 h肺泡灌洗液IL-6和TNF-α含量均显著升高,而48 h肺泡灌洗液IL-6和TNF-α含量较24 h分别下降28.3%和21.6%(P均< 0.05);与0 h组比较,12 h组、24 h组肺组织FLI-1蛋白表达水平分别下调20.4%和56.9%,而48 h组较24 h组上调18.2%(P均<0.05);与0 h组比较,12 h组、24 h组肺组织SRC蛋白表达水平分别上调76.8%和176.7%,而48 h组较24 h组下调33.4%(P均<0.05);肺血管内皮屏障通透性与FLI-1蛋白表达水平呈显著负相关(r= -0.8992,P<0.01),而与SRC蛋白表达水平呈显著正相关(r=0.8918,P<0.01),肺组织FLI-1与SRC蛋白表达呈显著负相关(r=-0.8087,P=0.0014)。结论: FLI-1可能参与LPS诱导的急性肺损伤肺血管内皮屏障功能失常过程。 相似文献