PDK1 Recruitment to the SHPS-1 Signaling Complex Enhances Insulin-like Growth Factor-I-stimulated AKT Activation and Vascular Smooth Muscle Cell Survival

In vascular smooth muscle cells, exposed to hyperglycemia and insulin-like growth factor-I (IGF-I), SHPS-1 functions as a scaffold protein, and a signaling complex is assembled that leads to AKT activation. However, the underlying mechanism by which formation of this complex activates the kinase that phosphorylates AKT (Thr³⁰⁸) is unknown. Therefore, we investigated the mechanism of PDK1 recruitment to the SHPS-1 signaling complex and the consequences of disrupting PDK1 recruitment for downstream signaling. Our results show that following IGF-I stimulation, PDK1 is recruited to SHPS-1, and its recruitment is mediated by Grb2, which associates with SHPS-1 via its interaction with Pyk2, a component of the SHPS-1-associated complex. A proline-rich sequence in PDK1 bound to an Src homology 3 domain in Grb2 in response to IGF-I. Disruption of Grb2-PDK1 by expression of either a Grb2 Src homology 3 domain or a PDK1 proline to alanine mutant inhibited PDK1 recruitment to SHPS-1, leading to impaired IGF-I-stimulated AKT Thr³⁰⁸ phosphorylation. Following its recruitment to SHPS-1, PDK1 was further activated via Tyr^373/376 phosphorylation, and this was required for a maximal increase in PDK1 kinase activity and AKT-mediated FOXO3a Thr³² phosphorylation. PDK1 recruitment was also required for IGF-I to prevent apoptosis that occurred in response to hyperglycemia. Assembly of the Grb2-PDK1 complex on SHPS-1 was specific for IGF-I signaling because inhibiting PDK1 recruitment to SHPS-1 had no effect on EGF-stimulated AKT Thr³⁰⁸ phosphorylation. These findings reveal a novel mechanism for recruitment of PDK1 to the SHPS-1 signaling complex, which is required for IGF-I-stimulated AKT Thr³⁰⁸ phosphorylation and inhibition of apoptosis.     

新型GATA-1相互作用蛋白质及其在造血调控中的作用

GATA-1在造血干细胞的谱系分化中起关键的调控作用,为红系和巨核系发育成熟必不可少,对肥大细胞系及嗜酸性粒细胞系发育也有一定的调控作用。GATA-1的转录活性受到多层次的精确调节,其调控不同谱系分化的功能大多通过与不同的蛋白质相互作用来实施。近年来,多种新的GTAT-1相互作用蛋白质被确定,特别是GATA-1复合体的研究,揭示了GATA-1转录活性及其调控造血分化的新机制。     

Regulation of Vascular Endothelial Growth Factor Expression by cAMP in Rat Aortic Smooth Muscle Cells

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen which stimulates angiogenesis. VEGF is regulated by multiple factors such as hypoxia, phorbol esters, and growth factors. However, data concerning the expression of VEGF in the different vascular cell types and its regulation by cAMP are not available. In the present study, we have investigated the effect of adenylate cyclase activation on VEGF mRNA expression in rat vascular cells in primary culture. Basal VEGF expression is greater in smooth muscle cells than in endothelial cells and fibroblasts. A 4-h treatment with forskolin (10⁻⁵M) induced a 2-fold stimulation of VEGF mRNA expression in smooth muscle cells and fibroblasts, but, in contrast, did not affect VEGF expression in endothelial cells. In smooth muscle cells, a pharmacologically induced increase in intracellular cAMP levels using iloprost or isoprenaline led to a rise in VEGF mRNA expression comparable to that induced by forskolin. Adenosine, which increases cAMP levels in smooth muscle cells, also increases VEGF expression. Moreover, the 2.2-fold stimulation of VEGF expression by adenosine was enhanced following a cotreatment with cobalt chloride (a hypoxia miming agent). The observed additive effect (4.3-fold increase) suggests that these two factors, hypoxia and adenosine, regulate VEGF mRNA expression in smooth muscle cells by independent mechanisms.     

PcG蛋白在干细胞调控中的作用

PcG (polycomb group)蛋白作为一种表观遗传修饰系统,在动物和植物中具有 保守性.从功能上讲,PcG蛋白可以分为PRC1（polycomb repressive complex 1）和 PRC2（polycomb repressive complex 2）两个核心蛋白复合体. PRC2含有组蛋白甲 基化酶的活性,而PRC1在泛素连接酶E3介导的组蛋白泛素化中发挥作用,二者通过对 组蛋白的修饰控制靶基因转录. 近来研究表明,PcG蛋白对干细胞数量维持和命运转变 有重要的调控作用,其成员表达失调或缺失导致许多恶性肿瘤的发生或导致植物细胞 丧失分化能力、形成愈伤组织. 本文简要综述了PcG蛋白的组成及其在干细胞调控中 的作用.     

血管舒-缩肽在血管平滑肌细胞中的表达与调控

为探讨血管舒 缩肽表达的调控机制及血管平滑肌细胞 (VSMC)在该网络平衡中的地位 ,以血管紧张素Ⅱ (AngⅡ )为诱发因素刺激培养的大鼠VSMC ,用RT PCR和放射免疫分析观察内皮素 1(ET 1)、AngⅡ、心钠素 (ANF)和肾上腺髓质素 (ADM)在VSMC中的表达与释放及相互关系 ,用电泳迁移率改变分析 (EMSA)和染色质免疫沉淀 (ChIP)分析揭示其分子机制 .在被AngⅡ处理的VSMC中 ,4种血管活性肽的表达活性均升高 ,其中缩血管肽基因表达被迅速诱导 ,而舒血管肽则是先降后升 .但刺激前后舒 缩血管肽之间的平衡关系无明显改变 .放免分析证实 ,AngⅡ可程度不同地促进 4种血管活性肽合成 ,使胞内 4种活性肽水平升高 ;对培养液中 4种活性肽进行检测的结果显示 ,AngⅡ可促进ET 1、AngⅡ释放 ,抑制舒血管肽释放 ,尤以ANF的胞内水平明显高于胞外 .EMSA分析显示 ,在AngⅡ诱导 4种肽表达的同时 ,与细胞增殖有关的转录调控因子转录激活蛋白(AP 1)与 4种活性肽基因启动子的结合活性明显增强 .ChIP结果表明 ,AP 1在染色质靶位点的募集与血管活性肽基因的表达上调有直接关系 .结果提示 ,AP 1与特异DNA顺式作用元件的相互作用参与了血管活性肽的转录激活 .VSMC不仅作为它们的效应器 ,而且还通过调节AP 1与靶基因中的共有顺式元件——     

Notch3在血管平滑肌细胞中对Erk1/2的调控作用研究

Erk1/2活性在血管许多细胞功能中具有重要影响,而Notch3主要表达在动脉平滑肌细胞中,并且是发育过程中动脉成熟所必需的.为了探讨Notch3在血管平滑肌细胞中对Erk1/2信号通路的调控作用,采用siRNA基因敲除Notch3,γ-分泌酶抑制剂DAPT抑制Notch信号通路,质粒转染过表达Notch3活性区等方法,用Western印迹检测Notch3对血管平滑肌细胞中Erk1/2磷酸化水平,即Erk1/2活性的影响.同时,利用活性氧自由基（ROS）诱导激活Erk1/2;siRNA敲除Notch3表达致使血管平滑肌细胞中Erk1/2的磷酸化水平显著降低,并且抑制了ROS诱导的Erk1/2激活;同样,Notch通路抑制剂DAPT也抑制了ROS诱导的Erk1/2激活;而Notch3活性区NICD的过表达并没有改变血管平滑肌细胞中Erk1/2的磷酸化水平,但其延缓了ROS激活后Erk1/2活性的衰减.上述结果表明,Notch3可在血管平滑肌细胞中调控Erk1/2活性以及ROS诱导的Erk1/2信号激活.     

Potassium Depletion and Regulation of Angiotensin II Receptors in Vascular Smooth Muscle Cells

Abstract

Mesenteric artery smooth muscle cells were grown in culture media containing high, normal, or low concentrations of potassium to study the effects on angiotensin II (Ang II) receptor regulation. Cell growth was similar among cells grown in the different culture media. Cells grown in high potassium media (K=5.8 mEq/L) had an equilibrium dissociation constant, K_d, of 1.59 ± 0.2 nM, whereas those grown in normal potassium media (K=4.1 mEq/L) had a K_d of 1.79 ± 0.2 nM and those grown in a low potassium media (K=2.9 mEq/L) had a K_d of 1.19 ± 0.12 nM (not significantly different, NS). Binding capacity of smooth muscle cells grown in high potassium media was 81 ± 16.7 fmol/mg prot, 95.1 ± 12.4 fmol/mg prot in those grown in normal potassium media and those grown in low potassium media 86.4 ± 24.1 fmol/mg prot (NS). Binding of radiolabelled Ang II was reduced by approximately 70% in cells exposed to unlabelled Ang II for 30 or 60 minutes. However, this effect of exposure to Ang II to reduce subsequent binding of Ang II was identical in cells grown in high and low potassium medium. Therefore, we were unable to identify a direct effect of low potassium to induce changes in Ang II receptor binding affinity or binding capacity. Previously observed changes in these Ang II binding parameters in potassium-depleted rats was probably a consequence of other factors which were simultaneously altered by potassium deficiency.     

Differential Regulation of NOTCH2 and NOTCH3 Contribute to Their Unique Functions in Vascular Smooth Muscle Cells

Notch signaling is a key regulator of vascular smooth muscle cell (VSMC) phenotypes, including differentiation, proliferation, and cell survival. However, the exact contribution of the individual Notch receptors has not been thoroughly delineated. In this study, we identify unique roles for NOTCH2 and NOTCH3 in regulating proliferation and cell survival in cultured VSMCs. Our results indicate that NOTCH2 inhibits PDGF-B-dependent proliferation and its expression is decreased by PDGF-B. In contrast, NOTCH3 promotes proliferation and receptor expression is increased by PDGF-B. Additionally, data show that NOTCH3, but not NOTCH2 protects VSMCs from apoptosis and apoptosis mediators degrade NOTCH3 protein. We identified three pro-survival genes specifically regulated by NOTCH3 in cultured VSMCs and in mouse aortas. This regulation is mediated through MAP kinase signaling, which we demonstrate can be activated by NOTCH3, but not NOTCH2. Overall, this study highlights discrete roles for NOTCH2 and NOTCH3 in VSMCs and connects these roles to specific upstream regulators that control their expression.     

KCl Cotransport Regulation and Protein Kinase G in Cultured Vascular Smooth Muscle Cells

K-Cl cotransport is activated by vasodilators in erythrocytes and vascular smooth muscle cells and its regulation involves putative kinase/phosphatase cascades. N-ethylmaleimide (NEM) activates the system presumably by inhibiting a protein kinase. Nitrovasodilators relax smooth muscle via cGMP-dependent activation of protein kinase G (PKG), a regulator of membrane channels and transporters. We investigated whether PKG regulates K-Cl cotransport activity or mRNA expression in normal, PKG-deficient-vector-only-transfected (PKG-) and PKG-catalytic-domain-transfected (PKG+) rat aortic smooth muscle cells. K-Cl cotransport was calculated as the Cl-dependent Rb influx, and mRNA was determined by semiquantitative RT-PCR. Baseline K-Cl cotransport was higher in PKG+ than in PKG- cells (p <0.01). At 0.5 mM, NEM stimulated K-Cl cotransport by 5-fold in PKG- but not in PKG+ cells. However, NEM was more potent although less effective to activate K-Cl cotransport in normal (passage 1-3) and PKG+ than in PKG- cells. In PKG- cells, [(dihydroindenyl) oxy] alkanoic acid (300 mM) but not furosemide (1 mM) inhibited K-Cl cotransport. Furthermore, no difference in K-Cl cotransport mRNA expression was observed between these cells. In conclusion, this study shows that manipulation of PKG expression in vascular smooth muscle cells affects K-Cl cotransport activity and its activation by NEM.     

蜗轴螺旋动脉平滑肌的培养及其鉴定

目的:本研究通过联合两种常用的细胞培养方法,建立了一个蜗轴螺旋动脉平滑肌细胞原代培养的模型.方法:分离的蜗轴螺旋动脉的平滑肌来自于豚鼠.切碎血管组织并且将其放入37度的0.1%胰蛋白酶溶液中消化20分钟.消化后,将这些消化后的组织块在35-mm的培养皿中贴壁.运用这种培养方法,混杂的成纤维细胞通过与血管平滑肌细胞不同的贴壁能力,可在传代的时候被去除.在7-10天后,细胞从组织块中长出来.大约3周,细胞可长满培养皿.在第三代培养的血管平滑肌细胞中,纯的并且活性好的细胞可以被活得.经过形态学,免疫荧光化学和电镜对这种方法得到的血管平滑肌细胞进行了鉴别.结果:显示了典型的平滑肌的细胞的特点:形态学的"峰-谷"样的生长形态,免疫荧光化学显示这些细胞表达平滑肌细胞的特异性标志物(α-SM-actin和myosin.结论:通过本文研究方法得到的大量的纯的蜗轴螺旋动脉血管平滑肌细胞是一个很好的研究血管平滑肌细胞在内耳循环紊乱过程生理功能的一个体外模型.除此之外,还可以是一些作用于血管平滑肌药物评价的体外模型.     

Prostaglandin Production by Mast Cells and the Regulation of Airway Smooth Muscle Responses

The role of mast cell activation and the generation of mediator release are important factors in determining the reactivity of lung tissue during allergic reactions and asthma. In this article, the advantages of our procedure for isolating mast cells from lung tissue over other methods are discussed. Our studies have demonstrated the importance of cross talk between mast cells and other cell types that also release mediators of bronchoconstriction during activation, thus amplifying the response. In addition, it has been found that inositol phospholipid turnover in response to various mast cell mediators is enhanced in hyperresponsive lung tissue. The consequences to the contractility of the tissue of such alterations in the cellular signaling system in lung tissue are discussed.     

Protein Tyrosine Phosphatase LMW-PTP Exhibits Distinct Roles Between Vascular Endothelial and Smooth Muscle Cells

Abstract

The present study examined the cellular functions of low-molecular-weight protein tyrosine phosphatase (LMW-PTP), which consists of two active isoforms IF-1 and IF-2, in vascular smooth muscle cells (VSMCs) and endothelial cells (ECs), focusing on cell growth and migration. We transduced recombinant IF-1 and IF-2, and ribozyme targeting both isoforms using an adenovirus vector in these cells. We detected the expression of IF-1 and IF-2 in both types of cells. IF-1 as well as IF-2 inhibited PDGF-induced DNA synthesis and migration in VSMCs. In contrast, both isoforms enhanced lysophosphatidic acid-stimulated cell migration without change in DNA synthesis in ECs. Whereas there is a report indicating that reactive oxygen species-dependent inactivation of LMW-PTP regulates actin cytoskeleton reorganization during cell spreading and migration, the isoforms conversely suppressed the PDGF-induced H₂O₂ generation with subsequent decrease in the p38 activity in VSMCs. Catalytically inactive LMW-PTP exerted the opposite and similar effects to the wild type in ECs and in VSMCs, respectively, suggesting that substrates for the phosphatase differ between these cells. Moreover, high concentrations of glucose suppressed the expression of LMW-PTP in both cells. These data suggest that LMW-PTP negatively regulates the pathogenesis of atherosclerosis and that glucose-dependent suppression of LMW-PTP expression may promote the development of atherosclerosis in diabetics.     

Steroid-sensitive Gene 1 Is a Novel Cyclic GMP-dependent Protein Kinase I Substrate in Vascular Smooth Muscle Cells

NO, via its second messenger cGMP, activates protein kinase GI (PKGI) to induce vascular smooth muscle cell relaxation. The mechanisms by which PKGI kinase activity regulates cardiovascular function remain incompletely understood. Therefore, to identify novel protein kinase G substrates in vascular cells, a λ phage coronary artery smooth muscle cell library was constructed and screened for phosphorylation by PKGI. The screen identified steroid-sensitive gene 1 (SSG1), which harbors several predicted PKGI phosphorylation sites. We observed direct and cGMP-regulated interaction between PKGI and SSG1. In cultured vascular smooth muscle cells, both the NO donor S-nitrosocysteine and atrial natriuretic peptide induced SSG1 phosphorylation, and mutation of SSG1 at each of the two predicted PKGI phosphorylation sites completely abolished its basal phosphorylation by PKGI. We detected high SSG1 expression in cardiovascular tissues. Finally, we found that activation of PKGI with cGMP regulated SSG1 intracellular distribution.     

新型大豆异黄酮磺酸酯抑制血管平滑肌细胞增殖活性研究

为了寻找对血管平滑肌细胞异常增殖有较强抑制作用的化合物,本文用MMT法考察新型大豆苷元磺酸酯体外抑制血管平滑肌细胞增殖活性.结果表明:该大豆苷元磺酸酯对血管平滑肌细胞增殖在10-7 mol/L时有抑制作用(P＜0.05),该浓度下的抑制率为56.06％,与先导化合物大豆苷元相比活性提高约1000倍.构效关系研究表明,大豆苷元经苯磺酸酯修饰,改变分子的空间结构、分子的可极化率从26.51增加到54.12,改变了药物的电荷分布,更有利于药物通过细胞膜到达靶标和与靶标更精确作用而导致药物药理作用大大增强.药理实验与构效关系研究初步表明,该大豆苷元磺酸酯有进一步研究价值.     

平滑肌细胞骨架结构及其信号调节途径

平滑肌细胞骨架是一个复杂的动态性网络,是细胞生命活动不可缺少的细胞结构。Rho通过活化其下游靶分子促进应力纤维的形成,其中Rho—associated coiled—coil kinase(ROCK)和Dial在该过程中起关键作用;PKC通过在细胞内不同定位的亚型使细胞骨架蛋白磷酸化,发挥其调节细胞骨架重构的作用。两条信号转导途径通过Src途径相互联系,共同参与细胞骨架动力学的调节。