14-3-3参与apelin-13促进大鼠血管平滑肌细胞增殖ERK1/2信号途径研究

本室以前已经报道了G蛋白偶联受体APJ的内源性配体多肽,apelin-13,通过激活ERK1/2促进大鼠血管平滑肌细胞增殖.本文研究14-3-3信号蛋白是否参与apelin-13促进大鼠血管平滑肌细胞增殖ERK1/2信号途径,探讨apelin/APJ系统的细胞信号转导机制.组织贴块法培养大鼠胸主动脉VSMCs;Western blotting方法检测14-3-3、pRaf-1、Raf-1、pERK1/2、ERK1/2、cyclinD1、cyclinE的表达;MTT方法观察14-3-3抑制剂Difopein对VSMCs的增殖作用;免疫共沉淀方法检测14-3-3和Raf-1蛋白复合物的形成.Western blotting方法结果显示,apelin-13(0、0.5、1、2、4μmol/L)浓度依赖性刺激大鼠VSMCs 14-3-3表达、Raf-1和ERK1/2磷酸化,以2μmol/L最为明显;2μmol/L apelin-13时间依赖性刺激大鼠VSMCs 14-3-3表达、Raf-1和ERK1/2磷酸化,在4 h增加最为显著;14-3-3蛋白抑制剂Difopein明显抑制apelin-13诱导的Raf-1磷酸化、ERK1/2磷酸化、cyclinD1及cyclinE表达;免疫共沉淀方法发现apelin-13诱导14-3-3与Raf-1结合增加,而Difopein明显抑制两者结合;MTT法显示Difopein明显抑制apelin-13诱导的血管平滑肌细胞增殖.上述结果表明,Apelin-13通过14-3-3/Raf-1复合物-ERK1/2信号转导通路促进大鼠血管平滑肌细胞增殖.     

Roles for p21waf1/cip1 and p27kip1 during the adaptation response to massive intestinal resection

The magnitude of gut adaptation is a decisive factor in determining whether patients are able to live independent of parenteral nutrition after massive small bowel loss. We previously established that the cyclin-dependent kinase inhibitor (CDKI) p21(waf1/cip1) is necessary for enterocyte proliferation and a normal adaptation response. In the present study, we have further elucidated the role of this CDKI in the context of p27(kip1), another member of the Cip/Kip CDKI family. Small bowel resections (SBRs) or sham operations were performed in control (C57/BL6), p21(waf1/cip1)-null, p27(kip1)-null, and p21(waf1/cip1)/p27(kip1) double-null mice. Morphological (villus height/crypt depth) alterations in the mucosa, the kinetics of enterocyte turnover (rates of enterocyte proliferation and apoptosis), and the protein expression of various cell cycle-regulatory proteins were recorded at various postoperative times. Enterocyte compartment-specific mRNA expression was investigated using laser capture microdissection. Resection-induced adaptation in control mice coincided with increased protein expression of p21(waf1/cip1) and decreased p27(kip1) within 3 days postoperatively. Identical changes in mRNA expression were detected in crypt but not in villus enterocytes. Adaptation occurred normally in control and p27(kip1)-null mice; however, mice deficient in both p21(waf1/cip1) and p27(kip1) failed to increase baseline rates of enterocyte proliferation and adaptation. The expression of p21(waf1/cip1) protein and mRNA in the proliferative crypt compartment is necessary for resection-induced enterocyte proliferation and adaptation. The finding that deficient expression of p27(kip1) does not affect adaptation suggests that these similar CDKI family members display distinctive cellular functions during the complex process of intestinal adaptation.     

MARCKS Signaling Differentially Regulates Vascular Smooth Muscle and Endothelial Cell Proliferation through a KIS-, p27kip1- Dependent Mechanism

Background

Overexpression of the myristolated alanine-rich C kinase substrate (MARCKS) occurs in vascular proliferative diseases such as restenosis after bypass surgery. MARCKS knockdown results in arrest of vascular smooth muscle cell (VSMC) proliferation with little effect on endothelial cell (EC) proliferation. We sought to identify the mechanism of differential regulation by MARCKS of VSMC and EC proliferation in vitro and in vivo.

Methods and Results

siRNA-mediated MARCKS knockdown in VSMCs inhibited proliferation and prevented progression from phase G₀/G₁ to S. Protein expression of the cyclin-dependent kinase inhibitor p27^kip1, but not p21^cip1 was increased by MARCKS knockdown. MARCKS knockdown did not affect proliferation in VSMCs derived from p27^kip1-/- mice indicating that the effect of MARCKS is p27^kip1-dependent. MARCKS knockdown resulted in decreased phosphorylation of p27^kip1 at threonine 187 and serine 10 as well as, kinase interacting with stathmin (KIS), cyclin D1, and Skp2 expression. Phosphorylation of p27^kip1 at serine 10 by KIS is required for nuclear export and degradation of p27^kip1. MARCKS knockdown caused nuclear trapping of p27^kip1. Both p27^kip1 nuclear trapping and cell cycle arrest were released by overexpression of KIS, but not catalytically inactive KIS. In ECs, MARCKS knockdown paradoxically increased KIS expression and cell proliferation. MARCKS knockdown in a murine aortic injury model resulted in decreased VSMC proliferation determined by bromodeoxyuridine (BrdU) integration assay, and inhibition of vascular wall thickening. MARCKS knockdown increased the rate of re-endothelialization.

Conclusions

MARCKS knockdown arrested VSMC cell cycle by decreasing KIS expression. Decreased KIS expression resulted in nuclear trapping of p27^kip1 in VSMCs. MARCKS knockdown paradoxically increased KIS expression in ECs resulting in increased EC proliferation. MARCKS knockdown significantly attenuated the VSMC proliferative response to vascular injury, but accelerated reestablishment of an intact endothelium. MARCKS is a novel translational target with beneficial cell type-specific effects on both ECs and VSMCs.     

p53 Down Regulates PDGF-Induced Formation of Circular Dorsal Ruffles in Rat Aortic Smooth Muscle Cells

The tumor suppressor, p53, negatively regulates cell migration and invasion in addition to its role in apoptosis, cell cycle regulation and senescence. Here, we study the roles of p53 in PDGF-induced circular dorsal ruffle (CDR) formation in rat aortic smooth muscle (RASM) cells. In primary and immortalized RASM cells, up-regulation of p53 expression or increase in activity with doxorubicin inhibits CDR formation. In contrast, shRNA-knockdown of p53 or inhibition of its activity with pifithrin α promotes CDR formation. p53 acts by up-regulating PTEN expression, which antagonizes Rac and Cdc42 activation. Both lipid and protein phosphatase activities of PTEN are required for maximal suppression of CDR, but the lipid activity clearly plays the dominant role. N-WASP, the downstream effector of Cdc42, is the major positive contributor to CDR formation in RASM, and is an indirect target of p53. The Rac effector, WAVE2, appears to also play a minor role, while WAVE1 has no significant effect in CDR formation. In sum, we propose that p53 suppresses PDGF-induced CDR formation in RASM cells by upregulating PTEN leading mainly to the inhibition of the Cdc42-N-WASP pathway.     

Analysis of cyclin D3-cdk4 complexes in fibroblasts expressing and lacking p27(kip1) and p21(cip1)

Our studies examined the effects of p27(kip1) and p21(cip1) on the assembly and activity of cyclin D3-cdk4 complexes and determined the composition of the cyclin D3 pool in cells containing and lacking these cyclin-dependent kinase inhibitors. We found that catalytically active cyclin D3-cdk4 complexes were present in fibroblasts derived from p27(kip1)-p21(cip1)-null mice and that immunodepletion of extracts of wild-type cells with antibody to p27(kip1) and/or p21(cip1) removed cyclin D3 protein but not cyclin D3-associated activity. Similar results were observed in experiments assaying cyclin D1-cdk4 activity. Data obtained using mixed cell extracts demonstrated that p27(kip1) interacted with cyclin D3-cdk4 complexes in vitro and that this interaction was paralleled by a loss of cyclin D3-cdk4 activity. In p27(kip1)-p21(cip1)-deficient cells, the cyclin D3 pool consisted primarily of cyclin D3 monomers, whereas in wild-type cells, the majority of cyclin D3 molecules were complexed to cdk4 and either p27(kip1) or p21(cip1) or were monomeric. We conclude that neither p27(kip1) nor p21(cip1) is required for the formation of cyclin D3-cdk4 complexes and that cyclin D3-cdk4 complexes containing p27(kip1) or p21(cip1) are inactive. We suggest that only a minor portion of the total cyclin D3 pool accounts for all of the cyclin D3-cdk4 activity in the cell regardless of whether the cell contains p27(kip1) and p21(cip1).     

Sphingosine 1-Phosphate Regulates Heat Shock Protein 27 Induction by a p38 MAP Kinase-Dependent Mechanism in Aortic Smooth Muscle Cells

In an aortic smooth muscle cell line, A10 cells, we investigated the effect of sphingosine 1-phosphate on the induction of heat shock protein 27 (HSP27), a low-molecular-weight heat shock protein. Sphingosine 1-phosphate significantly induced the accumulation of HSP27 in a pertussis toxin-sensitive manner. The effect was dose-dependent in the range between 0.1 and 30 microM. Sphingosine 1-phosphate stimulated an increase in the levels of mRNA for HSP27. Sphingosine 1-phosphate stimulated both p42/p44 mitogen-activated protein (MAP) kinase and p38 MAP kinase activation. PD98059, an inhibitor of the upstream kinase that activates p42/p44 MAP kinase, did not affect sphingosine 1-phosphate-stimulated HSP27 induction. In contrast, SB203580, an inhibitor of p38 MAP kinase, reduced sphingosine 1-phosphate-induced HSP27 induction. SB203580 reduced the levels of mRNA for HSP27 induced by sphingosine 1-phosphate. These results indicate that sphingosine 1-phosphate stimulates the induction of HSP27 via p38 MAP kinase activation in aortic smooth muscle cells.     

Adipose Stem Cells Promote Smooth Muscle Cells to Secrete Elastin in Rat Abdominal Aortic Aneurysm

Background

Abdominal aortic aneurysm (AAA) is a life-threatening disease and its prevalence rate increases with social aging. The degradation of elastic is an important factor in the formation of AAA.

Methods

Adipose derived stem cells (ADSCs) and bone marrow mesenchymal stem cells (BMSCs) were isolated from rats, and identified by Oil red O and alizarin red staining after adipogenesis and osteogenesis induction. In addition, ADSCs were also identified by flow cytometry with CD markers. AAA model in rats was established, and smooth muscle cells (SMCs) were isolated from AAA aortic wall and identified by immunohistochemistry. ADSCs or BMSCs were co-cultured with AAA aortic wall for in vitro experiment, and ADSCs were injected into AAA model for in vivo test. Then orcein staining was used for observing the morphology of elastic fiber, Western blot and real-time PCR were used respectively to detect the protein and gene expression of elastin, gelatinases spectrum analysis was used to detect the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9.

Results

Lots of red lipid droplets were visible by Oil red O staining after adipogenesis induction, and black calcium nodules appeared by alizarin red staining after osteogenesis induction. The results of flow cytometry showed that ADSCs expressed CD44 and CD105, but exhibited negligible expression of CD31 and CD45. SMCs exhibited spindle-like morphology and α-actin protein was positive in cytoplasm. After co-cultured with ADSCs or BMSCs, the elastic fiber recovered normal winding shape, both the gene and protein expression of elastin increased, and the activity of MMP-2 decreased. The in vivo result was similar to that of in vitro.

Conclusions

ADSCs promote the expression of elastin in SMCs and contribute to the reconstruction of elastic fiber, which may provide new ideas for treating AAA.     

p38信号通路参与LPS诱导的Raw264.7细胞骨架重构

应用免疫荧光标记、基因转染等方法,观察脂多糖（LPS）刺激对单核细胞系Raw264.7细胞骨架的影响,探讨p38家族不同亚型对LPS诱导的细胞骨架蛋白微管蛋白与肌动蛋白变化的调控作用结果显示,未受LPS刺激的细胞富含微管蛋白,微管蛋白交联形成辐射状的交联丝网,丝网在细胞中分布均匀;LPS刺激后,微管蛋白募集在细胞膜、核膜周围;p38α、p38β、p38γ亚型的特异性抑制剂FHPI对LPS诱导的微管蛋白募集无影响,而p38无活性突变体p38δ(AF)的基因转染,可抑制LPS诱导的细胞骨架微管蛋白的募集;肌动蛋白在静息的细胞内主要存在于细胞膜周围,LPS作用后,肌动蛋白在细胞中形成广泛分布的辐射状应激纤维;p38上游激酶活性诱变体MKK6b基因转染可诱导Raw细胞形成类似的应激纤维,而p38γ(AF)的基因转染,可抑制LPS诱导的细胞应激纤维的形成.上述结果表明,p38δ可能参与了LPS诱导的微管蛋白的重构;而LPS诱导Raw细胞应激纤维的形成,可能是通过p38γ蛋白激酶而发挥作用.     

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.     

ERK信号通道调控大鼠气道平滑肌细胞的增殖与凋亡

 为了了解ERK信号通道对正常大鼠气道平滑肌细胞(airway smooth muscle cells, ASMCs)增殖与凋亡的调控. 通过对正常大鼠ASMCs原代培养,4～7代用于实验,以ERK激动剂表皮生长因子（EGF）和抑制剂PD98059干预ASMCs生长,采用RT-PCR和免疫荧光染色观察ASMCs上ERK mRNA和蛋白的表达,MTT法、^３Ｈ-TdR掺入法检测ASMC增殖,Hoechst染色和Annexin-Ⅴ FITC PI双染色法检测细胞凋亡,Western免疫印迹检测ERK1/2、磷酸化ERK1/2和procaspase-3蛋白的表达.结果发现ASMCs上存在ERK mRNA和蛋白的表达,与空白对照组比较,PD98059干预后ASMCs的Ａ_４９０值和细胞DNA合成量均减少(Ｐ<0.05),细胞凋亡指数、早期凋亡细胞百分率均增高(Ｐ<0.05),ERK1/2、磷酸化ERK1/2表达和ERK活化率均降低, procaspase-3蛋白的表达增高.EGF干预后ASMCs的Ａ_４９０值和细胞DNA合成量均增高(Ｐ<0.05),细胞凋亡指数、早期凋亡细胞百分率均下降(Ｐ<0.05),ERK1/2、磷酸化ERK1/2表达和ERK活化率均增高, procaspase-3蛋白的表达降低.P+E组无明显差异(P>0.05).ERK信号通道参与大鼠ASMCs增殖和凋亡的调控,ERK对大鼠ASMCs凋亡的调控与procaspase-3蛋白有关,这一发现将有助于对哮喘ASMCs异常增殖调控机制的深入研究.     

低氧促进大鼠肺动脉平滑肌细胞Periostin表达及其信号转导机制

观察低氧对大鼠肺动脉平滑肌细胞（pulmonary artery smooth muscle cells,PASMCs）Periostin表达的影响及其相关信号转导机制。胶原酶I法原代培养PASMCs,经低氧（5％O2）分别处理PASMCs2,6,12,24h后,RT-PCR和Western blot法检测Periostin mRNA和蛋白表达。加入PI3K/Akt通路特异性抑制剂LY294002（10μmol／L）进行干预,Western blot分析比较不同条件下低氧处理24h后大鼠PASMCs中Periostin和Akt／P-Akt的蛋白表达。结果表日月,与常氧组比较,低氧处理6h组、12h组和24h纽Periostin mRNA和蛋白的表达均显著上升（P〈0．05,P〈0．01）,低氧处理后的PASMCs中Periostin mRNA和蛋白的表达逐渐升高：低氧处理2h组无显著差异（P〉0．05）。用LY294002对PASMCs处理,并低氧24h后,Periostin的表达被显著抑制（P〈0．01）,细胞P-Akt的表达下调（P〈0．05）,总Akt的蛋白表达没有明显差异（P〉0．05）。推测低氧可诱导大鼠PASMCs中Periostin mRNA和蛋白的表达上调。低氧可能通过激活P13K/Akt通路促进Akt的磷酸化,进而使Periostin在PASMCs中过表达,提示Periostin在低氧性PASMCs增殖过程中可能起着重要作用。     

The p27cip/kip ortholog dacapo maintains the Drosophila oocyte in prophase of meiosis I

Animal oocytes undergo a highly conserved developmental arrest in prophase of meiosis I. Often this marks a period of rapid growth for the oocyte and is necessary to coordinate meiotic progression with the developmental events of oogenesis. In Drosophila, the oocyte develops within a 16-cell germline cyst. Throughout much of oogenesis, the oocyte remains in prophase of meiosis I. By contrast, its 15 mitotic sisters enter the endocycle and become polyploid in preparation for their role as nurse cells. How germline cysts establish and maintain these two independent cell cycles is unknown. We demonstrate a role for the p21(CIP)/p27(Kip1)/p57(Kip2)-like cyclin-dependent kinase inhibitor (cki) dacapo in the maintenance of the meiotic cycle in Drosophila oocytes. Our data indicate that it is through the differential regulation of the cki Dacapo that two modes of cell-cycle regulation are independently maintained within the common cytoplasm of ovarian cysts.     

Genetic Dissection of the Vav2-Rac1 Signaling Axis in Vascular Smooth Muscle Cells

Vascular smooth muscle cells (vSMCs) are key in the regulation of blood pressure and the engagement of vascular pathologies, such as hypertension, arterial remodeling, and neointima formation. The role of the Rac1 GTPase in these cells remains poorly characterized. To clarify this issue, we have utilized genetically engineered mice to manipulate the signaling output of Rac1 in these cells at will using inducible, Cre-loxP-mediated DNA recombination techniques. Here, we show that the expression of an active version of the Rac1 activator Vav2 exclusively in vSMCs leads to hypotension as well as the elimination of the hypertension induced by the systemic loss of wild-type Vav2. Conversely, the specific depletion of Rac1 in vSMCs causes defective nitric oxide vasodilation responses and hypertension. Rac1, but not Vav2, also is important for neointima formation but not for hypertension-driven vascular remodeling. These animals also have allowed us to dismiss etiological connections between hypertension and metabolic disease and, most importantly, identify pathophysiological programs that cooperate in the development and consolidation of hypertensive states caused by local vascular tone dysfunctions. Finally, our results suggest that the therapeutic inhibition of Rac1 will be associated with extensive cardiovascular system-related side effects and identify pharmacological avenues to circumvent them.     

PP2A通过Akt/Skp2通路调控p27~(Kip1)的表达并抑制肺癌细胞的致瘤能力

蛋白磷酸酶2A(PP2A)是由36 k Da的催化亚基C(PP2Ac)和65 k Da的结构亚基A(PP2Aα/β)一起组成PP2A的核心酶,并且和各种不同的调节亚基B形成具有不同功能的PP2A全酶复合体。在细胞中PP2A发挥着重要作用,特别是在抑制肿瘤的形成当中,编码PP2Aα/β基因的突变将导致肿瘤的形成和其他疾病。当非小细胞肺癌细胞H1299中过表达PP2A-Aα时,细胞生长被抑制,细胞周期停留在G0/G1期,致瘤能力也同时被抑制。进一步研究证明当PP2A-Aα过表达时,Akt被去磷酸化失活使Skp2的表达下调,从而导致细胞周期抑制因子p27kip1的表达上调。肿瘤细胞软琼脂克隆形成实验的结果表明过表达PP2A-Aα之后H1299细胞的锚定非依赖性生长能力明显的降低,形成的克隆细胞团也较小,这些结果和裸鼠成瘤实验的结果是一致的。     

Glycosaminoglycans and Glucose Prevent Apoptosis in 4-Methylumbelliferone-treated Human Aortic Smooth Muscle Cells

Smooth muscle cells (SMCs) have a pivotal role in cardiovascular diseases and are responsible for hyaluronan (HA) deposition in thickening vessel walls. HA regulates SMC proliferation, migration, and inflammation, which accelerates neointima formation. We used the HA synthesis inhibitor 4-methylumbelliferone (4-MU) to reduce HA production in human aortic SMCs and found a significant increase of apoptotic cells. Interestingly, the exogenous addition of HA together with 4-MU reduced apoptosis. A similar anti-apoptotic effect was observed also by adding other glycosaminoglycans and glucose to 4-MU-treated cells. Furthermore, the anti-apoptotic effect of HA was mediated by Toll-like receptor 4, CD44, and PI3K but not by ERK1/2.