G蛋白耦联受体激酶结合蛋白1通过调节细胞外调节激酶1/2的活性促进成骨细胞迁移

目的探索G蛋白耦联受体激酶结合蛋白1(GITI)在成骨细胞迁移中的作用,并分析其机理。方法通过Western blot方法检测GIT1蛋白在鼠的成骨细胞内的表达；用免疫荧光染色方法确定：在血小板衍生生长因子(PDGF)不刺激和刺激的条件下,GIT1和细胞外调节激酶1/2(ERK1/2)在成骨细胞内的位置；用共同免疫沉淀的方法测定GIT1和ERK1/2相互结合,并且用免疫荧光双染的方法确定这两种蛋白相互结合的位置；用包含GIT1-RNA发夹结构的腺病毒感染成骨细胞后,用免疫荧光染色方法确定磷酸化ERK1/2(pERK1/2)在成骨细胞内的位置,用划痕愈合法检测在PDGF刺激下的迁移能力。结果在成骨细胞内,PDGF刺激导致了GIT1和ERK1/2的相互结合,并且这种结合发生在成骨细胞的局部粘附内。包含GIT1-RNA发夹结构的腺病毒明显抑制了pERK1/2招募至成骨细胞局部粘附内以及PDGF所刺激的成骨细胞的迁移。结论在PDGF刺激下,GIT1招募pERK1/2至成骨细胞的局部粘附内,从而促进成骨细胞的迁移。     

磷酸化细胞外信号调节激酶1/2定位于成骨细胞的局部黏附时需要Src活性

目的探讨血小板源性生长因子(platelet-derived growth factor,PDGF)刺激成骨细胞,对磷酸化细胞外信号调节激酶1/2(phosphorylation extracellular signal-regulated kinase1/2,pERK1/2)位置的影响。方法出生3d清洁级健康小鼠10只,雌雄不拘,体重6～9g。取小鼠颅骨,分离培养原代成骨细胞。取第6代成骨细胞,1%血清培养液培养12h后,随机分成经10μmol/L PP2处理30min组(实验组)和未处理组(对照组),每组再随机分成2个亚组:其中一组用PDGF(20ng/ml)刺激10min,另一组不用PDGF刺激,采用免疫组织化学染色检测pERK1/2分布。另取第6代成骨细胞,当细胞生长至80%融合时,用细胞刮随机分成2组,一组用10μmol/L PP2预处理30min(实验组),另一组不用PP2作用(对照组),再用20ng/ml PDGF培养12h,采用划痕愈合法检测PP2对成骨细胞在PDGF刺激下迁移能力的影响。另取第6代成骨细胞,调整细胞浓度1×106/ml,随机分成2组,分别经DMSO(对照组)和10μmol/L PP2(实验组)预处理30min,每组再随机分成2个亚组:其中一组用PDGF(20ng/ml)刺激10min,另一组不用PDGF剌激,采用Western blot检测细胞骨架蛋白内pERK1/2活性。结果免疫荧光染色结果显示,PDGF促进pERK1/2定位于成骨细胞的局部黏附和细胞核内;而PP2显著抑制了由PDGF刺激引起的pERK1/2定位于成骨细胞的局部黏附,但并不影响pERK1/2定位于细胞核内。细胞划痕愈合实验显示,PP2明显抑制了由PDGF所诱导的成骨细胞迁移。Western blot检测结果显示,PP2明显抑制了由PDGF所诱导的成骨细胞局部黏附内ERK1/2的磷酸化。结论PDGF通过激活Src活性,促进pERK1/2定位于成骨细胞的局部黏附内;PP2通过抑制pERK1/2定位于成骨细胞的局部黏附,从而抑制由PDGF所诱导的成骨细胞迁移。     

G蛋白偶联受体激酶结合蛋白1发夹结构通过影响Paxillin的功能抑制成骨细胞迁移

目的探索G蛋白偶联受体激酶结合蛋白1（Gprotein coupled receptor kinase interacting protein 1，GIT1）的RNA发夹结构（hairpin）（GIT1-RNAh）对成骨细胞迁移的影响，并分析其机制。方法取培养至第6代的鼠成骨细胞，随机分成两组，分别用包含GIT1-RNAh（实验组）和绿色荧光蛋白（green fluoresence protein，GFP）的RNA发夹结构（GFP—RNAh）的腺病毒（对照组）感染12h后，再将每组分成有、无血小板源性生长因子（platelet drived growth factor，PDGF）刺激的两组。免疫荧光染色方法检测成骨细胞内源性GIT1蛋白表达和Paxillin的位置。Western Blot检测Paxillin的磷酸化。构建包含蓝色荧光蛋白的GIT1-RNAh（CFP—GIT1-RNAh）实验组和GFP—RNAh（CFP—GFP—RNAh）（对照组），免疫荧光双染的方法检测CFP—GIT1-RNAh和CFP—GFP—RNAh对Paxillin位置的特异性作用。用划痕愈合法检测GIT1-RNAh（实验组）和GFP—RNAh（对照组）腺病毒对成骨细胞在PDGF刺激下的迁移能力的影响。结果免疫组织化学观察，实验组和对照组比较，明显抑制成骨细胞内源性的GIT1蛋白表达和扰乱Paxillin的分布。Western Blot观察，和对照组相比，实验组明显抑制了Paxiliin的磷酸化（P〈0．05）。划痕愈合法检测观察，和对照组相比，实验组明显抑制成骨细胞的迁移。结论GIT1-RNAh通过扰乱Paxillin的分布和其磷酸化从而抑制成骨细胞的迁移。     

CO_2气腹对胃癌MKN-45细胞黏着斑激酶的影响

目的 研究CO_2气腹对胃癌MKN-45细胞黏着斑激酶(FAK)的影响.方法 体外模拟不同压力CO_2气腹环境,实验组:人胃癌MKN-45细胞置于5、10、15 mm Hg(1mm Hg=0.133 kPa)CO_2气腹环境培养4 h.对照组:常规条件培养人胃癌MKN-45细胞.采用Western blot法检测各组细胞FAK及磷酸化FAK(FAK Tyr397)的表达量,且分别观察15 mm Hg CO_2作用0.5、2、4 h的情况.多组比较采用单因素方差分析,组间比较采用LSD法检验.结果 实验组5、10、15 mm Hg CO_2气腹环境下,FAK表达量分别为2.14±0.17、2.07±0.21、2.52±0.26;FAK Tyr397表达量分别为1.82±0.28、1.93±0.52、3.71±0.37;而对照组FAK表达量为2.43±0.46,FAK Tyr397表达量为1.71±0.23,两组比较差异有统计学意义(F=2.171,26.951,P<0.01).15 mm Hg CO_2作用0.5、2、4 h后,FAK Tyr397表达量分别为3.41±0.44、4.12±0.56、5.24±0.41,三者比较差异有统计学意义(F=116.119,P<0.01).脱离气腹后恢复至处理前水平(0.72±0.16).结论 不同压力CO_2气腹环境处理胃癌MKN-45细胞4 h不增加其FAK表达,但可使其磷酸化而激活,且压力越高,作用时间越长,磷酸化程度越高,但脱离气腹环境后,其活性迅速降至处理前水平.     

酪氨酸磷酸化蛋白在伤口愈合原胶原基因表达中的作用

目的　研究伤口愈合时成纤维细胞与纤维粘连蛋白黏附 ,以及黏附诱导的酪氨酸磷酸化蛋白在成纤维细胞表达原胶原mRNA中的作用。方法　采用RT PCR、免疫印迹法分别检测伤口愈合时成纤维细胞原胶原mRNA表达的变化及黏附诱导的酪氨酸磷酸化蛋白 ,并观察阻断酪氨酸磷酸化后原胶原mRNA和酪氨酸磷酸化蛋白的变化。结果　伤口愈合时成纤维细胞与纤维粘连蛋白黏附 ,可诱导 98kd、6 5kd酪氨酸磷酸化蛋白生成 ,且原胶原proα1 (Ⅰ )mRNA的表达显著增加 ;经酪氨酸激酶抑制后 ,原胶原proα1 (Ⅰ )mRNA显著降低。结论　成纤维细胞与纤维粘连蛋白黏附 ,在伤口愈合原胶原表达增加中具重要作用 ,由黏附诱导的酪氨酸磷酸化是其中的一个重要环节。     

E-钙黏蛋白及黏着斑激酶在直肠癌组织中的表达及意义

目的 探讨E-钙黏蛋白(E-CD)及黏着斑激酶(FAK)和磷酸化黏着斑激酶(FAK py397)在直肠癌组织中的表达水平及其临床意义.方法 收集2001年至2002年武汉大学人民医院手术切除的直肠癌及对应癌旁组织石蜡标本30例和2006年至2007年手术切除的直肠癌新鲜标本及对应癌旁组织35例,应用免疫组织化学法、Western blot检测E-CD、FAK和FAK py397的表达,采用X2检验分析相关数据.结果 正常直肠黏膜中E-CD呈细胞膜阳性表达,直肠癌组织中E-CD细胞膜表达缺失,直肠癌组织中E-CD、FAK及FAK py397的表达与肿瘤分化程度、浸润深度和淋巴结转移有关(χ~2=7.099、18.358、25.612,12.316、28.823、23.168,8.927、18.122、22.620,P＜0.05);与年龄、性别无关(χ~2=0.439、1.899、3.676,0.541、4.051、1.135,P＞0.05);癌组织和癌旁组织中FAK及FAK py397表达率分别为83%(54/65)、68%(44/65)和31%(20/65)、26%(17/65),两者比较差异有统计学意义(χ~2=33.707,34.163,20.897,P＜0.05).结论 直肠癌组织中E-CD细胞膜表达缺失、FAK和FAK py397的表达水平明显升高,与肿瘤分化程度、浸润深度和淋巴结转移有关,可预测肿瘤的生物学行为.     

胆管细胞癌酪氨酸激酶激活及其磷酸化酶分布

目的:探讨胆管细胞癌组织酪氨酸磷酸化信号通路变化及相关分子情况,以寻找新的治疗靶点。方法:免疫亲和、LC-MS/MS分析识辨胆管癌病人(n=23)750多种不同蛋白质中1 000多个酪氨酸磷酸化位点。结果:胆管细胞癌在DDR1、EPHA2、EGFR和ROS1表现最高水平酪氨酸激酶磷酸化,高表达DDR1、EPHA2、ROS1酪氨酸激酶活性(n=18,78%),低表达或不表达RTK活性(n=5,22%);癌旁组织EGFR、AXL、EPHB4和PDGFRA表现出最高水平酪氨酸磷酸化;肿瘤组织和癌旁组织磷酸化蛋白种类分布类似。结论:胆管细胞癌组织中酪氨酸激酶激活,DDR1、EPHA2、EGFR和ROS1酪氨酸激酶活性比癌旁组织高表达,可能致胆管细胞癌的发生。     

丝裂素激活蛋白激酶活化与肾小管上皮细胞生物学行为的关系

目的探讨丝裂素激活蛋白激酶(MAPK)信号通路对血小板源生长因子(PDGF)诱导肾小管上皮细胞表型转化的作用及其对细胞移行能力的影响.方法以PDGF(20 ng/ml)刺激培养的人近端肾小管上皮HK-2细胞,分别观察细胞形态、增殖和移行能力的变化.采用蛋白免疫印记法检测MAPK各亚类活性及α-平滑肌肌动蛋白(SMA)的表达,同时观察分别采用细胞外信号调节激酶(ERK)、c-Jun氨基末端激酶(JNK)和p38信号通路特异阻断剂PD98059、SP600125和SB203580后上述指标的变化.结果 PDGF刺激6～12 h使α-SMA表达上调近2倍,但刺激24～48 h反而使α-SMA表达低于基础水平.PDGF刺激24 h后HK-2细胞从立方形转变为梭形,细胞虽尚无增殖反应但移行能力增强至3倍(P＜0.01).PDGF诱导的ERK、JNK和p38活性均在2 min时迅速增高,分别在2～5 min达高峰,分别为基础水平的5.7倍、2.6倍和1.6倍(P＜0.05),ERK和JNK活性增高可持续至120 min,而p38活性则于60 min以后恢复至基础水平.在PDGF刺激24 h的情况下,PD98059和SP600125不影响基础状态及PDGF对α-SMA表达的作用,但SP600125可抑制PDGF上调的细胞移行能力(P＜0.01);而SB203580既可抑制α-SMA的基础表达(P＜0.01)及PDGF下调的α-SMA表达(P＜0.001),同时还可显著抑制PDGF诱导的细胞移行能力(P＜0.05).结论 PDGF可刺激MAPK的不同亚类信号通路磷酸化,并可诱导肾小管上皮细胞形态改变及其移行能力增强.在PDGF诱导的表型转化和移行功能改变中,ERK信号通路无介导作用,JNK通路主要与细胞移行能力变化相关,而p38可能是调控α-SMA表达和细胞移行改变的主要信号通路.     

酪氨酸激酶途径在成骨细胞增殖及c-fos表达中的作用

目的探讨酪氨酸激酶信息传递途径在成骨细胞增殖及ｃ－ｆｏｓ表达中的作用。方法采用阶段性酶消化法分离拳头新生大鼠颅盖骨居骨细胞,用ＭＴＴ法观察成骨细胞增殖,用免疫组化ＳＰ法结合计算机图像处理系统检测成骨细胞ｃ－ｆｏｓ表达。结果酪氨酸激酶抑制剂Ｇｅｎｉｓｔｅｉｎ和ＨｅｒｂｉｍｙｃｉｎＡ抑制成骨细胞增殖,且两者均部分阻断血清刺激的成骨细胞ｃ－ｆｏｓ表达。结论血清刺激和维持成骨细胞增殖及诱导ｃ－ｆｏｓ表达均     

黏着斑激酶在转化生长因子β1诱导的人肾小管上皮细胞转分化中的作用

目的 观察转化生长因子（TGF）β1诱导的正常人近端肾小管上皮细胞（HK-2）转分化（EMT）过程中黏着斑激酶（FAK）的表达及下调FAK的表达后对TGF-β1诱导的HK-2细胞转分化进程的影响。 方法 应用TGF-β1（10 μg/L）刺激HK-2细胞,采用RT-PCR、Western印迹和免疫荧光方法分别检测E钙黏蛋白（E-cadherin）、α平滑肌肌动蛋白（α-SMA）、FAK mRNA和蛋白的表达及磷酸化（p）-FAK（Tyr397）的蛋白表达。应用Lipofectmine2000将FAK siRNA转染HK-2细胞,采用Western印迹观察下调表达FAK对上述指标的影响。 结果 TGF-β1刺激后,HK-2细胞α-SMA蛋白和mRNA水平上调,E-cadherin蛋白和mRNA表达下调。FAK蛋白和mRNA随时间的延长表达逐渐增多,48 h达到高峰。p-FAK（Tyr397）蛋白表达趋势与FAK相同。脂质体转染siRNA后FAK的mRNA和蛋白分别下调了50%和41%,下调表达FAK后可以显著抑制TGF-β1诱导的HK-2细胞α-SMA蛋白的上调表达,逆转 E-cadherin蛋白的下调表达。 结论 在TGF-β1诱导的HK-2细胞转分化进程FAK蛋白表达上调,敲低FAK蛋白表达后可以部分减轻EMT的程度,提示FAK在TGF-β1诱导的肾小管上皮细胞转分化和肾脏纤维化中发挥一定的作用。     

Subsequent activation of mitogen-activated protein kinase after adhesion of transitional cell cancer cells to fibronectin

INTRODUCTION: In the process of tumor invasion and metastasis, interactions between tumor cells and extracellular matrix play a crucial role. Recently, it was shown that fibronectin binding to fibronectin receptor promotes mitogen-activated protein kinase (MAPK) activation after tyrosine phosphorylation of focal adhesion kinase (FAK). We investigated these signal transduction events in transitional cell cancer (TCC) cells. MATERIALS AND METHODS: (1) The adhesion of T24 cells, a fibronectin-receptor-positive TCC cell line, to fibronectin was investigated; (2) the MAPK activation after fibronectin stimulation in bladder cancer cell lines was examined by Western blotting using an antiactive MAPK antibody, and (3) FAK, Sos, and Grb-2 were also examined by Western blot analysis. RESULTS AND CONCLUSIONS: T24 cells adhered to fibronectin-coated dishes more quickly than to the noncoated dishes. Fibronectin stimulation induced activation of MAPK in T24, SCaBER, and HT1376 cells. However, activated MAPK was not detected in RT4 cells which do not express alpha(5)beta(1) integrin (major fibronectin receptor) after fibronectin stimulation. T24, SCaBER, and HT1376 expressed FAK and Sos. RT4 showed little FAK and Sos expression. Grb-2 was expressed in all cell lines. Adhesion of fibronectin-receptor-positive TCC cells to fibronectin activates the MAPK cascade, possibly resulting in activation of tumor cells.     

Focal adhesion kinase pp125FAK interacts with the large conductance calcium-activated hSlo potassium channel in human osteoblasts: potential role in mechanotransduction.

Molecular events of mechanotransduction in osteoblasts are poorly defined. We show that the mechanosensitive BK channels open and recruit the focal adhesion kinase FAK in osteoblasts on hypotonic shock. This could convert mechanical signals in biochemical events, leading to osteoblast activation. INTRODUCTION: Mechanical strains applied to the skeleton influence bone remodeling and architecture mainly through the osteoblast lineage. The molecular mechanisms involved in osteoblastic mechanotransduction include opening of mechanosensitive cation channels and the activation of protein tyrosine kinases, notably FAK, but their interplay remains poorly characterized. The large conductance K+ channel (BK) seems likely as a bone mechanoreceptor candidate because of its high expression in osteoblasts and its ability to open in response to membrane stretch or hypotonic shock. Propagation of the signals issued from the mechanosensitivity of BK channels inside the cell likely implies complex interactions with molecular partners involved in mechanotransduction, notably FAK. METHODS: Interaction of FAK with the C terminus of the hSlo alpha-subunit of BK was investigated using the yeast two-hybrid system as well as immunofluorescence microscopy and coimmunoprecipitation experiments with a rabbit anti-hslo antibody on MG63 and CAL72 human osteosarcoma cell lines and on normal human osteoblasts. Mapping of the FAK region interacting with hSlo was approached by testing the ability of hSlo to recruit mutated ot truncated FAK proteins. RESULTS: To the best of our knowledge, we provide the first evidence of the physical association of FAK with the intracellular part of hslo. We show that FAK/hSlo interaction likely takes place through the Pro-1-rich domain situated in the C-terminal region of the kinase. FAK/hSlo association occurs constitutively at a low, but appreciable, level in human osteosarcoma cells and normal human osteoblasts that express endogenous FAK and hSlo. In addition, we found that application of an hypo-osmotic shock to these cells induced a sustained activation of BK channels associated to a marked increase in the recruitment of FAK on hSlo. CONCLUSIONS: Based on these data, we propose that BK channels might play a triggering role in the signaling cascade induced by mechanical strains in osteoblasts.     

Integrin‐associated tyrosine kinase FAK affects Cbfa1 expression

Following cell adhesion, focal adhesion kinase (FAK) autophosphorylates on tyrosine and regulates intracellular signaling cascades that regulate cell growth and differentiation. The hypothesis of this study was FAK mediates osteoblast differentiation dependent Cbfa1 expression. Slowly mineralizing UI and rapidly mineralizing UMR‐106‐01 BSP osteoblasts formed focal adhesions; however, the level of FAK in UI focal adhesions was less than that seen in BSP cells. UI cultures had less FAK expression (p < 0.05) along with elevated levels of FAK phosphotyrosine in comparison to rapidly mineralizing BSP cultures. Mineralization decreased in a dose‐dependent manner in response to Herbimycin A, a tyrosine kinase inhibitor. Overexpression of FAK in UI cells led to a fourfold increase in Cbfa1 gene expression (p < 0.02), and an increase in Cbfa1 protein expression. These results suggest that the integrin‐associated tyrosine kinase FAK contributes to the regulation of the osteoblast differentiation in part through the regulation of Cbfa1 expression. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1443–1447, 2011     

Role of protein kinase C alpha in primary human osteoblast proliferation.

Protein kinase C (PKC) isoforms have been shown to have specific expression profiles and individual isoforms are believed to play distinct roles in the cells in which they are found. The goal here was to determine which specific isoform(s) is involved in proliferation of primary human osteoblasts. In primary human osteoblasts, 10 microM of acute sphingosine-1-phosphate (S1P) treatment induced an increase in proliferation that correlated with an increase in PKCalpha and PKCiota expression. To further delineate which isoforms are involved in osteoblastic cell proliferation, the effect of low versus high serum culture conditions on PKC isoform expression was determined. Likewise, the effect of antisense oligodeoxynucleotides (ODNs) to specific PKC isoforms on proliferation and MAPK activation was studied. The effect of S1P on intracellular translocation of activated PKC isoforms was also evaluated. The results indicated that in primary human osteoblasts, PKCalpha was not expressed under conditions of low proliferative rate while PKCdelta and PKCiota expression was not affected. The specific inhibition of PKCalpha by antisense ODNs resulted in inhibition of MAPK activity leading to a significant decrease in proliferation. S1P up-regulated antisense ODN inhibited PKCalpha expression and MAPK activity and led to an increase in proliferation. Subsequent experiments using platelet-derived growth factor (PDGF) as an additional mitogen generated similar data. PDGF stimulation resulted in a significant increase in proliferation that correlated with an up-regulation of inhibited PKCalpha expression in antisense ODN-treated cells. Immunofluorescence methods showed that mitogenic stimulation of PKCa resulted in nuclear translocation. Our findings present original data that PKCalpha is the isoform specifically involved in the proliferation of primary human osteoblasts.     

Thrombospondin-1-induced migration is functionally dependent upon focal adhesion kinase

Vascular smooth muscle cell migration is important in vascular disease. Previously, we showed thrombospondin-1 activates focal adhesion kinase in these cells. We hypothesized that focal adhesion kinase is important for thrombspondin-1-induced vascular smooth muscle cell migration. Bovine aortic smooth muscle cells were transfected with FAK397, FAK-wild type, pcDNA, or beta-Gal plasmids. Migration was assessed with thrombospondin-1 or serum-free medium in quiescent transfected cells or quiescent cells pretreated with the focal adhesion kinase inhibitor, geldanamycin. Number of cells migrated per 5 fields (x400) were recorded. Antihemagglutinin immunoprecipitation and Western blot were used to examine thrombospondin-1-induced focal adhesion kinase phosphorylation in transfected cells. FAK397 transfection inhibited thrombospondin-1-induced focal adhesion kinase phosphorylation and migration (P < .05). Geldanamycin inhibited thrombospondin-1-induced smooth muscle cell migration (P < .05). In conclusion, vascular smooth muscle cells transfected with FAK397 inhibited thrombosponin-1-induced migration and tyrosine phosphorylation. Further, geldanamycin also inhibited migration. These results suggest focal adhesion kinase is involved in thrombospondin-1-induced vascular smooth muscle cell migration.