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.     

人小肠三叶因子在大肠杆菌中的表达

利用PCR技术将人小肠三叶因子(hITF)基因重组入表达载体pGEX-4T-1,构建了融合蛋白GST-hITF的重组表达质粒pGTF,在大肠杆菌中诱导表达。表达的融合蛋白经亲和层析、凝血酶切和凝胶过滤层析得到纯化的hITF蛋白。测定了重组蛋白的氨基酸组成、分子量及其对酸和蛋白酶的抗性。Western印迹表明重组蛋白具有hITF的抗原性,并对大鼠胃溃疡具有明显的预防和保护作用。     

Secreted Trefoil Factor 2 Activates the CXCR4 Receptor in Epithelial and Lymphocytic Cancer Cell Lines

The secreted trefoil factor family 2 (TFF2) protein contributes to the protection of the gastrointestinal mucosa from injury by strengthening and stabilizing mucin gels, stimulating epithelial restitution, and restraining the associated inflammation. Although trefoil factors have been shown to activate signaling pathways, no cell surface receptor has been directly linked to trefoil peptide signaling. Here we demonstrate the ability of TFF2 peptide to activate signaling via the CXCR4 chemokine receptor in cancer cell lines. We found that both mouse and human TFF2 proteins (at ∼0.5 μm) activate Ca²⁺ signaling in lymphoblastic Jurkat cells that could be abrogated by receptor desensitization (with SDF-1α) or pretreatment with the specific antagonist AMD3100 or an anti-CXCR4 antibody. TFF2 pretreatment of Jurkat cells decreased Ca²⁺ rise and chemotactic response to SDF-1α. In addition, the CXCR4-negative gastric epithelial cell line AGS became highly responsive to TFF2 treatment upon expression of the CXCR4 receptor. TFF2-induced activation of mitogen-activated protein kinases in gastric and pancreatic cancer cells, KATO III and AsPC-1, respectively, was also dependent on the presence of the CXCR4 receptor. Finally we demonstrate a distinct proliferative effect of TFF2 protein on an AGS gastric cancer cell line that expresses CXCR4. Overall these data identify CXCR4 as a bona fide signaling receptor for TFF2 and suggest a mechanism through which TFF2 may modulate immune and tumorigenic responses in vivo.Trefoil factor 2 (TFF2),² previously known as spasmolytic polypeptide, is a unique member of the trefoil family that is expressed primarily in gastric mucous neck cells and is up-regulated in the setting of chronic inflammation. Experimental induction of ulceration in the rat stomach leads to rapid up-regulation of TFF2 expression with high levels observed 30 min after ulceration with persistence for up to 10 days (1). TFF2 is secreted into the mucus layer of the gastrointestinal tract of mammals where it stabilizes the mucin gel layer and stimulates migration of epithelial cells (2–4), suggesting an important role in restitution and in maintenance of the integrity of the gut. Exogenous administration of recombinant TFF2, either orally or intravenously, provides mucosal protection in several rodent models of acute gastric or intestinal injury (5, 6). A TFF2^-/- knock-out mouse model has confirmed the importance of TFF2 in the protection of gastrointestinal mucosa against chronic injury (7).It is widely accepted that trefoil factors exert their biological action through a cell surface receptor. This suggestion comes from studies on binding of ¹²⁵I-labeled TFF2 that demonstrated specific binding sites in the gastric glands, intestine, and colon that could be displaced by non-radioactive TFF2 (6, 8–10). Structural studies have revealed potential binding sites for receptors for all members of the trefoil factor family (11, 12). In concordance with this hypothesis, several membrane proteins were found to interact with TFF2. First it was shown that recombinant human TFF2 (and TFF3) could bind to a 28-kDa peptide from membrane fractions of rat jejunum and two human adenocarcinoma cell lines, MCF-7 and Colony-29 (13). Later it was found that recombinant TFF3 fused with biotin selectively bound with a 50-kDa protein from the membrane of rat small intestinal cells (14). However, these 28- and 50-kDa proteins were characterized only by their molecular size without further identification. Two TFF2-binding proteins that have been characterized include a 140-kDa protein, the β subunit of the fibronectin receptor, and a 224-kDa protein called muclin (15). Another TFF2-binding protein was isolated by probing two-dimensional blots of mouse stomach with a murine TFF2 fusion protein, leading to the identification of the gastric foveolar protein blottin, a murine homolog of the human peptide TFIZ1(16). Although these three proteins have now been well characterized, none of them has been shown to mediate responses to TFF2, and no activated signaling cascades have been shown.Despite the absence of an identified cell surface receptor for TFF2, there is nevertheless clear evidence that TFF2 and TFF3 rapidly activate signal transduction pathways (17, 18). TFF3 prevents cell death via activation of the serine/threonine kinase AKT in colon cancer cell lines (19). The TFF3 protein also activates STAT3 signaling in human colorectal cancer cells, thus providing cells with invasion potential (20). TFF3 treatment leads to EGF receptor activation and β-catenin phosphorylation in HT-29 cells (21) and to transient phosphorylation of ERK1/2 in oral keratinocytes (22). With respect to TFF2, recombinant peptide enhances the migration of human bronchial epithelial cell line BEAS-2B (4). TFF2 has been shown to induce phosphorylation of c-Jun NH₂-terminal kinase (JNK) and ERK1/2. Consistent with this observation, the motogenic effect of TFF2 is significantly inhibited by antagonists of ERK kinases and protein kinase C but not by inhibitors of p38 mitogen-activated protein kinase (MAPK). It is believed that the motogenic effect of trefoil factors and of TFF2 in particular, could contribute to in vivo restitution of gastric epithelium by enhancing cell migration.Although previous studies have suggested that TFF2 functions primarily in cytoprotection, accumulating evidence now suggests that TFF2 may also play a role in the regulation of host immunity. For example, recombinant TFF2 reduces inflammation in rat and mouse models of colitis (23, 24). In addition, TFF2 was detected in rat lymphoid tissues (spleen, lymph nodes, and bone marrow) (25). Recently we and others found TFF2 mRNA expression in primary and secondary lymphopoietic organs (26, 27). These data suggest that TFF2 may play some function in the immune system. In concordance with these findings, we detected an exacerbated inflammatory response to acute injury in TFF2 knock-out animals (27, 28). These observations prompted us to look at the possible function of TFF2 in immune cells. Unexpectedly we found that TFF2 modulates Ca²⁺ and AKT signaling in lymphoblastic Jurkat cells and that these effects appear to be mediated through the CXCR4 receptor.     

Synthesis of Indole Derived Protease-Activated Receptor 4 Antagonists and Characterization in Human Platelets

Protease activated receptor-4 (PAR4) is one of the thrombin receptors on human platelets and is a potential target for the management of thrombotic disorders. We sought to develop potent, selective, and novel PAR4 antagonists to test the role of PAR4 in thrombosis and hemostasis. Development of an expedient three-step synthetic route to access a novel series of indole-based PAR4 antagonists also necessitated the development of a platelet based high-throughput screening assay. Screening and subsequent structure activity relationship analysis yielded several selective PAR4 antagonists as well as possible new scaffolds for future antagonist development.     

人小肠三叶因子（hITF）基因在生菜中的整合与表达

用根癌土壤杆菌（Agrobacterium tumefaciens(Smith et Townsend)conn)介导的叶盘法,将人小肠三叶因子（hITF)导入生菜（Lactuca sativa L.)中,在含有除草上培养基上筛选,获得抗性植株,通过PCR和Southern印迹分析证明,hITF cDNA已整合到生菜基因组中,Western印迹分析证明hITF在生菜中的表达。ELISA检测表明,hITF在生菜新鲜叶片中的表达量为200－300ng/g,最高达700ng/g,约占总可溶性蛋白的0．1％。     

人三叶因子3在毕赤酵母中表达条件的研究

为提高人三叶因子 3 (HumanTrefoilfactor 3 ,hTFF3 )在毕赤酵母中的表达量 ,研究了转化子生长的培养条件 ,包括不同碳源对转化子生长的影响和接种量、甲醇浓度、pH值、摇瓶转速及不同诱导时间对人三叶因子 3表达的影响。结果表明转化子在生长阶段加入葡萄糖生长旺盛 ,培养 14h后OD₆₀₀ 就可达到 50。在 100mL生长培养基上的菌液以 1∶1接入诱导培养基时蛋白表达量最高 ;转化子在 1%的甲醇、pH60、摇瓶转速240r/min的条件下诱导4 8h ,菌体密度OD₆₀₀为 15 ,目的蛋白表达量达到 20mg L。用 5L发酵罐进行了高密度发酵 ,经2%甲醇32h诱导 ,最终菌体密度OD₆₀₀ 达到 120 ,每升发酵液中含目的蛋白100mg。     

人三叶因子3在毕氏酵母中的表达及生物活性分析

利用PCR方法在人胎儿胎盘cDNA中扩增了人三叶因子3基因（hTFF3）,插入到含有AOX1启动子和α－因子信号肽序列的表达载体pPIC9K中,采用毕氏酵母表达系统对其进行了高效的分泌表达,并用G418筛选高拷贝整合转化子。2％甲醇诱导酵母表达48h后,上清经SDS－PAGE和Western印迹证明重组蛋白质以双体的形式分泌表达,占总蛋白质的45％,能被抗hTFF3抗体所识别。表达产物经S－Sepharose、Q-Sepharose离子交换及Sephacryl S-100纯化后纯度达到95％以上。体内生物学活性研究证实hTFF3可有效的防止盐酸诱导的大鼠胃溃疡。     

Irinotecan Upregulates Fibroblast Growth Factor Receptor 3 Expression in Colorectal Cancer Cells,Which Mitigates Irinotecan-Induced Apoptosis

BACKGROUND: Irinotecan (IRI) is an integral part of colorectal cancer (CRC) therapy, but response rates are unsatisfactory and resistance mechanisms are still insufficiently understood. As fibroblast growth factor receptor 3 (FGFR3) mediates essential survival signals in CRC, it is a candidate gene for causing intrinsic resistance to IRI. METHODS: We have used cell line models overexpressing FGFR3 to study the receptor's impact on IRI response. For pathway blockade, a dominant-negative receptor mutant and a small molecule kinase inhibitor were employed. RESULTS: IRI exposure induced expression of FGFR3 as well as its ligands FGF8 and FGF18 both in cell cultures and in xenograft tumors. As overexpression of FGFR3 mitigated IRI-induced apoptosis in CRC cell models, this suggests that the drug itself activated a survival response. On the cellular level, the antiapoptotic protein bcl-xl was upregulated and caspase 3 activation was inhibited. Targeting FGFR3 signaling using a dominant-negative receptor mutant sensitized cells for IRI. In addition, the FGFR inhibitor PD173074 acted synergistically with the chemotherapeutic drug and significantly enhanced IRI-induced caspase 3 activity in vitro. In vivo, PD173074 strongly inhibited growth of IRI-treated tumors. CONCLUSION: Together, our results indicate that targeting FGFR3 can be a promising strategy to enhance IRI response in CRC patients.     

肠三叶因子在大肠杆菌中的表达、纯化及其抗体制备

目的:获得肠三叶因子(ITF)的原核表达产物及抗rITF抗体,为深入研究ITF的作用机制及其受体研究奠定基础。方法:常规提取人小肠组织总RNA,用RT-PCR获得ITF编码基因片段,克隆至质粒pET32a获得原核表达栽体,双酶切和测序后转化至Origami B(DE3)用IPTG诱导表达,优化条件获得最大表达产量;用SDS-PAGE、Western blot鉴定表达产物,亲和层析纯化获得的重组蛋白rITF皮下多点注射家兔,制备多克隆抗体,并用此抗体进行大鼠肠组织免疫组化研究。结果:测序证实PCR扩增获得ITF全长基因序列与基因文库中的完全一致,将该基因片段正确插入表达载体pET32a中、优化表达条件后,重组蛋白的表达量达到50mg/L;Western blot证明重组蛋白具有良好的抗原性和特异性;通过Ni-NTA亲和层析、超滤离心后,得到90%纯度的蛋白;收集兔血清,纯化后获得特异性良好的ITF抗体,免疫组化染色肠组织显示ITF表达的部位定位于杯状细胞。结论:成功构建了表达载体pET32a-ITF,在大肠杆菌中表达并纯化获得纯度较高的rITF,并获得了生物活性较高的ITF抗体,ITF主要在肠道杯状细胞分泌表达。     

Conformational Plasticity of Human Protease-Activated Receptor 1 upon Antagonist- and Agonist-Binding

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HER2胞外段的克隆与原核表达

应用RT-PCR技术从人乳腺癌细胞系SK-BR-3中克隆出人表皮生长因子受体2(human epidermal growth factorreceptor 2,HER2)基因的胞外段,并插入到表达载体pET-30a中,得到重组表达载体pET30-HER2(Ex)。将该载体转化至大肠杆菌BL21(DE3)细胞中,加入IPTG进行诱导表达,成功获得HER2胞外段蛋白。分别提取培养液上清、大肠杆菌周质腔、细胞质可溶性及不可溶性组分蛋白进行SDS-PAGE电泳分析,确定目的蛋白定位于大肠杆菌细胞质包涵体中。通过改变诱导温度、诱导物浓度、诱导起始菌体密度和诱导时间,寻找最佳表达条件,使目的蛋白的表达量达到最高。结果表明,在37℃下,OD600达到1.0时,经终浓度为0.1 mmol/L的IPTG诱导4 h,目的蛋白的表达量最高。将重组表达菌进行超声破碎,分离出包涵体组分,经Ni2+亲和层析纯化后获得了纯度>90%的HER2胞外段蛋白,从而为抗HER2抗体的制备及肿瘤疫苗的研究奠定了基础。     

IgG Expression in Human Colorectal Cancer and Its Relationship to Cancer Cell Behaviors

Increasing evidence indicates that various cancer cell types are capable of producing IgG. The exact function of cancer-derived IgG has, however, not been elucidated. Here we demonstrated the expression of IgG genes with V(D)J recombination in 80 cases of colorectal cancers, 4 colon cancer cell lines and a tumor bearing immune deficient mouse model. IgG expression was associated with tumor differentiation, pTNM stage, lymph node involvement and inflammatory infiltration and positively correlated with the expressions of Cyclin D1, NF-κB and PCNA. Furthermore, we investigated the effect of cancer-derived IgG on the malignant behaviors of colorectal cancer cells and showed that blockage of IgG resulted in increased apoptosis and negatively affected the potential for anchor-independent colony formation and cancer cell invasion. These findings suggest that IgG synthesized by colorectal cancer cells is involved in the development and growth of colorectal cancer and blockage of IgG may be a potential therapy in treating this cancer.     

人小肠三叶因子的理化性质及光谱学行为

酵母表达的重组人小肠三叶因子(rh-ITF)飞行质谱测定二聚体的分子量为13154,等电点约为4.5～4.75.紫外和荧光光谱表明rh-ITF在pH2.7～8.4和pH2.7～7.7时,吸收值增加。随pH进一步增加,吸收值降低。推测色氨酸和酪氨酸所处微环境发生了一定的变化。园二色谱表明在不同pH下,rh-ITF所含二级结构百分数有所变化,但仍保留有一定的二级结构,即含有一定数量的α-helix,β-sheet或β-turn,其三级结构基本不变。光电滴定和有机溶剂微扰法表明rh-ITF分子中有两个酪氨酸,一个处于分子表面,另一个参与氢键的形成或存在于一个非极性的环境中。rh-ITF中的色氨酸处于分子内部。另外,质谱测定rh-ITF在体外对酸和蛋白酶有一定的抗性     

人小肠三叶因子在糙皮侧耳中的整合、表达及检测

用原生质体法将含双 35S_AMV启动子及人小肠三叶因子 (hITF)cDNA的表达载体导入糙皮侧耳 (Pleurotusostreatus)中 ,原生质体转化子在含有除草剂的培养基上初步筛选 ,获得抗性菌株。通过PCR分析证明hITFcDNA已整合到侧耳基因组中。以hITF为抗原免疫家兔制备了抗血清 ,纯化后的IgG以直接型酶联免疫吸附检测 (ELISA)方法测定效价 ,并建立起hITF的竞争型酶联免疫吸附检测 (ELISA)方法 ,给出了标准工作曲线 ,以应用于大规模表达菌株的筛选确立。检测表明 ,hITF在侧耳新鲜菌丝体中分 3个表达量段 :10 0 0～ 12 5 0ng g、14 80～ 170 0ng g、最高达2 0 0 0～ 2 2 5 0ng g ,约占总可溶性蛋白的 0 7%～ 1 5 %。Western印迹进一步分析证明hITF在侧耳中的表达。