Bactrian camel (Camelus bactrianus) integrins αvβ3 and αvβ6 as FMDV receptors: Molecular cloning, sequence analysis and comparison with other species

Integrins are heterodimeric adhesion receptors that participate in a variety of cell–cell and cell–extracellular matrix protein interactions. Many integrins recognize RGD sequences displayed on extracellular matrix proteins and the exposed loops of viral capsid proteins. Four members of the αv integrin family of cellular receptors, αvβ3, αvβ6, αvβ1 and αvβ8, have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro, and integrins are believed to be the receptors used to target epithelial cells in the infected animals. To analyse the roles of the αv integrins from a susceptible species as viral receptors, we have cloned Bactrian camel αv, β3 and β6 integrin cDNAs and compared them to those of other species. The coding sequences for Bactrian camel integrin αv, β3 and β6 were found to be 3165, 2289 and 2367 nucleotides in length, encoding 1054, 762 and 788 amino acids, respectively. The Bactrian camel αv, β3 and β6 subunits share many structural features with homologues of other species, including the ligand binding domain and cysteine-rich region. Phylogenetic trees and similarity analyses showed the close relationships of integrin genes from Bactrian camels, pigs and cattle, which are each susceptible to FMDV infection, that were distinct from the orders Rodentia, Primates, Perissodactyla, Carnivora, Galliformes and Xenopus. We postulate that host tropism of FMDV may in part be related to the divergence in integrin subunits among different species.     

口蹄疫病毒受体猪源整联蛋白β6亚基配体结合域单克隆抗体识别的抗原表位分析

[目的]分析测定口蹄疫病毒受体猪源整联蛋白β6亚基配体结合域单克隆抗体（mAb）的抗原识别位点、饱和值、亲和力常数。[方法]利用叠加ELISA法、间接ELISA法、硫氰酸盐洗脱法测定C2D8、B786、B8C9、CAC7、E7C75株mAb的抗原识别位点、饱和值、亲和力常数。[结果15株mAb间具有相似或不同的抗原识剐位点,其中C4C7与E7C7、C4C7与B8C9、E7C7与B8C9等mAb间的识别位点相似．而C4C7与B786、E7C7与C2D8等mAb间的识别位点不同。5株mAbC2D8、E7C7、B8C9、C4C7、B7B6的饱和值分别为1：200、1：400、1：800、1：800、1：800,亲和力为B8C9〉C4C7〉B7B6≥E7C7〉C2D8。[结论]分析测定了5株mAb的抗原识别位点、饱和值、亲和力常数,从而为利用mAb深入研究整联蛋白OtV[36在口蹄疫病毒感染过程中的作用奠定基础。     

Effects of β3 integrin on adhesion and migration of vascular smooth muscle cells induced by growth factor

AIM: To investigate the effect of platelet-derived growth factor (PDGF) on β₃ integrin gene expression and the role of β₃ integrin on adhesion, migration and proliferation of vascular smooth muscle cells (VSMC) induced by PDGF. METHODS: β₃ integxin gene expression was detected by RT-PCr. After β₃ integrin extracellular do-main was blocks, VSMC adhesio, migration and proliferation were measured by adhesion assay awound-culture model an [³H]-TSR incorporation respectively.RESULTS: After the interaction between β₃ integrin and extracellular matrix was blocked, VSMC proliferation was inhibited in some degree and the rate of [³H]-TdR incorporation into VSMC decreased 39%. The cell adhesion and migration were significantly inhibited when 10 mg/L anti-β₃ integrin antibody was added (P＜0.05). When VSMC were treated by PDGF for 6 hours, the expression of β₃ integrin gene was 87% higher than that of control. CONCLUSION: PDGF significantly induces expression of β₃ integrin gene in VSMC, and the interaction between β₃ integrin and ECM protein may play an important role in VSMC adhesion and migration.     

αvβ6-ERK pathway participates in norcantharidin-induced apoptosis of HT-29 colon cancer cells

AIM: To investigate the pharmacological mechanism of norcantharidin (NCTD)-induced apoptosis of HT-29 colon cancer cells. METHODS: Hoechst 33258 staining was used to analyze the apoptosis of HT-29 cells treated with NCTD. The effects of NCTD on the expression of integrin in HT-29 cells were determined by flow cytometry. The effects of several functional blocking antibodies on HT-29 cells were detected by MTT method. The expression and the phosphorylation of mitogen activated protein kinases (MAPKs) in HT-29 cells were measured by Western blotting. Co-immunoprecipitation assay was used to detect the activity of αvβ6-extracellular signal-regulated kinase (ERK) direct linkage in HT-29 cells.RESULTS: NCTD induced the apoptosis of HT-29 colon cancer cells. The expression of integrin αvβ6 in HT-29 cells treated with NCTD was reduced, but the expression of αvβ3 and αvβ5 was not changed. A function-blocking antibody to αvβ6,10D5,strengthened the growth inhibitory effect of NCTD on HT-29 cells ,but LM609 (a function-blocking antibody to αvβ3) and P1F6 (a function-blocking antibody to αvβ5) did not. The level of phosphorylated ERK (p-ERK) was decreased substantially after treated with NCTD in a dose-and time-dependent manner. NCTD also affected the association of αvβ6 and ERK. CONCLUSION: NCTD decreases the expression of integrin αvβ6 and interferes with the phosphorylation of ERK. As a result, the formation of αvβ6-ERK direct linkage is affected and the signal transduction mediated by αvβ6 is disturbed. The mechanism of NCTD-induced HT-29 cell apoptosis is involved in the αvβ6-ERK signaling pathway.     

牛整合素β6基因的原核表达及其抗血清的制备研究

[目的]为深入研究ITGB6亚基的基因功能奠定基础。[方法]利用基因克隆技术获得整合素β6基因（ITGB6）胞外区,然后将其重组到pET-32a（＋）质粒中,经鉴定及序列分析确定获得了重组阳性克隆;转化大肠杆菌BL21感受态细胞,IPTG诱导及蛋白纯化。[结果]SDS-PAGE结果显示,在94 kD处出现特异性条带;对该融合蛋白进行分离纯化后免疫新西兰兔,制备ITGB6蛋白抗血清,Western-blot分析表明抗血清可与原核表达的ITGB6蛋白特异性结合,ELISA方法检测抗血清的效价可达到1∶2 560。[结论]成功表达了牛的重组整合素β6基因。     

Effect of integrin β1 on multidrug resistance in gastric cancer SGC-7901 cells

AIM: To study the effect of integrin β1 on multidrug resistance in gastric cancer and its possible mechanisms. METHODS: The expression of integrin β1 at mRNA and protein levels in the SGC-7901 cells and SGC-7901/DDP cells was determined by qPCR and Western blot. The expression of integrin β1 in the SGC-7901/DDP cells was silenced by antisense oligodeoxynucleotide. The cell viability was detected by the CCK-8 assay, the cell apoptosis were analyzed by flow cytometry, and the protein levels of integrin β1, Bcl-2/Bax, cleaved caspase-3/caspase-3, cytochrome C (Cyt-C) and p-AKT/AKT were determined by Western blot.RESULTS: The expression of integrin β1 at both mRNA and protein levels was significantly upregulated in SGC-7901/DDP cells. The expression of integrin β1 was increased in SGC-7901 cells treated with chemotherapeutic agents such as cisplatin, paclitaxel and 5-fluorouracil. Knockdown of integrin β1 induced apoptosis of SGC-7901/DDP cells with an increased sensitivity to the chemotherapeutic agents. Meanwhile, knockdown of integrin β1 downregulated the protein levels of Bcl-2/Bax, p-AKT^Ser473 and p-AKT^Thr308, while promoted the release of Cyt-C and upregulated the protein level of cleaved caspase-3. CONCLUSION: Knockdown of integrin β1 increases the sensitivity of SGC-7901/DDP cells to the chemotherapeutic agents, and promotes the cell apoptosis via mitochondrial apoptosis pathway. The mechanism may be related to the attenuation of AKT pathway by inhibiting phosphorylations of AKT at Ser473 and Thr308.     

Effect of lysophosphatidic acid on expression of integrin β6 in epithelial cells

AIM: To investigate the effect of lysophosphatidic acid (LPA) on the expression of integrin β₆ (ITGB6) for determining the role of transforming growth factor β (TGF-β) activation induced by LPA in this process. METHODS: Normal human bronchial epithelial (NHBE) cells were primarily cultured in 6 well plate and stimulated with LPA. The mRNA expression of ITGB6 and the level of cell surface ITGB6 protein were detected by RT-PCR and flow cytometry,respectively. The activity of active TGF-β induced by LPA was measured by the method of transformed mink lung epithelial cells (TMLC) transfected with TGF-β responsive plasminogen activator inhibitor 1(PAI-1) promoter fused with firefly luciferase reporter gene. RESULTS: After stimulated with LPA at concentration of 10 μmol/L for 2 h, the mRNA expression of ITGB6 in epithelial cells was significantly increased in a time-dependent manner. The results of flow cytometry showed that the protein level of ITGB6 on cell surface was obviously increased after treated with LPA at concentration of 10 μmol/L for 4 h. The active TGF-β induced by LPA in epithelial cells was blocked by an α_Vβ₆ blocking antibody. However, α_Vβ₆ blocking antibody failed to inhibit the mRNA expression of ITGB6 induced by LPA. CONCLUSION: LPA induces the mRNA and cell surface protein of ITGB6 in epithelial cells. The up-regulated ITGB6 expression by LPA is independent on LPA-induced TGF-β activation.     

Mouse early integral embryo induces expression of endometrial integrin β3 and leukaemia-inhibitory factor,and improves uterine receptivity in mice

AIM: To investigate the changes of endometrial receptivity under the effect of mouse embryo both in vitro and in vivo, and to figure out which part of the embryo induces the change. METHODS: Scanning electron microscope was applied to observe the pinpode formation on day 4 endometrium both in vitro and in vivo. The expression of integrin β₃ and leukaemia-inhibitory factor(LIF) on day 2 pregnant mouse endometrium , day 4 endometrium after co-culture for 2 d with day 2 embryo , blastomere and zona pellucida as well as control group in vitro were detected by the methods of fluorescent quantitative PCR, immunohistochemistry and Western blotting. The same tests were conducted in the in vivo part of the experiment with the integral embryo or different parts of the embryo being transferred to pseudopregnant mouse uterus. On day 4 of the pregnancy, the endometrium was extracted to carry out the tests. RESULTS: After co-cultured for 2 d with whole embryo, the experssion of integrin β₃ and LIF was higher than that in any other group in the in vitro part. The expression of integrin β₃ and LIF on day 4 of normal pregnancy was higher than that in any other group in the in vivo experiments. CONCLUSION: Mouse embryo as a whole is able to induce better endometrial receptivity, while any separated part of embryo, such as blastomere or zona pellucida, couldn't.