TEM8与uPA的相互作用及其生物学意义研究
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摘要
血管生成是从组织中已存在的成熟血管发展形成新的毛细血管网络的过程，在包括胚胎发育、伤口愈合、组织再生和重塑等许多重要的生理过程是必不可少的。然而失控的或有缺陷的血管生成也存在于各种疾病，包括肿瘤、心血管疾病以及增生性视网膜疾病的病理过程中。靶向血管生成疗法可以被用来终止或延缓这些疾病的发展，所以这种治疗方法已经引起了科学界极大的关注。然而在临床应用中靶向血管生成的抗肿瘤药物被证明只有有限的疗效，同时还存在很大的副作用，于是寻找更特异性的血管生成标志物变得更加迫切。肿瘤内皮标志物Ⅷ(tumor endothelial marker8，TEM8)是一种特异性表达于肿瘤脉管系统且物种间高度保守的Ⅰ型膜蛋白。后来的研究发现TEM8是炭疽毒素保护性抗原（PA）的受体，作为炭疽毒素的“帮凶”，介导其进入宿主细胞。关于TEM8生理功能方面的研究则相对滞后了很多，仅有一些研究发现TEM8在肿瘤细胞的迁移、粘附和肿瘤血管生成等生理过程中发挥关键性作用，tem8基因敲除小鼠实验也证明TEM8对促进肿瘤血管生成和肿瘤生长起到重要作用，而对正常的生长发育和伤口愈合没有任何影响。TEM8这种特异性的分布以及在正常和肿瘤血管形成中功能的差异性，引起了极大关注。在最近的一篇临床前研究中发现，靶向TEM8胞外结构域的抗体能够抑制肿瘤血管的生成以及多种植入到小鼠体内的人肿瘤细胞的生长和迁移，这种抗TEM8抗体能够增强其他抗血管生成剂、血管靶向剂以及传统化疗药物的活性，并且没有发现其存在细胞毒性。
本课题组在前期工作中开发了一种抗体样分子TEM8-Fc （TEM8N端与炭疽毒素保护性抗原PA结合的vWA结构域和IgG1Fc段组成的融合蛋白），发现它是一种具有抗血管生成能力的分子，能抑制多种肿瘤细胞的生长、迁移以及肿瘤血管的生成，然而其抑制肿瘤的机制尚未阐述清楚。为了弄清TEM8-Fc的抑瘤机制，首先需要弄清TEM8的生理性配体及其发挥生理功能的作用机制。本研究将研究焦点首先集中在关于TEM8生理性配体的研究上，因为只有找到它的生理性配体才能进一步阐明它在细胞生理条件下的功能作用，为靶向TEM8的肿瘤抗血管生成疗法提供理论依据。在找到TEM8的生理性配体后，又进一步研究了TEM8-Fc抗肿瘤血管生成的机制以及TEM8发挥生物学功能的机制。
在研究中我们得出了如下结论：
1.通过软琼脂克隆形成实验发现，TEM8和CMG2（特别是TEM8）能够使HEK293细胞的软琼脂克隆形成能力增加，即过表达TEM8或CMG2使HEK293细胞具有了恶性转化的特征。通过CCK8细胞增殖实验和划痕实验等验证了TEM8和CMG2的部分生理功能：TEM8促进细胞迁移，而CMG2促进细胞增殖。
2.利用本课题组前期构建的抗体样分子TEM8-Fc与人肝癌组织匀浆液进行免疫共沉淀和SDS-PAGE电泳，把共沉下的蛋白条带经生物质谱分析发现了TEM8的两个可能的相互作用蛋白——尿型纤溶酶原激活剂（uPA）与α-烯醇化酶（α-enolase）。因为uPA是胞外可溶性的信号分子，而α-enolase主要在胞内和胞膜上发挥功能，所以推测uPA即是TEM8的生理性配体，于是下一步工作主要围绕uPA与TEM8展开。为了进一步确定两者之间的相互作用，我们先后用ELISA、WesternBlot等方法进行了验证，并且通过表面等离子体共振（SPR）的方法测定了TEM8-Fc与uPA的结合常数。TEM8-Fc结合到高分子量单链uPA（HMW-scuPA），高分子量双链uPA（HMW-tcuPA）和低分子量uPA（LMW-uPA）的结合常数分别为16.6nM，29.7nM和31.1nM。另外还通过溶圈实验和S2444发色底物法发现与TEM8的结合不会影响uPA的酶活性。
3.构建了uPA的几种截短突变体与EGFP的融合蛋白表达载体，同时构建了TEM8胞外区（N端）与RFP的融合蛋白表达载体，然后将各种uPA截短突变体与EGFP的融合蛋白表达载体和TEM8胞外区（N端）与RFP的融合蛋白表达载体分别共转染到HEK293细胞中。最后通过FRET的方法确定了uPA与TEM8之间的相互作用区域。FRET的结果显示uPA的Kringle区和酶活性区的C端与TEM8的N端有FRET效应，即介导uPA与TEM8相互作用区域是它的Kringle结构域和催化结构域的C-末端。因此uPA与TEM8相互作用区域不同于uPA受体（uPAR）（uPA的EGF样结构域负责与uPAR相互作用）。
4.用IP的方法证明了uPA能够剂量依赖性的使TEM8磷酸化，而TEM8-Fc可拮抗uPA导致的TEM8磷酸化。通过Raybiotech公司的RTK磷酸化芯片和EGFR磷酸化位点芯片，发现uPA能够上调EGFR845位和1173位酪氨酸的磷酸化水平，而TEM8-Fc能拮抗它的作用。在细胞生物学方面研究了TEM8与uPA的相互作用对肿瘤生长和迁移的作用，通过迁移实验发现uPA能够显著地增加HepG2细胞的uPAR非依赖性的迁移，而TEM8-Fc能够封闭uPA的作用。最后观察到本组开发的两种抗体样分子TEM8-Fc和ATF-Fc具有协同抗肿瘤的作用。
综上所述，本研究结果表明TEM8是uPA一种新型的受体，在调节肿瘤的生长和转移中起到了重要的作用。本组开发的抗体样分子TEM8-Fc正是通过封闭TEM8与uPA的相互作用来达到抗肿瘤血管生成的作用。
Angiogenesis is the process new capillary network sprouts from those matureblood vessels that already exist, which is essential for many important physiologicalprocesses including wound healing, embryonic development, and tissue regenerationand remodeling. However, uncontrolled or defective angiogenesis is also present in avariety of pathological processes of diseases including cancer, cardiovascular diseaseand proliferate retinal disease. Therapies that target angiogenesis can be used toterminate or delay the development of these disorders, so this treatment has causedgreat concern in the scientific community. Anti-cancer drugs that target angiogenesisin clinical application, however, have been shown that they have only limited efficacy,but a lot of side effects, so it become more urgent to look for more specific angiogenicmarkers. Tumor endothelial marker8(TEM8) is a type I transmembrane proteinwhich is highly conserved between species and specifically expressed on humantumor vasculature. Later TEM8was found to be a receptor of the anthrax toxinprotective antigen (PA), as an "accomplice" of anthrax toxin, mediating anthrax toxininto the host cell. Research about TEM8physiological functions, however, is laggingbehind a lot, and only a few studies have found TEM8play a key role in thephysiological processes such as cell migration, adhesion, and tumor angiogenesis.Tem8gene knockout mice also demonstrated the TEM8play an important role inpromoting tumor angiogenesis and tumor growth, and have no effect on the normalgrowth and development and wound healing. While the specific distribution as well asdifferences in function between normal and tumor blood vessel formation of TEM8,caused the greatest attention of the scientists who were struggling to find the newtargets of tumor angiogenesis therapies. In a recent preclinical study, antibodiestargeted against the TEM8extracellular domain can inhibit tumor angiogenesis andblock the growth of a variety of human tumor xenografts. This antibody targetingTEM8enhanced the activity of other vascular targeting agents, anti-angiogenic agentsas well as conventional chemotherapy drugs, and found no cytotoxicity.
My group developed an antibody-like molecule in previous work, TEM8-Fc,which is a recombinant fusion protein comprising the extracellular domain of humanTEM8linked to the Fc portion of human IgG1, and found that it was ananti-angiogenic molecule, and could inhibit a variety of tumor cell growth, migrationand angiogenesis. However the mechanism that it suppressed tumor has not been clarified. In order to clarify the tumor suppression mechanism of TEM8-Fc, we firstneed to ascertain the TEM8physiological ligand and the mechanism how they playtheir physiological function. We will first focus our concern in the study of TEM8physiological ligand, because in order to clarify TEM8function under physiologicalconditions and further develop anti-angiogenesis therapy targeting TEM8, we mustfirst make clear its physiological ligand. When uPA was found to be TEM8physiological ligand, we further study the anti-angiogenic mechanism of TEM8-Fcand the mechanism how TEM8play its biological function.In this study we have come to the following conclusions:
1. It was shown that TEM8or CMG2overexpression can enhance the colonyformation ability of HEK293cells in soft agar by soft agar colony formationexperiment, which means that overexpression of TEM8and CMG2can make theHEK293cells acquired the characteristics of malignant transformation. We thenverified TEM8and CMG2physiological functions through CCK8cellproliferation assay and wound-healing assay, and we found that TEM8play a rolein endothelial cell migration, and CMG2in cell proliferation.
2. Using a TEM8-Fc-conjugated Protein A Sepharose affinity chromatography, aprotein band of approximately50kDa was detected in the homogenate of humanmalignant hepatoma tissue that was capable of binding TEM8-Fc. The50kDaband was excised, in-gel digested with trypsin and analyzed by massspectrometry.We found that two possible TEM8interacting protein:the urine typeplasminogen activator (uPA) and alpha-enolase. The uPA is a soluble extracellularsignaling molecule, and however alpha-enolase is present intracellular ormembrane, so we speculate the uPA is TEM8physiological ligand, and thereforeour next work mainly expand around uPA-TEM8interaction. In order to furtherdetermine the interaction between the two molecules,we successively validatetheir interaction with the ELLISA, Western methods, and then determinate thebinding constants between TEM8-Fc and uPA using surface plasmon resonance(SPR) method. The dissociation constants (KD) for binding of TEM8-Fc to highmolecular weight single-chain uPA (HMW-scuPA), high molecular weighttwo-chain uPA (HMW-tcuPA) or low molecular weight uPA (LMW-uPA) were16.6nM,29.7nM and31.1nM, respectively.In addition, we also found thebinding of uPA to TEM8didn’t affect its enzymatic activity through thefibrinogen agarose plate assay and the Chromogenic activity assay.
3. To identify the region in uPA that interacts with TEM8, several truncationmutants of uPA were constructed, all of which were fused to EGFP. A truncatedform of TEM8(the extracellular domain) was also fused to RFP. RFP-taggedTEM8was individually co-transfected into HEK293cells with each of theEGFP-tagged construct FRET analysis was conducted to determine which portionof uPA interacted with the extracellular domain of TEM8in living cells.TheFRET assay results showed binding of uPA to TEM8was mediated by the kringledomain and the C-terminal catalytic domain of uPA. Therefore the interactionbetween TEM8and uPA is distinct from the interaction between the uPA receptor(uPAR) and the EGF-like domain of uPA.
4. We found uPA treatment led to a dose-dependent phosphorylation of TEM8, andhowever, TEM8-Fc could block the phosphorylation of TEM8induced by uPA byimmunoprecipitation method. Then in order to investigate which of the classicalsignaling pathways could be activated following TEM8phosphorylation, aphosphorylation antibody array specifically designed to simultaneouslyinvestigate the relative phosphorylation states of seventy one different humanreceptor tyrosine kinases (RTKs) and an EGFR phosphorylation antibody arrayconsisting of seventeen EGFR phosphorylation sites were performed. We foundincubation of HepG2cells with uPA signifcantly increased the intensity of theTyr845and Tyr1173of EGFR receptor tyrosine kinase phosphorylation, andTEM8-Fc could antagonize this effect to a significant extent. On the other hand,the role of the TEM8/uPA interaction in tumor growth and metastasis wasinvestigated. By using migration assay, we found that uPA can significantlyincrease the uPAR-independent migration of HepG2cells; this activity wasinhibited significantly by TEM8-Fc. Finally, we observed administration ofTEM8-Fc with ATF-Fc yielded synergistic anti-tumor activity.
In summary, our data provide definitive evidence that TEM8is a novel receptorfor uPA, which plays a significant role in the regulation of tumor growth andmetastasis. TEM8-Fc, the antibody-like molecule we developed precisely, achievedthe purpose of anti-angiogenesis by blocking the interaction of TEM8and uPA.
本课题组在前期工作中开发了一种抗体样分子TEM8-Fc （TEM8N端与炭疽毒素保护性抗原PA结合的vWA结构域和IgG1Fc段组成的融合蛋白），发现它是一种具有抗血管生成能力的分子，能抑制多种肿瘤细胞的生长、迁移以及肿瘤血管的生成，然而其抑制肿瘤的机制尚未阐述清楚。为了弄清TEM8-Fc的抑瘤机制，首先需要弄清TEM8的生理性配体及其发挥生理功能的作用机制。本研究将研究焦点首先集中在关于TEM8生理性配体的研究上，因为只有找到它的生理性配体才能进一步阐明它在细胞生理条件下的功能作用，为靶向TEM8的肿瘤抗血管生成疗法提供理论依据。在找到TEM8的生理性配体后，又进一步研究了TEM8-Fc抗肿瘤血管生成的机制以及TEM8发挥生物学功能的机制。
在研究中我们得出了如下结论：
1.通过软琼脂克隆形成实验发现，TEM8和CMG2（特别是TEM8）能够使HEK293细胞的软琼脂克隆形成能力增加，即过表达TEM8或CMG2使HEK293细胞具有了恶性转化的特征。通过CCK8细胞增殖实验和划痕实验等验证了TEM8和CMG2的部分生理功能：TEM8促进细胞迁移，而CMG2促进细胞增殖。
2.利用本课题组前期构建的抗体样分子TEM8-Fc与人肝癌组织匀浆液进行免疫共沉淀和SDS-PAGE电泳，把共沉下的蛋白条带经生物质谱分析发现了TEM8的两个可能的相互作用蛋白——尿型纤溶酶原激活剂（uPA）与α-烯醇化酶（α-enolase）。因为uPA是胞外可溶性的信号分子，而α-enolase主要在胞内和胞膜上发挥功能，所以推测uPA即是TEM8的生理性配体，于是下一步工作主要围绕uPA与TEM8展开。为了进一步确定两者之间的相互作用，我们先后用ELISA、WesternBlot等方法进行了验证，并且通过表面等离子体共振（SPR）的方法测定了TEM8-Fc与uPA的结合常数。TEM8-Fc结合到高分子量单链uPA（HMW-scuPA），高分子量双链uPA（HMW-tcuPA）和低分子量uPA（LMW-uPA）的结合常数分别为16.6nM，29.7nM和31.1nM。另外还通过溶圈实验和S2444发色底物法发现与TEM8的结合不会影响uPA的酶活性。
3.构建了uPA的几种截短突变体与EGFP的融合蛋白表达载体，同时构建了TEM8胞外区（N端）与RFP的融合蛋白表达载体，然后将各种uPA截短突变体与EGFP的融合蛋白表达载体和TEM8胞外区（N端）与RFP的融合蛋白表达载体分别共转染到HEK293细胞中。最后通过FRET的方法确定了uPA与TEM8之间的相互作用区域。FRET的结果显示uPA的Kringle区和酶活性区的C端与TEM8的N端有FRET效应，即介导uPA与TEM8相互作用区域是它的Kringle结构域和催化结构域的C-末端。因此uPA与TEM8相互作用区域不同于uPA受体（uPAR）（uPA的EGF样结构域负责与uPAR相互作用）。
4.用IP的方法证明了uPA能够剂量依赖性的使TEM8磷酸化，而TEM8-Fc可拮抗uPA导致的TEM8磷酸化。通过Raybiotech公司的RTK磷酸化芯片和EGFR磷酸化位点芯片，发现uPA能够上调EGFR845位和1173位酪氨酸的磷酸化水平，而TEM8-Fc能拮抗它的作用。在细胞生物学方面研究了TEM8与uPA的相互作用对肿瘤生长和迁移的作用，通过迁移实验发现uPA能够显著地增加HepG2细胞的uPAR非依赖性的迁移，而TEM8-Fc能够封闭uPA的作用。最后观察到本组开发的两种抗体样分子TEM8-Fc和ATF-Fc具有协同抗肿瘤的作用。
综上所述，本研究结果表明TEM8是uPA一种新型的受体，在调节肿瘤的生长和转移中起到了重要的作用。本组开发的抗体样分子TEM8-Fc正是通过封闭TEM8与uPA的相互作用来达到抗肿瘤血管生成的作用。
Angiogenesis is the process new capillary network sprouts from those matureblood vessels that already exist, which is essential for many important physiologicalprocesses including wound healing, embryonic development, and tissue regenerationand remodeling. However, uncontrolled or defective angiogenesis is also present in avariety of pathological processes of diseases including cancer, cardiovascular diseaseand proliferate retinal disease. Therapies that target angiogenesis can be used toterminate or delay the development of these disorders, so this treatment has causedgreat concern in the scientific community. Anti-cancer drugs that target angiogenesisin clinical application, however, have been shown that they have only limited efficacy,but a lot of side effects, so it become more urgent to look for more specific angiogenicmarkers. Tumor endothelial marker8(TEM8) is a type I transmembrane proteinwhich is highly conserved between species and specifically expressed on humantumor vasculature. Later TEM8was found to be a receptor of the anthrax toxinprotective antigen (PA), as an "accomplice" of anthrax toxin, mediating anthrax toxininto the host cell. Research about TEM8physiological functions, however, is laggingbehind a lot, and only a few studies have found TEM8play a key role in thephysiological processes such as cell migration, adhesion, and tumor angiogenesis.Tem8gene knockout mice also demonstrated the TEM8play an important role inpromoting tumor angiogenesis and tumor growth, and have no effect on the normalgrowth and development and wound healing. While the specific distribution as well asdifferences in function between normal and tumor blood vessel formation of TEM8,caused the greatest attention of the scientists who were struggling to find the newtargets of tumor angiogenesis therapies. In a recent preclinical study, antibodiestargeted against the TEM8extracellular domain can inhibit tumor angiogenesis andblock the growth of a variety of human tumor xenografts. This antibody targetingTEM8enhanced the activity of other vascular targeting agents, anti-angiogenic agentsas well as conventional chemotherapy drugs, and found no cytotoxicity.
My group developed an antibody-like molecule in previous work, TEM8-Fc,which is a recombinant fusion protein comprising the extracellular domain of humanTEM8linked to the Fc portion of human IgG1, and found that it was ananti-angiogenic molecule, and could inhibit a variety of tumor cell growth, migrationand angiogenesis. However the mechanism that it suppressed tumor has not been clarified. In order to clarify the tumor suppression mechanism of TEM8-Fc, we firstneed to ascertain the TEM8physiological ligand and the mechanism how they playtheir physiological function. We will first focus our concern in the study of TEM8physiological ligand, because in order to clarify TEM8function under physiologicalconditions and further develop anti-angiogenesis therapy targeting TEM8, we mustfirst make clear its physiological ligand. When uPA was found to be TEM8physiological ligand, we further study the anti-angiogenic mechanism of TEM8-Fcand the mechanism how TEM8play its biological function.In this study we have come to the following conclusions:
1. It was shown that TEM8or CMG2overexpression can enhance the colonyformation ability of HEK293cells in soft agar by soft agar colony formationexperiment, which means that overexpression of TEM8and CMG2can make theHEK293cells acquired the characteristics of malignant transformation. We thenverified TEM8and CMG2physiological functions through CCK8cellproliferation assay and wound-healing assay, and we found that TEM8play a rolein endothelial cell migration, and CMG2in cell proliferation.
2. Using a TEM8-Fc-conjugated Protein A Sepharose affinity chromatography, aprotein band of approximately50kDa was detected in the homogenate of humanmalignant hepatoma tissue that was capable of binding TEM8-Fc. The50kDaband was excised, in-gel digested with trypsin and analyzed by massspectrometry.We found that two possible TEM8interacting protein:the urine typeplasminogen activator (uPA) and alpha-enolase. The uPA is a soluble extracellularsignaling molecule, and however alpha-enolase is present intracellular ormembrane, so we speculate the uPA is TEM8physiological ligand, and thereforeour next work mainly expand around uPA-TEM8interaction. In order to furtherdetermine the interaction between the two molecules,we successively validatetheir interaction with the ELLISA, Western methods, and then determinate thebinding constants between TEM8-Fc and uPA using surface plasmon resonance(SPR) method. The dissociation constants (KD) for binding of TEM8-Fc to highmolecular weight single-chain uPA (HMW-scuPA), high molecular weighttwo-chain uPA (HMW-tcuPA) or low molecular weight uPA (LMW-uPA) were16.6nM,29.7nM and31.1nM, respectively.In addition, we also found thebinding of uPA to TEM8didn’t affect its enzymatic activity through thefibrinogen agarose plate assay and the Chromogenic activity assay.
3. To identify the region in uPA that interacts with TEM8, several truncationmutants of uPA were constructed, all of which were fused to EGFP. A truncatedform of TEM8(the extracellular domain) was also fused to RFP. RFP-taggedTEM8was individually co-transfected into HEK293cells with each of theEGFP-tagged construct FRET analysis was conducted to determine which portionof uPA interacted with the extracellular domain of TEM8in living cells.TheFRET assay results showed binding of uPA to TEM8was mediated by the kringledomain and the C-terminal catalytic domain of uPA. Therefore the interactionbetween TEM8and uPA is distinct from the interaction between the uPA receptor(uPAR) and the EGF-like domain of uPA.
4. We found uPA treatment led to a dose-dependent phosphorylation of TEM8, andhowever, TEM8-Fc could block the phosphorylation of TEM8induced by uPA byimmunoprecipitation method. Then in order to investigate which of the classicalsignaling pathways could be activated following TEM8phosphorylation, aphosphorylation antibody array specifically designed to simultaneouslyinvestigate the relative phosphorylation states of seventy one different humanreceptor tyrosine kinases (RTKs) and an EGFR phosphorylation antibody arrayconsisting of seventeen EGFR phosphorylation sites were performed. We foundincubation of HepG2cells with uPA signifcantly increased the intensity of theTyr845and Tyr1173of EGFR receptor tyrosine kinase phosphorylation, andTEM8-Fc could antagonize this effect to a significant extent. On the other hand,the role of the TEM8/uPA interaction in tumor growth and metastasis wasinvestigated. By using migration assay, we found that uPA can significantlyincrease the uPAR-independent migration of HepG2cells; this activity wasinhibited significantly by TEM8-Fc. Finally, we observed administration ofTEM8-Fc with ATF-Fc yielded synergistic anti-tumor activity.
In summary, our data provide definitive evidence that TEM8is a novel receptorfor uPA, which plays a significant role in the regulation of tumor growth andmetastasis. TEM8-Fc, the antibody-like molecule we developed precisely, achievedthe purpose of anti-angiogenesis by blocking the interaction of TEM8and uPA.
引文
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