小鼠KyoT家族的新异构发现及其对血管内皮细胞的影响
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摘要
血管生成是多数情况下血管新生的重要方式——从创伤愈合到特定性生理周期（女性月经周期），从多种人类疾病的发生到肿瘤的生长和转移，血管生成参与众多重要生理性和病理性过程。
血管生成是由多步骤组成的复杂过程：首先细胞外基质（Extracellularmatrix, ECM）发生降解，接着血管内皮细胞（Endothelial cells, ECs）从已有血管中出芽，随后ECs经历增殖、迁移、分化而形成血管，最后平滑肌细胞被募集至新生的血管上。ECs不仅是构成血管的重要组成部分，更是血管生成过程重要的“执行者”。
近年来随着体外血管生成体系的应用、体内基因剔除/敲入小鼠模型的建立，对血管生成机制的研究也不断深入，发现多条信号途径在细胞、分子水平调控血管生成和ECs功能。Notch信号途径因参与调控ECs的多种功能，决定ECs的命运，而成为备受关注的调控信号。
Notch信号途径是进化中高度保守的、通过细胞与细胞之间直接接触而激活的信号途径，参与调控包括细胞增殖、分化、凋亡、命运决定等多种重要过程。当相邻细胞间Notch配体与其受体接触时，Notch受体的胞内段（Notch intracellular domain, NIC）就会释放进入细胞核，与核内DNA结合蛋白——重组信号结合蛋白-J（Recombination signal binding protein-Jκ,RBP-J）结合，从而激活下游基因的转录。在缺少NIC的情况下，RBP-J则通过募集多种转录共抑制分子而发挥抑制转录的作用。本室前期研究发现：RBP-J可通过与LIM蛋白KyoT2结合而募集多种转录共抑制分子。但是，KyoT是由选择性拼接形成的分子亚家族，与RBP-J结合的只有KyoT2吗？是否还有其它KyoT分子能和RBP-J结合？这种结合对Notch信号途径有什么样的调控作用？对ECs的功能又有怎样的影响？
基于上述设想，本课题发现了KyoT另一个剪接变异体KyoT3，深入研究了与RBP-J结合的LIM蛋白家族KyoT3的组织分布与细胞定位，寻找其与RBP-J结合的直接证据，阐明KyoT3对Notch信号途径的调控作用，探究KyoT家族成员对ECs的功能的影响。主要研究结果如下：
1.发现了KyoT家族的新异构体。
首先，通过以小鼠胚胎cDNA文库为模板，经聚合酶链反应（Polymerasechain reaction, PCR）获得KyoT家族另一剪接变异体，KyoT3。KyoT3，其全长为969bp，编码包含323个氨基酸的大小为36.26KD的蛋白质。KyoT3的N-端是与KyoT1相同的3个半LIM结构域，在LIM结构域之后为KyoT1所不具备的3个核定位信号（Nuclear localization signals, NLS）和1个出核序列（Nuclear export sequence, NES）。与KyoT2相同的是：KyoT3也拥有由C-端27个氨基酸构成的RBP-J结合基序，这提示KyoT3可能也具有与KyoT2相似的功能。利用位于KyoT3序列上的酶切位点XhoⅠ，将KyoT3分为前后2段，通过对2段分别扩增后再拼接的方式，在不改变KyoT3序列、不引入新碱基的情况下，成功构建pMD18-T-KyoT3，为进一步研究KyoT3的功能打下基础。
其次，探明了KyoT3在组织中的分布和细胞内的定位，并确认其在细胞核内的定位依赖于其NLS。提取小鼠不同组织器官的RNA，经过反转录为cDNA之后，采用KyoT3特异性引物，通过PCR的方法研究KyoT3在组织内的分布，发现KyoT3的mRNA在小鼠脾脏、胸腺、睾丸、卵巢、小肠、结肠、心脏、大脑、胎盘、肺脏、肝脏、骨骼肌、肾脏和胰腺都有表达，说明其组织分布十分广泛。为明确KyoT3在细胞内的定位，构建了含KyoT3全长的pEGFP-C2-KyoT3和pEGFP-N1-KyoT3；仅含NLS的pEGFP-C2-NLS和pEGFP-N1-NLS；不含有NLS的pEGFP-C2-KyoT3N和pEGFP-N1-KyoT3N质粒。Western blot的方法发现其中C2-KyoT3N的表达量很低后，通过其它5种质粒分别转染HeLa细胞，而确定了KyoT3主要分布于细胞核内，并且KyoT3在细胞核内的定位是依赖于其3个串联的NLS实现的。
最后，通过免疫共沉淀验证了KyoT3与RBP-J之间的相互作用，并进一步明确了KyoT3具有抑制RBP-J依赖的转录的作用。构建了带Myc标签的pCMV-Myc-KyoT3真核表达质粒，通过与带Flag标签的pCMV-RBP-J-Flag共转染HeLa细胞的方式，采用免疫共沉淀实验，利用不同的抗体检测，确认了KyoT3和RBP-J之间存在物理相互作用。随即使用双荧光报告基因系统，在HeLa细胞和HEK293细胞内证实KyoT3具有抑制RBP-J依赖的转录的作用，并且这种作用具有剂量依赖效应。此外，通过将KyoT3与NIC质粒共转染HeLa细胞，24h后用实时定量PCR的方法检测下游基因Hes-1的mRNA水平的方法，结果也表明：共转染KyoT3和NIC时，KyoT3能显著抑制NIC激活的Hes-1的转录。
2.阐明KyoT家族成员对血管内皮细胞的影响。
首先，成功分离和培养人脐静脉内皮细胞（Human umbilical veinendothelial cells, HUVECs），在其中检测到KyoT家族成员KyoT2的表达。为研究在血管生成中KyoT家族成员的作用，在本室建立了HUVECs的分离、培养的方法，并通过其形态表现为典型的铺路石样、表面CD31分子表达平均约为99%、具有形成管腔的能力，对分离、培养的细胞进行了确认。通过PCR的方法检测HUVECs中KyoT家族成员的表达。结果发现：在HUVECs中仅KyoT2表达，于是将研究聚焦于KyoT2。
其次，发现转染KyoT2能使HUVECs细胞系（HUVEC Cell line,HUVEC-CL）中管腔形成增多，tip细胞的数目增加，细胞增殖减少。在后续的实验中，使用脂质体LTXPLUS瞬时转染EGFP-KyoT2于HUVECs和HUVEC-CL，通过计数绿色细胞总数的方法，发现：无论在HUVECs或是HUVEC-CL中，与转染EGFP的对照组相比，转染KyoT2后细胞增殖减弱，然而转染KyoT2却能使HUVEC-CL中管腔形成增多，tip细胞的数目增加。
通过本课题的研究证实：KyoT另一剪接变异体KyoT3的存在，并明确了其在组织中的分布和细胞内的定位，并且KyoT3在细胞核内的定位是依赖于其3个串联的NLS的。KyoT3能够与RBP-J发生物理上的相互作用，也因此参与了RBP-J介导的Notch信号途径的调控。KyoT3能够抑制RBP-J介导的转录激活，并且这种抑制作用具有剂量依赖效应。为研究KyoT家族成员在血管生成中的作用，在本室建立了HUVECs的分离与培养方法，通过该方法能够获得纯度高、功能好的HUVECs，作为研究ECs的模型。使用PCR的方法，检测到仅KyoT2在HUVECs内的表达，因此也将研究重心转移到KyoT2对ECs的功能影响。进一步研究发现瞬时转染KyoT2能够抑制ECs增殖，促使管腔形成增多，tip细胞的数目增加。为更深入地研究KyoT2在血管生成过程中的作用奠定基础。
Angiogenesis, a critical pattern of new blood vessel formation has beenacknowledged as a main process from wound healing to certain physiologicalprocesses. Meanwhile, angiogenesis also participates in the pathological processof several human diseases and plays a pivotal role in tumor growth andmetastasis.
Angiogenesis is a complex process of many stages: Endothelial cells (ECs)sprout from pre-existing vessel after extracellular matrix (ECM) degrade.Subsequently, ECs undergo proliferation, migration, differentiation and recruitpericytes/smooth muscle cells. ECs are important components of blood vessel aswell as key element of angiogenesis.
With the extensive use of angiogenesis assays in vitro and the intensiveanalysis of mouse knock-in/knock-out model in vivo, the mechanism ofangiogenesis has been investigated more intensively. Scientists have found manysignaling pathways which regulate angiogenesis process and the function of ECs at cellular and molecular level. One of them is an evolutionarily conservedpathway, which is named Notch signaling pathway.
Notch signaling pathway is used by metazoans to control a broad range ofdevelopmental processes, including cell fate determination, differentiation,proliferation and apoptosis through local cell-cell interactions. When ligand insignal sending cell contacts its receptor in signal receiving cell, the intracellulardomain of Notch (NIC) is released into the nucleus and binds to an DNA bindingprotein RBP-J, which hence starts the transcription of downstream genes. On thecontrary, when NIC is absent, RBP-J recruits many co-repressors to repress thetranscription of these genes. In our previous studies we have found that RBP-Jrecruits many co-repressors through binding KyoT2.
Due to the fact that KyoT2belongs to the KyoT family which has splicingvariants, two hypotheses have been proposed:1) Whether there is anothermember of KyoT family, which could bind RBP-J?2) What kind of role does themember of KyoT family play in the process of angiogenesis and the function ofECs through these kinds of binding? Based on these questions, we found in thisproject another splicing variant of KyoT family beyond KyoT1and KyoT2,named KyoT3. The main results are as following:
1. Anew isoform of KyoT family, named KyoT3has been found.
Firstly, the full length of KyoT3has been obtained and the plasmid hasbeen constructed which containing the full length sequence of KyoT3. Thefull length cDNA of KyoT3were amplified from a murine embryonic cDNAlibrary by PCR. The murine KyoT3is969bp in length, encoding a polypeptideof323amino acids with a predicted molecular mass of36.26kDa. KyoT3has thesame3and a half LIM domain as KyoT1in its N-terminal, but it possesses the3tandem NLS and1NES which are located behind the LIM domain and are absent in KyoT1. As KyoT2, KyoT3has the same RBP-J binding motif of about27amino acids in its C-terminal, suggesting similar functions of these two isoforms.Joining the2fragments of KyoT3which are separated by the XhoⅠsite, thefull-length cDNAof KyoT3is obtained without changing a single base pair.
Secondly, the tissue distribution pattern and the cellular localization ofKyoT3has been detected. That the localization in the nucleus of KyoT3depends on its NLS has been further verified. The expression pattern ofKyoT3was examined by RT-PCR using specific PCR primers. The results showthat KyoT3distributes extensively in murine tissue, including spleen, thymus,testis, ovary, small intestine, colon, heart, brain, placenta, lung, liver, skeletalmuscle, kidney and pancreas. To further study its intracellular localization,plasmids of different structure containing different sequence of KyoT3wereconstructed as pEGFP-C2-KyoT3, pEGFP-N1-KyoT3, pEGFP-C2-NLS,pEGFP-N1-NLS, pEGFP-C2-KyoT3N and pEGFP-N1-KyoT3N. The expressionlevel of pEGFP-C2-KyoT3N was detected as very low through Western blotanalysis. Then by transfecting other5plasmids, that the nuclear localization ofKyoT3and the localization in the nucleus of KyoT3via its NLS have been found.
Thirdly, the physical interaction between KyoT3and RBP-J throughCo-ip has been proved. And the role of KyoT3in repressing RBP-Jdependent transcription has been further verified. pCMV-Myc-KyoT3wasconstructed by inserting full-length KyoT3into the pCMV-Myc vector. HeLacells were transfected by this plasmid and pCMV-RBP-J-Flag together.Co-precipitated proteins were analyzed by SDS-PAGE followed by Westernblotting which used the anti-Flag or the anti-Myc antibody, respectively. Theresult show that KyoT3could interact with RBP-J. Further studies usingdual-luciferase reporter assay were conducted in both HeLa and HEK293cells verifying that KyoT3inhibits the transactivation of RBP-J-dependent promoter.Real-time PCR method has been adopted to test the downstream gene of Notchsignaling pathway in HeLa cell which transfected with different combination ofNIC and KyoT3. The result shows that co-transfection with KyoT3could inhibitthe NIC-induced Hes-1up-regulation.
2. The role of KyoT family in regulating ECs function has been illustrated.
Firstly, HUVECs were isolated and cultured from the human umbilicalvein. The identity of the cells was conferred by its morphic character, expressionof CD31, and the ability of tube formation. Through RT-PCR, only KyoT2amongKyoT family is expressed in isolated HUVECs.
Secondly, comparing to the control transfection, the total number oftransfected cells decrease significantly while the tube formation and the tipcell number increase in cells transfection of KyoT2. LTXPLUSwas firstly usedto transfect EGFP-KyoT2or EGFP only into the HUVEC and HUVEC-CL, thetransfected cell number was counted by FACS, the proliferation of the cellstransfected with EGFP-KyoT2decline comparing to the cells transfected withEGFP. Simultaneously, the tube formation and the tip cell number increase inHUVEC-CL transfected with EGFP-KyoT2.
Conclusion
Current studies shows that Notch signaling pathway is critical in angiogenesis.Many components of Notch signaling pathway play a role in regulatingangiogenesis. The Notch signaling itself is a "complex" signaling pathwaybecause many components participate in regulating this pathyway, including anew member---an isform of KyoT family existing in the murine named KyoT3.This protein distributes extensively in many tissues and mainly locates in thenucleus. KyoT3could bind RBP-J and hence plays a role in inhibiting the RBP-J-dependent transcription. Interestingly, there is only KyoT2expressed inHUVECs, and it could inhibit proliferation as well as promotes tube formationand tip cell number of ECs.
血管生成是由多步骤组成的复杂过程：首先细胞外基质（Extracellularmatrix, ECM）发生降解，接着血管内皮细胞（Endothelial cells, ECs）从已有血管中出芽，随后ECs经历增殖、迁移、分化而形成血管，最后平滑肌细胞被募集至新生的血管上。ECs不仅是构成血管的重要组成部分，更是血管生成过程重要的“执行者”。
近年来随着体外血管生成体系的应用、体内基因剔除/敲入小鼠模型的建立，对血管生成机制的研究也不断深入，发现多条信号途径在细胞、分子水平调控血管生成和ECs功能。Notch信号途径因参与调控ECs的多种功能，决定ECs的命运，而成为备受关注的调控信号。
Notch信号途径是进化中高度保守的、通过细胞与细胞之间直接接触而激活的信号途径，参与调控包括细胞增殖、分化、凋亡、命运决定等多种重要过程。当相邻细胞间Notch配体与其受体接触时，Notch受体的胞内段（Notch intracellular domain, NIC）就会释放进入细胞核，与核内DNA结合蛋白——重组信号结合蛋白-J（Recombination signal binding protein-Jκ,RBP-J）结合，从而激活下游基因的转录。在缺少NIC的情况下，RBP-J则通过募集多种转录共抑制分子而发挥抑制转录的作用。本室前期研究发现：RBP-J可通过与LIM蛋白KyoT2结合而募集多种转录共抑制分子。但是，KyoT是由选择性拼接形成的分子亚家族，与RBP-J结合的只有KyoT2吗？是否还有其它KyoT分子能和RBP-J结合？这种结合对Notch信号途径有什么样的调控作用？对ECs的功能又有怎样的影响？
基于上述设想，本课题发现了KyoT另一个剪接变异体KyoT3，深入研究了与RBP-J结合的LIM蛋白家族KyoT3的组织分布与细胞定位，寻找其与RBP-J结合的直接证据，阐明KyoT3对Notch信号途径的调控作用，探究KyoT家族成员对ECs的功能的影响。主要研究结果如下：
1.发现了KyoT家族的新异构体。
首先，通过以小鼠胚胎cDNA文库为模板，经聚合酶链反应（Polymerasechain reaction, PCR）获得KyoT家族另一剪接变异体，KyoT3。KyoT3，其全长为969bp，编码包含323个氨基酸的大小为36.26KD的蛋白质。KyoT3的N-端是与KyoT1相同的3个半LIM结构域，在LIM结构域之后为KyoT1所不具备的3个核定位信号（Nuclear localization signals, NLS）和1个出核序列（Nuclear export sequence, NES）。与KyoT2相同的是：KyoT3也拥有由C-端27个氨基酸构成的RBP-J结合基序，这提示KyoT3可能也具有与KyoT2相似的功能。利用位于KyoT3序列上的酶切位点XhoⅠ，将KyoT3分为前后2段，通过对2段分别扩增后再拼接的方式，在不改变KyoT3序列、不引入新碱基的情况下，成功构建pMD18-T-KyoT3，为进一步研究KyoT3的功能打下基础。
其次，探明了KyoT3在组织中的分布和细胞内的定位，并确认其在细胞核内的定位依赖于其NLS。提取小鼠不同组织器官的RNA，经过反转录为cDNA之后，采用KyoT3特异性引物，通过PCR的方法研究KyoT3在组织内的分布，发现KyoT3的mRNA在小鼠脾脏、胸腺、睾丸、卵巢、小肠、结肠、心脏、大脑、胎盘、肺脏、肝脏、骨骼肌、肾脏和胰腺都有表达，说明其组织分布十分广泛。为明确KyoT3在细胞内的定位，构建了含KyoT3全长的pEGFP-C2-KyoT3和pEGFP-N1-KyoT3；仅含NLS的pEGFP-C2-NLS和pEGFP-N1-NLS；不含有NLS的pEGFP-C2-KyoT3N和pEGFP-N1-KyoT3N质粒。Western blot的方法发现其中C2-KyoT3N的表达量很低后，通过其它5种质粒分别转染HeLa细胞，而确定了KyoT3主要分布于细胞核内，并且KyoT3在细胞核内的定位是依赖于其3个串联的NLS实现的。
最后，通过免疫共沉淀验证了KyoT3与RBP-J之间的相互作用，并进一步明确了KyoT3具有抑制RBP-J依赖的转录的作用。构建了带Myc标签的pCMV-Myc-KyoT3真核表达质粒，通过与带Flag标签的pCMV-RBP-J-Flag共转染HeLa细胞的方式，采用免疫共沉淀实验，利用不同的抗体检测，确认了KyoT3和RBP-J之间存在物理相互作用。随即使用双荧光报告基因系统，在HeLa细胞和HEK293细胞内证实KyoT3具有抑制RBP-J依赖的转录的作用，并且这种作用具有剂量依赖效应。此外，通过将KyoT3与NIC质粒共转染HeLa细胞，24h后用实时定量PCR的方法检测下游基因Hes-1的mRNA水平的方法，结果也表明：共转染KyoT3和NIC时，KyoT3能显著抑制NIC激活的Hes-1的转录。
2.阐明KyoT家族成员对血管内皮细胞的影响。
首先，成功分离和培养人脐静脉内皮细胞（Human umbilical veinendothelial cells, HUVECs），在其中检测到KyoT家族成员KyoT2的表达。为研究在血管生成中KyoT家族成员的作用，在本室建立了HUVECs的分离、培养的方法，并通过其形态表现为典型的铺路石样、表面CD31分子表达平均约为99%、具有形成管腔的能力，对分离、培养的细胞进行了确认。通过PCR的方法检测HUVECs中KyoT家族成员的表达。结果发现：在HUVECs中仅KyoT2表达，于是将研究聚焦于KyoT2。
其次，发现转染KyoT2能使HUVECs细胞系（HUVEC Cell line,HUVEC-CL）中管腔形成增多，tip细胞的数目增加，细胞增殖减少。在后续的实验中，使用脂质体LTXPLUS瞬时转染EGFP-KyoT2于HUVECs和HUVEC-CL，通过计数绿色细胞总数的方法，发现：无论在HUVECs或是HUVEC-CL中，与转染EGFP的对照组相比，转染KyoT2后细胞增殖减弱，然而转染KyoT2却能使HUVEC-CL中管腔形成增多，tip细胞的数目增加。
通过本课题的研究证实：KyoT另一剪接变异体KyoT3的存在，并明确了其在组织中的分布和细胞内的定位，并且KyoT3在细胞核内的定位是依赖于其3个串联的NLS的。KyoT3能够与RBP-J发生物理上的相互作用，也因此参与了RBP-J介导的Notch信号途径的调控。KyoT3能够抑制RBP-J介导的转录激活，并且这种抑制作用具有剂量依赖效应。为研究KyoT家族成员在血管生成中的作用，在本室建立了HUVECs的分离与培养方法，通过该方法能够获得纯度高、功能好的HUVECs，作为研究ECs的模型。使用PCR的方法，检测到仅KyoT2在HUVECs内的表达，因此也将研究重心转移到KyoT2对ECs的功能影响。进一步研究发现瞬时转染KyoT2能够抑制ECs增殖，促使管腔形成增多，tip细胞的数目增加。为更深入地研究KyoT2在血管生成过程中的作用奠定基础。
Angiogenesis, a critical pattern of new blood vessel formation has beenacknowledged as a main process from wound healing to certain physiologicalprocesses. Meanwhile, angiogenesis also participates in the pathological processof several human diseases and plays a pivotal role in tumor growth andmetastasis.
Angiogenesis is a complex process of many stages: Endothelial cells (ECs)sprout from pre-existing vessel after extracellular matrix (ECM) degrade.Subsequently, ECs undergo proliferation, migration, differentiation and recruitpericytes/smooth muscle cells. ECs are important components of blood vessel aswell as key element of angiogenesis.
With the extensive use of angiogenesis assays in vitro and the intensiveanalysis of mouse knock-in/knock-out model in vivo, the mechanism ofangiogenesis has been investigated more intensively. Scientists have found manysignaling pathways which regulate angiogenesis process and the function of ECs at cellular and molecular level. One of them is an evolutionarily conservedpathway, which is named Notch signaling pathway.
Notch signaling pathway is used by metazoans to control a broad range ofdevelopmental processes, including cell fate determination, differentiation,proliferation and apoptosis through local cell-cell interactions. When ligand insignal sending cell contacts its receptor in signal receiving cell, the intracellulardomain of Notch (NIC) is released into the nucleus and binds to an DNA bindingprotein RBP-J, which hence starts the transcription of downstream genes. On thecontrary, when NIC is absent, RBP-J recruits many co-repressors to repress thetranscription of these genes. In our previous studies we have found that RBP-Jrecruits many co-repressors through binding KyoT2.
Due to the fact that KyoT2belongs to the KyoT family which has splicingvariants, two hypotheses have been proposed:1) Whether there is anothermember of KyoT family, which could bind RBP-J?2) What kind of role does themember of KyoT family play in the process of angiogenesis and the function ofECs through these kinds of binding? Based on these questions, we found in thisproject another splicing variant of KyoT family beyond KyoT1and KyoT2,named KyoT3. The main results are as following:
1. Anew isoform of KyoT family, named KyoT3has been found.
Firstly, the full length of KyoT3has been obtained and the plasmid hasbeen constructed which containing the full length sequence of KyoT3. Thefull length cDNA of KyoT3were amplified from a murine embryonic cDNAlibrary by PCR. The murine KyoT3is969bp in length, encoding a polypeptideof323amino acids with a predicted molecular mass of36.26kDa. KyoT3has thesame3and a half LIM domain as KyoT1in its N-terminal, but it possesses the3tandem NLS and1NES which are located behind the LIM domain and are absent in KyoT1. As KyoT2, KyoT3has the same RBP-J binding motif of about27amino acids in its C-terminal, suggesting similar functions of these two isoforms.Joining the2fragments of KyoT3which are separated by the XhoⅠsite, thefull-length cDNAof KyoT3is obtained without changing a single base pair.
Secondly, the tissue distribution pattern and the cellular localization ofKyoT3has been detected. That the localization in the nucleus of KyoT3depends on its NLS has been further verified. The expression pattern ofKyoT3was examined by RT-PCR using specific PCR primers. The results showthat KyoT3distributes extensively in murine tissue, including spleen, thymus,testis, ovary, small intestine, colon, heart, brain, placenta, lung, liver, skeletalmuscle, kidney and pancreas. To further study its intracellular localization,plasmids of different structure containing different sequence of KyoT3wereconstructed as pEGFP-C2-KyoT3, pEGFP-N1-KyoT3, pEGFP-C2-NLS,pEGFP-N1-NLS, pEGFP-C2-KyoT3N and pEGFP-N1-KyoT3N. The expressionlevel of pEGFP-C2-KyoT3N was detected as very low through Western blotanalysis. Then by transfecting other5plasmids, that the nuclear localization ofKyoT3and the localization in the nucleus of KyoT3via its NLS have been found.
Thirdly, the physical interaction between KyoT3and RBP-J throughCo-ip has been proved. And the role of KyoT3in repressing RBP-Jdependent transcription has been further verified. pCMV-Myc-KyoT3wasconstructed by inserting full-length KyoT3into the pCMV-Myc vector. HeLacells were transfected by this plasmid and pCMV-RBP-J-Flag together.Co-precipitated proteins were analyzed by SDS-PAGE followed by Westernblotting which used the anti-Flag or the anti-Myc antibody, respectively. Theresult show that KyoT3could interact with RBP-J. Further studies usingdual-luciferase reporter assay were conducted in both HeLa and HEK293cells verifying that KyoT3inhibits the transactivation of RBP-J-dependent promoter.Real-time PCR method has been adopted to test the downstream gene of Notchsignaling pathway in HeLa cell which transfected with different combination ofNIC and KyoT3. The result shows that co-transfection with KyoT3could inhibitthe NIC-induced Hes-1up-regulation.
2. The role of KyoT family in regulating ECs function has been illustrated.
Firstly, HUVECs were isolated and cultured from the human umbilicalvein. The identity of the cells was conferred by its morphic character, expressionof CD31, and the ability of tube formation. Through RT-PCR, only KyoT2amongKyoT family is expressed in isolated HUVECs.
Secondly, comparing to the control transfection, the total number oftransfected cells decrease significantly while the tube formation and the tipcell number increase in cells transfection of KyoT2. LTXPLUSwas firstly usedto transfect EGFP-KyoT2or EGFP only into the HUVEC and HUVEC-CL, thetransfected cell number was counted by FACS, the proliferation of the cellstransfected with EGFP-KyoT2decline comparing to the cells transfected withEGFP. Simultaneously, the tube formation and the tip cell number increase inHUVEC-CL transfected with EGFP-KyoT2.
Conclusion
Current studies shows that Notch signaling pathway is critical in angiogenesis.Many components of Notch signaling pathway play a role in regulatingangiogenesis. The Notch signaling itself is a "complex" signaling pathwaybecause many components participate in regulating this pathyway, including anew member---an isform of KyoT family existing in the murine named KyoT3.This protein distributes extensively in many tissues and mainly locates in thenucleus. KyoT3could bind RBP-J and hence plays a role in inhibiting the RBP-J-dependent transcription. Interestingly, there is only KyoT2expressed inHUVECs, and it could inhibit proliferation as well as promotes tube formationand tip cell number of ECs.
引文
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