第二型富含半胱胺酸蛋白質在血管平滑肌細胞表現之調節機制以及其調控細胞移動之機轉

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研究生：	陳忠煌 Chen, Chung-Huang
論文名稱：	第二型富含半胱胺酸蛋白質在血管平滑肌細胞表現之調節機制以及其調控細胞移動之機轉 Cysteine-rich Protein 2 Expression in Vascular Smooth Muscle Cells and Its Role in Regulating Cellular Migration
指導教授：	林秀芳 Yet, Shaw-Fang 莊永仁 Chuang, Yung-Jen
口試委員:	陳令儀 Linyi Chen 李秀珠 Shiow-Ju Lee 郭呈欽 Cheng-Chin Kuo
學位類別：	博士 Doctor
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2014
畢業學年度：	103
語文別：	英文
論文頁數：	100
中文關鍵詞：	平滑肌細胞、第二型富含半胱胺酸蛋白質、血管疾病、細胞遷徙、p130Cas蛋白質、轉殖基因小鼠
外文關鍵詞：	VSMC, Cysteine-rich Protein2, Vascular disease, Cell migration, p130 Crk-associated substrate, Transgenic mice
相關次數：	點閱：3 下載：0
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動脈血管壁中層的平滑肌細胞(vascular smooth muscle cells, VSMCs)在血管栓塞病變的過程中扮演著重要的角色。第二型富含半胱胺酸蛋白質(cysteine-rich protein2, CRP2)屬於LIM蛋白族的一員，其蛋白結構含有兩個LIM結構域。CRP2在血管受創病變的過程扮演著重要的角色。CRP2主要會表現在平滑肌細胞，並藉由抑制細胞的遷徙能力而減低了血管栓塞損傷的形成。本論文有兩個主要的研究目的，第一是研究CRP2在VSMCs表現的調控機制，第二是研究CRP2調節VSMC遷徙能力的分子調控機制。
先前的研究已經證實了，在老鼠發育中的血管內CRP2基因的表現必需要有5’端基因啟動子的存在，而在成鼠血管內只有啟動子則是不夠的。而此研究論文的第一個研究目的是要探討CRP2在成鼠血管VSMCs表現的調控機制。我們構築了一系列含有CRP2可能的基因調控序列並帶有乳糖酵素報導基因(LacZ reporter gene)的轉殖載體，並且生產了這一系列轉殖基因小鼠。我們發現CRP2基因的第一內含子(intron1)在成鼠血管是必需的序列，但是在發育中血管則不是必需的。序列分析顯示CRP2 intron1的6.3-kb區段內含有兩個CArG (CC(A/T)6GG)序列，而在凝膠阻滯分析(gel mobility shift assay)以及染色質免疫沉澱分析(chromatin immunoprecipitation)實驗結果發現血清反應因子(SRF)比較趨向與CArG2序列結合，另外也發現SRF的輔因子心肌素(myocardin)及相關分子主要是透過CArG2序列來誘發CRP2的表現。針對不同突變型的轉殖基因老鼠血管研究，我們進一步發現CArG2是調控LacZ報導基因在成鼠血管平滑肌細胞表現的主要調控序列。所以在發育中血管CRP2的表現雖然與CArG序列沒有相關，但是在成鼠血管平滑肌細胞CRP2則需要第一內含子的CArG2序列的存在。總合這部分的研究結果，顯示CRP2在發育中血管與在成鼠血管的VSMCs表現是經由不同的分子機制進行調控。
由之前的研究已經知道缺少CRP2會促使VSMC遷徙能力增加，因而在血管受創傷時會使得血管內膜明顯增生。因此，第二部分的研究目的是要找出CRP2調節VSMCs細胞遷徙的分子機制。在平滑肌細胞內轉殖表現CRP2-EGFP綠色螢光蛋白的結果顯示出CRP2主要是表現在VSMCs內與肌動蛋白相關聯的細胞骨架上，這透露出CRP2具有細胞骨架相關的功能。由我們的實驗發現VSMCs對於不同細胞外基質的貼附能力與延展能力並沒有因為缺乏CRP2而有不同。而CRP2剔除的細胞在化學引誘劑的刺激下會表現出較多且明顯的層狀偽足(lamellipodia)。在CRP2剔除VSMCs重新轉殖表現CRP2之後，化學引誘劑所引發的細胞層狀偽足以及細胞遷徙能力則明顯降低。哺乳動物細胞蛋白質交互作用分析(mammalian two-hybrid assay) 以及免疫共沈澱法(co-immunoprecipitation)的實驗證實了CRP2與細胞遷徙以及偽足形成調控相關的蛋白質p130Cas有交互作用。有趣地，我們發現血管創傷會影響p130Cas的磷酸化程度。另外，進一步地抑制p130Cas的表現或是磷酸化也發現血管受創後內膜增生的情況減弱了，這暗示著p130Cas以及其磷酸化在血管創傷修復扮演著重要的角色。免疫螢光染色分析進一步發現，在靜止的細胞內CRP2與p130Cas會一同表現在細胞骨架纖維束末端的附著點(FAs)。有趣的是，磷酸化p130Cas在遷徙中的細胞內會表現在附著點以及偽足的前緣上，而CRP2則僅會表現於附著點以及骨架纖維束。總而言之，我們的研究結果顯示CRP2會箝制p130Cas在細胞的附著點，因而降低了偽足的形成並且減弱了VSMC的遷徙能力。

Vascular smooth muscle cells (VSMCs) of the arterial wall play a critical role in the development of occlusive vascular lesions. Cysteine-rich protein (CRP) 2, a member of the LIM-only CRP family that contains two LIM domains, plays an important role in vascular remodeling. CRP2 is expressed in VSMCs and functions to reduce vascular lesion formation by inhibiting cellular migration. The goals of this study are (i) to investigate the molecular mechanisms that control CRP2 expression in VSMCs, and (ii) to define the molecular mechanisms by which CRP2 regulates VSMC migration.
We previously demonstrated that the 5’-flanking Csrp2 (gene symbol of the mouse CRP2 gene) promoter is sufficient for gene expression in the developing vessels but not sufficient for adult vasculature. In the present study, the first goal was to elucidate the molecular mechanisms that control CRP2 expression in the adult vasculature. By generating and analyzing a series of transgenic mice harboring potential Csrp2 regulatory regions with a lacZ reporter, we determined that the 12-kb first intron was necessary for transgene activity in adult but not in developing vasculature. Within the intron we identified a 6.3-kb region that contains 2 CArG boxes (CC(A/T)6GG). Serum response factor (SRF) preferentially bound to CArG2 box in gel mobility shift and chromatin immunoprecipitation assays; additionally, SRF coactivator myocardin and the related factors activated CRP2 expression via the CArG2 box. Mutational analysis revealed that CArG2 box was important in directing lacZ expression in VSMCs of adult vessels. Although CRP2 expression during development is independent of CArG box regulatory sites, CRP2 expression in adult VSMCs requires CArG2 element within the first intron. Our results suggest that distinct mechanisms regulate CRP2 expression in VSMCs that are controlled by separate embryonic and adult regulatory modules.
Given that an absence of CRP2 enhances VSMC migration and increases neointima formation following arterial injury, the second specific goal of this study was to define the molecular mechanisms by which CRP2 regulates VSMC migration. Transfection of VSMCs with CRP2-EGFP constructs revealed that CRP2 is associated with the actin cytoskeleton, suggesting a cytoskeletal function of CRP2. Lack of CRP2 did not affect cell’s ability to adhere to or spread on extracellular matrix. In response to chemoattractant stimulation, Csrp2-deficient (Csrp2–/–) VSMCs exhibited increased lamellipodia formation. Re-introduction of CRP2 abrogated the enhanced lamellipodia formation and migration of Csrp2–/– VSMCs following chemoattractant stimulation. Mammalian two-hybrid and co-immunoprecipitation assays demonstrated that CRP2 interacts with p130Cas, a scaffold protein important for lamellipodia formation and cell motility. Intriguingly, vascular injury modulated p130Cas phosphorylation levels. Furthermore, suppression of p130Cas expression or its phosphorylation attenuated neointima formation following arterial injury, suggesting an important role of p130Cas and its phosphorylation in vascular remodeling. Immunofluorescence staining showed that CRP2 colocalized with phospho-p130Cas at focal adhesions (FAs)/terminal ends of stress fibers in non-migrating cells. Interestingly, in migrating cells phospho-p130Cas localized to the leading edge of lamellipodia and FAs, whereas CRP2 was restricted to FAs and stress fibers. Taken together, our results indicate that CRP2 sequesters p130Cas at FAs, thereby reducing lamellipodia formation and blunting VSMC migration.

Abstract    1
中文摘要    3
Abbreviations    5
Chapter 1. General Introduction    6
1.1 Vascular smooth muscle cells    7
1.2 Cysteine-rich protein 2    8
1.2.1 LIM protein family    8
1.2.2 Cysteine-rich protein family    8
1.2.3 Cysteine-rich protein 2    9
1.3 p130Cas (Crk-associated substrate)    12
1.4 Objectives    13
Chapter 2. Material and Methods    16
2.1 Transgenic Mice    17
2.2 Cell Culture and Transient Transfection Assays    18
2.3 Construction of Expression Plasmids    19
2.3.1 Luciferase reporter plasmids    19
2.3.2 CRP2 expression plasmids    20
2.4 Electrophoretic Mobility Shift Assays    21
2.5 Chromatin Immunoprecipitation Assays    21
2.6 Adhesion, Spreading, and Migration Assays    22
2.7 Immunofluorescence    24
2.8 Mammalian Two-hybrid Assays    25
2.9 Mouse Vascular Remodeling Model and Histological Analysis    25
2.10 Coimmunoprecipitation    27
2.11 Statistical Analysis    27
Chapter 3. Intronic CArG Box Regulates Cysteine-rich Protein 2 Expression in the Adult but Not in Developing Vasculature    28
3.1 Introduction    29
3.2 Results    30
3.2.1 Csrp2 intron 1 is required for lacZ transgene expression in VSMCs of adult blood vessels    30
3.2.2 Two CArG elements are present in intron 1 of the Csrp2 gene    31
3.2.3 SRF coactivators upregulate Csrp2 transcription via intron 1 sequences in VSMCs    33
3.2.4 Intronic CArG elements are important for lacZ transgene expression in VSMCs of adult vessels     34
3.2.5 CArG2 is important for Csrp2 expression in adult blood vessels     35
3.3 Discussion    36
Chapter 4. Cysteine-rich Protein 2 Alters p130Cas Localization and Inhibits Vascular Smooth Muscle Cell Migration    40
4.1 Introduction    41
4.2 Results    43
4.2.1 Effects of CRP2 on cellular adhesion, spreading, and lamellipodia formation    43
4.2.2 CRP2 associates with actin stress fibers in VSMCs    44
4.2.3 Restoring CRP2 expression in Csrp2–/– VSMCs inhibits lamellipodia formation and migration following PDGF-BB stimulation    45
4.2.4 CRP2 interacts with scaffold molecule p130Cas    46
4.2.5 Phospho-p130Cas expression in the normal and injured arterial wall    47
4.2.6 PDGF-BB stimulated Csrp2–/– VSMCs exhibit increased p-p130Cas at the leading edges of lamellipodia    48
4.2.7 CRP2 colocalizes with p-p130Cas at FAs but not at leading edges of lamellipodia in migratory VSMCs    48
4.2.8 The substrate domain of p130Cas is required for CRP2 interaction    50
4.3 Discussion    50
Chapter 5. Conclusions and Perspectives    55
References    58
Tables    70
Table  I. Summary of Csrp2-lacZ transgenic mice    70
Figures    71
Figure  1. Csrp2 intron 1 is required for lacZ transgene expression in adult blood vessels    71
Figure  2. SRF binds to CArG1 and CArG2 elements of Csrp2 intron 1 in electrophoretic mobility shift assays    73
Figure  3. SRF preferentially binds to CArG2 element of Csrp2 intron 1 in Chromatin immunoprecipitation assays    74
Figure  4. The first intron of the Csrp2 gene contains SRF/CArG-dependent activity    75
Figure  5. SRF coactivators contribute to Csrp2 expression via intronic sequences in VSMCs     77
Figure  6. Csrp2 intronic CArG are important for transgene expression in adult vessels    79
Figure  7. CArG2 of Csrp2 intron 1 is essential for lacZ transgene expression in adult blood vessels    80
Figure  8. Whole mount -galactosidase staining of major arteries in adult –825Int1-Csrp2-lacZ and CArG mutant transgenic mice from different founder lines    81
Figure  9. CRP2 on VSMC adhesion, spreading, and lamellipodia formation     83
Figure 10. CRP2 is associated with actin stress fibers of vascular smooth muscle cells    85
Figure 11. Re-expression of CRP2 in Csrp2–/– VSMCs inhibits PDGF-BB-induced   lamellipodia formation and migration     87
Figure 12. CRP2 interacts with p130Cas by mammalian two-hybrid assays    89
Figure 13. The role of p130Cas in vascular remodeling    91
Figure 14. Suppression of p130Cas expression levels and inhibition of its phosphorylation    92
Figure 15. Subcellular localization of p-p130Cas in non-migrating and migratory Csrp2+/+ and Csrp2–/– VSMCs    93
Figure 16. Colocalization of p-p130Cas and paxillin at focal adhesions in VSMCs    96
Figure 17. CRP2 colocalizes with p-p130Cas at FAs but not leading edge of lamellipodia in VSMCs    97
Figure 18. The substrate domain of p130Cas is required for interaction with CRP2    99


                                

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