The role of GRIM 19 in xenopus embryo development

THE ROLE OF GRIM-19 IN XENOPUS EMBRYO DEVELOPMENT CHEN YONG (M.Med Wuhan Univ.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY INSTITUTE OF MOLECULAR AND CELL BIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2006 Acknowledgements Acknowledgments I would like to express my sincere gratitude to my supervisor, Dr Xinmin Cao, for providing me with the opportunity to pursue my Ph.D research work in her laboratory I am grateful to Dr Xinmin Cao for her guidance and support throughout my graduate studies I am thankful to my graduate supervisory committee, Drs Alan G Porter, Walter Hunziker and Yun-jin Jiang for their constructive suggestions and critical comments I would especially like to thank Dr Jianlin Fu, Wai Hong Yuen and all the other staff in the transgenic frog facility for providing excellent technical support and an ideal working environment for animal model generation and phenotype analysis I also thank Ke Guo, Jie Li and Zeng Qi for histological analysis, and Chee Peng Ng for EM I am grateful to Drs Alirio J Melendez and Farazeela Bte Mohod Ibrahim in the Department of Physiology, National University of Singapore, and Dr Andrew L Miller in the Department of Biology, Hong Kong University of Science and Techlology, for helpful discussion, technical assistance and collaboration in the area of calcium signaling I also thank Dr Katherine E Yutzey in the Children’s Hospital Research Foundation, Cincinnati, OH, for providing Nkx 2.5 promoter constructs Thanks also go to all past and present members of the CXM laboratory for their discussion, good suggestions, technical assistance and friendship I am deeply grateful to Xing Chen and John Tng for their critical comments on my thesis writing Finally, my deepest appreciation goes to my parents and my wife for their consistent love, support and encouragement through out the years i List of publications List of Publications Chen Y, Yuen W., Fu J., Huang G., Melendez A J., Ibrahim F.B., Lu H., and Cao X Mitochondrial respiratory chain controls intracellular calcium signaling and NFAT activity essential for heart formation in Xenopus Mol Cell Biol (under revision) Emerald B.S.*, Chen Y.*, Zhu T., Zhu Z., Lee K.O., Gluckman P.D and Lobie P.E (2007) alpha CP1 mediates stabilization of hTERT mRNA by autocrine human growth hormone J Bio Chem (Published online on 2006 Nov 3) * Authors contributed equally to this work Huang G., Chen Y., Lu H., and Cao X (2006) Coupling mitochondrial respiratory chain to cell death: an essential role of mitochondrial complex I in the interferon-beta and retinoic acid-induced cancer cell death Cell Death Differ (Published online on 2006 Jul 7) Zhang X., Zhu T., Chen Y., Mertani H.C., Lee K.O., and Lobie P.E (2003) Human growth hormone-regulated HOXA1 is a human mammary epithelial oncogene J Biol Chem 278, 7580-7590 ii Table of Contents Table of Contents Acknowledgements……………………………………………………………………….i List of Publications……………………………………………………………………….ii Table of Contents………………………………………………………………… …….iii Summary……………………………………………………………………………… viii Abbreviation……………………………………………………………………….…… x List of Figures and Tables………………………………………………………… … xiv Chapter General introduction………………………………….…………………… …1 1.1.Mitochondria respiratory chain…………………………………… ………………2 1.1.1 Oxidative phosphorylation……………………………………………………2 1.1.2 Components of MRC……………………… ……………………………….4 1.1.2.1 NADH:ubiquinone oxidoreductase (Complex I)…………………………5 1.1.2.2 Succinate:ubiquinone oxidoreductase ( complex II)………………….… 1.1.2.3 Ubiquinol:cytochrome c oxidoreductase (Complex III)……………….…7 1.1.2.4 Cytochrome c oxidase (Complex IV)……………………………….……9 1.1.2.5 ATP synthase (Complex V)………………………………………………9 1.1.3 MRC diseases………………………………………………… ……………10 1.1.4 GRIM19 - a subunit of MRC complex 1……………………………………13 1.2 Intracellular calcium signaling ……………………………… …………………15 1.2.1 Regulation of calcium mobilization ……………………………………… 16 1.2.1.1 Calcium ON mechanism……………… ………………………………17 1.2.1.2 Calcium OFF mechanism……………… …………… ………………20 iii Table of Contents 1.2.2 Calcium-calcineurin-NFAT signalling pathway ……………………………23 1.2.2.1 Structure and function of calcineurin……………………………………24 1.2.2.2 Structure and function of NFAT…………………………………… …25 1.2.3 Role of NFAT in cardiogenesis…………………………… ………………28 1.3 Cardiogenesis………… …………………………………………………………31 1.3.1 Molecular pattern in cardiaogenesis…………… ………………………….32 1.3.2 The role of Nkx2.5 in cardiogenesis ……………………………………… 37 1.3.3 Transcriptional regulation of Nkx2.5 ……………………………………….39 1.4 Rationale of this thesis ………………………………………… ……………….41 Chapter Material and Methods…………………………………………………………43 2.1 Materials …………………………………………………………………… …44 2.2 Constructtion of plasmids…………………………………… …………… …44 2.3 Cell culture ……………………………………………………………… …… 45 2.4 Preparation of DH5α Escherichia coli competent cells…………………… … 45 2.5 DNA transformation ……………………………………………………………46 2.6 LIPOFECTAMINE™ DNA transfection………………………………… …46 2.7 Xenopus embryo manipulation …………………………………………… …47 2.8 Isolation of cDNA clones of Xenopus laevis GRIM-19………… ……… …48 2.8.1 Prepare Xenopus tropicalis GRIM-19 cDNA probe………… ……… .48 2.8.2 Screening of Xenopus laevis oocyte cDNA library………… ……… …48 2.9 QuikChange™ Site-Directed Mutagenesis………… …………………… …49 2.10 Prepare RNA probe or caped mRNA by in vitro transcription……………… 50 2.11 Whole-mount in situ hybridization………… ……… …51 iv Table of Contents 2.12 Histological analysis ………… ……… …52 2.13 Transmission electron microscopy… …53 2.14 In vitro transcription and translation… …53 2.15 Si RNA… …54 2.16 Western blotting… …54 2.17 Intracellular calcium measurement… …55 2.18 Luciferase reporter assay… …56 2.19 RT-PCR… …56 2.20 Electrophoretic mobility shift assay (EMSA) … …57 2.21 Mitochondrial complex I oxidative phosphorylation assay……………………58 2.22 Whole-mount in situ TUNEL staining…………………………………………59 2.23 Statistical Analysis…………………………………………………………… 59 Chapter Mitochondrial respiratory chain complex I is essential for heart formation in Xenopus……………………………………………………………………….60 3.1 Introduction……………………………………………………………………….61 3.2 Results…………………………………………………………………………….64 3.2.1 Cloning and expression pattern of XGRIM-19 in Xenopus laevis……………64 3.2.2 Knockdown of XGRIM-19 impairs MRC complex I activity in Xenopus embryos.………………………………………………………………….….66 3.2.3 Knockdown of XGRIM-19 causes heart defect in Xenopus embryos……… 69 3.2.4 Knockdown of XGRIM-19 down-regulates cardiac gene expression and NFAT activity……………………………………………………………………….74 v Table of Contents 3.2.5 Constitutively activated NFATc4 rescues the heart defect in XGRIM-19 KD embryos …………………………………………………………………….78 3.2.6 NFATc4 rescues the defects of sarcomere formation in the heart muscles… 80 3.2.7 Knockdown of XGRIM-19 or NDUFS3 impairs calcium mobilization and calcium-induced NFAT activity…………………………………………… 82 3.3 Discussion………………………………………………………………………….87 Chapter NFAT regulated Nkx2.5 expression in transcriptional level…………………91 4.1 Introduction………………………………………………………………….……92 4.2 Results…………………………………………………………………………….95 4.2.1 Constitutively active NFATc4 rescued Nkx2.5 expression in GRIM-19 KD Xenopus embryos………………………………………………………………95 4.2.2 Nkx2.5 gene expression is NFAT dependent during RA-induced cardiac differentiation of P19 cells…………………………………………………….96 4.2.3 Predicted conserved NFAT and its cofactor binding elements are localized in the promoter region of Nkx2.5 genes.………………………………… …… 100 4.2.4 NFATc4 interacted with NFAT binding elements in Nkx2.5 gene promoter.103 4.2.5 NFATc4 up-regulates Nkx2.5 expression on transcriptional level……… …106 4.3 Discussion…………………………… ……………………………………….110 Chapter General discussion………………………………… ………………………114 5.1 GRM-19 knocking-down Xenopus as a model for studying the MRC functions in early embryonic development……………………………………………… ….115 5.2 MRC activity is crucial for triggering intracellular calcium mobilization and NFAT activity……………………………………………………………………………116 vi Table of Contents 5.3 NFAT is a transcriptional regulator of Nkx2.5…………………… ……………117 5.4 A model of regulation of heart development by MRC……………………… …118 References………………………………………………………………………………121 vii Summary Summary The mitochondrial respiratory chain (MRC) plays a crucial role in cellular energy production, which is needed for cell division, movement, secretion, and activation of signaling pathways MRC mutations cause diseases with multi-system disorders including encephalopathies, myopathies and cardiomyopathies, which occur in per 10,000 live births in humans (Triepels et al., 2001) Depletion of MRC activity results in severe abnormalities in embryo development and leads to embryonic lethality (Huang et al., 2004; Larsson et al., 1998) The lack of an adequate animal model imposes limits on our current understanding of molecular processes in MRC-dependent embryonic development and the pathogenesis of these MRC diseases To address this issue, GRIM19, a newly identified MRC complex I subunit, was knocked down in Xenopus embryos The embryos exhibited typical phenotypes associated with mitochondrial diseases including retarded growth, mitochondrial proliferation, and moderately serious levels of neural, eye, and muscle tissue disorders However, the most striking phenotype exhibited is that of defective heart formation This can be rescued by reintroduction of human GRIM-19 mRNA The heart tube failed to loop in most of GRIM19 knocked-down embryos, and the expression of several cardiac markers such as Nkx2.5 and its downstream gene, MLC2, and cardiac actin, were also reduced Upon further investigation, we found that the activity of NFAT, a family of transcription factors that contributes to early organ development, was down-regulated in GRIM-19 knockdown embryos Furthermore, expression of a constitutively active form of mouse NFATc4 in these embryos could restore normal heart development NFAT activity is controlled by viii Summary the calcium-dependent phosphatase protein, calcineurin, which suggests that calcium signaling 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(>0.3µM) inhibits the opening of InsP3Rs Thus, during the onset of InsP3Rs opening, the release of Ca2+ increases the sensitivity of InsP3Rs, resulting in a rapid rise in Ca2+ levels Once the Ca2+... to the low-affinity sites of calcineurin B and affects the conformation change of both CnB and the regulatory domain of CnA, resulting in the exposure of the calmodulin-binding domain (Sikkink... al., 199 5) The Ca2+/calmodulin then bind to the CaM-binding domain and causes further conformational changes The conformation change in the flexible CaM-binding domain displaces the auto-inhibitory