RhoA prevents apoptosis during zebrafish embryogenesis through activation of Mek/Erk pathway

RhoA small GTPase, as a key regulator for actin cytoskeletal rearrangement, plays pivotal roles during morphogenesis, cytokinesis, phagocytosis and cell migration, but little is known about its signaling mechanism that controls cell survival in vivo. Using zebrafish as a model, we show that non-overlapping antisense morpholinos that block either translation or splicing of rhoA lead to extensive apoptosis during embryogenesis, resulting in overall reduction of body size and body length. These defects are associated with reduced activation of growth-promoting Erk and decreased expression of anti-apoptotic bcl-2. Moreover, ectopic expression of rhoA, Mek or BCL-2 mRNA rescues such phenotypes. Consistently, combined suppression of RhoA and Mek/Erk or Bcl-2 pathways by sub-optimal dose of rhoA morpholino and pharmacological inhibitors for either Mek (U0126) or Bcl-2 (HA 14-1) can induce developmental abnormalities and enhanced apoptosis, similar to those caused by effective RhoA knockdown. Furthermore, U0126 abrogates the rescue by RhoA and MEK but not BCL-2. In contrast, HA 14-1 effectively abolishes all functional rescues by RhoA, MEK or BCL-2, supporting that RhoA prevents apoptosis by activation of Mek/Erk pathway and requiring Bcl-2. These findings reveal an important genetic and functional relationship between RhoA with Mek/Erk and Bcl-2 for cell survival control during embryogenesis.

[1]  B. Shilo,et al.  In situ activation pattern of Drosophila EGF receptor pathway during development. , 1997, Science.

[2]  T. Takano,et al.  Actin cytoskeleton regulates extracellular matrix-dependent survival signals in glomerular epithelial cells. , 2005, American journal of physiology. Renal physiology.

[3]  C. Kimmel,et al.  Stages of embryonic development of the zebrafish , 1995, Developmental dynamics : an official publication of the American Association of Anatomists.

[4]  J. Rossant,et al.  Spatial and temporal patterns of ERK signaling during mouse embryogenesis , 2003, Development.

[5]  M. Endres,et al.  Neuronal gelsolin prevents apoptosis by enhancing actin depolymerization , 2004, Molecular and Cellular Neuroscience.

[6]  Ming Zhao,et al.  Regulation of CXCR 4-Mediated Nuclear Translocation of Extracellular Signal-Related Kinases 1 and 2 , 2005 .

[7]  Xianwu Li,et al.  Inhibition of Protein Geranylgeranylation and RhoA/RhoA Kinase Pathway Induces Apoptosis in Human Endothelial Cells* , 2002, The Journal of Biological Chemistry.

[8]  U. Monani,et al.  Molecular, Cellular and Developmental Biology Program – Specialization 2018/2019 , 2017 .

[9]  C. Xing,et al.  Structure-activity relationship studies of ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (HA 14-1), an antagonist for antiapoptotic Bcl-2 proteins to overcome drug resistance in cancer. , 2006, Journal of medicinal chemistry.

[10]  A. Hall,et al.  Rho GTPases in cell biology , 2002, Nature.

[11]  M. Wullimann,et al.  Proliferation pattern changes in the zebrafish brain from embryonic through early postembryonic stages , 2000, Anatomy and Embryology.

[12]  D. Illingworth,et al.  3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors , 1994 .

[13]  S. Srinivasula,et al.  Structure-based discovery of an organic compound that binds Bcl-2 protein and induces apoptosis of tumor cells. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[14]  R. Peterson,et al.  Artery/Vein Specification Is Governed by Opposing Phosphatidylinositol-3 Kinase and MAP Kinase/ERK Signaling , 2006, Current Biology.

[15]  D. Stainier,et al.  In vivo cell biology: following the zebrafish trend. , 2006, Trends in cell biology.

[16]  P. Leder,et al.  Human MCF10A Mammary Epithelial Cells Undergo Apoptosis following Actin Depolymerization That Is Independent of Attachment and Rescued by Bcl-2 , 2001, Molecular and Cellular Biology.

[17]  B. Bierer,et al.  Actin Stabilization by Jasplakinolide Enhances Apoptosis Induced by Cytokine Deprivation* , 1999, The Journal of Biological Chemistry.

[18]  Ming Zhao,et al.  Regulation of CXCR4-Mediated Nuclear Translocation of Extracellular Signal-Related Kinases 1 and 2 , 2006, Molecular Pharmacology.

[19]  A. Hall,et al.  The GTPase Rho has a critical regulatory role in thymus development , 1997, The EMBO journal.

[20]  U. Langheinrich,et al.  Zebrafish as a Model Organism for the Identification and Characterization of Drugs and Genes Affecting p53 Signaling , 2002, Current Biology.

[21]  Á. Pascual,et al.  3-Hydroxy-3-methyl-glutaryl coenzyme A reductase inhibitors, atorvastatin and simvastatin, induce apoptosis of vascular smooth muscle cells by downregulation of Bcl-2 expression and Rho A prenylation. , 2002, Atherosclerosis.

[22]  L. S. Ross,et al.  Apoptosis in the developing zebrafish embryo. , 2001, Developmental biology.

[23]  P. Leder,et al.  Bcl-2-mediated cell survival promotes metastasis of EpH4 betaMEKDD mammary epithelial cells. , 2004, Molecular cancer research : MCR.

[24]  J. Blagburn,et al.  Fibroblast growth factor 2 applied to the optic nerve after axotomy increases Bcl‐2 and decreases Bax in ganglion cells by activating the extracellular signal‐regulated kinase signaling pathway , 2005, Journal of neurochemistry.

[25]  V. Prince,et al.  Knockdown of duplicated zebrafish hoxb1 genes reveals distinct roles in hindbrain patterning and a novel mechanism of duplicate gene retention. , 2002, Development.

[26]  U. Hämmerling,et al.  F-actin as a functional target for retro-retinoids: a potential role in anhydroretinol-triggered cell death. , 1999, Journal of cell science.

[27]  Kazuto Kobayashi,et al.  Survival of Developing Motor Neurons Mediated by Rho GTPase Signaling Pathway through Rho-Kinase , 2004, The Journal of Neuroscience.

[28]  S. Kornblau,et al.  Regulation of Bcl2 phosphorylation and potential significance for leukemic cell chemoresistance. , 2000, Journal of the National Cancer Institute. Monographs.

[29]  K. Giehl.  Oncogenic Ras in tumour progression and metastasis , 2005, Biological chemistry.

[30]  H. Spaink,et al.  Characterization and expression patterns of the MAPK family in zebrafish. , 2006, Gene expression patterns : GEP.

[31]  Emilios Tahinci,et al.  Distinct functions of Rho and Rac are required for convergent extension during Xenopus gastrulation. , 2003, Developmental biology.

[32]  David A. Cheresh,et al.  Regulation of Cell Motility by Mitogen-activated Protein Kinase , 1997, The Journal of cell biology.

[33]  H. Loh,et al.  Adenylyl Cyclase Superactivation Induced by Long-Term Treatment with Opioid Agonist Is Dependent on Receptor Localized within Lipid Rafts and Is Independent of Receptor Internalization , 2006, Molecular Pharmacology.

[34]  M. Benito,et al.  Activated Ha-ras Induces Apoptosis by Association with Phosphorylated Bcl-2 in a Mitogen-activated Protein Kinase-independent Manner* , 1999, The Journal of Biological Chemistry.

[35]  P. Auberger,et al.  RhoA GTPase inactivation by statins induces osteosarcoma cell apoptosis by inhibiting p42/p44-MAPKs-Bcl-2 signaling independently of BMP-2 and cell differentiation , 2006, Cell Death and Differentiation.

[36]  K. Hirata,et al.  Lysophosphatidylcholine inhibits endothelial cell migration and proliferation via inhibition of the extracellular signal-regulated kinase pathway. , 2000, Arteriosclerosis, thrombosis, and vascular biology.

[37]  S. Miyamoto,et al.  RhoA/Rho Kinase Up-regulate Bax to Activate a Mitochondrial Death Pathway and Induce Cardiomyocyte Apoptosis* , 2007, Journal of Biological Chemistry.

[38]  H. Miyake,et al.  Overexpression of Bcl-2 enhances metastatic potential of human bladder cancer cells , 1999 .

[39]  J. Lacal,et al.  Rho signals to cell growth and apoptosis. , 2001, Cancer letters.

[40]  J. Dichgans,et al.  BCL‐2 promotes migration and invasiveness of human glioma cells , 1998, FEBS letters.

[41]  C. Martínez-A,et al.  Rho prevents apoptosis through Bcl‐2 expression: Implications for interleukin‐2 receptor signal transduction , 1997, European journal of immunology.

[42]  Z. Gong,et al.  RhoA acts downstream of Wnt5 and Wnt11 to regulate convergence and extension movements by involving effectors Rho kinase and Diaphanous: use of zebrafish as an in vivo model for GTPase signaling. , 2006, Cellular signalling.

[43]  Wei Li,et al.  Extracellular signal-regulated kinase 2 is necessary for mesoderm differentiation , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[44]  E. Sahai,et al.  Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I , 2001, Nature Cell Biology.