The NADPH Oxidase NOX4 Drives Cardiac Differentiation: Role in Regulating Cardiac Transcription Factors and MAP Kinase Activation

Abstract

Reactive oxygen species (ROS) generated by the NOX family of NADPH oxidases have been described to act as second messengers regulating cell growth and differentiation. However, such a function has hitherto not been convincingly demonstrated. We investigated the role of NOX-derived ROS in cardiac differentiation using mouse embryonic stem cells. ROS scavengers prevented the appearance of spontaneously beating cardiac cells within embryoid bodies. Downregulation of NOX4, the major NOX isoform present during early stages of differentiation, suppressed cardiogenesis. This was rescued by a pulse of low concentrations of hydrogen peroxide 4 d before spontaneous beating appears. Mechanisms of ROS-dependent signaling included p38 mitogen-activated protein kinase (MAPK) activation and nuclear translocation of the cardiac transcription factor myocyte enhancer factor 2C (MEF2C). Our results provide first molecular evidence that the NOX family of NADPH oxidases regulate vertebrate developmental processes. INTRODUCTIONReactive oxygen species (ROS) are generated either in a nonregulated manner as side products of several enzymatic systems (e.g., cyclooxygenases, nitric oxide [NO] synthases, mitochondrial cytochromes) or in a regulated way as main products of superoxide producing enzymes, the NADPH oxidases. In the mouse, the family of NADPH oxidases includes NOX1, NOX2 (gp91 phox ), NOX3, and NOX4.Excessive cellular generation of ROS, such as superoxide anions (O 2 Ϫ ) and hydrogen peroxide (H 2 O 2 ), is potentially destructive and is used by phagocytes to kill invading microorganisms. Under normal conditions, scavenging mechanisms (e.g., superoxide dismutase, catalase, glutathioneglutathione peroxidase system) remove excessive amounts of ROS. Under stress conditions, however, the production of ROS may exceed the reducing capacity of the cell and damage cellular functions. Small amounts of ROS, on the other hand, can function as intracellular second messengers and activate signaling cascades involved in growth and differentiation of many cell types (for review see Rhee, 1999;Laloi et al., 2004). For example, the MAP kinase-signaling pathway is sensitive to ROS (Torres and Forman, 2003). Moreover, distinct signaling pathways have differential sensitivity to oxidative stress, leading to dose-dependent effect on, for example, cardiomyocytes on which ROS can induce hypertrophy or apoptosis (Kwon et al., 2003). Transcription factors such as NF-B, p53, and AP-1 are redox-sensitive and can be directly modified by ROS, providing a link with the control of gene expression (Morel and Barouki, 1999).Cardiac differentiation can be studied by differentiating mouse embryonic stem cells (ESC) into embryoid bodies (EB), where the appearance of spontaneously beating cardiomyocytes is observed after 7-8 d of culture. This system thus provides a unique experimental model to study the role of ROS and ROS-generating enzymes in the regulation of cardiomyocyte growth and differentiation in vitro. Previous reports have shown a link between ROS and...

Effect of inhibitors of NADPH oxidase complex and mitochondrial ATP‐sensitive potassium channels on generation of dopaminergic neurons from neurospheres of mesencephalic precursors

“…The present results suggest that Apocynin and 5-HD act by stimulating differentiation of mesencephalic precursors toward the DA phenotype. The results are consistent with those of previous studies, which have suggested that the redox state modulates differentiation of mesencephalic precursors (Studer et al, 2000;Rodriguez-Pallares et al, 2001Tsatmali et al, 2005), and with those of studies that have suggested that ROS levels affect the phenotype of cells in culture (Li et al, 2006;Xiao et al, 2009), including neurons (Tsatmali et al, 2006).…”

Mitochondrial Reactive Oxygen Species Mediate Cardiomyocyte Formation from Embryonic Stem Cells in High Glucose

“…4F), indicating that an extrinsic source of ROS can signal cardiogenesis in the absence of a mitochondrial signal. Indeed, extrinsic peroxides have been shown to be cardiogenic in various models of stem cell differentiation [15][16][17][18], and hydrogen peroxide (10 lM) was also cardiogenic during differentiation in low glucose (Supporting Information Fig. 2).…”

“…The ES cell line R1 was provided by Dr. Sagrario Ortega (CNIO, Madrid, Spain). The CGR8 ES cell line and a clonal derivative containing a ribozyme targeting NOX4 (R19 cell line) were gifted from Dr. Marisa Jaconi (Universite de Geneve, Genève, Switzerland) and have been described previously [18]. The Brachyury-green fluorescent protein (Brachyury-GFP) ES cell line was provided by Dr. Gordon Keller (McEwen Center for Regenerative Medicine, Toronto, ON, Canada).…”

“…Indeed NOX4, which is a major Nox isoform in nonphagocytic cells, has recently been shown to play a pivotal role during serumdirected differentiation of ES cells to cardiomyogenesis [18], suggesting a role for ROS as signaling molecules during in vitro differentiation to the cardiomyocyte lineage.…”

Nox enzymes, ROS, and chronic disease: An example of antagonistic pleiotropy

“…Nox4 is necessary for the differentiation of mouse embryonic stem cells or fibroblasts into myocytes [53,54]. In addition, Nox4 may contribute to the pathological activation of cardiac fibroblasts in cardiac fibrosis associated with heart failure [54].…”