The International Journal of Biochemistry & Cell Biology
Carbonyl reductase 1 is an essential regulator of skeletal muscle differentiation and regeneration☆
Introduction
Muscle differentiation is a highly coordinated process involving cell cycle arrest, expression of myogenic genes, myoblast elongation and cell fusion to form multinucleated myotubes (Weintraub et al., 1991). All myogenic precursor cells express the paired-homeodomain transcription factor 3 and 7 (Pax3 and 7). When myogenesis begins, expression of Pax3 and 7 gradually decreases, but expression of a group of basic helix–loop–helix muscle regulatory transcription factors, such as MyoD, myocyte enhancer factor 2 (MEF2), myogenic factor 5 (Myf5), myogenin, and myogenic regulatory factor 4 (MRF4), significantly increases during muscle differentiation (Arnold and Winter, 1998, Molkentin and Olson, 1996). In these complex processes, two key signaling pathways are required for myogenesis: PI3K (phosphatidylinositol 3-kinase) and p38 mitogen-activated protein kinase (p38 MAPK). These two kinases activate MEF2 transcription factors and MyoD. PI3K is an important mediator of tyrosine kinase receptor signal transduction and affects downstream targets such as Akt, Rac, p70 S6 kinase, phospholipase C-γ1 and mTOR (Lim et al., 2007), all of which are major signaling molecules that mediate myogenesis. Insulin-like growth factor 1 and 2 are the best characterized ligands that stimulate muscle differentiation by activating PI3K pathway (Tureckova et al., 2001). p38 MAPK is also one of the key signaling molecules in the expression of muscle-specific genes as well as in the fusion of myoblasts (Zetser et al., 1999). For the activation of p38 MAPK during myogenic differentiation, ROS generated by myogenic precursor cells disrupt the complex of thioredoxin and apoptosis signal-regulating kinase 1 (ASK1), and then the activated ASK1 phosphorylates MKK3/6 which in turn phosphorylates p38 MAPK (Choi et al., 2011). The Raf/MEK/Erk pathway has been shown to stimulate cell proliferation rather than muscle differentiation (Lee et al., 2002, Rommel et al., 1999).
Reactive oxygen species (ROS) generally consist of superoxide (O2−), hydrogen peroxide (H2O2) and the hydroxyl radical (OH) (Hoffman and Brookes, 2009). All are involved in a variety of cellular processes including cell proliferation, differentiation, and apoptosis. High levels of ROS induce oxidative modification to cellular macromolecules, such as proteins, lipids, and nucleic acids, and subsequently high levels of ROS promote cell death (Oppermann, 2007). In particular, lipid peroxidation plays a central role in the pathogenesis of oxidative stress (Obulesu et al., 2011, Reed, 2011). The acyl chain of polyunsaturated fatty acids (PUFAs), such as linoleic or arachidonic acid that can be found in cell membranes and lipoproteins, is particularly susceptible to free radical-mediated oxidation and leads to the release of reactive aldehydes. Unsaturated aldehydes such as 4-oxonon-2-enal (ONE) and 4-hydroxynon-2-enal (HNE) can cause modifications of proteasomes, alterations of the cytoskeleton, changes in transcriptional activities, as well as diffuse cytotoxic effects (Niki, 2009).
Low levels of ROS on the other hand play an important role as “redox messengers” in intracellular signaling. Several proteins have been shown to function as ROS effectors that are sensitively and reversibly oxidized by ROS. These ROS-effector proteins commonly possess highly reactive cysteine (Cys) residues, and enable signal transmission to downstream targets through oxidative changes in the protein. Most importantly, protein tyrosine phosphatase (PTP), thioredoxin (TRX) and peroxiredoxin (PRX) family proteins possess special domains/motifs that maintain the reactivity of Cys and allow responsiveness to low levels of ROS (Miki and Funato, 2012). Various redox systems, such as the glutathione, thioredoxin, and pyridine nucleotide redox couples, also participate in cell function, regulation, and adaptation to diverse growth conditions (Oka et al., 2012).
Carbonyl reductase 1 (CBR1) is a NADPH-dependent, monomeric, and cytosolic enzyme belonging to a family of short-chain dehydrogenases/reductases (SDR) (Forrest and Gonzalez, 2000), and is involved in protecting the cell against oxidative stress. Furthermore, CBR1 catalyzes physiologically and pharmacologically active substrates, including a variety of endogenous and xenobiotic carbonyl compounds (Hoffmann and Maser, 2007). CBR1 reduces highly reactive lipid aldehydes, such as ONE, HNE, and acrolein, all of which can induce protein and DNA damage within cells (Oppermann, 2007). Overexpression of human CBR1 in NIH3T3 cells was shown to provide protection from ROS-induced cellular damage (Kelner et al., 1997) and CBR1 was also shown to regulate apoptosis and cell survival in insulin-secreting cells by reducing oxidative stress (Rashid et al., 2010). Recently, it was discovered that HIF1α, AP-1, and Nrf2 could all regulate CBR1 at the transcriptional level (Jang et al., 2012, Miura et al., 2012, Tak et al., 2011). Collectively, these findings indicate that transcriptionally regulated CBR1 is a major contributor to the control of oxidative stress.
In this study, we provide evidence for the first time that CBR1 is upregulated by the transcription factor Nrf2 during muscle differentiation and regeneration. Also, we show that CBR1 contributes directly to muscle differentiation by serving as a regulator of oxidative stress.
Section snippets
Materials
Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Lonza (Walkersville, MD, USA). 2′7′-dichlorofluorescein diacetate (DCF-DA), actinomycin D and N-acetyl l-cysteine (NAC) were purchased from Sigma–Aldrich Inc. (St. Louis, MO, USA). Antibody against CBR1 was purchased from AVIVA Systems Biology. Antibodies specific to Nrf2, myosin heavy chain (MHC), myogenin, β-tubulin and lamin B were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
CBR1 expression and ROS generation increases during muscle differentiation
To investigate the role of CBR1 in the process of muscle differentiation, we first examined the morphological changes (Fig. 1A) and fusion index changes (Fig. 1B) associated with the differentiation process in C2C12 cells. Replacement of proliferation medium (PM) with differentiation medium (DM) resulted in the formation of multinucleated myotubes in a time-dependent manner (Fig. 1A and B). To determine whether CBR1 expression is induced during muscle differentiation, we observed the expression
Discussion
In this study, we demonstrate for the first time that CBR1 plays a crucial role in muscle differentiation and regeneration. Furthermore, we clearly showed that Nrf2 induces CBR1 expression and that CBR1 controls excessive ROS generation and lipid peroxidation during muscle differentiation.
In response to both endogenous and exogenous stimuli, ROS are generated by the mitochondrial respiratory chain, cytochrome p450 enzymes, NADPH oxidase, etc. (Zangar et al., 2004). When ROS overwhelm the
Acknowledgements
All authors read and approved the final manuscript. There are no conflicts of interest.
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Supported by the National Research Foundation of Korea (NRF) grant funded by the Korean Government (MEST) (No. 20120009380) to S.S. Kim.