Tiny transporters: how exosomes and calcineurin signaling regulate miR-23a levels during muscle atrophy. Focus on "miR-23a is decreased during muscle atrophy by a mechanism that includes calcineurin signaling and exosome-mediated export".

SKELETAL MUSCLE ATROPHY is characterized by an increase in protein degradation that is accomplished through a variety of cellular mechanisms. Significant attention has been given to the ubiquitin-proteasome pathway and to expression of the E3 ubiquitin ligases muscle-specific RING finger-1 (MuRF1) and atrogin-1/muscle atrophy F box (MAFbx) during muscle atrophy. The increased expression of these ubiquitin ligases has been documented in multiple models of muscle atrophy, and an attenuation of atrophy has been shown after their knockdown (2). Induction of MuRF1 and atrogin-1 expression by the transcription factor forkhead box O3a (FOXO3a) is an integral step in enhanced protein degradation through the ubiquitinproteasome pathway; however, regulation of MuRF1/atrogin-1 expression can also occur through the binding of microRNAs. MicroRNAs are small, noncoding RNAs that regulate protein expression through translation inhibition and/or degradation of specific transcripts. Recent work identified microRNA23a (miR-23a) as capable of binding MuRF1 and atrogin-1 transcripts and inhibiting their translation (10). Overexpression of miR-23a also protected muscles from atrophy in vitro and in vivo (10). miR-23a expression is regulated in part through the transcription factor cytoplasmic nuclear factor of activated T cells 3 (NFATc3), which is activated by the phosphatase calcineurin (Cn) (6). This holds true in skeletal muscle as well, with expression or reporter activity of miR-23a responsive to Cn activity and NFATc3 expression (1, 10). Inhibition of Cn activity following an atrophy-inducing stimulus such as dexamethasone administration leads to decreased expression of miR-23a, thus relieving repression of MuRF1/atrogin-1 expression by miR-23a. Together with increased transcription through FOXO3a, this mechanism provides for increased expression of these E3 ubiquitin ligases, leading to enhanced degradation of proteins through the ubiquitin-proteasome pathway. In addition to regulation of microRNA at the transcriptional level, microRNA levels may also be controlled by their packaging into extracellular vesicles. Exosomes are small (30to 100-nm) vesicles released from cells that contain proteins, mRNA, and microRNA. A recent study characterized exosomes released from C2C12 myoblasts, focusing primarily on protein content (3). Exosomes have also been suggested to serve as paracrine factors, whereby an exosome released from a donor cell can travel and fuse with a target cell, releasing its contents, which can influence activity of the target cell (9). This novel hypothesis is supported by a study demonstrating that microRNAs present in exosomes released by dendritic cells are capable of repressing mRNA translation in target cells (7). The packaging and subsequent release of microRNAcontaining exosomes by donor cells could represent a new mechanism through which cells are capable of regulating microRNA levels. In this issue of American Journal of Physiology-Cell Physiology, Hudson et al. (4) report on two mechanisms through which skeletal muscle cells regulate intracellular levels of miR-23a: 1) Cn-NFATc3 transcriptional control and 2) rapid packaging and release into exosomes (4) (Fig. 1). The authors take a multifaceted approach in demonstrating transcriptional regulation of miR-23a through Cn. In a rat diabetic model of muscle atrophy, Cn/NFATc3 signaling was depressed, resulting in decreased levels of miR-23a (4). These findings provide evidence for an association of Cn activity and miR-23a levels on which the authors expand to demonstrate a direct relationship. Pharmacological activation of Cn in C2C12 myotubes increased miR-23a expression, and this increase in miR-23a expression was prevented with the addition of cyclosporine A to inhibit Cn. Knockout of either catalytic subunit of Cn in primary mouse myotubes led to decreased levels of miR-23a, and further reductions in miR-23a levels were prevented with the addition of dexamethasone, demonstrating Cn specificity in the expression of miR-23a (4). The authors make an interesting observation that constitutive activation of Cn does not increase miR-23a expression but does offer protection from dexamethasone-induced reduction of miR-23a expression (4). This finding is consistent with previous work (10) and provides evidence that a narrow range of Cn activity is required to regulate miR-23a levels. The breadth of experimental approaches employed by Hudson and colleagues demonstrates a direct link between Cn signaling and miR-23a expression. Hudson et al. (4) propose a novel hypothesis that miR-23a levels are also regulated in part through the release of exosomes containing miR-23a. While exosomes have been shown to contain viable microRNAs capable of influencing protein expression in target cells (7), little is known regarding microRNA exosomal incorporation, and previous work in C2C12 myoblasts focused heavily on the protein content of exosomes (3). Hudson et al. demonstrate for the first time that dexamethasone treatment of C2C12 myotubes results in the release of exosomes enriched with microRNA (miR-1 and miR-23a). Exosomal microRNA content, not total number of exosomes, was increased with dexamethasone treatment, providing exciting evidence for the specificity of microRNA packaging into exosomes under differing physiological conditions. The increase in exosomal miR-23a content mirrors the Address for reprint requests and other correspondence: C. S. Fry, Dept. of Rehabilitation Sciences, College of Health Sciences, Univ. of Kentucky, Lexington, KY 40503 (e-mail: [email protected]). Am J Physiol Cell Physiol 306: C529–C530, 2014; doi:10.1152/ajpcell.00022.2014. Editorial Focus