Jae-Sung You

The role of skeletal muscle mTOR in the regulation of mechanical load-induced growth

Non‐Technical Summary  Chronic mechanical loading (CML) of skeletal muscle induces growth and this effect can be blocked by the drug rapamycin. Rapamycin is considered to be a highly specific inhibitor of the mammalian target of rapamycin (mTOR), and thus, many have concluded that mTOR plays a key role in CML‐induced growth. However, direct evidence that mTOR confers the CML‐induced activation of growth promoting events such as hypertrophy, hyperplasia and ribosome biogenesis is lacking. This study addressed that gap in knowledge by using a specialized line of transgenic mice. Surprisingly, the results indicate that only a few of the growth promoting events induced by CML are fully dependent on mTOR signalling (e.g. hypertrophy). These results advance our understanding of the molecular mechanisms that regulate skeletal muscle mass and should help future studies aimed at identifying targets for therapies that can prevent the loss of muscle mass during conditions such as bedrest, immobilization, and ageing.

The Role of Diacylglycerol Kinase ζ and Phosphatidic Acid in the Mechanical Activation of Mammalian Target of Rapamycin (mTOR) Signaling and Skeletal Muscle Hypertrophy

Background: Diacylglycerol kinases (DGKs) synthesize phosphatidic acid (PA), and PA can activate growth-regulatory mTOR signaling. Results: The ζ isoform of DGK is necessary for a mechanically induced increase in PA-mTOR signaling, and overexpression of DGKζ induces skeletal muscle hypertrophy. Conclusion: PA synthesized by DGKζ regulates the mechanical activation of mTOR signaling and hypertrophy. Significance: DGKζ is a potential target for treating muscle atrophy/wasting. The activation of mTOR signaling is essential for mechanically induced changes in skeletal muscle mass, and previous studies have suggested that mechanical stimuli activate mTOR (mammalian target of rapamycin) signaling through a phospholipase D (PLD)-dependent increase in the concentration of phosphatidic acid (PA). Consistent with this conclusion, we obtained evidence which further suggests that mechanical stimuli utilize PA as a direct upstream activator of mTOR signaling. Unexpectedly though, we found that the activation of PLD is not necessary for the mechanically induced increases in PA or mTOR signaling. Motivated by this observation, we performed experiments that were aimed at identifying the enzyme(s) that promotes the increase in PA. These experiments revealed that mechanical stimulation increases the concentration of diacylglycerol (DAG) and the activity of DAG kinases (DGKs) in membranous structures. Furthermore, using knock-out mice, we determined that the ζ isoform of DGK (DGKζ) is necessary for the mechanically induced increase in PA. We also determined that DGKζ significantly contributes to the mechanical activation of mTOR signaling, and this is likely driven by an enhanced binding of PA to mTOR. Last, we found that the overexpression of DGKζ is sufficient to induce muscle fiber hypertrophy through an mTOR-dependent mechanism, and this event requires DGKζ kinase activity (i.e. the synthesis of PA). Combined, these results indicate that DGKζ, but not PLD, plays an important role in mechanically induced increases in PA and mTOR signaling. Furthermore, this study suggests that DGKζ could be a fundamental component of the mechanism(s) through which mechanical stimuli regulate skeletal muscle mass.

Eccentric contractions increase the phosphorylation of tuberous sclerosis complex‐2 (TSC2) and alter the targeting of TSC2 and the mechanistic target of rapamycin to the lysosome

•  Mechanical stimuli play a major role in the regulation of skeletal muscle mass. •  Signalling through a protein kinase called the mechanistic target of rapamycin (mTOR) is essential for mechanically induced changes in muscle mass; however, the mechanism(s) via which mechanical stimuli regulate mTOR signalling have not been defined. •  In this study, mouse skeletal muscles were stimulated with eccentric contractions (ECs) to determine if the mechanical activation of mTOR signalling is associated with changes in the phosphorylation of the tuberous sclerosis complex‐2 (TSC2) and the targeting of both mTOR and TSC2 to the lysosome. •  Our results demonstrate that ECs induce hyper‐phosphorylation of TSC2, enhanced lysosomal targeting of mTOR and nearly abolish the lysosomal targeting of TSC2. •  These novel observations suggest that alterations in the lysosomal targeting of mTOR/TSC2 could play a fundamental role in the mechanism via which mechanical stimuli regulate mTOR signalling and ultimately skeletal muscle mass.

The role of mTOR signaling in the regulation of protein synthesis and muscle mass during immobilization in mice

ABSTRACT The maintenance of skeletal muscle mass contributes substantially to health and to issues associated with the quality of life. It has been well recognized that skeletal muscle mass is regulated by mechanically induced changes in protein synthesis, and that signaling by mTOR is necessary for an increase in protein synthesis and the hypertrophy that occurs in response to increased mechanical loading. However, the role of mTOR signaling in the regulation of protein synthesis and muscle mass during decreased mechanical loading remains largely undefined. In order to define the role of mTOR signaling, we employed a mouse model of hindlimb immobilization along with pharmacological, mechanical and genetic means to modulate mTOR signaling. The results first showed that immobilization induced a decrease in the global rates of protein synthesis and muscle mass. Interestingly, immobilization also induced an increase in mTOR signaling, eIF4F complex formation and cap-dependent translation. Blocking mTOR signaling during immobilization with rapamycin not only impaired the increase in eIF4F complex formation, but also augmented the decreases in global protein synthesis and muscle mass. On the other hand, stimulating immobilized muscles with isometric contractions enhanced mTOR signaling and rescued the immobilization-induced decrease in global protein synthesis through a rapamycin-sensitive mechanism that was independent of ribosome biogenesis. Unexpectedly, the effects of isometric contractions were also independent of eIF4F complex formation. Similar to isometric contractions, overexpression of Rheb in immobilized muscles enhanced mTOR signaling, cap-dependent translation and global protein synthesis, and prevented the reduction in fiber size. Therefore, we conclude that the activation of mTOR signaling is both necessary and sufficient to alleviate the decreases in protein synthesis and muscle mass that occur during immobilization. Furthermore, these results indicate that the activation of mTOR signaling is a viable target for therapies that are aimed at preventing muscle atrophy during periods of mechanical unloading. Summary: The activation of mTOR signaling is both necessary and sufficient to alleviate the decreases in protein synthesis and muscle mass that occur during immobilization.

Lipid domain-dependent regulation of single cell wound repair

Cell repair is a conserved and medically important process. Cell damage triggers the rapid accumulation of several different lipids around wounds, and the lipids sort into distinct domains around them. One of these lipids—diacylglycerol—is required for activation of Rho and Cdc42 and healing.

Yes-Associated Protein is up-regulated by mechanical overload and is sufficient to induce skeletal muscle hypertrophy

Mechanically‐induced skeletal muscle growth is regulated by mammalian/mechanistic target of rapamycin complex 1 (mTORC1). Yes‐Associated Protein (YAP) is a mechanically‐sensitive, and growth‐related, transcriptional co‐activator that can regulate mTORC1. Here we show that, in skeletal muscle, mechanical overload promotes an increase in YAP expression; however, the time course of YAP expression is markedly different from that of mTORC1 activation. We also show that the overexpression of YAP induces hypertrophy via an mTORC1‐independent mechanism. Finally, we provide preliminary evidence of possible mediators of YAP‐induced hypertrophy (e.g. increased MyoD and c‐Myc expression, and decreased Smad2/3 activity and muscle ring finger 1 (MuRF1) expression).

A map of the phosphoproteomic alterations that occur after a bout of maximal‐intensity contractions

Mechanical signals play a critical role in the regulation of muscle mass, but the molecules that sense mechanical signals and convert this stimulus into the biochemical events that regulate muscle mass remain ill‐defined. Here we report a mass spectrometry‐based workflow to study the changes in protein phosphorylation that occur in mouse skeletal muscle 1 h after a bout of electrically evoked maximal‐intensity contractions (MICs). Our dataset provides the first comprehensive map of the MIC‐regulated phosphoproteome. Using unbiased bioinformatics approaches, we demonstrate that our dataset leads to the identification of many well‐known MIC‐regulated signalling pathways, as well as to a plethora of novel MIC‐regulated events. We expect that our dataset will serve as a fundamentally important resource for muscle biologists, and help to lay the foundation for entirely new hypotheses in the field.

Identification of mechanically regulated phosphorylation sites on tuberin (TSC2) that control mechanistic target of rapamycin (mTOR) signaling

Mechanistic target of rapamycin (mTOR) signaling is necessary to generate a mechanically induced increase in skeletal muscle mass, but the mechanism(s) through which mechanical stimuli regulate mTOR signaling remain poorly defined. Recent studies have suggested that Ras homologue enriched in brain (Rheb), a direct activator of mTOR, and its inhibitor, the GTPase-activating protein tuberin (TSC2), may play a role in this pathway. To address this possibility, we generated inducible and skeletal muscle-specific knock-out mice for Rheb (iRhebKO) and TSC2 (iTSC2KO) and mechanically stimulated muscles from these mice with eccentric contractions (EC). As expected, the knock-out of TSC2 led to an elevation in the basal level of mTOR signaling. Moreover, we found that the magnitude of the EC-induced activation of mTOR signaling was significantly blunted in muscles from both inducible and skeletal muscle-specific knock-out mice for Rheb and iTSC2KO mice. Using mass spectrometry, we identified six sites on TSC2 whose phosphorylation was significantly altered by the EC treatment. Employing a transient transfection-based approach to rescue TSC2 function in muscles of the iTSC2KO mice, we demonstrated that these phosphorylation sites are required for the role that TSC2 plays in the EC-induced activation of mTOR signaling. Importantly, however, these phosphorylation sites were not required for an insulin-induced activation of mTOR signaling. As such, our results not only establish a critical role for Rheb and TSC2 in the mechanical activation of mTOR signaling, but they also expose the existence of a previously unknown branch of signaling events that can regulate the TSC2/mTOR pathway.

Insights into the role and regulation of TCTP in skeletal muscle

The translationally controlled tumor protein (TCTP) is upregulated in a range of cancer cell types, in part, by the activation of the mechanistic target of rapamycin (mTOR). Recently, TCTP has also been proposed to act as an indirect activator of mTOR. While it is known that mTOR plays a major role in the regulation of skeletal muscle mass, very little is known about the role and regulation of TCTP in this post-mitotic tissue. This study shows that muscle TCTP and mTOR signaling are upregulated in a range of mouse models (mdx mouse, mechanical load-induced hypertrophy, and denervation- and immobilization-induced atrophy). Furthermore, the increase in TCTP observed in the hypertrophic and atrophic conditions occurred, in part, via a rapamycin-sensitive mTOR-dependent mechanism. However, the overexpression of TCTP was not sufficient to activate mTOR signaling (or increase protein synthesis) and is thus unlikely to take part in a recently proposed positive feedback loop with mTOR. Nonetheless, TCTP overexpression was sufficient to induce muscle fiber hypertrophy. Finally, TCTP overexpression inhibited the promoter activity of the muscle-specific ubiquitin proteasome E3-ligase, MuRF1, suggesting that TCTP may play a role in inhibiting protein degradation. These findings provide novel data on the role and regulation of TCTP in skeletal muscle in vivo.

A DGKζ-FoxO-ubiquitin proteolytic axis controls fiber size during skeletal muscle remodeling

Increasing DGKζ abundance could enhance skeletal muscle growth or prevent muscle wasting under atrophy-promoting conditions. More muscle definition with DGKζ The failure to maintain muscle mass is a leading risk factor for morbidity and mortality in the elderly and in cancer patients experiencing cachexia. You et al. found that the ζ isoform of diacylglycerol kinase (DGKζ) not only enhanced exercise-induced skeletal muscle growth but also limited muscle wasting in response to denervation or food deprivation. DGKζ exerted these effects independently of its DAG kinase activity. In rodents, DGKζ prevented the activation of the protein-degrading machinery that causes atrophy. Thus, increasing the abundance of DGKζ could accelerate muscle growth in response to exercise and block muscle wasting and its adverse effects. Skeletal muscle rapidly remodels in response to various stresses, and the resulting changes in muscle mass profoundly influence our health and quality of life. We identified a diacylglycerol kinase ζ (DGKζ)–mediated pathway that regulated muscle mass during remodeling. During mechanical overload, DGKζ abundance was increased and required for effective hypertrophy. DGKζ not only augmented anabolic responses but also suppressed ubiquitin-proteasome system (UPS)–dependent proteolysis. We found that DGKζ inhibited the transcription factor FoxO that promotes the induction of the UPS. This function was mediated through a mechanism that was independent of kinase activity but dependent on the nuclear localization of DGKζ. During denervation, DGKζ abundance was also increased and was required for mitigating the activation of FoxO-UPS and the induction of atrophy. Conversely, overexpression of DGKζ prevented fasting-induced atrophy. Therefore, DGKζ is an inhibitor of the FoxO-UPS pathway, and interventions that increase its abundance could prevent muscle wasting.