Leucyl-tRNA Synthetase Contributes to Muscle Weakness through Mammalian Target of Rapamycin Complex 1 Activation and Autophagy Suppression in a Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD), caused by loss-of-function mutations in the dystrophin gene, results in progressive muscle weakness and early fatality. Impaired autophagy is one of the cellular hallmarks of DMD, contributing to the disease progression. Molecular mechanisms underlying the inhibition of autophagy in DMD are not well understood. In the current study, the DMD mouse model mdx is used for the investigation of signaling pathways leading to suppression of autophagy. Mammalian target of rapamycin complex 1 (mTORC1) is found to be hyperactive in the DMD muscles, accompanying muscle weakness and autophagy impairment. Surprisingly, Akt, a well-known upstream regulator of mTORC1, is not responsible for mTORC1 activation or the dystrophic muscle phenotypes. Instead, leucyl-tRNA synthetase (LeuRS) is found to be overexpressed in mdx muscles compared with the wild type. LeuRS is known to activate mTORC1 in a noncanonical mechanism that involves interaction with RagD, an activator of mTORC1. Disrupting LeuRS interaction with RagD by the small-molecule inhibitor BC-LI-0186 reduces mTORC1 activity, restores autophagy, and ameliorates myofiber damage in the mdx muscles. Furthermore, inhibition of LeuRS by BC-LI-0186 improves dystrophic muscle strength in an autophagy-dependent manner. Taken together, our findings uncover a noncanonical function of the housekeeping protein LeuRS as a potential therapeutic target in the treatment of DMD.

A non-translational role of threonyl-tRNA synthetase in regulating JNK signaling during myogenic differentiation.

Aminoacyl-tRNA synthetases (aaRSs) are house-keeping enzymes that are essential for protein synthesis. However, it has become increasingly evident that some aaRSs also have non-translational functions. Here we report the identification of a non-translational function of threonyl-tRNA synthetase (ThrRS) in myogenic differentiation. We find that ThrRS negatively regulates myoblast differentiation in vitro and injury-induced skeletal muscle regeneration in vivo. This function is independent of amino acid binding or aminoacylation activity of ThrRS, and knockdown of ThrRS leads to enhanced differentiation without affecting the global protein synthesis rate. Furthermore, we show that the non-catalytic new domains (UNE-T and TGS) of ThrRS are both necessary and sufficient for the myogenic function. In searching for a molecular mechanism of this new function, we find the kinase JNK to be a downstream target of ThrRS. Our data further reveal MEKK4 and MKK4 as upstream regulators of JNK in myogenesis and the MEKK4-MKK4-JNK pathway to be a mediator of the myogenic function of ThrRS. Finally, we show that ThrRS physically interacts with Axin1, disrupts Axin1-MEKK4 interaction and consequently inhibits JNK signaling. In conclusion, we uncover a non-translational function for ThrRS in the maintenance of homeostasis of skeletal myogenesis and identify the Axin1-MEKK4-MKK4-JNK signaling axis to be an immediate target of ThrRS action.

Redefining the specificity of phosphoinositide-binding by human PH domain-containing proteins.

Pleckstrin homology (PH) domains are presumed to bind phosphoinositides (PIPs), but specific interaction with and regulation by PIPs for most PH domain-containing proteins are unclear. Here we employ a single-molecule pulldown assay to study interactions of lipid vesicles with full-length proteins in mammalian whole cell lysates. Of 67 human PH domain-containing proteins initially examined, 36 (54%) are found to have affinity for PIPs with various specificity, the majority of which have not been reported before. Further investigation of ARHGEF3 reveals distinct structural requirements for its binding to PI(4,5)P2 and PI(3,5)P2, and functional relevance of its PI(4,5)P2 binding. We generate a recursive-learning algorithm based on the assay results to analyze the sequences of 242 human PH domains, predicting that 49% of them bind PIPs. Twenty predicted binders and 11 predicted non-binders are assayed, yielding results highly consistent with the prediction. Taken together, our findings reveal unexpected lipid-binding specificity of PH domain-containing proteins.

ARHGEF3 Regulates Skeletal Muscle Regeneration and Strength through Autophagy.

Skeletal muscle regeneration after injury is essential for maintaining muscle function throughout aging. ARHGEF3, a RhoA/B-specific GEF, negatively regulates myoblast differentiation through Akt signaling independently of its GEF activity in vitro. Here, we report ARHGEF3's role in skeletal muscle regeneration revealed by ARHGEF3-KO mice. These mice exhibit indiscernible phenotype under basal conditions. Upon acute injury, however, ARHGEF3 deficiency enhances the mass/fiber size and function of regenerating muscles in both young and regeneration-defective middle-aged mice. Surprisingly, these effects occur independently of Akt but via the GEF activity of ARHGEF3. Consistently, overexpression of ARHGEF3 inhibits muscle regeneration in a Rho-associated kinase-dependent manner. We further show that ARHGEF3 KO promotes muscle regeneration through activation of autophagy, a process that is also critical for maintaining muscle strength. Accordingly, ARHGEF3 depletion in old mice prevents muscle weakness by restoring autophagy. Taken together, our findings identify a link between ARHGEF3 and autophagy-related muscle pathophysiology.

Lentivirus-Mediated RNAi in Skeletal Myogenesis.

RNA interference (RNAi) has greatly facilitated investigation of gene functions in vitro as well as in vivo. Recombinant lentivirus is widely used to deliver small hairpin RNA (shRNA) because of its high transduction capacity into diverse cell types and tissues. Here, we describe methods of lentivirus-mediated delivery of shRNA for the study of skeletal muscle cell differentiation in vitro and injury-induced muscle regeneration in mice.

Lanthionine synthetase C-like protein 2 (LanCL2) is important for adipogenic differentiation.

Adipogenic differentiation is a highly regulated process that is necessary for metabolic homeostasis and nutrient sensing. The expression of PPARγ and the subsequent activation of adipogenic genes is critical for the process. In this study, we identified lanthionine synthetase C-like protein 2 (LanCL2) as a positive regulator of adipogenesis in 3T3-L1 cells. Knockdown of LanCL2, but not LanCL1, inhibited adipogenic differentiation, and this effect was not mediated through cAMP or Akt signaling pathways. The expression of early adipogenic markers CCAAT enhancer binding protein ß (C/EBPß) and C/EBPδ remained intact in LanCL2 knockdown cells, but levels of late adipogenic markers PPARγ and C/EBPα were suppressed. The addition of the naturally occurring PPARγ activator 15-deoxy-Δ12,14-prostaglandin J2 or conditioned medium from differentiating cells did not restore differentiation, implying that LanCL2 may not be involved in the production of a secreted endogenous PPARγ ligand. Pulldown assays demonstrated a direct physical interaction between LanCL2 and PPARγ. Consistent with a regulatory role of LanCL2, luciferase reporter assays revealed that full transcriptional activation by PPARγ was dependent on LanCL2. Taken together, our study reveals a novel role of LanCL2 in adipogenesis, specifically involved in PPARγ-mediated transactivation of downstream adipogenic genes.

Effects of rapamycin on growth hormone receptor knockout mice.

It is well documented that inhibition of mTORC1 (defined by Raptor), a complex of mechanistic target of rapamycin (mTOR), extends life span, but less is known about the mechanisms by which mTORC2 (defined by Rictor) impacts longevity. Here, rapamycin (an inhibitor of mTOR) was used in GHR-KO (growth hormone receptor knockout) mice, which have suppressed mTORC1 and up-regulated mTORC2 signaling, to determine the effect of concurrently decreased mTORC1 and mTORC2 signaling on life span. We found that rapamycin extended life span in control normal (N) mice, whereas it had the opposite effect in GHR-KO mice. In the rapamycin-treated GHR-KO mice, mTORC2 signaling was reduced without further inhibition of mTORC1 in the liver, muscle, and s.c. fat. Glucose and lipid homeostasis were impaired, and old GHR-KO mice treated with rapamycin lost functional immune cells and had increased inflammation. In GHR-KO MEF cells, knockdown of Rictor, but not Raptor, decreased mTORC2 signaling. We conclude that drastic reduction of mTORC2 plays important roles in impaired longevity in GHR-KO mice via disruption of whole-body homeostasis.

Cxcl14 depletion accelerates skeletal myogenesis by promoting cell cycle withdrawal.

Skeletal muscle in adults retains a robust ability to regenerate after injury, which progressively declines with age. Many of the regulators of skeletal myogenesis are unknown or incompletely understood. Intriguingly, muscle cells secrete a wide variety of factors, such as cytokines, which can influence muscle development and regeneration in an autocrine or paracrine manner. Here we describe chemokine (C-X-C motif) ligand 14 (Cxcl14) as a novel negative regulator of skeletal myogenesis. We found that Cxcl14 expression in myoblasts prevented cell cycle withdrawal, thereby inhibiting subsequent differentiation. Knockdown of Cxcl14 in vitro enhanced myogenic differentiation through promoting cell cycle withdrawal in an ERK1/2-dependent manner. Recapitulating these in vitro observations, the process of muscle regeneration following injury in young adult mice was accelerated by Cxcl14 depletion, accompanied by reduced cell proliferation. Furthermore, impaired capacity for muscle regeneration in aging mice was fully restored by Cxcl14 depletion. Our results indicate that Cxcl14 may be a promising target for development of therapeutics to treat muscle disease, especially aging-related muscle wasting.