PRMT7 ablation in cardiomyocytes causes cardiac hypertrophy and fibrosis through ß-catenin dysregulation.

Angiotensin II (AngII) has potent cardiac hypertrophic effects mediated through activation of hypertrophic signaling like Wnt/ß-Catenin signaling. In the current study, we examined the role of protein arginine methyltransferase 7 (PRMT7) in cardiac function. PRMT7 was greatly decreased in hypertrophic hearts chronically infused with AngII and cardiomyocytes treated with AngII. PRMT7 depletion in rat cardiomyocytes resulted in hypertrophic responses. Consistently, mice lacking PRMT7 exhibited the cardiac hypertrophy and fibrosis. PRMT7 overexpression abrogated the cellular hypertrophy elicited by AngII, while PRMT7 depletion exacerbated the hypertrophic response caused by AngII. Similar with AngII treatment, the cardiac transcriptome analysis of PRMT7-deficient hearts revealed the alteration in gene expression profile related to Wnt signaling pathway. Inhibition of PRMT7 by gene deletion or an inhibitor treatment enhanced the activity of ß-catenin. PRMT7 deficiency decreases symmetric dimethylation of ß-catenin. Mechanistic studies reveal that methylation of arginine residue 93 in ß-catenin decreases the activity of ß-catenin. Taken together, our data suggest that PRMT7 is important for normal cardiac function through suppression of ß-catenin activity.

Kv4.1, a Key Ion Channel For Low Frequency Firing of Dentate Granule Cells, Is Crucial for Pattern Separation.

The dentate gyrus (DG) in the hippocampus may play key roles in remembering distinct episodes through pattern separation, which may be subserved by the sparse firing properties of granule cells (GCs) in the DG. Low intrinsic excitability is characteristic of mature GCs, but ion channel mechanisms are not fully understood. Here, we investigated ionic channel mechanisms for firing frequency regulation in hippocampal GCs using male and female mice, and identified Kv4.1 as a key player. Immunofluorescence analysis showed that Kv4.1 was preferentially expressed in the DG, and its expression level determined by Western blot analysis was higher at 8-week than 3-week-old mice, suggesting a developmental regulation of Kv4.1 expression. With respect to firing frequency, GCs are categorized into two distinctive groups: low-frequency (LF) and high-frequency (HF) firing GCs. Input resistance (Rin) of most LF-GCs is lower than 200 MΩ, suggesting that LF-GCs are fully mature GCs. Kv4.1 channel inhibition by intracellular perfusion of Kv4.1 antibody increased firing rates and gain of the input-output relationship selectively in LF-GCs with no significant effect on resting membrane potential and Rin, but had no effect in HF-GCs. Importantly, mature GCs from mice depleted of Kv4.1 transcripts in the DG showed increased firing frequency, and these mice showed an impairment in contextual discrimination task. Our findings suggest that Kv4.1 expression occurring at late stage of GC maturation is essential for low excitability of DG networks and thereby contributes to pattern separation.SIGNIFICANCE STATEMENT The sparse activity of dentate granule cells (GCs), which is essential for pattern separation, is supported by high inhibitory inputs and low intrinsic excitability of GCs. Low excitability of GCs is thought to be attributable to a high K+ conductance at resting membrane potentials, but this study identifies Kv4.1, a depolarization-activated K+ channel, as a key ion channel that regulates firing of GCs without affecting resting membrane potentials. Kv4.1 expression is developmentally regulated and Kv4.1 currents are detected only in mature GCs that show low-frequency firing, but not in less mature high-frequency firing GCs. Furthermore, mice depleted of Kv4.1 transcripts in the dentate gyrus show impaired pattern separation, suggesting that Kv4.1 is crucial for sparse coding and pattern separation.

Intracellular Zn2+ Signaling Facilitates Mossy Fiber Input-Induced Heterosynaptic Potentiation of Direct Cortical Inputs in Hippocampal CA3 Pyramidal Cells.

Repetitive action potentials (APs) in hippocampal CA3 pyramidal cells (CA3-PCs) backpropagate to distal apical dendrites, and induce calcium and protein tyrosine kinase (PTK)-dependent downregulation of Kv1.2, resulting in long-term potentiation of direct cortical inputs and intrinsic excitability (LTP-IE). When APs were elicited by direct somatic stimulation of CA3-PCs from rodents of either sex, only a narrow window of distal dendritic [Ca2+] allowed LTP-IE because of Ca2+-dependent coactivation of PTK and protein tyrosine phosphatase (PTP), which renders non-mossy fiber (MF) inputs incompetent in LTP-IE induction. High-frequency MF inputs, however, could induce LTP-IE at high dendritic [Ca2+] of the window. We show that MF input-induced Zn2+ signaling inhibits postsynaptic PTP, and thus enables MF inputs to induce LTP-IE at a wide range of [Ca2+]i values. Extracellular chelation of Zn2+ or genetic deletion of vesicular zinc transporter abrogated the privilege of MF inputs for LTP-IE induction. Moreover, the incompetence of somatic stimulation was rescued by the inhibition of PTP or a supplement of extracellular zinc, indicating that MF input-induced increase in dendritic [Zn2+] facilitates the induction of LTP-IE by inhibiting PTP. Consistently, high-frequency MF stimulation induced immediate and delayed elevations of [Zn2+] at proximal and distal dendrites, respectively. These results indicate that MF inputs are uniquely linked to the regulation of direct cortical inputs owing to synaptic Zn2+ signaling.SIGNIFICANCE STATEMENT Zn2+ has been mostly implicated in pathological processes, and the physiological roles of synaptically released Zn2+ in intracellular signaling are little known. We show here that Zn2+ released from hippocampal mossy fiber (MF) terminals enters postsynaptic CA3 pyramidal cells, and plays a facilitating role in MF input-induced heterosynaptic potentiation of perforant path (PP) synaptic inputs through long-term potentiation of intrinsic excitability (LTP-IE). We show that the window of cytosolic [Ca2+] that induces LTP-IE is normally very narrow because of the Ca2+-dependent coactivation of antagonistic signaling pairs, whereby non-MF inputs become ineffective in inducing excitability change. The MF-induced Zn2+ signaling, however, biases toward facilitating the induction of LTP-IE. The present study elucidates why MF inputs are more privileged for the regulation of PP synapses.

Long non-coding RNA ChRO1 facilitates ATRX/DAXX-dependent H3.3 deposition for transcription-associated heterochromatin reorganization.

Constitutive heterochromatin undergoes a dynamic clustering and spatial reorganization during myogenic differentiation. However the detailed mechanisms and its role in cell differentiation remain largely elusive. Here, we report the identification of a muscle-specific long non-coding RNA, ChRO1, involved in constitutive heterochromatin reorganization. ChRO1 is induced during terminal differentiation of myoblasts, and is specifically localized to the chromocenters in myotubes. ChRO1 is required for efficient cell differentiation, with global impacts on gene expression. It influences DNA methylation and chromatin compaction at peri/centromeric regions. Inhibition of ChRO1 leads to defects in the spatial fusion of chromocenters, and mislocalization of H4K20 trimethylation, Suv420H2, HP1, MeCP2 and cohesin. In particular, ChRO1 specifically associates with ATRX/DAXX/H3.3 complex at chromocenters to promote H3.3 incorporation and transcriptional induction of satellite repeats, which is essential for chromocenter clustering. Thus, our results unveil a mechanism involving a lncRNA that plays a role in large-scale heterochromatin reorganization and cell differentiation.

PRMT7 deficiency enhances adipogenesis through modulation of C/EBP-ß.

Obesity that is critically correlated with the initiation and development of metabolic syndrome and cardiovascular diseases has increased worldwide. Adipogenesis is coordinated through multi-steps involving adipogenic commitment, mitotic clonal expansion (MCE) and differentiation. Recently, protein arginine methyltransferase 4 (PRMT4) and PRMT5 have been implicated in modulation of adipogenesis via regulation of C/EBP-ß activity or PPAR-γ2 expression. In the current study, we demonstrate a suppressive role of PRMT7 in adipogenesis. PRMT7-depleted preadipocytes or PRMT7-/- mouse embryonic fibroblasts (MEFs) displayed increased adipogenesis while PRMT7 overexpression attenuated it. PRMT7 depletion in preadipocytes promoted MCE, an initial step of adipogenesis. Furthermore, we found that PRMT7 interacted with and methylated a key adipogenic factor C/EBP-ß upon adipogenic induction and modulated the accumulation of C/EBP-ß at its target sites in the PPAR-γ2 promoter. Taken together, our data suggest that PRMT7 suppresses adipogenesis through modulation of C/EBP-ß activity.

Vitamin D ameliorates age-induced nonalcoholic fatty liver disease by increasing the mitochondrial contact site and cristae organizing system (MICOS) 60 level.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease. Despite intensive research, considerable information on NAFLD development remains elusive. In this study, we examined the effects of vitamin D on age-induced NAFLD, especially in connection with mitochondrial abnormalities. We observed the prevention of NAFLD in 22-month-old C57BL/6 mice fed a vitamin D3-supplemented (20,000 IU/kg) diet compared with mice fed a control (1000 IU/kg) diet. We evaluated whether vitamin D3 supplementation enhanced mitochondrial functions. We found that the level of mitochondrial contact site and cristae organizing system (MICOS) 60 (Mic60) level was reduced in aged mice, and this reduction was specifically restored by vitamin D3. In addition, depletion of Immt, the human gene encoding the Mic60 protein, induced changes in gene expression patterns that led to fat accumulation in both HepG2 and primary hepatocytes, and these alterations were effectively prevented by vitamin D3. In addition, silencing of the vitamin D receptor (VDR) decreased the Mic60 levels, which were recovered by vitamin D treatment. To assess whether VDR directly regulates Mic60 levels, we performed chromatin immunoprecipitation and reporter gene analysis. We discovered that VDR directly binds to the Immt 5' promoter region spanning positions -3157 to -2323 and thereby upregulates Mic60. Our study provides the first demonstration that a reduction in Mic60 levels due to aging may be one of the mechanisms underlying the development of aging-associated NAFLD. In addition, vitamin D3 could positively regulate Mic60 expression, and this may be one of the important mechanisms by which vitamin D could ameliorate age-induced NAFLD.

Protein Arginine Methyltransferase 1 Ablation in Motor Neurons Causes Mitochondrial Dysfunction Leading to Age-related Motor Neuron Degeneration with Muscle Loss.

Neuromuscular dysfunction is tightly associated with muscle wasting that occurs with age or due to degenerative diseases. However, the molecular mechanisms underlying neuromuscular dysfunction are currently unclear. Recent studies have proposed important roles of Protein arginine methyltransferase 1 (Prmt1) in muscle stem cell function and muscle maintenance. In the current study, we set out to determine the role of Prmt1 in neuromuscular function by generating mice with motor neuron-specific ablation of Prmt1 (mnKO) using Hb9-Cre. mnKO exhibited age-related motor neuron degeneration and neuromuscular dysfunction leading to premature muscle loss and lethality. Prmt1 deficiency also impaired motor function recovery and muscle reinnervation after sciatic nerve injury. The transcriptome analysis of aged mnKO lumbar spinal cords revealed alterations in genes related to inflammation, cell death, oxidative stress, and mitochondria. Consistently, mnKO lumbar spinal cords of sciatic nerve injury model or aged mice exhibited elevated cellular stress response in motor neurons. Furthermore, Prmt1 inhibition in motor neurons elicited mitochondrial dysfunction. Our findings demonstrate that Prmt1 ablation in motor neurons causes age-related motor neuron degeneration attributing to muscle loss. Thus, Prmt1 is a potential target for the prevention or intervention of sarcopenia and neuromuscular dysfunction related to aging.

Farnesol prevents aging-related muscle weakness in mice through enhanced farnesylation of Parkin-interacting substrate.

Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a master regulator of mitochondrial biogenesis. Reduced PGC-1α abundance is linked to skeletal muscle weakness in aging or pathological conditions, such as neurodegenerative diseases and diabetes; thus, elevating PGC-1α abundance might be a promising strategy to treat muscle aging. Here, we performed high-throughput screening and identified a natural compound, farnesol, as a potent inducer of PGC-1α. Farnesol administration enhanced oxidative muscle capacity and muscle strength, leading to metabolic rejuvenation in aged mice. Moreover, farnesol treatment accelerated the recovery of muscle injury associated with enhanced muscle stem cell function. The protein expression of Parkin-interacting substrate (PARIS/Zfp746), a transcriptional repressor of PGC-1α, was elevated in aged muscles, likely contributing to PGC-1α reduction. The beneficial effect of farnesol on aged muscle was mediated through enhanced PARIS farnesylation, thereby relieving PARIS-mediated PGC-1α suppression. Furthermore, short-term exercise increased PARIS farnesylation in the muscles of young and aged mice, whereas long-term exercise decreased PARIS expression in the muscles of aged mice, leading to the elevation of PGC-1α. Collectively, the current study demonstrated that the PARIS-PGC-1α pathway is linked to muscle aging and that farnesol treatment can restore muscle functionality in aged mice through increased farnesylation of PARIS.

Cytoplasmic LMO2-LDB1 Complex Activates STAT3 Signaling through Interaction with gp130-JAK in Glioma Stem Cells.

The oncogenic role of nuclear LIM domain only 2 (LMO2) as a transcriptional regulator is well established, but its function in the cytoplasm is largely unknown. Here, we identified LMO2 as a cytoplasmic activator for signal transducer and activator of transcription 3 (STAT3) signaling in glioma stem cells (GSCs) through biochemical and bioinformatics analyses. LMO2 increases STAT3 phosphorylation by interacting with glycoprotein 130 (gp130) and Janus kinases (JAKs). LMO2-driven activation of STAT3 signaling requires the LDB1 protein and leads to increased expression of an inhibitor of differentiation 1 (ID1), a master regulator of cancer stemness. Our findings indicate that the cytoplasmic LMO2-LDB1 complex plays a crucial role in the activation of the GSC signaling cascade via interaction with gp130 and JAK1/2. Thus, LMO2-LDB1 is a bona fide oncogenic protein complex that activates either the JAK-STAT signaling cascade in the cytoplasm or direct transcriptional regulation in the nucleus.

Generation of reproductive transgenic pigs of a CRISPR-Cas9-based oncogene-inducible system by somatic cell nuclear transfer.

Alternative cancer models that are close to humans are required to create more valuable preclinical results during oncology studies. Here, a new onco-pig model via developing a CRISPR-Cas9-based Conditional Polycistronic gene expression Cassette (CRI-CPC) system to control the tumor inducing simian virus 40 large T antigen (SV40LT) and oncogenic HRASG12V . After conducting somatic cell nuclear transfer (SCNT), transgenic embryos were transplanted into surrogate mothers and five male piglets were born. Umbilical cord analysis confirmed that all piglets were transgenic. Two of them survived and they expressed a detectable green fluorescence. The test was made whether CRI-CPC models were naturally fertile and whether the CRI-CPC system was stably transferred to the offspring. By mating with a normal female pig, four offspring piglets were successfully produced. Among them, only three male piglets were transgenic. Finally, their applicability was tested as cancer models after transduction of Cas9 into fibroblasts from each CRI-CPC pig in vitro, resulting in cell acquisition of cancerous characteristics via the induction of oncogene expression. These results showed that our new CRISPR-Cas9-based onco-pig model was successfully developed.

The oncogenic JAG1 intracellular domain is a transcriptional cofactor that acts in concert with DDX17/SMAD3/TGIF2.

Jagged1 (JAG1) is a Notch ligand that contact-dependently activates Notch receptors and regulates cancer progression. The JAG1 intracellular domain (JICD1) is generated from JAG1, like formation of the NOTCH1 intracellular domain (NICD1); however, the role of JICD1 in tumorigenicity has not been comprehensively elucidated. Here we show that JICD1 induces astrocytes to acquire several cancer stem cell properties, including tumor formation, invasiveness, stemness, and resistance to anticancer therapy. The transcriptome, chromatin immunoprecipitation sequencing (ChIP-seq), and proteomics analyses show that JICD1 increases SOX2 expression by forming a transcriptional complex with DDX17, SMAD3, and TGIF2. JICD1-driven tumorigenicity is directly regulated by SOX2. Our results demonstrate that, like NICD1, JICD1 acts as a transcriptional cofactor in formation of the DDX17/SMAD3/TGIF2 transcriptional complex, leading to oncogenic transformation.

Impaired pattern separation in Tg2576 mice is associated with hyperexcitable dentate gyrus caused by Kv4.1 downregulation.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that causes memory loss. Most AD researches have focused on neurodegeneration mechanisms. Considering that neurodegenerative changes are not reversible, understanding early functional changes before neurodegeneration is critical to develop new strategies for early detection and treatment of AD. We found that Tg2576 mice exhibited impaired pattern separation at the early preclinical stage. Based on previous studies suggesting a critical role of dentate gyrus (DG) in pattern separation, we investigated functional changes in DG of Tg2576 mice. We found that granule cells in DG (DG-GCs) in Tg2576 mice showed increased action potential firing in response to long depolarizations and reduced 4-AP sensitive K+-currents compared to DG-GCs in wild-type (WT) mice. Among Kv4 family channels, Kv4.1 mRNA expression in DG was significantly lower in Tg2576 mice. We confirmed that Kv4.1 protein expression was reduced in Tg2576, and this reduction was restored by antioxidant treatment. Hyperexcitable DG and impaired pattern separation in Tg2576 mice were also recovered by antioxidant treatment. These results highlight the hyperexcitability of DG-GCs as a pathophysiologic mechanism underlying early cognitive deficits in AD and Kv4.1 as a new target for AD pathogenesis in relation to increased oxidative stress.

Phage PPPL-1, A New Biological Agent to Control Bacterial Canker Caused by Pseudomonas syringae pv. actinidiae in Kiwifruit.

Pseudomonas syringae pv. actinidiae (Psa) is a Gram-negative bacterium that causes bacterial canker disease in kiwifruit. Copper or antibiotics have been used in orchards to control this disease, but the recent emergence of antibiotic-resistant Psa has called for the development of a new control agent. We previously reported that the bacteriophage (or phage) PPPL-1 showed antibacterial activity for both biovar 2 and 3 of Psa. To investigate the possibility of PPPL-1 to control bacterial canker in kiwifruit, we further tested the efficacy of PPPL-1 and its phage cocktail with two other phages on suppressing disease development under greenhouse conditions using 6 weeks old kiwifruit plants. Our results showed that the disease control efficacy of PPPL-1 treatment was statistically similar to those of phage cocktail treatment or AgrimycinTM, which contains streptomycin and oxytetracycline antibiotics as active ingredients. Moreover, PPPL-1 could successfully kill streptomycin-resistant Psa isolates, of which the treatment of BuramycinTM carrying only streptomycin as an active ingredient had no effect in vitro. The phage PPPL-1 was further characterized, and stability assays showed that the phage was stable in the field soil and at low temperature of 0 ± 2 °C. In addition, the phage could be scaled up quickly up to 1010 pfu/mL at 12 h later from initial multiplicity of infection of 0.000005. Our results indicate that PPPL-1 phage is a useful candidate as a biocontrol agent and could be a tool to control the bacterial canker in kiwifruit by Psa infection in the field conditions.

Calbindin regulates Kv4.1 trafficking and excitability in dentate granule cells via CaMKII-dependent phosphorylation.

Calbindin, a major Ca2+ buffer in dentate granule cells (GCs), plays a critical role in shaping Ca2+ signals, yet how it regulates neuronal function remains largely unknown. Here, we found that calbindin knockout (CBKO) mice exhibited dentate GC hyperexcitability and impaired pattern separation, which co-occurred with reduced K+ current due to downregulated surface expression of Kv4.1. Relatedly, manipulation of calbindin expression in HT22 cells led to changes in CaMKII activation and the level of surface localization of Kv4.1 through phosphorylation at serine 555, confirming the mechanism underlying neuronal hyperexcitability in CBKO mice. We also discovered that Ca2+ buffering capacity was significantly reduced in the GCs of Tg2576 mice to the level of CBKO GCs, and this reduction was restored to normal levels by antioxidants, suggesting that calbindin is a target of oxidative stress. Our data suggest that the regulation of CaMKII signaling by Ca2+ buffering is crucial for neuronal excitability regulation.

Cdo Is Required for Efficient Motor Neuron Generation of Embryonic Stem Cells.

BACKGROUND AND OBJECTIVES: The directed differentiation of pluripotent stem cells into motor neurons is critical for the development of disease modelling and therapeutics to intervene degenerative motor neuron diseases. Cell surface receptor Cdo functions as a coreceptor for Sonic hedgehog (Shh) with Boc and Gas1 in the patterning of ventral spinal cord neurons including motor neurons. However, the discrete function of Cdo is not fully understood. METHODS AND RESULTS: In this study, we examined the role of Cdo in motor neuron generation by utilizing in vitro differentiation of Cdo＋/＋ and Cdo-/- embryonic stem cells (ESCs). In response to Shh, Cdo-/- ESCs exhibited impaired expression of motor neuron specification markers while dorsal interneuron specification markers were significantly increased, compared to Cdo＋/＋ ESCs. Reactivation of Shh signalling pathway with Smoothened (Smo) agonist (SAG) restored motor neuron specification in Cdo-/- ESCs. In addition, electrophysiological analysis revealed the immature electrical features of Cdo-/- ESCs-derived neurons which was restored by SAG. CONCLUSIONS: Taken together, these data suggest that Cdo as a Shh coreceptor is required for the induction of motor neuron generation by fully activating Shh signalling pathway and provide additional insights into the biology of motor neuron development.

Correction: PRMT1 suppresses ATF4-mediated endoplasmic reticulum response in cardiomyocytes.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

PRMT7 methylates and suppresses GLI2 binding to SUFU thereby promoting its activation.

Cellular senescence is implicated in aging or age-related diseases. Sonic hedgehog (Shh) signaling, an inducer of embryonic development, has recently been demonstrated to inhibit cellular senescence. However, the detailed mechanisms to activate Shh signaling to prevent senescence is not well understood. Here, we demonstrate that Protein arginine methyltransferase 7 (PRMT7) promotes Shh signaling via GLI2 methylation which is critical for suppression of cellular senescence. PRMT7-deficient mouse embryonic fibroblasts (MEFs) exhibited a premature cellular senescence with accompanied increase in the cell cycle inhibitors p16 and p21. PRMT7 depletion results in reduced Shh signaling activity in MEFs while PRMT7 overexpression enhances GLI2-reporter activities that are sensitive to methylation inhibition. PRMT7 interacts with and methylates GLI2 on arginine residues 225 and 227 nearby a binding region of SUFU, a negative regulator of GLI2. This methylation interferes with GLI2-SUFU binding, leading to facilitation of GLI2 nuclear accumulation and Shh signaling. Taken together, these data suggest that PRMT7 induces GLI2 methylation, reducing its binding to SUFU and increasing Shh signaling, ultimately leading to prevention of cellular senescence.

Satellite cell-specific ablation of Cdon impairs integrin activation, FGF signalling, and muscle regeneration.

BACKGROUND: Perturbation in cell adhesion and growth factor signalling in satellite cells results in decreased muscle regenerative capacity. Cdon (also called Cdo) is a component of cell adhesion complexes implicated in myogenic differentiation, but its role in muscle regeneration remains to be determined. METHODS: We generated inducible satellite cell-specific Cdon ablation in mice by utilizing a conditional Cdon allele and Pax7 CreERT2 . To induce Cdon ablation, mice were intraperitoneally injected with tamoxifen (tmx). Using cardiotoxin-induced muscle injury, the effect of Cdon depletion on satellite cell function was examined by histochemistry, immunostaining, and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assay. Isolated myofibers or myoblasts were utilized to determine stem cell function and senescence. To determine pathways related to Cdon deletion, injured muscles were subjected to RNA sequencing analysis. RESULTS: Satellite cell-specific Cdon ablation causes impaired muscle regeneration with fibrosis, likely attributable to decreased proliferation, and senescence, of satellite cells. Cultured Cdon-depleted myofibers exhibited 32 ± 9.6% of EdU-positive satellite cells compared with 58 ± 4.4% satellite cells in control myofibers (P < 0.05). About 32.5 ± 3.7% Cdon-ablated myoblasts were positive for senescence-associated ß-galactosidase (SA-ß-gal) while only 3.6 ± 0.5% of control satellite cells were positive (P < 0.001). Transcriptome analysis of muscles at post-injury Day 4 revealed alterations in genes related to mitogen-activated protein kinase signalling (P < 8.29 e-5 ) and extracellular matrix (P < 2.65 e-24 ). Consistent with this, Cdon-depleted tibialis anterior muscles had reduced phosphorylated extracellular signal-regulated kinase (p-ERK) protein levels and expression of ERK targets, such as Fos (0.23-fold) and Egr1 (0.31-fold), relative to mock-treated control muscles (P < 0.001). Cdon-depleted myoblasts exhibited impaired ERK activation in response to basic fibroblast growth factor. Cdon ablation resulted in decreased and/or mislocalized integrin ß1 activation in satellite cells (weak or mislocalized integrin1 in tmx = 38.7 ± 1.9%, mock = 21.5 ± 6%, P < 0.05), previously linked with reduced fibroblast growth factor (FGF) responsiveness in aged satellite cells. In mechanistic studies, Cdon interacted with and regulated cell surface localization of FGFR1 and FGFR4, likely contributing to FGF responsiveness of satellite cells. Satellite cells from a progeria model, Zmpste24-/- myofibers, showed decreased Cdon levels (Cdon-positive cells in Zmpste24-/- = 63.3 ± 11%, wild type = 90 ± 7.7%, P < 0.05) and integrin ß1 activation (weak or mislocalized integrin ß1 in Zmpste24-/- = 64 ± 6.9%, wild type = 17.4 ± 5.9%, P < 0.01). CONCLUSIONS: Cdon deficiency in satellite cells causes impaired proliferation of satellite cells and muscle regeneration via aberrant integrin and FGFR signalling.

ZNF746/PARIS overexpression induces cellular senescence through FoxO1/p21 axis activation in myoblasts.

Various stresses, including oxidative stress, impair the proliferative capacity of muscle stem cells leading to declined muscle regeneration related to aging or muscle diseases. ZNF746 (PARIS) is originally identified as a substrate of E3 ligase Parkin and its accumulation is associated with Parkinson's disease. In this study, we investigated the role of PARIS in myoblast function. PARIS is expressed in myoblasts and decreased during differentiation. PARIS overexpression decreased both proliferation and differentiation of myoblasts without inducing cell death, whereas PARIS depletion enhanced myoblast differentiation. Interestingly, high levels of PARIS in myoblasts or fibroblasts induced cellular senescence with alterations in gene expression associated with p53 signaling, inflammation, and response to oxidative stress. PARIS overexpression in myoblasts starkly enhanced oxidative stress and the treatment of an antioxidant Trolox attenuated the impaired proliferation caused by PARIS overexpression. FoxO1 and p53 proteins are elevated in PARIS-overexpressing cells leading to p21 induction and the depletion of FoxO1 or p53 reduced p21 levels induced by PARIS overexpression. Furthermore, both PARIS and FoxO1 were recruited to p21 promoter region and Trolox treatment attenuated FoxO1 recruitment. Taken together, PARIS upregulation causes oxidative stress-related FoxO1 and p53 activation leading to p21 induction and cellular senescence of myoblasts.

Prmt7 promotes myoblast differentiation via methylation of p38MAPK on arginine residue 70.

MyoD functions as a master regulator to induce muscle-specific gene expression and myogenic differentiation. Here, we demonstrate a positive role of Protein arginine methyltransferase 7 (Prmt7) in MyoD-mediated myoblast differentiation through p38MAPK activation. Prmt7 depletion in primary or C2C12 myoblasts impairs cell cycle withdrawal and myogenic differentiation. Furthermore, Prmt7 depletion decreases the MyoD-reporter activities and the MyoD-mediated myogenic conversion of fibroblasts. Together with MyoD, Prmt7 is recruited to the Myogenin promoter region and Prmt7 depletion attenuates the recruitment of MyoD and its coactivators. The mechanistic study reveals that Prmt7 methylates p38MAPKα at the arginine residue 70, thereby promoting its activation which in turn enhances MyoD activities. The arginine residue 70 to alanine mutation in p38MAPKα impedes MyoD/E47 heterodimerization and the recruitment of Prmt7, MyoD and Baf60c to the Myogenin promoter resulting in blunted Myogenin expression. In conclusion, Prmt7 promotes MyoD-mediated myoblast differentiation through methylation of p38MAPKα at arginine residue 70.