Regulation of the Antioxidant Response by MyoD Transcriptional Coactivator in Castration-resistant Prostate Cancer Cells.

OBJECTIVE: To reveal the potential role of the basic helix-loop-helix myogenic transcription regulator MyoD in the regulation of castration-resistant prostate cancer. METHODS: Expression level of MyoD was assessed in prostate cancer tissues using quantitative reverse transcription polymerase chain reaction and immunohistochemistry and in experimentally induced castration-resistant LNCaP/R cells using quantitative reverse transcription polymerase chain reaction and immunoblotting. Effect of MyoD knockdown on LNCaP/R cell progression was determined by assessing cell proliferation, apoptosis, and colony formation rate. The effect of MyoD knockdown on the oxidative stress state in PC3 cells was determined by assessing antioxidant response gene expression and glutathione synthetase-to-glutathione ratio. Finally, the functional link between the nuclear factor erythroid-derived 2-related factor 1 (NRF1) and the regulation of antioxidant response element-driven transcription by MyoD was studied at both molecular and functional levels. RESULTS: MyoD expression was significantly upregulated in hormone-refractory prostate cancer tissues and in experimentally induced castration-resistant LNCaP/R cells, and MyoD knockdown effectively impaired LNCaP/R cell proliferation and promoted apoptosis under androgen-depleted condition. Moreover, MyoD enhanced the glutathione production and protected against oxidative stress by positively regulating a cluster of antioxidant genes known to be the downstream targets of NRF1. Mechanistically, MyoD could augment the antioxidant response element-driven transcription in an NRF1-dependent manner, and the stimulatory effect of MyoD on the antioxidant response was substantially compromised in the presence of NRF1 small interfering RNA treatment. CONCLUSION: We have identified an unexpected collaboration between MyoD and NRF1 under androgen-depleted condition, which may serve as an important adaptive mechanism during the pathogenesis of castration-resistant prostate cancer.

Developmental dosing with a MEK inhibitor (PD0325901) rescues myopathic features of the muscle-specific but not limb-specific Nf1 knockout mouse.

Neurofibromatosis Type 1 (NF1) is a common autosomal dominant genetic disorder While NF1 is primarily associated with predisposition for tumor formation, muscle weakness has emerged as having a significant impact on quality of life. NF1 inactivation is linked with a canonical upregulation Ras-MEK-ERK signaling. This in this study we tested the capacity of the small molecule MEK inhibitor PD0325901 to influence the intramyocellular lipid accumulation associated with NF1 deficiency. Established murine models of tissue specific Nf1 deletion in skeletal muscle (Nf1MyoD-/-) and limb mesenchyme (Nf1Prx1-/-) were tested. Developmental PD0325901 dosing of dams pregnant with Nf1MyoD-/- progeny rescued the phenotype of day 3 pups including body weight and lipid accumulation by Oil Red O staining. In contrast, PD0325901 treatment of 4 week old Nf1Prx1-/- mice for 8 weeks had no impact on body weight, muscle wet weight, activity, or intramyocellular lipid. Examination of day 3 Nf1Prx1-/- pups showed differences between the two tissue-specific knockout strains, with lipid staining greatest in Nf1MyoD-/- mice, and fibrosis higher in Nf1Prx1-/- mice. These data show that a MEK/ERK dependent mechanism underlies NF1 muscle metabolism during development. However, crosstalk from Nf1-deficient non-muscle mesenchymal cells may impact upon muscle metabolism and fibrosis in neonatal and mature myofibers.

MyoD- and FoxO3-mediated hotspot interaction orchestrates super-enhancer activity during myogenic differentiation.

Super-enhancers (SEs) are cis-regulatory elements enriching lineage specific key transcription factors (TFs) to form hotspots. A paucity of identification and functional dissection promoted us to investigate SEs during myoblast differentiation. ChIP-seq analysis of histone marks leads to the uncovering of SEs which remodel progressively during the course of differentiation. Further analyses of TF ChIP-seq enable the definition of SE hotspots co-bound by the master TF, MyoD and other TFs, among which we perform in-depth dissection for MyoD/FoxO3 interaction in driving the hotspots formation and SE activation. Furthermore, using Myogenin as a model locus, we elucidate the hierarchical and complex interactions among hotspots during the differentiation, demonstrating SE function is propelled by the physical and functional cooperation among hotspots. Finally, we show MyoD and FoxO3 are key in orchestrating the Myogenin hotspots interaction and activation. Altogether our results identify muscle-specific SEs and provide mechanistic insights into the functionality of SE.

miR-195/497 induce postnatal quiescence of skeletal muscle stem cells.

Skeletal muscle stem cells (MuSCs), the major source for skeletal muscle regeneration in vertebrates, are in a state of cell cycle arrest in adult skeletal muscles. Prior evidence suggests that embryonic muscle progenitors proliferate and differentiate to form myofibres and also self-renew, implying that MuSCs, derived from these cells, acquire quiescence later during development. Depletion of Dicer in adult MuSCs promoted their exit from quiescence, suggesting microRNAs are involved in the maintenance of quiescence. Here we identified miR-195 and miR-497 that induce cell cycle arrest by targeting cell cycle genes, Cdc25 and Ccnd. Reduced expression of MyoD in juvenile MuSCs, as a result of overexpressed miR-195/497 or attenuated Cdc25/Ccnd, revealed an intimate link between quiescence and suppression of myogenesis in MuSCs. Transplantation of cultured MuSCs treated with miR-195/497 contributed more efficiently to regenerating muscles of dystrophin-deficient mice, indicating the potential utility of miR-195/497 for stem cell therapies.

Retinoblastoma protein and MyoD function together to effect the repression of Fra-1 and in turn cyclin D1 during terminal cell cycle arrest associated with myogenesis.

The acquisition of skeletal muscle-specific function and terminal cell cycle arrest represent two important features of the myogenic differentiation program. These cellular processes are distinct and can be separated genetically. The lineage-specific transcription factor MyoD and the retinoblastoma protein pRb participate in both of these cellular events. Whether and how MyoD and pRb work together to effect terminal cell cycle arrest is uncertain. To address this question, we focused on cyclin D1, whose stable repression is required for terminal cell cycle arrest and execution of myogenesis. MyoD and pRb are both required for the repression of cyclin D1; their actions, however, were found not to be direct. Rather, they operate to regulate the immediate early gene Fra-1, a critical player in mitogen-dependent induction of cyclin D1. Two conserved MyoD-binding sites were identified in an intronic enhancer of Fra-1 and shown to be required for the stable repression of Fra-1 and, in turn, cyclin D1. Localization of MyoD alone to the intronic enhancer of Fra-1 in the absence of pRb was not sufficient to elicit a block to Fra-1 induction; pRb was also recruited to the intronic enhancer in a MyoD-dependent manner. These observations suggest that MyoD and pRb work together cooperatively at the level of the intronic enhancer of Fra-1 during terminal cell cycle arrest. This work reveals a previously unappreciated link between a lineage-specific transcription factor, a tumor suppressor, and a proto-oncogene in the control of an important facet of myogenic differentiation.

Cellular reprogramming.

Nuclear reprogramming technology was first established more than 50 years ago. It can rejuvenate somatic cells by erasing the epigenetic memories and reconstructing a new pluripotent order. The recent discovery reviewed here that induced pluripotency can be achieved by a small set of transcription factors has opened up unprecedented opportunities in the pharmaceutical industry, the clinic, and laboratories. This technology allows us to access pathological studies by using patient-specific induced pluripotent stem (iPS) cells. In addition, iPS cells are also expected to be a rising star for regenerative medicine, as sources of transplantation therapy.

MyoD is a tumor suppressor gene in medulloblastoma.

While medulloblastoma, a pediatric tumor of the cerebellum, is characterized by aberrations in developmental pathways, the majority of genetic determinants remain unknown. An unbiased Sleeping Beauty transposon screen revealed MyoD as a putative medulloblastoma tumor suppressor. This was unexpected, as MyoD is a muscle differentiation factor and not previously known to be expressed in cerebellum or medulloblastoma. In response to deletion of one allele of MyoD, two other Sonic hedgehog-driven mouse medulloblastoma models showed accelerated tumor formation and death, confirming MyoD as a tumor suppressor in these models. In normal cerebellum, MyoD was expressed in the proliferating granule neuron progenitors that are thought to be precursors to medulloblastoma. Similar to some other tumor suppressors that are induced in cancer, MyoD was expressed in proliferating medulloblastoma cells in three mouse models and in human medulloblastoma cases. This suggests that although expression of MyoD in a proliferating tumor is insufficient to prevent tumor progression, its expression in the cerebellum hinders medulloblastoma genesis.

MyoD regulates apoptosis of myoblasts through microRNA-mediated down-regulation of Pax3.

The molecules that regulate the apoptosis cascade are also involved in differentiation and syncytial fusion in skeletal muscle. MyoD is a myogenic transcription factor that plays essential roles in muscle differentiation. We noticed that MyoD(-/-) myoblasts display remarkable resistance to apoptosis by down-regulation of miR-1 (microRNA-1) and miR-206 and by up-regulation of Pax3. This resulted in transcriptional activation of antiapoptotic factors Bcl-2 and Bcl-xL. Forced MyoD expression induces up-regulation of miR-1 and miR-206 and down-regulation of Pax3, Bcl-2, and Bcl-xL along with increased apoptosis in MyoD(-/-) myoblasts. In contrast, MyoD gene knockdown increases cell survival of wild-type myoblasts. The 3' untranslated region of Pax3 mRNA contains two conserved miR-1/miR-206-binding sites, which are required for targeting of these microRNAs (miRNAs). Therefore, these data suggest that MyoD not only regulates terminal differentiation but also apoptosis through miRNA-mediated down-regulation of Pax3. Finally, MyoD, miR-1, and miR-206 are all down-regulated in quiescent satellite cells, which may be required for maintenance of muscle stem cells.

Enhancement of myogenic and muscle repair capacities of human adipose-derived stem cells with forced expression of MyoD.

Muscle disorders such as Duchenne muscular dystrophy (DMD) still need effective treatments, and mesenchymal stem cells (MSCs) may constitute an attractive cell therapy alternative because they are multipotent and accessible in adult tissues. We have previously shown that human multipotent adipose-derived stem (hMADS) cells were able to restore dystrophin expression in the mdx mouse. The goal of this work was to improve the myogenic potential of hMADS cells and assess the impact on muscle repair. Forced expression of MyoD in vitro strongly induced myogenic differentiation while the adipogenic differentiation was inhibited. Moreover, MyoD-expressing hMADS cells had the capacity to fuse with DMD myoblasts and to restore dystrophin expression. Importantly, transplantation of these modified hMADS cells into injured muscles of immunodepressed Rag2(-/-)gammaC(-/-) mice resulted in a substantial increase in the number of hMADS cell-derived fibers. Our approach combined the easy access of MSCs from adipose tissue, the highly efficient lentiviral transduction of these cells, and the specific improvement of myogenic differentiation through the forced expression of MyoD. Altogether our results highlight the capacity of modified hMADS cells to contribute to muscle repair and their potential to deliver a repairing gene to dystrophic muscles.

MyoD and E-protein heterodimers switch rhabdomyosarcoma cells from an arrested myoblast phase to a differentiated state.

Rhabdomyosarcomas are characterized by expression of myogenic specification genes, such as MyoD and/or Myf5, and some muscle structural genes in a population of cells that continues to replicate. Because MyoD is sufficient to induce terminal differentiation in a variety of cell types, we have sought to determine the molecular mechanisms that prevent MyoD activity in human embryonal rhabdomyosarcoma cells. In this study, we show that a combination of inhibitory Musculin:E-protein complexes and a novel splice form of E2A compete with MyoD for the generation of active full-length E-protein:MyoD heterodimers. A forced heterodimer between MyoD and the full-length E12 robustly restores differentiation in rhabdomyosarcoma cells and broadly suppresses multiple inhibitory pathways. Our studies indicate that rhabdomyosarcomas represent an arrested progress through a normal transitional state that is regulated by the relative abundance of heterodimers between MyoD and the full-length E2A proteins. The demonstration that multiple inhibitory mechanisms can be suppressed and myogenic differentiation can be induced in the RD rhabdomyosarcomas by increasing the abundance of MyoD:E-protein heterodimers suggests a central integrating function that can be targeted to force differentiation in muscle cancer cells.

A highly conserved molecular switch binds MSY-3 to regulate myogenin repression in postnatal muscle.

Myogenin is the dominant transcriptional regulator of embryonic and fetal muscle differentiation and during maturation is profoundly down-regulated. We show that a highly conserved 17-bp DNA cis-acting sequence element located upstream of the myogenin promoter (myogHCE) is essential for postnatal repression of myogenin in transgenic animals. We present multiple lines of evidence supporting the idea that repression is mediated by the Y-box protein MSY-3. Electroporation in vivo shows that myogHCE and MSY-3 are required for postnatal repression. We further show that, in the C2C12 cell culture system, ectopic MSY-3 can repress differentiation, while reduced MSY-3 promotes premature differentiation. MSY-3 binds myogHCE simultaneously with the homeodomain protein Pbx in postnatal innervated muscle. We therefore propose a model in which the myogHCE motif operates as a switch by specifying opposing functions; one that was shown previously is regulated by MyoD and Pbx and it specifies a chromatin opening, gene-activating function at the time myoblasts begin to differentiate; the other includes MYS-3 and Pbx, and it specifies a repression function that operates during and after postnatal muscle maturation in vivo and in myoblasts before they begin to differentiate.

Pharmacological activation of PPARbeta promotes rapid and calcineurin-dependent fiber remodeling and angiogenesis in mouse skeletal muscle.

Recent studies have shown that administration of peroxisome proliferator-activated receptor-beta (PPARbeta) agonists enhances fatty acid oxidation in rodent and human skeletal muscle and that muscle-restricted PPARbeta overexpression affects muscle metabolic profile by increasing oxidative myofiber number, which raises the possibility that PPARbeta agonists alter muscle morphology in adult animals. This possibility was examined in this study in which adult mice were treated with a PPARbeta agonist, and the resulting changes in myofiber metabolic phenotype and angiogenesis were quantified in tibialis anterior muscles. The findings indicate a muscle remodeling that is completed within 2 days and is characterized by a 1.63-fold increase in oxidative fiber number and by a 1.55-fold increase in capillary number. These changes were associated with a quick and transient upregulation of myogenic and angiogenic markers. Both myogenic and angiogenic responses were dependent on the calcineurin pathway, as they were blunted by cyclosporine A administration. In conclusion, the data indicate that PPARbeta activation is associated with a calcineurin-dependent effect on muscle morphology that enhances the oxidative phenotype.

Different autonomous myogenic cell populations revealed by ablation of Myf5-expressing cells during mouse embryogenesis.

The development of myogenic cells is mainly determined by expression of two myogenic factors, Myf5 and Myod1 (MyoD), which genetically compensate for each other during embryogenesis. Here, we demonstrate by conditional cell ablation in mice that Myf5 determines a distinct myogenic cell population, which also contains some Myod1-positive cells. Ablation of this lineage uncovers the presence of a second autonomous myogenic lineage, which superseded Myf5-dependent myogenic cells and expressed Myod1. By contrast, ablation of myogenin-expressing cells erased virtually all differentiated muscle cells, indicating that some aspects of the myogenic program are shared by most skeletal muscle cells. We conclude that Myf5 and Myod1 define different cell lineages with distinct contributions to muscle precursor cells and differentiated myotubes. Individual myogenic cell lineages seem to substitute for each other within the developing embryo.

Malignant transformation of multipotent muscle-derived cells by concurrent differentiation signals.

Recent studies have shown that germ-line determination occurs early in development and that extracellular signaling can alter this fate. This denial of a cell's fate by counteracting its intrinsic signaling pathways through extrinsic stimulation is believed to be associated with oncogenesis. Using specific populations of multipotent skeletal muscle-derived stem cells (MDSCs), we have been able to generate tumors by subjecting cells with specific lineage predilections to concomitant differentiation signals. More specifically, when a stem cell that had a predilection toward osteogenesis was implanted into a skeletal muscle, tumors formed in 25% of implanted mice. When cells predilected to undergo myogenesis were pretreated with bone morphogenetic protein 4 (BMP4) for 4 days prior to implantation, they formed tumors in 25% of mice. These same myogenic predilected cells, when transduced to express BMP4 and implanted into either a long-bone or cranial defect, formed bone, but they formed tumors in 100% of mice when implanted into the skeletal muscle. The tumors generated in this latter study were serially transplantable as long as they retained BMP4 expression. Furthermore, when we impeded the ability of the cells to undergo myogenic differentiation using small interfering RNA to the myogenic regulator MyoD1, we stopped transformation. Based on our findings, we postulate that specific MDSC populations can undergo concomitant signal-induced transformation and that the initial stages of transformation may be due to changes in the balance between the inherent nature of the cell and extrinsic signaling pathways. This theory represents a potential link between somatic stem cells and cancer and suggests an involvement of the niche/environment in transformation.

Macrophages promote muscle membrane repair and muscle fibre growth and regeneration during modified muscle loading in mice in vivo.

Muscle injury or modified muscle use can stimulate muscle invasion by leucocytes that have the potential to increase tissue damage or promote tissue growth and repair. In the present investigation, we examined the role of macrophages in muscle injury, repair and regeneration during modified muscle loading. Weight-bearing was removed from the hindlimbs of mice for 10 days followed by reloading through normal ambulation. During the unloading period, soleus muscle fibre cross-section decreased by 38%. Prior to the onset of reloading, mice received a series of intraperitoneal injections of anti-F4/80, which binds a mouse macrophage surface antigen. Although anti-F4/80 injections did not affect macrophage numbers in soleus muscles at 2 days of reloading, macrophages were reduced by 86% at 4 days of reloading. Muscle membrane lysis during the reloading period did not differ at 2 days of reloading between anti-F4/80-treated mice and mice that received isotype control antibody. However, control animals showed large decreases in the number of fibres with membrane lesions at 4 days of reloading, but this membrane repair did not occur in macrophage-depleted mice. Macrophage-depletion also reduced muscle regeneration (indicated by central nucleation) and satellite cell differentiation (indicated by reductions in MyoD-expressing satellite cells) and prevented growth of muscle fibres that normally occurred in control animals between days 2 and 4 of reloading. These findings collectively show that macrophages play a significant role in muscle fibre membrane repair, regeneration and growth during increased muscle use after a period of atrophy.

MyoD-positive epiblast cells regulate skeletal muscle differentiation in the embryo.

MyoD mRNA is expressed in a subpopulation of cells within the embryonic epiblast. Most of these cells are incorporated into somites and synthesize Noggin. Ablation of MyoD-positive cells in the epiblast subsequently results in the herniation of organs through the ventral body wall, a decrease in the expression of Noggin, MyoD, Myf5, and myosin in the somites and limbs, and an increase in Pax-3-positive myogenic precursors. The addition of Noggin lateral to the somites compensates for the loss of MyoD-positive epiblast cells. Skeletal muscle stem cells that arise in the epiblast are utilized in the somites to promote muscle differentiation by serving as a source of Noggin.

Avian MyoD and c-Jun coordinately induce transcriptional activity of the 3,5,3'-triiodothyronine nuclear receptor c-ErbAalpha1 in proliferating myoblasts.

Although physical interactions with other receptors have been reported, heterodimeric complexes of T(3) nuclear receptors (TR) with retinoid X receptors (RXRs) are considered as major regulators of T(3) target gene expression. However, despite the potent T(3) influence in proliferating myoblasts, RXR isoforms are not expressed during proliferation, raising the question of the nature of the complex involved in TRalpha transcriptional activity. We have previously established that c-Jun induces TRalpha1 transcriptional activity in proliferating myoblasts not expressing RXR. This regulation is specific to the muscle lineage, suggesting the involvement of a muscle-specific factor. In this study, we found that MyoD expression in HeLa cells stimulates TRalpha1 activity, an influence potentiated by c-Jun coexpression. Similarly, in the absence of RXR, MyoD or c-Jun overexpression in myoblasts induces TRalpha1 transcriptional activity through a direct repeat 4 or an inverted palindrome 6 thyroid hormone response element. The highest rate of activity was recorded when c-Jun and MyoD were coexpressed. Using c-Jun-negative dominants, we established that MyoD influence on TRalpha1 activity needs c-Jun functionality. Furthermore, we demonstrated that TRalpha1 and MyoD physically interact in the hinge region of the receptor and the transactivation and basic helix loop helix domains of MyoD. RXR expression (spontaneously occurring at the onset of myoblast differentiation) in proliferating myoblasts abrogates these interactions. These data suggest that in the absence of RXR, TRalpha1 transcriptional activity in myoblasts is mediated through a complex including MyoD and c-Jun.

Myogenic basic helix-loop-helix proteins regulate the expression of peroxisomal proliferator activated receptor-gamma coactivator-1alpha.

Peroxisomal proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), a transcriptional coactivator, is selectively expressed in slow-twitch fibers in skeletal muscle. Ectopic expression of the PGC-1alpha gene in either a cell or an animal has been shown to promote fast to slow fiber-type switch. The expression of PGC-1alpha in muscle is regulated by myocyte enhancer factor 2 and Forkhead in rhabdomyosarcoma, two transcription factors implicated in terminal muscle differentiation. In this study we found that PGC-1alpha expression was activated during terminal muscle differentiation in both C2C12 and Sol8 myoblasts. Using retrovirus-mediated MyoD overexpression in C3H10T1/2 cells, we also demonstrated that MyoD, the master regulator of terminal differentiation, could activate PGC-1alpha expression in vivo. Our transient transfection results also show that myogenic basic helix-loop-helix (bHLH) proteins, especially MyoD, can activate PGC-1alpha expression by targeting its promoter. Myogenic bHLH protein target sites on PGC-1alpha promoter were localized to a short region (-49 to approximately +2) adjacent to the transcription start site, which contains two putative E boxes. Mutation of either site significantly reduced MyoD-mediated transactivation in the cells, suggesting that both sites are required for myogenic bHLH protein-mediated activation. However, only one site, the E2 box, was directly bound by glutathione-S-transferase-MyoD protein in EMSAs. Our results indicate that myogenic bHLH proteins not only are involved in lineage determination and terminal differentiation, but also are directly implicated in activation of the key fiber-type and metabolic switch gene, PGC-1alpha.

p57Kip2 is induced by MyoD through a p73-dependent pathway.

The cyclin-dependent-kinase inhibitors p21 and p57 are highly expressed in skeletal muscle where they redundantly control cell cycle arrest during differentiation. We have previously shown that p57 is a target of the myogenic factor MyoD in cells lacking p21. Here we show that MyoD induces p57 at the transcriptional level through a mechanism different from that involved in p21 regulation, since it is E-box-independent and requires new synthesized protein(s). We have identified p73 family members as the factors that mediate the activation of p57 through a 165bp promoter region. The levels of p73 alpha, beta and delta isoforms increase during muscle differentiation both in MyoD-expressing fibroblasts and in spontaneously differentiating C2 myoblasts. Moreover, the expression of a p73 dominant negative mutant interferes with the induction of p57. Finally, each of the isoforms up-regulated by MyoD, even when over-expressed alone, is capable of inducing p57 in p21-lacking fibroblasts. In contrast, the same p73 isoforms, either induced by MyoD or exogenously over-expressed, are unable to activate the expression of p57 in p21-expressing fibroblasts. Our finding that a transfected p57 promoter-reporter construct, unlike the endogenous gene, is responsive to both MyoD and p73 even in these cells, suggests that a cis-acting mechanism, probably involving a repressive chromatin structure, prevents the induction of p57 in p21-expressing fibroblasts.

Ran binding protein 9 interacts with Raf kinase but does not contribute to downstream ERK1/2 activation in skeletal myoblasts.

Raf kinase is the upstream activator of MEK1/2 leading to phosphorylation and activation of ERK1/2. Sustained activation of Raf represses skeletal muscle-specific reporter gene transcription and formation of multinucleated myofibers. Inhibition of myogenesis by activated Raf involves downstream ERK1/2 as well as undefined mediators. To identify Raf-interacting proteins that may influence repression of muscle formation, a yeast two-hybrid screen was performed using a MEK1-binding defective Raf (RafBXB-T481A) as bait. Twenty cDNAs coding for Raf-interacting proteins were identified including Ran binding protein 9 (RanBP9), a protein previously reported to interact with receptor tyrosine kinases. Forced expression of RanBP9 in myogenic cells did not alter myogenesis. Co-expression of RanBP9 with constitutively active RafBXB, but not RafBXB-T481A, synergistically inhibited MyoD-directed muscle reporter gene transcription. Knockdown of RanBP9 expression did not restore the differentiation program to Raf-expressing myoblasts. Thus, RanBP9 physically associates with Raf but does not substantially contribute to the inhibitory actions of the kinase.