Inhibits Muscle Cell Differentiation Research Articles

Excess PrPC inhibits muscle cell differentiation via miRNA-enhanced liquid–liquid phase separation implicated in myopathy

The cellular prion protein (PrPC) is required for skeletal muscle function. Here, we report that a higher level of PrPC accumulates in the cytoplasm of the skeletal muscle of six myopathy patients compared to controls. PrPC inhibits skeletal muscle cell autophagy, and blocks myoblast differentiation. PrPC selectively binds to a subset of miRNAs during myoblast differentiation, and the colocalization of PrPC and miR-214-3p was observed in the skeletal muscle of six myopathy patients with excessive PrPC. We demonstrate that PrPC is overexpressed in skeletal muscle cells under pathological conditions, inhibits muscle cell differentiation by physically interacting with a subset of miRNAs, and selectively recruits these miRNAs into its phase-separated condensate in living myoblasts, which in turn enhances liquid–liquid phase separation of PrPC, promotes pathological aggregation of PrP, and results in the inhibition of autophagy-related protein 5-dependent autophagy and muscle bundle formation in myopathy patients characterized by incomplete muscle regeneration.

Indoxyl sulfate inhibits muscle cell differentiation via Myf6/MRF4 and MYH2 downregulation.

Chronic kidney disease (CKD) is associated with a significant decrease in muscle strength and mass, possibly related to muscle cell damage by uremic toxins. Here, we studied in vitro and in vivo the effect of indoxyl sulfate (IS), an indolic uremic toxin, on myoblast proliferation, differentiation and expression of myogenic regulatory factors (MRF)-myoblast determination protein 1 (MyoD1), myogenin (Myog), Myogenic Factor 5 (Myf5) and myogenic regulatory factor 4 (Myf6/MRF4)-and expression of myosin heavy chain, Myh2. C2C12 myoblasts were cultured in vitro and differentiated in myotubes for 7 days in the presence of IS at a uremic concentration of 200µM. Myocytes morphology and differentiation was analyzed after hematoxylin-eosin staining. MRF genes' expression was studied using reverse transcription polymerase chain reaction in myocytes and 5/6th nephrectomized mice muscle. Myf6/MRF4 protein expression was studied using enzyme-linked immunosorbent assay; MYH2 protein expression was studied using western blotting. The role of Aryl Hydrocarbon Receptor (AHR)-the cell receptor of IS-was studied by adding an AHR inhibitor into the cell culture milieu. In the presence of IS, the myotubes obtained were narrower and had fewer nuclei than control myotubes. The presence of IS during differentiation did not modify the gene expression of the MRFs Myf5, MyoD1 and Myog, but induced a decrease in expression of Myf6/MRF4 and MYH2 at the mRNA and the protein level. AHR inhibition by CH223191 did not reverse the decrease in Myf6/MRF4 mRNA expression induced by IS, which rules out the implication of the ARH genomic pathway. In 5/6th nephrectomized mice, the Myf6/MRF4 gene was down-regulated in striated muscles. In conclusion, IS inhibits Myf6/MRF4 and MYH2 expression during differentiation of muscle cells, which could lead to a defect in myotube structure. Through these new mechanisms, IS could participate in muscle atrophy observed in CKD.

Dual-specificity phosphatase 29 is induced during neurogenic skeletal muscle atrophy and attenuates glucocorticoid receptor activity in muscle cell culture.

Skeletal muscle atrophy is caused by a decrease in muscle size and strength and results from a range of physiological conditions, including denervation, immobilization, corticosteroid exposure and aging. Newly named dual-specificity phosphatase 29 (Dusp29) has been identified as a novel neurogenic atrophy-induced gene in skeletal muscle. Quantitative PCR analysis revealed that Dusp29 expression is significantly higher in differentiated myotubes compared with proliferating myoblasts. To determine how Dusp29 is transcriptionally regulated in skeletal muscle, fragments of the promoter region of Dusp29 were cloned, fused to a reporter gene, and found to be highly inducible in response to ectopic expression of the myogenic regulatory factors (MRF), MyoD and myogenin. Furthermore, site-directed mutagenesis of conserved E-box elements within the proximal promoter of Dusp29 rendered a Dusp29 reporter gene unresponsive to MRF overexpression. Dusp29, an atypical Dusp also known as Dupd1/Dusp27, was found to attenuate the ERK1/2 branch of the MAP kinase signaling pathway in muscle cells and inhibit muscle cell differentiation when ectopically expressed in proliferating myoblasts. Interestingly, Dusp29 was also found to destabilize AMPK protein while simultaneously enriching the phosphorylated pool of AMPK in muscle cells. Additionally, Dusp29 overexpression resulted in a significant increase in the glucocorticoid receptor (GR) protein and elevation in GR phosphorylation. Finally, Dusp29 was found to significantly impair the ability of the glucocorticoid receptor to function as a transcriptional activator in muscle cells treated with dexamethasone. Identifying and characterizing the function of Dusp29 in muscle provides novel insights into the molecular and cellular mechanisms for skeletal muscle atrophy.

Methylmercury modifies temporally expressed myogenic regulatory factors to inhibit myoblast differentiation

Methylmercury (MeHg) is a pervasive environmental toxicant, with known detrimental effects on neurodevelopment. Despite a longstanding paradigm of neurotoxicity, where motor deficits are prevalent among those developmentally exposed, consideration of muscle as a MeHg target has received minimal investigation. Recent evidence has identified muscle-specific gene networks that modulate developmental sensitivity to MeHg toxicity. One such network is muscle cell differentiation. Muscle cell differentiation is a coordinated process regulated by the myogenic regulatory factors (MRFs): Myf5, MyoD, MyoG, and MRF4. A previous study demonstrated that MeHg inhibits muscle cell differentiation in vitro, concurrent with reduced MyoG expression. The potential for MeHg to modify the temporal expression of the MRFs to alter differentiation, however, has yet to be fully explored. Using the C2C12 mouse myoblast model, we examined MRF expression profiles at various stages subsequent to MeHg exposure to proliferating myoblasts. MeHg was seen to persistently alter myoblast differentiation capacity, as myod, myog, and mrf4 gene expression were all affected. Myog exhibited the most robust changes in expression across the various culture conditions, while myf5 was unaffected. Following MeHg exposure to myoblasts, where elevated p21 expression indicated departure from proliferation, cells failed to subsequently differentiate, even in the absence of MeHg, as reflected by a concurrent reduction in MRF4 and myosin heavy chain (MHC), markers of terminal differentiation. Our results indicate that within a brief window of exposure MeHg can disrupt the intrinsic myogenic differentiation program of proliferative myoblasts.

Dual Specificity Phosphatase and Pro Isomerase Domain Containing 1 (Dupd1) is Upregulated During Neurogenic Skeletal Muscle Atrophy and is Differentially Expressed in MuRF1‐null Mice

Skeletal muscle atrophy is caused by a decrease in muscle size and strength and results from a range of physiological conditions, including denervation, immobilization, corticosteroid exposure and aging. Dual Specificity Phosphatase and Pro Isomerase Domain Containing 1 (Dupd1) has been identified as a novel atrophy‐induced gene in skeletal muscle and has been found to be differentially regulated in wild‐type mice compared to Muscle RING Finger 1 (MuRF1) knockout mice in response to denervation. Muscle RING Finger 1 (MuRF1), an E3 ubiquitin ligase, has previously been shown to be upregulated under almost all conditions of skeletal muscle wasting. Preliminary data described in this study provides evidence that MuRF1 may play a more complex role in the muscle atrophy cascade than previously believed and appears to be necessary for full activation of a subset of neurogenic atrophy‐induced genes, including Dupd1. A recent study that utilized microarray analysis of skeletal muscle isolated from wild‐type and MuRF1‐null mice revealed that Dupd1 expression is significantly induced at 3 days and 14 days post‐denervation in wild‐type mice, while Dupd1 expression is dramatically blunted in the MuRF1‐null mice at both 3 days and 14 days post‐denervation. qPCR analysis revealed that Dupd1 is significantly upregulated during differentiation of cultured muscle cells. To determine how Dupd1 might be transcriptionally regulated in skeletal muscle, fragments of the promoter region of Dupd1 were cloned, fused to a reporter gene and found to be highly inducible in response to ectopic expression of MyoD and myogenin. Furthermore, site‐directed mutagenesis of conserved E‐box elements in the proximal promoter of Dupd1 rendered the Dupd1 reporter genes incapable of being induced by overexpression of myogenic regulatory factors. The Dupd1 gene codes for a dual specificity phosphatase (DUSP) that can recognize and dephosphorylate tyrosine, serine and threonine amino acids of substrate proteins. Finally, we have found that Dupd1, an atypical DUSP, can modulate the ERK branch of the MAP Kinase signaling in muscle cells and inhibits muscle cell differentiation when ectopically expressed in proliferating muscle cells. Elucidating the role of Dupd1 in muscle will help contribute new insight into the molecular mechanisms for skeletal muscle atrophy.Support or Funding InformationThe work was support by University of North Florida Transformational Learning Opportunity grants and a University of North Florida Foundation Board Grant to D.W.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MicroRNA-29a promotes smooth muscle cell differentiation from stem cells by targeting YY1

MicroRNA-29a (miR-29a) has been extensively studied in tumor biology and fibrotic diseases, but little is known about its functional roles in vascular smooth muscle cell (VSMC) differentiation from embryonic stem cells (ESCs). Using well-established VSMC differentiation models, we have observed that miR-29a induces VSMC differentiation from mouse ESCs by negatively regulating YY1, a transcription factor that inhibits muscle cell differentiation and muscle-specific gene expression. Moreover, gene expression levels of three VSMC specific transcriptional factors were up-regulated by miR-29a over-expression, but down-regulated by miR-29a inhibition or YY1 over-expression. Taken together, our data demonstrate that miR-29a and its target gene, YY1, play a regulatory role in VSMC differentiation from ESCs in vitro and in vivo.

MiR-30-5p Regulates Muscle Differentiation and Alternative Splicing of Muscle-Related Genes by Targeting MBNL.

MicroRNAs (miRNAs), a class of single stranded, small (~22 nucleotides), non-coding RNAs, play an important role in muscle development. We focused on the role of the miR-30-5p family during bovine muscle development from previous high-throughput sequencing results and analyzed their expression profiles. MHC and MyoG mRNAs expression as well as their proteins were suppressed in differentiated C2C12 cells, suggesting the importance of miR-30-5p in muscle development. MBNL, the candidate target of miR-30-5p, is an alternative splicing regulation factor. MBNL1 and MBNL3 have opposite effects on muscle differentiation. Our results confirmed that miR-30a-5p and miR-30e-5p repress the expression of MBNL1, MBNL2 and MBNL3, whereas miR-30b-5p inhibits MBNL1 and MBNL2 expression. This provides direct evidence that MBNL expression can be flexibly regulated by miR-30-5p. Previous studies showed that MBNL1 promotes exon inclusion of two muscle-related genes (Trim55 and INSR). Through RNA splicing studies, we found that miR-30-5p had an effect on their alternative splicing, which means miR-30-5p via MBNL1 could be integrated into muscle signaling pathways in which INSR or Trim55 are located. In conclusion, miR-30-5p could inhibit muscle cell differentiation and regulate the alternative splicing of Trim55 and INSR by targeting MBNL. These results promote the understanding of the function of miRNAs in muscle development.

Nutlin-3 down-regulates retinoblastoma protein expression and inhibits muscle cell differentiation

The p53 tumor suppressor gene plays a critical role in regulation of proliferation, cell death and differentiation. The MDM2 oncoprotein is a major negative regulator for p53 by binding to and targeting p53 for proteasome-mediated degradation. The small molecule inhibitor, nutlin-3, disrupts MDM2-p53 interaction resulting in stabilization and activation of p53 protein. We have previously shown that nutlin-3 activates p53, leading to MDM2 accumulation as concomitant of reduced retinoblastoma (Rb) protein stability. It is well known that Rb is important in muscle development and myoblast differentiation and that rhabdomyosarcoma (RMS), or cancer of the skeletal muscle, typically harbors MDM2 amplification. In this study, we show that nutlin-3 inhibited myoblast proliferation and effectively prevented myoblast differentiation, as evidenced by lack of expression of muscle differentiation markers including myogenin and myosin heavy chain (MyHC), as well as a failure to form multinucleated myotubes, which were associated with dramatic increases in MDM2 expression and decrease in Rb protein levels. These results indicate that nutlin-3 can effectively inhibit muscle cell differentiation.

USP19 deubiquitinating enzyme inhibits muscle cell differentiation by suppressing unfolded-protein response signaling.

The USP19 deubiquitinating enzyme modulates the expression of myogenin and myofibrillar proteins in L6 muscle cells. This raised the possibility that USP19 might regulate muscle cell differentiation. We therefore tested the effects of adenoviral-mediated overexpression or small interfering RNA (siRNA)-mediated silencing of either the cytoplasmic or endoplasmic reticulum (ER)-localized isoforms of USP19. Only the ER-localized isoform of USP19 (USP19-ER) modulated myoblast fusion as well as the expression of myogenin and myofibrillar proteins, and these effects were also dependent on USP19 catalytic activity. USP19-ER inhibited muscle cell differentiation and the induction of CHOP, a transcription factor in the unfolded-protein response (UPR) that is activated during differentiation. Inducing the UPR by creating mild ER stress with thapsigargin was able to reverse the defect in myoblast fusion caused by the overexpression of USP19-ER, suggesting strongly that USP19 exerts its effects on fusion through its effects on UPR signaling. USP19 also functions similarly in vivo, as USP19(-/-) mice display improved muscle regeneration concomitant with enhanced expression of CHOP. Collectively these results implicate a deubiquitinating enzyme as a regulator of the UPR. They also suggest that inhibition of USP19 may be a therapeutic approach for the enhancement of muscle growth following injury.

MiR-186 Inhibits Muscle Cell Differentiation through Myogenin Regulation

The complex process of skeletal muscle differentiation is organized by the myogenic regulatory factors (MRFs), Myf5, MyoD, Myf6, and myogenin, where myogenin plays a critical role in the regulation of the final stage of muscle differentiation. In an effort to investigate the role microRNAs (miRNAs) play in regulating myogenin, a bioinformatics approach was used and six miRNAs (miR-182, miR-186, miR-135, miR-491, miR-329, and miR-96) were predicted to bind the myogenin 3'-untranslated region (UTR). However, luciferase assays showed only miR-186 inhibited translation and 3'-UTR mutagenesis analysis confirmed this interaction was specific. Interestingly, the expression of miR-186 mirrored that of its host gene, ZRANB2, during development. Functional studies demonstrated that miR-186 overexpression inhibited the differentiation of C2C12 and primary muscle cells. Our findings therefore identify miR-186 as a novel regulator of myogenic differentiation.

Abstract 4786: A role for increased Fra-1 expression in cachexia: Novel mechanisms of increased apoptosis in C2C12 myotubes

Abstract Over 60% of patients with advanced cancer have cancer-induced cachexia, and the prevalence of weight loss reaches 86% in end-stage cancer. While cachexia is directly responsible for approximately 25% of cancer deaths and is inversely correlated with survival time, current treatments are limited and primarily palliative. Tumor necrosis factor (TNFα) is elevated in cancer patients, and is strongly associated with cachexia as it produces pleiotropic cachectic effects including apoptosis, inhibition of myogenesis and increased protein degradation. These effects are mediated in part by the pro-inflammatory transcription factor nuclear factor kappa B (NFκB) which upregulates components of the ubiquitin-proteasome pathway, and inhibits muscle cell differentiation through post-translational suppression of the muscle specific transcription factor, myogenic determination factor 1. We believe a critical role of AP-1 in cancer cachexia precedes NFκB-activated proteasome degradation. AP-1, and more specifically a switch to Jun:Fra-1 heterodimers, induces caspase 3 activity which is necessary to cleave large actomyosin complexes prior to further degradation by the ubiquitin-proteasome system. In C2C12 mouse muscle cells treated with TNFα, we see increased expression of the AP-1 subunits cJun, JunB and Fra-1 along with the expected decrease in myosin heavy chain (MyHC) expression. Inhibiting the mitogen activated protein kinase (MAPK) pathway with U0126 in TNFα-treated cells reduces the expression of these AP-1 subunits and partially restores expression of MyHC. When we overexpress the Fra-1 subunit in C2C12 cells, we see increased mRNA expression of FAS and caspases 1, 8 and 11 (ortholog of human caspase 4). Caspase 11-mediated activation of caspase 1 appears to activate IL-1β, a catabolism-inducing cytokine implicated in cachexia. Downstream of Fra-1 expression, we also see increased caspase 3 activity and an increase in the number of TUNEL-positive apoptotic cells. We hypothesize that TNFα signaling through the MAPK pathway leads to phosphorylation-dependent stabilization of Fra-1, and leads to a switch to Jun:Fra-1 heterodimer composition which, in turn, transcriptionally up-regulates FAS and caspases 1, 8 and 11. We think increased expression of FAS, and caspases 8 and 11 lead to FAS-mediated apoptosis and increased caspase 3 activity. We used high-throughput screening to identify AP-1 inhibitors, and then tested these inhibitors for their ability to restore MyHC expression in TNFα-treated cells. We have identified a novel compound, LS474, which specifically inhibits Fra-1 expression and restores MyHC expression. This suggests that the Fra-1 subunit of AP-1 plays an important role in mediating cachexia and is a good therapeutic target in the treatment of cancer cachexia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4786. doi:10.1158/1538-7445.AM2011-4786

Myostatin reduces Akt/TORC1/p70S6K signaling, inhibiting myoblast differentiation and myotube size

Myostatin is a negative regulator of skeletal muscle size, previously shown to inhibit muscle cell differentiation. Myostatin requires both Smad2 and Smad3 downstream of the activin receptor II (ActRII)/activin receptor-like kinase (ALK) receptor complex. Other transforming growth factor-beta (TGF-beta)-like molecules can also block differentiation, including TGF-beta(1), growth differentiation factor 11 (GDF-11), activins, bone morphogenetic protein 2 (BMP-2) and BMP-7. Myostatin inhibits activation of the Akt/mammalian target of rapamycin (mTOR)/p70S6 protein synthesis pathway, which mediates both differentiation in myoblasts and hypertrophy in myotubes. Blockade of the Akt/mTOR pathway, using small interfering RNA to regulatory-associated protein of mTOR (RAPTOR), a component of TOR signaling complex 1 (TORC1), increases myostatin-induced phosphorylation of Smad2, establishing a myostatin signaling-amplification role for blockade of Akt. Blockade of RAPTOR also facilitates myostatin's inhibition of muscle differentiation. Inhibition of TORC2, via rapamycin-insensitive companion of mTOR (RICTOR), is sufficient to inhibit differentiation on its own. Furthermore, myostatin decreases the diameter of postdifferentiated myotubes. However, rather than causing upregulation of the E3 ubiquitin ligases muscle RING-finger 1 (MuRF1) and muscle atrophy F-box (MAFbx), previously shown to mediate skeletal muscle atrophy, myostatin decreases expression of these atrophy markers in differentiated myotubes, as well as other genes normally upregulated during differentiation. These findings demonstrate that myostatin signaling acts by blocking genes induced during differentiation, even in a myotube, as opposed to activating the distinct "atrophy program." In vivo, inhibition of myostatin increases muscle creatine kinase activity, coincident with an increase in muscle size, demonstrating that this in vitro differentiation measure is also upregulated in vivo.

RAGE Expression in Rhabdomyosarcoma Cells Results in Myogenic Differentiation and Reduced Proliferation, Migration, Invasiveness, and Tumor Growth

Activation of receptor for advanced glycation end products (RAGE) by its ligand, HMGB1, stimulates myogenesis via a Cdc42-Rac1-MKK6-p38 mitogen-activated protein kinase pathway. In addition, functional inactivation of RAGE in myoblasts results in reduced myogenesis, increased proliferation, and tumor formation in vivo. We show here that TE671 rhabdomyosarcoma cells, which do not express RAGE, can be induced to differentiate on transfection with RAGE (TE671/RAGE cells) but not a signaling-deficient RAGE mutant (RAGEDeltacyto) (TE671/RAGEDeltacyto cells) via activation of a Cdc42-Rac1-MKK6-p38 pathway and that TE671/RAGE cell differentiation depends on RAGE engagement by HMGB1. TE671/RAGE cells also show p38-dependent inactivation of extracellular signal-regulated kinases 1 and 2 and c-Jun NH(2) terminal protein kinase and reduced proliferation, migration, and invasiveness and increased apoptosis, volume, and adhesiveness in vitro; they also grow smaller tumors and show a lower tumor incidence in vivo compared with wild-type cells. Two other rhabdomyosarcoma cell lines that express RAGE, CCA and RMZ-RC2, show an inverse relationship between the level of RAGE expression and invasiveness in vitro and exhibit reduced myogenic potential and enhanced invasive properties in vitro when transfected with RAGEDeltacyto. The rhabdomyosarcoma cell lines used here and C2C12 myoblasts express and release HMGB1, which activates RAGE in an autocrine manner. These data suggest that deregulation of RAGE expression in myoblasts might concur in rhabdomyosarcomagenesis and that increasing RAGE expression in rhabdomyosarcoma cells might reduce their tumor potential.

Activated raf kinase inhibits muscle cell differentiation through a MEF2-dependent mechanism.

Muscle cell development is dependent on the activity of cell type-specific basic-helix-loop-helix transcription factors, MyoD, Myf-5, myogenin, and MRF4 which collaborate with myocyte enhancer factor 2 proteins to activate muscle-specific gene expression. Growth factors and activated Ras prevent differentiation of myoblasts in culture but the downstream signalling pathways are not well understood. Here, we demonstrate that active Raf kinase (Raf-BxB) completely inhibits myogenic conversion of 10T1/2 cells mediated by Myf-5 and differentiation of L6 myoblasts as indicated by the absence of myotubes, lack of myogenin expression, and markedly reduced expression of myosin heavy chain. However, activated Raf inhibits transcriptional activation by Myf-5 only partially suggesting that other potential targets of Ras/Raf signalling may be involved. Significantly, we observed that elevated Raf kinase activity in L6 muscle cells suppresses the accumulation of MEF2 protein in nuclei, while MEF2 transcription appears unaffected. Moreover, forced expression of MEF2A in 10T1/2 cells rescues MyoD dependent myogenic conversion in the presence of constitutively active Raf kinase and partially restores transactivation of a myogenin promoter-dependent reporter gene in L6 muscle cells containing activated Raf kinase. From these observations we conclude that persistent activation of Raf signalling affects nuclear MEF2 functions which may explain why myogenin expression and myoblast differentiation are inhibited.

Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway.

Notch controls cell fate by inhibiting cellular differentiation, presumably through activation of the transcriptional regulator human C promoter Binding Factor (CBF1), which transactivates the hairy and Enhancer of split (HES-1) gene. However, we describe constitutively active forms of Notch1, which inhibit muscle cell differentiation but do not interact with CBF1 or upregulate endogenous HES-1 expression. In addition, Jagged-Notch interactions that prevent the expression of muscle cell specific genes do not involve the upregulation of endogenous HES-1. In fact, exogenous expression of HES-1 in C2C12 myoblasts does not block myogenesis. Our data demonstrate the existence of a CBF1-independent pathway by which Notch inhibits differentiation. We therefore propose that Notch signaling activates at least two different pathways: one which involves CBF1 as an intermediate and one which does not.

The regulation of α 5β 1 integrin expression in human muscle cells

The expression of alpha 5 beta 1 integrin was examined in either cloned or fluorescence-activated cell-sorted satellite cells derived from human biceps muscle. Removal of serum and factors required for muscle cell growth and proliferation both induced terminal differentiation and resulted in a coordinate downregulation of mRNA transcripts encoding alpha 5 and beta 1 integrin subunits. A corresponding downregulation of the alpha 5 subunit occurred at the protein level. Treatment of cultures with 5-bromo-2'-deoxyuridine (BUdR), a thymidine analog which inhibits muscle cell differentiation, resulted in increased expression of alpha 5 integrin subunit at both the mRNA and protein levels. However, levels of alpha 5 subunit message and protein were still markedly downregulated on removal of serum and growth factors from BUdR-treated cultures, indicating that downregulation of alpha 5 beta 1 integrin during myogenesis does not require and is not a consequence of muscle cell terminal differentiation. Downregulation of alpha 5 integrin subunit expression could be prevented by maintenance of cells in medium supplemented with serum and growth factors, although no single defined component of this medium could on its own prevent the downregulation of alpha 5 integrin subunit expression. Collectively, these results suggest that downregulation of alpha 5 beta 1 integrin expression is not a consequence of muscle cell terminal differentiation, but is dependent on a combination of exogenous growth factors which are also required for muscle cell growth and proliferation.

Overexpression of Id protein inhibits the muscle differentiation program: in vivo association of Id with E2A proteins.

The helix-loop-helix (HLH) protein Id lacks the basic DNA-binding domain common to this class of proteins. In vitro experiments suggested that Id could associate tightly with two other HLH proteins encoded by the E2A gene, E12 and E47 (referred to here collectively as E proteins) and prevent their binding to a sequence present in the muscle creatine kinase (MCK) enhancer either as homo-oligomers or hetero-oligomers with MyoD. In this report we present evidence for the in vivo roles of Id and E proteins: (1) Id and E proteins co-fractionate and co-immunoprecipitate in whole-cell extracts prepared from myoblasts; (2) the loss of Id protein observed during the conversion of proliferating myoblasts into mature myotubes correlates with the formation of MyoD/E hetero-oligomeric complexes in whole-cell extracts (these complexes do not form when purified Id protein is added to the extracts); and (3) stable overexpression of Id mRNA and protein in the C2C12 muscle cell line inhibits differentiation in these cells 16 hr post-induction. The myotubes that do eventually form 48 hr post-induction have no detectable Id protein in the nucleus despite the persistence of exogenous Id mRNA. These data support a model in which Id can inhibit muscle cell differentiation by associating with E proteins and preventing them from forming active hetero-oligomeric complexes with the muscle determination gene products.

Fibroblast growth factor inhibits insulin-like growth factor-II (IGF-II) gene expression and increases IGF-I receptor abundance in BC3H-1 muscle cells.

Muscle is an important target tissue for insulin-like growth factor (IGF) action. We have previously reported that muscle cell differentiation is associated with down-regulation of the IGF-I receptor at the level of gene expression that is concomitant with an increase in the expression and secretion of IGF-II. Furthermore, treatment of myoblasts with IGF-II resulted in a similar decrease in IGF-I receptor mRNA abundance, suggesting an autocrine role of IGF-II in IGF-I receptor regulation. To explore further the role of IGF-II in IGF-I receptor regulation, BC3H-1 mouse muscle cells were exposed to differentiation medium in the presence of basic fibroblast growth factor (FGF), a known inhibitor of myogenic differentiation. FGF treatment of cells resulted in a 50% inhibition of IGF-II gene expression compared to that in control myoblasts and markedly inhibited IGF-II secretion. Concomitantly, FGF resulted in a 60-70% increase in IGF-I binding compared to that in control myoblasts. Scatchard analyses and studies of gene expression demonstrated that the increased IGF-I binding induced by FGF reflected parallel increases in IGF-I receptor content and mRNA abundance. These studies indicate that FGF may up-regulate IGF-I receptor expression in muscle cells through inhibition of IGF-II peptide expression and further support the concept of an autocrine role of IGF-II in IGF-I receptor regulation. In addition, these studies suggest that one mechanism by which FGF inhibits muscle cell differentiation is through inhibition of IGF-II expression.

Temperature sensitivity of muscle and neuron differentiation in embryonic cell cultures from the Drosophila mutant, shibirets1

The sex-linked temperature-sensitive mutation, shibire ts1 , which causes, at the restrictive temperature, adult paralysis and pleiotropic morphological defects in embryonic, larval, and pupal development, has been shown to exhibit temperature-sensitive inhibition of differentiation in embryonic cultures in vitro. When shi cultures were incubated at 30°C for 24 hr, both muscle and neuron differentiation were inhibited more than 90% compared to control shi cultures incubated at 20°C. Heat shift experiments showed that the temperature-sensitive periods for neuron and muscle differentiation occurred at 11 to 18 and 14 to 16 hr, respectively, where zero time was the initiation of gastrulation in donor embryos. Short heat pulses (4 and 8 hr) which extended into the temperature-sensitive period resulted in moderate inhibition of differentiation; greater inhibition occurred as the duration of the pulses increased. In contrast, heating wild-type Oregon-R cultures at 30°C for 24 hr did not inhibit muscle cell differentiation and inhibited neuron differentiation relatively little. The temperature-sensitive period in shibire for muscle differentiation occurred well after myoblast division, during the period of myocyte elongation, aggregation, and fusion, whereas that for neuron differentiation took place during a period of enzyme synthesis (acetylcholinesterase and choline acetyltransferase) and axon elongation. Thus, the shi temperature-sensitive gene product affects at least two different cell types, in vitro, at different times during differentiation.