Proliferating C2C12 Cells Research Articles

Peptide Assembly of Different Dimensions and Their Effect on Myoblast Proliferation.

Variations in the functionalities of materials of different dimensions containing the same functional groups can be attributed to the structural stability and morphology of the materials. The morphology of peptide assemblies can influence their interactions with biological systems and ultimately modulate their bioactivity. Among reported Arg-Gly-Asp (RGD)-based supramolecular materials, two-dimensional (2-D) peptide assembly has been rarely studied. Herein, we report the fabrication of RGD-based supramolecular one-dimensional (1-D) and 2-D assemblies as peptide-based myoblast growth accelerators. The 2-D assembly was more effective in proliferating C2C12 cells than the 1-D assembly. These findings provide insights into the construction of optimal RGD-based supramolecular functional materials of different dimensions.

EFFECT OF FORCE ACTIVATION OF NOTCH SIGNALING ON MTOR

PURPOSE: Notch signaling is a prominent pathway necessary for repair of injured muscle. The interactions of Notch with other signaling pathways, specifically mechanistic/ mammalian target of rapamycin (mTOR), in regulating myogenesis is not well known. Studies have been conducted on Notch inhibition, but little research has been performed on activated Notch and the interactions with mTOR. This study was implemented to develop a Notch force activation protocol and to measure the effect of overexpressed Notch on C2C12 proliferation, differentiation and mTOR signaling. METHODS: Notch signaling was force activated via suspension or adhesion. For suspension, Notch-1 antibody was introduced to a 12-well plate with C2C12 cells at a concentration of 1:10. For adhesion, Notch ligand, Delta-like Ligand 1 (DLL1), was mixed with Extracellular Matrix (ECM) and coated on a 12-well plate at different concentrations (control, 2.5ug, 5ug, and 10 ug) for 12 hours. C2C12 cells were seeded at a concentration of 15,000 cells/well and differentiated for 96 hours. Following designated time period, lysates were collected for Western Blots. Primary antibodies probed for Notch (Hes1), total mTOR signaling (TmTOR), and β-actin. RESULTS: Preliminary data for suspension was analyzed via a two-way ANOVA (time x treatment). There is a significant difference in percent change of proliferation at 48 hours (p=0.02). Preliminary data of adhesion was analyzed via one-way ANOVA. There is a trend between Hes1 concentration in ligand concentration of 2.5 ug (p=0.0739). It shows no significant difference in TmTOR concentrations between the various Notch force activation concentrations in proliferating C2C12 cells (p=.4298). CONCLUSIONS: Preliminary data shows a stronger effect of suspension over adhesion for force activating Notch. A concentration of 2.5ug of ligand is possibly sufficient to force activate Notch, any higher appears too concentrated to activate Notch. Preliminary data also suggests that force activating Notch does not affect mTOR signaling. Experiments are currently testing the ideal DLL1 to activate Notch signaling, as well as examining the effect of force activating Notch on proliferation, differentiation and other markers of mTOR signaling. Different time points for differentiation should be tested in future research.

P0707UREMIC TOXINS IMPAIR SKELETAL MUSCLE REGENERATION PROCESS INDUCING CELL CYCLE ARREST AND APOPTOSIS IN CULTURED MYOBLASTS

Abstract Background and Aims The loss of muscle mass and function has been related to chronic kidney disease (CKD). About 37% of dialysis patients show symptoms of sarcopenia and this has been related to an increased risk of mortality. Changes in sarcopenic muscle include the loss of its regenerative capacity due to a reduction in the number and function of satellite cells, the muscle stem cells. The concentration of serum uremic toxins (UT) increases in parallel to a decline in the glomerular filtration rate in patients with CKD and this uremia may be involved in the development of sarcopenia. Previous studies showed as serum concentration of UT found in the early stages of CKD inhibits myogenic differentiation of cultured myoblasts. Nevertheless, the effect of those concentrations found in the advanced stages of CKD has not been described. The study aimed to analyse whether UT affect the muscular regeneration process by modifying the proliferation capacity of myoblasts (activated satellite cells). Method Cultured mouse myoblasts C2C12 cells were used for all experiments. Cells were grown with 0% or 10% FBS culture media in the presence or absence of indoxyl sulphate and para-cresol at doses of 100µg/ml each one, which are similar to ones found in the advanced stages of CKD. Proliferation was evaluated by scratch wound healing and cell cycle by flow cytometry with propidium iodide and the fluorescent probe CFSE, an intracellular protein binding dye that is divided equally between daughter cells, allowing the discrimination of successive rounds of cell division. Chromosome condensation was assessed by immunofluorescence staining by confocal microscopy. Apoptosis was analysed by annexin V staining. Results C2C12 cells treated with UT shown a significant decrease in the proliferation rate. A significant delay in wound closure was observed in cells treated with UT compared to control cells. Myoblasts treated with UT suffered a significant decrease in the proliferation rate since the probe remained higher than in the vehicle-treated cells. Proliferating cells treated with UT suffered a dramatic cell cycle arrest between the phases S and G2/M. Chromosome condensation was also analysed, finding that in the presence of colcemid, vehicle-treated cells condensed their chromosomes, as expected, whereas UT-treated cells did not, suggesting that UT stop the cell cycle at any point before the entry of cells in the mitosis phase. Besides, there was strong phosphorylation of cdc2 in the presence of UT indicating that cdc2 and the complex cdc2-cyclin B were inactive. This result explains why cells did not enter in the mitosis phase under UT exposition. Finally, UT induced the death of proliferating C2C12 cells by apoptosis. Conclusion In the advanced stages of CKD, uremic toxins concentration increases, thereby inducing a dramatic arrest in the cell cycle of myoblasts, inactivating the cdc2-cyclin B complex, interrupting their proliferation and leading them towards cell apoptosis. These results point to a role of uremic toxins impairing the skeletal muscle regeneration process, which could be involved in CKD-related sarcopenia and frailty.

MLL1 promotes myogenesis by epigenetically regulating Myf5.

ObjectivesMixed lineage leukaemia protein‐1 (MLL1) mediates histone 3 lysine 4 (H3K4) trimethylation (me3) and plays vital roles during early embryonic development and hematopoiesis. In our previous study, we found its expression was positively correlated with embryonic myogenic ability in pigs, indicating its potential roles in mammalian muscle development. The present work aimed to explore the roles and regulation mechanisms of MLL1 in myogenesis.Materials and methodsThe expression of MLL1 in C2C12 cells was experimentally manipulated using small interfering RNAs (siRNA). 5‐ethynyl‐2′‐deoxyuridine (EdU) assay, cell cycle assay, immunofluorescence, qRT‐PCR and Western blot were performed to assess myoblast proliferation and differentiation. Chromatin immunoprecipitation assay was conducted to detect H3K4me3 enrichment on myogenic factor 5 (Myf5) promoter. A cardiotoxin (CTX)‐mediated muscle regeneration model was used to investigate the effects of MLL1 on myogenesis in vivo.ResultsMLL1 was highly expressed in proliferating C2C12 cells, and expression decreased after differentiation. Knocking down MLL1 suppressed myoblast proliferation and impaired myoblast differentiation. Furthermore, knockdown of MLL1 resulted in the arrest of cell cycle in G1 phase, with decreased expressions of Myf5 and Cyclin D1. Mechanically, MLL1 transcriptionally regulated Myf5 by mediating H3K4me3 on its promoter. In vivo data implied that MLL1 was required for Pax7‐positive satellite cell proliferation and muscle repair.ConclusionMLL1 facilitates proliferation of myoblasts and Pax7‐positive satellite cells by epigenetically regulating Myf5 via mediating H3K4me3 on its promoter.

Function analysis of Mef2c promoter in muscle differentiation

Regeneration of adult skeletal muscle following injury occurs through the activation of satellite cells that proliferates, differentiates, and fuses with injured myofibers. Myocyte enhancer factor 2 (MEF2) proteins are reported to have the potential contributions to adult muscle regeneration. To further understand Mef2c gene, the promoter of pig Mef2c gene was analyzed in this paper. Quantitative real-time PCR (qRT-PCR) revealed the expression pattern of Mef2c gene in muscle of eight tissues. The Mef2c promoter had the higher transcriptional activity in differentiated C2C12 cells than that in proliferating C2C12 cells, which was accompanied by the upregulation of mRNA expression of Mef2c gene. Function deletion and mutation analyses showed that MyoD and MEF2 binding sites within the Mef2c promoter were responsible for the regulation of Mef2c transcription. MEF2C could upregulate the transcriptional activities of Mef2c promoter constructs, which contained a 3'-end nucleotide sequence with p300 binding site. The electrophoretic mobility shift assays and chromatin immunoprecipitation assays determined the MyoD binding site in Mef2c promoter. These results advanced our knowledge of the promoter of the pig Mef2c gene, and the study of Mef2c promoter regulator elements helped to elucidate the regulation mechanisms of Mef2c in muscle differentiation or muscle repair and regeneration.

A novel D2O tracer method to quantify RNA turnover as a biomarker of de novo ribosomal biogenesis, in vitro, in animal models, and in human skeletal muscle.

Current methods to quantify in vivo RNA dynamics are limited. Here, we developed a novel stable isotope (D2O) methodology to quantify RNA synthesis (i.e., ribosomal biogenesis) in cells, animal models, and humans. First, proliferating C2C12 cells were incubated in D2O-enriched media and myotubes ±50 ng/ml IGF-I. Second, rat quadriceps (untrained, n = 9; 7-wk interval-"like" training, n = 13) were collected after ~3-wk D2O (70 atom %) administration, with body-water enrichment monitored via blood sampling. Finally, 10 (23 ± 1 yr) men consumed 150-ml D2O followed by 50 ml/wk and undertook 6-wk resistance exercise (6 × 8 repetitions, 75% 1-repetition maximum 3/wk) with body-water enrichment monitored by saliva sampling and muscle biopsies (for determination of RNA synthesis) at 0, 3, and 6 wk. Ribose mole percent excess (r-MPE) from purine nucleotides was analyzed via GC-MS/MS. Proliferating C2C12 cell r-MPE exhibited a rise to plateau, whereas IGF-I increased myotube RNA from 76 ± 3 to 123 ± 3 ng/μl and r-MPE by 0.39 ± 0.1% (both P < 0.01). After 3 wk, rat quadriceps r-MPE had increased to 0.25 ± 0.01% (P < 0.01) and was greater with running exercise (0.36 ± 0.02%; P < 0.01). Human muscle r-MPE increased to 0.06 ± 0.01 and 0.13 ± 0.02% at 3/6 wk, respectively, equating to synthesis rates of ~0.8%/day, increasing with resistance exercise to 1.7 ± 0.3%/day (P < 0.01) and 1.2 ± 0.1%/day (P < 0.05) at 3/6 wk, respectively. Therefore, we have developed and physiologically validated a novel technique to explore ribosomal biogenesis in a multimodal fashion.

Characterization of Zinc Finger Protein 593 (Zfp593) in Skeletal Muscle

Skeletal muscle atrophy is a debilitating medical condition which can arise due to aging, cancer, corticosteroid use, and denervation. In order to better characterize the molecular genetic events of neurogenic atrophy, the gastrocnemius muscle was isolated from mice following 3 days and 14 days of sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue in order to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that zinc finger protein 593 (Zfp593) is expressed in skeletal muscle and is significantly induced in response to denervation. Furthermore, Western blot analysis confirmed that Zfp593 is expressed at the protein level in C2C12 mouse muscle cells. In order to characterize the transcriptional regulation of Zfp593, fragments of the proximal promoter located immediately upstream from the start of transcription were cloned and fused to a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells alone or in combination with MyoD1 or myogenin expression plasmids, resulting in a differential effect on reporter activity. Furthermore, analysis of the promoter region of Zfp593 has revealed four conserved E‐box elements, which are known myogenic regulatory factor (MRF) binding sites. Zfp593 is predicted to possess a zinc finger domain and is believed to function as a transcription factor. Furthermore, two predicted putative nuclear localization signal sequences have been identified in the Zfp593 protein sequence, further suggesting that this protein is localized to the nucleus. Therefore, in order to determine the sub‐cellular localization of Zfp593 in muscle cells, the Zfp593 cDNA was isolated from C2C12 cells, cloned into a green fluorescent protein (GFP) expression plasmid and transfected into proliferating C2C12 cells. Transfected cells were visualized by confocal microscopy revealing that Zfp593 is localized to the nucleus of proliferating myoblasts. The discovery that Zfp593 is expressed in skeletal muscle combined with the observation that this gene, a potential modulator of gene expression itself, is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of skeletal muscle wasting.Support or Funding InformationThe work was supported by University of North Florida Transformational Learning grants to D.W.

Caspase activity and apoptotic signaling in proliferating C2C12 cells following cisplatin or A23187 exposure.

Investigating cell death signaling using cell culture is commonly performed to examine the effects of novel pharmaceuticals or to further characterize discrete cellular signaling pathways. Here, we provide data regarding the cell death response to either cisplatin or A23187 in sub-confluent C2C12 cells, by utilizing several concentrations and incubation times for each chemical. These data include an assessment of the activation of the proteolytic enzymes caspase-3, caspase-8, caspase-9, calpain, and cathepsin B/L. Additionally, the expression of the apoptosis-regulating proteins Bax, Bcl2, and p53 are presented.

Intracellular Distribution and Involvement of GPR30 in the Actions of E2 on C2C12 Cells.

G-protein-coupled receptor 30 (GPR30) is an estrogen receptor that initiates several rapid, non-genomic signaling events triggered by E2. GPR30 has recently been identified in C2C12 cells; however, little is known about the intracelular distribution and its role in C2C12 myoblasts and myotubes. By western blotting and immunohistochemistry, we evidenced expression of GPR30. While in C2C12 myoblasts, the receptor was present in nucleus, mitochondria, and endoplasmic reticulum, in C2C12 myotubes, it was additionally found in cytoplasm. Using trypan blue uptake assay to determine cellular death and fluorescent microscopy to evaluate picnotic nuclei and mitochondrial distribution, we demonstated that treatment of C2C12 myoblasts with G1 (GPR30 agonist) did not protect the cells against apoptosis induced by H2O2 as E2. However, when G15 (GPR30 antagonist) was used, E2 could not prevent the damage caused by the oxidative stress. Further, some of the molecular mechanisms involved were investigated by wertern blot assays. Thus, E2 was able to induce AKT phosphorylation in apoptotic conditions and ERK phosphorylation in proliferating C2C12 cells but not when the cultures were incubated with G15. Additionally, using G15 antagonist we have found that GPR30 participates in the myogenin expression and creatine kinase activity stimulated by E2 in the first steps of C2C12 differentiation. Althogether these findings provide evidences showing that GPR30 is expressed in diverse intracellular compartments in undifferentiated and differentiated C2C12 cells and mediates E2 actions.

Potential Role of PYY in Skeletal Muscle Cells?

PYY is characteristically considered an appetite hormone with identified roles in metabolism, inflammation, and proliferation. The expression of PYY and its receptors has been demonstrated in the skeletal muscle; however, the role of PYY in the skeletal muscle is unknown.ObjectiveTo investigate the role of PYY on proliferating and differentiating/differentiated skeletal myoblasts and to determine the effect of PYY on inflammatory and metabolic signaling pathways.MethodsExpression of the PYY gene was determined in cultured murine C2C12 cells incubated in growth or differentiation media (GM and DM, respectively) using quantitative PCR. Additionally, changes in proteins of interest were determined from C2C12 cells that were cultured in DM for 96 h then switched to DM containing 0.1 mM palmitate for 72 h, followed by exposure to 500 pM murine PYY3‐36 for 6 h prior to lysis.ResultsPYY gene expression, which was identified in proliferating C2C12 cells, decreased in differentiating cells. Relative to myotubes that received only palmitate, the presence of PYY decreased phosphorylation of STAT3 (signal transducer and activator of transcription 3) serine‐727 (‐37%, p = 0.03) and tyrosine‐705 (‐33%, p = 0.01). There was no change in the total STAT3 protein, the ratio of phospho‐STAT3 to total STAT3, or NF‐kB‐p65 levels.ConclusionsThese results indicate that the PYY gene is not only detectable in mouse myoblasts, but it also changes with different physiological states (i.e. proliferating vs. differentiating). Additionally, PYY appears to play a role in skeletal muscle STAT3 signaling. Follow‐up research is necessary to confirm these results and identify downstream targets of PYY through the STAT3 pathway and functional outcomes. Support: NIDDK P30DK056336

Setdb1 is required for myogenic differentiation of C2C12 myoblast cells via maintenance of MyoD expression.

Setdb1, an H3-K9 specific histone methyltransferase, is associated with transcriptional silencing of euchromatic genes through chromatin modification. Functions of Setdb1 during development have been extensively studied in embryonic and mesenchymal stem cells as well as neurogenic progenitor cells. But the role of Sedtdb1 in myogenic differentiation remains unknown. In this study, we report that Setdb1 is required for myogenic potential of C2C12 myoblast cells through maintaining the expressions of MyoD and muscle-specific genes. We find that reduced Setdb1 expression in C2C12 myoblast cells severely delayed differentiation of C2C12 myoblast cells, whereas exogenous Setdb1 expression had little effect on. Gene expression profiling analysis using oligonucleotide micro-array and RNA-Seq technologies demonstrated that depletion of Setdb1 results in downregulation of MyoD as well as the components of muscle fiber in proliferating C2C12 cells. In addition, exogenous expression of MyoD reversed transcriptional repression of MyoD promoter-driven lucif-erase reporter by Setdb1 shRNA and rescued myogenic differentiation of C2C12 myoblast cells depleted of endogenous Setdb1. Taken together, these results provide new insights into how levels of key myogenic regulators are maintained prior to induction of differentiation.

Possible role of TIEG1 as a feedback regulator of myostatin and TGF-β in myoblasts

Myostatin and TGF-β negatively regulate skeletal muscle development and growth. Both factors signal through the Smad2/3 pathway. However, the regulatory mechanism of myostatin and TGF-β signaling remains unclear. TGF-β inducible early gene (TIEG) 1 is highly expressed in skeletal muscle and has been implicated in the modulation of TGF-β signaling. These findings prompted us to investigate the effect of TIEG1 on myostatin and TGF-β signaling using C2C12 myoblasts. Myostatin and TGF-β induced the expression of TIEG1 and Smad7 mRNAs, but not TIEG2 mRNA, in proliferating C2C12 cells. When differentiating C2C12 myoblasts were stimulated by myostatin, TIEG1 mRNA was up-regulated at a late stage of differentiation. In contrast, TGF-β enhanced TIEG1 expression at an early stage. Overexpression of TIEG1 prevented the transcriptional activation of Smad by myostatin and TGF-β in both proliferating or differentiating C2C12 cells, but the expression of Smad2 and Smad7 mRNAs was not affected. Forced expression of TIEG1 inhibited myogenic differentiation but did not cause more inhibition than the empty vector in the presence of myostatin or TGF-β. These results demonstrate that TIEG1 is one possible feedback regulator of myostatin and TGF-β that prevents excess action in myoblasts.

Reproducibility of miRNA Expression by Proliferating C2C12 Cells Using RT‐PCR Array

RT‐PCR array is a new technique to study microRNA (miRNA) expression; however, minimal data regarding intra‐ and inter‐assay reproducibility are available. This study examined the variability of the TaqMan MicroRNA Array (Applied Biosystems) in the evaluation of RNA obtained from proliferating murine myoblast C2C12 cells. The same RNA was used for two separate reverse transcription (RT) reactions and the RT products were used in duplicate arrays. The log2 expression levels of most genes (66%) in the four replicates had a standard deviation (SD) below 0.5 with median SD=0.2. When cycle threshold (CT) exceeded 32 there was a significant SD increase from 0.23 to 1.07, thus, genes with an average CT&gt;32 (129/381) were excluded. With CT&lt;32, 82% of miRNA exhibited a SD&lt;0.2. The endogenous control RNA Mammu6 correlated well in the four replicates with a coefficient of variation of 0.5% (SD&lt;0.1) and average CT=19.6. Thus, the variability due to different RT reactions on replicate samples was low and approximately equal. We estimate that the platform can detect a fold change of 2 with 99% power and a type I error rate 0.0005, assuming 4 biological replicates under 2 conditions given the gene has an SD of 0.1. In conclusion, this real‐time PCR based array method provided highly reproducibly estimates of miRNA expression by proliferating myoblasts in culture. Supported by the Veterans Administration and USPHS HL07446 and HL074236.

17β‐estradiol exerts protective effects in skeletal muscle cells at the level of the apoptotic ASK1‐MAP kinase pathway

17β‐estradiol is a steroid hormone that promotes cell survival and prevents from apoptosis in a great variety of tissues. Previous investigations with the murine skeletal muscle C2C12 cell line demonstrated that the action of 17β‐estradiol involves stimulation of PI3K‐Akt. Studies with other cell types have shown that upon its activation Akt can inhibit apoptosis by alternative mechanisms. In this report, we investigated whether the antiapoptotic effect of estrogen in muscle cells is mediated by Akt through interaction and dephosphorylation at Thr 845 of ASK‐1, a MAPKKK pro‐apoptotic protein. When proliferating C2C12 cells were treated with 17β‐estradiol (10−8 M), phosphorylation of ASK1 (Thr 845) and JNK was decreased. However, no significant changes occurred in ASK1 and JNK 1/2 protein levels. On the other hand, treatment with the apoptic agent H2O2 caused a sustained (30 min ‐ 24 h) increase in ASK1 levels and phosphorylation at Thr 845 (associated to stimulation of kinase activity) and, in addition, of JNK 1/2 phosphorylation. Furthermore, a 17β‐estradiol‐dependent interaction between p‐Akt and ASK1 was detected by coimmunoprecipitation. The association of both proteins was diminished by H2O2. In conclusion, these results suggest that Akt interaction with ASK1 followed by dephosphorylation of the latter is part of the mechanism by which 17β‐estradiol exerts its antiapoptotic action in skeletal muscle cells challenged with stress stimuli.

Generation and analysis of an androgen-responsive myoblast cell line indicates that androgens regulate myotube protein accretion

Androgens have anabolic actions in skeletal muscle and could potentially act to: (a) increase proliferation of myoblasts; (b) delay differentiation to myotubes; and (c) induce protein accretion in post-proliferative myofibers. To identify the site of androgens action, we investigated the proliferative response of the C2C12 mouse myoblast cell line to testosterone and dihydrotestosterone (DHT) treatment. Neither androgens affected cell proliferation after up to 7 days treatment, nor was there a synergistic effect of androgens on the proliferative response of C2C12 cells to IGF-I treatment. However, proliferating C2C12 cells expressed 0.1% of the level of androgen receptor (AR) mRNA found in adult mouse gastrocnemius muscle (p<0.01). Therefore, we generated mouse C2C12 myoblast cell lines stably transfected with the mouse AR cDNA driven by the SV40 promoter (C2C12-AR). C2C12-AR cell proliferation, differentiation, and protein content were analyzed in response to androgen treatment. Our data demonstrated that androgen treatment does not alter either proliferation rate or differentiation rate of C2C12-AR cells. However, treatment of differentiated C2C12-AR myotubes with 100 nM DHT for 3 days caused a 20% increase in total protein content vs vehicle treatment (p<0.05). This effect was not observed in control C2C12 cells transfected with empty vector. These data suggest that androgens act via the AR to upregulate myotube protein content. This model cell line will be useful to further investigate the molecular mechanisms via which androgens regulate protein accretion.

Down-regulation of CIBZ, a Novel Substrate of Caspase-3, Induces Apoptosis

We previously identified and characterized a murine BTB domain-containing protein, CIBZ (ZBTB38 in human), that interacts with CtBP and binds to methylated CpGs. However, its physiological function remained unknown. As CtBP is reportedly involved in p53-independent programmed cell death, we examine here whether CIBZ is associated with apoptosis. We found that CIBZ was highly expressed in proliferating C2C12 cells but that its expression levels decreased upon induction of apoptosis by serum starvation. Knockdown of CIBZ by small interfering RNA in C2C12 cells induced apoptosis, as determined by an increase of annexin V/propidium iodide labeling, activation of caspase-3, and cleavage of poly(ADP-ribose) polymerase. CIBZ inhibition also activated caspase-7 and caspase-9, suggesting that CIBZ-associated apoptosis occurs through the mitochondrial pathway. Notably, knockdown of CIBZ in p53(-/-) mouse embryonic fibroblast cells also activated caspase-3 and cleavage of poly(ADP-ribose) polymerase, indicating that CIBZ-associated apoptosis is mediated by a p53-independent pathway; however, because both common and distinct targets are regulated by CIBZ- and CtBP-associated apoptosis, we conclude that more than one pathway is involved. Finally, using mutagenesis and an in vitro caspase cleavage assay, we show that CIBZ is a novel substrate of caspase-3 and identify two caspase-3 recognition sites. These findings indicate, collectively, that CIBZ plays an important role by participating in the negative regulation of apoptosis in murine cells.

The transcriptional repressor ZEB regulates p73 expression at the crossroad between proliferation and differentiation.

The newly discovered p73 gene encodes a nuclear protein that has high homology with p53. Furthermore, ectopic expression of p73 in p53(+/+) and p53(-/-) cancer cells recapitulates some of the biological activities of p53 such as growth arrest, apoptosis, and differentiation. p73(-/-)-deficient mice exhibit severe defects in proper development of the central nervous system and pheromone sensory pathway. They also suffer from inflammation and infections. Here we studied the transcriptional regulation of p73 at the crossroad between proliferation and differentiation. p73 mRNA is undetectable in proliferating C2C12 cells and is expressed at very low levels in undifferentiated P19 and HL60 cells. Conversely, it is upregulated during muscle and neuronal differentiation as well as in response to tetradecanoyl phorbol acetate-induced monocytic differentiation of HL60 cells. We identified a 1-kb regulatory fragment located within the first intron of p73, which is positioned immediately upstream to the ATG codon of the second exon. This fragment exerts silencer activity on p73 as well as on heterologous promoters. The p73 intronic fragment contains six consensus binding sites for transcriptional repressor ZEB, which binds these sites in vitro and in vivo. Ectopic expression of dominant-negative ZEB (ZEB-DB) restores p73 expression in proliferating C2C12 and P19 cells. Thus, transcriptional repression of p73 expression by ZEB binding may contribute to the modulation of p73 expression during differentiation.

Identification of a role for the sialomucin CD164 in myogenic differentiation by signal sequence trapping in yeast.

Determination and differentiation of skeletal muscle precursors requires cell-cell contact, but the full range of cell surface proteins that mediate this requirement and the mechanisms by which they work are not known. To identify participants in cell contact-mediated regulation of myogenesis, genes that encode secreted proteins specifically upregulated during differentiation of C2C12 myoblasts were identified by the yeast signal sequence trap method (K. A. Jacobs, L. A. Collins-Racie, M. Colbert, M. Duckett, M. Golden-Fleet, K. Kelleher, R. Kriz, E. R. La Vallie, D. Merberg, V. Spaulding, J. Stover, M. J. Williamson, and J. M. McCoy, Gene 198:289-296, 1997), followed by RNA expression analysis. We report here the identification of CD164 as a gene expressed in proliferating C2C12 cells that is upregulated during differentiation. CD164 encodes a widely expressed cell surface sialomucin that has been implicated in regulation of cell proliferation and adhesion during hematopoiesis. Stable overexpression of CD164 in C2C12 and F3 myoblasts enhanced their differentiation, as assessed by both morphological and biochemical criteria. Furthermore, expression of antisense CD164 or soluble extracellular regions of CD164 inhibited myogenic differentiation. Treatment of C2C12 cells with sialidase or O-sialoglycoprotease, two enzymes previously reported to destroy functional epitopes on CD164, also inhibited differentiation. These data indicate that (i) CD164 may play a rate-limiting role in differentiation of cultured myoblasts, (ii) sialomucins represent a class of potential effectors of cell contact-mediated regulation of myogenesis, and (iii) carbohydrate-based cell recognition may play a role in mediating this phenomenon.

Genomic organization and promoter analysis of the murine Id3 gene

Overexpression of the helix–loop–helix motif-containing transcription inhibitor Id3 has been shown to repress muscle-specific gene expression. Consistent with its putative negative regulatory role in the myogenic process, Id3 is highly expressed in proliferating myoblasts but down regulated when myoblasts are induced to differentiate. To investigate how Id3 expression may be transcriptionally regulated, we isolated a mouse Id3 genomic DNA fragment and characterized its organization and promoter activity. Comparison of the Id3 gene from human and mouse demonstrated a conserved exon–intron organization in which the first intron interrupts the C-terminal protein coding region and the second intron interrupts the 3′ untranslated region at analogous positions in the two species. Sequence analysis of the 5′-flanking region revealed an unexpected mouse strain-specific genetic polymorphism due to a single base substitution. Deletion analysis revealed that as little as 180 base pairs of the mouse Id3 promoter upstream of the transcription start site is sufficient for a high level of gene expression in proliferating C2C12 myoblasts. In particular, the region between the nucleotide position −180 and −34 appeared to be crucial for maximal reporter gene activity and interacted specifically with C2C12 nuclear proteins. Finally, we showed that, despite the creation of a putative transcription factor-binding site, the genetic polymorphism observed did not affect Id3 promoter activity in proliferating C2C12 cells.

Expression of Neurofilament Proteins in Proliferating C2C12 Mouse Skeletal Muscle Cells

Expression of neurofilament proteins, NF140K, NF68K, and NF200K, in C2C12 mouse skeletal muscle cells was studied. Immunofluorescence and immunoblot analyses revealed that NF140K was expressed in proliferating C2C12 cells and its localization was similar to desmin, a muscle-specific intermediate filament protein. NF140K became undetectable in C2C12 cells as muscle cell differentiation proceeded. Reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analyses confirmed the expression of NF140K, NF68K, and NF200K in proliferating C2C12 cells. Sequences of the RT-PCR products of NF140K and NF68K were identical to that of authentic mouse NF140K and NF68K, respectively. The NF140K and NF68K mRNA were down-regulated during myogenesis in contrast to the up-regulation of mRNA encoding troponin-T. Furthermore, subcloned C2C12 cells, which express NF140K at a higher level, exhibited retarded myogenesis, i.e., delayed onset of myosin heavy chain synthesis and myoblast fusion. These results suggest that neurofilament proteins may play an inhibitory role in commitment of muscle cell differentiation. The presence of neurofilament proteins in skeletal muscle cells indicates that desmin, vimentin, and neurofilament proteins can be expressed in a single muscle cell.