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Ankyrin repeat and suppressor of cytokine signaling (SOCS) box-containing protein (ASB) 15 alters differentiation of mouse C₂C₁₂ myoblasts and phosphorylation of mitogen-activated protein kinase and Akt¹

Department of Animal Science, Iowa State University, Ames 50011

	Abstract

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Ankyrin repeat and suppressor of cytokine signaling box-containing protein (ASB) 15 is a novel ASB gene family member predominantly expressed in skeletal muscle. We have previously reported that overexpression of ASB15 delays differentiation and alters protein turnover in mouse C₂C₁₂ myoblasts. However, the extent of ASB15 regulation of differentiation and molecular pathways underlying this activity are unknown. The extracellular signal-regulated kinase (Erk) 1/2 and phosphatidylinositol-3 kinase-Akt (PI3K/Akt; Akt is also known as protein kinase B) signaling pathways have a role in skeletal muscle growth. Activation (phosphorylation) of the Erk1/2 signaling pathway promotes proliferation, whereas activation of the PI3K/Akt signaling pathway promotes myoblast differentiation. Accordingly, we tested the hypothesis that ASB15 controls myoblast differentiation through its regulation of these kinases. Stably transfected myoblasts overexpressing ASB15 (ASB15+) demonstrated decreased differentiation, whereas attenuation of ASB15 expression (ASB15-) increased differentiation. However, ASB15+ cells had less abundance of the phosphorylated mitogen-activated protein kinase (active) form, despite decreased differentiation relative to control myoblasts (ASB15Con). The mitogen-activated protein kinase kinase inhibitor, U0126, effectively decreased mitogen-activated protein kinase phosphorylation and stimulated differentiation in ASB15- and ASB15Con cells. However, inhibition of the Erk1/2 pathway was unable to overcome the inhibitory effect of overexpressing ASB15 on differentiation (ASB15+), suggesting that the Erk1/2 pathway is likely not the predominant mediator of ASB15 activity on differentiation. Expression of ASB15 also altered phosphorylation of the PI3K/Akt pathway, as ASB15+ and ASB15- cells had decreased and increased Akt phosphorylation, respectively. These data were consistent with observed differences in differentiation. Administration of IGF-I, a PI3K/Akt activator, in ASB15+ was able to partially override the previously observed phenotype of delayed differentiation, whereas administration of the PI3K/ Akt inhibitor, LY294002, decreased phosphorylation of Akt and differentiation of all cell lines similar to the untreated ASB15+ myoblasts. These results provide initial evidence that ASB15 has a role in early myoblast differentiation and that its effects may be mediated in part by the PI3K/Akt signal transduction pathway.

Key Words: ankyrin • differentiation • muscle • myoblast

	INTRODUCTION

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Increasing efficiency of muscle growth is an important goal in the production of meat animals. Ankyrin repeat and suppressor of cytokine signaling box-containing protein (ASB) 15 is a member of the ASB gene family (Hilton et al., 1998; Kile et al., 2000). The ASB15 is expressed predominantly in muscle and is rapidly downregulated in response to ß-adrenergic receptor agonists (McDaneld et al., 2004; Spurlock et al., 2006). In C₂C₁₂ myoblasts, ASB15 causes delayed differentiation and altered protein synthesis, and local overexpression of ASB15 in skeletal muscle in vivo stimulates a significant increase in muscle fiber diameter (McDaneld et al., 2006).

Whereas previous research in our lab suggests that ASB15 may play a role in myogenesis and muscle metabolism, the underlying molecular mechanisms that facilitate its effects are unknown. Current literature suggests that ASB family members are functional in multiple biological processes relating to cell growth (Guibal et al., 2002), tissue development (Kohroki et al., 2001; Guibal et al., 2002), and insulin signaling (Wilcox et al., 2004). Mitogen-activated protein kinase (MAPK) activates a series of signal transduction pathways that are important regulators of proliferation, differentiation, and protein turnover in myogenic cells (Pena et al., 2000; Jones et al., 2001; Rommel et al., 2001). Two pathways in particular, involving extracellular signal-regulated kinase (Erk) 1/2 and phosphati-dylinositol-3 kinase-Akt (PI3K/Akt; Akt is also known as protein kinase B), play a coordinated role in regulating proliferation, differentiation, and protein turnover throughout the development of myotubes (Bennett and Tonks, 1997; Coolican et al., 1997; Jiang et al., 1998). Therefore, the objective of the research reported herein was to determine whether ASB15 alters activation of the Erk1/2 or PI3K/Akt pathway to affect early differentiation of myoblasts.

	MATERIALS AND METHODS

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Animal Care and Use Committee approval was not obtained for this study because all experiments were done in immortalized cell culture models.

C₂C₁₂ Cell Culture

Proliferating C₂C₁₂ myoblasts (American Type Culture Collection, Manassas, VA; CRL-1772) were maintained in growth media consisting of Dulbecco’s Modified Eagle’s Medium adjusted to contain 10% fetal bovine serum (Life Technologies, Bethesda, MD) and selected antibiotics (50 mg of gentamicin, Invitrogen, Carlsbad, CA; 1% antibiotic-antimycotic solution, Sigma, St. Louis, MO). Differentiation was induced in all experiments by replacing fetal bovine serum with 2% horse serum in confluent monolayers of myoblasts. Culture conditions were maintained at 37°C and 5% CO₂. Differentiation was evaluated both qualitatively by visual evaluation of cells and quantitatively by measuring creatine kinase activity as described previously (Florini, 1989). Total protein was measured by bicinchoninic acid assay (Sigma), and DNA was quantified using PicoGreen (Molecular Probes, Eugene, OR). Creatine kinase activity was adjusted for both protein and DNA, with similar results.

Regulation of ASB15 Expression

Increased expression of ASB15 (ASB15+) in C₂C₁₂ cells was achieved using a stable cell line constitutively expressing full-length bovine ASB15, described previously as ASB15-FL (McDaneld et al., 2006). Reduced expression of ASB15 (ASB15-) was achieved by transfection of small interfering RNA (siRNA) oligonucleotides (Ambion, Austin, TX). Initially, 3 ASB15 siRNA oligonucleotides (GCAUCUGUGCCGAUUAAAAtt, GC-CAGGCUAUAUUUCAUCCtt, and GGAGUAUGUG-CAAUAUAAtt) were evaluated. Cells were transfected at approximately 50% confluency with individual siR-NA (50 µM) using Lipofectamine2000 (Invitrogen). To confirm a decrease in expression, ASB15 messenger RNA (mRNA) abundance was evaluated after 1 and 2 d of incubation in differentiation media by quantitative PCR (SYBR Green; BioRad, Hercules, CA), as described previously (McDaneld et al., 2006), using a standard curve to express data as the log of the starting copy number. The siRNA with greatest reduction at d 2 of differentiation, GCCAGGCUAUAUUUCAUCCtt, was used for all subsequent experiments. Cells with unaltered levels of ASB15 expression (ASB15Con) were cultured as described for ASB15- cells, except that a negative control siRNA oligonucleotide (AM4611; Ambion, Austin, TX) was transfected. Cells were evaluated in subsequent experiments after 1 and 2 d of incubation in differentiation media. However, only data from d 2 are reported because this time point had the greatest decrease of ASB15 mRNA abundance, as well as the greatest change in differentiation in ASB15+ and ASB15- cells.

Western Blotting

Activation of Erk1/2 and PI3K/Akt signaling proteins was evaluated by Western blotting. Cell lysates were collected 1 (data not shown) and 2 d after the stimulation of differentiation. Lysates were separated by SDS-PAGE and transferred to membranes by electrotransfer, following standard protocols. The Erk1/2 pathway was investigated using phosphorylated-MAPK and MAPK primary antibodies (No. 9101 and No. 9102, respectively; Cell Signaling Technologies Inc., Beverly, MA). The PI3K/Akt signaling cascade was investigated using phosphorylated-Akt and Akt primary antibodies (no. 9271 and no. 9272, respectively; Cell Signaling Technologies Inc.). Anti-rabbit immunoglobulin G secondary antibodies with horseradish peroxidase conjugate (Cell Signaling Technologies Inc.) were used for detection, along with the ECL Plus Western Blotting Detection Reagents (GE Health Care, Buckingham-shire, UK). An imaging system (Alpha Innotech, San Leandro, CA) was used to select and quantify detected proteins.

Evaluation of Erk1/2 and PI3K/Akt Signaling Pathways

Activation of MAPK signal transduction pathways was evaluated in ASB15+, ASB15-, and ASB15Con C₂C₁₂ cells, in the presence and absence of known regulators of the Erk1/2 and PI3K/Akt pathways. Inhibition of the Erk1/2 pathway was achieved by pretreating cells with U0126 (10 µM) in growth media for 2 h before induction of differentiation. Growth media was then replaced with differentiation media, and 24 h later, cells were treated with U0126 (10 µM) in differentiation media for 2 h. Cells representing the control treatment received an equal volume of carrier in place of U0126. Regulation of the PI3K/Akt pathway was achieved using 4 experimental treatments: inhibition by LY294002, activation by IGF-I, IGF-I and LY294002 combination (IGF-I + LY), and control. The LY294002 inhibitor was administered as described previously for U0126, except at 50 µM. The IGF-I (50 ng/ mL) was administered continually by addition to differentiation media. The control treatment received an equal volume of carrier in place of LY294002 and IGF-I. Treatments were applied to cells such that each cell type (ASB15+, ASB15-, and ASB15Con) by treatment (U0126 and control; LY294002, IGF-I, IGF-I + LY, and control) combination was represented by 3 wells of a 6-well plate (ThermoFisher, Waltham, MA). This experimental design was replicated across 5 independent experiments for each pathway investigated.

Statistical Analysis

Phosphorylation of proteins in the Erk1/2 and PI3K/ Akt pathways was evaluated by ANOVA using the PROC MIXED procedure (SAS Inst. Inc., Cary, NC). Fixed effects included experiment, cell type, treatment, and their interactions. Expression of the total protein (MAPK or Akt) was used as a covariate in the analysis to correct for variation in sample loading. Least squares means were calculated for cell type by treatment combinations, and differences between means were determined using the PDIFF option. Treatment differences were deemed significant at a P-value 0.05.

	RESULTS AND DISCUSSION

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Differentiation Altered by ASB15 Expression

Understanding specific cellular mechanisms that regulate growth and muscle accretion is critical to the development of novel methods to improve the efficiency of muscle accretion. We reported previously (McDaneld et al., 2006) that mRNA abundance of ASB15 is low in proliferating myoblasts, but increases rapidly upon stimulation of differentiation. We have also demonstrated that overexpression of ASB15 in cell culture increases protein synthesis and increases skeletal muscle fiber diameter in vivo (McDaneld et al., 2006). The objectives of this experiment were to further examine the role of ASB15 by characterizing differentiation in myoblasts that differentially express ASB15 and by identifying signaling pathways that participate in mediating the effects of ASB15 in myoblast differentiation.

Consistent with our previous data, ASB15 mRNA abundance was significantly increased by stable transfection of a constitutively active ASB15 expression vector (Table 1; McDaneld et al., 2006). Overexpression of ASB15 also resulted in a significant inhibition of differentiation (Figure 1; McDaneld et al., 2006). This effect was evident upon visual evaluation of cell morphology (Figure 1A) and a significant decrease in creatine kinase activity in ASB15+ vs. ASB15Con cells at both 1 and 2 d of differentiation (Figure 1B). The delay in differentiation that accompanies increased ASB15 mRNA abundance in ASB15+ cells may appear contradictory to our previous report that endogenous levels of ASB15 mRNA increase with induction of differentiation (McDaneld et al., 2006). However, these results suggest that ASB15 functions to moderate or limit cellular processes that promote differentiation. Thus, when ASB15 expression is artificially high before the stimulation of differentiation in As15+ cells, additional time is needed for these cells to overcome the inhibitory role of ASB15, and differentiation is delayed. To explore this hypothesis further, we evaluated C₂C₁₂ myoblasts with decreased ASB15 abundance (ASB15-) through transfection of ASB15 siRNA. The ASB15 siRNA significantly decreased mRNA abundance of ASB15 through the first 48 h of differentiation (Table 1). This inhibition of ASB15 mRNA abundance was also associated with a significant increase in differentiation that was apparent visually (Figure 1, panel A) and confirmed by a significant increase in creatine kinase activity (Figure 1, panel B) after 2 d of differentiation. The lack of a significant effect of ASB15- on differentiation at d 1 is likely due to the low expression of endogenous ASB15 at this time point (Table 1), such that further reduction caused by ASB15 siRNA had little additional effect. Together, these results replicate our previous findings that increased ASB15 expression inhibits early differentiation of myoblasts (McDaneld et al., 2006) and provide substantial evidence that the presence of ASB15 effectively limits the progression of differentiation during the first 48 h in C₂C₁₂ myoblasts. Additionally, data from ASB15- cells support our hypothesis that ASB15 functions to limit differentiation.

View larger version (75K): [in this window] [in a new window]   Figure 1. Expression of ankyrin repeat and suppressor of cytokine signaling box-containing protein (ASB) 15 alters differentiation of C2C12 myoblasts. (A) Images of C2C12 cells with decreased (ASB15-), increased (ASB15+), and unaltered (ASB15Con) ASB15 expression were captured 1 (Day 1) and 2 (Day 2) d following the stimulation of differentiation. (B) Creatine kinase activity, adjusted for DNA content, from cells described in (A). Bars represent mean ± SE, n = 3 replicates from each of 3 experiments. a–dMeans without a common letter differ, P < 0.05.

Activation of the Erk1/2 signal transduction pathway has been associated with a delay in differentiation of myoblasts (Yang et al., 2006). Therefore, we hypothesized initially that activation of this pathway may account for the regulation of differentiation by ASB15. However, increased expression of ASB15 actually decreased activation of the Erk1/2 pathway after 2 d of differentiation, as indicated by a 42% decrease in phosphorylated MAPK, a regulatory subunit of the Erk1/2 signaling pathway, in ASB15+ vs. ASB15Con cells (Figure 2, panels A and B). Additionally, down-regulation of ASB15 by siRNA had no significant effect on the Erk1/2 pathway. These results strongly suggest that the effects of ASB15 on early differentiation of C₂C₁₂ cells are not mediated by activation of the Erk1/2 pathway and that ASB15 alters differentiation through another pathway that may also inhibit Erk1/2. The MAPK inhibitor U0126, which inhibits at the level of MAPK, was used to investigate ASB15 in the absence of a fully functional Erk1/2 signal transduction pathway. As expected, U0126 significantly decreased phosphorylated-MAPK (Figure 2, panels A and B). Additionally, the Erk1/2 inhibitor significantly stimulated differentiation and subsequent creatine kinase activity in ASB15Con and ASB15- cells (Figure 2, panel C), confirming the expected response to Erk1/2 inhibition in our cell culture model. However, it is interesting to note that inhibition of the Erk1/2 pathway did not alter creatine kinase activity in cells overexpressing ASB15 (Figure 2, panel C). These data support our conclusion that overexpression of ASB15 does not alter differentiation through the Erk1/2 pathway because further inhibition of the Erk1/2 signaling pathway does not override the effect of overexpression of ASB15 (ASB15+) on differentiation. These observations of differential ASB15 expression on phosphorylation of the Erk1/2 pathway, coupled with the effects of ASB15 on differentiation, imply that ASB15 may be a potent regulator of a different physiological pathway that has a stronger impact on early differentiation of myoblasts than the Erk1/2 pathway.

View larger version (24K): [in this window] [in a new window]   Figure 2. Increased ankyrin repeat and suppressor of cytokine signaling box-containing protein (ASB) 15 expression decreases phosphorylation of mitogen-activated protein kinase (MAPK) of the extracellular signal-regulated kinase (Erk) 1/2 pathway in differentiating C2C12 myoblasts. Phosphorylation of MAPK was measured by Western blotting for phosphorylated MAPK (P-MAPK) 2 d after the stimulation of differentiation. Cells with decreased (ASB15-), increased (ASB15+), and unaltered (ASB15Con) expression of ASB15 were evaluated in the presence and absence of the Erk1/2 inhibitor U0126, as described in the text. (A) An example Western blot, showing a single replicate from 1 experiment. (B) Abundance of P-MAPK, adjusted for abundance of inactive MAPK, as determined by quantitation of Western blot bands. (C) Creatine kinase activity, adjusted for DNA content. Bars represent mean ± SE, n = 3 replicates in each of 3 experiments. a–dMeans within a graph without a common letter differ, P < 0.05.

Activation of the PI3K/Akt pathway has opposing effects to the Erk1/2 pathway and stimulates myoblast differentiation (Xu and Wu, 2000; Moelling et al., 2002; Sumitani et al., 2002). Therefore, we hypothesized that ASB15 is an inhibitor of the PI3K/Akt pathway, such that increased expression of ASB15 inhibits differentiation via decreased activity of the PI3K/Akt pathway, and vice versa. This hypothesis was supported by results showing decreased and increased abundance of phosphorylated-Akt, a regulatory subunit of the PI3K/ Akt signaling pathway, with ASB15+ and ASB15-, respectively, at d 2 of differentiation (Figure 3, panels A and B). Differences in creatine kinase activity observed among ASB15Con, ASB15-, and ASB15+ cells were consistent with these changes in PI3K/Akt activation (Figure 3, panel C).

View larger version (24K): [in this window] [in a new window]   Figure 3. Increased and decreased expression of ankyrin repeat and suppressor of cytokine signaling box-containing protein (ASB) 15 alter differentiation of C2C12 myoblasts via regulation of the phosphatidylinos-itol-3 kinase (PI3K)/Akt pathway (Akt is also known as protein kinase B). Activation of the PI3K/Akt pathway was measured by Western blotting for phosphorylated Akt (P-Akt) 2 d after the stimulation of differentiation. Cells with decreased (ASB15-), increased (ASB15+), and unaltered (ASB15Con) expression of ASB15 were evaluated. Four experimental treatments were applied to all cells, including a vehicle control (control), an activator of the PI3K/Akt pathway (IGF-I), an activator plus inhibitor of the PI3K/Akt pathway (IGF-I+LY), and a PI3K/Akt inhibitor (LY294002), as described in the text. (A) An example Western blot, showing a single replicate from 1 experiment. (B) Abundance of P-Akt, adjusted for abundance of inactive Akt, as determined by quantitation of Western blot bands. (C) Creatine kinase activity, adjusted for DNA content. Bars represent mean ± SE, n = 3 replicates in each of 3 experiments. a–gMeans within a graph without a common letter differ, P < 0.05.

The LY294002 is a potent inhibitor of the PI3K/Akt signal transduction pathway. It effectively inhibited activation of Akt and blocked almost all differentiation across cell types (Figure 3). Thus, a treatment combination of IGF-I and LY294002 was used to more effectively evaluate the effect of ASB15 in the presence of partial PI3K/Akt inhibition. In the presence of both IGF-I and LY294002, activation of Akt and creatine kinase activity were similar in ASB15Con and ASB15- cells. Conversely, phosphorylated-Akt and creatine kinase activity were significantly less in ASB15+ vs. ASB15Con cells, suggesting LY294002 and ASB15 act in an additive manner to inhibit activation of the PI3K/ Akt pathway by IGF-I.

In summary, the data reported herein provide preliminary evidence that ASB15 has a role in regulation of early myoblast differentiation and that this effect may be mediated in part by the PI3K/Akt signal transduction pathway. Currently, specific molecules within the PI3K/Akt pathway that interact with ASB15, and the nature of their interaction, remain to be determined. Additionally, the potential impact of the timeline of differentiation on regulation of the PI3K/Akt pathway by ASB15 remains to be explored. However, the data reported herein are the first to describe alteration in activation of the PI3K/Akt signal transduction pathway by differential expression of an ASB protein, and they provide initial insight to the mechanism of action of a novel regulator of myogenesis that may affect complex physiological processes important to muscle growth.

	Footnotes

 
¹ This research was supported in part by the National Research Initiative Competitive Grant 003-35206-13664 from the United States Department of Agriculture Cooperative State Research, Education, and Extension Service.

² Current address: US Meat Animal Research Center, Clay Center, NE 68933.

³ Corresponding author: moodyd{at}iastate.edu

Received for publication March 31, 2008. Accepted for publication June 30, 2008.

	LITERATURE CITED

Top Abstract INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

 


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This Article Abstract Full Text (PDF) All Versions of this Article: jas.2008-1076v1 86/11/2897    most recent Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by McDaneld, T. G. Articles by Spurlock, D. M. Search for Related Content PubMed PubMed Citation Articles by McDaneld, T. G. Articles by Spurlock, D. M.