Elsevier

Cellular Signalling

Volume 35, July 2017, Pages 48-60
Cellular Signalling

Ubiquitin-specific protease 4 (USP4) suppresses myoblast differentiation by down regulating MyoD activity in a catalytic-independent manner

https://doi.org/10.1016/j.cellsig.2017.03.008Get rights and content

Highlights

  • Knockdown of USP4 promotes myotube formation in myoblast differentiation.

  • USP4 knockdown increases the transcriptional activity of MyoD in C2C12 cells.

  • The effect of USP4 on myogenesis is independent of its deubiquitinase activity.

  • USP4 is associated with the phosphorylation of AKT and p38.

Abstract

For myotube formation, proliferation and differentiation of myoblasts must be tightly regulated by various myogenic regulatory factors (MRFs) such as MyoD, myogenic factor 5 (Myf5), myogenin, and muscle-specific regulatory factor 4 (MRF4). However, it is not clear how the expression or activity of these MRFs is controlled during myogenesis. In this study, we identified ubiquitin-specific protease 4 (USP4), one of deubiquitinating enzymes, as a suppressor of MRFs by demonstrating that a knockdown of USP4 enhances myogenesis by controlling MyoD and the level of myogenesis marker proteins in C2C12 cells. However, it was revealed that the effect of USP4 on myogenesis is independent of its deubiquitinase activity because the catalytic-site mutant has the same inhibitory effects as the wild-type USP4 on myogenesis. We observed that the activity and protein levels of both HDAC1 and HDAC4 are decreased when myoblast differentiation is promoted by the USP4 knockdown. We also found that the role of USP4 in muscle differentiation is correlated with two major signaling pathways in myogenesis, AKT and the p38 mitogen-activated protein kinase pathways. According to these results, we propose that USP4 is a key player in myogenic differentiation; it controls myogenic regulatory factors in a catalytic-independent manner.

Introduction

During myogenesis, myoblasts differentiate into myotubes through the cell fusion from mononucleated muscle fibers into multinucleated myofibers, and proliferation and differentiation of myoblasts are tightly regulated by myogenic regulatory factors (MRFs) such as myoblast determination protein (MyoD), myogenic factor 5 (Myf5), myogenin, and MRF4 [25]. At the earliest stage of differentiation, expression of Myf5 is responsible for myoblast proliferation, but subsequently, expressions of Myf5 and MyoD drive the differentiation of myoblasts [26]. Myogenin is involved in the fusion of myogenic precursor cells and their maturation [20]. Finally, for the completion of myogenesis, differentiated myoblasts express mechanical muscle proteins such as myosin heavy chain (MHC) and tropomyosin. Among various MRFs, MyoD, a key transcription factor promoting myogenic differentiation, binds to the promoters of numerous muscle-specific genes [32]. For example, MyoD binds directly to the promoter of myogenin [19] and enhances expression of the myogenin protein, which is essential for morphological maturation of myotubes and terminal differentiation of skeletal muscle. Generally, E-proteins (E2A, E12, and E47) and proteins of the myocyte enhancer factor 2 (MEF2) family (MEF2A, MEF2B, MEF2C, and MEF2D) work as co-transcription factors with MyoD by forming a heterodimer with MyoD.

Transcriptional activation is often linked to hyperacetylation of histones by histone acetyltransferase (HAT). Acetylated histones are deacetylated by histone deacetylases (HDACs), and this action leads to inactivation of transcription. In this context, it is known that skeletal myogenesis is controlled by histone deacetylases: HDAC1, one of class I HDACs, inhibits the myogenic program by direct interaction with MyoD [18], [24], and HDAC4 and HDAC5 (class II histone deacetylases) inhibit myogenesis by binding to MEF2 [21], [22].

AKT and p38 mitogen-activated protein (MAP) kinase signaling pathways also enhance muscle differentiation by increasing the expression of p300, a transcriptional coactivator of MRF [4] and by activating MEF2 through phosphorylation [14], [37]. In addition, the involvement of AKT and p38 signaling in myogenesis can be explained by the role of AKT in the enhancement of protein stability of prohibitin 2 (PHB2), which recruits HDAC1 for the transcriptional repression of MyoD and MEF2 [11], [28]. Furthermore, it is known that p38 causes caspase-mediated cleavage of HDAC4 [41]. Accordingly, both AKT and p38 signaling cascades enhance myogenesis by increasing the transcriptional activity of MyoD.

There is some evidence that myogenesis is also controlled at protein level by the ubiquitin-proteasome system (UPS). For example, E3 ubiquitin ligases, Mdm2 and Praja1 (PJA1), have a role in promoting skeletal myogenesis by removing the negative regulator of muscle differentiation [5], [9]. However, myogenesis is negatively regulated by UPS since MyoD and myogenin are degraded by UPS through an ubiquitin E3 ligase such as MAFbx/Atrogin-1 (Fbxo32) and MuRF1 (Trim63) [13], [31]. Accordingly, UPS is activated during muscle atrophy in which Fbxo32 and Trim63 are highly expressed [2]. This UPS-dependent protein degradation can be reversed by deubiquitinating enzymes which work as a feedback mechanism maintaining the protein levels. Many differentiation processes are controlled by ubiquitin-specific proteases (USPs), members of the deubiquitinase protein family. For example, USP27x, USP22, and USP51 are associated with neuronal differentiation through their regulation of the Hes stability [16], and USP7 controls adipogenesis by deubiquitinating Tip60, a transcriptional cofactor and acetyltransferase involved in adipogenesis [6]. Therefore, it can be expected that myogenesis is also controlled by deubiquitinases. A recent study showed that endoplasmic-reticulum-localized USP19 decreases myoblast fusion during muscle cell differentiation [33]. Nevertheless, little is known about the relevance of deubiquitinases for myogenesis.

Wnt/β-catenin signaling also controls multiple steps at some stages of muscle cell proliferation and differentiation by regulating gene expression [29], [30]. Specifically, it is reported that β-catenin increases the transcriptional activity of MyoD by enhancing the binding of MyoD to E box elements [15]. Because USP4 was identified as one of positive regulators of Wnt/β-catenin signaling in cancer cells [12], [38] owing to its effect on the stability of β-catenin, we hypothesized that USP4 plays a role in myogenesis. To test this hypothesis, we evaluated the effect of USP4 on myoblast differentiation in C2C12 cells, a mouse myoblast cell line, and confirmed that a USP4 knockdown promotes myoblast differentiation. By further studying the effects of USP4 on myogenesis, we found that USP4-mediated myoblast differentiation is not related to the deubiquitinating activity of USP4. To understand the role of USP4 in myogenesis, in this study, we investigated how USP4 controls myoblast differentiation.

Section snippets

Reagents

For immunoblotting, anti-MyoD (sc-304) and anti-α-tubulin (sc-53646) antibodies were purchased from Santa Cruz Biotechnology; an anti-USP4 antibody (A300-830) from Bethyl Laboratory System; an anti-myogenin antibody (ab1835) from Abcam; an anti-Ac-Histone H3 antibody (06-599) from Millipore; and anti-p-AKT (S473, 9271), anti-AKT (9272), anti-p-p38 (4511), anti-p38 (9212), anti-HDAC1 (5356), anti-HDAC4 (15164) and anti-GAPDH (5174) antibodies from Cell Signaling Technology. An anti-MHC antibody

The knockdown of USP4 induces myoblast differentiation

Because USP4 is known to increase the stability of β-catenin in cancer cells [12], [38] and that Wnt/β-catenin signaling stimulates myogenic differentiation, we hypothesized that USP4 upregulates myogenesis. To prove this hypothesis, we examined the effect of USP4 knockdown and overexpression on myogenesis by measuring the protein levels of α-MHC, MyoD and myogenin, representative myogenic markers, in C2C12 cells. For the knockdown experiment, we first confirmed the knockdown efficiency of USP4

Discussion

The ubiquitin proteasome system plays a critical role in muscle differentiation by controlling the concentration of key proteins in myogenesis through ubiquitin E3 ligases such as Mdm2, Praja1 [5], [9], MuRF1 (Trim63), and MAFbx/Atrogin-1 (Fbxo32) [2], [3], [7], followed by proteasome-dependent degradation. Therefore, deubiquitinating enzymes are also expected to be involved in myogenic differentiation by stabilizing myogenic regulatory factors. USP4 is known to control many signaling pathways

Acknowledgments

This research was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2016R1A2B2008081 and 2016R1A6A3A11930924).

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