HMGB2 regulates satellite-cell-mediated skeletal muscle regeneration ...

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downstream target of HMGB2, as previously shown for HMGA2. IGF2BP2 binds to mRNAs of Myf5 or cyclin A2, resulting in translation enhancement or mRNA ...
© 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 4305-4316 doi:10.1242/jcs.189944

RESEARCH ARTICLE

HMGB2 regulates satellite-cell-mediated skeletal muscle regeneration through IGF2BP2

ABSTRACT Although the mechanism underlying modulation of transcription factors in myogenesis has been well elucidated, the function of the transcription cofactors involved in this process remains poorly understood. Here, we identified HMGB2 as an essential nuclear transcriptional co-regulator in myogenesis. HMGB2 was highly expressed in undifferentiated myoblasts and regenerating muscle. Knockdown of HMGB2 inhibited myoblast proliferation and stimulated its differentiation. HMGB2 depletion downregulated Myf5 and cyclin A2 at the protein but not mRNA level. In contrast, overexpression of HMGB2 promoted Myf5 and cyclin A2 protein upregulation. Furthermore, we found that the RNA-binding protein IGF2BP2 is a downstream target of HMGB2, as previously shown for HMGA2. IGF2BP2 binds to mRNAs of Myf5 or cyclin A2, resulting in translation enhancement or mRNA stabilization, respectively. Notably, overexpression of IGF2BP2 could partially rescue protein levels of Myf5 and cyclin A2, in response to HMGB2 decrease. Moreover, depletion of HMGB2 in vivo severely attenuated muscle repair; this was due to a decrease in satellite cells. Taken together, these results highlight the previously undiscovered and crucial role of the HMGB2– IGF2BP2 axis in myogenesis and muscle regeneration. KEY WORDS: HMGB2, Myogenesis, IGF2BP2, Muscle regeneration

INTRODUCTION

Myogenesis is the term describing the process of myofiber formation, which occurs during embryonic development, postnatal growth and muscle regeneration (Chargé and Rudnicki, 2004; Tidball and Villalta, 2010). This process is a highly coordinated event and implicated in a series of transcriptional networks, among which Pax7, Myf5, MyoD (also known as MYOD1), myogenin and Mrf4 (also known as MYF6) are crucial and indispensable (Buckingham and Rigby, 2014; Mok and Sweetman, 2011). Skeletal muscle repair from injury involves in a series of complicated cascade events, which can be divided into the following steps. First, quiescent satellite cells, a pool of adult muscle stem cells, are activated and become committed myoblasts in response to the damage (Kang and Krauss, 2010; Kuang

State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510006, China. *These authors contributed equally to this work ‡ Authors for correspondence: ([email protected]; [email protected])

X.Z., 0000-0001-7589-9928; M.L., 0000-0003-3019-3387; H.H., 0000-00016455-676X; K.C., 0000-0002-3871-7651; Z.Y., 0000-0001-6192-6905; Y.Z., 0000-0002-5861-2355; Y.N., 0000-0003-4456-0651; H.C., 0000-0001-8136-7442; L.C., 0000-0001-7802-9215; Y.C., 0000-0002-3871-7651; D.M., 0000-00028738-4486 Received 18 April 2016; Accepted 17 September 2016

and Rudnicki, 2008). Then, these myoblasts begin to proliferate to expand the population of progenitor for repair (Chargé and Rudnicki, 2004; Kuang and Rudnicki, 2008). Finally, proliferating myoblasts withdraw from the cell cycle, undergo terminal differentiation and fuse with each other to form renascent myofibers that replace the damaged and dead ones (Apponi et al., 2011; Kang and Krauss, 2010). Sets of transcription factors have been identified that regulate the proliferation of satellite cells and terminal differentiation. However, more precise molecular mechanism and alternative key regulators are required to be further elucidated. The high-mobility group (HMG) superfamily consists of three families, HMGA, HMGB and HMGN, all of which are chromatinbinding proteins that share similarities in structural and functional properties (Agresti and Bianchi, 2003; Hock et al., 2007). It has been well documented that HMG proteins not only act as dynamic modulators of chromatin architecture but also especially influence DNA replication, recombination, repair and transcriptional regulation (Agresti and Bianchi, 2003; Bianchi and Agresti, 2005; Ueda and Yoshida, 2010). Multiple transcription factors have been verified to interact with HMG proteins at specific gene locus during the process of transcription (Ueda and Yoshida, 2010). Moreover, it has been reported that both HMGA and HMGB families are mainly expressed in stem cells or progenitors to maintain proliferation and undifferentiated status (Hock et al., 2007). In particularly, HMGA2 can induce mouse embryonic stem cells to commit to the myogenic lineage and regulate myoblast proliferation and skeletal muscle development (Caron et al., 2005; Li et al., 2012). There are three members of HMGB proteins in mammals, HMGB1, HMGB2 and HMGB3. All of them contain two DNAbinding domains (basic HMG boxes) followed by a long acidic C-terminal tail and have more than 80% identity in protein sequences (Bustin, 1999; McCauley et al., 2005; Catena et al., 2009). HMGB1 has been demonstrated to play a key role in multiple biochemical and molecular activities, such as the innate immunity response, necrosis, arthritis and tumorigenesis (Hock et al., 2007; Yanai et al., 2012). Although it has been demonstrated that as a transcriptional coregulator, HMGB2 regulates various differentiation programs, including erythropoiesis, chondrogenesis and spermatogenesis, its role in myogenesis and muscle regeneration remains largely unclear (Laurent et al., 2010; Ronfani et al., 2001; Taniguchi et al., 2011). Post-transcriptional regulation of myogenesis is an important process for muscle development and regeneration (Apponi et al., 2011). Insulin-like growth factor-II mRNA-binding proteins (IGF2BP2), also known as IMP2, is a member of IMP family that contains two RNA-binding domains and four K homology motifs and functions in mRNA localization, turn over and translation modulation (Nielsen et al., 2002, 2003). The involvement of RNAbinding proteins, such as IGF2BP2, HuR (also known as ELAVL1), CUGBP1 and Lin-28, in muscle biology has been extensively highlighted in recent investigations (Apponi et al., 2011; Li et al., 4305

Journal of Cell Science

Xingyu Zhou*, Mingsen Li*, Huaxing Huang, Keren Chen, Zhuning Yuan, Ying Zhang, Yaping Nie, Hu Chen, Xumeng Zhang, Luxi Chen, Yaosheng Chen‡ and Delin Mo‡

2012). A previous study found that IGF2BP2 functions by binding to the 5′ UTR of insulin-like growth factor 2 (IGF2) mRNA and controlling IGF2 translation (Dai et al., 2011). More recently, IGF2BP2 was also suggested to regulate myoblasts proliferation through binding to various target mRNAs and modulating their translation, such as Myc, Sp1, IGF1R, Ccng1 and Nras (Gong et al., 2015; Li et al., 2012). Interestingly, IGF2BP2 has been identified as a direct target of HMGA2 in proliferating myoblasts, NIH/3T3 fibroblasts and mouse embryos (Brants et al., 2004; Cleynen et al., 2007; Li et al., 2012). However, whether or not HMGB2 is able to regulate the expression of IGF2BP2 during myogenesis is unknown. In the present study, we first found that HMGB2 was upregulated significantly in the early stage of muscle regeneration and highly expressed in proliferating myoblasts. Then, we used gain- and lossof-function approaches to demonstrate that HMGB2 maintains myoblast proliferation and impedes myogenic differentiation by controlling IGF2BP2, which in return enhanced the protein production of Myf5 and cyclin A2. It was further demonstrated that IGF2BP2 could bind either to Myf5 or to cyclin A2 mRNA to improve the mRNA stability or enhance the translation, respectively. In vivo data also implied that HMGB2 was required for satellite cell proliferation and muscle repair after injury. In summary, we have identified and characterized the role of the HMGB2–IGF2BP2 axis in myogenesis and muscle regeneration. RESULTS The expression pattern of HMGB2 during muscle development and generation

To determine the expression pattern of potential genes in the early stage of muscle repair after injury. RNA sequencing (RNA-seq) was employed to analyze the genome-wide gene expression of damaged muscle tissues at various time points after injury. Among sets of differentially expressed genes, HMGB2 was focused on because of its upregulated expression (Fig. 1A). Real-time quantitative PCR (qPCR) and western blotting also confirmed that the expression of HMGB2 significantly increased in the early period of muscle repair, peaking at day 3 after cardiotoxin (CTX) injection, indicating its potential role in muscle regeneration (Fig. 1B). Furthermore, large numbers of HMGB2-positive cells were found in the damaged adult tibialis anterior muscle but not in the uninjured one (Fig. 1C,D). It has been well documented that activation and proliferation of quiescent satellite cells are required for adult muscle regeneration (Kang and Krauss, 2010). Importantly, immunofluorescence revealed that most of HMGB2-positive cells were simultaneously Pax7-positive, a marker of muscle stem cell or progenitor (Fig. 1D). Further evidence revealed that both mRNA and protein of HMGB2 were highly expressed in neonatal muscle but decreased during the progress of postnatal development and were not detectable in mature muscle (Fig. 1E). Collectively, these data support that HMGB2 is expressed mainly in myoblasts but not in mature myofibers. Intriguingly, the myogenic genes Myf5, myogenin and Mrf4 showed a similar expression pattern during muscle development, implying that HMGB2 might be expressed during myogenesis (Fig. S1A). However, MyoD and Pax7 have differing expression patterns to HMGB2 in muscle development, which indicates that they potentially operate through a different mechanism. In addition, the expression of HMGB2 in other tissues of adult mice was also detected. We found that HMGB2 was widely expressed in various adult tissues (Fig. S1B), raising the possibility that HMGB2 might participate in maintaining the physiological function and homeostasis of these organs. Taken together, HMGB2 might play a vital role in muscle development and regeneration. 4306

Journal of Cell Science (2016) 129, 4305-4316 doi:10.1242/jcs.189944

HMGB2 locates in nuclei of myoblast and functions as a myogenic repressor

In order to further investigate the impact of HMGB2 on myogenesis, the C2C12 myoblast differentiation model was studied. The expression trend of HMGB2 was evaluated during myogenic differentiation. Similar to the results in vivo, both HMGB2 and Pax7 were highly expressed in proliferating C2C12 myoblasts but promptly downregulated after differentiation (Fig. 2A,B). Few HMGB2 proteins were detected in mature myotubes (Fig. 2C). Both in vivo and in vitro analyses showed that HMGB2 proteins were mainly located in Pax7-positive muscle stem cells or progenitors but rarely in mature myofibers (Figs 1C,D, 2C), indicating that HMGB2 is mostly expressed in undifferentiated myoblasts. As HMGB2 is a chromatin-binding protein and also interacts with transcription regulators in the nucleus, its location in C2C12 myoblasts and primary satellite cells isolated from adult mice was detected in this work. Primary satellite cells were successfully isolated and became activated myoblasts. As expected, immunofluorescence staining showed that HMGB2 proteins were only located in the nuclei of C2C12 and primary myoblasts (Fig. 2D,E), which prompted us to speculate that HMGB2 operates with other myogenic regulators in the nucleus. Next, three small interfering RNAs (siRNAs) were designed to knock down HMGB2 in C2C12 myoblasts (denoted si-HM-1, siHM-2 and si-HM-3), two of which were efficient (Fig. S2A). SiHMGB2, a mixture of si-HM-2 and si-HM-3, caused a substantial knockdown of HMGB2 proteins and was used in all of the following analysis (Fig. S2B). As shown, depletion of HMGB2 accelerated the initiation of myogenic program, because of a significant increase in the mRNA expression of early myogenic marker myogenin at 24 h after differentiation induction (Fig. 3A). Myogenin-positive cells also increased prominently at 24 h after differentiation when HMGB2 was knocked down (Fig. 3B). A similar increase in the expression of fast myosin skeletal heavy chain (MyHC, also known as Myh1) was also observed in differentiating C2C12 cells after HMGB2 knockdown (Fig. 3C,E). Consistently, addition of si-HMGB2 in C2C12 cells caused a significant enhancement of myotube formation (Fig. 3D). However, ectopic expression of HMGB2 led to a remarkable concomitant repression of myotube formation (Fig. 3F). Thus, HMGB2 was identified as a crucial myogenic regulator. HMGB2 regulates myoblast proliferation through cell-cycle proteins

It has been well established that HMG proteins are mainly specific to stem cells, and they are implicated in the regulation of stem cell proliferation and differentiation (Agresti and Bianchi, 2003; Hock et al., 2007). More recently, Zhizhong Li and his colleagues have reported that the HMGA2–IGF2BP2 axis affects muscle development through controlling myoblast proliferation (Li et al., 2012). Simultaneously, the expression of HMGA2 decreased significantly when HMGB2 was downregulated (Fig. S2C–E). These results drove us to hypothesize that HMGB2 probably suppresses myogenesis by means of regulating myoblast proliferation and self-renewal. To test this, the proliferation rate of C2C12 cells and muscle satellite cells transfected with si-HMGB2 and si-NT was examined, respectively. Data from a real-time monitoring system revealed that the proliferation of C2C12 myoblasts and primary satellite cells was reduced after HMGB2 depletion with siRNAs (Fig. 4A; Fig. S4B). Flow cytometry analysis followed by propidium iodide staining further revealed that an arrest of cell cycle in S phase occurred in response to a HMGB2 decrease in C2C12 myoblasts (Fig. 4B).

Journal of Cell Science

RESEARCH ARTICLE

RESEARCH ARTICLE

Journal of Cell Science (2016) 129, 4305-4316 doi:10.1242/jcs.189944

To address the molecular mechanism underlying the control of cell cycle progression through HMGB2, HMGB2 was knocked down in myoblasts. The expression of several myogenic-associated factors (Myf5, MyoD, Pax7, id2 and YY1) and cell cycle regulators [cyclin A2, cyclin E, CDK2, CDK4, Rb, P27 (CDKN1B) and Akt (Akt1 isoform)] was detected by qPCR. A significant difference in mRNA level between the si-HMGB2 group and the control group was not observed for any of the detected genes (Fig. 4C,D). Intriguingly, a pronounced reduction of Myf5 and cyclin A2 proteins occurred in response to si-HMGB2 treatment both in C2C12 myoblasts and primary muscle satellite cells. However, there was no significant difference for YY1, Pax7 and MyoD proteins (Fig. 4E; Fig. S4C). In addition, overexpression of HMGB2 did not affect the mRNA levels of Myf5 and cyclin A2, but promoted their protein expression (Fig. S3A,B). Consistent with the siRNA experiment, overexpression of HMGB2 did not affect either the RNA or protein levels of Pax7 and MyoD (Fig. S3A,B).

Next, a model of CTX-mediated muscle injury concomitant with HMGB2 depletion was used to examine the expression of the above regulators (Fig. 4F). Both mRNA and protein levels of HMGB2 were successfully downregulated in tibialis anterior at day 3 after injury by intramuscular injection of si-HMGB2 (Fig. 4G,H), which resulted in significant reduction in Myf5, cyclin A2 and Pax7 proteins (Fig. 4H). In addition, the number of EdU (5-ethynyl-2′deoxyuridine)-positive cells decreased substantially in tibialis anterior when HMGB2 was downregulated (Fig. 4I), implying that the proliferation of satellite cells was restrained after HMGB2 was depleted. Based on in vitro and in vivo results, we conclude that HMGB2 maintains myoblast proliferation and self-renewal by controlling protein expression of Myf5 and cyclin A2. IGF2BP2 acts as a downstream target of HMGB2

We found that HMGB2 regulates Myf5 and cyclin A2 at the protein but not mRNA level, suggesting that a post-transcriptional 4307

Journal of Cell Science

Fig. 1. HMGB2 expression gradually decreases during postnatal muscle development and is induced in the early of muscle regeneration. (A) Some of the differentially expressed genes identified by RNA-seq during muscle regeneration (n=2 mice per group) represented by a heat map. (B) The tibialis anterior muscle was subjected to cardiotoxin (CTX) injury and then qPCR (left) and western blotting (right) were performed to detect the expression of HMGB2 at day 0, day 1, day 2, day 3 and day 4 after injury, respectively. Data are presented as mean±s.e.m.; n=3 mice per group. (C) Immunofluorescence staining for DAPI (blue), HMGB2 (green) and Pax7 (red) on tibialis anterior muscle sections after 3 days of NaCl (top) or CTX (bottom) injection. White arrows indicate Pax7and HMGB2-double-positive cells. Scale bar: 100 μm. (D) Immunohistochemistry analysis for HMGB2 on tibialis anterior sections 3 days after CTX or NaCl injection. Brown, HMGB2; blue, nuclei. Scale bar: 100 μm. (E) qPCR (left) and western blotting (right) analysis for HMGB2 expression in tibialis anterior at postnatal day 1, day 14 and day 140. Data are presented as mean±s.e.m.; n=3 mice per group.

RESEARCH ARTICLE

Journal of Cell Science (2016) 129, 4305-4316 doi:10.1242/jcs.189944

Fig. 2. The expression and location of HMGB2 in satellite cells and differentiating C2C12 cells. (A) qPCR analysis for the expression of HMGB2 in C2C12 cells cultured in growth medium (GM) and in the first 3 days after differentiation induction (DM). Data are presented as mean±s.e.m.; n=3. (B) Western blotting analysis for the expression of HMGB2, Pax7 and MyHC in C2C12 cells cultured in growth medium and in the first 3 days after differentiation induction. (C) C2C12 cells were induced to differentiate for 3 days before immunofluorescence staining for DAPI, HMGB2 and MyHC (left). The percentage of HMGB2-positive or HMGB2-negative nuclei in MyHC-positive myofibers was calculated (right). White arrows indicate unfused HMGB2-positive nuclei. Data are presented as mean± s.e.m.; n=3. Scale bar: 50 μm. **P