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Originally published In Press as doi:10.1074/jbc.M308889200 on August 27, 2003

J. Biol. Chem., Vol. 278, Issue 47, 47119-47128, November 21, 2003
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RNAi-mediated HuR Depletion Leads to the Inhibition of Muscle Cell Differentiation*

Kate van der Giessen, Sergio Di-Marco, Eveline Clair, and Imed Eddine Gallouzi{dagger}

From the Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada

Received for publication, August 12, 2003 , and in revised form, August 27, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The formation of muscle fibers involves the sequential expression of many proteins that regulate key steps during myoblast-to-myotube transition. MyoD, myogenin, and the cyclin-dependent kinase inhibitor p21cip1 are major players in the initiation and maintenance of the differentiated state of mouse embryonic muscle cells (C2C12). The messenger RNAs encoding these three proteins contain typical AU-rich elements (AREs) in their 3'-untranslated regions (3'-UTRs), which are known to affect the half-life of many short-lived mRNAs. HuR, an RNA-binding protein that regulates both the stability and cellular movement of ARE-containing mRNAs, interacts and stabilizes the p21cip1 message under UV stress in human RKO colorectal carcinoma cells. Here, by the use of gel shift experiments and immunoprecipitation followed by reverse transcription-PCR analysis, we show that HuR interacts with MyoD, myogenin, and p21cip1 mRNAs through specific sequences in their 3'-UTRs. To demonstrate the implication of endogenous HuR in myogenesis, we knocked down its expression in myoblasts using RNA interference and observed a significant reduction of HuR expression, associated with complete inhibition of myogenesis. Moreover, the expression of MyoD and myogenin mRNAs, as well as proteins, is significantly reduced in the HuR knockdown C2C12 cells. We were able to completely re-establish the myogenic process of these defective cells by introducing back HuR protein conjugated to a cell-permeable peptide. Finally, HuR accumulates in the cytoplasm during myogenesis. Thus, our results clearly demonstrated that endogenous HuR plays a crucial role in muscle differentiation by regulating the expression and/or the nuclear export of ARE-containing mRNAs that are essential for this process.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The first step of skeletal muscle formation (also named myogenesis) is the cell cycle arrest of myoblasts, which leads to cell fusion and the formation of multinucleated myotubes. This transition is controlled by muscle-specific transcriptional regulators that respond to external signals to couple myogenesis to the development and growth of the organism (1). MyoD, Myf-5, myogenin, and MRF4 represent the main myogenic transcription factors and belong to the MRF (myogenic regulatory factor) family. MyoD and Myf-5 are expressed in proliferating myoblasts, whereas myogenin expression is induced only upon muscle differentiation (2). MyoD activates the cyclin-dependent kinase inhibitor p21cip1 (3), which, along with p57 (another Cip1/Kip1 family member), leads to cell cycle arrest (4). It is widely accepted that p21cip1 induction follows myogenin expression during the myoblast-to-myotube transition and that a high level of p21cip1 is required for myotube maintenance (5).

C2C12 myoblasts (a mouse embryonic muscle cell line) provide a powerful model to study skeletal muscle differentiation (6). They overexpress the myogenic factors described above and fuse to form muscle fibers when induced to differentiate. This overexpression is due not only to an increase in the transcription of their corresponding genes, but also to the stabilization of their mRNAs (7, 8). Therefore, preventing the degradation of mRNAs that encode key factors of muscle cell differentiation is crucial for maintaining the integrity of the whole process. However, the mechanisms that affect the turnover and the cellular movement of these messages remain elusive.

MyoD, myogenin, and p21cip1 mRNAs each harbor an AU-rich element (ARE)1 in their 3'-untranslated regions (3'-UTRs) (9). These AREs contain one or two AU3A sequences that trigger the rapid degradation of short-lived mRNAs, such as many oncogenes, lymphokines, and cytokines (10, 11). AREs do not always target mRNAs for degradation (12) but may also serve as an anchor for some of the shuttling AU-binding proteins (AUBPs) that monitor the nuclear export of ARE-containing mRNAs (13, 14). Therefore, it is conceivable that the half-lives and cellular movement of MyoD, myogenin, and p21cip1 mRNAs may be regulated through their AREs in association with AUBPs.

HuR is a well-studied RNA-binding protein and AUBP that specifically interacts and stabilizes AU3A-containing mRNAs (15-19). It is a 32-kDa protein that belongs to the Hu/ELAV family (embryonic lethal, abnormal vision (20, 21)) of RNA-binding proteins. Unlike the other three ELAV proteins, HuB, HuC, and HuD (which are expressed primarily in the brain), HuR is ubiquitously expressed (22) and functions as an adaptor protein for the nuclear export of many ARE-containing mRNAs (14, 23).

The elav locus was first discovered as a gene that is essential for the development and maintenance of the nervous system of Drosophila (20, 21). Recently, it has been shown that the ELAV human counterparts, HuB, HuC, and HuD (22), are involved in mouse embryonic development (24) and the differentiation of a variety of embryonic cell lines (25-29). The first demonstration that an Hu/ELAV protein is involved in differentiation came from the work of Jain and coworkers (26). They showed that the ectopic overexpression of HuB in the preadipocyte cell line 3T3 L1 leads to the enhancement of adipocyte differentiation and the stabilization, as well as the translation induction of the glucose transporter 1 mRNA. Furthermore, HuB induces neurite formation of human embryonic tetracarcinoma (hNT2) cells by stabilizing and/or regulating the translation of the neurofilamin mRNA (27).

Together, these observations suggest that the tissue-specific ELAV proteins, through their ability to regulate the stability and/or the translation of many mRNAs, play a crucial role in cell differentiation (30). Therefore, it is possible that HuR, which is ubiquitously expressed, could have similar effects on the differentiation of other embryonic cell lines. Establishing this role for endogenous HuR would be unexpected and of high interest, because these regulatory functions are usually associated with tissue-specific proteins. However, there are many examples in the literature suggesting that many RNA-binding proteins may have different functions in different tissues (31-36). These different effects are due to a variation in their expression or in their regulation, through protein-protein associations or post-translational modifications, which will modulate the association with their end targets (such as an mRNA).

The stabilizing function of HuR in mammalian cells was only demonstrated in experiments whereby cells were co-transfected with the recombinant HuR protein as well as an artificial ARE-containing message (15, 17, 19). Thus, the question of whether endogenous HuR exercises the same function in vivo remains unanswered. Recently, it was observed that the overexpression of HuR in myoblasts correlates with the stabilization of MyoD, myogenin, and p21cip1 messages, as well as with the acceleration of muscle cell differentiation (9). Because this effect was observed only upon the overexpression of exogenous HuR, we still do not know whether the observed stabilization and differentiation stimulation reflect the true function of endogenous HuR during myogenesis. Therefore, the best approach to define the implication of HuR in this process is to disturb its expression in myoblasts by RNA interference, then stimulate muscle differentiation and monitor the effect on myotube formation.

HuR shuttles between the nuclear and cytoplasmic compartments by virtue of its HNS (HuR nucleocytoplasmic shuttling domain (37)). We recently used a novel approach for delivering protein domains to the interior of mammalian cells to delineate the shuttling pathways of HuR (14). Specifically, the HNS and the CRM1-dependent nuclear export signal (38) domains were fused to the cell-permeable peptide antennapedia (AP). AP facilitates the cellular uptake of such chimeras into mammalian cells via a non-endocytic, non-degradative pathway with high efficiency (>90%). Using this approach, we demonstrated that, unlike other known shuttling proteins, HuR utilizes two pathways to export its mRNA targets from the nucleus to the cytoplasm (14). However, these findings (14) did not address whether the stabilizing function of HuR and/or its nucleocytoplasmic movement may affect vital processes such as cell growth and differentiation. To address these issues, C2C12 myoblasts were used to define the role of HuR during cell differentiation. Our results demonstrate that HuR plays an essential role in myogenesis and regulates the expression of mRNAs that encode key myogenic factors.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Construction and Protein Purification—The AP PCR product was produced by self-amplification using AP-N-sense (5'-ggg gac aag ttt gta caa aaa agc agg ctt cga agg aga tag aac cat gcg tca aat taa gat ttg gtt cca gaa ccg tcg cat g-3') and AP-N-antisense (5'-ggg gac cac ttt gta caa gaa agc tgg gtc cat atg aag ctt aga tat ctc gag tgc ggc cgc ttt ctt cca ttt cat gcg acg gtt ctg-3') oligonucleotides. To create the AP-C entry vector, a binding protein recombination reaction between pDONR1 (Invitrogen) and the AP PCR product was performed using the Gateway system (Invitrogen).

PCR amplification of HuR was performed using the GST-HuR plasmid as a template (39) with the primers HuR-TAG-For (5'-ccc ctc gag tct aat ggt tat gaa gac cac-3') and AP-HuR-Rev (5'-ggg aag ctt tta ttt gtg gga ctt gtt gg-3'). The AP-HuR-GST plasmid was created by recombination between the AP-HuR-TAG entry vector and the pDEST15 plasmid (Invitrogen). The XhoI/HindIII fragment of the HuR PCR product was inserted into the XhoI/HindIII sites of the AP-N entry vector to produce the AP-HuR entry vector. The GST-AP plasmid was generated by recombination reaction between the AP-C entry vector and pDEST15 (Invitrogen).

The fusion proteins were purified as described (39) with the following modifications. The proteins were eluted from the glutathione-agarose beads with three applications of 500-µl glutathione elution buffer (10 mM for the first elution, and 20 mM for the second and third elutions). Proteins were then dialyzed overnight against phosphate-buffered saline at 4 °C. The 20 mM glutathione eluates were the most pure (as determined by SDS-PAGE) and were used in all experiments.

Cell Culture and Transfection—C2C12 cells (ATCC) were grown and maintained in Dulbecco's modified eagle medium (DMEM, Invitrogen) containing 20% fetal bovine serum (Invitrogen), penicillin/streptomycin, and L-glutamine, following the manufacturer's directions (Invitrogen). Differentiation was induced immediately upon 100% confluency on plates previously coated with 0.1% gelatin. To induce differentiation, growth media were replaced with differentiation media containing DMEM, 2% horse serum, penicillin/streptomycin antibiotics, and 10 µg/ml insulin (Invitrogen), 10 µg/ml transferrin (Invitrogen), and 50 mM HEPES, pH 7.4 (Invitrogen).

The transfection of siRNA into C2C12 cells was performed in six-well plates using twice the number of wells that were eventually needed. The first transfection with HuSi-1, HuSi-C, or mock (transfection reagents only, no oligonucleotide) was performed when cells were 20-30% confluent. At 24 h post-transfection, when cells were 50-60% confluent, the procedure was repeated. At 6-8 h after the second transfection, two wells (with the same siRNA treatment) were combined into one by trypsinizing one plate and moving the cells from each well to corresponding wells on the second plate. For all transfections, LipofectAMINE Plus (Invitrogen) was used, following the manufacturer's protocol. siRNA duplexes were ordered from Dharmacon. The siRNA duplex (0.12 µM) was used for each well of a six-well plate. Differentiation was induced 2 days after the second transfection. For rescue of siRNA-treated cells, 25 nM purified protein was added to the C2C12 growth media. This protein/media solution was added to the cells twice, for 2 days, following the second siRNA transfection. Differentiation was induced the day after the protein was added for the second time.

Immunoblotting, Immunofluorescence, and Preparation of Cell Extracts—Total cell extracts, as well as nuclear and cytoplasmic fractions, were prepared as described (40, 41). Western blotting was performed as described previously (39). The blots were probed variously with antibodies to HuR (42), hnRNP A1 (kindly provided by Dr. G. Dreyfuss, University of Pennsylvania School of Medicine, Philadelphia), myogenin, MyoD, tubulin (Santa Cruz Biotechnology), myoglobin (DAKO), MF-20 (Developmental Studies Hybridoma Bank), and {beta}-actin (Sigma).

Immunofluorescence was performed as previously described (37). A polyclonal antibody to myoglobin was used at a 1:500 dilution in 1% goat serum/phosphate-buffered saline, and was incubated simultaneously with a monoclonal antibody to HuR (1:1500). Myoglobin was detected using a rhodamine-labeled secondary antibody, and HuR using an fluorescein isothiocyanate secondary antibody, both at a 1:500 dilution. Hoechst stain, 1:1500, was also incubated with the secondary antibodies. A Zeiss Axiovision 3.1 microscope was used to observe the cells using a 40x oil objective, and an Axiocam HR (Zeiss) digital camera was used for immunofluorescence photography.

Northern (RNA) Blot Analysis—Northern blot analysis was performed as described (43) using 7 µg of total RNA prepared using TRIzol reagent (Invitrogen). After transferring to a Hybond-N membrane (Amersham Biosciences) and UV-cross-linking, the blot was hybridized with murine p21cip1, MyoD, or myogenin cDNA probes generated by random primer labeling (Roche Applied Science) according to the manufacturer's instructions (44). PCR-amplified fragments from MyoD, myogenin, and p21cip1 cDNAs (kindly supplied by Dr. A. Lassar, at Harvard Medical School) were used to generate labeled probes. After hybridization, the membranes were washed and subsequently exposed on BioMax films.

Preparation of mRNA (mRNP) Complexes and Analysis with RTPCR—Immunoprecipitation and RNA preparation were performed as previously described (45) using antibodies to HuR (3A2) and MF-20, and cell extracts were prepared from differentiating C2C12 cells. mRNP lysate (400 µl) was added to beads (protein-A-Sepharose) for each immunoprecipitation. Specific messages associated with HuR were defined using RT-PCR as described below.

Purified RNA was resuspended in 10 µl of water, and 1 µl was reverse-transcribed using the Thermoscript RT-PCR system (Invitrogen) according to the manufacturer's protocol in 20 µl of final volume. Subsequently, 2 µl of cDNA was PCR-amplified using platinum TaqDNA polymerase (Invitrogen) for 22 cycles using MyoD, myogenin, or p21cip1 cDNA-specific primers. The sequences of the primers, as well as the PCR conditions, were described previously (44).

Preparation of RNA Transcripts, RNA Binding Assay, and Gel Shift Assay—The MyoD, myogenin, and p21cip1 cRNA probes were produced by in vitro transcription as previously described (40). The p21cip1 cRNA probe was transcribed from a synthesized oligonucleotide subcloned into pGEM 3Zf(+) vector (Promega), which was linearized with BamH1 prior to the reaction. The myogenin cRNA probe was generated from a synthetic oligonucleotide (spanning nucleotides 1244-1279 of myogenin 3'-UTR) fused to a T7 promoter (5'-gaattgtaatacgactcactatagggcga-3'). The MyoD cRNA probe was generated by PCR amplification of a region of the MyoD 3'-UTR spanning nucleotides 1301-1408 using a forward primer fused to the T7 promoter as well as a MyoD cDNA expression vector as the template. The RNA binding assay and the HuR supershift assays were performed as previously described (41).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
HuR Expression during C2C12 Differentiation—To investigate HuR function in regulating gene expression during muscle differentiation, mouse embryonic myoblasts (C2C12, also termed C2, myoblasts) were used as a system model. Myotubes and differentiation markers such as the myosin heavy chain (MF-20) and myoglobin become detectable in these cells ~48-72 h after serum starvation (46, 47). HuR expression was first monitored during myogenesis (Fig. 1). Western blot analysis using total extracts from differentiating myoblasts and a monoclonal antibody against HuR demonstrated that HuR expression levels did not change throughout myogenesis and remained high in mature myotubes (Fig. 1, B, lanes 4 and 5, and A, lanes d3 and d5). Both light microscopy and Western blots showed that the C2C12 cells differentiated normally and expressed normal levels of myoglobin and MF-20 (Fig. 1, A and B) as previously described (48). These results show that HuR protein levels do not change during the myoblast-to-myotube transition.



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  		FIG. 1.
HuR is expressed in differentiating C2C12 cells. A, C2C12 (C2) myoblasts were induced to differentiate when cells reached 100% confluency (defined as day 0, or d0). Multinucleated myotubes were seen 3 days after induction of differentiation (d3), with maximum differentiation at day 5. B, Western blot analyses were performed using 10 µg of total cell extract from differentiating C2C12 cells. The blot was probed with antibodies to HuR, MF-20, and myoglobin.

 
HuR Associates with MyoD, Myogenin, and p21cip1 Messages during C2C12 Differentiation through Specific Sequences Located in Their 3'-UTR—Nucleotide sequence analysis of MyoD, myogenin, and p21cip1 indicated that each mRNA contains an AU/GU-rich sequences in its 3'-UTR (Ref. 9 and Fig. 2A). Moreover, it was recently shown that HuR associates with the 3'-UTR of myogenin and p21cip1 during C2C12 differentiation (9). However, the association with the MyoD 3'-UTR, as well as the precise sequences that interact with HuR, remain unknown. To delineate the exact HuR binding sites in these messages, we performed RNA gel shift assays, using radiolabeled probes for the 3'-UTR of p21cip1, MyoD, and myogenin, on nuclear and cytoplasmic extracts prepared from differentiating C2C12 cells (Fig. 2B). The HuR antibody was incubated with the extracts in the presence of p21cip1 (Fig. 2A, c), MyoD (Fig. 2A, a), or myogenin (Fig. 2A, b) cRNA probes. With the p21cip1 cRNA probe (Fig. 2B, lanes 20-27), a complex was evident in both the cytoplasmic (Cc) (Fig. 2B, lanes 21-23) and the nuclear (data not shown) extracts. When the HuR antibody was added to these extracts, a shift of an HuR-associated complex (HC) was observed throughout differentiation (Fig. 2B, lanes 24-26). This shift was absent in both nuclear and cytoplasmic extracts incubated with an antibody to p38 (Fig. 2B, lane 27). HuR therefore interacts in vitro with the p21cip1 ARE in both the nuclear (data not shown) and cytoplasmic compartments during C2C12 differentiation. The same result was obtained with the myogenin cRNA probe (spanning nucleotides 1244-1279), which forms complexes with both nuclear (data not shown) and cytoplasmic extracts on all days (Fig. 2B, lanes 12-14, Cb). Cb shifts to form HC in the presence of the anti-HuR antibody (Fig. 2B, lanes 15-17). The HC was also observed using another cRNA probe from the myogenin 3'-UTR spanning nucleotides 1352-1471, but not with a cRNA probe spanning nucleotides 992-1024 (data not shown). The same approach was used with the 3'-UTR of MyoD, where two cRNA probes were prepared and formed the HC in the presence of the anti-HuR antibody (Fig. 2B, lanes 5-7 and data not shown). However, a probe containing the iron-responsive element (IRE) (49) forms a complex with differentiating C2C12 extracts (Fig. 2B, lanes 29-31), but did not form the HC in the presence of the anti-HuR antibody (Fig. 2B, lanes 32-34). This last observation indicates that the HC obtained with the p21cip1, MyoD, and myogenin probes reflects a specific association between HuR and these 3'-UTRs.



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  		FIG. 2.
HuR associates with the 3'-UTR of MyoD, myogenin, and p21cip1 mRNAs during differentiation. A, schematic representations of mRNA sequences of MyoD, myogenin, and p21cip1. All three messages contain an ARE in their 3'-untranslated regions (3'UTR). The dashed boxes a, b, and c represent the location of the respective AREs used as probes for the gel shift experiments. B, gel shift RNA binding assays on cytoplasmic fractions (Ct.Ext) prepared from C2C12 cells during differentiation, using a MyoD (a), myogenin (b), or p21cip1 cRNA (c) probe. Complexes containing HuR were shifted with a monoclonal antibody to HuR (solid bar). An antibody to p38 was incubated with day 3 cytoplasmic extracts as a negative control (lanes 10, 19, and 27). HC refers to HuR-shifted complexes. Ca, Cb, and Cc represent the complexes of MyoD, myogenin, and p21cip1, respectively, without antibodies. The gel shift experiment was also performed using an iron-responsive element (IRE) probe. CI represents the complex with and without antibodies. We did not detect any HC complexes with the IRE probe. C, immunoprecipitation of HuR or MF-20 followed by RT-PCR of the associated RNA. RNAs were reverse-transcribed and then PCR-amplified used MyoD-, myogenin-, or p21cip1-specific primers as indicated. PCR reactions (15 µl) were loaded onto a 10% Tris acetate/EDTA (TAE) acrylamide gel and stained with ethidium bromide.

 
To confirm that HuR interacts with these messages in vivo during differentiation, HuR and MF-20 were immunoprecipitated, and the associated RNAs were analyzed by RT-PCR. Total extracts prepared from differentiating C2C12 cells were incubated with either an anti-HuR or anti-MF-20 antibody. The precipitated RNAs were isolated and analyzed using specific primers for MyoD, myogenin, and p21cip1 mRNAs (Fig. 2C). As expected, at day 0 there was no detectable interaction between HuR and either myogenin or p21cip1 mRNAs, because their genes are expressed only later in the differentiation process (50-52). However, at day 3 it was clear that all three mRNAs interact with HuR, but not with MF-20 (Fig. 2C, lanes 3 and 4); the association with HuR was maintained with myogenin and p21cip1 at day 5 (Fig. 2C, lane 6). Despite the fact that MyoD mRNA is expressed at early stages of differentiation (Fig. 2C, lane 1), it did not interact with HuR at day 0 (Fig. 2C, lanes 2). This observation suggests that the implication of HuR in MyoD mRNA expression is likely to be indirect at early stages of differentiation. However, HuR association with the three mRNAs at days 3 and 5 of differentiation clearly suggests a more direct role in the post-transcriptional events undergone by MyoD, myogenin, and p21cip1 mRNAs at late stages of differentiation. Our data, and the fact that these messages are regulated at the level of their stabilization in C2C12 (9), show that HuR could play a prominent role in regulating their half-lives during myogenesis.

HuR Knockdown Inhibits C2C12 Differentiation—To define the essential role of HuR in C2C12 differentiation, RNA interference was employed to specifically knockdown the HuR mRNA (53). Two siRNA duplexes were synthesized, HuSi-1 and HuSi-C. HuSi-1 represents a target sequence located at the 5'-region of the HuR cDNA (Fig. 3A). The negative control HuSi-C was obtained by mutating four specific nucleotides in the HuSi-1 sequence (Fig. 3A, underlined nucleotides). Two successive transfections of HuSi-1 were necessary to decrease HuR by >80% (see "Materials and Methods"). Total extracts from these HuSi-1-treated myoblasts were analyzed by Western blot using antibodies to HuR, {beta}-actin, and myoglobin (Fig. 3B). 24 h after the second transfection with HuSi-1 (Fig. 3B, lanes 1-3), HuR expression decreased by ~83% compared with the control. Moreover, under these conditions, HuR expression was reduced by ~25% at day 0 compared with the other siRNA treatments (Fig. 3B, lanes 4-6). By day 3 of differentiation, HuR levels were back to normal and remained equivalent throughout differentiation, thus demonstrating that HuR knockdown at the protein level is only effective for ~3 days. The observed HuR knockdown led to a significant reduction in myoglobin expression (Fig. 3B, lanes 8 and 9), consistent with an inhibition of C2C12 differentiation. Indeed, multinucleated myotubes were clearly visible in control cells under the light microscope but were not observed in the cells treated with HuSi-1 (data not shown).



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  		FIG. 3.
Silencing HuR expression inhibits myogenesis. A, the siRNA duplex HuSi-1 (Dharmacon) targets HuR mRNA at the 111-131 sequence. HuSi-C represents a mutant version of HuSi-1 (underlined nucleotides), which was used as a negative control. B, Western blot analysis of 2.5 µg of total cell extracts from RNAi-treated C2C12 cells. The extracts were harvested 48 h post-RNAi transfection (48h pt), as well as days 0, 3, and 5 of differentiation. The membrane was probed with antibodies to HuR, {beta}-actin, and myoglobin. C and D, the fusion index of myotubes was calculated to assess the percentage decrease in differentiation efficiency of C2C12 cells treated with HuSi-1. Immunofluorescence staining with anti-myoglobin was performed to determine the differentiation status of the transfected cells (D). Shown are antimyoglobinand DAPI-stained images of a single representative field of view. The quantitative analysis of differentiation was performed by determining the number of nuclei in each microscopic field in relation to the number of nuclei in myotubes in the same field. The percentage of differentiation is indicated (see D). On day 0, there were no myotubes detected and therefore there was 0% differentiation for all treatments.

 
To assess the effect of the HuR knockdown on the differentiation efficiency of C2C12 cells, the fusion index for HuSi-1-treated and mock or control-treated cells was measured as described (54). The expression of myoglobin during myogenesis was followed by immunofluorescence using an anti-myoglobin antibody (Fig. 3C); the same inhibition of C2C12 differentiation was observed as described above. The percentage of differentiated cells was determined for all treatments throughout the course of myogenesis (Fig. 3D). By day 3 of differentiation, the control cells showed ~86% differentiation efficiency, whereas only ~7% differentiation was measured for HuSi-1-treated cells. At day 5, differentiation was 97% in normal cells but only 26% in those treated with the HuSi-1. These data showed that differentiation of HuR-knockdown C2C12 cells was delayed until the level of HuR was back to normal.

Differentiation of C2C12 Cells Treated with HuSi-1 Is Reestablished by the Addition of AP-conjugated HuR—To ensure that the observed inhibition of C2C12 differentiation was due specifically to the HuR knockdown, AP-HuR-GST and GST-AP (negative control) fusion proteins were prepared and tested in a rescue experiment on HuSi-1-treated myoblasts (Fig. 4A). These AP-conjugated proteins were added to the siRNA-treated cells 48 and 24 h prior to the stimulation of differentiation. When we followed the differentiation of these cells treated with AP-conjugated proteins by immunofluorescence, using DAPI staining and anti-myoglobin, we observed that AP-HuR-GST, but not GST-AP, rescued C2C12 differentiation with the appearance of visible myotubes (Fig. 4B, panels 5 and 7). This result clearly demonstrates that the rescue was due specifically to the added HuR. Moreover, the addition of AP-HuR-GST to HuSi-C-treated C2C12 cells, which mimics HuR overexpression, enhanced differentiation (Fig. 4B, panels 6 and 8). Indeed, the myotubes become visible as soon as days 1-2 of differentiation initiation (data not shown). Furthermore, the effect of AP-HuR-GST also appeared to be dose-dependent, because the addition of 25 nM AP-HuR-GST, but not 10 nM, to exponentially growing HuSi-1-treated myoblasts was required to induce a normal level of myoglobin expression at day 5 of differentiation (Fig. 4C, lane 6). However, the expression of {beta}-actin protein was not affected by the addition of the AP-conjugated proteins (Fig. 4C). Due to the early addition of the recombinant protein, we were not able to detect the AP-HuR-GST by Western blotting at day 5, although endogenous HuR (which has reached normal levels by this point after RNAi treatment) was readily detected (Fig. 4C, lanes 3-6). However, Western blot on extracts from exponentially growing myoblasts shows that APHuR-GST was taken up by the cells with high efficiency (Fig. 4C, lanes 1-2). Together, these results demonstrate the specificity of HuR to counteract siRNA treatment and confirm its essential role at early stages of myogenesis.



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  		FIG. 4.
Differentiation can be restored in HuSi-1-treated cells by the addition of AP-HuR-GST. In all experiments described below, the AP-conjugated proteins were added twice to siRNA-treated C2C12 cells 48 and 24 h prior to differentiation initiation. A, schematic representation of the constructs used to rescue siRNA-treated cells. HuR-GST was conjugated to AP (antennapedia). GST-AP was used as a control. B, AP-HuR-GST rescues C2C12 myogenesis. C2C12 cells at day 5 of differentiation, treated with GST-AP or AP-HuR-GST, were stained with DAPI and an antibody to myoglobin to determine their differentiation status. Immunofluorescence images of a single representative field are shown. C, the expression levels of myoglobin in HuSi-1 and AP-HuR-GST-treated or GST-AP-treated cells were examined by Western blot. C2C12 cells, treated with 10 or 25 nM of these recombinant proteins, were used to prepare total cell extracts the day after the second addition of AP-HuR-GST protein (exponentially growing cells-Exp, lanes 1 and 2), or at day 5 (lanes 3-6) of differentiation. Western blot analysis was performed using anti-myoglobin, anti-HuR, and anti-{beta}-actin antibodies.

 
The Expression of MyoD and Myogenin mRNAs Requires Normal HuR Levels during the Early Steps of Myogenesis— HuR affects the expression of many genes at the post-transcriptional level (13). Thus, we hypothesized that HuR may affect myogenesis by regulating the expression of MyoD, myogenin, and p21cip1 mRNAs. To address this question, Northern blotting was performed on total RNA prepared from exponentially growing and differentiating cells that were either mock or HuSi-1-treated. Both MyoD and myogenin mRNAs were significantly decreased throughout differentiation in cells treated with HuSi-1 (Fig. 5) but not in the mock (Fig. 5) or with HuSi-C-treated cells (data not shown). This effect on MyoD was seen at 48 h post-transfection and was still prominent at day 3. By day 5, MyoD RNA levels were restored (Fig. 5, lanes 5 and 6). Myogenin displayed a similar profile. It was not expressed at 48 h post-transfection, but there was a clear decrease in myogenin RNA levels at days 3 and 5 in the HuSi-1-treated cells compared with the control (Fig. 5, lanes 3-6). The HuR knockdown, however, did not affect the expression of the GAPDH message (Fig. 5). Furthermore, we also observed that p21cip1 mRNA expression was not significantly affected in HuSi-1-treated cells (data not shown). These results were confirmed by RT-PCR (data not shown). The expression of MyoD and myogenin RNAs in control cells was consistent with previous reports (50-52). Western blot analysis using extracts from differentiated C2C12 treated or not with HuSi-1 demonstrated a decrease in MyoD and myogenin proteins that is consisting with the decrease observed at the mRNA levels (Fig. 5 and data not shown). Together, these data clearly show that normal HuR levels in the early stages of myogenesis are required for the proper expression and translation of MyoD and myogenin mRNAs but not that of p21cip1.



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  		FIG. 5.
HuR regulates the expression of some myogenic mRNAs. Total RNA from differentiating myoblasts mock treated (-)or treated with HuSi-1 duplex were prepared and subjected to Northern blot analysis. RNA was detected with 32P-labeled cDNA probes encoding MyoD, myogenin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins as indicated. The films were scanned using an Arcus 1200 scanner, and the Image 1.61 computer program (National Institutes of Health) was used to measure band intensity to determine changes in RNA levels. The -fold increase/decrease for each RNA was calculated relative to the loading control used for each experiment.

 
HuR Accumulates in the Cytoplasm during Differentiation— HuR regulates the stability and/or the nuclear export of its mRNA targets (14, 15, 23). Because its expression does not change during the myoblast-to-myotube transition, we hypothesized that its cellular localization changes. To address this point, both immunocytochemistry and cell fractionation approaches were used to monitor HuR cellular movement during C2C12 differentiation. In the immunofluorescence experiments, C2C12 cells were fixed and stained using DAPI, antiHuR, and anti-myoglobin (Fig. 6A). HuR localizes primarily to the nucleus in proliferating cells (Fig. 6A, panel 2) but becomes more cytoplasmic during myogenesis (panels 5, 8, and 11). The cytoplasmic accumulation of HuR correlates with its association with p21cip1 and myogenin mRNAs at the late stages of differentiation, because it is at this time that these messages must be stabilized and translated.



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  		FIG. 6.
HuR accumulates in the cytoplasm during myogenesis. A, the subcellular distribution of HuR during the differentiation process. Cells were fixed during the exponential (Exp) growth phase as well as during the differentiation process (on the days indicated), and the localization of endogenous HuR was determined by double immunofluorescence staining. DAPI-, HuR (fluorescein isothiocyanate)-, and myoglobin (rhodamine)-stained images of a single representative field are shown. B, Western blot analysis of the subcellular distribution of HuR during differentiation. Nuclear and cytoplasmic extracts were prepared on the indicated days. Relative amounts of HuR as well as the nuclear and cytoplasmic markers hnRNP A1 and tubulin, respectively, were determined by Western blotting. Actin was used as a loading control, and the presence of myoglobin indicates the differentiation status of the cells. C, quantification of HuR cytoplasmic accumulation during differentiation. The level of cytoplasmic HuR is given as a relative ratio of the intensity of HuR to actin bands in each lane. Ratios were measured (using National Institutes of Health Image, 1.61) during differentiation for three independent fractionation experiments. The increase in cytoplasmic HuR over the course of differentiation was ~5-fold. Error bars indicate standard deviation.

 
The accumulation of HuR in the cytoplasm was confirmed, and its level was defined using biochemical cell fractionation and Western blotting (Fig. 6, B and C). Exponentially growing and differentiating C2C12 cells were lysed and separated into nuclear and cytoplasmic fractions, which were analyzed by Western blot using antibodies to HuR, hnRNP A1, tubulin, {beta}-actin, and myoglobin. Actin was used as a loading control for both nuclear and cytoplasmic fractions (55), whereas tubulin and hnRNP A1 were used as cytoplasmic and nuclear markers, respectively. HuR levels increased between 9and 13-fold at days 3 and 5 (Fig. 6, B, lanes 58, and C). These results are consistent with the idea that HuR uses its shuttling ability to protect and escort its associated mRNAs to the translation machinery, allowing their expression at the appropriate time. The fact that HuR mRNA levels are significantly reduced at day 5 of differentiation (data not shown) supports the idea that the observed cytoplasmic accumulation of HuR is due to an increase in export rather than to a block of the import of de novo translated protein.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our data indicate that HuR plays a critical role in myogenesis by regulating the expression and possibly the nuclear export of MyoD, myogenin, and p21cip1 mRNAs. Here we demonstrated, both in vivo and in vitro, that HuR accumulates in the cytoplasm and associates with MyoD, myogenin, and p21cip1 mRNAs during differentiation. Likewise, depletion of HuR from myoblasts leads to a significant reduction in the expression of MyoD and myogenin mRNAs and to a complete inhibition of myogenesis (Figs. 3 and 4). Although the steadystate level of p21cip1 mRNA is not affected in HuR knockdown cells, this message interacts with HuR through its AREs (Fig. 2). Previous studies have shown that sequential translation of MyoD, myogenin, and p21cip1 mRNAs is required to ensure muscle fiber formation (1, 56, 57). Therefore, protecting these mRNAs from degradation, as well as exporting them from the nucleus at the proper time, is a prerequisite for normal myogenesis. Moreover, the function of HuR as an mRNA stabilizer and adaptor for nuclear export was suggested in other systems (14, 15, 19, 23). Thus, we suggest a model in which HuR could regulate the expression of MyoD, myogenin, and p21cip1 proteins during muscle formation.

HuR is ubiquitous, and thus it was not surprising to detect it in C2C12 cells (Fig. 1). Our experiments show that its level does not change during myogenesis. If HuR plays an important role in myogenesis, we expected that it would be differentially expressed; however, our results clearly show that this is not the case. One possible explanation is that the function of HuR is so crucial for C2C12 differentiation that these cells cannot afford to reduce its level at any time.

Because C2C12 cells already have a high level of HuR, defining the functions of HuR during myogenesis using the overexpression approach could lead to effects that might not reflect the true function of the endogenous proteins. To address this concern, we disrupted the expression of endogenous HuR in myoblasts using RNA interference (Fig. 3). Interestingly, HuR knockdown via siRNA led to a complete inhibition of C2C12 differentiation. HuR expression was significantly reduced (>80%) 24 h prior to the initiation of the differentiation process (Fig. 3B). Even though HuR expression recovers in HuSi-1treated C2C12 cells 3 days after differentiation initiation, few myoblasts become detectable in these cells even on day 5. These data suggest that HuR affects the initial stages of myogenesis. Although in some of our RNAi experiments we reduced the expression of HuR by less than 40%, we still observed complete inhibition of differentiation (data not shown). Thus, it is reasonable to consider that myoblasts need to maintain HuR above a threshold that allows the initiation of muscle differentiation.

We used AP-conjugated HuR to re-establish differentiation in C2C12 cells treated with RNAi (Fig. 4). This observation clearly indicates that HuR exerts its function by directly regulating important pathways during myogenesis and confirms that the observed inhibition of myogenesis with the HuSi-1 duplex is due specifically to a knockdown of the HuR protein. Furthermore, treatment of normal C2C12 cells with the APHuR chimera (which mimics HuR overexpression) led to a significant increase in the efficiency of myotube formation, producing larger muscle fibers capable of contraction (data not shown). The fact that AP-HuR-GST was added to exponentially growing myoblasts just before the initiation of differentiation is a clear indication that HuR affects the early stages of myogenesis. This observation supports our previous findings and suggests a prominent role for HuR in muscle differentiation. Our results are consistent with the fact that other Hu/ELAV family members, HuB, HuC, and HuD, are involved the differentiation of neuronal cells (30, 58). It will be very interesting to determine if HuR affects the differentiation of other embryonic cell lines.

HuR knockdown led to a significant reduction in the steadystate levels of MyoD and myogenin mRNAs (Fig. 5). The reduction of MyoD mRNA was observed in exponentially growing RNAi-treated C2C12 cells and was maintained 72 h after induction of myogenesis. On the other hand, myogenin mRNA was detected 48-72 h after the induction of differentiation, but it was significantly reduced in the absence of HuR expression. These results suggest that HuR first participates in the induction of MyoD expression, which in turn stimulates the transcription of both myogenin and p21cip1 genes (2, 3).

The AREs of MyoD, myogenin, and p21cip1 mRNAs (Fig. 2A) contain one or two AU3A sequences that constitute HuR binding sites (9, 16). The destabilizing activity of AREs is vital for maintaining a precise level of key cell cycle and cell differentiation factors to prevent cell transformation and to ensure proper cell development (11). Here we show that HuR associates in vivo with MyoD, myogenin, and p21cip1 mRNAs (Fig. 2C). Moreover, this association remains almost unchanged for myogenin and p21cip1 mRNAs at day 5. However, the interaction of HuR with MyoD mRNA is significantly reduced at the late stages of myogenesis. Gel shift experiments confirmed that HuR interacts with MyoD, myogenin, and p21cip1 mRNAs via specific sequences located in their 3'-UTRs (Fig. 2B). Surprisingly, some of the sequences that are AU-rich did not bind to HuR; however, others, which are only U-rich and often GUrich, bind with high affinity (Fig. 2B and data not shown). Despite our efforts, we did not detect any HuR-MyoD association in vivo at an early stage of myogenesis (exponential and day 0, see Fig. 2C). One plausible explanation is that, in myoblasts, HuR regulates the expression of other messages that in turn activate the transcription of the MyoD gene. This explanation is supported by the fact that AP-HuR-GST is needed at early stages to rescue the differentiation of HuSi-1-treated cells (Fig. 4). Our data are consistent with a role for HuR in myogenesis, but whether all of the interactions of HuR with its mRNA targets are direct or indirect remains to be clarified.

Interestingly, HuR was shown to participate in the rapid export of some of its mRNA targets using two shuttling pathways (14, 23). The involvement of HuR in myogenesis as an adaptor protein for mRNA export should predicate its partial or complete localization in the cytoplasm during this process. Indeed, we show that HuR partially accumulates in the cytoplasm during C2C12 differentiation (Fig. 6). Our immunofluorescence experiments clearly demonstrate that, in some myotubes, HuR localizes almost exclusively to the cytoplasm (Fig. 6). Moreover, addition of AP-HuR enhances myogenesis and leads to a complete relocalization of HuR to the cytoplasm (Fig. 4B, panel 8). This localization correlates with the high expression of MyoD, myogenin, and p21cip1 mRNAs and proteins (9). The fact that HuR is maintained at a high level and accumulates in the cytoplasm could argue for its role in the rapid export of its mRNA targets during myotube formation. Although import inhibition of the de novo translated HuR at days 3-5 of differentiation could explain the observed cytoplasmic accumulation, the significant reduction in HuR mRNA levels at day 5 (data not shown) argues for export enhancement instead. However, two key points that remain to be determined are the HuR export pathways during myogenesis and the mechanisms that link the cellular movement of HuR with its effect on the expression of key messages such as MyoD, myogenin, and p21cip1. Defining these pathways and mechanisms is of significant interest and will promote our understanding of how the mRNAs of key myogenic factors are transported from the nucleus in a manner that synchronizes their translation during muscle formation.


   FOOTNOTES
 
* This work was supported by a Canadian Institutes for Health Research (CIHR) Cancer Consortium Training Grant Fellowship Award (to S. D.) and CIHR Operating Grant Mop-57680 and a Fond de Recherche Scientifique Quebecois "Subvention d'etablissement de jeune chercheur" Frsq 23516-2760 (to I. E. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{dagger} To whom correspondence should be addressed: Dept. of Biochemistry, McGill University, McIntyre Bldg., Rm. 904, 3655 Promenade Sir William Osler, Montreal, Quebec H3G 1Y6, Canada. Tel.: 514-398-4537; Fax: 514-398-7384; E-mail: imed.gallouzi{at}mcgill.ca.

1 The abbreviations used are: ARE, AU-rich element; UTR, untranslated region; AUBP, AU-binding protein; ELAV, embryonic lethal, abnormal vision; AP, antennapedia; GST, glutathione S-transferase; DMEM, Dulbecco's modified Eagle's medium; siRNA, small interference RNA; RT, reverse transcription; HC, HuR-associated complex; IRE, iron-responsive element; DAPI, 4',6-diamidino-2-phenylindole; hnRNP, heterogeneous nuclear ribonucleoprotein; RNAi, RNA interference. Back


   ACKNOWLEDGMENTS
 
We are grateful to Dr. J. Pelletier and L. Roudaia for helpful discussions and comments on the manuscript. We thank Dr. A. B. Lassar (Harvard Medical School, Boston) for MyoD, myogenin, and p21cip1 plasmids and G. Dreyfuss for the anti-hnRNP A1 antibody. The monoclonal anti-MF20 antibody developed by Dr. D. Fischman was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD, National Institutes of Health and maintained by the Department of Biological Sciences, The University of Iowa, Iowa City, IA.



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