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The role of the transcription factors sterol regulatory element binding protein 1a (SREBP-1a) and SREBP-1c in the regulation of cholesterol and fatty acid metabolism has been well studied; however, little is known about their specific function in muscle. In the present study, analysis of recent microarray data from muscle cells overexpressing SREBP1 suggested that they may play a role in the regulation of myogenesis. We then demonstrated that SREBP-1a and -1c inhibit myoblast-to-myotube differentiation and also induce in vivo and in vitro muscle atrophy. Furthermore, we have identified the transcriptional repressors BHLHB2 and BHLHB3 as mediators of these effects of SREBP-1a and -1c in muscle. Both repressors are SREBP-1 target genes, and they affect the expression of numerous genes involved in the myogenic program. Our findings identify a new role for SREBP-1 transcription factors in muscle, thus linking the control of muscle mass to metabolic pathways.
The sterol regulatory element binding protein (SREBP) transcription factors belong to the basic helix-loop-helix (bHLH) leucine zipper family of DNA-binding proteins. The three isoforms identified thus far in mammalian tissues are coded by two distinct genes, Srebf1 and Srebf2, and vary in structure, regulation, and functions (14). SREBP-1a and SREBP-1c proteins are produced by alternative promoter usage of the SREBF1 gene and are key actors of the regulation of genes related to lipid metabolism, especially those involved in lipogenesis and triglyceride deposition. In contrast, SREBP-2 has been more closely associated with cholesterol synthesis and accumulation (20, 52).
In agreement with these known functions, the SREBP-1 proteins are strongly expressed in tissues with high lipogenic capacities, such as liver and adipose tissues. However, significant expression has been also reported in skeletal muscle, both in vivo and in vitro, in cultured muscle cells (12, 13, 18). In muscle, SREBP-1 expression is induced by activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and the mitogen-activated protein (MAP) kinase pathways by insulin and growth factors (6, 12, 18, 28, 38), suggesting additional functions of these transcription factors in a tissue with a low rate of lipid synthesis. Using microarray analysis to characterize the role of SREBP-1a and -1c in skeletal muscle, we have recently identified some of their potential target genes in primary cultures of human myotubes overexpressing SREBP-1a or SREBP-1c (43). In the present study, we found that SREBP-1a and -1c regulate more than 1,000 genes, indicating that they are potentially involved in the regulation of a large variety of biological functions in muscle cells. Quite unexpectedly, we observed a dramatic reduction in the expression of a number of muscle-specific genes and markers of muscle differentiation in cells overexpressing SREBP-1 proteins. This led us to investigate their potential role in the regulation of myogenesis and muscle development.
The early stages of muscle development are regulated by muscle-specific bHLH transcription factors (e.g., MYF5, MYOD1, MYOG [myogenin], and MYF6 [MRF4]), which are also involved in the differentiation of satellite cells during the regeneration process in adult muscle. Recently, the transcriptional factor BHLHB3 was shown to inhibit in vitro muscle cell differentiation by interacting with MYOD1 (2). BHLHB3 (also named DEC1/SHARP1) is a transcriptional repressor closely related (97% homology in amino acid sequence in the bHLH domain) to BHLHB2 (also named Stra13/DEC2/SHARP2). They both repress the expression of target genes by binding to E-Box sequences, as well as through protein-protein interactions with other transcription factors (reviewed in reference 51). BHLHB2 and BHLHB3 genes are widely expressed in both embryonic and adult tissues and their expression is regulated in cell type-specific manner in various biological processes, including circadian rhythms (19), hypoxia (35), or cellular differentiation (7). Their involvement in the regulation of developmental processes during embryogenesis has been largely studied (4, 7, 24, 34, 44). We demonstrate here that BHLHB2 and BHLHB3 mediate negative effects of SREBP-1 transcription factors on myogenesis, acting at both the myoblast and the myotube stages. The SREBP-1-mediated effects on BHLHB2 and BHLHB3 activity thus defines a novel negative regulation pathway in skeletal muscle cell development.
Muscle biopsies were taken from healthy lean subjects during surgical procedure, with the approval of the Ethics Committee of Lyon Hospitals. Myoblasts were purified, and differentiated myotubes were prepared according to a procedure previously described in detail (11).
For the construction of expression vector encoding BHLHB2, a verified sequence IMAGE clone (cloneID 4860809) was purchased from Geneservice (Cambridge, United Kingdom) and subcloned into the pcDNA 3.1 expression vector (Invitrogen). The expression vector encoding BHLH3 was generated by PCR amplification and ligated into PCDNA3.1. Expression vector encoding the dominant-negative form of SREBP-1 (ADD1-DN) is a generous gift of B. Spiegelman (Dana-Farber Cancer Institute/Harvard Medical School, Boston, MA) (27). Recombinant adenoviral genomes carrying the human BHLHB2 or BHLHB3 or ADD1-DN were generated by homologous recombination in the VmAdcDNA3 plasmid (a gift from S. Rusconi, Fribourg, Switzerland) and amplified as described previously (9, 12).
Construction of expression vectors encoding mature nuclear forms of human SREBP-1a (named pCMV-hSREBP1a) and SREBP-1c (named pCMV-hSREBP1c) was described previously (12). A fragment of the pIRES plasmid (Clontech, Mountain View, CA) containing the internal ribosome entry site (IRES) and enhanced green fluorescent protein (EGFP) sequence was cloned into pCMV-hSREBP1a and pCMV-hSREBP1c to obtain pCMV-hSREBP1a-IRES-GFP and pCMV-hSREBP1c-IRES-GFP. Recombinant adenoviruses expressing simultaneously nuclear forms of either SREBP-1a or SREBP-1c and GFP as a marker were generated by homologous recombination in the VmAdcDNA3 plasmid and amplified.
The construction of recombinant adenoviruses encoding nuclear SREBP-1a and SREBP-1c was described previously (12). Human muscle cells were infected as myoblasts or myotubes. Myoblasts were grown in six-well plates. Myoblasts at 70% confluence or myotubes after 5 days of differentiation were infected for 48 h with the recombinant adenovirus encoding BHLHB2 or BHLHB3 or nuclear forms of SREBP-1a or SREBP-1c or GFP as a control.
Inhibition of BHLHB2 and BHLHB3 expression was performed by RNA interference using small interfering RNA (siRNA) against BHLHB2 and against BHLHB3 (Qiagen). A rhodamine labeled GFP-22 siRNA was used as control. Myoblasts at 70% confluence were transfected with siRNAs using the Hiperfect transfection reagent (Qiagen, Courtaboeuf, France) according to the manufacturer's protocol.
All animal procedures were conducted according to the national guidelines for the care and use of laboratory animals. Adult (12- to 14-week-old) BALB/c male mice (Harlan, France) were subjected to adenoviral delivery according to the procedure described by Sapru et al. (45). Briefly, right tibialis anterior muscles of mice were injected with 1010 infectious units of recombinant adenovirus expressing either SREBP-1a/GFP, SREBP-1c/GFP, BHLHB2, or BHLHB3. As a control, the contralateral tibialis anterior muscles were also injected with 1010 infectious units of recombinant adenovirus expressing GFP. Mice were sacrificed 7 days after injection. The tibialis anterior muscle was removed and immediately snap-frozen in liquid nitrogen. Sections (10 μm) were cut, and every tenth section was collected onto glass slides for examination under fluorescence illumination using an Axiovert 200 microscope, an Axiocam MRm camera, and Axiovision 4.1 image acquisition software (Carl Zeiss, Göttingen, Germany). Muscle fiber sizes and fluorescence intensities were measured by using NIH ImageJ software.
Cells were fixed in 10% formaldehyde and permeabilized with 0.1% Triton X-100. Nonspecific binding sites were blocked with 1% bovine serum albumin in 1× phosphate-buffered saline for 1 h at room temperature. Cells were then incubated overnight at 4°C with specific primary antibodies (anti-TNNI1, C-19; Santa Cruz Biotechnology, Santa Cruz, CA; antimyogenin, F5D; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA). Detection was achieved by using Alexa 555-conjugated donkey anti-goat and goat anti-mouse IgG (Molecular Probes/Invitrogen).
Cells were mounted with Vectashield with DAPI Fluoprep mounting medium (H1200; Vector Laboratories, Peterborough, England) and examined by fluorescence microscopy using an Axiovert 200 microscope, an Axiocam MRm camera, and Axiovision 4.1 image acquisition software. The area of TNNI1 immunostained differentiated myotubes was measured by using NIH ImageJ software.
Classical Western blot experiments were performed as described previously (12). After transfer, gels were stained with Coomassie blue. Membranes were then incubated overnight at 4°C with the following specific primary antibodies: anti-SREBP-1 (H160), anti-MYOD1 (M316), anti-MEF2C (E17), anti-MYOG (M225), anti-TNNI1 (C-19), and anti-TNNI2 (C-19) from Santa Cruz Biotechnology (Santa Cruz, CA); anti-BHLHB2 (M01; 5B1); and anti-BHLHB3 (M01; 4H6) from Abnova (Taipei, Taiwan).
The signal was detected by using a horseradish peroxidase-conjugated secondary antibody and revealed with the enhanced chemiluminescence system (Pierce, Rockford, IL). Signal was quantified by using NIH ImageJ software. The intensity of Coomassie blue staining was used to normalize the total amount of proteins.
Total RNA was isolated by using the TRIzol reagent (Invitrogen, Courtaboeuf, France) according to the manufacturer's instructions. First-strand cDNAs were synthesized from 500 ng of total RNAs in the presence of 100 U of Superscript II (Invitrogen) and a mixture of random hexamers and oligo(dT) primers (Promega). Real-time PCR assays were performed with Rotor-Gene 6000 (Corbett Research, Mortlake, Australia). A list of the primers and real-time PCR assay conditions are available from the authors upon request. The results were normalized by using RPLP0 or HPRT (hypoxanthine phosphoribosyltransferase) mRNA concentration, measured as reference gene in each sample.
The chromatin immunoprecipitation (ChIP) experiments were performed as previously described (43) using a ChIP It Express enzymatic kit from Active Motif (Rixensart, Belgium) according to the manufacturer's instructions. ChIP products were analyzed by quantitative and classical PCR using specific primers for BHLHB2 and BHLHB3 promoter (PCR primers are available on request).
A human genomic clone (NR5-IH18RS), which contains NotI flanking regions corresponding to the BHLHB2 promoter was obtained from E. R. Zabarovsky (Microbiology and Tumor Biology Center and Center for Genomics and Bioinformatics, Karolinska Institute, Stockholm, Sweden). The −408/+75 (according to the transcription starting site) fragment was then subcloned into the luciferase reporter gene vector pGL3-Enhancer (Promega) to obtain pB21 (−408/+75). The −951/-407 fragment was generated by PCR and ligated into pB21 to obtain pB22 (−951/+75). The constructs pB23 (−264/+75) and pB26 (−187/+75) were generated by deletion of pB21. To obtain pB32, two genomic fragments, corresponding to the −940/−289 and −524/+238 regions of the BHLHB3 gene, were generated by PCR and combined to obtain the −940/+238 fragment into pGL3-E vector. Mutations of the SRE motifs were performed as described previously (12). Mutagenesis was performed to replace bases 2, 4, and 6 of each identified SRE by thymidine residues (QuikChange mutagenesis kit; Qiagen).
Transfection studies were carried out on myoblasts or myotubes plated in 12-well plates as previously described (12). Firefly and Renilla luciferase activities (dual luciferase reporter assay system; Promega) were measured by using a Centro LB 960 Luminometer (Berthold Technology, Thoiry, France).
The procedure used to obtain and analyze microarray data has previously been described (43). Briefly, total RNA extracted from BHLHB2 and BHLHB3 overexpressing myotubes were hybridized on oligonucleotide microarrays produced by the French Genopole Network (RNG) consisting of 25,342 oligonucleotides of 50-mers printed on glass slides. Only spots with recorded data on the eight slides (four for BHLHB2 and four for BHLHB3) were selected for further analysis. With these selection criteria, 12,825 spots were retrieved. The data were analyzed by using the one-class significance analysis of microarray (SAM) procedure. Microarrays data are available in the GEO database under accession number GSE12947 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi).
We recently reported that adenovirus-mediated expression of the mature nuclear forms of either SREBP-1a or SREBP-1c triggered the regulation of more than 1,300 genes in human differentiated myotubes (43). Using FATiGO software (Babelomics) to analyze these microarray data, three Gene Ontology (GO) classes showed significant over-representation in the list of genes found to be regulated in the presence of SREBP-1 proteins compared to their representation in the human genome: “muscle contraction” (GO 0006936, adjusted P value = 2.84 e−5), the subclass “striated muscle contraction” (GO 0006941, adjusted P value = 2.46 e−5), and “muscle development” (GO 0007517, adjusted P value = 6.27 e−5). The corresponding genes with fold change values upon SREBP-1a or -1c expression are listed in Table Table1.1. These genes encode transcription factors involved in muscle differentiation (i.e., MYOD1, MYOG, and MEF2C), as well as a large number of muscle contraction proteins (i.e., heavy and light chains of myosin, troponins, and titin). Most of them were downregulated in the presence of SREBP-1a or -1c (28 of 38 for “muscle contraction” and 26 of 39 for “muscle development”).
The SREBP-1s microarray data obtained on differentiated myotubes contain two bHLH family members that are upregulated upon SREBP-1s overexpression. The transcriptional repressors BHLHB2 and BHLHB3 show an ~2-fold increase in their expression levels (see the supplemental data in reference 43). Since recent report indicated that BHLHB3 is a potent inhibitor of muscle cell differentiation (2), we decided to focus on these factors. To assess SREBP-1a and -1c effects on BHLHB2 and BHLHB3 expression, we overexpressed nuclear SREBP-1 in human primary muscle cells at both myoblast and myotube stages and also in vivo in mouse tibialis anterior muscle. As shown in Fig. Fig.1,1, overexpression of SREBP-1 in myoblasts, myotubes, and mouse muscle induced significant increases in both BHLHB2 and BHLHB3 mRNA and protein levels in all situations. As a control, we verified that overexpression of ADD1-DN, a dominant-negative mutant of SREBP-1 (27), does not significantly affect BHLHB2 and BHLHB3 expression levels in cultured muscle cells.
The promoter sequences of the human BHLHB2 and BHLHB3 genes contain putative SRE motifs for SREBP-1 binding (located at −839/−830 and −32/−23 for BHLHB2; −651/−642 and + 43/+52 for BHLHB3 relative to the respective transcription start sites). In addition, a degenerate motif was identified at −248/−238 (TCACAGGGT) in the BHLHB2 promoter. To investigate whether SREBP-1a and -1c increase BHLHB2 and BHLHB3 expression through promoter activation, we performed gene reporter experiments in muscle and nonmuscle cell lines transiently transfected with SREBP-1a- and/or SREBP-1c-expressing plasmids. Measurements of luciferase activities confirm that overexpression of SREBP-1 proteins strongly increases both BHLHB2 and BHLHB3 promoter activities in myoblasts, myotubes, and nonmuscle HepG2 cells (Fig. 2A and B, left). Activation of the promoters in nonmuscle cells excluded the participation of additional muscle-specific factors in the induction of BHLHB2 and BHLHB3 by SREBP-1 proteins. To assess the involvement of the identified putative SREs in both promoters, we performed mutations and deletions of the various sites (Fig. 2A and B, right). Concerning the BHLHB2 promoter, deletion of the distal motif, as well as mutation of the proximal motif, did not modify enhancement of promoter activity by SREBP-1 proteins, whereas the deletion of the SRE-like motif suppressed SREBP-1 activation. Concerning the BHLHB3 promoter, mutation of either distal or proximal SREs suppressed promoter activation, showing that they are both are involved in the response to SREBP-1. Finally, ChIP experiments further confirmed that SREBP-1 proteins directly bind the BHLHB2 and BHLHB3 promoters (Fig. (Fig.2C2C).
We then conclude that transcriptional repressors BHLHB2 and B3 are new direct target genes of SREBP-1, the expression of which is increased by SREBP-1 binding on their promoters.
We performed microarray analysis in human primary myotubes overexpressing either BHLHB2 or BHLHB3 after adenovirus infection. FATiGO analysis revealed that the same biological processes identified after SREBP-1 overexpression (“muscle contraction,” “striated muscle contraction,” and “muscle development”) were significantly enriched (adjusted P values < 0.05) in the lists of regulated genes. We found that BHLHB2 and BHLHB3 downregulated 69 and 65 genes with muscle annotation, respectively (Table (Table2).2). Furthermore, the comparison with the SREBP-1 microarray data showed that a large proportion (34%) of the muscle-specific genes that were downregulated by SREBP-1 expression were also downregulated by BHLHB2/B3 overexpression.
Overlapping downregulated genes for the two GO biological processes “muscle development” and “muscle contraction” are represented in Fig. Fig.3.3. Among genes involved in muscle differentiation, MYOD1, MYOG, and MEF2C show a decrease in their expression upon both SREBP-1 and BHLHB2/B3 overexpression.
Because the expression of specific markers of muscle differentiation was decreased in myotubes overexpressing SREBP-1, we first examined the expression of the four studied transcription factors during the differentiation of human primary muscle cells (Fig. (Fig.4A).4A). All four present a similar pattern of expression with an increase during proliferation and a decrease after induction of differentiation. To further examine whether SREBP-1 could directly affect myogenic differentiation, primary human myoblasts were thus infected with recombinant adenoviruses expressing GFP, SREBP-1a, or SREBP-1c. After 48 h, SREBP-1-expressing myoblasts showed a dramatic decrease in MYOD1, MYOG, and MEF2C levels (Fig. (Fig.4B).4B). When the cells were induced to differentiate (medium change and serum starvation) for 5 days, only Ad-GFP-infected cells underwent differentiation (Fig. (Fig.4C).4C). The presence of SREBP-1 totally blocked the differentiation of myoblasts into myotubes.
To determine the implication of BHLHB2 and/or BHLHB3 in this process, human primary myoblasts were infected with recombinant adenovirus expressing either BHLHB2 or BHLHB3. As shown in Fig. Fig.4D,4D, 48 h of BHLHB2 and BHLHB3 overexpression also induced a marked decrease in the expression of muscle regulatory factors (MYOD1, MYOG, and MEF2C). After 5 days of differentiation, we observed a dramatic decrease in the number and the size of polynucleated cells, correlated with the reduced expression of myogenin and troponin (Fig. (Fig.4E4E).
To finally demonstrate the involvement of BHLHB2 and BHLHB3 in the effects of SREBP-1 on myoblasts, SREBP-1-overexpressing myoblasts were transfected with siRNA against GFP (control), BHLHB2, or BHLHB3, resulting in a partial gene extinction of BHLHB2 and BHLHB3 expression (Fig. (Fig.5A).5A). As shown in Fig. Fig.5B,5B, inhibition of either BHLHB2 or BHLHB3 can restore, at least partially, the expression of MYOD1, MYOG, and MEF2C proteins that are downregulated upon SREBP-1 overexpression. Depletion of BHLHB2/B3 was sufficient to restore differentiation and myogenin and troponin expression in cells overexpressing SREBP-1 (Fig. (Fig.5C5C).
Altogether, these data led us to propose that SREBP-1a and -1c block myoblast-to-myotube differentiation via an increase in BHLHB2 and BHLHB3 expression, the latter repressing the expression of muscle regulatory factors (MRFs).
We next examined the consequences of nuclear accumulation of SREBP-1 proteins in differentiated muscle cells. To confirm and expand the microarray data, we measured the expression levels of several transcription factors and sarcomeric protein genes using quantitative PCR in primary myotubes overexpressing the SREBP-1 factors for 48 h. Figure Figure66 shows that both SREBP-1a and -1c decreased the expression of myogenic regulatory factors (MYOD1, MYOG, and MEF2C) (Fig. (Fig.6A).6A). A significant reduction in the mRNA levels of muscle contractile proteins (TTN, TNNI1, TNNI2, and MYL1) was also observed. These data were further confirmed at the protein level (Fig. (Fig.6B).6B). Therefore, the mature forms of SREBP-1a and -1c clearly induced a dramatic decrease in the expression of major actors of skeletal muscle function, involved in either formation or contractility.
Direct observation of myotubes overexpressing SREBP-1 showed a decrease in cell surfaces. Troponin immunostaining confirmed a considerable reduction in sarcomeric protein content (Fig. (Fig.6C).6C). Cell sizes measurements showed that SREBP-1 proteins induced an ~6-fold decrease in cell surface (Fig. (Fig.6D).6D). These observations indicated thus that nuclear accumulation of SREBP-1 led to myotube atrophy, with a severe decrease in the expression of muscle regulatory factors and sarcomeric proteins. To assess whether the observed SREBP1-induced atrophy involved known atrophic factors, we measured the mRNA levels of FBXO32 (Atrogin1), MURF1 (MuRF-1/TRIM63), and FOXO1. As shown in Fig. Fig.6A,6A, with the exception of MURF1, the expression of these factors was reduced in the presence of SREBP-1a and -1c. The upregulation of MURF1 mRNA, however, is in agreement with our previous microarray data (43).
As observed with SREBP-1a and -1c, infection of fully differentiated myotubes with adenoviruses expressing BHLHB2 or BHLHB3 strongly repressed the expression of myogenic factors (MYOD1, MYOG, and MEF2C) and sarcomeric proteins (MYL1, TNNI1, and TTN) (Fig. (Fig.7A).7A). Overexpression of BHLHB2 and BHLHB3 also provoked the atrophy of muscle cells (Fig. (Fig.7B),7B), as evidenced by cell size measurements indicating a >60% reduction in myotube areas (Fig. (Fig.7C).7C). However, in contrast to SREBP-1, BHLHB2 and BHLHB3 overexpression induced a marked decrease in MURF1 expression level (Fig. (Fig.7A7A).
To confirm the involvement of BHLHB2 and BHLHB3 in the atrophic effect of SREBP-1 on differentiated myotubes, SREBP-1 overexpressing myotubes were transfected with siRNA against GFP, BHLHB2, or BHLHB3. As shown in Fig. Fig.7D,7D, gene extinction of either BHLHB2 or BHLHB3 restored the expression of troponin. Depletion of BHLHB2/B3 also restored, at least partially, the size of myotubes, with a greater effect of BHLHB3 silencing (Fig. (Fig.7E7E).
Altogether, these data indicated that, as observed for inhibition of myoblast differentiation, the transcriptional repressors BHLHB2 and BHLHB3 are directly involved in the atrophy induced by SREBP-1 in differentiated myotubes.
To investigate the effects of SREBP-1 factors on muscle phenotype in vivo, we overexpressed SREBP-1a or SREBP-1c in limb muscle of mice using recombinant adenovirus. Adenoviruses expressing either GFP only, or both SREBP-1a and GFP (or SREBP-1c and GFP) were generated using dual expression properties of constructs containing an IRES element (26). Twelve-week-old BALB/c male mice were separated into two groups, and adenoviral suspensions were injected in tibialis anterior muscle with 1010 infectious units of recombinant adenoviruses expressing only GFP (Ad-GFP) in the left limb of all animals and either SREBP-1a and GFP (Ad-1a/GFP, first group) or SREBP-1c and GFP (Ad-1c/GFP, second group) in the right limb. Animals were sacrificed 7 days after injections, and tibialis anterior muscles were removed for analysis. When comparing the two groups, no differences were found in GFP-only expressing muscles of the left limbs (weight, fiber sizes, and fluorescence intensity); we thus considered the data concerning Ad-GFP-infected muscles as a unique set. As shown in Fig. Fig.8A,8A, tibialis anterior weight showed a significant decrease of 17.5% (SREBP-1a/GFP versus GFP, n = 7, P = 0.001) and 18.6% (SREBP-1c/GFP versus GFP, n = 7, P = 0.002) when expressing either of the SREBP-1 proteins. When we performed a similar experiment with intramuscular injection of recombinant adenoviruses overexpressing either BHLHB2 or BHLHB3, muscle weight showed a decrease of 17.1% (BHLHB2 versus GFP, n = 7, P = 0.001) and 24.8% (BHLHB3 versus GFP, n = 7, P = 0.001), respectively (Fig. (Fig.8A).8A). We next examined fiber size in histological sections of treated muscles. Quantitative analysis revealed a significant decrease in average cross-sectional area (CSA) of myofibers for both SREBP-1a (mean ± the standard error of the mean [SEM] = 1,998.3 ± 19.7 μm2) and SREBP-1c (mean ± the SEM = 1,950.2 ± 21.0 μm2) compared to GFP (mean ± the SEM = 2,378.6 ± 21.7 μm2, P < 0.001 for both) (Fig. (Fig.8B).8B). Size distribution of muscle fiber CSA was different between GFP-only and SREBP-1/GFP-expressing muscles, the latter presenting a marked displacement of distribution toward smaller sizes of fibers (Fig. (Fig.8C).8C). Representative histological sections are shown in Fig. Fig.8D8D with the expected mosaic pattern of fluorescence. Because of the dual expression strategy, fluorescence intensities in the muscle fibers of the right limbs reflect the level of expression of the SREBP-1 recombinant proteins. We therefore examined fiber CSA as a function of the fluorescence distribution (Fig. (Fig.8E).8E). Although uninfected fibers (lowest fiber fluorescence category) showed similar myofiber CSA means, the reduction in mean fiber CSA of Ad-1a/GFP and Ad-1c/GFP-infected fibers increased with fluorescence intensity, reaching a maximum ca. 20% reduction of mean CSA compared to Ad-GFP-infected fibers.
SREBP-1a and SREBP-1c are bHLH transcription factors first identified as adipocyte determination and differentiation factors (49). Their functions have been extensively studied in hepatocytes and in mouse liver. By activation of specific target genes involved in lipogenesis, SREBP-1 increase triglycerides synthesis, and to a lesser extent cholesterol synthesis (8, 20, 21, 47). SREBP-1c was also shown to mediate the action of insulin on the expression of lipogenic genes in liver (16). SREBP-1 proteins are also expressed in skeletal muscle (13, 38, 39) and in cultured muscle cells (12, 18). In the present study we identified a new role for these transcription factors and demonstrated that both SREBP-1a and SREBP-1c can block myoblast to myotube differentiation, and also induce myotube atrophy in vitro and in vivo.
The results of the present study also demonstrate that the transcriptional repressors BHLHB2 and BHLHB3 are SREBP-1 target genes and that they mediate the observed SREBP-1 action on human muscle cell. Both BHLHB2 and BHLHB3 have been involved in the regulation of differentiation and growth of several cell types. BHLHB2 promotes the differentiation of trophoblast stem cells to trophoblast giant cells (22), induces neuronal differentiation of pheochromocytoma P19 cell (7) and promotes chondrocyte differentiation of ATDC5 cells (46). BHLHB2 can also block adipocyte differentiation through direct transcriptional repression of PPARγ gene expression (53). Concerning muscle cells, BHLHB2 is expressed in embryonic and adult skeletal muscle cells and has been recently proposed as a possible regulator of satellite cell activation since BHLHB2 knockout mice exhibit increased cellular proliferation and degenerated myotubes during muscle regeneration process (48). BHLHB3 mRNA is expressed in proliferating C2C12 cells and is downregulated during myogenic differentiation (2). Moreover, its overexpression blocks myoblast-to-myotube differentiation in C2C12 cells, through either E-Box occupancy, direct interaction with MYOD1 protein, or both (3).
We have thus demonstrated that both BHLHB2 and BHLHB3 can inhibit muscle cell differentiation and induce myotube atrophy, reproducing the observed SREBP-1 effects in cultured muscle cells, notably a marked decrease in the expression of muscle specific transcription factors and sarcomeric proteins. Furthermore, silencing of BHLHB2 and BHLHB3 protein levels using siRNA fully restored the myogenic differentiation process in the presence of SREBP-1, and rescued, even if not completely, myotubes from atrophy induced by SREBP-1 overexpression. These data therefore establish a novel regulatory pathway of muscle cell differentiation implicating SREBP-1, BHLHB2, and BHLHB3. Interestingly, it is also known that the transcriptional repressors BHLHB2 and BHLHB3 can antagonize each other's effects (3, 32), and the scheme of this novel pathway can be completed with a negative-feedback loop that has recently been described in which both BHLHB2 and BHLHB3 inhibit SREBP-1c expression in a HIF-dependent mechanism (10).
Muscle differentiation is under the control of two families of transcription factors, named MRFs: the myogenic bHLH proteins (i.e., MYF5, MYOD1, MYOG, and MYF6), and the myocyte enhancer factor2 (MEF2) family of MADS domain-containing proteins (i.e., MEF2A, -2B, -2C, and -2D) (5, 40). Moreover, the myogenic bHLH factors interact with MEF2 proteins to cooperatively activate muscle specific genes (36). We have demonstrated here that nuclear accumulation of SREBP-1 proteins led to a coordinated inhibition of the expression of the MRF in myoblasts. This decrease, which results from BHLHB2/B2 transcriptional repressors activation, is sufficient to explain the blockade of differentiation. How BHLHB2/B3 repress the expression of MRF remains to be precisely examined, but this may occur through competitive binding to E-Box on MRF promoters. Moreover, a direct interaction of the transcriptional repressors with MRF proteins may participate in the inhibition of differentiation, as already demonstrated with BHLHB3 and MYOD1 in C2C12 cells (3).
Overexpression of SREBP-1 proteins, and also of BHLHB2/B3, induces both in vitro and in vivo myotube atrophy. The maintenance of muscle protein content results from intricately regulated anabolic and catabolic pathways. Examining genes regulated by both transcription factors reveals that MRFs and sarcomeric proteins are jointly downregulated, whereas only SREBP-1 induces MURF1, an actor in the proteolytic pathway. The ubiquitin proteasome system has been described as the main regulator of muscle atrophy (30), and the role of MURF1, FBXO32 (atrogin-1), and FOXO1 in this process has been recently reviewed (37). The marked reduction in sarcomeric protein, the induction of myotube atrophy, and the in vivo muscle wasting observed in the presence of SREBP-1 proteins could also have resulted from activation of this pathway. Since the reversion of atrophy by BHLHB2/B3 silencing is only partial, a specific action of SREBP-1 proteins on the ubiquitin proteasome system involving other effectors than BHLHB2/B3 might thus be considered. Nevertheless, a significant part of the atrophic effect is due to BHLHB2/B3 action, through inhibition of sarcomeric proteins expression. This decrease in protein synthesis may be due to a direct action of BHLHB2/B3 on contractile protein promoters or may also involve the decrease in MRF expression. MRFs are still expressed in differentiated myotubes (50) and participate in the expression of sarcomeric proteins (31). Whether MRFs are involved in the maintenance of the fully differentiated phenotype is still debated, but a combined decrease in MRF expression in differentiated myotubes may affect muscle protein synthesis and thus participate in the observed atrophy. Further studies are needed to characterize this atrophic process in terms of fiber type change, mitochondrial content, and oxidation capacity.
The control of the amount of SREBP-1 proteins in the nucleus involves regulation at several levels, including SREBP-1 gene expression, proteolytic cleavage in the endoplasmic reticulum, nuclear import, and activation/degradation within the nucleus (for a review, see reference 42). It has been recently demonstrated that SREBP-1 expression is enhanced through the PKB/mTOR pathway and could participate in the regulation of cell size through the control of lipid and cholesterol metabolism (41). The inflammatory cytokine tumor necrosis factor alpha, which is known to induce muscle atrophy (33), has been shown to increase SREBP-1 levels in hepatocytes (15). Growth factors such as insulin and IGF-1 are potent inducers of SREBP-1 expression in various cell types and tissues (1, 13, 38). In muscle, SREBP-1c nuclear content can be dramatically increased by insulin through activation of both the PI3K/PKB (38) and the MAPK (28, 38) pathways. Furthermore, the SREBP-1 proteins can control and enhance their own expression in human muscle cells (12). Due to the major and clearly demonstrated role of insulin, growth factors and the PI3K/PKB signaling pathway on muscle development and hypertrophy (23, 29), the atrophic effect of SREBP-1 proteins overexpression demonstrated in the present study likely represents a negative feedback loop to control muscle hypertrophy. In the same context, it is also interesting to notice that SREBP-1a and -1c enhance the expression of the p55 subunit of the PI3K (25, 43), which is regarded as a positive regulator of the PI3K/PKB pathway (17). The SREBP-1 proteins may thus regulate the hypertrophic effects of growth factors not only negatively through induction of the BHLHB2 and BHLHB3 repressors but also positively through the control of PI3K/PKB signaling pathway. Further investigations are required to study the impact of SREBP-1 on signaling pathways in skeletal muscle cells.
In summary, the data presented here identify a new role for the SREBP-1 transcription factors in the regulation of myogenesis and muscle tissue maintenance. Since SREBP-1a and -1c are master regulators of fatty acids and cholesterol synthesis, this new function can justify to consider them as integrators of signals coming from growth factors, inflammation, and nutritional status toward a control of muscle mass. It will therefore be of particular interest to further study these transcription factors in pathological situations inducing muscle wasting, but also in metabolic diseases where abnormalities in SREBP-1 have already been reported such as insulin-resistance and type 2 diabetes.
This study was supported by the Programme National de Recherche sur le Diabète (grant to E.L.). V.L. is supported by a doctoral fellowship from the Fondation pour la Recherche Médicale.
We thank Annie Durand, Cyrille Debard, and Aurelie Granjon for technical assistance and E. R. Zabarowsky for the generous gift of the human genomic clone containing the BHLHB2 promoter region.
Published ahead of print on 22 December 2009.