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Originally published In Press as doi:10.1074/jbc.M408391200 on August 9, 2004

J. Biol. Chem., Vol. 279, Issue 42, 43625-43633, October 15, 2004
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E2F-1 Regulates the Expression of a Subset of Target Genes during Skeletal Myoblast Hypertrophy*{boxs}

Myint Hlaing{ddagger}§, Paul Spitz{ddagger}, Krishnan Padmanabhan{ddagger}, Blanca Cabezas¶, Christopher S. Barker¶, and Harold S. Bernstein{ddagger}||**{ddagger}{ddagger}

From the {ddagger}Cardiovascular Research Institute, the ||Cancer Center, and the **Department of Pediatrics, University of California, San Francisco, California 94143 and the Genomics Laboratory, The J. David Gladstone Institutes, San Francisco, California 94103

Received for publication, July 26, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cellular hypertrophy, or growth without division, is an adaptive response to various physiological and pathological stimuli in postmitotic muscle. We demonstrated previously that angiotensin II stimulates hypertrophy in C2C12 myoblasts by transient activation of the cyclin-dependent kinase 4 complex, subsequent phosphorylation of retinoblastoma protein, release of histone deacetylase 1 from the retinoblastoma protein inhibitory complex, and partial activation of the transcription factor E2F-1. These observations led us to propose a model in which partial inactivation of the retinoblastoma protein complex leads to the derepression of a subset of E2F-1 targets necessary for cell growth without division during hypertrophy. We now present data that support this model and suggest the mechanism by which E2F-1 regulates hypertrophy. We examined expression profiles of angiotensin II-stimulated myoblasts and identified a subset of E2F-1 target genes that are specifically regulated during the hypertrophic response. We showed that the expression of E2F-1 targets involved in G1/S transit, DNA replication, and mitosis is not altered during the hypertrophic response, while the expression of E2F-1-regulated genes controlling early G1 progression, cytoskeletal organization, protein synthesis, mitochondrial function, and programmed cell death is up-regulated. Furthermore, we demonstrated that activation of cytochrome c oxidase genes occurs during the development of hypertrophy and that cytochrome c oxidase IV is a direct transcriptional target of E2F-1. These studies demonstrated that E2F-1 activity at specific promoters is dependent on physiological circumstances and that E2F-1 should be considered a potential target in the treatment of pathologic hypertrophy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Cellular hypertrophy, or growth without division, is an adaptive response to various physiological and pathological stresses in postmitotic muscle. The decision to undergo DNA synthesis and proliferation or to cease proliferation is determined in late G1 of the cell cycle (1). G1 progression and G1/S transit are governed by G1 cyclins and cyclin-dependent kinase (Cdk)1 complexes through their actions on the E2F-1·retinoblastoma protein (Rb) complex (2,3). Cdks 4 and 6, assembled with their regulatory subunits, the D-type cyclins, are activated in response to mitogenic stimuli, heralding cell cycle entry and G1 progression (4). Active Cdk4/6·cyclin D1 phosphorylates Rb during early G1; this leads to the up-regulation of cyclin E, its assembly with Cdk2, and activation of the Cdk2·cyclin E complex, which in turn hyperphosphorylates Rb (5). Hyperphosphorylated Rb releases and thereby activates the transcription factor E2F-1, allowing the expression of genes necessary for G1/S transit, DNA replication, and mitosis (6).

The E2F family of transcription factors plays an important role in regulating the expression of genes required for G1/S transit, DNA replication, and mitosis. The E2F family can be divided into distinct subgroups on the basis of their structural and functional similarities. E2F-1, E2F-2, and E2F-3 are primarily transcriptional activators; their expression peaks in late G1 and coincides with activation of G1/S-specific genes (7). In contrast, E2F-4 and E2F-5 act mainly as transcriptional repressors by primarily complexing with the Rb-related proteins p107 and p130 (8). E2F-6 is a transcriptional repressor that lacks domains for Rb binding and for transactivation (9, 10) and is thought to exert its transcriptional repression through the recruitment of polycomb group proteins, histone methyltransferase, and Mga and Max proteins (11). E2F-7 recently was identified and is thought to be a transcriptional repressor of cell cycle progression (12, 13).

While details about the E2F family are being elucidated, precise functional divisions among individual members and the level of redundancy among them are only starting to be made clear. Recent knock-out experiments have provided some insight. Studies in E2F-1-deficient mouse embryonic fibroblasts have demonstrated that E2F-1 is required for timely cell cycle reentry in G0-arrested cells but not for G1/S transit in cycling cells (14). In contrast, E2F-4/E2F-5 single or double nullizygous mouse embryonic fibroblasts show no defect in cell cycle reentry from G0 and proliferate normally (15). Studies with E2F-6-deficient mouse embryonic fibroblasts have demonstrated that E2F-6 is not required either for growth arrest following serum withdrawal or for timely reentry into the cell cycle from G0 (16). In whole animals, E2F-1-null mice have a higher incidence of tumor development but exhibit testicular atrophy, suggesting tissue-specific functions for E2F-1 in both suppressing and stimulating cell proliferation (17). In muscle, E2F-1 overexpression has been shown to drive postmitotic rat ventricular myocytes into G1 both in vitro and in vivo (18–20). Taken together, these observations suggest that E2F-1, as distinct from other E2F family members, promotes cell cycle reentry in G0-arrested cells. Previous experiments have suggested that differentiating, cell cycle-arrested myoblasts treated with hypertrophic stimuli, such as angiotensin II (AngII), undergo a process with biochemical features similar to cell cycle reentry (21). E2F-1, therefore, would appear to be the most likely among E2F family members to be involved.

Over the past several years, the role of E2F-1 in the transcriptional regulation of other cellular functions, such as apoptosis, has been revealed (22, 23). However, little is known about the role of E2F-1 during muscle development or in response to pathophysiological conditions. Recently others have reported a role for E2F-1 during skeletal muscle regeneration (24). Although both E2F-1 and E2F-2 expression were induced in response to skeletal muscle injury, regeneration was severely compromised in E2F-1-null mice but not in E2F-2-null mice. In addition, others have demonstrated that inhibition of E2F-1 prevents the development of myocyte hypertrophy in vitro, suggesting that E2F-1 is necessary for hypertrophy (25). The mechanism by which E2F-1 controls the hypertrophic response, however, has not been described.

We demonstrated previously that AngII stimulates hypertrophy in C2C12 myoblasts by transient activation of Cdk4, subsequent phosphorylation of Rb, release of histone deacetylase 1 from the Rb inhibitory complex, and partial activation of E2F-1 (21). We hypothesized that partial inactivation of the Rb complex leads to the derepression of a subset of E2F-1-responsive genes necessary for cell growth without division during hypertrophy (21). We now present data that support this model and suggest the mechanism by which E2F-1 regulates hypertrophy. We examined expression profiles of AngII-stimulated myoblasts and identified subsets of E2F-1 target genes that are specifically regulated during the hypertrophic response. These data revealed that E2F-1-responsive genes involved in protein synthesis, cytoskeletal organization, and mitochondrial function were up-regulated during the hypertrophic response. However, the expression patterns of E2F-1 targets that regulate G1/S transit, DNA replication, and mitosis were not altered. To determine whether our data would identify novel E2F-1 targets involved in the hypertrophy response, we examined the transcriptional regulation of members of the cytochrome c oxidase (Cox) gene family. We demonstrated that Cox IV is a direct transcriptional target of E2F-1 and mapped the E2F-1-regulatory element within the Cox IV promoter.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—C2C12 skeletal myoblasts (ATCC) were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum and 100 IU of penicillin/streptomycin. Cells were synchronized in the quiescent state by starvation for 36–48 h with serum-free Dulbecco's modified Eagle's medium and stimulated with 100 nM AngII (Sigma) to render hypertrophy for the indicated times at 37 °C.

Microarray Analysis—The Qiagen/Operon version 2.0 mouse oligonucleotide library, comprised of oligonucleotides (70-mers) representing 16,443 unique mouse genes, was printed on poly-L-lysine-coated glass slides. The library also included sets of positive and negative controls that were used for quality control purposes. Oligonucleotides were prepared in sets of 384-well plates at a concentration of 40 µM. Arrays were prepared using a custom-built array printer equipped with Majer MicroQuill-2000 split tip pins with a capacity of 255 slides per print run. The printed arrays were numbered sequentially during each run and tracked by a unique identification number. After completion of printing and air drying of the arrays, all slides were washed in 0.2% SDS to remove excess oligonucleotide followed by a brief rinse in 70% ethanol. Slides selected from throughout the run were tested for correct printing by nonspecific hybridization of Cy3-labeled, random 9-mers. An additional test to confirm DNA attachment and availability of the oligonucleotides was performed by hybridization of Cy3- and Cy5-labeled cDNAs prepared from total RNA isolated from previously characterized tissues or cell lines.

For microarray analysis, total RNA was purified from quiescent C2C12 myoblasts and myoblasts that were stimulated with AngII for 12 or 24 h using the RNeasy minikit (Qiagen). The quality of RNA was analyzed using the RNA 6000 nanokit on an Agilent BioAnalyzer 2100 system (Agilent Technologies); as expected, discreet 28 S rRNA and 18 S rRNA peaks were observed. RNA samples with an A260/A280 ratio of 1.8–2.1 were used. Targets to be hybridized to the arrays were prepared by synthesis of single-stranded cDNA from total RNA using an oligo(dT) primer in the presence of aminoallyl-modified dUTP. After synthesis, RNA was removed by hydrolysis in alkali followed by neutralization and ethanol precipitation. Either monofunctional N-hydroxysuccinimide ester Cy3 or Cy5 fluors were chemically coupled to the free amine groups incorporated into the cDNA. After coupling, the uncoupled fluors were quenched by the addition of hydroxylamine and then removed by purification using a Qiagen QIAquick cleanup kit. Efficiency of cDNA synthesis and Cy3 and Cy5 labeling was monitored by measurement of the absorbance at 260, 550, and 649 nm, respectively.

Arrays were hybridized using a Genomic Solutions GeneTAC Hybridization Station according to the manufacturer's instructions. Slides were scanned for the presence of Cy3 and Cy5 signal using an Axon GenePix4000 scanner, and the images were quantified using GenePix Pro 3.0 software. Normalization of arrays was performed using the Locally Weighted Scatter plot Smooth (LOWESS) normalization algorithm in the Bioconductor package (www.bioconductor.org). Expression ratios were calculated using the SCANALYZE software package (26). Ratios had a mean close to 1 and a S.D. of 0.4 for each array, which is comparable to the variation seen in other array studies (27–29).

Each array experiment was performed at least four times with independently isolated RNA target pools and arrays. To correct for any gene-, sequence-, or intensity-specific artifacts, a set of three arrays comparing quiescent, serum-deprived cells was used to determine normal variation in expression level for each gene as described previously (29). The median expression ratio for each gene over the three control arrays was compared with each subsequent experimental array. Statistical significance for multiple comparisons of changes in expression was determined using two-sided, unpaired t tests (30). Absolute -fold change in expression greater than 1.5 with p < 0.05 was considered significant. GenMAPP and MAPPFinder (www.GenMAPP.org) were used to cluster microarray data into functional groups and biological pathways, respectively (31, 32). The array data were deposited in Gene Expression Omnibus (GEO) and can be viewed or downloaded under accession number GSE1592 [NCBI GEO] .

Semiquantitative RT-PCR—Total RNA was extracted using the RNeasy minikit (Qiagen). RT-PCR was performed, following DNase I treatment, using the Titanium one-step RT-PCR kit (BD Biosciences) as described by the manufacturer. A minimum amount of cycles was carried out to ensure linear amplification, and reactions were repeated at least three times. The following sequences of 5' and 3' primers were used: calreticulin, 5'-TGTGGGGGCGGCTACGTGAA and 5'-GCCTCATCATTGGTGATGAG; cyclin E, 5'-GCAGAAGGTCTCAGGTTATC and 5'-GTGGCCTCCTTAACTTCAAG; Cox VIIc, 5'-ACCGAAGGAAGTTAGGTGGTACGGCCA TTT and 5'-CATAGCCAGCAACCGCCACTTGTTT; cellular retinoic acid-binding protein 2, 5'-AAGATCGCTGTGGCTGCAGC and 5'-CAAATGGCGGTGGGAGGGTT; {beta}-actin, 5'-GTGGGCCGCTCTAGGCACCAA and 5'-CTCTTTGATGTCACGCACGATTTC; cyclin B2, 5'-AACAGAACCACTCAGGTGGC and 5'-GGCACGCATACGTCCATTTA; cyclin B1, 5'-ACTGCTCTTGGAGACATTGG and 5'-CGTCACTCACTGCAAGGATT; cyclin A2, 5'-TAACAGCATGAGGGCGATCC and 5'-AAGGCAGCTCCAGCAATGAG; thymidine kinase, 5'-CAGCATCTTGAACCTGGTGC and 5'-CTGAGAGGCAAAGAGCTTCC; topoisomerase II{alpha}, 5'-AAGACAGAAGACAGCGGAAG and 5'-TCTGAGTCCACGTTCGGATA; Cox Va, 5'-GGGTCACACGAGACAGATGAGGAGTTT and 5'-AGTCCTTAGGAAGCCCATCGAAGGGAGTTT; Cox IV, 5'-ACTTACGCTGATCGGCGTGA and 5'-GCAGCGGGCTCTCACTTCTT.

Plasmids—Mouse Cox IV promoter fragments were generated by PCR from mouse genomic DNA. The PCR primers were designed to amplify 1501 bp (–500 to +1051) of Cox IV promoter: 5'-TGCCGTGGAAGAGCGGGATGCAGTCCACGAA and 5'-TCTTGCCAATCAGGCTCAGCGCTCTGGAAG. The PCR product was cloned into pCR 2.1-TOPO (Invitrogen), and then a KpnI-XhoI fragment was subcloned into pGL3 (Promega) upstream of the Photinus pyralis luciferase reporter gene (pGL3-CoxIVpro). 5' deletion fragments of the Cox IV promoter (–288/+1051, –193/+1051, +195/+1051, and +821/+1051) were generated using the reverse primer mentioned above with the following forward primers, respectively: 5'-ACCCTCCGCTGCACGCCGTGACGCTCTCGCA, 5'-ACCCGCTCGGCCTTTCGCGACAGTTACCGC, 5'-CTTCCCCGAGCGGGTCTGTTGTCTCGGGTC, and 5'-AGTGGAAGCTTTCAGGCCAGTCCGTGCCTTA. Mutations in the E2F-1 binding sites were generated in pGL3-CoxIVpro using the QuikChange multisite-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions that changed CTTGGGGC at position –81 to CTTGATAC, ATTCCCGC at position –61 to ATTCATAG, CTTCGCGG at position –49 to CTTCATAG, and GTTGCGGG at position –23 to GTTGATAG.

Transcriptional Reporter Assay—Adenoviral constructs expressing E2F-1 (AdE2F1) and green fluorescent protein (AdG) were the generous gifts of Joseph Nevins (Duke University) (33). C2C12 myoblasts were infected with adenovirus or transiently transfected with reporter plasmids using LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. E2F-1 expression was confirmed by immunoblot with monoclonal anti-E2F-1 antibody (Santa Cruz Biotechnology) using methods described previously (21). Luciferase assays were performed in whole cell lysates using the dual luciferase reporter assay system (Promega) as described previously (34). pRL-TK (Promega) encoding constitutively expressed Renilla reniformis luciferase was included in each transfection to normalize for transfection efficiency. Briefly C2C12 cells were transfected with 4 µg of pGL3, pGL3-CoxIVpro, deletions, or point mutants of pGL3-CoxIVpro together with 0.1 µg of pRL-TK, and following 24 h recovery infected with AdE2F1 or AdG virus. For assays to determine the effect of AngII, cells were transfected with the reporter plasmids described above but without adenovirus infection. For all reporter assays, cells were maintained in serum-free media for 36–48 h prior to stimulation with 100 nM AngII. Following stimulation for 12 h, cell lysates were assayed for Photinus and Renilla luciferase activities. Photinus luciferase activity was normalized against Renilla luciferase activity and expressed as relative light units. All conditions were assayed in triplicate, and each assay was performed at least three times.

Electrophoretic Mobility Shift Assay (EMSA)—EMSA was performed as described previously (34). Nuclear extract from asynchronously dividing C2C12 myoblasts were prepared according to standard methods (35). All buffers contained protease inhibitor mixture (Sigma catalog number P2714) and phosphatase inhibitor cocktails I and II (Sigma catalog numbers P2850 and P5726). Wild type and mutant EMSA probes, consisting of 179-bp fragments that spanned the region from –177 to +2 of the Cox IV promoter, were generated by NruI digestion of Cox IV wild type and mutant reporter plasmids described above. The probes were labeled with [{alpha}-32P]dATP using the DNA 5' end-labeling system (Promega) according to the manufacturer's instructions. The wild type and mutant E2F-1 consensus oligonucleotides used for competition experiments were obtained commercially (catalog number sc-2507, Santa Cruz Biotechnology) or amplified from mutant plasmids described above, respectively. 10 µg of nuclear extract protein were incubated with 300 ng of poly(dI-dC) (Amersham Biosciences) for 10 min at 4 °C in a 20-µl reaction volume containing 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1 mM MgCl2, 4% glycerol, 0.5 mM dithiothreitol, 0.5 mM EDTA with or without a 100-fold molar excess of unlabeled competitor oligonucleotide. After incubation, 105 cpm labeled probe were added, and the DNA binding reaction was continued for an additional 20 min. DNA·protein complexes were separated by electrophoresis on a 4% nondenaturing polyacrylamide gel. Following electrophoresis, the gels were dried, and DNA·protein complexes were visualized by autoradiography at –80 °C.

Chromatin Immunoprecipitation (ChIP) Assay—ChIP assays were performed as described previously (36, 37). Briefly asynchronously dividing C2C12 cells were treated with formaldehyde to cross-link chromatin complexes and then lysed in 50 mM Tris, pH 8.1, 10 mM EDTA, 1% SDS with protease inhibitor mixture (Sigma catalog number P2714). Cross-linked chromatin was sonicated to generate fragments of ~500 –900 bp in length and diluted with 1% Triton X-100, 0.01% SDS, 1.5 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl followed by preclearing with salmon sperm DNA/protein A-agarose. Immunoprecipitation was performed at 4 °C overnight with either anti-E2F1 antibody (catalog number sc-193, Santa Cruz Biotechnology), control IgG (catalog number sc-2027, Santa Cruz Biotechnology), or no antibody. The immunoprecipitates were washed once with 1% Triton X-100, 0.1% SDS, 2 mM EDTA, 20 mM Tris, pH 8.1, 150 mM NaCl; once with the same buffer except that it contained 500 mM NaCl; once with 0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris, pH 8.1; and three times with 10 mM Tris, 1 mM EDTA, pH 8 and then eluted with 1% SDS, 0.1 M NaHCO3. Eluates were heated at 65 °C for 4–6 h to reverse cross-linking, and DNA fragments were purified using the QIAquick spin kit (Qiagen) according to the manufacturer's instructions. The presence of specific promoter sequences was detected by PCR using primers spanning the region –198 to +72 of the Cox IV promoter (5'-CTGCTACCCGCTCGGCCTTTCGCGACAGTTA and 5'-TCCAAGGCCGTCACCTGCCACCGCTGCCCAG) and {beta}-actin (5'-GTGGGCCGCTCTAGGCACCAA and 5'-CTCTTTGATGTCACGCACGATTTC) as control.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Genes Involved in Cellular Metabolism Are Specifically Regulated during the Hypertrophic Response in C2C12 Myoblasts—To test our hypothesis that a subset of E2F-1 target genes responsible for cell growth without division are activated during the induction of hypertrophy, C2C12 mouse myoblasts were stimulated with AngII for 12 or 24 h, and total RNA extracted from these cells was used to probe oligonucleotide microarrays containing ~16,000 unique mouse sequences. Hypertrophy-responsive genes were identified that demonstrated greater than 1.5-fold change in expression (p ≤ 0.05) with AngII stimulation (see Supplemental Table). Consistent with previous reports of expression profiling in various models of hypertrophy (28, 38–41), we demonstrated that ~180 genes regulating growth factor signaling, cytoskeletal organization and biogenesis, ribosome biogenesis, protein biosynthesis, mitochondrial function, cell cycle progression, and programmed cell death were specifically regulated during AngII-stimulated hypertrophy in C2C12 myoblasts. We performed RT-PCR to confirm the expression patterns of five genes (cyclins A2, B1, and B2; thymidine kinase 1; and DNA topoisomerase II{alpha}) with unaltered expression (Fig. 1A) and four up-regulated genes (calreticulin, cyclin E1, cytochrome c oxidase VIIc subunit, and cellular retinoic acid-binding protein 2) (Fig. 1B) as identified by microarray screen. These observations demonstrate the reliability of our system for examining gene expression patterns during hypertrophy.



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  		FIG. 1.
Expression of known E2F-1 target genes during the hypertrophic response. C2C12 myoblasts were made quiescent by starvation for 48 h with Dulbecco's modified Eagle's medium lacking serum (Q) and stimulated with 100 nM AngII to render hypertrophy for the indicated times at 37 °C. RT-PCR analysis of RNA was performed in triplicate using primers corresponding to indicated genes. Representative expression patterns are shown. {beta}-Actin served as a control. A, expression of E2F-1 targets necessary for DNA replication, G1/S, and G2/M was unchanged. TK-1, thymidine kinase 1; Topo II{alpha}, DNA topoisomerase II{alpha}. B, E2F-1 targets regulating cell growth were up-regulated in response to AngII. Calretic, calreticulin; CRABP2, cellular retinoic acid-binding protein 2.

 
Expression of E2F-1 Target Genes Promoting Cell Division Is Not Altered during the Hypertrophic Response—We have shown previously that hypertrophic C2C12 cells are arrested in G1 (21) and expected that expression of E2F-1 targets involved in cell cycle progression beyond G1 would remain unchanged. Indeed expression patterns of known E2F-1 target genes represented in our microarrays and involved in G1/S transit (cyclin A and Cdk2), DNA synthesis and replication (thymidine kinase 1, thymidylate synthase, DNA topoisomerase II{alpha}, proliferating cell nuclear antigen, DNA polymerase II, and ribonucleotide reductase M2), and G2/M transit (cyclin B1) were unaltered during the hypertrophic response (Table I); we confirmed five of these by semiquantitative RT-PCR (Fig. 1A). However, 26 known E2F-1 target genes involved in processes necessary for cell growth were specifically regulated in this system (Table II); we confirmed four of these by semiquantitative RT-PCR (Fig. 1B). These studies support our model that despite E2F-1 transcriptional activity in response to hypertrophic stimuli the majority of E2F-1 targets responsible for cell division are not activated, while the expression of a subset of E2F-1-regulated genes involved in cell growth is induced. The exceptions to this observation, cyclins E1 and E2 and Cdc25a, demonstrated increased expression in response to AngII despite the absence of Cdk2 activity or G1/S transit (Table II). Why the expression of these regulators was induced and what effects they might have beyond G1/S transit will require further investigation.


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  		TABLE I
Known E2F-1 target genes represented on arrays that were unchanged in response to AngII

 


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  		TABLE II
Known E2F-1 target genes represented on arrays that were specifically regulated in response to AngII

 
Expression of Genes Controlling Mitochondrial Activity Is Up-regulated during the Hypertrophic Response—Cellular energy requirements increase with cell growth and hypertrophic stress (42). Cox is the terminal component of the electron transport chain located in the inner mitochondrial membrane responsible for catalyzing the oxidation of cytochrome c that generates the proton gradient necessary for ATP synthesis. Mammalian Cox contains 13 subunits, 10 of which are encoded by the nuclear genome (IV, Va, Vb, VIa, VIb, VIc, VIIa, VIIb, VIIc, and VIII) (43, 44). Recent studies have suggested that mitochondrial proliferation and an increase in Cox activity are associated with the hypertrophic response (45, 46). Consistent with these observations, we identified elevated expression of mRNA for Cox IV, Va, Vb, VIc, VIIa, and VIIc by microarray analysis (Table III) and confirmed three of these by RT-PCR (Fig. 2). These data support a role for Cox enzymes during cellular hypertrophy, a state of increased energy demand (42).


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  		TABLE III
Cytochrome c oxidase gene expression is induced by AngII stimulation

 



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  		FIG. 2.
Transcripts for cytochrome c oxidase subunits IV, V, and VIIc are up-regulated during the hypertrophic response. C2C12 myoblasts were treated as described for Fig. 1. RT-PCR analysis of RNA was performed in triplicate using primers corresponding to indicated genes. Representative expression patterns are shown. {beta}-Actin served as a control. Expression of Cox subunits is induced during hypertrophy. Q, quiescent cells.

 
Cytochrome c Oxidase IV Is a Direct Transcriptional Target of E2F-1—Microarray and semiquantitative PCR analysis indicated that expression of the Cox IV gene was up-regulated in response to hypertrophic stimuli (Table III and Fig. 2); however, transcriptional activation of Cox IV by E2F-1 has not been demonstrated previously. The Cox VIIc promoter has been shown to bind E2F-1 (47), and Cox IV transcript is up-regulated in Rat-1a cells with E2F-1 overexpression (48). We wanted to determine whether Cox IV expression is induced by E2F-1 in myoblasts and whether it is in fact a transcriptional target of E2F-1 during hypertrophy. We overexpressed E2F-1 in C2C12 myoblasts using an adenovirus expressing full-length E2F-1 and stimulated cells with AngII (Fig. 3A). We observed an increase in the level of Cox IV expression in response to AngII in E2F-1-overexpressing cells compared with AngII-stimulated cells infected with control adenovirus (Fig. 3B). This observation supported a role for E2F-1 in the pathway leading from hypertrophic stimulus to Cox IV induction.



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  		FIG. 3.
E2F-1 induces cytochrome c oxidase subunit IV transcription during hypertrophy. A, proliferating C2C12 myoblasts (P) were infected with adenovirus expressing green fluorescent protein (AdG) or E2F-1 (AdE2F1) at a multiplicity of infection from 400 to 600 and harvested after 24 h. Cell lysates were separated by SDS-PAGE and analyzed by immunoblot with anti-E2F-1 antibody. Robust overexpression of E2F-1 was achieved. B, C2C12 myoblasts were made quiescent by serum-deprivation (Q), infected with AdG or AdE2F1, and stimulated with 100 nM AngII for the indicated times to render hypertrophy. RT-PCR analysis of RNA was performed using primers corresponding to indicated genes. E2F-1 overexpression amplified AngII-induced Cox IV expression compared with {beta}-actin control.

 
To determine whether Cox IV is a direct target of E2F-1, we tested the responsiveness of the Cox IV promoter to E2F-1. Previous analysis mapped the Cox IV promoter to nucleotides –500 to + 1051 relative to the major transcription start site (49). The Cox IV promoter is TATA-less, a finding similar to many known E2F-1 target genes (50–52). We transfected quiescent C2C12 myoblasts with a reporter plasmid containing a 1.5-kb genomic fragment spanning the murine Cox IV promoter upstream of P. pyralis luciferase, infected these cells with E2F-1-expressing adenovirus, and assayed for luciferase activity (Fig. 4A). E2F-1 overexpression induced a 6-fold increase in luciferase activity from the 1.5-kb Cox IV promoter (–500 to +1051) compared with control virus. This suggested that E2F-1 is specifically active at the Cox IV promoter. To further define the E2F-1-regulatory region of the Cox IV promoter, we generated reporter constructs containing Cox IV promoter deletions upstream of the P. pyralis luciferase (Fig. 4A). Promoter constructs starting at or after +195 showed no response to E2F-1 overexpression, suggesting that sequences between –193 to +195 are necessary for E2F-1 induction of the Cox IV gene.



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  		FIG. 4.
The cytochrome c oxidase IV promoter is activated by E2F-1 overexpression and AngII stimulation. C2C12 cells were co-transfected with pGL3 containing only the luciferase gene or pGL-CoxIV plasmids containing the 1.5-kb promoter region of the Cox IV gene or deletion fragments upstream of the luciferase gene and pRL-TK as a control for transfection efficiency. Following serum starvation for 24 h, cells were treated with 10 mM AngII for 24 h (+AngII) or infected with adenovirus containing E2F-1 (AdE2F1) or green fluorescent protein (AdG) and assayed for luciferase activity. A, AdE2F1- or AdG-infected cells were assayed 24 h following infection. Data represent mean ± S.E. (n = 3). The E2F-1-responsive region of the Cox IV promoter maps to –193 to +195 relative to the transcriptional start site (arrow). B, schematic representation of four potential E2F-1 binding sites in the E2F-1-responsive region of the Cox IV promoter (–193 to +195). C, inactivating point mutations were introduced into E2F-1 binding sites I, II, III, or IV of the Cox IV promoter individually or in combination (as indicated by X). Data represent mean ± S.E. (n = 3). Only reporter constructs containing E2F-1 binding site II conferred E2F-1-inducible luciferase activity. D, AngII-treated cells were assayed after 24 h stimulation. Deletion and point mutants are as described above. Data represent mean ± S.E. (n = 3). Only reporter constructs containing E2F-1 binding site II conferred AngII-inducible luciferase activity. TSS, transcription start site; Luc, luciferase.

 
Inspection of the promoter segment revealed four potential E2F-1 binding sites at positions –81 (site I), –61 (site II), –49 (site III), and –23 (site IV) relative to the transcriptional start site (Fig. 4B). The four potential sites were tested for their ability to mediate Cox IV promoter activity (Fig. 4C). When disrupting mutations were introduced into each site individually or in combination, the induction of promoter activity in response to E2F-1 overexpression was reduced only by the E2F-1 site II mutation (at –61) but not by the remaining three potential E2F-1 sites (I, III, and IV) (Fig. 4C). This demonstrated that E2F-1 site II within the minimal promoter element (–193 to +195 nucleotides) is essential for E2F-1-induced Cox IV promoter activation and strongly suggests that Cox IV is a direct transcriptional target of E2F-1.

To demonstrate that AngII stimulation activates the Cox IV promoter in an E2F-1 binding site-dependent manner, we conducted reporter assays using wild type and mutant Cox IV reporter constructs in C2C12 myoblasts stimulated with AngII (Fig. 4D). AngII stimulation resulted in 2-fold activation of the reporter containing the wild type Cox IV promoter, and that activation was dependent on the region between –193 and +195 containing the four putative E2F-1 binding sites. In addition, E2F-1 site II (at –61) within the minimal promoter element was essential for Cox IV promoter activation by AngII. Together these experiments indicate that E2F-1 binds and activates the Cox IV promoter in an E2F-1 binding site-dependent manner and that AngII stimulation of Cox IV promoter activity also is dependent on the same E2F-1 binding site sequences.

To determine whether endogenous E2F-1 interacts directly with the Cox IV promoter, we performed EMSA and ChIP assays in C2C12 cells (Fig. 5). EMSA demonstrated that E2F-1 binds the Cox IV promoter region spanning –177 to +2, which contains the four putative E2F-1 binding sites, but not to a mutant promoter fragment in which the E2F-1 binding site II was mutated (Fig. 5A). These binding interactions were effectively competed by an oligonucleotide containing a consensus E2F-1 binding site but not by the mutant E2F-1 oligonucleotide (Fig. 5A). These studies indicated that E2F-1 binding to the Cox IV promoter sequence was dependent upon the presence of an intact E2F-1 binding site. To verify the E2F-1/Cox IV promoter interaction in vivo, we performed a ChIP assay (Fig. 5B). PCR analysis of E2F-1-immunoprecipitated chromatin from C2C12 cells using primers that span the region of –198 to +72 of the Cox IV promoter demonstrated that E2F-1 bound to the endogenous promoter under physiological condition (Fig. 5B).



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  		FIG. 5.
E2F-1 directly binds the cytochrome c oxidase promoter in a site-specific manner. Quiescent C2C12 myoblasts were stimulated with 100 nM AngII for 24 h. A, nuclear extracts were prepared from AngII-stimulated C2C12 cells, and mobility shift assays were performed using 32P-labeled oligonucleotides consisting of Cox IV promoter sequence spanning –177 to +2 (32P-wild type) or mutant sequence in which E2F-1 binding site II was disrupted by changing ATTCCCGC at positions –61 to –54 to ATTCATAG (32P-mutant). Unlabeled oligonucleotides (wild type and mutant) were used to compete for E2F-1 binding at 100-fold molar excess. Nuclear extracts from AngII-stimulated C2C12 cells specifically shifted 32P-labeled wild type Cox IV promoter sequences but not mutant sequences in which the E2F-1 binding site at –61 was disrupted. B, cross-linked chromatin complexes were immunoprecipitated with anti-E2F-1, control IgG, or no antibody. Co-precipitated DNA sequences were amplified using pairs of primers specific for the Cox IV and {beta}-actin promoters. Cox IV promoter sequences were immunoprecipitated from C2C12 myoblasts in an anti-E2F-1 antibody-dependent manner. *, E2F-1·oligonucleotide; ns, nonspecific band.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Hypertrophy is an adaptive response of cardiac, smooth, and skeletal muscle cells to various stresses. The hypertrophic response is initiated by a range of stimuli such as growth factors, hormones, cytokines, vasoactive peptides, and catecholamines (53–58), and while the intracellular pathways that are activated may distinguish pathologic from physiologic cell growth (59, 60) it is likely that these pathways converge on common events affecting cell growth, architecture, and metabolic requirements. We previously demonstrated that several hypertrophic stimuli, including AngII, isoproterenol, prostaglandin F2{alpha}, insulin-like growth factor-1, and 1,25-dihydroxyvitamin D3, are capable of inducing cell growth without division in C2C12 myoblasts and that all of these result in Rb phosphorylation specifically at serine 780 (10).2 Using an AngII-stimulated model of hypertrophy in C2C12 skeletal myoblasts, we identified genes involved in growth factor signaling, cytoskeletal organization, protein synthesis, mitochondrial function, and protection against apoptosis. These findings support our current understanding of events responsible for hypertrophy across many systems (28, 38–41) and promote the concept of a unified output from diverse stimuli.

An increase in cell size must be accompanied by an increase in cell mass. Generally protein synthesis is regulated through the translational machinery of the ribosome (61, 62) and factors involved in translation initiation (63) and elongation (64). Our microarray data identified up-regulation of nucleolin, eukaryotic elongation factors 1{alpha} and 1{beta}, and several large and small subunit ribosomal proteins (L9, L21, L28, L44, S7, and S24), linking their expression to an AngII-stimulated increase in cell mass.

An increase in mitochondrial protein expression has been associated with exercise training or chronic electrical stimulation in skeletal muscle (65). In addition, overexpression of the serotonin 5-HT2B receptor in the heart in vivo results in hypertrophy associated with mitochondrial proliferation and increase in mitochondrial Cox activity (46). The simultaneous increase in mitochondrial number and activity has been shown to coincide with elevation of mRNA for nuclear encoded mitochondrial proteins cytochrome c and Cox subunit Va in cardiomyocyte hypertrophy induced by electrical stimulation (45). These results suggest that an increase in mitochondrial number and enzyme activity enables myocytes to cope with the increased energy demand of the hypertrophic state.

We identified elevated expression of mRNA for nuclear encoded Cox subunits IV, Va, Vb, VIc, VIIa3, and VIIc. Of these, a recent report has suggested that Cox VIIc may be a target of E2F-1 because E2F-1 binding to the promoter region of Cox VIIc can be demonstrated by chromatin immunoprecipitation (47). Cox IV expression also has been coupled indirectly to E2F-1 since it was shown to be up-regulated upon E2F-1 overexpression in Rat-1a cells (48). In this study, we demonstrated that mRNA expression of Cox IV is up-regulated during the hypertrophic response and that this activation is amplified by ectopic E2F-1 overexpression. Using deletion analysis, we demonstrated that the Cox IV promoter region –193 to +195 is essential for E2F-1 induction of Cox IV gene expression. In addition, sequence analysis revealed that this promoter segment contained four potential E2F-1 binding sites. Furthermore, mutation analysis demonstrated that only the site at position –61 substantially regulates the induction of promoter activity by E2F-1 overexpression. ChIP assays showed that binding of endogenous E2F-1 to the Cox IV promoter occurs in vivo, and EMSA demonstrated that this binding is dependent on an intact E2F-1 binding site. In addition, AngII stimulated Cox IV reporter activity, and that activity was dependent on E2F-1 site II within the minimal promoter element (–193 to +195 nucleotides) as well. Taken together, these data suggest that Cox IV is a direct transcriptional target of E2F-1 and that this site (–61) is necessary for E2F-1 regulation of the Cox IV promoter.

Although the prevailing paradigm predicts that phosphorylated Rb should dissociate from E2F-1 complexes during late G1 (66), recent work has suggested that Rb-associated E2F-1 remains bound to DNA at specific promoters during S phase (67). Moreover, while phosphorylation of Rb abolishes binding at some promoters, apparently it does not diminish binding at others, suggesting that the effect of phosphorylation may be DNA binding site-dependent (67). Specific mechanisms for E2F-1 promoter discrimination have been determined for some E2F-1 target genes such as MYCN (68), and it is likely that this occurs through cooperative interactions with other factors present at the specific promoter, including transcription factors, such as Sp1 (69), chromatin-remodeling enzymes (70), protein acetyltransferases (71), and histone deacetylases (72, 73).

What can be concluded about the role of E2F-1 in determining muscle cell size from animal studies? E2F-1-null mice showed significant growth retardation and weighed 17% less than their wild type littermates for at least the first 8 months of postnatal development despite the absence of obvious structural abnormalities in specific organs (17, 74). E2F-1/E2F-2 double knock-out mice also showed growth retardation compared with wild type littermates throughout postnatal development (75). While it has not been reported whether growth failure and decreased body weight were the result of a defect in physiological muscle hypertrophy or hyperplasia (i.e. smaller cells or fewer cells), it is known that most skeletal and cardiac growth beyond midgestation occurs by hypertrophy (76, 77), suggesting a possible defect in physiological, hypertrophic growth in E2F-1-deficient animals.

Previously we demonstrated that AngII-induced hypertrophy results in activation of E2F-1, without the release of phosphorylated Rb, and subsequent up-regulation of cyclin E expression (21). This led us to propose a model in which incomplete release from Rb inhibition results in the derepression of a subset of E2F-1 targets necessary for cell growth but not division (21). Subsequently it has been shown that blocking E2F-1·DP heterodimerization inhibits the development of myocyte hypertrophy in vitro (25), supporting a functional role for E2F-1-regulated gene expression during hypertrophy. We now present expression profiling evidence that AngII-induced myoblast hypertrophy results in activation of a subset of E2F-1 target genes that specifically enhance protein synthesis and the capacity for energy production but does not induce the expression of genes promoting cell division (Fig. 6). Thus, E2F-1 appears to regulate the hypertrophic response through its actions upon specific genes. These studies suggest that E2F-1 may be an important therapeutic target in the treatment of pathologic hypertrophy.



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  		FIG. 6.
E2F-1 activates cell growth but not division in muscle cells stimulated to undergo hypertrophy. See text for discussion.

 

   FOOTNOTES
 
* This work was supported in part by National Institutes of Health Grants HL62174 (to H. S. B.) and HL72301 supporting the UCSF NHLBI, National Institutes of Health Shared Microarray Facility. 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.

The array data were deposited in Gene Expression Omnibus (GEO) under accession number GSE1592 [NCBI GEO] . Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains a supplemental table. Back

§ Supported by National Institutes of Health Grant F32 HL 72571. Back

{ddagger}{ddagger} An established investigator of the American Heart Association. To whom correspondence should be addressed: University of California, 505 Parnassus Ave., Box 0130, San Francisco, CA 94143-0130. Fax: 415-514-0235; E-mail: hsbernstein{at}pedcard.ucsf.edu.

1 The abbreviations used are: Cdk, cyclin-dependent kinase; Rb, retinoblastoma protein; AngII, angiotensin II; Cox, cytochrome c oxidase; RT, reverse transcription; Ad, adenovirus; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation. Back

2 M. Hlaing and H. Bernstein, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Joseph Nevins (Duke University) for sharing adenoviral constructs; Michael Salazar and David Erle (University of California, San Francisco (UCSF) NHLBI, National Institutes of Health Shared Microarray Facility) for assistance with analyzing microarray data; and Paul Simpson (UCSF), Daniel Lerner (Cornell), and members of our laboratory for helpful discussion and critical reading of this manuscript.



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