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Am J Physiol Cell Physiol 291: C699-C709, 2006. First published May 3, 2006; doi:10.1152/ajpcell.00056.2006
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GROWTH, DIFFERENTIATION, AND APOPTOSIS

Muscle cell survival mediated by the transcriptional coactivators p300 and PCAF displays different requirements for acetyltransferase activity

David Kuninger, Alistair Wright, and Peter Rotwein

Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon

Submitted 6 February 2006 ; accepted in final form 24 April 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Normal skeletal muscle development requires the proper orchestration of genetic programs by myogenic regulatory factors (MRFs). The actions of the MRF protein MyoD are enhanced by the transcriptional coactivators p300 and the p300/CBP-associated factor (PCAF). We previously described C2 skeletal myoblasts lacking expression of insulin-like growth factor-II (IGF-II) that underwent progressive apoptotic death when incubated in differentiation-promoting medium. Viability of these cells was sustained by addition of IGF analogs or unrelated peptide growth factors. We now show that p300 or PCAF maintains myoblast viability as effectively as added growth factors through mechanisms requiring the acetyltransferase activity of PCAF but not of p300. The actions of p300 to promote cell survival were not secondary to increased expression of known MyoD targets, as evidenced by results of gene microarray experiments, but rather appeared to be mediated by induction of other genes, including fibroblast growth factor-1 (FGF-1). Conditioned culture medium from cells expressing p300 increased myoblast viability, and this was blocked by pharmacological inhibition of FGF receptors. Our results define a role for p300 in promoting cell survival, which is independent of its acetyltransferase activity and acts at least in part through FGF-1.

myoblast; microarray; fibroblast growth factor-I

THE FORMATION AND MAINTENANCE of skeletal muscle throughout the life span is dependent on transcriptional networks that are established by myogenic regulatory factors (MRFs) and modulated through signal transduction pathways regulated by growth factors and hormones. The MRF family is composed of four basic helix-loop-helix (bHLH) transcription factors: MyoD, Myf5, myogenin, and MRF4 (34). These muscle-specific regulatory molecules play essential roles in myoblast determination and differentiation through control of muscle gene expression programs (34). Studies in mice have shown that MyoD or Myf5 is critical for myogenic specification of uncommitted somatic mesodermal cells, whereas myogenin is required for terminal differentiation, and MRF4 plays a more auxiliary role (3, 42). Overexpression of individual MRFs in cultured cells can convert fibroblasts and other cell types to the myogenic lineage (42). MRFs function as heterodimers with ubiquitously expressed bHLH E proteins and bind to E-box elements found in the promoters of many muscle-specific genes. Members of the MEF2 family of transcription factors can enhance actions of MRFs through binding to AT-rich sequences, which often are located adjacent to E-box elements (2).

Gene expression regulated by MRFs and MEF2 proteins, like many other transcription factors, depends on interactions with transcriptional coregulatory molecules. Coactivators promote transcription through reversible posttranslational alterations of core histones and chromatin remodeling, leading to a more permissive template (49). Corepressors also modify histones and alter chromatin structure, but to prevent gene expression (32). Transcriptional coactivators like p300, cAMP response element-binding protein (CREB)-binding protein (CBP), and p300/CBP-associated factor (PCAF) possess intrinsic lysine acetyltransferase activities and have been shown to acetylate both histone and nonhistone proteins, including MyoD and other sequence-specific transcription factors (58). Acetylation of transcription factors may increase their DNA binding affinity or alter stability, coregulator association, or subcellular localization and is generally associated with increased transcriptional potential (58). In addition to directly modulating the activity of transcription factors, both p300 and CBP may interact with components of the basal transcription complex, thereby enhancing transcription through a "bridging" function (14, 19).

Results from several groups have indicated roles for p300, CBP, and PCAF in the regulation of muscle differentiation (30). Cell culture studies have shown that MyoD binds p300 and that this interaction appears to be required for robust differentiation (39, 44). Acetylation of MyoD on lysine-99, -102, and -104, residues adjacent to its bHLH domain, is an important functional modification, because nonacetylatable mutants have impaired transcriptional activity (36, 45). It has been suggested that the acetyltransferase (AT) activity of p300 is dispensable for activation of MyoD and that this role is fulfilled by PCAF, which physically associates with p300 (45). However, both p300 and PCAF are able to acetylate MyoD in vitro (36), and more recent genetic studies have shown that the AT activity of p300 is essential for activation of MyoD and Myf5 (41). Roth et al. (41) demonstrated in cell culture that mouse embryonic stem cells lacking p300 or with a nonfunctional AT domain failed to form skeletal muscle (41). In contrast, only p300 null cells had a defect in proliferation. These results suggest that there are different mechanisms of action of p300 in myogenic differentiation than during cell proliferation.

The insulin-like growth factors IGF-I and -II play key roles in normal somatic growth and are important for muscle development, repair, and regeneration (1, 28, 33, 37). The actions of both IGFs are mediated by the IGF-I receptor (IGF-IR), a transmembrane tyrosine protein kinase (31). Ligand binding stimulates receptor autophosphorylation, leading to activation of several intracellular signaling pathways (31). Work from several laboratories has demonstrated key roles for the phosphatidylinositol 3-kinase (PI3-kinase)/Akt pathway in IGF-regulated metabolism, survival, and differentiation (7, 10, 18, 21, 40, 43, 48). We have found that sustained activation of this pathway by muscle-derived IGF-II is critical for myoblast viability and differentiation (47). In muscle cells engineered to lack IGF-II, forced expression of either MyoD or the cyclin-dependent kinase inhibitor p21, a MyoD target gene, promoted viability in the absence of IGF signaling, whereas blocking their expression prevented IGF-stimulated myoblast survival, thus implicating MyoD and p21 as mediators of IGF action during early events leading to differentiation (26, 27). Other peptide growth factors, including platelet-derived growth factor (PDGF) BB and fibroblast growth factor-2 (FGF-2), which did not promote differentiation, also could maintain muscle cell viability (25, 47). PDGF-stimulated myoblast survival required sustained activation of the MAP kinases ERK-1 and -2, because inhibition of the upstream activator Mek1 blocked its effects, and forced expression of constitutively active ERK-1 promoted muscle cell survival in the absence of PDGF (25). These results demonstrate that distinct growth factor-regulated signaling pathways may independently control myoblast viability.

The current experiments are based on our observations that MyoD expression was required for IGF-mediated muscle cell survival and on work from several laboratories demonstrating that the transcriptional coactivators p300 and PCAF play important roles in promoting MyoD-dependent gene activation. We now find that both p300 and PCAF can maintain muscle cell viability in the absence of added growth factors. The survival-promoting actions of p300 appear to be independent of its AT activity, whereas those of PCAF are not. A combination of gene microarray studies and selective signaling experiments suggest that at least part of the survival effects of p300 are mediated through the production and secretion of FGF-1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. IGF-II-deficient murine C2 myoblasts (C2AS12 cells) were used between passages 6 and 12 and were grown on gelatin-coated tissue culture dishes in growth medium [GM: Dulbecco's modified Eagle's medium (DMEM; Mediatech-Cellgrow) with 10% fetal calf serum (FCS; Hyclone) and 10% newborn calf serum (Hyclone)] plus 400 µg/ml G418 (Fisher Scientific), as described previously (26). Myoblast viability was assessed after incubation for 24 or 48 h in differentiation medium [DM: DMEM with 2% horse serum (Invitrogen)], with or without additions as indicated in text and legends, by counting nuclei of fixed, adherent cells stained with Hoechst 33258 nuclear dye using NIH Image 1.62 software. The IGF-I analog R3-IGF-I (GroPep) was used at a final concentration of 2 nM, and bovine basic FGF (R&D Systems) was used at a concentration of 10 ng/ml. Murine C3H10T1/2 mouse embryonic fibroblasts (ATCC catalog no. CCL-226) were grown in DMEM with 10% FCS. Cells were incubated at 37°C in humidified air with 5% CO2. The FGF receptor inhibitor SU5402 was used at a final concentration of 20 µM.

Preparation and use of recombinant adenoviruses encoding human p300 and PCAF. A plasmid containing wild-type human p300 with a COOH-terminal Flag epitope tag (pcDNA3-p300Flag) was a gift from Dr. James Lundblad (Oregon Health & Science University; OHSU). The coding region was excised by digestion with HindIII and NotI endonucleases and was subcloned into identically digested pBluescriptSK (Stratagene). To generate pShuttleTetR-p300wt, we subcloned the p300 coding region into the SalI and EcoRV sites of the adenoviral transfer vector pShuttleTetR (55) after first modifying the NotI site of p300wt DNA by filling in the overhanging end using the Klenow fragment of DNA polymerase I and then digesting with SalI. A plasmid containing the human p300 AT2 mutation was obtained from Dr. James Kadonaga (University of California, San Diego, CA) (23). The encoded protein contains substitutions at the following amino acids within the catalytic domain: His1415, Glu1423, Tyr1423, Tyr1430, and His1434 to Ala, and Leu1428 to Ser (23). An ~2-kb DNA fragment spanning the catalytic region was excised and purified after digestion with BglII and PmlI and was subcloned using the same restriction sites of the pShuttleTetR-p300wt plasmid to generate pShuttleTetR-p300AT2. The transcriptional adaptor putative zinc finger {Delta}TAZ2 mutation spans amino acids 1739–1871 of human p300 (nt 5221–5619); it was prepared by PCR with the use of primers flanking this segment, followed by subcloning into the analogous part of pShuttleTetR-p300wt. The p300DN (dominant negative) mutant encodes amino acids 1869–2414 and was generated by PCR, followed by subcloning into pShuttleTetR as described for p300wt. The 5' primer contained a restriction site for XbaI followed by an ATG codon engineered to be in frame with codon 1869. All DNA fragments generated by PCR were verified by DNA sequencing.

A plasmid containing human PCAF (58) was obtained from Dr. Hua Lu (OHSU). The coding region was subcloned into pBluescriptSK using XmaI and Asp718 restriction sites, and the NH2-terminal Flag epitope tag was replaced with an influenza hemagglutinin (HA) tag by PCR. Because generation of adenoviruses by recombination in Escherichia coli using the AdEasy system (Q Biogene) requires that the target gene contain no sites for the restriction enzyme PacI, the single PacI site in PCAF spanning codons 448–450 was replaced by site-directed mutagenesis (QuickChange kit; Stratagene) while maintaining the same encoded amino acids, Leu-Ile-Lys (nucleotides TTA ATT AAA changed to CTA ATC AAA; PacI site is underlined and mutated residues are bold). Site-directed mutagenesis also was used to produce the AT mutant PCAFmutAT by changing codons for Phe568, Thr569, and Glu570 within the catalytic domain to Ala (nucleotides TTC ACA GAG altered to GCG GCC GCG). A NotI site (underlined) also was added for identification of the modified DNA segment. All mutations were verified by DNA sequencing. The coding regions of the two modified PCAFs were subcloned into pShuttleTetR using SalI and XbaI restriction sites.

Recombinant adenoviral DNAs were produced in E. coli by homologous recombination of PacI-linearized pShuttleTetR plasmids and pAdEasy-1 after electroporation into the BJ5183 bacterial strain, following steps recommended by the supplier. Recombinant adenoviruses were generated after transfection of PacI-linearized adenoviral DNA into 293T cells, as described previously (55). Individual plaques were picked and screened by PCR, and recombinant viruses were amplified and purified by banding on discontinuous CsCl gradients (55). Viral titers were determined by optical density. Adenoviruses encoding the tetracycline transactivator protein (Ad-tTA) and enhanced green fluorescent protein (Ad-EGFP) have been described elsewhere (55, 56). For infections of myoblasts, recombinant adenoviruses were diluted in DMEM containing 2% FCS [Ad-tTA at a multiplicity of infection (MOI) of 1,500 and all others at an MOI of 3,000], filtered through a Pall Acrodisk syringe filter (0.45 µM), and added to cells at 37°C for 90–120 min. After addition of an equal volume of DMEM with 20% FCS and 20% newborn calf serum and incubation for 18 h, cells were washed and incubated in DM with or without doxycycline (500 ng/ml) for up to 48 h. For infections of C3H10T1/2 cells, Ad-tTA was used at an MOI of 150 and other viruses at MOIs of 250. The infection rate ranged from 50 to 60% of cells in all experiments, as established by immunocytochemistry.

Immunocytochemistry. Cells were fixed in 4% paraformaldehyde for 15 min at 20°C and permeabilized with a 50:50 mixture of ethanol and acetone for 2 min before blocking in 0.25% normal goat serum for at least 1 h at 20°C. Primary antibodies were added for 16 h at 4°C in blocking buffer [anti-Flag M2 (GE Biosciences) or anti-HA.11 (Covance), each at 1:1,000 dilution]. After a washing step, cells were incubated for 2 h at 20°C in goat anti-mouse IgG-Alexa 488 (Molecular Probes) diluted to 1:2,000 in blocking buffer containing Hoechst 33258 nuclear dye (Polysciences). Images were captured with a Roper Scientific Cool Snap FX charge-coupled device camera attached to a Nikon Eclipse T300 fluorescent microscope using IP Labs 3.5 software. Adobe Photoshop was used for image processing and editing.

Cell lysates and immunoblotting. Cells were placed on ice, washed with cold Tris-buffered saline, scraped into RIPA buffer [50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1 mM DTT, 0.1% SDS, 0.5% Na-deoxycholate, 1% Nonidet P-40, and a protease inhibitor cocktail (Mini-Complete; Roche) plus 1 mM NaF, 0.5 mM Na-orthovanadate, and 1 µM okadaic acid as phosphatase inhibitors], passed through a 22-gauge syringe needle, and vigorously shaken for 15 min at 4°C. Insoluble material was removed by centrifugation, and protein concentrations were determined using the BCA protein assay (Pierce). Cell lysates (40 µg) were separated by SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes, and incubated overnight at 4°C with primary antibodies to Flag M2 (1:1,000 dilution), HA (1:1,000 dilution), {alpha}-tubulin (1:4,000 dilution; Sigma), phospho-ERK-1 and -2 (1:1,000 dilution; Cell Signaling), or ERK-1 and -2 (1:1,000 dilution; Cell Signaling). After addition of secondary antibodies for 2 h at 15°C [Alexa 680-conjugated anti-mouse IgG (1:4,000 dilution; Molecular Probes) or IRD 800-conjugated anti-rabbit IgG (1:4,000 dilution; Rockland)], images were acquired using an LiCoR Odyssey infrared imaging system and version 1.2 analysis software.

In vitro acetylation assays. C3H10T1/2 fibroblasts were infected with adenoviruses encoding p300 or PCAF, and nuclear protein extracts (NE) were prepared 24 h later, as described previously (47). Recombinant p300 or PCAF proteins were immunoprecipitated from 100 µg of NE with the use of either anti-Flag or anti-HA antibodies plus protein A-agarose (Sigma). Immunoprecipitates were washed twice in PBS containing 0.1% Tween 20 (PBS-T) and once in AT assay buffer (50 mM Tris-Cl, pH 8, 10% glycerol, 10 mM butyric acid, 0.1 mM EDTA, 1 mM DTT, and 1 mM PMSF). Individual reactions contained immunoprecipitated proteins from 50 µg of NE in 50 µl of assay buffer with 10 µM acetyl-CoA (Sigma) and purified substrate. For assays employing p300, the substrate consisted of 500 ng of a purified recombinant protein containing the 20 NH2-terminal amino acids of Tetrahymena histone H4 fused to the NH2-terminus of maltose binding protein with a COOH-terminal Flag tag. For PCAF assays the substrate was 10 µg of acid extracted calf thymus core histones (Sigma). Glutathione S-transferase-PCAF (UBI) was used as a positive control. Acetylation reactions were incubated for 45 min at 30°C on a rotating platform, followed by addition of SDS-PAGE sample buffer, electrophoresis through 10% SDS-PAGE gels, and transfer to PVDF membranes. Enzymatic activity was assessed by immunoblotting using primary antibodies recognizing acetylated residues of histone H3 or H4 (rabbit anti-acH3 and anti-acH4, each at a 1:1,000 dilution; UBI) and IRD 800-conjugated anti-rabbit IgG (1:4,000 dilution). Substrate concentrations were determined by immunoblotting using anti-Flag antibodies at 1:3,000 dilution or by staining of core histones with Coomassie dye. The presence of p300 or PCAF in each immunoprecipitate was established using primary antibodies to Flag or HA, respectively, with detection by anti-mouse Alexa 680 (1:4,000 dilution). Image acquisition and quantification was performed using a LiCor Odyssey infrared imaging system, as stated above. The extent of acetylation was normalized to total levels of substrate.

RNA isolation, analysis of gene expression by oligonucleotide microarrays, and RT-PCR. C2AS12 myoblasts were infected with Ad-p300wt or Ad-p300{Delta}TAZ2 plus Ad-tTA at the MOIs indicated above. At 18 h after infection, cells were harvested or incubated in DM for an additional 8 or 24 h. Total RNA was extracted using Trizol reagent (Invitrogen), followed by an additional sodium acetate-ethanol precipitation. RNA concentrations were determined spectrophotometrically [absorbance at a ratio of 260 to 280 nm (A260/280) ≥1.95], and quality was assessed using agarose gel electrophoresis. The preparation of cDNAs, labeling, hybridization, quality control, and data acquisition were all performed at the OHSU Micro-array Core Facility using the Affymetrix murine genome U74A chip (MG-U74Av2). Chip data were normalized using global scaling values (200) and analyzed with Affymetrix Micro-array Suite 5.0 software with the use of default parameters except for a tau setting of 0.015. Filtered pairwise comparisons were performed with the following criteria: expression signal value ≥100 (vs. baseline) and a mean degree of change ≥4-fold. Gene annotation and functional classification was performed using online Gene Ontology (GO) tools provided by the NetAffx Analysis Center (http://www.affymetrix.com). The complete microarray data set (series record GSE4224 [NCBI GEO] ) can be found at the National Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/projects/geo). RNA (2.5 µg) was reverse transcribed with oligo-dT primers using SuperScript III (Stratagene). Products were diluted 10-fold in nuclease-free water, and PCR reactions were performed using specific single-stranded primers for mouse FGF-1 (sense, 5'-CAGCTCAGTGCGGAAAGTGCG-3'; antisense, 5'- TAGCGCAGCCAATGGTCAAGG-3'), FGF binding protein-1 (sense, 5'-CACCTGGATCTGCACACTCAC-3'; antisense, 5'-CGGGCAACCTGTTTCCAGTAG-3'), and FGF receptor 1 (FGFR1: sense, 5'-CAGCTCAGTGCGGAAAGTGCG-3'; antisense, 5'-TAGCGCAGCCAATGGTCAAGG-3'), following a protocol supplied with the Advantage2 GC kit (BD Biosciences-Clontech). PCR products were separated on 1.2% agarose gels, and images were captured on a Gel Doc imager and quantified with Quantity One software (Bio-Rad).

Generation and use of conditioned culture medium from C2AS12 cells infected with Ad-p300wt. Conditioned medium (CM) was collected from C2AS12 myoblasts that were infected with Ad-p300wt and incubated in DM with or without doxycycline for 24 h. CM was centrifuged at 3,000 g for 5 min at 4°C to pellet debris and was added immediately with or without the FGF receptor inhibitor SU5402 (20 µM) to confluent C2AS12 cells that had been preincubated for 2 h in serum-free DMEM. Cells were incubated in CM for up to 30 min for analysis of ERK phosphorylation or for 24 or 48 h for assessment of viability.

Statistical analysis. Data are presented as means ± SE. Statistical significance was determined using ANOVA between groups. Results are considered statistically significant when P < 0.005.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Ectopic expression of p300 promotes myoblast survival. In previous studies we have shown that C2 myoblasts engineered to lack IGF-II (C2AS12 cells) underwent rapid apoptotic cell death under differentiation conditions in the absence of added growth factors (26, 47). As seen in Fig. 1A, after 24 h of incubation in DM, only ~50% of C2AS12 myoblasts remained viable, and at 48 h <20% were alive. As established previously and depicted in Fig. 1A, addition of the IGF-I analog R3IGF-I maintained myoblast viability, with 85% of cells remaining alive after 48 h in DM (and over 70% being incorporated into multinucleated myotubes). Growth factor-mediated myoblast survival also has been observed with IGF-II, PDGF, and FGF-2 (47), and we found that the PI3-kinase/Akt pathway and downstream effector molecules MyoD and p21 are critical for IGF-stimulated myoblast viability (25–27). The transcriptional coactivator p300 plays an important facilitating role in muscle differentiation by enhancing the actions of MyoD and other muscle transcription factors (29, 44). We have used a two component adenoviral expression system to test the hypothesis that p300 can promote muscle cell viability.


Figure 1
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  		Fig. 1. p300 promotes survival of insulin-like growth factor-I (IGF-I)-deficient myoblasts. A: IGF-I maintains myoblast viability and promotes muscle differentiation. Representative phase-contrast micrographs are shown at x200 magnification of C2AS12 cells grown to ~95% confluent density in growth medium (GM) and incubated in differentiation medium (DM) ± IGF-I for 24 or 48 h. B: regulated expression of p300 after adenoviral infection. C2AS12 myoblasts were infected in GM with adenoviruses encoding p300 (Ad-p300) and the tetracycline transactivator protein (Ad-tTA) ± doxycycline (Dox). One day later, cells were fixed and p300 was detected by immunocytochemistry using anti-Flag antibodies (green), and nuclei were visualized with Hoechst dye (blue). C: Ad-p300 promotes myoblast survival. C2AS12 cells were infected with Ad-p300 plus Ad-tTA (shaded bars) or adenovirus encoding enhanced green fluorescent protein (Ad-EGFP) plus Ad-tTA (solid bars). One day later, cells were incubated in DM ± Dox for 24 or 48 h. Uninfected control cells were incubated in DM ± IGF-I (open bars). Cell viability was determined by counting, as described in MATERIALS AND METHODS. Results are graphed as means ± SE of 3 independent experiments. Statistical significance was determined by ANOVA (P < 0.0001 for 24- and 48-h groups).

 
As shown in Fig. 1B, infection with recombinant adenoviruses encoding human p300 (Ad-p300wt) and the tetracycline transactivator protein (Ad-tTA) resulted in the production and nuclear accumulation of p300 as assessed by immunocytochemistry (top), whereas addition of the tetracycline analog doxycycline prevented p300 expression (bottom). To determine whether p300 can maintain myoblast survival, we coinfected C2AS12 cells with Ad-p300wt and Ad-tTA or with Ad-EGFP and Ad-tTA, and cells were incubated in DM in the presence or absence of doxycycline for up to 48 h. Uninfected cells incubated in DM with or without IGF-I were included as controls. As graphed in Fig. 1C, expression of p300 significantly enhanced myoblast survival (85 ± 9% at 24 h and 67 ± 8% at 48 h) compared with EGFP (47 ± 3 and 33 ± 6%, respectively) or no viral infection. Cell viability with Ad-p300wt was similar to that seen with IGF-I. Treatment with doxycycline completely eliminated this effect of p300 and reduced cell survival to background levels (47 ± 9% at 24 h and 26 ± 8% at 48 h). Thus p300 can sustain myoblast viability under conditions that otherwise lead to cell death.

Enzymatic activity of p300 is not essential for myoblast survival. It has been reported that expression of muscle-specific genes regulated by MyoD is enhanced by p300, which physically associates with MyoD through a region encompassed within the TAZ2 domain (39) (see Fig. 2). However, it remains controversial whether the AT activity of p300 is required for augmentation of MyoD-dependent gene transcription directly or whether that function is mediated by an associated AT, PCAF (35). To determine which domains of p300 were required to promote myoblast survival, we constructed a series of Tet-regulated recombinant adenoviruses encoding modified versions of p300, including a form deficient in catalytic activity (AT2 mutant) or lacking the TAZ2 domain ({Delta}TAZ2) (Fig. 2A). In addition, we generated p300 encoding only the COOH-terminal ~550 residues, which has been found previously to act in a dominant negative fashion in transfection-based promoter-reporter assays (39). Doxycycline- and tTA-regulated expression of each virus was confirmed by immunoblotting extracts of infected cells (Fig. 2B). Immunocytochemical analysis demonstrated that like p300wt, p300AT2, and p300{Delta}TAZ2 were located in the nucleus, whereas the p300DN was broadly distributed throughout the cell (not shown). Both p300wt and p300{Delta}TAZ2 demonstrated histone acetyltransferase (HAT) activity as shown by in vitro HAT assays, whereas the p300AT2 and p300DN did not, as expected (Fig. 2, C and D). Cell survival studies showed that both p300wt and p300AT2 were able to maintain myoblast viability compared with EGFP, although p300wt was more effective (85 ± 6 vs. 73 ± 3% at 24 h and 72 ± 8 vs. 54 ± 6% at 48 h; Fig. 3). In contrast, p300{Delta}TAZ2 and p300DN were completely ineffective. These results indicate that enzymatic activity of p300 was partially dispensable for myoblast survival but that an intact TAZ2 domain was essential. This latter domain (also termed CH3) has been described as an interface for protein-protein interactions between p300 and other factors, including but not limited to MyoD and PCAF (14, 19).


Figure 2
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  		Fig. 2. Regulated expression of p300 variants. A: structure of p300 wild type (p300wt) and variants. Domains are as follows: TAZ1 and TAZ2, transcriptional adaptor putative zinc finger; KIX, phospho-cAMP response element-binding protein (CREB) interacting domain; Br, bromodomain; PHD, small zinc finger-like domain; AT, acetyltransferase (catalytic domain); ZZ, zinc finger, type ZZ domain; Q, glutamine-rich region. Mutations are described in detail in MATERIALS AND METHODS. DN, dominant negative. All recombinant p300 proteins have an COOH-terminal Flag tag. B: expression of adenovirus-derived p300wt (wt) or variants (AT2, {Delta}TAZ, and DN) in C2AS12 myoblasts is shown by immunoblotting with anti-Flag antibodies (top). Dox treatment is indicated. Tubulin was used as a protein loading control (bottom). C: assessment of AT activity of adenovirus-derived p300wt and variants. The indicated p300 proteins were immunoprecipitated (IP) from nuclear extracts of Ad-p300-infected 10T1/2 cells and used in AT assays with Flag-tagged histone H4-maltose binding protein (MBP) fusion proteins as substrates, as described in MATERIALS AND METHODS. Acetylation was detected by immunoblotting with anti-acetyl H4 (acH4) antibodies, and total H4-MBP and p300 levels were detected with anti-Flag antibodies. Asterisk indicates a nonspecific band. D: relative H4 acetylation.

 

Figure 3
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  		Fig. 3. Effects of p300wt and variants on myoblast survival: the AT activity of p300 is partially dispensable for myoblast survival. C2AS12 cells were infected with Ad-tTA and the indicated Ad-p300 constructs or Ad-EGFP and were incubated in DM for 24 or 48 h. Cell viability was determined as indicated in MATERIALS AND METHODS. Results are means ± SE of 3 independent experiments. Statistical significance was determined by ANOVA (P < 0.0001 for 24- and 48-h groups).

 
PCAF-mediated myoblast survival requires its AT activity. On the basis of results shown in Fig. 3 and reports describing a role for PCAF in p300-mediated MyoD-dependent muscle-specific gene transcription (39, 45), we next investigated whether PCAF also could enhance muscle cell survival. For these studies we generated and expressed Tet-regulated recombinant adenoviruses encoding HA-tagged PCAF and a mutant lacking AT activity (PCAFwt and PCAFmutAT; Fig. 4, A and B). The PCAFmutAT harbors three amino acid substitutions within its catalytic domain and was based on an enzymatically inert mutant of the related GCN5 protein (52). Immunohistochemical analysis demonstrated that both proteins accumulated in the nucleus of infected cells (not shown), and biochemical studies showed that only PCAFwt contained AT activity toward histone substrates in in vitro assays (Fig. 4, C and D). Myoblast survival was assessed after infection of C2AS12 cells. As shown in Fig. 5, PCAFwt was able to maintain cell viability and was as effective as p300wt (82 ± 5% survival at 24 h and 81 ± 8% at 48 h). In contrast, PCAFmutAT was no better than EGFP or incubation in DM alone. Thus PCAF-mediated myoblast survival requires its enzymatic activity.


Figure 4
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  		Fig. 4. Regulated expression of PCAF and a catalytically inert variant. A: structure of the p300/CREB-binding protein-associated factor wild type (PCAFwt) and an AT-deficient mutant (PCAFmutAT). Domains are indicated as follows: A-G, alanine-glycine-rich region; AT, acetyltransferase (catalytic domain); Br, bromodomain. Generation of the AT mutant is described in MATERIALS AND METHODS. Recombinant PCAF proteins contain an NH2-terminal hemagglutinin (HA) epitope. B: expression of adenovirus-derived PCAF in C2AS12 myoblasts is shown by immunoblotting with anti-HA antibodies (top). Tubulin was detected as a protein loading control (bottom). C: assessing AT activity of PCAFwt or PCAFmutAT. Proteins were immunoprecipitated from infected 10T1/2 cells and used in AT assays with calf thymus histones as substrates, as described in MATERIALS AND METHODS. Acetylation of histone H3 was detected with specific antibodies (top), and PCAF was detected with anti-HA antibodies (bottom). Total histone substrate levels are shown by direct staining (middle); glutathione S-transferase (GST)-PCAF was included as a positive control. D: relative H3 acetylation.

 

Figure 5
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  		Fig. 5. Catalytic activity of PCAF is required for myoblast survival. Effects of PCAF and an AT-deficient variant on myoblast survival. C2AS12 cells were infected with Ad-tTA and the indicated Ad-PCAF constructs or Ad-EGFP and were incubated 24 h later in DM for 24 or 48 h. Cell viability was determined as described in MATERIALS AND METHODS. Results are means ± SE of 3 independent experiments. Statistical significance was determined by ANOVA (P < 0.0002 for 24 h group; P < 0.0016 for 48 h group).

 
Identification of genes specifically induced by p300wt during myoblast survival. In skeletal muscle, p300 has been shown to enhance the transcriptional activity of MyoD toward target genes, such as the cyclin-dependent kinase inhibitor p21 (11, 45). Because our group had previously characterized a role for p21 in IGF-mediated myoblast survival (26, 27), we surmised that one mechanism by which p300 might mediate muscle cell viability would be through induction of p21 mRNA and protein expression. However, we were unable to detect an increase in p21 transcript or protein abundance in C2AS12 myoblasts infected with Ad-p300wt (not shown). We thus hypothesized that the increase in muscle cell viability caused by p300wt might be secondary to induction of other genes that encode prosurvival molecules. To test this idea, we performed microarray analysis of RNA isolated from C2AS12 myoblasts infected with Ad-p300wt, Ad-p300wt plus doxycycline (Ad-p300wt+Dox), and Ad-p300{Delta}TAZ2 at three different time points, just before the addition of DM and 8 or 24 h later. These RNAs were used to interrogate Affymetrix MG-U74 Av2 gene chips, which contain ~12,000 probe sets for annotated genes and expressed sequence tags. A series of pairwise comparisons was used to identify transcripts induced by p300wt compared with the other treatment groups. Stringent filtering criteria were used to limit genes of interest to those with at least a moderate level of expression (signal ≥100) and a change in abundance of at least fourfold in each pairwise comparison. We identified 29 transcripts fulfilling these criteria that were uniquely upregulated in cells expressing p300wt versus both p300wt+Dox and p300{Delta}TAZ2 (Table 1). This group included 23 annotated genes and 6 uncharacterized mRNAs and represented ~0.03% of interrogated transcripts. These mRNAs could be classified into five distinct subpopulations by employing the Gene Ontology terms (15), as assigned by Affymetrix (6). Among the induced transcripts were seven mRNAs encoding proteins with potential roles in signaling, including FGF-1, FGF binding protein 1, and the C-C chemokines CCL-2, -5, and -7 (also known as MCP-1/MCAF, RANTES, and MCP-3, respectively). To independently confirm our microarray results, we performed semiquantitative RT-PCR analysis on a subset of genes identified as upregulated by p300wt expression. As shown in Fig. 6, steady-state mRNA levels for both FGF-1 and FGF binding protein-1 are increased by p300wt compared with p300wt+Dox and p300{Delta}TAZ. In contrast, transcripts for FGFR1 remained relatively constant. These results led us to examine whether p300wt regulated myoblast viability through secreted survival factor(s).


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  		Table 1. Genes induced more than four fold in myoblasts expressing p300wt vs. p300wt+Dox or p300{Delta}TAZ2

 

Figure 6
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  		Fig. 6. Induction of fibroblast growth factor-1 (FGF-1) and FGF binding protein-1 (FGFBP-1) gene expression by p300wt. The abundance of mRNAs for FGF-1, FGFBP-1, and FGF receptor-1 (FGFR1) was assessed in C2AS12 myoblasts infected with Ad-p300wt (wt), Ad-p300wt+Dox (wt+Dox), or Ad-p300{Delta}TAZ2 ({Delta}TAZ) and incubated in DM for 0 (T0) or 24 h (T24).

 
p300 induces expression of secreted factor(s) that enhance myoblast viability. To test the idea that secreted proteins produced by p300wt-infected cells mediated muscle cell survival, we collected CM from C2AS12 myoblasts infected with Ad-p300wt in the presence or absence of doxycycline after incubation in DM (±Dox) for 24 h. Subsequently, confluent C2AS12 cells were incubated in freshly isolated CM for 48 h, and surviving myoblasts were counted. As seen in Fig. 7, incubation in p300 CM resulted in a 40% increase in cell viability over that measured using DM or CM prepared in the presence of doxycycline.


Figure 7
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  		Fig. 7. p300 induces expression of soluble factors that enhance myoblast viability. C2AS12 cells were infected with Ad-p300wt and Ad-tTA ± Dox and were incubated in DM for 48 h. Freshly harvested CM or nonconditioned DM was added to confluent C2AS12 myoblasts, as outlined in MATERIALS AND METHODS, and viability was measured 48 h later. Results are means ± SE of 3 independent experiments. Statistical significance was determined by ANOVA among DM, CM, and CM (Dox) groups (P < 0.0001).

 
Previous results from our laboratory have showed that FGF-2 was a potent myoblast survival factor (47); in our microarray studies, transcripts for FGFR1 (probe set 97509_f_at) were highly expressed (signal >850) under all conditions sampled (see Fig. 6), whereas FGFR2 was minimally detected (probe set 93090_at; all values <130) and mRNAs for FGFR3 and FGFR4 were below detection limits (probe sets 160919_at and 92937_at, respectively). In contrast, expression of known C-C chemokine receptors for CCL-2, -5, and -7, specifically CCR-1, -2, -3, and -5 (probe sets 93397_at, 99413_at, 99412_at, and 102718_at, respectively) were near or below detection limits in all samples (not shown). Together, these results implicated FGF-1 but not C-C chemokines in mediating C2AS12 myoblast survival.

FGFR1 is a high affinity receptor for many members of the FGF family, including FGF-1 and -2 (38). Ligand binding leads to receptor tyrosine phosphorylation and activation of several signal transduction pathways, including the Ras-Raf-Mek-ERK cascade (22, 38). As shown in Fig. 8A, addition of FGF-2 to C2AS12 myoblasts stimulated the phosphorylation of ERK-1 and -2 within 15 min, an effect that was largely blocked by the FGFR1 inhibitor SU5402. As shown in Fig. 8B, in myoblast survival assays, FGF-2 was as effective as IGF-I in maintaining viability but was inhibited by SU5402 (compare Figs. 8 and 1). As shown in Fig. 8C, p300 CM also induced ERK phosphorylation within 15 min, whereas CM from myoblasts incubated with doxycycline had no stimulatory effect. In addition, the enhancement of myoblast survival by CM was eliminated in the presence of SU5402. Together, these results indicate that p300-mediated myoblast survival depends at least in part on FGF-1.


Figure 8
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  		Fig. 8. FGF mediates part of the survival effects of p300 in myoblasts. A: FGF-2 induces ERK phosphorylation in C2AS12 cells. Results are shown as immunoblots (top) after incubation of C2AS12 myoblasts with 10 ng/ml FGF-2 for 15 or 30 min. Coincubation with the FGF receptor inhibitor SU5402 (SU) diminishes this effect. Total ERK levels are shown at bottom. B: SU5402 blunts FGF-stimulated myoblast survival. C2AS12 cells were incubated for 48 h in DM ± 10 ng/ml FGF-2 in the presence or absence of SU5402. Results are means ± SE of 3 independent experiments. Statistical significance was assessed by ANOVA (P < 0.0001 among all groups). C: CM induces ERK phosphorylation in C2AS12 cells, whereas CM conditioned in the presence of Dox does not (top). Total ERK levels are depicted at bottom. D: SU5402 blocks CM-stimulated myoblast survival. C2AS12 cells were incubated for 48 h in CM conditioned ± Dox in the presence or absence of SU5402. Results are means ± SE of 3 independent experiments. Statistical significance was determined by ANOVA (P < 0.0001 for all groups).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The experiments presented in this manuscript demonstrate that the transcriptional coactivators p300 and PCAF are able to maintain the viability of cultured skeletal myoblasts under conditions that otherwise lead to apoptotic cell death. The survival-promoting functions of p300 are largely independent of its enzymatic properties, because a molecule lacking AT activity was ~80% as effective as the wild-type protein. In contrast, the AT activity of PCAF was absolutely required for muscle cell viability, because the enzymatically deficient protein was inert. Because PCAF and p300 are known to interact with each other via the NH2-terminal region of PCAF and the TAZ2 domain of p300 (39), one interpretation of our results is that both proteins cooperate to maintain viability through mechanisms that are dependent on formation of a complex with intact AT function. In support of this idea, deletion of the TAZ2 domain of p300 eliminated its survival-promoting properties.

The coactivator p300 and the related molecule CBP are large, multidomain proteins with roles in multiple biological processes including cell growth and differentiation (14, 19). Both coactivators have been shown to interact with a wide array of sequence-specific transcription factors and with components of the basal transcriptional machinery and are thought to enhance transcription by physically linking these different regulatory molecules in a bridging or scaffolding function. In addition, the intrinsic AT activity of p300 and CBP facilitates transcriptional activation through direct acetylation of histone and nonhistone proteins (14, 19).

Despite similar activities in vitro, cell-based and in vivo studies have indicated that p300 and CBP have distinct functions (20, 24, 59). This seems particularly true in skeletal muscle. Mice heterozygous for AT-deficient p300 exhibited defective embryonic muscle development, with reduced and disorganized myofibers, and diminished MRF expression, whereas in contrast, mice with the identical heterozygous mutation in CBP had no muscle phenotype (41). Embryonic stem cells from mice lacking p300 or with 50% reduced AT activity showed little myogenic differentiation in vitro and had minimal expression of muscle-specific genes, whereas CBP null or equivalently AT-deficient cells formed normal skeletal muscle under the same conditions. The requirement for a full complement of AT-replete p300 for normal muscle development did not extend to other tissues derived from mesenchymal stem cells, because osteoblast differentiation was normal in cells deficient or defective in either p300 or CBP (41).

Studies in cultured myoblasts have demonstrated that p300 could enhance the transcriptional actions of MyoD toward the gene for p21 (39, 44), a cyclin-dependent protein kinase inhibitor that is able to maintain the viability of C2AS12 cells and other myoblast lines (26, 27). Surprisingly, in our experiments forced expression of p300 did not lead to an increase in p21 mRNA or protein accumulation (Table 1 and data not shown), thus leading us to look for other mediators of p300-stimulated myoblast survival. Because p300 is capable of enhancing the activity of a wide array of transcription factors, we hypothesized that its survival-promoting effects were modulated by the actions of these regulatory proteins, leading to activation of genes whose encoded protein products sustained muscle cell viability. By employing transcriptional profiling using oligonucleotide microarrays, we identified 29 mRNAs of ~12,000 screened that showed a greater than fourfold increase in mRNA abundance in the presence of p300wt versus p300{Delta}TAZ2 or when the production of p300 expression was blocked by doxycycline. Among the transcripts induced were the growth factors FGF-1 and FGF binding protein-1 and the C-C chemokines CCL-2, -5, and -7.

Given that both in vitro and in vivo studies have defined a functional requirement for p300 for normal muscle development, where it appears to act as a transcriptional coactivator for MyoD and for other MRFs (39, 41, 44), it was thus surprising that the expression of muscle-specific genes was not induced in our microarray analysis, even with a reduced threshold of twofold (Table 1 and data not shown). One explanation for this apparent conundrum may relate to the requirement for an intact IGF-mediated amplification network for muscle differentiation. This requirement has manifested itself in vivo in mice lacking the IGF receptor, which die at birth because profound muscle weakness prevents normal inflation of the lungs, secondary to muscle hypoplasia (28, 37). In cultured myoblasts, interference with IGF signaling, including blockade of production of endogenous IGF-II, appears to impair both muscle gene expression and morphological differentiation (47, 55). We therefore hypothesize that the lack of IGF-II in the C2AS12 cells used in the present studies blocked signaling through the IGF-I receptor and limited the transcriptional actions of MRFs.

FGFs comprise a family of heparin-binding proteins with diverse biological effects on growth, development, angiogenesis, and wound healing, among other processes (38). The term FGF was first applied to two proteins, originally named acidic and basic FGF (and now FGF-1 and -2, respectively) based on their chemical properties and on their ability to stimulate DNA synthesis in fibroblasts (38). The family now contains at least 22 members, whose actions are mediated by transmembrane receptors and extracellular heparin sulfate proteoglycans (38). FGF receptors comprise a family of four related genes in mammals termed FGFR-1 to -4 that are expressed as a series of protein isoforms derived through alternative RNA splicing (38). Binding of FGFs to FGFRs rapidly leads to receptor dimerization and tyrosine autophosphorylation. Receptor dimerization appears to be mediated by a complex of heparin sulfate proteoglycans containing several FGFs (38). Heparin sulfate proteoglycans also serve as a storage pool for FGFs and, along with other FGF binding proteins, additionally modulate biological effects by sequestration, enhancement of local growth factor concentrations in the extracellular matrix, or regulation of access to cell-surface FGFRs.

Several reports have described antiapoptotic actions of FGF-1 in cardiac and vascular smooth muscle (4, 8, 9), as well as in other cell types (12, 51), in part through receptor-stimulated activation of the MAP kinases ERK-1 and -2 (4). In agreement with the hypothesis that local production of FGF-1 could account for some of the effects of p300 on muscle cell survival, we found that conditioned culture medium from cells expressing p300wt but not p300wt+Dox increased myoblast viability and could activate ERK-1 and -2. The enhanced cell survival caused by conditioned medium was prevented by the FGFR-1 inhibitor SU5402, thus further implicating FGF-1 as an agent of myoblast viability. The prosurvival effects of p300-conditioned medium were less pronounced than infection of myoblasts with Ad-p300. Several factors may account for this difference, including sequestration of FGF-1 in the extracellular matrix by heparin sulfate proteoglycans and actions of other proteins whose gene expression was induced by p300.

Although the expression of several C-C chemokine genes was increased in myoblasts infected with Ad-p300, including CCL-2, -5, and -7, it is unlikely that these factors contributed to muscle cell survival, because the relevant receptors were not expressed. Chemokines are small secreted proteins of 70–90 amino acids in length that have been categorized into four families (C, C-C, CXC, and CX3C) and that act as chemoattractant molecules for a number of cell types (60) after binding to specific seven-membrane-spanning G protein-coupled receptors (17). Recent work from several groups has reported increases in C-C chemokine gene expression during skeletal muscle regeneration (5, 13, 16, 50), and there is evidence that CCL-2 and its receptor CCR-2 are required for full muscle regeneration after traumatic injury (53, 54). These reports, together with our results, suggest a potential role for p300 in the repair of damaged muscle tissue.

In summary, we have defined potential mechanisms of action for the transcriptional coactivators p300 and PCAF in promoting muscle cell survival. The acetyltransferase function of p300 was largely dispensable for sustaining myoblast viability, but this activity may have been supplied by PCAF interacting through p300's TAZ2 domain. At least some of the survival-promoting actions of p300 were mediated by induction of a limited subset of genes, and several lines of evidence implicate FGF-1 as one agent of p300-stimulated cell viability. In aggregate, our results define a new potentially physiologically significant role for p300 and PCAF as cell survival agents that may extend to other cell types in addition to skeletal myoblasts.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were supported in part by National Institutes of Health Grants F32 AR049131 (to D. Kuninger) and R01 DK-42748 (to P. Rotwein) and research grants from the Muscular Dystrophy Foundation (to P. Rotwein).


   ACKNOWLEDGMENTS
 
We thank Drs. James Lundblad, James Kadonaga, and Hua Lu for plasmids, Dr. William Horton for SU5402, Ryan Kuzmickas for technical assistance, and the OHSU Affymetrix Micro-array Core Facility for gene chip analysis and software.

Present address for A. Wright: Dalhousie Medical School, School of Medicine, Halifax, Nova Scotia, Canada B3H 4R2.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. Rotwein, Dept. of Biochemistry and Molecular Biology, L224, Oregon Health & Sciences Univ., 3181 SW Sam Jackson Park Rd., Portland, OR 97239-3098 (e-mail: rotweinp{at}ohsu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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