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Am J Physiol Cell Physiol. Jun 2011; 300(6): C1490–C1501.
Published online Mar 9, 2011. doi:  10.1152/ajpcell.00255.2010
PMCID: PMC3118617

p300 Acetyltransferase activity differentially regulates the localization and activity of the FOXO homologues in skeletal muscle

Abstract

The Forkhead Box O (FOXO) transcription factors regulate diverse cellular processes, and in skeletal muscle are both necessary and sufficient for muscle atrophy. Although the regulation of FOXO by Akt is well evidenced in skeletal muscle, the current study demonstrates that FOXO is also regulated in muscle via the histone acetyltransferase (HAT) activities of p300/CREB-binding protein (CBP). Transfection of rat soleus muscle with a dominant-negative p300, which lacks HAT activity and inhibits endogenous p300 HAT activity, increased FOXO reporter activity and induced transcription from the promoter of a bona fide FOXO target gene, atrogin-1. Conversely, increased HAT activity via transfection of either wild-type (WT) p300 or WT CBP repressed FOXO activation in vivo in response to muscle disuse, and in C2C12 cells in response to dexamethasone and acute starvation. Importantly, manipulation of HAT activity differentially regulated the expression of various FOXO target genes. Cotransfection of FOXO1, FOXO3a, or FOXO4 with the p300 constructs further identified p300 HAT activity to also differentially regulate the activity of the FOXO homologues. Markedly, decreased HAT activity strongly increased FOXO3a transcriptional activity, while increased HAT activity repressed FOXO3a activity and prevented its nuclear localization in response to nutrient deprivation. In contrast, p300 increased FOXO1 nuclear localization. In summary, this study provides the first evidence to support the acetyltransferase activities of p300/CBP in regulating FOXO signaling in skeletal muscle and suggests that acetylation may be an important mechanism to differentially regulate the FOXO homologues and dictate which FOXO target genes are activated in response to varying atrophic stimuli.

Keywords: muscle atrophy, disuse, cachexia, gene regulation, atrogin-1, Forkhead Box O

forkhead box o (FOXO) signaling has been implicated in skeletal muscle atrophy associated with sepsis (9), starvation (24, 27), diabetes (28), cancer (27), aging (16, 27), and heart failure (44). More direct work, using genetic approaches, demonstrates that at least two of the FOXO homologues, FOXO1 (23) and FOXO3a (43), are sufficient to cause skeletal muscle atrophy in vivo. Perhaps more importantly, blocking FOXO transactivation prevents at least 40% of disuse muscle fiber atrophy (39, 45), further demonstrating the requirement of FOXO for the normal atrophy phenotype in a physiological model of muscle atrophy. Given the significance of FOXO in the regulation of muscle mass, identifying the immediate upstream regulators of FOXO may lead to the development of specific countermeasures to prevent muscle wasting.

The regulation of FOXO signaling by Akt has been extensively characterized in a variety of cell types, including skeletal muscle (2, 26, 43). In response to growth conditions or growth factor stimuli, the IGF-I/phosphatidylinositol 3-kinase (PI3K)/Akt pathway is activated, which leads to FOXO phosphorylation by Akt on specific residues, which promotes the cytosolic retention and inactivation of FOXO (2, 42). In skeletal muscle, direct evidence to support the IGF-I/PI3K/Akt pathway in regulating FOXO can be found in several studies (26, 43, 48). Decreases in this signaling pathway in skeletal muscle during physiological conditions of muscle atrophy such as starvation and muscle disuse are thought to contribute to FOXO activation. However, increasing evidence demonstrates FOXO signaling to be controlled via additional posttranslational modifications and protein-protein interactions which are distinct from Akt-mediated phosphorylation (19, 51). Yet, many of these additional regulatory mechanisms have yet to be thoroughly explored in skeletal muscle. If similar control mechanisms indeed exist in skeletal muscle to modulate FOXO activity, this could potentially open up new avenues for therapeutically blocking FOXO function and the associated muscle atrophy during physiological conditions of muscle wasting.

One such mechanism of FOXO regulation identified in multiple cell types involves the regulation of FOXO-dependent transcription by the histone acetyltransferase (HAT) proteins, p300 and CREB-binding protein (CBP) (11, 14, 35). These HAT proteins each possess an intrinsic acetyltransferase activity that catalyzes the transfer of an acetyl group to specific lysine residues on target proteins (10, 11, 32). Although HATs are most well known for regulating gene transcription through histone acetylation and relaxation of chromatin structure at gene promoters (12, 31), HATs also play an important role in regulating the activity of a variety of transcription factors, including p53, MyoD, hypoxia-inducible factor-1α, as well as the FOXO transcription factors (47, 52). HATs may regulate transcription factor activity through various mechanisms which include interaction and recruitment of factors to target gene promoters, via acting as adaptor molecules facilitating protein-protein interactions, and through direct acetylation of transcription factors or other necessary cofactors which thereby alter transcription factor activity (25). Evidence for HAT-mediated regulation of the FOXO transcription factors can be found in multiple cell types. Interestingly, however, the resulting effect of HATs on FOXO appears to be cell-type specific and/or specific to the FOXO homologue. For example, p300 increases FOXO1-dependent transcription from the IGF binding protein-1 promoter reporter in H4IIE rat hepatoma cells, which requires p300 HAT activity (35). Similarly, p300 increases FOXO3a-dependent transcription from the Bim promoter in human embryonic kidney cells (HEK293T) (34). In contrast, p300 represses FOXO4-induced transcription of Gadd45, p27, p21, and MnSOD in HEK293 cells (11). Therefore depending on the cell type, FOXO homologue, and target gene measured, HATs may either repress or activate FOXO-induced transcription, which may reflect an important fine-tuning mechanism of FOXO target gene regulation. However, despite the importance of understanding the mechanisms which lead to FOXO-dependent transcription in skeletal muscle due to its known role in causing muscle atrophy, no data currently exist to suggest whether the acetyltransferase activities of p300/CBP regulate FOXO in skeletal muscle. Therefore, the purpose of the current study was to determine whether HAT proteins regulate the FOXO transcription factors in skeletal muscle, and whether this is altered during conditions of muscle wasting.

MATERIALS AND METHODS

Animals.

Sprague-Dawley male rats (200–225 g) were ordered from Charles River Laboratories (Wilmington, MA). The University of Florida Institutional Animal Care and Use Committee approved all animal procedures. University of Florida is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (no. A3377-01). Animals were maintained in a temperature and humidity-controlled facility with a 12-h light/dark cycle. Water and standard diet were provided ad libitum.

Plasmids and reporter gene assays.

Expression plasmids for wild-type (WT) p300 and the dominant-negative (d.n.) p300 mutant (which lacks acetyltransferase activity due to an inactivating point mutation, converting aspartic acid 1399 to tyrosine) were obtained from Dr. Tso-Pang Yao (Duke University, Durham, NC) and have been previously described (21). The FOXO1 expression plasmid was a gift from Dr. Akiyoshi Fukamizu (University of Tsukuba, Ibaraki, Japan) and has previously been used and described (32). The tagged FOXO1-enhanced green fluorescent protein (EGFP) plasmid was obtained from Addgene (plasmid 9022) and was deposited by Dr. Domenico Accili (Columbia University, New York, NY) and has previously been described (13). The FOXO4 expression plasmid was a gift from Dr. Boudewijn Burgering (University Medical Center, Utrecht, The Netherlands) and has been previously used and described (53). The FOXO3a expression plasmid was obtained from Addgene (plasmid 10710), was deposited by Dr. William Sellers (Novartis, Cambridge, MA), and has been previously used and described (37). The WT FOXO3a-DsRed fusion construct was created via PCR amplification of the FOXO3a cDNA out of the parent vector using primers to create HindIII and SalI restriction sites on the 5′- and 3′-ends of the FOXO3a coding region, respectively. FOXO3a cDNA was then subcloned, in frame, into the DsRed2-c1 plasmid. Verification that FOXO3a cDNA was in frame was confirmed via DNA sequencing (DNA Sequencing Core, University of Florida). The d.n.Akt and constitutively active (c.a.)Akt expression plasmids were obtained from Addgene (plasmids 12643 and 16244, respectively) and were deposited by Dr. Mien-Chie Hung (The University of Texas, M. D. Anderson Cancer Center, Houston, TX) and have previously been described (58). The DAF-16/FOXO responsive reporter plasmid, the atrogin-1-GL2 promoter reporter plasmid, and the d.n.FOXO construct have also been previously used and described (46). pRL-TK-Renilla was purchased from Promega (Madison, WI). Plasmid DNA was amplified and isolated from bacterial cultures using Endotoxin-Free Maxi or Mega Prep Kits (Qiagen, Valencia, CA), precipitated in ethanol and resuspended in 1× sterile filtered phosphate-buffered saline (PBS) for in vivo transfections, or Tris-EDTA (TE) buffer for transfections in culture.

In vivo plasmid injection and electroporation.

Transfection of plasmid DNA into skeletal muscle in vivo has been detailed previously (22, 46). For rat experiments, 10 μg each of the expression or control plasmid(s) and 40 μg of the reporter plasmid were diluted in a total of 50 μl 1× PBS for each solei injection. Standard procedures were used to determine luciferase activity on skeletal muscle homogenates using a Modulus single tube multimode reader (Promega) and have been described previously (46).

Animal models and muscle preparation.

Disuse muscle atrophy via cast immobilization of both hindlimbs was induced in rats 4 days following plasmid injection and has been detailed previously (46). After 3 days of immobilization or weight-bearing activity, soleus muscles were either removed and processed immediately for RNA isolation or frozen in liquid nitrogen and stored at −80°C until further biochemical analyses. For experiments using exclusively genetic manipulations, muscles were harvested 7 days post plasmid injection.

Cell culture experiments.

C2C12 cells were cultured on 0.1% gelatin-coated six-well plates in high-glucose DMEM (Invitrogen), 10% fetal bovine serum, and 5% CO2. Muscle cells were transfected with plasmid DNA at ~80% confluence using FuGENE HD Transfection Reagent (Promega) at a 3.5:1 ratio of reagent to total DNA. Sixteen hours following transfection, muscle cells were differentiated into myotubes by incubation in differentiation medium (2% horse serum in DMEM). For dexamethasone studies, 6-day differentiated myotubes were treated with either vehicle (water) or 1 μM water-soluble dexamethasone (Sigma, St. Louis, MO) in differentiation media for 6 h and harvested in Passive Lysis Buffer (Promega). In the nutrient deprivation groups, differentiation media were removed from 4-day differentiated cells, and Hanks' balanced salt solution (HBSS) was added for either 2 h (localization experiments) or 6 h (reporter assays and gene expression) before harvest. To inhibit PI3K, 10 μM LY294002 (Calbiochem, Merck, Darmstadt, Germany) or vehicle (ethanol) was added to 4-day differentiated myotubes for 6 h. For reporter experiments, cells were harvested in Passive Lysis Buffer, and luciferase activity was determined by normalizing firefly luciferase activity to pRL-TK-Renilla luciferase activity using a Dual-Luciferase Reporter Assay (Promega).

RNA isolation, cDNA synthesis, and RT-PCR.

RNA isolation and cDNA synthesis from whole muscle was performed using a TRIzol-based method as previously described (46). RNA isolation from C2C12 myotubes was performed similarly, following addition of 250 μl TRIzol/well and vigorous scraping, as previously described (33). cDNA was generated from 1 μg of RNA and was used as a template for quantitative RT-PCR using primers for atrogin-1, GenBank NM_133521; MuRF1, GenBank NM_080903; cathepsin-L, GenBank NM_013156; 4E-BP1, GenBank NM_053857; LC3b, GenBank NM_022867; p21, GenBank NM_080782; Gadd45α, GenBank NM_024127; FOXO1, GenBank NM_001191846; FOXO3a, GenBank NM_001106395; FOXO4, GenBank NM_001106943; or 18S, GenBank X03205.1, which were ordered from Applied Biosystems (Austin, TX). TaqMan probe-based chemistry was used to allow detection of PCR products using a 7300 real-time PCR system (Applied Biosystems), and quantification of gene expression was performed using the relative standard curve method.

Western blotting and coimmunoprecipitation assays.

Preparation of muscle homogenates and Western blotting were performed according to standard procedures and have been described previously (46). Primary antibodies for p300 (no. 554215, BD Pharmingen, San Jose, CA); FOXO1 (no. 9454S, Cell Signaling Technology, Boston, MA); phospho-FOXO1 (Ser256) (no. 9461, Cell Signaling Technology); FOXO3a (SC-11351, Santa Cruz Biotechnology, Santa Cruz, CA); phospho-FOXO3a (Thr32) (SC-12357, Santa Cruz Biotechnology); and FOXO4 (07-1720, Millipore, Billerica, MA) were used according to the manufacturer's directions. Tubulin primary antibody (T6074 from Sigma-Aldrich) was used to control for equal protein loading and protein transfer. For coimmunoprecipitation assays, 500 μg of muscle protein were incubated overnight with either 4 μg of Anti-Acetyl Lysine antibody (no. 05–515) or 4 μg of anti-p300 (no. 05-257) using a Catch and Release Reversible Immunoprecipitation System (no. 17-500), all from Millipore. The following day, precipitated proteins were washed and subsequently eluted in denaturing buffer and boiled, and Western blotting was performed for endogenous FOXO3a and FOXO1.

Fluorescence microscopy.

C2C12 myoblasts were seeded on six-well plates containing 0.1% gelatin-coated glass coverslips, transfected, and differentiated for 4 days. Following treatment, cells were rinsed with PBS and fixed for 30 min in 4% paraformaldehyde. Following three washes in PBS, two drops of Vectashield Mounting Medium for Fluorescence with DAPI (no. H-1200, Vector Laboratories, Burlingame, CA) were added to each coverslip. A Leica DM5000B microscope (Leica Microsystems, Bannockburn, IL) containing GFP (green) and rhodamine (red) filter cubes was used to visualize FOXO1-EGFP or FOXO3a-DsRed-positive myotubes, respectively. A DAPI (blue) filter was used to visualize DAPI-stained nuclei. Images were captured and merged using Leica Application Suite software (version 3.5.0).

Statistical analysis.

Data were analyzed using a two-way ANOVA followed by Bonferroni post hoc comparisons when appropriate (GraphPad Software, San Diego, CA). All data are expressed as means ± SE, and significance was set at P < 0.05.

RESULTS

p300 Acetyltransferase activity is necessary and sufficient to repress FOXO signaling in skeletal muscle.

To determine whether p300 HAT activity regulates FOXO signaling in skeletal muscle, we injected and electrotransferred a FOXO-responsive luciferase reporter plasmid plus either a control plasmid, WT p300, or d.n.p300 (which lacks acetyltransferase activity) expression plasmid into the skeletal muscle of rats before either normal weight-bearing activity or 3 days of muscle disuse induced via hindlimb cast immobilization. As shown in Fig. 1A, FOXO activity was increased (~3-fold) in response to immobilization, which is in agreement with our previous findings (46). However, in muscles overexpressing WT p300, FOXO reporter activation in response to immobilization was prevented. Furthermore, expression of the d.n.p300 mutant, which outcompetes and inhibits endogenous p300 HAT activity, was sufficient to increase FOXO activity in normal weight-bearing muscle (~6-fold) and further enhanced FOXO activity during immobilization (~14-fold). To determine whether another protein which possesses HAT activity and is highly homologous to p300, CBP, also inhibits disuse-induced activation of the FOXO reporter, we collected additional data using a control or a WT CBP expression plasmid. Similar to WT p300, overexpression of WT CBP prevented the increase in FOXO activity during disuse (Fig. 1B).

Fig. 1.
p300 Acetyltransferase activity is necessary and sufficient to repress Forkhead Box O (FOXO) transcriptional activity in skeletal muscle. A and B: FOXO-dependent luciferase reporter activity from weight-bearing (WB) and 3-day cast immobilized (Imm) solei ...

To test whether p300 and CBP could similarly prevent the activation of FOXO in response to a stimulus distinct from muscle disuse, we determined the effect of p300/CBP on FOXO activation in vitro, in response to treatment with the glucocorticoid dexamethasone (Dex). C2C12 myoblasts were transfected with the FOXO reporter and pRL-TK-Renilla plus an empty vector, WT p300, or WT CBP plasmid. Cells were differentiated for 6 days and treated with either vehicle (water) or Dex at a concentration of 1 μM for 6 h. Dex treatment decreases phospho-FOXO protein levels in C2C12 myotubes (43), which is widely used as a marker of FOXO activation and, indeed, we found that Dex increased FOXO reporter activity 1.7-fold over control. However, this increase in FOXO activity due to Dex treatment was abolished in C2C12 cells transfected with either WT p300 or WT CBP (Fig. 1C). These findings provide the first evidence that FOXO activity is regulated in skeletal muscle via the acetyltransferase activity of p300/CBP and further indicate that HAT activity is both necessary and sufficient to repress FOXO activity in skeletal muscle.

p300 HAT activity differentially regulates FOXO target gene transcription.

Owing to the pronounced effect that p300 HAT activity has on FOXO transcriptional activity in skeletal muscle, we next determined whether p300 could also block the increased transcription of a bona fide FOXO target gene, atrogin-1/MAFbx, during 3 days of muscle disuse. Atrogin-1 mRNA was increased 4.7-fold in response to muscle disuse, which was attenuated by 65% in muscles overexpressing WT p300 (Fig. 2A). This repression by p300 required its HAT activity since expression of d.n.p300 did not similarly repress atrogin-1 mRNA levels. To further confirm that p300 can repress atrogin-1 transcription during muscle disuse, we conducted similar experiments to test the effect of WT p300 or d.n.p300 on a luciferase reporter construct driven by 2.4 kb of the atrogin-1 promoter. As shown in Fig. 2B, similar to the effect of p300 on atrogin-1 mRNA, WT p300 prevented disuse-induced activation of the atrogin-1 promoter reporter, which required its HAT activity. Notably, the effects of p300 HAT activity were more pronounced on the atrogin-1 promoter reporter when compared with atrogin-1 mRNA levels. This finding is likely explained by the muscle fiber transfection efficiency in whole muscle with the p300 constructs, which in our hands is ~50%. Therefore, the effects of WT and d.n.p300 on mRNA levels in transfected fibers are diluted by mRNA levels from nontransfected fibers. In contrast, when coinjecting two plasmids (p300 and the atrogin-1 promoter reporter constructs), cotransfection of a muscle fiber occurs nearly 100% of the time (38). As a result, the atrogin-1 promoter reporter is only reporting from those fibers which also took up the p300 (or empty vector) constructs—thereby eliminating the dilution effect.

Fig. 2.
p300 HAT activity differentially regulates the transcription of FOXO-target genes during muscle disuse. A: relative atrogin-1 mRNA levels from weight-bearing or 3-day cast immobilized solei injected with an EV, WT p300, or d.n.p300 plasmid. B: atrogin-1 ...

Since d.n.p300 was sufficient to increase both the FOXO reporter and the atrogin-1 promoter reporter during normal weight-bearing conditions, we further determined whether the increase in atrogin-1 promoter activity by d.n.p300 required active FOXO. Coexpression of d.n.p300 with a d.n.FOXO construct [which inhibits FOXO activity (46)] blocked the ability of d.n.p300 to activate the atrogin-1 promoter reporter (Fig. 2C). Collectively, these data demonstrate that p300 HAT activity represses atrogin-1 transcription, which is mediated through the repression of FOXO.

We next examined the effect of p300 HAT activity on the mRNA levels of additional FOXO target genes during normal weight-bearing conditions and 3 days of immobilization. As shown in Fig. 2D, and in agreement with others (1, 4, 22, 30, 57), 3 days of muscle disuse induced a significant increase in the mRNA levels of MuRF1 (3.2-fold), p21 (3.9-fold), 4E-BP1 (2-fold), Gadd45α (1.7-fold), cathepsin L (2.4-fold), and LC3b (2.1-fold). However, increasing HAT activity via overexpression of WT p300 significantly repressed the disuse-induced increase in MuRF1 by 55%, and the bona fide FOXO target gene, p21, by 54%, both of which required p300's HAT activity. In contrast, WT p300 further enhanced the disuse-induced increases in 4E-BP1 by 48% and another bona fide FOXO target gene, Gadd45α, by 75%, which required p300's HAT activity. Importantly, reducing HAT activity via expression of d.n.p300 significantly repressed the disuse-induced increases in cathepsin-L by 78%, and 4E-BP1 by 45%. Alterations in p300 HAT activity did not significantly affect the levels of LC3b mRNA. Three days of muscle disuse also significantly increased the mRNA levels of FOXO1 (2.2-fold) and FOXO3 (1.9-fold), which has previously been demonstrated (40, 45), and also increased the mRNA levels of FOXO4 (2.3-fold) (Fig. 2E). Notably, increased p300 HAT activity had a significant repressive effect on FOXO4 mRNA levels during both weight-bearing and immobilization conditions and on FOXO1 mRNA levels during immobilization. There was no significant effect of p300 HAT activity on FOXO3a mRNA levels. Together, these data indicate that p300 HAT activity differentially regulates the expression of FOXO target genes, as well as the FOXO genes themselves.

p300 Acetylates and represses FOXO3a transcriptional activity.

Data thus far demonstrate that p300 HAT activity represses the increase in FOXO transcriptional activity in response to two different atrophic stimuli and differentially regulates the gene expression of FOXO target genes. Because the FOXO reporter may respond to each of the skeletal muscle FOXO family members, the regulation of total FOXO activity and target gene expression by p300 may be related to the regulation of FOXO1, FOXO3a, FOXO4, or some combination of FOXO factors. We therefore tested the extent to which p300 HAT activity regulates the transcriptional activity of FOXO1, -3a, or -4 in skeletal muscle. To do this, we injected and electrotransferred rat solei with the FOXO reporter plasmid plus FOXO1, FOXO3a, or FOXO4 expression plasmids (Fig. 3A), each with an empty vector, WT p300, or d.n.p300. As shown earlier in Fig. 1A, transfection of WT p300 alone had no effect on basal levels of the FOXO reporter, while transfection of d.n.p300 was sufficient to increase FOXO activity 4.6-fold. Transfection of FOXO1 alone did not significantly increase the FOXO reporter and did not significantly alter reporter activity in the presence of WT p300 or d.n.p300 (Fig. 3B). In contrast, the 2.5-fold increases in FOXO reporter activity induced individually by FOXO3a and FOXO4 were abolished by WT p300. Moreover, the increases in FOXO reporter activity induced individually by FOXO3a (2.5-fold) and d.n.p300 (4.6-fold) were synergistically increased to 16-fold when FOXO3a and d.n.p300 were coexpressed together. Coexpression of FOXO4 plus d.n.p300 resulted in a sevenfold increase in FOXO activity, demonstrating an additive effect on FOXO activity. Together, these findings show that p300 is sufficient to repress both FOXO3a and FOXO4 activity via its HAT activity and suggest that a decrease in p300 acetyltransferase activity potently increases the transcriptional activity of FOXO3a.

Fig. 3.
p300 HAT activity differentially regulates the transcriptional activity of the FOXO homologues. AE: rat soleus muscles were injected and electroporated with a FOXO-responsive luciferase reporter plus an EV, FOXO1, FOXO3a, or FOXO4 expression ...

Because of the pronounced effect that p300 HAT activity has on the repression of FOXO3a transcriptional activity, we determined whether we could detect changes in endogenous FOXO3a acetylation in muscles injected with the WT p300 and d.n.p300 constructs. Total acetylated proteins were immunoprecipitated from equal amounts of protein extract using an anti-acetyl-lysine antibody and subsequently immunoblotted for FOXO3a. As shown in Fig. 3C, muscles injected with WT p300 demonstrated an increase in acetylated FOXO3a when compared with muscles injected with an empty vector, while d.n.p300-injected muscles showed a decrease in acetylated FOXO3a. These findings show that FOXO3a is a target of p300 HAT activity and suggest that p300 may regulate FOXO3a transcriptional activity through direct acetylation.

Protein modification via acetylation has previously been demonstrated to regulate protein stability (41). Therefore, we determined whether p300 HAT activity regulates FOXO3a protein levels. Because of the relatively low levels of endogenous FOXO3a protein in skeletal muscle and the difficulty that this poses in quantifying a p300 effect on FOXO3a, we measured the effect of either WT or d.n.p300 on ectopically expressed FOXO3a. As shown in Fig. 3D, cotransfection of WT p300 significantly reduced FOXO3a protein, which required its HAT activity, since cotransfection of d.n.p300 did not similarly reduce FOXO3a protein levels. Although the decrease in FOXO3a protein by WT p300 may explain our finding that WT p300 repressed FOXO3a-induced transcription of the FOXO reporter (as shown in Fig. 3B), the levels of FOXO3a protein in muscles cotransfected with d.n.p300 were not significantly increased. Therefore, the synergistic increase in FOXO3a-induced reporter activity when HAT activity was decreased (by d.n.p300) cannot be fully accounted for by higher levels of FOXO3a protein. To demonstrate this, we normalized FOXO reporter activity from Fig. 3B (underlined groups) to total FOXO3a protein levels. Data were normalized such that the ratio of FOXO activity to total FOXO3a protein levels in muscles transfected with FOXO3a alone was set to baseline (Fig. 3E). Since cotransfection of WT p300 with FOXO3a blocked FOXO3a-induced reporter activation and concomitantly decreased total FOXO3a levels, the ratio of FOXO activity to total FOXO3a protein did not change in the presence of WT p300. However, the 16-fold increase in FOXO activity in muscles transfected with FOXO3a and d.n.p300 was still increased by more than 4-fold when normalized to total FOXO3a protein levels (Fig. 3E). Therefore, regulation of FOXO3a protein levels by p300 HAT activity cannot fully account for the effect of p300 on FOXO3a transcriptional activity. In summary, these data demonstrate that p300 HAT activity increases FOXO3a acetylation and represses the transcriptional capacity of FOXO3a.

p300 and CBP differentially regulate FOXO1 and FOXO3a cellular localization.

One mechanism whereby acetylation has been shown to regulate FOXO transcriptional activity is through the regulation of FOXO cellular localization (13, 36). To determine whether p300/CBP regulate FOXO cellular localization, we transfected C2C12 cells with a FOXO3a-DsRed fusion construct plus an empty vector, WT p300, or WT CBP expression plasmid. Following differentiation into myotubes, we induced FOXO3a-DsRed nuclear localization via nutrient deprivation for 2 h, which has been shown previously (7, 43), and determined the effect of p300 and CBP on FOXO3a-DsRed cellular localization via fluorescent microscopy using a rhodamine (red) filter. The redistribution of FOXO3a-DsRed from the cytosol to the nucleus in response to nutrient deprivation (Fig. 4, A and B) was confirmed via DAPI staining (blue) and was prevented by p300 (Fig. 4D) and attenuated by CBP (Fig. 4F). Similarly, during control conditions, FOXO3a-DsRed was more predominantly cytosolic in myotubes coexpressing p300 (Fig. 4C) or CBP (Fig. 4E). Since FOXO1 signaling is also increased in response to nutrient deprivation (15), we further tested the ability of p300 and CBP to regulate FOXO1 cellular localization, using the same experimental design just described, but replacing FOXO3a-DsRed with FOXO1-GFP and using a GFP (green) filter. Two hours of nutrient deprivation induced a modest but visible increase in FOXO1-GFP nuclear localization (Fig. 4, G and H), which was potentiated by WT p300 (Fig. 4J) and attenuated by WT CBP (Fig. 4L). Though less visible, WT p300 similarly increased FOXO1-GFP nuclear localization during control conditions. This effect of p300 on FOXO1-GFP localization in response to starvation is in stark contrast to its effect on FOXO3a-DsRed, further indicating that p300 differentially regulates the FOXO homologues.

Fig. 4.
p300 and CBP differentially regulate FOXO3a and FOXO1 cellular localization. AL: FOXO1 and FOXO3a localization in C2C12 cells transfected with FOXO3a-DsRed or FOXO1-green fluorescent protein (GFP) plus an EV, WT p300, or WT CBP plasmid, differentiated ...

Since both p300 and CBP were found to regulate FOXO1 and FOXO3a cellular localization, we further measured their ability to regulate total FOXO reporter activation in response to nutrient deprivation. To do this, we transfected C2C12 cells with the FOXO reporter plasmid plus an empty vector, WT p300, or WT CBP plasmid. Following 4 days of differentiation, cells either remained in differentiation media (control conditions) or were nutrient deprived by removal of the media and incubation in HBSS for 6 h. FOXO-dependent luciferase activity was increased by ~20% following nutrient deprivation and was not altered by p300 during either condition (Fig. 4M). In contrast, transfection of CBP reduced FOXO-dependent luciferase activity during both control and nutrient deprivation conditions when compared with empty vector groups. To determine the physiological relevance of this decrease in total FOXO activity by CBP, we measured the ability of CBP to regulate the gene transcription of the atrophy-related FOXO target gene, atrogin-1, following 6 h of nutrient deprivation. As shown in Fig. 4N, 6 h of HBSS treatment induced a 2.5-fold increase in atrogin-1 transcription, which was attenuated by ~60% in HBSS-treated cells transfected with CBP. Therefore overexpression of CBP can attenuate both FOXO3a and FOXO1 nuclear localization in response to nutrient deprivation and prevent the full activation of atrogin-1.

Our findings in Fig. 3B using the FOXO1 and p300 constructs suggest that alterations in p300 HAT activity are not sufficient to regulate FOXO1 transcriptional activity (on the FOXO reporter) in vivo. However, in our localization experiments shown in Fig. 4, I and J, p300 strongly induced FOXO1 nuclear localization. Since p300 is predominantly localized to the nucleus, we tested the extent to which FOXO1 and p300 interact, which may potentially explain the increased nuclear residence of FOXO1 in p300-transfected cells. Since p300 also regulated FOXO3a localization, though in an opposite manner, we also measured the extent to which p300 interacts with FOXO3a. To do this, we used a coimmunoprecipitation assay kit in which we immunoprecipitated p300 from protein extracts of soleus muscles injected with an empty vector or WT p300 plasmid. Following p300 protein precipitation, we subsequently immunoblotted for endogenous FOXO3a or FOXO1. As shown in Fig. 4, O and P, p300 interacts with both FOXO3a and FOXO1, and this interaction is increased when p300 is overexpressed. The interaction of p300 was found to be greater with FOXO1 than FOXO3a, which may therefore explain our finding that p300 potentiates FOXO1 nuclear localization during nutrient deprivation.

p300/CBP repression of FOXO is mediated via Akt.

The ability of HATs to regulate FOXO signaling has previously been shown in other cell types to occur through enhancing Akt-mediated repression of FOXO. This led us to question whether p300/CBP could repress FOXO activation when Akt signaling is specifically inhibited. Treatment of C2C12 myotubes with 10 μM LY294002 for 6 h to inhibit PI3K/Akt signaling resulted in a 40% increase in FOXO reporter activation (Fig. 5A). Neither CBP nor p300 were able to repress FOXO reporter activation induced by LY294002 treatment. These findings suggest that p300/CBP may require Akt to inhibit FOXO signaling. To further test this, we transfected C2C12 cells with the FOXO reporter plasmid plus an empty vector, d.n.Akt, or d.n.Akt plus WT p300 to determine whether p300 can repress FOXO activation when Akt activity is directly and chronically inhibited. Following 3 days of differentiation, luciferase activity was measured. As shown in Fig. 5B, transfection of d.n.Akt was sufficient to significantly increase FOXO reporter activity by ~50%, which trended toward a further increase (P = 0.087) in cells cotransfected with WT p300. This finding confirms that p300 cannot repress FOXO activation when Akt activity is chronically inhibited and suggests that p300 HAT activity may inhibit FOXO through promotion of FOXO inhibition by Akt. On the basis of these data, we hypothesized that d.n.p300-induced activation of FOXO during normal conditions in vivo (as shown in Fig. 1A) may have occurred through decreasing FOXO sensitivity to Akt. To test this, we determined whether increased Akt activity could overcome FOXO activation induced by d.n.p300. To do this, we electroporated rat soleus muscles with the FOXO reporter plasmid and either empty vector, d.n.p300, c.a.Akt, or d.n.p300 plus c.a.Akt plasmids. As shown in Fig. 5C, the increase in FOXO activity induced by d.n.p300 was repressed in muscles cotransfected with c.a.Akt. Collectively, these data demonstrate that p300 HAT activity is necessary for the normal repression of FOXO in skeletal muscle under baseline conditions and that increasing HAT activity is sufficient to repress FOXO activation during atrophic conditions, both of which appear to be mediated via Akt signaling. To identify whether p300 can increase FOXO phosphorylation at the known Akt sites, we measured endogenous FOXO1 and FOXO3a phosphorylation from soleus muscles injected with an empty vector or WT p300 plasmid. Overexpression of p300 increased both FOXO1 and FOXO3a phosphorylation (Fig. 5D), providing further evidence that p300 may increase FOXO sensitivity to Akt. Importantly, since we found in previous experiments that p300 increases FOXO3a acetylation (Fig. 3C), we similarly determined whether p300 also acetylates FOXO1. To do this, we precipitated total acetylated proteins from muscles injected with either an empty vector or a WT p300 plasmid, as described previously, and subsequently immunoblotted for FOXO1. Similar to the effect on FOXO3a, overexpression of WT p300 increased FOXO1 acetylation (Fig. 5E).

Fig. 5.
HAT-induced repression of FOXO is mediated via Akt. A: C2C12 cells were transfected with a FOXO-responsive reporter, pRL-TK-Renilla, and either an EV, WT p300, or WT CBP expression plasmid. Following 4 days of differentiation, myotubes were treated with ...

DISCUSSION

The current study provides the first evidence to support the acetyltransferase (HAT) activities of p300/CBP in regulating FOXO signaling in skeletal muscle. We demonstrate that 1) p300 HAT activity is necessary to repress FOXO transcriptional activity in vivo during normal physiological conditions; 2) increasing CBP or p300 HAT activity is sufficient to block FOXO activation in vivo in response to skeletal muscle disuse and in C2C12 cells in response to nutrient deprivation or dexamethasone treatment; 3) increased p300/CBP HAT activity can repress the activation of a subset of FOXO target genes in response to catabolic stimuli; and 4) p300 interacts with and acetylates both FOXO1 and FOXO3a and differentially regulates their cellular localization and transcriptional activity. Together, these findings are the first to identify p300/CBP-mediated acetylation as a mechanism to regulate the FOXO transcription factors in skeletal muscle.

The findings in the current study suggest that p300/CBP HAT activity plays an important role in repressing FOXO activity in skeletal muscle during normal physiological conditions (Fig. 6A). Importantly, since neither p300 nor CBP overexpression reduced basal levels of FOXO activity during normal conditions, this suggests that endogenous HAT activity already maintains a maximal inhibitory effect on total FOXO signaling. In contrast, because increased HAT activity via either p300 or CBP overexpression repressed FOXO activation in vivo in response to muscle disuse and in vitro in response to nutrient deprivation and Dex treatment, these data indicate that FOXO regulation by HAT activity is altered during catabolic conditions (Fig. 6B). The insufficiency of endogenous HAT proteins to repress FOXO during these conditions may potentially be explained by multiple scenarios, one of which could be a decrease in HAT activity. Although the regulation of p300/CBP HAT activity is multifactorial, their activity is regulated in part via direct Akt-mediated phosphorylation (20), and active Akt is reduced during muscle disuse, (6), nutrient deprivation (43), and Dex treatment (43). In addition, acetyl-CoA is a substrate for p300/CBP-mediated acetylation of target substrates and is a key intermediate in numerous metabolic processes (25). Therefore, decreases in the availability of acetyl-CoA during catabolic conditions could also reduce p300-mediated acetylation of target proteins.

Fig. 6.
Proposed regulation of FOXO by p300/CBP acetyltransferase (HAT) activity. A: during normal physiological conditions, HAT proteins acetylate FOXO and promote FOXO retention in the cytosol by Akt. B: in response to catabolic conditions, disruptions in both ...

Alternatively, the insufficiency of HAT proteins to repress FOXO during catabolic conditions could be due to an increase in histone deacetylase (HDAC) activity. HDAC proteins counteract the activities of HATs by removing acetyl groups from target proteins. There are five different classes of HDACs [class I, class IIa, class IIb, class III (Sirtuins or SIRTs) and class IV] which each contain multiple family members (17). Similarly, there are multiple proteins which possess HAT activity, including p300, CBP, p300/CBP-associated factor (PCAF), and GCN5. Since HAT and HDAC proteins acetylate/deacetylate specific protein substrates, the inability of endogenous HAT proteins to suppress FOXO during catabolic conditions may result from the altered expression, cellular localization, and/or activity of any one or combination of the HAT or HDAC proteins. While measurement of these variables was beyond the scope of the current study, other studies have shown that SIRT1 (5), HDAC2 (55), HDAC4 (1, 8, 50), HDAC5 (8), and HDAC6 (50) are increased in response to skeletal muscle disuse. Although we are unaware of any published data to support FOXO regulation by any of these HDACs in skeletal muscle, there is certainly evidence in other cell types to support FOXO regulation by the NAD+-dependent SIRTs (3, 10, 49). In response to low-nutrient conditions when NAD+ levels are elevated, FOXO deacetylation by the Sirtuins increases FOXO-dependent transcription of various genes involved in both glucose metabolism (13, 29) as well as autophagy (18). Since the activities of both SIRTs and HATs are regulated via bioenergetic factors (NAD+ and acetyl-CoA, respectively), it seems plausible that alterations in the energy state of the muscle during catabolic conditions could dictate which FOXO-target genes are activated, through altering HAT/HDAC-mediated regulation of the FOXO factors.

In the current study we measured the effects of p300 HAT activity on various FOXO target genes known to be elevated during muscle disuse. Interestingly, increased p300 HAT activity was found to repress the disuse-induced activation of some FOXO targets (atrogin-1, MuRF1, and p21), yet contributed to the increased expression of others (Gadd45α, 4E-BP1, and cathepsin-L). Subsequent experiments further found that p300 also differentially regulated the activity and localization of the FOXO homologues, which could potentially explain the differential effect of p300 and its HAT activity on FOXO target gene expression. Although the relative contribution of each endogenous FOXO homologue to the regulation of these genes in skeletal muscle during physiological muscle wasting is not known, FOXO overexpression and transgenic studies have yielded important information in this regard. Several studies have now demonstrated that FOXO3a is sufficient to increase atrogin-1, MuRF1, and LC3 gene transcription (43, 54), both in C2C12s and in whole muscle. In contrast, the data are conflicting on whether FOXO1 is sufficient to increase atrogin-1 and/or MuRF1 in skeletal muscle (23, 48, 54). Importantly, however, cathepsin-L and Gadd45α mRNA levels are increased in the skeletal muscle of FOXO1 transgenic mice and decreased in mice transgenically expressing d.n.FOXO1 (56). These genetic studies therefore suggest that atrogin-1 and MuRF1 are primary targets of FOXO3a and that cathepsin-L and Gadd45α are primary targets of FOXO1. Given our collective findings that 1) p300 HAT activity represses FOXO3a transcriptional activity and nuclear localization and represses atrogin-1 and MuRF1 gene expression and 2) p300 increases FOXO1 nuclear localization and contributes to the increased gene expression of cathepsin-L and Gadd45α, it may be speculated that p300 regulates these genes through differentially regulating FOXO3a- and FOXO1-dependent gene transcription.

It is important to mention, however, that p300 also regulates histone configuration, can act as a transcriptional coactivator, and may regulate additional transcription factors other than FOXO. Therefore the changes in mRNA levels observed with the p300 constructs may also reflect changes in these variables. However, our use of the atrogin-1 promoter reporter plasmid circumnavigates at least one of these issues. Since plasmid DNA constructs remain extrachromosomal, their regulation does not therefore depend on an open histone configuration for active gene transcription (as does genomic DNA). Therefore, at least for atrogin-1, p300-mediated regulation of its promoter activity is not mediated through changes in histone configuration.

While the current study provides evidence that p300/CBP-mediated repression of FOXO may require intact Akt signaling, p300/CBP overexpression still repressed the physiological activation of FOXO during skeletal muscle disuse. Therefore, although the levels of active Akt are reduced in vivo during periods of muscle disuse (6), these reduced levels of Akt were presumably sufficient for overexpressed WT p300/CBP to repress the disuse-induced activation of FOXO in the current study. Furthermore, since reduction of endogenous HAT activity via expression of d.n.p300 in control muscle was sufficient to activate FOXO, this also demonstrates that FOXO can be activated without directly manipulating Akt levels. Collectively, these findings suggest that it may be possible to therapeutically manipulate Akt-mediated repression of FOXO indirectly, via targeting HAT activity, which would have important ramifications for the muscle wasting field.

In summary, these findings demonstrate that p300/CBP acetyltransferase activity is both necessary and sufficient to repress FOXO transcriptional activity in skeletal muscle in vivo. Furthermore, this study offers new insight into the differential regulation of the FOXO homologues in skeletal muscle and highlights new therapeutic possibilities for blocking specific FOXO target genes during conditions of muscle wasting.

GRANTS

This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant R03AR056418 (to A. R. Judge). S. M. Senf is supported by a T32 from the National Institute of Child Health and Human Development Grant T32-HD-043730.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

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