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.

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 injected with wild-type (WT) p300 or dominant-negative (d.n.)p300 [lacks histone acetyltransferase (HAT) activity] (A) or WT CREB-binding protein (WT CBP) (B), or the respective control plasmids (empty vector; EV). A representative Western blot for p300 from EV, WT p300, and d.n.p300-injected muscles is shown in A. Data are reported as means ± SE, normalized to the weight-bearing, EV-injected group; n = at least 5 muscles/group. C: C2C12 myoblasts were transfected with a FOXO-responsive reporter, pRL-TK-Renilla, and either an EV, WT p300, or WT CBP expression plasmid. Following 6 days of differentiation, myotubes were treated with vehicle or 1 μM dexamethasone (Dex) for 6 h and assayed for luciferase activity. Data represent n = 3 and are reported as means ± SE, normalized to the absolute control group. Experiments were repeated at least three times. Significance was established at P < 0.05. *Significantly different from absolute control group. †Significantly different from EV within respective treatment group.

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 promoter reporter activity from weight-bearing or 3-day cast immobilized rat solei injected with an atrogin-1 promoter luciferase reporter plus an EV, WT p300, or d.n.p300 plasmid. C: atrogin-1 promoter reporter activity from weight-bearing rat solei coinjected with an EV or d.n.p300 plus either an EV or a d.n.FOXO construct. D and E: relative mRNA levels of MuRF1, p21, 4E-BP1, Gadd45α, cathepsin-L, LC3b, and 18S (D) and FOXO1, FOXO3a, and FOXO4 (E) from rat solei injected with an EV, WT p300, or d.n.p300 and exposed to weight bearing or 3 days of cast immobilization. All data are reported as means ± SE, normalized to the absolute control group; n = at least 5 muscles/group. Significance was established at P < 0.05. *Significantly different from absolute control group. †Significantly different from EV within respective treatment group.

Fig. 3.

p300 HAT activity differentially regulates the transcriptional activity of the FOXO homologues. A–E: rat soleus muscles were injected and electroporated with a FOXO-responsive luciferase reporter plus an EV, FOXO1, FOXO3a, or FOXO4 expression plasmid, each with an EV, WT p300, or d.n.p300 plasmid. A: representative Western blots for total FOXO1, FOXO3a, and FOXO4 overexpression. B: 7 days following plasmid injection, muscles were removed and assayed for FOXO-dependent luciferase activity. All data are reported as means ± SE from at least 6 muscles/group, normalized to the absolute control-injected group. Significance was established at P < 0.05. *Significantly different from absolute control group (EV only). †Significantly different from FOXO + EV within respective FOXO group. #Significantly different. C: endogenous FOXO3a acetylation was determined in soleus muscles injected with an EV, WT p300, or d.n.p300 plasmid via immunoprecipitation (IP) of total acetylated proteins from protein extracts using an anti-acetyl-lysine antibody followed by Western blotting for FOXO3a. Experiments were independently repeated three times. D: representative Western blot showing FOXO3a protein levels in soleus muscles injected with FOXO3a plus an EV, WT p300, or d.n.p300, using α-tubulin as loading control. E: FOXO activity (underlined in B) normalized to total FOXO3a protein levels from muscles injected with FOXO3a plus either an EV, WT p300, or d.n.p300 (from D).

Fig. 4.

p300 and CBP differentially regulate FOXO3a and FOXO1 cellular localization. A–L: 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 for 4 days and either left in differentiation media (control) or nutrient deprived by replacing media with HBSS for 2 h. FOXO3a-DsRed localization was visualized using a rhodamine (Red) filter and was merged with DAPI-stained (blue) nuclei (A–F). Arrows in B point to FOXO3a-DsRed-positive nuclei, which are pink in the merged image. FOXO1-GFP positive myotubes were visualized using a GFP (green) filter and were merged with DAPI-stained (blue) nuclei (G–L). Arrows in H and J point to nuclei that are FOXO1-GFP positive and which are light blue in merged images. M: C2C12 myoblasts transfected with a FOXO-responsive reporter, pRL-TK-Renilla, and an EV, WT p300, or WT CBP expression plasmid. Following 4 days of differentiation, myotubes were left in differentiation media (control) or nutrient deprived (HBSS) for 6 h and harvested for luciferase activity. Data are expressed as means ± SE, normalized to the absolute control group. N: atrogin-1 gene expression following 6 h of nutrient deprivation (HBSS) of C2C12 cells. Myoblasts were transfected with either an EV or WT CBP and differentiated for 4 days before treatment. Data are expressed as means ± SE, normalized to the respective control group. All data represent n = 3, and experiments were independently repeated at least three times. Significance was established at P < 0.05. *Significantly different from the respective control group. †Significantly different from EV + HBSS group. O and P: the ability of p300 to interact with endogenous FOXO3a (O) and FOXO1 (P) was determined in soleus muscles injected with either an EV or WT p300 expression plasmid. Equal amounts of protein extract were used to immunoprecipitate p300, followed by subsequent immunoblot for either FOXO3a or FOXO1. Western blots for endogenous FOXO3a and FOXO1 indicate the relative input for IP experiments. Experiments were independently repeated three times.

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 either vehicle (ethanol) or the phosphatidylinositol 3-kinase inhibitor LY294002 (10 μM) for 6 h and harvested for luciferase activity. Data were normalized to their respective vehicle-treated group and are therefore expressed as (+/−) LY294002. B: C2C12 cells were transfected with a FOXO-responsive reporter, pRL-TK-Renilla, and either an EV, WT p300, d.n.Akt, or WT p300 + d.n.Akt. Following 3 days of differentiation, myotubes were harvested for luciferase activity. Data were normalized to the absolute control group. C: rat soleus muscles were injected and electroporated with a FOXO-responsive reporter plus either an EV, d.n.p300, constitutively active (c.a.)Akt, or c.a.Akt + d.n.p300. Seven days following injections, muscles were removed and harvested for luciferase activity. Data were normalized to the respective EV group (black bars) within the EV or c.a.Akt groups. All cell culture data represent n = 3, are reported as means ± SE, and were repeated at least three times. Significance was established at P < 0.05. *Significantly different from absolute control group. D: representative Western blots showing phospho- and total FOXO1 and FOXO3a from soleus muscles injected with either an EV or WT p300 plasmid. E: endogenous FOXO1 acetylation from muscle extracts in D was determined via IP of total acetylated proteins using an anti-acetyl-lysine antibody followed by Western blotting for FOXO1. Experiments represent n = 3.

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 Akt and HAT signaling contribute to FOXO nuclear localization and transcriptional activation of target genes.