Articles in PresS. Am J Physiol Cell Physiol (March 9, 2011). doi:10.1152/ajpcell.00255.2010
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p300 Acetyltransferase Activity Differentially Regulates the
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Localization and Activity of the FOXO Homologues in Skeletal
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Muscle
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Sarah M. Senf1, Pooja B. Sandesara2, Sarah A. Reed2, Andrew R. Judge1,2
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1Department of Applied Physiology and Kinesiology, University of Florida, Gainesville, Florida,
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USA; 2 Department of Physical Therapy, University of Florida, Gainesville, Florida, USA
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Corresponding author:
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Andrew R. Judge,
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Department of Physical Therapy
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J303, Biomedical Sciences Building
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1275 Center Drive
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University of Florida,
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Gainesville, FL 32610
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USA
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Tel: 352-273-9220
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Fax: 352-273-6109
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E-mail:
[email protected]
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Running title: p300 HAT activity represses FOXO in muscle
Copyright © 2011 by the American Physiological Society.
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ABSTRACT
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The Forkhead Box O (FOXO) transcription factors regulate diverse cellular processes, and in
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skeletal muscle are both necessary and sufficient for muscle atrophy. Although the regulation of
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FOXO by Akt is well evidenced in skeletal muscle, the current study demonstrates that FOXO is
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also regulated in muscle via the acetyltransferase (HAT) activities of p300/CBP. Transfection of
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rat soleus muscle with a dominant-negative (d.n.) p300, which lacks HAT activity and inhibits
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endogenous p300 HAT activity, increased FOXO reporter activity and induced transcription
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from the promoter of a bona fide FOXO target gene, atrogin-1. Conversely, increased HAT
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activity via transfection of either WT p300 or WT CBP repressed FOXO activation in vivo in
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response to muscle disuse, and in C2C12 cells in response to dexamethasone & acute starvation.
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Importantly, manipulation of HAT activity differentially regulated the expression of various
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FOXO target-genes. Co-transfection of FOXO1, FOXO3a or FOXO4 with the p300 constructs
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further identified p300 HAT activity to also differentially regulate the activity of the FOXO
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homologues. Markedly, decreased HAT activity strongly increased FOXO3a transcriptional
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activity, while increased HAT activity repressed FOXO3a activity and prevented its nuclear
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localization in response to nutrient deprivation. In contrast, p300 increased FOXO1 nuclear
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localization. In summary, this study provides the first evidence to support the acetyltransferase
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activities of p300/CBP in regulating FOXO signaling in skeletal muscle, and suggests that
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acetylation may be an important mechanism to differentially regulate the FOXO homologues and
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dictate which FOXO target-genes are activated in response to varying atrophic stimuli.
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Key Words: HAT, muscle atrophy, disuse, cachexia, gene regulation, atrogin-1
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INTRODUCTION
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FOXO signaling has been implicated in skeletal muscle atrophy associated with sepsis
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(9), starvation (24, 27), diabetes (28), cancer (27), aging (16, 27) and heart failure (44). More
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direct work, using genetic approaches, demonstrate that at least two of the FOXO homologues,
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FOXO1 (23) and FOXO3a (43), are sufficient to cause skeletal muscle atrophy, in vivo. Perhaps
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more importantly, blocking FOXO transactivation prevents at least 40% of disuse muscle fiber
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atrophy (39, 45), further demonstrating the requirement of FOXO for the normal atrophy
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phenotype in a physiological model of muscle atrophy. Given the significance of FOXO in the
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regulation of muscle mass, identifying the immediate upstream regulators of FOXO may lead to
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the development of specific countermeasures to prevent muscle wasting.
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The regulation of FOXO signaling by Akt has been extensively characterized in a variety
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of cell types, including skeletal muscle (2, 26, 43). In response to growth conditions or growth
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factor stimuli the IGF-1/PI3K/Akt pathway is activated, which leads to FOXO phosphorylation
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by Akt on specific residues, which promotes the cytosolic retention and inactivation of FOXO (2,
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42). In skeletal muscle, direct evidence to support the IGF-1/PI3K/Akt pathway in regulating
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FOXO can be found in several studies (26, 43, 48). Decreases in this signaling pathway in
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skeletal muscle during physiological conditions of muscle atrophy such as starvation and muscle
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disuse are thought to contribute to FOXO activation. However, increasing evidence demonstrate
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FOXO-signaling to be controlled via additional post-translational modifications and protein-
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protein interactions which are distinct from Akt-mediated phosphorylation (19, 51). Yet, many of
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these additional regulatory mechanisms have yet to be thoroughly explored in skeletal muscle. If
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similar control mechanisms indeed exist in skeletal muscle to modulate FOXO activity, this
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could potentially open up new avenues for therapeutically blocking FOXO function and the
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associated muscle atrophy during physiological conditions of muscle wasting.
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One such mechanism of FOXO regulation identified in multiple cell types, involves the
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regulation of FOXO-dependent transcription by the histone acetyltransferase (HAT) proteins,
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p300 and CREB binding protein (CBP) (11, 14, 35). These HAT proteins each possess an
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intrinsic acetyltransferase activity which catalyzes the transfer of an acetyl group to specific
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lysine residues on target proteins (10, 11, 32). Although HATs are most well known for
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regulating gene transcription through histone acetylation and relaxation of chromatin structure at
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gene promoters (12, 31), HATs also play an important role in regulating the activity of a variety
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of transcription factors, including p53, MyoD, HIF-1alpha, as well as the FOXO transcription
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factors (47, 52). HATs may regulate transcription factor activity through various mechanisms
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which include interaction and recruitment of factors to target gene promoters, via acting as
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adaptor molecules facilitating protein-protein interactions, and through direct acetylation of
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transcription factors or other necessary co-factors which thereby alter transcription factor activity
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(25). Evidence for HAT-mediated regulation of the FOXO transcription factors can be found in
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multiple cell types. Interestingly however, the resulting effect of HATs on FOXO appears to be
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cell-type specific and/or specific to the FOXO homologue.
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FOXO1-dependent transcription from the IGFBP-1 promoter reporter in H4IIE rat hepatoma
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cells, which requires p300 HAT activity (35). Similarly, p300 increases FOXO3a-dependent
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transcription from the Bim promoter in human embryonic kindey cells (HEK293T) (34). In
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contrast, p300 represses FOXO4-induced transcription of GADD45, p27, p21 and MnSOD in
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HEK293 cells (11). Therefore depending on the cell type, FOXO homologue, and target gene
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measured, HATs may either repress or activate FOXO-induced transcription, which may reflect
For example, p300 increases
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an important fine-tuning mechanism of FOXO-target gene regulation. However, despite the
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importance of understanding the mechanisms which lead to FOXO-dependent transcription in
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skeletal muscle due to its known role in causing muscle atrophy, no data currently exist to
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suggest whether the acetyltransferase activities of p300/CBP regulate FOXO in skeletal muscle.
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Therefore, the purpose of the current study was to determine whether HAT proteins regulate the
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FOXO transcription factors in skeletal muscle, and whether this is altered during conditions of
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muscle wasting.
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MATERIALS AND METHODS
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Animals
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Sprague-Dawley male rats (200-225g) were ordered from Charles River Laboratories
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(Wilmington, MA, USA).
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Committee approved all animal procedures. UF is accredited by the Association for Assessment
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and Accreditation of Laboratory Animal Care (#A3377-01). Animals were maintained in a
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temperature and humidity-controlled facility with a 12-hour light/dark cycle. Water and standard
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diet were provided ad libitum.
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Plasmids and Reporter Gene Assays
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Expression plasmids for WT p300 and the dominant negative (d.n.) p300 mutant (which lacks
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acetyltransferase activity due to an inactivating point mutation, converting aspartic acid 1399 to
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tyrosine ) were obtained from Dr. Tso-Pang Yao (Duke University, Durham, NC, USA) and have
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been previously described (21). The FOXO1 expression plasmid was a gift from Dr. Akiyoshi
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Fukamizu (University of Tsukuba, Ibaraki, Japan) and has previously been used and described
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(32). The tagged FOXO1-EGFP plasmid was obtained from Addgene (plasmid 9022), and was
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deposited by Dr. Domenico Accili (Columbia University, New York, NY, USA) and has
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previously been described (13). The FOXO4 expression plasmid was a gift from Dr. Boudewijn
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Burgering (University Medical Center, Utrecht, The Netherlands) and has been previously used
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and described (53). The FOXO3a expression plasmid was obtained from Addgene (plasmid
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10710), was deposited by Dr. William Sellers (Novartis, Cambridge, MA) and has been
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previously used and described (37). The WT FOXO3a-DsRed fusion construct was created via
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PCR amplification of the FOXO3a cDNA out of the parent vector using primers to create
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HindIII and SalI restriction sites on the 5’ and 3’ ends of the FOXO3a coding region,
The University of Florida Institutional Animal Care and Use
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respectively. FOXO3a cDNA was then sub-cloned, in frame, into the DsRed2-c1 plasmid.
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Verification that FOXO3a cDNA was in frame was confirmed via DNA sequencing (DNA
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Sequencing Core, University of Florida). The d.n.Akt and c.a.Akt expression plasmids were
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obtained from Addgene (plasmids 12643 & 16244, respectively) and were deposited by Dr.
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Mien-Chie Hung (The University of Texas, M. D. Anderson Cancer Center, Houston, TX) and
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have previously been described (58). The DAF-16/FOXO responsive reporter plasmid, the
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atrogin-1-GL2 promoter reporter plasmid and the d.n.FOXO construct have also been previously
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used and described (46). pRL-TK-Renilla was purchased from Promega. Plasmid DNA was
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amplified and isolated from bacterial cultures using Endotoxin-Free Maxi or Mega Prep Kits
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(Qiagen, Valencia, CA, USA), precipitated in ethanol and re-suspended in 1X sterile filtered
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phosphate buffered saline (PBS) for in vivo transfections, or Tris-EDTA (TE) buffer for
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transfections in culture.
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In vivo, Plasmid Injection and Electroporation.
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Transfection of plasmid DNA into skeletal muscle, in vivo, has been detailed previously (22, 46).
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For rat experiments, 10μg each of the expression or control plasmid(s) and 40μg of the reporter
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plasmid were diluted in a total of 50μl 1XPBS for each solei injection. Standard procedures
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were used to determine luciferase activity on skeletal muscle homogenates using a Modulus
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single tube multimode reader (Promega) and have been described previously (46).
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Animal Models and Muscle Preparation
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Disuse muscle atrophy via cast immobilization of both hind limbs was induced in rats four days
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following plasmid injection and has been detailed previously (46).
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immobilization or weight bearing activity soleus muscles were removed and processed either
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immediately for RNA isolation or frozen in liquid nitrogen and stored at -80°C until further
After three days of
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biochemical analyses. For experiments using exclusively genetic manipulations, muscles were
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harvested 7 days post-plasmid injection.
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Cell Culture Experiments
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C2C12 cells were cultured on 0.1% gelatin coated 6-well plates in high-glucose DMEM
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(Invitrogen), 10% fetal bovine serum and 5% CO2. Muscle cells were transfected with plasmid
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DNA at ~80% confluence using FuGENE® HD Transfection Reagent (Promega Corp, Madison,
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WI, USA) at a 3.5:1 ratio of reagent to total DNA. Sixteen hours following transfection muscle
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cells were differentiated into myotubes by incubation in differentiation medium (2% Horse
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Serum in DMEM). For dexamethasone studies, 6-day differentiated myotubes were treated with
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either vehicle (water) or 1uM water-soluble dexamethasone (Sigma, St. Louis, MO, USA) in
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differentiation media for 6 hours and harvested in Passive Lysis Buffer (Promega). In the
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nutrient deprivation groups, differentiation media was removed from 4-day differentiated cells
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and Hanks Balanced Salt Solution (HBSS) added for either 2 hours (localization experiments) or
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6 hours (reporter assays and gene expression) prior to harvest. To inhibit PI3Kinase, 10μM
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LY294002 (Calbiochem, Merck KGaA, Darmstadt, Germany) or vehicle (ethanol) was added to
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4-day differentiated myotubes for 6 hours. For reporter experiments, cells were harvested in
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Passive Lysis Buffer (Promega) and luciferase activity determined by normalizing firefly
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luciferase activity to pRL-TK Renilla luciferase activity using a Dual-Luciferase® Reporter
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Assay (Promega).
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RNA isolation, cDNA synthesis, and RT-PCR
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RNA isolation and cDNA synthesis from whole muscle was performed using a Trizol-based
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method as previously described (46). RNA isolation from C2C12 myotubes was performed
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similarly, following addition of 250uL Trizol per well and vigorous scraping, as previously
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described (33) . cDNA was generated from 1ug of RNA and was used as a template for qRT-
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PCR using primers for atrogin-1, GenBank NM_133521; MuRF1, GenBank NM_080903;
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Cathepsin-L, GenBank NM_013156; 4E-BP1, GenBank NM_053857; LC3b, GenBank
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NM_022867; p21, Genbank NM_080782; Gadd45α, GenBank NM_024127; Foxo1, GenBank
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NM_001191846; Foxo3a, GenBank NM_001106395; Foxo4, GenBank NM_001106943; or 18S,
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GenBank X03205.1, which were ordered from Applied Biosystems (Austin, TX, USA). TaqMan
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probe-based chemistry was used to allow detection of PCR products using a 7300 real-time PCR
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system (Applied Biosystems), and quantification of gene expression was performed using the
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relative standard curve method.
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Western Blotting and Co-Immunoprecipitation Assays
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Preparation of muscle homogenates and western blotting were performed according to standard
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procedures and have been described previously (46). Primary antibodies for p300 (#554215, BD
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Pharmingen, San Jose, CA, USA), FOXO1 (#9454S, Cell Signaling Technology, Boston, MA,
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USA); phospho-FOXO1 (Ser256) (#9461, Cell Signaling Technology); FOXO3a (SC-11351,
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Santa Cruz Biotechnology, Santa Cruz, CA, USA); phospho-FOXO3a (Thr32) (SC-12357, Santa
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Cruz Biotechnology) and FOXO4 (07-1720, Millipore, Billerica, MA), were used according to
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manufacturer’s directions.
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Louis, MO, USA) was used to control for equal protein loading and protein transfer. For co-
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immunoprecipitation assays proteins, 500ug of muscle protein were incubated overnight with
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either 4ug of Anti-Acetyl Lysine antibody (#05-515) or 4ug of anti-p300 (#05-257) using a
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Catch and Release Reversible Immunoprecipitation System (#17-500), all from Millipore. The
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following day, precipitated proteins were washed and subsequently eluted in denaturing buffer,
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boiled, and western blotting performed for endogenous FOXO3a and FOXO1.
Tubulin primary antibody, (T6074 from Sigma-Aldrich Inc, St.
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Fluorescent Microscropy
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C2C12 myoblasts were seeded on 6-well plates containing 0.1% gelatin-coated glass coverslips,
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transfected and differentiated for 4 days. Following treatment, cells were rinsed with PBS and
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fixed for 30 minutes in 4% paraformaldehyde. Following three washes in PBS, two drops of
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Vectashield Mounting Medium for Fluorescence, with Dapi, (#H-1200, Vector Laboratories, Inc,
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Burlingame, CA, USA) was added to each coverslip. A Leica DM5000B microscope (Leica
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Microsystems Inc., Bannockburn, IL USA) containing GFP (green) and Rhodamine (red) filter
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cubes was used to visualize FOXO1-EGFP or FOXO3a-DsRed positive myotubes, respectively.
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A Dapi (blue) filter was used to visualize Dapi-stained nuclei. Images were captured and merged
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using Leica Application Suite, version 3.5.0.
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Statistics
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Data were analyzed using a two-way ANOVA followed by Bonferroni post-hoc comparisons
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when appropriate (GraphPad Software, San Diego, CA). All data are expressed as the mean ±
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SEM, and significance was set at P
