Proteasome-mediated degradation of the coactivator p300 impairs cardiac transcription

Abstract

The transcription of tissue-specific genes is controlled by regulatory factors and cofactors and is suppressed in cardiac cells by the antineoplastic agent doxorubicin. Here we show that exposure of cultured cardiomyocytes to doxorubicin resulted in the rapid depletion of transcripts for MEF2C, dHAND, and NKX2.5, three pivotal regulators of cardiac gene expression. Delivery of exogenous p300, a coactivator of MEF2C and NKX2.5 in cardiomyocytes, restored cardiac transcription despite the presence of doxorubicin. Furthermore, p300 also restored the accumulation of transcripts for MEF2C itself. Importantly, cardiocytes exposed to doxorubicin displayed reduced levels of p300 proteins. This was not due to alterations in the level of p300 transcripts; rather, and surprisingly, doxorubicin promoted selective degradation of p300 mediated by the 26S-proteasome machinery. Doxorubicin had no effect on the general level of ubiquitinated proteins or on the levels of beta-catenin, a protein known to be degraded by proteasome-mediated degradation. These results provide evidence for a new mechanism of transcriptional repression caused by doxorubicin in which the selective degradation of p300 results in reduced p300-dependent transcription, including production of MEF2C mRNA.

FIG. 1

Inhibition of expression of cardiac transcription factors in neonatal cardiomyocytes treated with doxorubicin. (A) Doxorubicin inhibits MEF2C mRNA in cardiocytes. Quantitative RT-PCR analysis of MEF2C and dHAND in control (CT) and doxorubicin-treated cardiocytes. Total RNA (2 μg) from either control neonatal rat cardiocytes or cardiocytes exposed to 1 μM doxorubicin for 24 or 48 h was reverse transcribed and PCR amplified with gene-specific primers for the indicated transcripts. PCR products were separated on a 2% agarose gel and quantitated with AlphaImager software. In all PCRs, the level of expression of the gene of interest was normalized against 18S cDNA. Each experiment was repeated at least twice with RNA template from two independent cardiocyte preparations. (B) Doxorubicin downregulates NKX2.5 transcription in cardiocytes. Northern blot analysis of NKX2.5 transcription in control (CT) cardiocytes and cardiocytes exposed to doxorubicin (Dox) for 48 h. Cardiac troponin I (cTnI) was measured in parallel to ensure that doxorubicin treatment was effective. The methylene blue staining of the blot shows 28S and 18S RNA and indicates that an equal amount of RNA was loaded in each lane. (C) Doxorubicin inhibits MEF2C transcriptional activity in cardiocytes. Neonatal rat cardiocytes were transfected with 2 μg of 2xA/Temb-CAT plasmid DNA, which contains two copies of the MEF2 binding site from the embryonic myosin heavy-chain enhancer placed upstream of a CAT reporter gene, and the cells were then were exposed to 1 μM doxorubicin (Dox). CAT activity was measured after 48 h of drug exposure as described in the text and corrected for protein content. The data represent the mean ± standard deviation of three independent experiments carried out in triplicate.

FIG. 2

(A) p300 coactivates MEF2C in cardiocytes. Rat neonatal cardiocytes were cotransfected with 2 μg of the 2xA/Temb-CAT construct and with increasing amounts of a plasmid encoding either full-length p300 or just the CMV backbone. In all transfections, the total amount of DNA was kept constant. CAT activity measurements represent the means of three independent experiments carried out in triplicate and normalized to protein content. (B and C) Stimulation of MEF2C transcriptional activity by p300 is dependent on the MADS domain of MEF2C. Neonatal cardiocytes were cotransfected with a GAL4 construct fused to full-length MEF2C [Gal-MEF2C(1-465)] or with a construct lacking the MADS domain [Gal-MEF2C(247-327)] but retaining transcriptional activity and with increasing concentration of CMV-p300. Transcriptional activity was measured 48 h later on a GAL4-dependent reporter construct, G5E1b-luciferase. (D and E) MEF2C binds p300 in cardiocytes. A binding assay was performed with a DNA fragment containing an MEF2 binding site derived from the muscle creatine kinase promoter and with nuclear extracts prepared from cardiac cells. A major complex corresponding to the binding of MEF2C protein to the DNA template was observed (D). The binding reaction was specific to MEF2C, since an antibody specifically recognizing MEF2C resulted in a further reduction in the mobility of the complex (supershift). An antibody directed against large T antigen (TAg) had no effect. The binding of p300 with MEF2C was detected in cardiac cells, since the complex was supershifted by an anti-p300 antibody (E).

FIG. 3

(A and B) Disruption of p300 function inhibits MEF2C activity. Neonatal cardiocytes were cotransfected with 2 μg of 3xMEF2-luciferase construct or GAL-MEF2C(1-465) and with the indicated amount of expression vectors. After 48 h, transcriptional activity was measured in the transfected cells. Each value represents the mean ± standard deviation (SD) of three independent experiments carried out in triplicate and normalized to protein content. (C) p300 enhances NKX2.5-dependent transcription. Neonatal cardiocytes were transiently transfected with plasmids encoding either or both full-length NKX2.5 or p300 plus the ANF promoter. Luciferase activity was measured 24 h later and corrected for protein content. The data represent the mean ± SD of three independent experiments carried out in triplicate. (D) Overexpression of p300 rescues MEF2C inhibition by doxorubicin. Control cardiocytes or cardiocytes exposed to 1 μM doxorubicin (Dox) were cotransfected with 2 μg of 2xA/Temb-CAT and with increasing concentrations of CMV-p300 or CMV backbone. CAT activity was measured 48 h after transfection. The total amount of DNA was kept constant in all transfections. The data represent the mean ± SD of three independent experiments carried out in triplicate. (E) Forced expression of p300 in doxorubicin-treated cardiocytes upregulates endogenous MEF2C. Neonatal cardiocytes untreated or treated with 1 μM doxorubicin (Dox) were cotransfected with a plasmid expressing GFP and CMV-p300 or CMV alone. At 72 h after transfection, GFP-positive cells were sorted by FACS. Total RNA was extracted from the transfected cells, and the relative amounts of MEF2C and p300 mRNAs were determined by RT-PCR. 18S RNA mixed with competimers was used as an internal control. This experiment was done twice with two independent cardiac cell preparations.

FIG. 4

p300 protein levels but not mRNA levels are decreased by doxorubicin. (A) Nuclear extracts (100 μg) from neonatal cardiomyocytes cultured in the absence (control, CT) or presence of 1 μM doxorubicin (Dox) for 48 h were electrophoresed on a 4 to 20% gradient gel. After transfer of the proteins, the membranes were incubated with a primary antibody directed against p300 or p53 overnight at 4°C. After incubation with a secondary antibody, visualization of antigen-antibody reaction was performed with a chemiluminescent reagent. ∗, unknown protein not sensitive to doxorubicin. (B) Nuclear extracts (100 μg) from control and doxorubicin-treated cardiocytes, C₂C₁₂ cells, and 293 cells were incubated with an anti-p300 antibody (power clonal [UBI] or N-15X [Santa Cruz]). The extracts were then preadsorbed on protein A/G PLUS-agarose and washed. After centrifugation, the supernatant was analyzed by SDS-PAGE on a 5% polyacrylamide gel. After transfer of the proteins, membranes were blocked and incubated with anti-p300 antibody. Immunocomplexes were detected by chemiluminescence. (C) Relative quantitative RT-PCR of p300 in control and doxorubicin-treated cardiocytes and C₂C₁₂ cells. Total RNA (2 μg) was reverse transcribed and PCR amplified with specific primers for p300. PCR products were separated on a 2% agarose gel and quantitated with AlphaImager software. In all PCRs, the level of expression of p300 was normalized against 18S cDNA. Each experiment was repeated at least twice with RNA template from two independent cardiocyte preparations.

FIG. 5

Doxorubicin increases p300 degradation and inhibitors of the proteasome stabilize p300 protein and prevent p300 degradation by doxorubicin. (A) Neonatal cardiomyocytes were treated with 10 μM cycloheximide for 5 h to inhibit general protein synthesis. Half of the cells were exposed to doxorubicin. Cycloheximide was always present in the medium. Nuclear proteins (30 μg) were separated by SDS-PAGE on a 4 to 20% gradient gel. After transfer of the proteins, the levels of p300 protein were determined by Western blot analysis using an anti-p300 antibody. (B) Relative amounts of p300 protein were determined using an AlphaImager. □, p300 protein level in cardiocytes treated with cycloheximide only; ◊, p300 protein level in cardiocytes treated with cycloheximide plus doxorubicin. (C) Coomassie staining of the nuclear proteins separated by SDS-PAGE. (D) Neonatal cardiomyocytes exposed to 10 μM cycloheximide were treated with DMSO or with 30 μM MG-132 proteasome inhibitor for the indicated times. Nuclear extracts were prepared and resolved on a 4 to 20% gradient gel. Following transfer of the protein to nitrocellulose membranes, p300 levels were determined by Western blot using an anti-p300 antibody. (E) Two different inhibitors of the proteasome stabilize p300 protein. Cardiomyocytes treated with cycloheximide were coincubated with 30 μM MG-132 or 30 μM PSI. Nuclear extracts were prepared before the addition of inhibitors of the proteasome (time zero) or after 16 or 24 h of incubation. Proteins were analyzed on a 4 to 20% gradient gel, and p300 expression was detected by Western blot using an anti-p300 antibody. These experiments were repeated three times with three independent cardiocyte preparations. (F) Neonatal cardiocytes were exposed to 1 μM doxorubicin. Half the cells were also exposed to MG-132. Nuclear extracts were prepared after various times of incubation and resolved on a 4 to 20% gradient gel. Following transfer of the protein on nitrocellulose membranes, p300 levels were determined by Western blot using an anti-p300 antibody. (G) Quantitative analysis of experiment C. □, p300 protein level in cardiocytes treated with doxorubicin only; ◊, p300 protein level in cardiocytes treated with doxorubicin plus MG-132.

FIG. 6

Proteasome-mediated degradation of p300 is a specific process. Neonatal cardiocytes were maintained in culture in doxorubicin-free medium (CT), in medium containing 10 μM MG-132, in medium supplemented with 1 μM doxorubicin (Dox), or in medium containing both doxorubicin and MG-132 for 48 h. Equal amounts of nuclear extracts were electrophoresed on 4 to 20% gradient gels. After transfer of the proteins, ubiquitinated proteins were detected with an antiubiquitin antibody (A). β-Catenin levels were determined by Western blot using an anti-β-catenin antibody (B and C).

FIG. 7

Hypothetical mechanism of tissue-specific transcriptional repression mediated by doxorubicin. A tissue-specific transcription factor complex (A) comprising tissue-specific and general transcription factors binds to cognate DNA enhancer sites, including bHLH factors and MEF2C. These transcription factors are capable of acting as a scaffold for the coactivator p300; the stoichiometry of the factors depicted is purely hypothetical. The curved line represents the chromosomal DNA, and the arrow marks the start of transcription. Doxorubicin exposure rapidly leads to degradation of p300 via the proteasome pathway (B). Doxorubicin also more slowly leads to induction of the bHLH inhibitor Id (22). As a result, p300-dependent transcription is diminished, including the transcription of the MEF2C gene itself (C), which in turn further inhibits MEF2C-dependent transcription (D). PolII, polymerase II.