Selective class II HDAC inhibitors impair myogenesis by modulating the stability and activity of HDAC-MEF2 complexes - PubMed (original) (raw)

doi: 10.1038/embor.2009.88. Epub 2009 Jun 5.

Fabio Manzo, Marco Miceli, Mariarosaria Conte, Lucrezia Manente, Alfonso Baldi, Antonio De Luca, Dante Rotili, Sergio Valente, Antonello Mai, Alessandro Usiello, Hinrich Gronemeyer, Lucia Altucci

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Selective class II HDAC inhibitors impair myogenesis by modulating the stability and activity of HDAC-MEF2 complexes

Angela Nebbioso et al. EMBO Rep. 2009 Jul.

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Abstract

Histone deacetylase (HDAC) inhibitors are promising new epi-drugs, but the presence of both class I and class II enzymes in HDAC complexes precludes a detailed elucidation of the individual HDAC functions. By using the class II-specific HDAC inhibitor MC1568, we separated class I- and class II-dependent effects and defined the roles of class II enzymes in muscle differentiation in cultured cells and in vivo. MC1568 arrests myogenesis by (i) decreasing myocyte enhancer factor 2D (MEF2D) expression, (ii) by stabilizing the HDAC4-HDAC3-MEF2D complex, and (iii) paradoxically, by inhibiting differentiation-induced MEF2D acetylation. In vivo MC1568 shows an apparent tissue-selective HDAC inhibition. In skeletal muscle and heart, MC1568 inhibits the activity of HDAC4 and HDAC5 without affecting HDAC3 activity, thereby leaving MEF2-HDAC complexes in a repressed state. Our results suggest that HDAC class II-selective inhibitors might have a therapeutic potential for the treatment of muscle and heart diseases.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

Figure 1

Enzymatic inhibition of class specific histone deacetylase inhibitors. (A) The chemical structures of suberoyl anilide hydroxamic acid (SAHA), MS275 and MC1568. (B) Left: cell-based histone deacetylase (HDAC)4 and HDAC1 assay with HDAC inhibitors (all used at 5 μM, except valproic acid (VPA), which was used at 1 mM); right: in vitro HDAC4 and HDAC1 assay with HDAC inhibitors on histone substrate (all used at 5 μM, except VPA, which was used at 1 mM). (C) In vitro HDAC2, 3, 5 and 6 assay with HDAC inhibitors (all used at 5 μM, except VPA, which was used at 1 mM on histone substrate). (D) HDAC4 and HDAC6 assay on the substrate trifluoroacetyl-lysine and the specific HDAC6 substrate. DPM, distintegrations per minute; IP, immunoprecipitation; RFU, rate fluorescence unit; rhHDAC, recombinant human histone deacetylase.

Figure 2

Figure 2

MC1568 stabilize the MEF2–HDAC4–HDAC3 complex and blocks myogenesis of C2C12 cells. (A) Western blot of myogenin and αMHC in the absence and presence of differentiation medium (DM) with or without MC1568 added to the DM at the start of differentiation (lane 3) or after 24 h (lane 4); HSP70 indicates equal loading. (B) Western blot of MEF2D and HDAC4 expression in C2C12 cells; α-tubulin (Tub) indicates equal loading. (C) Immunoprecipitation (IP) assays using MEF2D antibodies for MEF2D IPs and HDAC4 co-IPs in C2C12 cells. Acetyl-MEF2D (AcMEF2D) levels were revealed with antibodies against acetylated lysines. (D) IPs using MEF2D and HDAC3 antibodies to show MEF2D, HDAC3 and HDAC4 complexes in differentiating C2C12 cells with or without treatment with MC1568; note that IgG-negative control IP did not show detectable bands. HDAC, histone deacetylase; HSP70, heat-shock protein 70; MEF2, myocyte enhancer factor 2; αMHC, α-myosin heavy chain.

Figure 3

Figure 3

MC1568 blocks MEF2D transcriptional activity in C2C12 cells and shows inhibitory activities in mice. (A) Histone deacetylase (HDAC) assay on myocyte enhancer factor 2D (MEF2D) immunoprecipitation (IP) from C2C12 cells in the presence of MC1568 (5 μM) with or without valproic acid (VPA; 1 mM); the inset shows the quantities of co-immunoprecipitated MEF2D, HDAC4 and HDAC3. (B) Chromatin immunoprecipitation (ChIP) assay of acetyl-H3 (AcH3) levels on the myogenin promoter in C2C12 cells. (C) ChIP assay of MEF2D on the myogenin (Myo) and muscle creatine kinase (MCK) promoters in C2C12 cells. (D) MC1568 increases acetylation of tubulin (AcTub) in selected organs of mice in a dose-dependent manner. (E) HDAC4 and HDAC5 expression levels in skeletal muscle and heart after MC1568 treatment (50 mg/kg); note that although differentially expressed both HDACs are present. Bottom: IPs using HDAC4 and HDAC5 antibodies to reveal MEF2D and HDAC3 complexes in skeletal muscle and heart. (F) Heart-extract-HDAC5 immunoprecipitation assay on treatment with 1, 10 and 50 mg/kg MC1568. (G) HDAC assay from heart extracts on MEF2D IP in the presence of MC1568 with or without 1 mM VPA. DM, differentiation medium; DPM, disintegrations per minute; ERK, extracellular signal regulated kinase.

Figure 4

Figure 4

Regulatory complexes formed with MEF2 at responsive genes. Myocyte enhancer factor 2 (MEF2) recruits class II histone deacetylases (HDACs) such as HDAC4 to responsive genes, resulting in repression. HDAC4 binds to HDAC3 on a co-repressor (nuclear receptor corepressor (NCOR)/silencing mediator of retinoic acid and thyroid hormone receptor (SMRT)) platform. Differentiation medium (DM) results in the dissociation of the MEF2–HDAC4 interaction, allowing association of a co-activator complex and target gene expression, as CREB (cyclic AMP response element binding)-binding protein (CBP)/p300 and HDAC4 bindings to MEF2 are mutually exclusive. The miR-1 achieves the same effect by down regulating HDAC4. HDAC inhibition does not result in the same events as the removal of HDAC4. Pan-histone decetylase inhibitors (HDACi) block both HDAC3 and HDAC4 and, depending on the stage of myogenesis, might block or stimulate differentiation. By contrast, MC1568 blocks HDAC4 activity but enhances the HDAC4–MEF2 interaction, thus resulting in enforced repression. SAHA, suberoyl anilide hydroxamic acid; TSA, tricostatin A.

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