Abstract
Ligand activation of peroxisome proliferator-activated receptors (PPARs) prevents cardiac myocyte hypertrophy, and we previously reported that diacylglycerol kinase zeta (DGKζ) is critically involved. DGKζ is an intracellular lipid kinase that catalyzes phosphorylation of diacylglycerol; by attenuating DAG signaling, DGKζ suppresses protein kinase C (PKC) and G-protein signaling. Here, we investigated how PPAR-DGKζ signaling blocks activation of the hypertrophic gene program. We focused on export of histone deacetylase 5 (HDAC5) from the nucleus, a key event during hypertrophy, since crosstalk occurs between PPARs and other members of the HDAC family. Using cardiac myocytes isolated from Sprague-Dawley rats, we determined that liganded PPARs disrupt endothelin-1 (ET1)-induced nuclear export of HDAC5 in a manner that is dependent on DGKζ. When DGKζ-mediated PKC inhibition was circumvented using a constitutively-active PKCε mutant, PPARs failed to block ET1-induced nuclear retention of HDAC5. Liganded PPARs also prevented (i) activation of protein kinase D (the downstream effector of PKC), (ii) HDAC5 phosphorylation at 14-3-3 protein chaperone binding sites (serines 259 and 498), and (iii) physical interaction between HDAC5 and 14-3-3, all of which are consistent with blockade of nucleo-cytoplasmic shuttling of HDAC5. Finally, the ability of PPARs to prevent neutralization of HDAC5 activity was associated with transcriptional repression of hypertrophic genes. This occurred by first, reduced MEF2 transcriptional activity and second, augmented deacetylation of histone H3 associated with hypertrophic genes expressing brain natriuretic peptide, β-myosin heavy chain, skeletal muscle α-actin, and cardiac muscle α-actin. Our findings identify spatial regulation of HDAC5 as a target for liganded PPARs, and to our knowledge, are the first to describe a mechanistic role for nuclear DGKζ in cardiac myocytes. In conclusion, these results implicate modulation of HDAC5 as a mechanism by which liganded PPARs suppress the hypertrophic gene program.
Highlights
Cardiac hypertrophy is the increase in myocardial mass provoked by hemodynamic stress or myocardial injury, and is a convergence point for many risk factors leading to heart failure
Because protein kinase C (PKC)/protein kinase D (PKD) activity opposes the anti-hypertrophic actions of histone deacetylase 5 (HDAC5) [16], and liganded peroxisome proliferator-activated receptors (PPARs) suppress myocyte hypertrophy by interfering with PKC signaling [24], we considered whether PPAR activation might preserve HDAC5 function
Liganded PPARs suppressed ET1-dependent histone acetylation, and again, these PPAR actions were abolished by small hairpin RNAs (shRNA) knockdown of DGKf. These findings provide insight into the mechanisms by which liganded PPARs suppress hypertrophic gene activation in cardiac myocytes
Summary
Cardiac hypertrophy is the increase in myocardial mass provoked by hemodynamic stress or myocardial injury, and is a convergence point for many risk factors leading to heart failure. Attenuation of hypertrophy is a promising therapeutic target to prevent heart failure. At the cardiac myocyte level, hypertrophy is characterized by increases in cell size, protein synthesis, and changes in gene expression [3]. The latter includes sequential activation of immediate early response genes (e.g. proto-oncogenes such as c-fos and c-jun), a fetal gene program (e.g. atrial natriuretic peptide, skeletal muscle a-actin, and b-myosin heavy chain), and sarcomeric genes (e.g. cardiac muscle a-actin) [3]. Re-induction of fetal genes such as brain natriuretic peptide (BNP) is one of the most consistent markers of transcriptional activation in hypertrophy [4]
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