ABSTRACT
Reversible proline-directed phosphorylation at Ser/Thr-Pro motifs has an essential role in myogenesis, a multistep process strictly regulated by several signaling pathways that impinge on two families of myogenic effectors, the basic helix-loop-helix myogenic transcription factors and the MEF2 (myocyte enhancer factor 2) proteins. The question of how these signals are deciphered by the myogenic effectors remains largely unaddressed. In this study, we show that the peptidyl-prolyl isomerase Pin1, which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds to induce conformational changes of its target proteins, acts as an inhibitor of muscle differentiation because its knockdown in myoblasts promotes myotube formation. With the aim of clarifying the mechanism of Pin1 function in skeletal myogenesis, we investigated whether MEF2C, a critical regulator of the myogenic program that is the end point of several signaling pathways, might serve as a/the target for the inhibitory effects of Pin1 on muscle differentiation. We show that Pin1 interacts selectively with phosphorylated MEF2C in skeletal muscle cells, both in vitro and in vivo. The interaction with Pin1 requires two novel critical phospho-Ser/Thr-Pro motifs in MEF2C, Ser(98) and Ser(110), which are phosphorylated in vivo. Overexpression of Pin1 decreases MEF2C stability and activity and its ability to cooperate with MyoD to activate myogenic conversion. Collectively, these findings reveal a novel role for Pin1 as a regulator of muscle terminal differentiation and suggest that Pin1-mediated repression of MEF2C function could contribute to this function.
Subject(s)
Cell Proliferation , Muscle Development/physiology , Muscle Fibers, Skeletal/metabolism , Myogenic Regulatory Factors/metabolism , Peptidylprolyl Isomerase/metabolism , Signal Transduction/physiology , Animals , COS Cells , Cell Line , Chlorocebus aethiops , Humans , MEF2 Transcription Factors , Mice , MyoD Protein/genetics , MyoD Protein/metabolism , Myogenic Regulatory Factors/genetics , NIMA-Interacting Peptidylprolyl Isomerase , Peptides/genetics , Peptides/metabolism , Peptidylprolyl Isomerase/genetics , Phosphorylation/physiology , Protein StabilityABSTRACT
Myocyte enhancer factor 2 (MEF2) proteins play a key role in promoting the expression of muscle-specific genes in differentiated muscle cells. MEF2 activity is regulated by the association with several transcriptional co-factors and by post-translational modifications. In the present report, we provide evidence for a novel regulatory mechanism of MEF2C activity, which occurs at the onset of skeletal muscle differentiation and is based on Lys4 acetylation. This covalent modification results in the enhancement of MEF2C binding to DNA and chromatin. In particular, we report that the kinetic parameters of MEF2/DNA association change substantially upon induction of differentiation to give a more stable complex and that this effect is mediated by Lys4 acetylation. We also show that Lys4 acetylation plays a prominent role in the p300-dependent activation of MEF2C.
Subject(s)
Lysine/metabolism , Muscle, Skeletal/metabolism , Myogenic Regulatory Factors/metabolism , Protein Processing, Post-Translational , Acetylation , Amino Acid Sequence , Animals , Binding Sites , Cell Differentiation , Cell Line , Chromatin/metabolism , DNA/metabolism , Electrophoretic Mobility Shift Assay , Fluorescence Recovery After Photobleaching , MEF2 Transcription Factors , Mice , Molecular Sequence Data , Muscle, Skeletal/cytology , Myogenic Regulatory Factors/chemistry , Myogenin/genetics , Protein Binding , Regulatory Elements, Transcriptional , Transcriptional Activation , p300-CBP Transcription Factors/metabolismABSTRACT
Alternative splicing of the locus AbetaH-J-J generates three functionally distinct proteins: an enzyme, AbetaH (aspartyl-beta-hydroxylase), a structural protein of the sarcoplasmic reticulum membrane (junctin), and an integral membrane calcium binding protein (junctate). Junctin and junctate are two important proteins involved in calcium regulation in eukaryotic cells. To understand the regulation of these two proteins, we identified and functionally characterized one of the two promoter sequences of the AbetaH-J-J locus. We demonstrate that the P2 promoter of the AbetaH-J-J locus contains (i) a minimal sequence localized within a region -159 bp from the transcription initiation site, which is sufficient to activate transcription of both mRNAs; (ii) sequences which bind known transcriptional factors such as those belonging to the myocyte enhancer factor 2 (MEF-2), MEF-3, and NF-kappaB protein families; and (iii) sequences bound by unknown proteins. The functional characterization of the minimal promoter in C2C12 cells and in the rat soleus muscle in vivo model indicates the existence of cis elements having positive and negative effects on transcription. In addition, our data demonstrate that in striated muscle cells the calcium-dependent transcription factor MEF-2 is crucial for the transcription activity directed by the P2 promoter. The transcription directed by the AbetaH-J-J P2 promoter is induced by high expression of MEF-2, further stimulated by calcineurin and Ca2+/calmodulin-dependent protein kinase I, and inhibited by histone deacetylase 4.
Subject(s)
Calcium-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Membrane Proteins/genetics , Mixed Function Oxygenases/genetics , Muscle Proteins/genetics , Promoter Regions, Genetic/genetics , Transcription Factors/physiology , Transcriptional Activation , Alternative Splicing , Animals , Base Sequence , Calcineurin/metabolism , Calcineurin/physiology , Calcium-Binding Proteins/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Calcium-Calmodulin-Dependent Protein Kinases/physiology , Cells, Cultured , Chromatin/metabolism , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , Histone Deacetylases/metabolism , Histone Deacetylases/physiology , Humans , MEF2 Transcription Factors , Male , Membrane Proteins/metabolism , Mixed Function Oxygenases/metabolism , Molecular Sequence Data , Muscle Cells/metabolism , Muscle Proteins/metabolism , Muscle, Skeletal/metabolism , Myogenic Regulatory Factors , NF-kappa B/metabolism , NF-kappa B/physiology , Rats , Repressor Proteins/metabolism , Repressor Proteins/physiology , Sequence Deletion , Transcription Factors/antagonists & inhibitors , Transcription Factors/metabolism , Transcription Initiation SiteABSTRACT
A soluble form of human low-density lipoprotein receptor (LDL-R) fused in frame with rabbit transferrin (LDL-Rs(hu)/Tf(rab)) is assessed in vivo as a therapeutic tool for lowering plasma LDL cholesterol. The cDNA encoding LDL-Rs(hu)/Tf(rab) is expressed in mice, using a hydrodynamics-based gene transfer procedure. The transgene is transcribed in the liver of transduced animals and the corresponding protein is secreted into the bloodstream. Circulating LDL-Rs(hu)/Tf(rab) binds LDL specifically, thus indicating that it is correctly processed through the cellular compartments in vivo. More importantly, the expression of LDL-Rs(hu)/Tf(rab) allows the removal of injected human (125)I-labeled LDL ((123)I-LDL) from the bloodstream of transduced CD1 mice, which show faster LDL plasma clearance, anticipating by approximately 90 min the same clearance value observed in control animals. A similar effect is observed in transduced LDL-R(-/-) mice, in which the clearance of injected human LDL depends solely on the presence of circulating LDL-Rs(hu) /Tf(rab). In these animals the extent of plasma LDL clearance is directly related to the concentration of LDL-Rs(hu)/Tf(rab) in the blood. Finally, LDL-Rs(hu)/Tf(rab) does not alter the pattern of LDL organ distribution: in transduced animals, as well as in control animals, liver and bladder are the predominantly labeled organs after (123)I-LDL injection. However, LDL-Rs(hu)/Tf(rab) has a quantitative effect on LDL tissue deposition: in treated animals LDL-Rs(hu)/Tf(rab) determines an increase in radioactivity in the liver at early times after (123)I-LDL injection and a progressive labeling of the bladder, starting 20 min after injection.