Small leucine zipper protein (sLZIP) negatively regulates skeletal muscle differentiation via interaction with α-actinin-4

Abstract

The small leucine zipper protein (sLZIP) plays a role in transcriptional regulation in various types of cells. However, the role of sLZIP in myogenesis is unknown. We identified α-actinin-4 (ACTN4) as a sLZIP-binding protein. ACTN4 functions as a transcriptional regulator of myocyte enhancer factor (MEF)2, which plays a critical role in expression of muscle-specific genes during skeletal muscle differentiation. We found that ACTN4 translocates to the nucleus, induces myogenic gene expression, and promotes myotube formation during myogenesis. The myogenic process is controlled by an association between myogenic factors and MEF2 transcription factors. ACTN4 increased expression of muscle-specific proteins via interaction with MEF2. However, sLZIP decreased myogenic gene expression and myotube formation during myogenesis via disruption of the association between ACTN4 and MEF2. ACTN4 increased the promoter activities of myogenic genes, whereas sLZIP abrogated the effect of ACTN4 on transcriptional activation of myogenic genes in myoblasts. The C terminus of sLZIP is required for interaction with the C terminus of ACTN4, based on deletion mutant analysis, and sLZIP plays a role in regulation of MEF2 transactivation via interaction with ACTN4. Our results indicate that sLZIP negatively regulates skeletal muscle differentiation via interaction with ACTN4 and that sLZIP can be used as a therapeutic target molecule for treatment of muscle hypertrophy and associated diseases.

Keywords: Actinin-4; Differentiation; Gene Transcription; Muscle; Myogenesis; Transcription Repressor; sLZIP.

FIGURE 1.

Identification of ACTN4 as a novel sLZIP-binding protein. A, protein was identified from silver-stained 10% SDS-polyacrylamide gel for automated LC-MS/MS. His pulldown assay was performed using purified His-sLZIP fusion proteins (His-sLZIP) in HeLa cells, and proteins were separated by 10% SDS-PAGE. The arrowheads indicate the candidate proteins that bind to His-sLZIP. B, His-sLZIP proteins were purified using nickel-nitrilotriacetic acid beads for a His pulldown assay in the absence or presence of differentiated C2C12 nuclear extracts. Equal volumes of purified fusion proteins and pulldown proteins were separated by 10% SDS-PAGE, followed by silver staining. Pulled down proteins were analyzed by Western blotting using anti-ACTN4 antibody. Wb, Western blotting. C, amino acid sequences of ACTN4 are shown. The peptide sequences obtained by LC-MS/MS are indicated in red.

FIGURE 2.

ACTN4 enhances differentiation of C2C12 myoblasts. A and B, C2C12 cells were transfected with the ACTN4 (0, 0.25, 0.5, and 1 μg) or sh-ACTN4 (0, 0.25, 0.5 and 1 μg) plasmids, and the MCK and myogenin luciferase reporter gene plasmids (0.5 μg). The luciferase activity was determined after 48 h of transfection. The luciferase activity was normalized to β-galactosidase activity, and the experiments were performed in triplicate. Data are expressed as the mean ± S.D. (error bars) and are presented as the relative luciferase activity. C, microscopy of undifferentiated proliferating myoblasts and differentiated myotubes in control (empty vector) and ACTN4-transfected C2C12 cells is shown. Cells were observed at same magnification, and the scale bars indicate 100 μm. D, C2C12 cells transfected with the empty vector (control) and ACTN4 (1 μg) were incubated in differentiation medium (DMEM supplemented with 2% horse serum) for 4 days, and the morphology was recorded using fluorescence microscopy against anti-MyHC antibody and DAPI. The myogenic fusion index was determined at 4 days of differentiation in control and ACTN4-transfected C2C12 myoblasts. *, p < 0.05. Error bars, S.D. E, lysates from C2C12 cells cultured in differentiation medium (0, 2, and 4 days) were immunoblotted with anti-FLAG, anti-MyHC, and anti-myogenin antibodies in ACTN4- (1.5 μg) and sh-ACTN4- (1.5 μg) transfected myoblasts. All experiments were repeated at least in triplicate with similar results. F, cytosolic and nuclear fractions were prepared, and ACTN4 expression levels were determined at the indicated time points using Western blotting. Cytosolic proteins were blotted for β-actin. Nuclear fraction was blotted for proliferating cell nuclear antigen (PCNA). Differentiated cells were fixed with 4% paraformaldehyde and immunofluorescence stained with DAPI and anti-ACTN4 antibody. Arrowheads indicate ACTN4 in the nucleus.

FIGURE 3.

sLZIP suppresses expression of muscle-specific proteins and myotube formation. A, C2C12 cells were transfected with the sLZIP (0, 0.25, 0.5, and 1 μg) expression plasmid, and the MCK and myogenin luciferase reporter gene plasmids (0.5 μg). The luciferase activity was determined after 48 h of transfection. The luciferase activity was normalized to β-galactosidase activity, and the experiments were performed in triplicate. Data are expressed as the mean ± S.D. and are presented as the relative luciferase activity. B, C2C12 cells transfected with the empty vector (control) and sLZIP (1 μg) were incubated in differentiation medium for 4 days, and the morphology was recorded using fluorescence microscopy against anti-MyHC antibody and DAPI. The myogenic fusion index was determined at 4 days of differentiation in control and sLZIP-transfected C2C12 myoblasts. *, p < 0.05. Error bars, S.D. C, cell lysates of the empty vector (control), ACTN4- (1 μg), and sLZIP- (1 μg) transfected myoblasts were extracted from cells cultured in differentiation medium (0 and 3 days), and were immunoblotted. D, the myogenic fusion index was determined at 4 days of differentiation in control, sLZIP-, and ACTN4-transfected C2C12 myoblasts. All experiments were repeated at least in triplicate with similar results. *, p < 0.05; **, p < 0.01. Error bars, S.D.

FIGURE 4.

sLZIP negatively regulates transcriptional activation of MEF2 in C2C12 cells. A, the empty vector (control), ACTN4 (1 μg), and sLZIP (1 μg) expression plasmids were co-transfected with the MCK luciferase reporter gene plasmid (0.5 μg) into C2C12 cells and were assayed for luciferase activity after 48 h. CM, complete medium; DM, differentiation medium. B, C2C12 cells were transfected with 0.5 μg of MCK-Luc and sh-ACTN4 (0, 0.25, 0.5, 1 μg). C, HEK-293T cells were transfected with 0.5 μg of MCK-Luc and si-sLZIP (0, 25, 50, 100 nm). *, p < 0.05. Error bars, S.D. D, HEK-293T cells were transfected with the MEF2^x3 reporter gene plasmid, combinations of MEF2A, C, D (1 μg) and ACTN4 (1 μg) as indicated. E, HEK-293T cells were transfected with the MEF2^x3 reporter gene plasmid (0.5 μg), ACTN4 (1 μg), and sLZIP (1 μg), and MEF2 (1 μg) expression plasmids were co-transfected and were assayed for luciferase activity after 48 h. The luciferase activity was normalized to β-galactosidase activity, and the experiments were performed in triplicate. Data are expressed as the mean ± S.D. and are presented as the relative luciferase activity. F, C2C12 myoblasts were transfected with the empty vector (control), sLZIP (1 μg), and ACTN-4 (1 μg), and subjected to myogenic differentiation conditions for 3 days. Total RNA was isolated and used for reverse transcription and real-time PCR analysis. The relative expression levels were determined after normalization with β-actin level. All experiments were repeated at least three times with similar results. *, p < 0.05; **, p < 0.01. Error bars, S.D.

FIGURE 5.

The C terminus of sLZIP is required for interaction with the C terminus of ACTN4. A, HEK-293T cells were transfected with sLZIP (4 μg) and ACTN4 (4 μg), and subjected to a GST pulldown assay and Western blotting against anti-FLAG and anti-GST antibodies. B, schematic represents ACTN4 deletion mutants. HEK-293T cells were transfected with mGST-sLZIP and FLAG-ACTN4 deletion mutants and subjected to a GST pulldown assay and Western blotting against anti-FLAG and anti-GST antibodies. C, schematic represents sLZIP deletion mutants. MEF2^x3 luciferase assay was performed in HEK-293T cells. ACTN4 (1 μg) was co-transfected with sLZIP deletion mutants (1 μg) and the MEF2^x3 luciferase reporter plasmids (0.5 μg). The luciferase activity was normalized to β-galactosidase activity, and the experiments were performed in triplicate. Data are expressed as the mean ± S.D. and are presented as the relative luciferase activity. Error bars, S.D. D, HEK-293T cells were transfected with mGST-mock (2 μg), mGST-sLZIP-CC (2 μg) and FLAG-ACTN4-C (2 μg) and subjected to a GST pulldown assay and Western blotting (WB) against anti-FLAG and anti-GST antibodies. E, MEF2^x3 luciferase assay was performed in HEK-293T cells. sLZIP-CC (0.5 μg) was co-transfected with MEF2 (0.5 μg), ACTN4 deletion mutants (0.5 μg), and the MEF2^x3 luciferase reporter plasmids (0.5 μg). The luciferase activity was normalized to β-galactosidase activity, and the experiments were performed in triplicate. Data are expressed as the mean ± S.D. and are presented as the relative luciferase activity.

FIGURE 6.

sLZIP disrupts the association between MEF2D and ACTN4. A and B, HEK-293T cells were transfected with FLAG-ACTN4 (2 μg), mGST-sLZIP (2 μg), and GFP-MEF2D (2 μg) and subjected to an immunoprecipitation (IP) assay. Result shown is representative of two independent experiments. Arrowhead indicates mGST-sLZIP. C, C2C12 cells were transfected with or without mGST-sLZIP (3 μg). Cells were differentiated for 3 days and subjected to an immunoprecipitation assay using anti-ACTN4 antibody. D, schematic represents the roles of sLZIP and ACTN4 in myoblast differentiation. During the myogenesis, ACTN4 translocates to the nucleus, binds to MEF2, and increases the transcriptional activity of MEF2, leading to expressions of MEF2-induced myogenic genes. sLZIP interacts with ACTN4 in the nucleus and disrupts the association between ACTN4 and MEF2, resulting in suppression of myogenic gene expression. Therefore, sLZIP functions as a negative regulator of MEF2-mediated gene expression and skeletal muscle differentiation.