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First published online 27 March 2007
doi: 10.1242/jcs.004739

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FHL3 binds MyoD and negatively regulates myotube formation

Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton, 3800, Australia

View larger version (46K): [in this window] [in a new window]   Fig. 1. FHL3 overexpression retards C2C12 cell differentiation. (A) C2C12 myoblast cell lines that stably express hemagglutinin (HA) vector [Vector (1) and Vector (2)] or HA-FHL3 [HA-FHL3 (1) and HA-FHL3 (2)] were generated in duplicate. Lysates were immunoblotted for FHL3 or total (pan) actin. Immunoblots are representative of four independent experiments. (B) The mean level of FHL3 protein expression in cell lines stably expressing HA-FHL3 versus those expressing Vector was quantified by densitometry and standardised to total actin (n=4, *P<0.05, **P<0.01). FHL3 protein levels standardised to actin were expressed relative to vector levels that were arbitrarily set at 1. (C) Lysates from differentiating C2C12 myoblasts stably expressing vector or HA-FHL3 were immunoblotted using anti-HA or anti-actin antibodies. Immunoblots are representative of three independent experiments. (D) C2C12 myoblasts stably expressing vector or HA-FHL3 were differentiated, stained with anti-MHC antibodies (green) and propidium iodide (red), and then imaged by confocal microscopy. Bar, 40 µm. Shown are representative images following 120 hours of differentiation. Immunofluorescence images are representative of three experiments for all cell lines. (E) Cells were co-stained with anti-MHC antibodies and propidium iodide. The differentiation index was determined by scoring the proportion of nuclei, of the total number of nuclei within MHC-positive cells (myocytes and myotubes). A minimum of 116 nuclei were scored per stable cell line per time point for each of three independent experiments (*P<0.05). Dashed line represents combined results of Vector (1) and (2) lines and black line shows combined results from HA-FHL3 (1) and (2) cell lines. (F) Cells were co-stained with anti-MHC antibodies and propidium iodide. The degree of cell fusion was quantified by determining the average number of nuclei per MHC-positive cell. A minimum of 42 and 69 MHC-positive cells were scored following 96 and 120 hours differentiation respectively, for each of three independent experiments, (*P<0.05, **P<0.01). Dashed line shows combined results of Vector (1) and (2) lines, and black line represents HA-FHL3 (1) and (2) cell lines combined results. Error bars represent ± s.e.m.

View larger version (40K): [in this window] [in a new window]   Fig. 2. FHL3 overexpression retards myogenin induction following 24 hours differentiation. (A) Lysates from differentiating C2C12 myoblasts stably expressing Vector or HA-FHL3 were immunoblotted for MyoD, myogenin or total actin using specific antibodies. Immunoblots are representative of three independent experiments. (B) The mean relative level of myogenin protein in C2C12 myoblasts stably expressing Vector or HA-FHL3 after 24 hours of differentiation was quantified by densitometry and presented as the combined data from Vector or HA-FHL3 (1) and (2) cell lines (n=3, **P<0.01). The myogenin protein levels in vector cell lines standardised to actin were arbitrarily set at 1 and myogenin levels in HA-FHL3 lines expressed relative to vector cell lines. Error bars represent ± s.e.m. (C) C2C12 myoblasts were differentiated for 24 hours, stained with anti-myogenin antibodies (blue), and propidium iodide (red) and imaged by confocal microscopy. Bar, 20 µm. Yellow arrowheads indicate myogenin-positive nucleus. White arrowheads indicate myogenin-negative nucleus. Shown are representative images for Vector (2) and HA-FHL3 (2) cell lines. Vector (1) and HA-FHL3 (1) cell lines showed equivalent results (data not shown). Results shown are representative of three independent experiments. (D) The mean percentage of nuclei expressing myogenin was quantified following 24 hours of differentiation and is presented as the combined data from Vector and HA-FHL3 (1) and (2) cell lines. A minimum of 116 nuclei were scored for each stable cell line per time point for each of three independent experiments (**P<0.01). Error bars represent ± s.e.m.

View larger version (78K): [in this window] [in a new window]   Fig. 3. FHL3 siRNA-knockdown accelerates C2C12 cell differentiation. C2C12 myoblasts were transfected with siRNA oligonucleotides, grown for 48 hours and then differentiated for up to 72 hours. In B,D,E, control siRNA-transfected cells are represented by dashed lines, siRNA FHL3 #2 by black solid lines and siRNA FHL3 #3 by grey solid lines, error bars represent ± s.e.m. *P<0.05, ***P<0.001. (A) Cell lysates from siRNA transfected C2C12 cells differentiated for up to 72 hours were immunoblotted for FHL3 or total actin. (B) FHL3 protein levels were quantified by densitometry and standardised to total actin (n=3). (C) siRNA-transfected C2C12 cells were differentiated, stained with anti-MHC antibodies (green) and propidium iodide (red) and imaged by confocal microscopy. Bars, 40 µm. Shown are representative images following 72 hours of differentiation. (D) The differentiation index was quantified using a minimum of 320 nuclei scored per transfected siRNA oligonucleotide species, per time point, for each of four independent experiments. (E) The average number of nuclei per MHC-positive cell was determined for siRNA-transfected cells, a minimum of 56 and 40 MHC-positive cells were scored following 48 and 72 hours differentiation, respectively, per transfected siRNA oligonucleotide species (n=4).

View larger version (32K): [in this window] [in a new window]   Fig. 4. FHL3 siRNA knockdown accelerates myogenin protein expression during differentiation. (A) Lysates from differentiating siRNA-transfected C2C12 cells were immunoblotted for MyoD, myogenin or actin. (B) Myogenin protein levels in siRNA-transfected C2C12 cells were quantified after 24 hours of differentiation by densitometry of immunoblots standardised to actin as a loading control and vector levels arbitrarily set at 1 (n=4, *P<0.05, **P<0.01). Error bars represent ± s.e.m. (C) siRNA-transfected C2C12 cells were differentiated for 24 hours, stained with anti-myogenin antibodies (blue) and propidium iodide (red) and imaged by confocal microscopy. Bar, 20 µm. Yellow arrowheads indicate myogenin-positive nucleus. White arrowheads indicate myogenin-negative nucleus. (D) The mean percentage of nuclei expressing myogenin was quantified following 24 hours of differentiation, a minimum of 320 nuclei were scored per transfected siRNA oligonucleotide species, per time point (n=4, *P<0.05, **P<0.01). Error bars represent ± s.e.m.

View larger version (37K): [in this window] [in a new window]   Fig. 5. Rescue of FHL3 siRNA knockdown phenotype. (A) siRNA-transfected C2C12 Vector (2) or HA-FHL3 (2) cell lines were harvested 48 hours post transfection (0 hours differentiation) and lysates immunoblotted for FHL3, HA or actin. Western blots are representative of four experiments. (B) siRNA-transfected C2C12 Vector (2) or HA-FHL3 (2) cell lines were differentiated for 72 hours and stained with anti-MHC antibodies (green) and propidium iodide (red), and imaged by confocal microscopy. Images are representative of four independent experiments. Bar, 20 µm. (C) siRNA-transfected C2C12 Vector (2) and HA-FHL3 (2) cell lines were harvested following 24 hours of differentiation and lysates immunoblotted for myogenin or actin. Western blots are representative of three experiments. (D) The mean relative level of myogenin protein during siRNA-mediated knockdown of endogenous FHL3 in Vector (2) or HA-FHL3 (2) cell lines was quantified by densitometry expressed relative to actin loading and standardised to vector levels set at 1 arbitrarily. C2C12 cells transfected with Vector (2) are represented by white bars, HA-FHL3 (2) by black bars, error bars represent ± s.e.m. (n=3, *P<0.05, **P<0.01). siRNA transfected is shown below figure as indicated.

View larger version (44K): [in this window] [in a new window]   Fig. 6. FHL3 forms a complex with MyoD and other bHLH proteins. (A) C2C12 cells were co-transfected with various combinations of expression vectors for Flag-vector, MyoD-Flag, Gal4DBD and/or Gal4DBD-FHL3, together with Gal4-luciferase and Renilla luciferase reporters. Transfected myoblasts were maintained in growth media for 24 hours then differentiated for 48 hours and assayed for luciferase activity. Luciferase values were corrected for background luminescence, normalised to Renilla luciferase activity to adjust for transfection efficiency and standardised relative to Gal4DBD expressing samples (arbitrarily defined as 1 relative luciferase unit RLU). Error bars represent ± s.e.m. (n=4, ***P<0.001). White boxes represent GAL4DBD-FHL3 and black-line just above baseline represents GAL4DBD. (B) Lysates from C2C12 myoblasts transfected with pCMX-Gal4DBD or pCMX-Gal4DBD-FHL3 for 24 hours were immunoblotted to confirm recombinant protein expression. (C) Lysates from C2C12 myoblasts transfected with pEFBOS-Flag or pEFBOS-MyoD-Flag for 24 hours were immunoblotted to confirm recombinant protein expression. (D) GST-FHL3 and His-MyoD were coexpressed in E. coli. GST-proteins and associated proteins were bound to glutathione Sepharose, washed extensively and eluted with reduced glutathione. Eluted proteins (bound) and whole-cell extracts (input fractions) were immunoblotted for recombinant GST, or His protein expression. Western blots are representative of three experiments. (E) GST or GST-FHL3 captured on glutathione-Sepharose was incubated for 4 hours, with cell lysates from C2C12 cells differentiated (24 hours), washed extensively and immunoblotted for endogenous MyoD, or recombinant GST and/or GST-FHL3 using antibodies as indicated. Results shown are representative of three similar experiments. S/N and input fractions represent unbound lysate and C2C12 cell lysates prior to incubation with GST or GST-FHL3 respectively. (F) GST or GST-FHL3 pull-downs described above in (E) were also immunoblotted for endogenous Myf5, myogenin, and E47 using indicated antibodies. Western blots are representative of three experiments. (G) C2C12 myoblasts were stained with anti-FHL3 (green), anti-MyoD antibodies (red), and with the nuclear stain To-Pro-3 (blue) and imaged by confocal microscopy. Bar, 20 µm.

View larger version (22K): [in this window] [in a new window]   Fig. 7. FHL3 overexpression reduces MyoD transcription of luciferase reporter genes. (A) C2C12 cells were co-transfected with various combinations of expression vectors for Vector (HA-vector), HA-FHL3, Gal4DBD and/or Gal4DBD-MyoD, together with a Gal4-luciferase and Renilla luciferase reporters. Transfected myoblasts were maintained in growth media for 48 hours and assayed for luciferase activity. Luciferase values were corrected for background luminescence, normalised to Renilla luciferase activity to adjust for transfection efficiency and standardised relative to Gal4DBD-expressing samples (arbitrarily defined as 1 relative luciferase unit-RLU). Gal4DBD activity is represented by grey bars, Gal4DBD-MyoD by white bars, error bars represent ± s.e.m. (n=6, *P<0.05). Co-transfection with Vector or HA-FHL3 is shown below graph. (B) C2C12 cells were co-transfected as above. Transfected myoblasts were maintained in growth media for 24 hours and switched to differentiation media for 48 hours, and then assayed. Gal4DBD activity is represented by grey bars, Gal4DBD-MyoD by white bars, error bars represent ± s.e.m. (n=7, *P<0.05). Co-transfection with Vector or HA-FHL3 is indicated below figure. (C) Lysates from C2C12 myoblasts transfected with pCMX-Gal4DBD or pCMX-Gal4DBD-MyoD for 24 hours, were immunoblotted to confirm recombinant protein expression. (D) Lysates from C2C12 myoblasts transfected with pCGN or pCGN-FHL3 for 24 hours, were immunoblotted to confirm recombinant protein expression. (E) C2C12 cells were co-transfected with various combinations of expression vectors for Vector, HA-FHL3, Flag-vector and/or MyoD-Flag, with a pGL3-MCK (muscle creatine kinase luciferase reporter) and renilla luciferase. Assays were performed under differentiation conditions as outlined above except values were standardised relative to Flag-vector and Vector coexpressing samples (arbitrarily defined as 1 RLU). Flag-vector activity is represented by grey bars, MyoD-Flag by white bars, error bars represent ± s.e.m. (n=3, *P<0.05, **P<0.01). Co-transfection with Vector or HA-FHL3 is shown below the graph.

View larger version (16K): [in this window] [in a new window]   Fig. 8. siRNA-mediated FHL3 knockdown increases MyoD transcription of luciferase reporter genes. (A) C2C12 cells were co-transfected with various combinations of expression vectors for Gal4DBD or Gal4DBD-MyoD and siRNA oligonucleotides as indicated, with a Gal4-luciferase and Renilla luciferase reporters. Assays were performed under differentiation conditions. Gal4DBD activity is represented by grey bars, Gal4DBD-MyoD by white bars, error bars represent ± s.e.m. (n=4 *P<0.05, **P<0.01). Co-transfection with specific siRNAs is shown below the graph. (B) Cellular extracts (20 µl) prepared from C2C12 cells transfected with the indicated constructs and used for the luciferase assay shown in A), were also immunoblotted with the anti-FHL3 antibody to confirm siRNA-mediated FHL3 knockdown and with total actin antibody as a protein loading control. (C) C2C12 cells were co-transfected with various combinations of expression vectors for Flag-tagged vector or MyoD-Flag and siRNA oligonucleotides as indicated with a pGL3-MCK and Renilla luciferase reporters. Assays were performed under differentiation conditions. Flag-tagged vector activity is represented by grey bars, MyoD-Flag by white bars, error bars represent ± s.e.m. (n=8, *P<0.05, ***P<0.001). Co-transfection with specific siRNAs is shown below the graph. (D) Cellular extracts (20 µl) prepared from C2C12 cells transfected with the indicated constructs and used for the luciferase assay shown in C), were also immunoblotted with the anti-FHL3 antibody to confirm siRNA-mediated knockdown of FHL3 protein and with total actin antibody as a protein loading control.

View larger version (20K): [in this window] [in a new window]   Fig. 9. Model for FHL3 regulation of MyoD-dependent myogenesis. FHL3 physically interacts with MyoD to negatively regulate MyoD-dependent transcription of differentiation genes such as myogenin, retarding myotube formation and myogenesis.

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