Limits...
Syntaxin 4 regulates the surface localization of a promyogenic receptor Cdo thereby promoting myogenic differentiation.

Yoo M, Kim BG, Lee SJ, Jeong HJ, Park JW, Seo DW, Kim YK, Lee HY, Han JW, Kang JS, Bae GU - Skelet Muscle (2015)

Bottom Line: Stx4 depletion decreases specifically the cell surface localization of Cdo without changes in surface N-Cadherin levels.Interestingly, Cdo depletion reduces the level of GLUT4 and Stx4 at cell surface.Consistently, overexpression of Cdo in C2C12 myoblasts generally increases glucose uptake, while Cdo depletion reduces it.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women's University, Seoul, 140-742 Republic of Korea.

ABSTRACT

Background: Syntaxins are a family of membrane proteins involved in vesicle trafficking, such as synaptic vesicle exocytosis. Syntaxin 4 (Stx4) is expressed highly in skeletal muscle and plays a critical role in insulin-stimulated glucose uptake by promoting translocation of glucose transporter 4 (GLUT4) to the cell surface. A cell surface receptor cell adhesion molecule-related, down-regulated by oncogenes (Cdo) is a component of cell adhesion complexes and promotes myoblast differentiation via activation of key signalings, including p38MAPK and AKT. In this study, we investigate the function of Stx4 in myoblast differentiation and the crosstalk between Stx4 and Cdo in myoblast differentiation.

Methods: The effects of overexpression or shRNA-based depletion of Stx4 and Cdo genes on C2C12 myoblast differentiation are assessed by Western blotting and immunofluorescence approaches. The interaction between Cdo and Stx4 and the responsible domain mapping are assessed by coimmunoprecipitation or pulldown assays. The effect of Stx4 depletion on cell surface localization of Cdo and GLUT4 in C2C12 myoblasts is assessed by surface biotinylation and Western blotting.

Results: Overexpression or knockdown of Stx4 enhances or inhibits myogenic differentiation, respectively. Stx4 binds to the cytoplasmic tail of Cdo, and this interaction seems to be critical for induction of p38MAPK activation and myotube formation. Stx4 depletion decreases specifically the cell surface localization of Cdo without changes in surface N-Cadherin levels. Interestingly, Cdo depletion reduces the level of GLUT4 and Stx4 at cell surface. Consistently, overexpression of Cdo in C2C12 myoblasts generally increases glucose uptake, while Cdo depletion reduces it.

Conclusions: Stx4 promotes myoblast differentiation through interaction with Cdo and stimulation of its surface translocation. Both Cdo and Stx4 are required for GLUT4 translocation to cell surface and glucose uptake in myoblast differentiation.

No MeSH data available.


Related in: MedlinePlus

Overexpression of Stx4 can override the block of myoblast differentiation in Cdo-depleted cells. a, b C2C12 cells were transfected with control, Stx4, or shStx4 expression vectors, and the lysates were analyzed for the levels of phospho-p38MAPK (p-p38) relative to total p38MAPK (p38). The relative levels of p-p38 are quantified and added under each well. c C2C12/pSuper and C2C12/Cdo shRNA cells were transiently transfected with pcDNA or Stx4 expression vector, plus GFP expression vector to mark transfectants. Confluent cultures were then fixed and stained with antibody to p-p38 (red). Cell nuclei were visualized by staining with DAPI (blue). The white arrows in p-p38 panels mark the transfected cells. Size bar = 10 μm. d Quantification of cultures shown in c. GFP+ cells were scored as positive or negative for p-p38 staining. These experiments were repeated three times with similar results. Significant difference from control, *p < 0.01. e Quantification of the relative signal strength of p-p38 in GFP-positive cells. Values are determinants of 10 fields and experiments were repeated three times with similar results. *p < 0.01. f C2C12/pSuper and C2C12/Cdo shRNA cells were transiently transfected with pcDNA or Stx4 expression vector, and the lysates were analyzed for the levels of phospho-p38MAPK (p-p38) relative to total p38MAPK (p38). The relative levels of p-p38 are quantified and added under each well. g C2C12 cells were transfected with pSuper or shStx4 expression vectors and were immunoprecipitated with control IgG or anti-Cdo antibody and immunoblotted with antibodies to JLP, Bnip2, Cdo, Stx4, and pan-Cadherin as a loading control. h Similar sets of cells as shown in panel c were induced to differentiate for 3 days and immunostained for MHC and GFP expressions. Size bar = 100 μm. i Quantification of myotube formation from panel e. GFP+ myotubes were quantified for MHC expression and the number of nuclei present in myotubes. Values are determinants of more than 10 fields and these experiments were repeated three times with similar results. Significant difference from control, *p < 0.01, **p < 0.005
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4561423&req=5

Fig5: Overexpression of Stx4 can override the block of myoblast differentiation in Cdo-depleted cells. a, b C2C12 cells were transfected with control, Stx4, or shStx4 expression vectors, and the lysates were analyzed for the levels of phospho-p38MAPK (p-p38) relative to total p38MAPK (p38). The relative levels of p-p38 are quantified and added under each well. c C2C12/pSuper and C2C12/Cdo shRNA cells were transiently transfected with pcDNA or Stx4 expression vector, plus GFP expression vector to mark transfectants. Confluent cultures were then fixed and stained with antibody to p-p38 (red). Cell nuclei were visualized by staining with DAPI (blue). The white arrows in p-p38 panels mark the transfected cells. Size bar = 10 μm. d Quantification of cultures shown in c. GFP+ cells were scored as positive or negative for p-p38 staining. These experiments were repeated three times with similar results. Significant difference from control, *p < 0.01. e Quantification of the relative signal strength of p-p38 in GFP-positive cells. Values are determinants of 10 fields and experiments were repeated three times with similar results. *p < 0.01. f C2C12/pSuper and C2C12/Cdo shRNA cells were transiently transfected with pcDNA or Stx4 expression vector, and the lysates were analyzed for the levels of phospho-p38MAPK (p-p38) relative to total p38MAPK (p38). The relative levels of p-p38 are quantified and added under each well. g C2C12 cells were transfected with pSuper or shStx4 expression vectors and were immunoprecipitated with control IgG or anti-Cdo antibody and immunoblotted with antibodies to JLP, Bnip2, Cdo, Stx4, and pan-Cadherin as a loading control. h Similar sets of cells as shown in panel c were induced to differentiate for 3 days and immunostained for MHC and GFP expressions. Size bar = 100 μm. i Quantification of myotube formation from panel e. GFP+ myotubes were quantified for MHC expression and the number of nuclei present in myotubes. Values are determinants of more than 10 fields and these experiments were repeated three times with similar results. Significant difference from control, *p < 0.01, **p < 0.005

Mentions: Previously, we have reported that Cdo promotes myoblast differentiation via activation of a key promyogenic kinase p38MAPK (p38) [13], and this is required for the efficient myoblast differentiation [13, 32]. Therefore, we examined the effect of Stx4 on p38 activation in C2C12 cells. Control or Stx4-overexpressing C2C12 cells were induced to differentiate for 2 days, and the status of p38 activation was analyzed by Western blot analysis with antibodies to an active phosphorylated form of p38 (p-p38) or total p38. Overexpression of Stx4 led to a substantial increase in p-p38 levels relative to that of control cells, while the level of total p38 was unchanged (Fig. 5a). In addition, C2C12/pSuper or C2C12/shStx4 cells were induced to differentiate for 3 days and analyzed for p38 activation. Stx4 knockdown in C2C12 cells caused a notable decrease in p-p38 levels relative to that of the control cells (Fig. 5b), suggesting that Stx4 is required for p38 activation during myoblast differentiation. We next asked whether the decreased p38 activation in Cdo-depleted C2C12 cells can be rescued by Stx4 expression. C2C12/pSuper and C2C12/shCdo cells were transiently transfected with pcDNA or Stx4 expression vector, plus GFP expression vector to label the transfectants. After 2 days of transfection, cells were immunostained with antibodies to p-p38 and GFP followed by DAPI staining to visualize nuclei. The representative pictures are shown in Fig. 5c. Roughly 36 % of control-transfected C2C12/pSuper cells were positive for the nuclear p-p38 accumulation (marked with a white arrow), whereas 63 % of Stx4-transfected C2C12/pSuper cells were positive for p-p38. On the other hand, only 17 % of control-transfected C2C12/shCdo cells were weakly positive for p-p38, whereas Stx4-expressing C2C12/shCdo cells displayed restored p-p38 levels with 31 % which is similar to control-transfected C2C12/pSuper cells (Fig. 5d). The quantification of relative p-p38 signal intensities in GFP-positive cells revealed the increased signal intensity in Stx4-overexpressing control cells. Furthermore, the overexpression of Stx4 restored p38 activation in C2C12/shCdo cells (Fig. 5e).Fig. 5


Syntaxin 4 regulates the surface localization of a promyogenic receptor Cdo thereby promoting myogenic differentiation.

Yoo M, Kim BG, Lee SJ, Jeong HJ, Park JW, Seo DW, Kim YK, Lee HY, Han JW, Kang JS, Bae GU - Skelet Muscle (2015)

Overexpression of Stx4 can override the block of myoblast differentiation in Cdo-depleted cells. a, b C2C12 cells were transfected with control, Stx4, or shStx4 expression vectors, and the lysates were analyzed for the levels of phospho-p38MAPK (p-p38) relative to total p38MAPK (p38). The relative levels of p-p38 are quantified and added under each well. c C2C12/pSuper and C2C12/Cdo shRNA cells were transiently transfected with pcDNA or Stx4 expression vector, plus GFP expression vector to mark transfectants. Confluent cultures were then fixed and stained with antibody to p-p38 (red). Cell nuclei were visualized by staining with DAPI (blue). The white arrows in p-p38 panels mark the transfected cells. Size bar = 10 μm. d Quantification of cultures shown in c. GFP+ cells were scored as positive or negative for p-p38 staining. These experiments were repeated three times with similar results. Significant difference from control, *p < 0.01. e Quantification of the relative signal strength of p-p38 in GFP-positive cells. Values are determinants of 10 fields and experiments were repeated three times with similar results. *p < 0.01. f C2C12/pSuper and C2C12/Cdo shRNA cells were transiently transfected with pcDNA or Stx4 expression vector, and the lysates were analyzed for the levels of phospho-p38MAPK (p-p38) relative to total p38MAPK (p38). The relative levels of p-p38 are quantified and added under each well. g C2C12 cells were transfected with pSuper or shStx4 expression vectors and were immunoprecipitated with control IgG or anti-Cdo antibody and immunoblotted with antibodies to JLP, Bnip2, Cdo, Stx4, and pan-Cadherin as a loading control. h Similar sets of cells as shown in panel c were induced to differentiate for 3 days and immunostained for MHC and GFP expressions. Size bar = 100 μm. i Quantification of myotube formation from panel e. GFP+ myotubes were quantified for MHC expression and the number of nuclei present in myotubes. Values are determinants of more than 10 fields and these experiments were repeated three times with similar results. Significant difference from control, *p < 0.01, **p < 0.005
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4561423&req=5

Fig5: Overexpression of Stx4 can override the block of myoblast differentiation in Cdo-depleted cells. a, b C2C12 cells were transfected with control, Stx4, or shStx4 expression vectors, and the lysates were analyzed for the levels of phospho-p38MAPK (p-p38) relative to total p38MAPK (p38). The relative levels of p-p38 are quantified and added under each well. c C2C12/pSuper and C2C12/Cdo shRNA cells were transiently transfected with pcDNA or Stx4 expression vector, plus GFP expression vector to mark transfectants. Confluent cultures were then fixed and stained with antibody to p-p38 (red). Cell nuclei were visualized by staining with DAPI (blue). The white arrows in p-p38 panels mark the transfected cells. Size bar = 10 μm. d Quantification of cultures shown in c. GFP+ cells were scored as positive or negative for p-p38 staining. These experiments were repeated three times with similar results. Significant difference from control, *p < 0.01. e Quantification of the relative signal strength of p-p38 in GFP-positive cells. Values are determinants of 10 fields and experiments were repeated three times with similar results. *p < 0.01. f C2C12/pSuper and C2C12/Cdo shRNA cells were transiently transfected with pcDNA or Stx4 expression vector, and the lysates were analyzed for the levels of phospho-p38MAPK (p-p38) relative to total p38MAPK (p38). The relative levels of p-p38 are quantified and added under each well. g C2C12 cells were transfected with pSuper or shStx4 expression vectors and were immunoprecipitated with control IgG or anti-Cdo antibody and immunoblotted with antibodies to JLP, Bnip2, Cdo, Stx4, and pan-Cadherin as a loading control. h Similar sets of cells as shown in panel c were induced to differentiate for 3 days and immunostained for MHC and GFP expressions. Size bar = 100 μm. i Quantification of myotube formation from panel e. GFP+ myotubes were quantified for MHC expression and the number of nuclei present in myotubes. Values are determinants of more than 10 fields and these experiments were repeated three times with similar results. Significant difference from control, *p < 0.01, **p < 0.005
Mentions: Previously, we have reported that Cdo promotes myoblast differentiation via activation of a key promyogenic kinase p38MAPK (p38) [13], and this is required for the efficient myoblast differentiation [13, 32]. Therefore, we examined the effect of Stx4 on p38 activation in C2C12 cells. Control or Stx4-overexpressing C2C12 cells were induced to differentiate for 2 days, and the status of p38 activation was analyzed by Western blot analysis with antibodies to an active phosphorylated form of p38 (p-p38) or total p38. Overexpression of Stx4 led to a substantial increase in p-p38 levels relative to that of control cells, while the level of total p38 was unchanged (Fig. 5a). In addition, C2C12/pSuper or C2C12/shStx4 cells were induced to differentiate for 3 days and analyzed for p38 activation. Stx4 knockdown in C2C12 cells caused a notable decrease in p-p38 levels relative to that of the control cells (Fig. 5b), suggesting that Stx4 is required for p38 activation during myoblast differentiation. We next asked whether the decreased p38 activation in Cdo-depleted C2C12 cells can be rescued by Stx4 expression. C2C12/pSuper and C2C12/shCdo cells were transiently transfected with pcDNA or Stx4 expression vector, plus GFP expression vector to label the transfectants. After 2 days of transfection, cells were immunostained with antibodies to p-p38 and GFP followed by DAPI staining to visualize nuclei. The representative pictures are shown in Fig. 5c. Roughly 36 % of control-transfected C2C12/pSuper cells were positive for the nuclear p-p38 accumulation (marked with a white arrow), whereas 63 % of Stx4-transfected C2C12/pSuper cells were positive for p-p38. On the other hand, only 17 % of control-transfected C2C12/shCdo cells were weakly positive for p-p38, whereas Stx4-expressing C2C12/shCdo cells displayed restored p-p38 levels with 31 % which is similar to control-transfected C2C12/pSuper cells (Fig. 5d). The quantification of relative p-p38 signal intensities in GFP-positive cells revealed the increased signal intensity in Stx4-overexpressing control cells. Furthermore, the overexpression of Stx4 restored p38 activation in C2C12/shCdo cells (Fig. 5e).Fig. 5

Bottom Line: Stx4 depletion decreases specifically the cell surface localization of Cdo without changes in surface N-Cadherin levels.Interestingly, Cdo depletion reduces the level of GLUT4 and Stx4 at cell surface.Consistently, overexpression of Cdo in C2C12 myoblasts generally increases glucose uptake, while Cdo depletion reduces it.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women's University, Seoul, 140-742 Republic of Korea.

ABSTRACT

Background: Syntaxins are a family of membrane proteins involved in vesicle trafficking, such as synaptic vesicle exocytosis. Syntaxin 4 (Stx4) is expressed highly in skeletal muscle and plays a critical role in insulin-stimulated glucose uptake by promoting translocation of glucose transporter 4 (GLUT4) to the cell surface. A cell surface receptor cell adhesion molecule-related, down-regulated by oncogenes (Cdo) is a component of cell adhesion complexes and promotes myoblast differentiation via activation of key signalings, including p38MAPK and AKT. In this study, we investigate the function of Stx4 in myoblast differentiation and the crosstalk between Stx4 and Cdo in myoblast differentiation.

Methods: The effects of overexpression or shRNA-based depletion of Stx4 and Cdo genes on C2C12 myoblast differentiation are assessed by Western blotting and immunofluorescence approaches. The interaction between Cdo and Stx4 and the responsible domain mapping are assessed by coimmunoprecipitation or pulldown assays. The effect of Stx4 depletion on cell surface localization of Cdo and GLUT4 in C2C12 myoblasts is assessed by surface biotinylation and Western blotting.

Results: Overexpression or knockdown of Stx4 enhances or inhibits myogenic differentiation, respectively. Stx4 binds to the cytoplasmic tail of Cdo, and this interaction seems to be critical for induction of p38MAPK activation and myotube formation. Stx4 depletion decreases specifically the cell surface localization of Cdo without changes in surface N-Cadherin levels. Interestingly, Cdo depletion reduces the level of GLUT4 and Stx4 at cell surface. Consistently, overexpression of Cdo in C2C12 myoblasts generally increases glucose uptake, while Cdo depletion reduces it.

Conclusions: Stx4 promotes myoblast differentiation through interaction with Cdo and stimulation of its surface translocation. Both Cdo and Stx4 are required for GLUT4 translocation to cell surface and glucose uptake in myoblast differentiation.

No MeSH data available.


Related in: MedlinePlus