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First published online 30 October 2007
doi: 10.1242/jcs.010009

Journal of Cell Science 120, 4050-4059 (2007)
Published by The Company of Biologists 2007
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The chemokine SDF1 controls multiple steps of myogenesis through atypical PKC{zeta}

Veysel Ödemis1,*, Karina Boosmann1,*, Maja Theresa Dieterlen2 and Jürgen Engele1,{ddagger}

1 Institute of Anatomy, Medical Faculty, University of Leipzig, Liebigstr. 13, 04103 Leipzig, Germany
2 Department of Neurology, University of Leipzig, Liebigstr. 22a, 04103 Leipzig, Germany


Figure 1
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  		Fig. 1. SDF1 is a chemoattractant for C2C12 cells. (A) C2C12 cells were grown in 90% DMEM and 10% FCS to subconfluency and subsequently tested for SDF1-dependent (10 ng/ml) and HGF-dependent (HGF, 10 ng/ml) chemotaxis in a modified Boyden chamber assay as described in Materials and Methods. Data represent the mean ± s.d. from three independent experiments. Note that the migratory response elicited by SDF1 was similar to that induced by HGF. *P<0.001; Co, control. (B) Checkerboard analysis demonstrating a directed migratory (chemotactic) response of C2C12 cells towards SDF1. Numbers of migrated cells were determined in a Boyden chamber in which either only the lower well received SDF1 (10 ng/ml) or in which the concentration gradient was eliminated by adding identical concentrations of SDF1 to both the lower and the upper well (10 ng/ml each). Note that C2C12 cells fail to migrate through the filter when no SDF1 concentration gradient is present. *P<0.001; Co, control.

 

Figure 2
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  		Fig. 2. SDF1 is a mitogen for C2C12 cells. C2C12 cells were maintained in DMEM containing 1% HS and varying concentrations of SDF1. FGF2 (20 ng/ml) served as a positive control. After 24 hours, cells were stained with antibodies against the cell-proliferation marker Ki67. The number of Ki67-expressing cells was quantified by FACS analysis and expressed as the percentage of total cell numbers. Data represent the mean ± s.d. from four independent experiments. SDF1 activated C2C12 proliferation in a dose-dependent manner (F=10.9, dF=13, P<0.002; ANOVA). Proliferative response was maximal with SDF1 at concentrations of ≥10 ng/ml. A comparable proliferative response was induced by FGF2 (P<0.002).

 

Figure 3
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  		Fig. 3. Serum-deprivation induces myogenic differentiation of C2C12 cells. MyoD and myogenin protein levels were analyzed by western blotting either in C2C12 cells maintained in DMEM containing 10% FCS (growth condition), or 24 hours and 48 hours after switching cultures to DMEM supplemented with 1% HS (differentiation condition). Protein loading was controlled by staining blots with anti-β-actin antibodies. MyoD and myogenin protein levels gradually increased after switching cultures to differentiation conditions.

 

Figure 4
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  		Fig. 4. SDF1 attenuates myogenic differentiation of C2C12 cells and primary myoblasts. (A,C) At the switch from growth to differentiation conditions, C2C12 cells were treated with the indicated concentrations of SDF1 for 24 hours or 48 hours and then analyzed for (A) MyoD or (C) myogenin expression, respectively, by western blotting. Numbers represent average MyoD or myogenin protein levels (arbitrary units), corrected for β-actin levels, as determined by densitometric analysis of blots from five independent experiments. SDF1 significantly attenuates myogenic differentiation of C2C12 cells, maintained under differentiation conditions (MyoD, F=7.9, dF=15, P<0.005; myogenin, F=8.6, dF=15, P<0.005, ANOVA). (B,D) Real time (RT)-PCR analysis of C2C12 cells, maintained for 24 hours under differentiation conditions and the additional presence of SDF1 at different concentrations, for (B) MyoD and (D) myogenin mRNA levels. Data represent the mean ±s.d. from four independent experiments. Note that SDF1-induces a significant loss in myogenin mRNA levels (F=13.7, dF=12, P<0.001; ANOVA) but does not affect MyoD mRNA levels. (E) C2C12 cells were treated at the switch from growth to differentiation conditions with SDF1 (10 ng/ml) and analyzed for MHC expression after 2 days and 5 days by western blotting using the MF20 antibody. Staining for β-actin served as a loading control. MHC was undetectable in C2C12 cells maintained with SDF1 for 5 days. (F) Cultures of primary myoblasts and myogenic precursors were established from rodent hindlimb musculature as described in Materials and Methods. Cells were maintained for 4 days in the presence or absence of SDF1 (10 ng/ml) and subsequently analyzed for myogenin expression levels by western blotting. As found for C2C12 cells, SDF1 inhibits differentiation of primary myoblasts.

 

Figure 5
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  		Fig. 5. Immunocytochemical analysis of C2C12 cells for myogenin and MHC expression. C2C12 cells were maintained for 72 hours under differentiation conditions in the presence or absence of SDF1 (10 ng/ml) and subsequently stained with antibodies against myogenin and MHC (MF20) as indicated. Trypan Blue was used for counterstaining. Note that SDF1 clearly reduces the number of myotubes that express myogenin or MHC. Bar, 100 µm.

 

Figure 6
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  		Fig. 6. Interaction of SDF1 with other extracellular factors controlling myogenesis. C2C12 cells were treated at the switch from growth to differentiation conditions with either SDF1 (10 ng/ml), FGF2 (10 ng/ml) or IGF1 (10 ng/ml) alone or in combination as indicated. After 48 hours, myogenin protein levels were analyzed by western blotting. Immunoreactive protein bands were measured by densitometry and corrected for β-actin. Data represent average myogenin protein levels ± s.d. as determined in four independent experiments. SDF1 did not further modify the known inhibitory influences of FGF2 on myogenic differentiation. By apparent contrast, SDF1 completely prevented the established stimulatory influences of IGF1 on myogenesis. C, control; aP<0.05, IGF1 vs control; bP<0.05, IGF1+SDF1 vs IGF1.

 

Figure 7
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  		Fig. 7. Characterization of signaling proteins or pathways activated by SDF1 in C2C12 cells. C2C12 cells were stimulated for 10 minutes with SDF1 at the indicated concentrations and subsequently analyzed for activation (phosphorylation) of Akt, p38, Erk and different PKC isoforms by western blotting using phosphospecific antibodies. Protein loading was controlled by staining blots with (pan) antibodies that recognize the non-phosphorylated forms of the respective signaling molecules. In case of PKC isoforms, β-actin staining in combination with staining for all phosphorylated PKC isoforms using P-PKC-pan served as a loading control. SDF1 induced a dose-dependent increase in levels of phosphorylated Erk1, Erk2 or PKC{zeta}, PKC{lambda} [(p)-Erk1, (p)-Erk2 or (p)-PKC{zeta}, (p)-PKC{lambda}, respectively], but showed no effects on phosphorylated levels of Akt, p38 or pPKC{alpha}, PKC β [(p)-Akt, (p)-p38 or (p)-PKC{alpha}, (p)-PKCβ, respectively].

 

Figure 8
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  		Fig. 8. PKC{zeta} mediates the inhibitory influences of SDF1 on myogenic differentiation. (A) C2C12 cells were maintained for 48 hours with either myristoylated PKC{zeta} peptide inhibitor (2 µM) alone or in combination with the indicated concentrations of SDF1, and subsequently analyzed for myogenin expression by western blotting. Protein loading was controlled by GAPDH staining. Myogenin levels increased with myristoylated PKC{zeta} peptide inhibitor and remained unaffected by SDF1. Data are given in Table 2. (B) C2C12 cells were transfected overnight with PKC{zeta} siRNA or non-homologous (nh) siRNA, and maintained further in DMEM containing 1% HS and SDF1 (10 ng/ml). After 48 hours, cells were lysed and myogenin expression was determined by western blotting. Immunoreactive protein bands were measured by densitometry and corrected for GAPDH. Numbers represent average protein level ± s.d. (arbitrary units) as determined in three independent experiments. Myogenin protein levels increased in C2C12 cells transfected with PKC{zeta} siRNA. This increase was not affected by the additional presence of SDF1. By contrast, C2C12 cells transfected with nh siRNA showed unchanged expression levels of myogenin which declined in the presence of SDF1. aP<0.05, PKC{zeta} siRNA vs control; bP<0.05, PKC{zeta} siRNA+SDF1 vs control. (C) Assessment of PKC{zeta} expression in cells transfected with PKC{zeta} siRNA. C2C12 cells were transfected with PKC{zeta} siRNA or non-homologous (nh) siRNA. Levels of PKC{zeta} were determined by western blotting. Numbers indicate average levels of PKC{zeta} ± s.d. (arbitrary units) as determined in five independent experiments. Note that PKC{zeta} levels significantly decline (P<0.001) in C2C12 cells transfected with PKC{zeta} siRNA as compared with cells transfected with nh siRNA.

 

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© The Company of Biologists Ltd 2007