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Pannexin 1 and pannexin 3 channels regulate skeletal muscle myoblast proliferation and differentiation.

Langlois S, Xiang X, Young K, Cowan BJ, Penuela S, Cowan KN - J. Biol. Chem. (2014)

Bottom Line: Using HSMM, we found that Panx1 expression promotes this process, whereas it was impaired in the presence of probenecid or carbenoxolone.Reduction of its endogenous expression using two Panx3 shRNAs significantly inhibited HSMM proliferation without triggering their differentiation.In summary, our results demonstrate that Panx1 and Panx3 are co-expressed in human skeletal muscle myoblasts and play a pivotal role in dictating the proliferation and differentiation status of these cells.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Surgery, Division of Paediatric Surgery, University of Ottawa, Children's Hospital of Eastern Ontario, Ottawa, Ontario K1H 8L1, Canada, Apoptosis Research Center, Children's Hospital of Eastern Ontario, Ottawa, Ontario K1H 8L1, Canada.

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Probenecid and carbenoxolone inhibit human primary skeletal muscle myoblast differentiation. HSMM were placed in differentiation medium for 6 days in the presence or absence of 1 mm probenecid (or its vehicle control, PBS) or 100 μm CBX (or its vehicle control, DMSO), and examined for MHC (labeled in green) expression and myotube formation. Representative pictures of three independent experiments are shown in A. Probenecid and CBX significantly reduced the percentage of MHC-positive cells after 6 days of differentiation (B) as well as the number of nuclei in MHC-positive cells (C). After 6 days in differentiation medium, HSMM were also analyzed for MHC levels. The increase in MHC seen after 6 days in differentiation medium was significantly reduced when cells were incubated with probenecid or CBX (D and E) without affecting the levels of endogenous Panx1 and Panx3 (D). Tubulin was used as a loading control. Blue = nuclei; bar = 100 μm; *, p < 0.05 compared with Day 0.
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Figure 6: Probenecid and carbenoxolone inhibit human primary skeletal muscle myoblast differentiation. HSMM were placed in differentiation medium for 6 days in the presence or absence of 1 mm probenecid (or its vehicle control, PBS) or 100 μm CBX (or its vehicle control, DMSO), and examined for MHC (labeled in green) expression and myotube formation. Representative pictures of three independent experiments are shown in A. Probenecid and CBX significantly reduced the percentage of MHC-positive cells after 6 days of differentiation (B) as well as the number of nuclei in MHC-positive cells (C). After 6 days in differentiation medium, HSMM were also analyzed for MHC levels. The increase in MHC seen after 6 days in differentiation medium was significantly reduced when cells were incubated with probenecid or CBX (D and E) without affecting the levels of endogenous Panx1 and Panx3 (D). Tubulin was used as a loading control. Blue = nuclei; bar = 100 μm; *, p < 0.05 compared with Day 0.

Mentions: To confirm the role of Panx1 channels in differentiation, HSMM were treated with probenecid and CBX (32, 33) and tested for their effect on this process. As shown in Fig. 6, A and B, <10% of HSMM were positive for MHC at day 0, which increased to 20–45% after 6 days in differentiation media (DM). Among those MHC-positive cells, about 15% contained 3–5 nuclei after 6 days in DM, which is indicative of fusion, compared with <1% at day 0 (Fig. 6C). However, when cells were treated with 1 mm probenecid or 100 μm CBX during the 6-day incubation in DM, the percentage of cells that were positive for MHC was drastically reduced (Fig. 6B) as well as the percentage of MHC-positive cells that had 3–5 nuclei (Fig. 6C). Taken together, these results suggest that inhibition of Panx1 channels impairs skeletal muscle myoblast differentiation.


Pannexin 1 and pannexin 3 channels regulate skeletal muscle myoblast proliferation and differentiation.

Langlois S, Xiang X, Young K, Cowan BJ, Penuela S, Cowan KN - J. Biol. Chem. (2014)

Probenecid and carbenoxolone inhibit human primary skeletal muscle myoblast differentiation. HSMM were placed in differentiation medium for 6 days in the presence or absence of 1 mm probenecid (or its vehicle control, PBS) or 100 μm CBX (or its vehicle control, DMSO), and examined for MHC (labeled in green) expression and myotube formation. Representative pictures of three independent experiments are shown in A. Probenecid and CBX significantly reduced the percentage of MHC-positive cells after 6 days of differentiation (B) as well as the number of nuclei in MHC-positive cells (C). After 6 days in differentiation medium, HSMM were also analyzed for MHC levels. The increase in MHC seen after 6 days in differentiation medium was significantly reduced when cells were incubated with probenecid or CBX (D and E) without affecting the levels of endogenous Panx1 and Panx3 (D). Tubulin was used as a loading control. Blue = nuclei; bar = 100 μm; *, p < 0.05 compared with Day 0.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215249&req=5

Figure 6: Probenecid and carbenoxolone inhibit human primary skeletal muscle myoblast differentiation. HSMM were placed in differentiation medium for 6 days in the presence or absence of 1 mm probenecid (or its vehicle control, PBS) or 100 μm CBX (or its vehicle control, DMSO), and examined for MHC (labeled in green) expression and myotube formation. Representative pictures of three independent experiments are shown in A. Probenecid and CBX significantly reduced the percentage of MHC-positive cells after 6 days of differentiation (B) as well as the number of nuclei in MHC-positive cells (C). After 6 days in differentiation medium, HSMM were also analyzed for MHC levels. The increase in MHC seen after 6 days in differentiation medium was significantly reduced when cells were incubated with probenecid or CBX (D and E) without affecting the levels of endogenous Panx1 and Panx3 (D). Tubulin was used as a loading control. Blue = nuclei; bar = 100 μm; *, p < 0.05 compared with Day 0.
Mentions: To confirm the role of Panx1 channels in differentiation, HSMM were treated with probenecid and CBX (32, 33) and tested for their effect on this process. As shown in Fig. 6, A and B, <10% of HSMM were positive for MHC at day 0, which increased to 20–45% after 6 days in differentiation media (DM). Among those MHC-positive cells, about 15% contained 3–5 nuclei after 6 days in DM, which is indicative of fusion, compared with <1% at day 0 (Fig. 6C). However, when cells were treated with 1 mm probenecid or 100 μm CBX during the 6-day incubation in DM, the percentage of cells that were positive for MHC was drastically reduced (Fig. 6B) as well as the percentage of MHC-positive cells that had 3–5 nuclei (Fig. 6C). Taken together, these results suggest that inhibition of Panx1 channels impairs skeletal muscle myoblast differentiation.

Bottom Line: Using HSMM, we found that Panx1 expression promotes this process, whereas it was impaired in the presence of probenecid or carbenoxolone.Reduction of its endogenous expression using two Panx3 shRNAs significantly inhibited HSMM proliferation without triggering their differentiation.In summary, our results demonstrate that Panx1 and Panx3 are co-expressed in human skeletal muscle myoblasts and play a pivotal role in dictating the proliferation and differentiation status of these cells.

View Article: PubMed Central - PubMed

Affiliation: From the Department of Surgery, Division of Paediatric Surgery, University of Ottawa, Children's Hospital of Eastern Ontario, Ottawa, Ontario K1H 8L1, Canada, Apoptosis Research Center, Children's Hospital of Eastern Ontario, Ottawa, Ontario K1H 8L1, Canada.

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Related in: MedlinePlus