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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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Panx1 and Panx3 levels are modulated during skeletal muscle myogenic differentiation.A, Panx1 and Panx3 were detected in human fetal and adult skeletal muscle tissue lysates, and their various species were quantified (B and C). For both Panx1 and Panx3, the higher Mr species are more abundant in the fetal skeletal muscle, and the banding pattern switches toward lower Mr forms in the adult tissue. Lysates of HEK293T cells expressing either Panx1 or Panx3 were used as positive controls. MHC and PCNA were detected to show the differentiation and proliferation status of the cells within the tissue, respectively. HSMM (D) and SkMC (G) were induced to differentiate for 6 days. Panx1 was found at very low levels in undifferentiated cells and was drastically induced during differentiation (E and H), whereas the ∼70-kDa immunoreactive species of Panx3 was abundant in undifferentiated and proliferative skeletal muscle cells and down-regulated during differentiation (F and I). As expected, MHC was increased during differentiation, and PCNA levels were reduced. Lysates of HEK293T cells expressing either Panx1 or Panx3 were used as positive controls. Tubulin was used as a loading control.
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Figure 2: Panx1 and Panx3 levels are modulated during skeletal muscle myogenic differentiation.A, Panx1 and Panx3 were detected in human fetal and adult skeletal muscle tissue lysates, and their various species were quantified (B and C). For both Panx1 and Panx3, the higher Mr species are more abundant in the fetal skeletal muscle, and the banding pattern switches toward lower Mr forms in the adult tissue. Lysates of HEK293T cells expressing either Panx1 or Panx3 were used as positive controls. MHC and PCNA were detected to show the differentiation and proliferation status of the cells within the tissue, respectively. HSMM (D) and SkMC (G) were induced to differentiate for 6 days. Panx1 was found at very low levels in undifferentiated cells and was drastically induced during differentiation (E and H), whereas the ∼70-kDa immunoreactive species of Panx3 was abundant in undifferentiated and proliferative skeletal muscle cells and down-regulated during differentiation (F and I). As expected, MHC was increased during differentiation, and PCNA levels were reduced. Lysates of HEK293T cells expressing either Panx1 or Panx3 were used as positive controls. Tubulin was used as a loading control.

Mentions: To start assessing the potential roles of Panx1 and Panx3 in skeletal muscle, we compared Panx expression in human fetal and adult skeletal muscle lysates. During embryonic development, muscle mass increases predominantly by proliferative growth of myoblasts, and genetic programs regulate their differentiation. Postnatally, the contribution of cell proliferation decreases in differentiated myofibers (30). The diminution in cell proliferation in adult compared with fetal skeletal muscle is indicated here by the reduction of proliferating cell nuclear antigen (PCNA) levels, whereas the increase in differentiation is shown by the elevated levels of a marker of terminal differentiation, MHC (Fig. 2A). We found that the higher molecular weight species of Panx1 (∼50 kDa) is the main form present in both fetal and adult skeletal muscle. However, it decreases in the adult tissue, whereas the lower molecular forms of Panx1, known as Gly1 and Gly2 (4), become more abundant (Fig. 2, A and B). These results suggest that the banding pattern of Panx1 shifts from the higher Mr species in the fetal tissue toward its lower forms in the adult skeletal muscle. Similarly, the main Panx3 species found in the fetal skeletal muscle is at ∼51 kDa. The level of this species decreases in adult tissue, whereas the lower Mr forms (Gly1 and Gly2) of Panx3 become more abundant (Fig. 2C). Interestingly, the levels of the ∼70-kDa immunoreactive species of Panx3 were very low in adults but found in the fetal skeletal muscle at a slightly lower molecular weight (∼62 kDa) (Fig. 2, A and C). Thus, similar to Panx1, the banding pattern of Panx3 shifts toward lower Mr species in the adult skeletal muscle tissue. Altogether, these data indicate that the various Mr species of Panx1 and Panx3 are differentially expressed in fetal compared with adult human skeletal muscle, which may suggest a role in skeletal muscle development possibly linked to proliferation and differentiation processes.


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)

Panx1 and Panx3 levels are modulated during skeletal muscle myogenic differentiation.A, Panx1 and Panx3 were detected in human fetal and adult skeletal muscle tissue lysates, and their various species were quantified (B and C). For both Panx1 and Panx3, the higher Mr species are more abundant in the fetal skeletal muscle, and the banding pattern switches toward lower Mr forms in the adult tissue. Lysates of HEK293T cells expressing either Panx1 or Panx3 were used as positive controls. MHC and PCNA were detected to show the differentiation and proliferation status of the cells within the tissue, respectively. HSMM (D) and SkMC (G) were induced to differentiate for 6 days. Panx1 was found at very low levels in undifferentiated cells and was drastically induced during differentiation (E and H), whereas the ∼70-kDa immunoreactive species of Panx3 was abundant in undifferentiated and proliferative skeletal muscle cells and down-regulated during differentiation (F and I). As expected, MHC was increased during differentiation, and PCNA levels were reduced. Lysates of HEK293T cells expressing either Panx1 or Panx3 were used as positive controls. Tubulin was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Panx1 and Panx3 levels are modulated during skeletal muscle myogenic differentiation.A, Panx1 and Panx3 were detected in human fetal and adult skeletal muscle tissue lysates, and their various species were quantified (B and C). For both Panx1 and Panx3, the higher Mr species are more abundant in the fetal skeletal muscle, and the banding pattern switches toward lower Mr forms in the adult tissue. Lysates of HEK293T cells expressing either Panx1 or Panx3 were used as positive controls. MHC and PCNA were detected to show the differentiation and proliferation status of the cells within the tissue, respectively. HSMM (D) and SkMC (G) were induced to differentiate for 6 days. Panx1 was found at very low levels in undifferentiated cells and was drastically induced during differentiation (E and H), whereas the ∼70-kDa immunoreactive species of Panx3 was abundant in undifferentiated and proliferative skeletal muscle cells and down-regulated during differentiation (F and I). As expected, MHC was increased during differentiation, and PCNA levels were reduced. Lysates of HEK293T cells expressing either Panx1 or Panx3 were used as positive controls. Tubulin was used as a loading control.
Mentions: To start assessing the potential roles of Panx1 and Panx3 in skeletal muscle, we compared Panx expression in human fetal and adult skeletal muscle lysates. During embryonic development, muscle mass increases predominantly by proliferative growth of myoblasts, and genetic programs regulate their differentiation. Postnatally, the contribution of cell proliferation decreases in differentiated myofibers (30). The diminution in cell proliferation in adult compared with fetal skeletal muscle is indicated here by the reduction of proliferating cell nuclear antigen (PCNA) levels, whereas the increase in differentiation is shown by the elevated levels of a marker of terminal differentiation, MHC (Fig. 2A). We found that the higher molecular weight species of Panx1 (∼50 kDa) is the main form present in both fetal and adult skeletal muscle. However, it decreases in the adult tissue, whereas the lower molecular forms of Panx1, known as Gly1 and Gly2 (4), become more abundant (Fig. 2, A and B). These results suggest that the banding pattern of Panx1 shifts from the higher Mr species in the fetal tissue toward its lower forms in the adult skeletal muscle. Similarly, the main Panx3 species found in the fetal skeletal muscle is at ∼51 kDa. The level of this species decreases in adult tissue, whereas the lower Mr forms (Gly1 and Gly2) of Panx3 become more abundant (Fig. 2C). Interestingly, the levels of the ∼70-kDa immunoreactive species of Panx3 were very low in adults but found in the fetal skeletal muscle at a slightly lower molecular weight (∼62 kDa) (Fig. 2, A and C). Thus, similar to Panx1, the banding pattern of Panx3 shifts toward lower Mr species in the adult skeletal muscle tissue. Altogether, these data indicate that the various Mr species of Panx1 and Panx3 are differentially expressed in fetal compared with adult human skeletal muscle, which may suggest a role in skeletal muscle development possibly linked to proliferation and differentiation processes.

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.

Show MeSH
Related in: MedlinePlus