Limits...
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.

Show MeSH

Related in: MedlinePlus

Treatment with N-glycosidase F and sialidase A alter the electrophoretic motility of the ∼70-kDa immunoreactive species of Panx3.A, deglycosylation using a mix of enzymes (N-glycosidase F, O-glycanase, neuraminidase (sialidase), β(1,4)-galactosidase, and β-N-acetylglucosaminidase) caused a shift in electrophoretic mobility (arrow) of the higher Mr form of Panx1 (∼50 kDa) expressed in differentiated HSMM and adult skeletal muscle tissue, similar to Panx1 transfected in HEK293T cells. B, incubation with that same mix of deglycosylation enzymes also caused a shift in the electrophoretic motility (arrow) of the ∼70-kDa immunoreactive species of Panx3 expressed in undifferentiated HSMM and human fetal skeletal muscle tissue and to the lower Mr form of Panx3 transfected in HEK293T cells. C, treatment with N-glycosidase F (PNGase F) and sialidase A independently resulted in a shift in the electrophoretic mobility of the ∼70-kDa immunoreactive species of Panx3 in HSMM but not with O-glycanase, β(1–4)-galactosidase, or β-N-acetylglucosaminidase.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4215249&req=5

Figure 3: Treatment with N-glycosidase F and sialidase A alter the electrophoretic motility of the ∼70-kDa immunoreactive species of Panx3.A, deglycosylation using a mix of enzymes (N-glycosidase F, O-glycanase, neuraminidase (sialidase), β(1,4)-galactosidase, and β-N-acetylglucosaminidase) caused a shift in electrophoretic mobility (arrow) of the higher Mr form of Panx1 (∼50 kDa) expressed in differentiated HSMM and adult skeletal muscle tissue, similar to Panx1 transfected in HEK293T cells. B, incubation with that same mix of deglycosylation enzymes also caused a shift in the electrophoretic motility (arrow) of the ∼70-kDa immunoreactive species of Panx3 expressed in undifferentiated HSMM and human fetal skeletal muscle tissue and to the lower Mr form of Panx3 transfected in HEK293T cells. C, treatment with N-glycosidase F (PNGase F) and sialidase A independently resulted in a shift in the electrophoretic mobility of the ∼70-kDa immunoreactive species of Panx3 in HSMM but not with O-glycanase, β(1–4)-galactosidase, or β-N-acetylglucosaminidase.

Mentions: As the lower molecular weight forms of Panxs were faint or below detectable levels in human primary skeletal muscle myoblasts in culture, we wanted to confirm that the higher molecular weight bands detected correspond to Panx1 and Panx3. To this end, HSMM and adult skeletal muscle tissue lysates were treated with a mixture of deglycosylation enzymes (N-glycosidase F, O-glycanase, neuraminidase (Sialidase), β(1,4)-galactosidase, and β-N-acetylglucosaminidase) and submitted to Western blotting. After deglycosylation, the endogenous ∼50-kDa protein species detected by anti-Panx1 in HSMM migrated further at ∼38 kDa (Fig. 3A). As a positive control, we show that the more classical Panx1 species also migrated further after treatment of Panx1-expressing HEK293T cell lysate with deglycosylation enzymes (Fig. 3A) (4). As for Panx3, the ∼70-kDa immunoreactive species detected by anti-Panx3 in HSMM and human fetal skeletal muscle lysates also migrated further into a predominant band at ∼50–51 kDa after treatment with deglycosylation enzymes (Fig. 3B). As a positive control, we show that this band was also present after submitting HEK293T over-expressing Panx3 to deglycosylation in addition to bands ranging from ∼35–40 kDa (Fig. 3B) (7). Because these results suggest that the ∼70-kDa immunoreactive species of Panx3 may have post-translational modifications that are different from the classical ∼43 kDa form, we wanted to better understand and characterize the glycosylation status of this higher Mr species. To this end, HSMM lysates were treated with the same enzymes but individually. As shown in Fig. 3C, the ∼70-kDa species of Panx3 migrated further after treatment with N-glycosidase F and sialidase A but not with O-glycanase, β(1,4)-galactosidase, or β-N-acetylglucosaminidase. However, O-glycosylation cannot be completely ruled out as the presence of sialic acids can block the action of O-glycanase. These results thus indicate that the ∼70-kDa immunoreactive species of Panx3 is modified by N-glycosylation and sialylation. Taken together these data suggest that the higher molecular weight species detected in HSMM and human skeletal muscle correspond to highly glycosylated species of endogenous human Panx1 and Panx3.


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)

Treatment with N-glycosidase F and sialidase A alter the electrophoretic motility of the ∼70-kDa immunoreactive species of Panx3.A, deglycosylation using a mix of enzymes (N-glycosidase F, O-glycanase, neuraminidase (sialidase), β(1,4)-galactosidase, and β-N-acetylglucosaminidase) caused a shift in electrophoretic mobility (arrow) of the higher Mr form of Panx1 (∼50 kDa) expressed in differentiated HSMM and adult skeletal muscle tissue, similar to Panx1 transfected in HEK293T cells. B, incubation with that same mix of deglycosylation enzymes also caused a shift in the electrophoretic motility (arrow) of the ∼70-kDa immunoreactive species of Panx3 expressed in undifferentiated HSMM and human fetal skeletal muscle tissue and to the lower Mr form of Panx3 transfected in HEK293T cells. C, treatment with N-glycosidase F (PNGase F) and sialidase A independently resulted in a shift in the electrophoretic mobility of the ∼70-kDa immunoreactive species of Panx3 in HSMM but not with O-glycanase, β(1–4)-galactosidase, or β-N-acetylglucosaminidase.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Treatment with N-glycosidase F and sialidase A alter the electrophoretic motility of the ∼70-kDa immunoreactive species of Panx3.A, deglycosylation using a mix of enzymes (N-glycosidase F, O-glycanase, neuraminidase (sialidase), β(1,4)-galactosidase, and β-N-acetylglucosaminidase) caused a shift in electrophoretic mobility (arrow) of the higher Mr form of Panx1 (∼50 kDa) expressed in differentiated HSMM and adult skeletal muscle tissue, similar to Panx1 transfected in HEK293T cells. B, incubation with that same mix of deglycosylation enzymes also caused a shift in the electrophoretic motility (arrow) of the ∼70-kDa immunoreactive species of Panx3 expressed in undifferentiated HSMM and human fetal skeletal muscle tissue and to the lower Mr form of Panx3 transfected in HEK293T cells. C, treatment with N-glycosidase F (PNGase F) and sialidase A independently resulted in a shift in the electrophoretic mobility of the ∼70-kDa immunoreactive species of Panx3 in HSMM but not with O-glycanase, β(1–4)-galactosidase, or β-N-acetylglucosaminidase.
Mentions: As the lower molecular weight forms of Panxs were faint or below detectable levels in human primary skeletal muscle myoblasts in culture, we wanted to confirm that the higher molecular weight bands detected correspond to Panx1 and Panx3. To this end, HSMM and adult skeletal muscle tissue lysates were treated with a mixture of deglycosylation enzymes (N-glycosidase F, O-glycanase, neuraminidase (Sialidase), β(1,4)-galactosidase, and β-N-acetylglucosaminidase) and submitted to Western blotting. After deglycosylation, the endogenous ∼50-kDa protein species detected by anti-Panx1 in HSMM migrated further at ∼38 kDa (Fig. 3A). As a positive control, we show that the more classical Panx1 species also migrated further after treatment of Panx1-expressing HEK293T cell lysate with deglycosylation enzymes (Fig. 3A) (4). As for Panx3, the ∼70-kDa immunoreactive species detected by anti-Panx3 in HSMM and human fetal skeletal muscle lysates also migrated further into a predominant band at ∼50–51 kDa after treatment with deglycosylation enzymes (Fig. 3B). As a positive control, we show that this band was also present after submitting HEK293T over-expressing Panx3 to deglycosylation in addition to bands ranging from ∼35–40 kDa (Fig. 3B) (7). Because these results suggest that the ∼70-kDa immunoreactive species of Panx3 may have post-translational modifications that are different from the classical ∼43 kDa form, we wanted to better understand and characterize the glycosylation status of this higher Mr species. To this end, HSMM lysates were treated with the same enzymes but individually. As shown in Fig. 3C, the ∼70-kDa species of Panx3 migrated further after treatment with N-glycosidase F and sialidase A but not with O-glycanase, β(1,4)-galactosidase, or β-N-acetylglucosaminidase. However, O-glycosylation cannot be completely ruled out as the presence of sialic acids can block the action of O-glycanase. These results thus indicate that the ∼70-kDa immunoreactive species of Panx3 is modified by N-glycosylation and sialylation. Taken together these data suggest that the higher molecular weight species detected in HSMM and human skeletal muscle correspond to highly glycosylated species of endogenous human Panx1 and Panx3.

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