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Integrated functions of Pax3 and Pax7 in the regulation of proliferation, cell size and myogenic differentiation.

Collins CA, Gnocchi VF, White RB, Boldrin L, Perez-Ruiz A, Relaix F, Morgan JE, Zammit PS - PLoS ONE (2009)

Bottom Line: We now show that constitutive expression of Pax3 or Pax7 in either satellite cells or C2C12 myoblasts results in an increased proliferative rate and decreased cell size.Similarly to the effects of Pax7, retroviral expression of Pax3 increases levels of Myf5 mRNA and MyoD protein, but does not result in sustained inhibition of myogenic differentiation.Our results show that in muscle progenitor cells, Pax3 and Pax7 function to maintain expression of myogenic regulatory factors, and promote population expansion, but are also required for myogenic differentiation to proceed.

View Article: PubMed Central - PubMed

Affiliation: Dubowitz Neuromuscular Centre, UCL Institute of Child Health, London, United Kingdom.

ABSTRACT
Pax3 and Pax7 are paired-box transcription factors with roles in developmental and adult regenerative myogenesis. Pax3 and Pax7 are expressed by postnatal satellite cells or their progeny but are down regulated during myogenic differentiation. We now show that constitutive expression of Pax3 or Pax7 in either satellite cells or C2C12 myoblasts results in an increased proliferative rate and decreased cell size. Conversely, expression of dominant-negative constructs leads to slowing of cell division, a dramatic increase in cell size and altered morphology. Similarly to the effects of Pax7, retroviral expression of Pax3 increases levels of Myf5 mRNA and MyoD protein, but does not result in sustained inhibition of myogenic differentiation. However, expression of Pax3 or Pax7 dominant-negative constructs inhibits expression of Myf5, MyoD and myogenin, and prevents differentiation from proceeding. In fibroblasts, expression of Pax3 or Pax7, or dominant-negative inhibition of these factors, reproduce the effects on cell size, morphology and proliferation seen in myoblasts. Our results show that in muscle progenitor cells, Pax3 and Pax7 function to maintain expression of myogenic regulatory factors, and promote population expansion, but are also required for myogenic differentiation to proceed.

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Related in: MedlinePlus

Quantitative analysis of the effects of Pax3 and Pax7 on myogenic gene expression.C2C12 were infected with control RV, Pax3 RV, Pax7 RV, Pax3DN RV or Pax7DN RV and analysed for mRNA (72 h later) and protein levels either 72 h or 1 week later. Semi-quantitative RT-PCR showed that constitutive Pax7 expression did not induce endogenous Pax3 mRNA expression, while Pax3DN did not down-regulate endogenous Pax7 mRNA (a – M: DNA ladder; NTC: no template control; amplicon sizes – Pax3: 151 bp; Pax7: 247 bp; Gapdh: 250 bp, representative experiments shown in duplicate). QPCR analysis of Myf5, MyoD and myogenin expression levels 72 h post-infection demonstrated that constitutive Pax3 or Pax7 expression both increased Myf5 levels, whereas the dominant-negative versions had the opposite effects and decreased Myf5 expression (b). Pax3DN or Pax7DN also suppressed both MyoD and myogenin expression (b). Data show mean fold change from control RV-infected cells (mean±SEM, from duplicate measurements from 2 independent experiments n = 2+2). Hypothesis testing performed using REST analysis where an asterisk denotes significant difference at p<0.05, while two asterisks denotes significant difference at p<0.01. Western blot analysis of infected cells showed that constitutive Pax3 or Pax7 expression increased MyoD protein levels, while the dominant-negative versions both resulted in less MyoD protein being present (c). Western blotting demonstrated that although the infection rates were similar, each construct produced eGFP with differing efficiencies from the IRES-eGFP in the retroviral backbone.
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pone-0004475-g005: Quantitative analysis of the effects of Pax3 and Pax7 on myogenic gene expression.C2C12 were infected with control RV, Pax3 RV, Pax7 RV, Pax3DN RV or Pax7DN RV and analysed for mRNA (72 h later) and protein levels either 72 h or 1 week later. Semi-quantitative RT-PCR showed that constitutive Pax7 expression did not induce endogenous Pax3 mRNA expression, while Pax3DN did not down-regulate endogenous Pax7 mRNA (a – M: DNA ladder; NTC: no template control; amplicon sizes – Pax3: 151 bp; Pax7: 247 bp; Gapdh: 250 bp, representative experiments shown in duplicate). QPCR analysis of Myf5, MyoD and myogenin expression levels 72 h post-infection demonstrated that constitutive Pax3 or Pax7 expression both increased Myf5 levels, whereas the dominant-negative versions had the opposite effects and decreased Myf5 expression (b). Pax3DN or Pax7DN also suppressed both MyoD and myogenin expression (b). Data show mean fold change from control RV-infected cells (mean±SEM, from duplicate measurements from 2 independent experiments n = 2+2). Hypothesis testing performed using REST analysis where an asterisk denotes significant difference at p<0.05, while two asterisks denotes significant difference at p<0.01. Western blot analysis of infected cells showed that constitutive Pax3 or Pax7 expression increased MyoD protein levels, while the dominant-negative versions both resulted in less MyoD protein being present (c). Western blotting demonstrated that although the infection rates were similar, each construct produced eGFP with differing efficiencies from the IRES-eGFP in the retroviral backbone.

Mentions: In order to better understand the dynamics of myogenic gene expression in response to constitutively expressed Pax3 and Pax7, we also examined the mRNA levels. RNA was isolated from C2C12 cells that had been infected with control RV, Pax3 RV, Pax7 RV, Pax3DN RV or Pax7DN RV, and then cultured for 72 h. The results of semi-quantitative RT-PCR for Pax3 and Pax7 transcripts confirmed our immunocytochemical data. Pax3 mRNA was only present at high levels in cultures infected with the Pax3 RV, whereas Pax7 mRNA was detected in all cultures, but was present at much higher levels in cultures infected with Pax7 RV (Figure 5a). The Pax7 primers also recognised transcript produced from the Pax7DN construct, but Western blotting using the Pax7 antibody confirmed that only cultures infected with Pax7 RV contained high levels of Pax7 protein (Figure 5c). We could not assess Pax3 protein levels by Western blot due to the unsuitability of the antibody for this application.


Integrated functions of Pax3 and Pax7 in the regulation of proliferation, cell size and myogenic differentiation.

Collins CA, Gnocchi VF, White RB, Boldrin L, Perez-Ruiz A, Relaix F, Morgan JE, Zammit PS - PLoS ONE (2009)

Quantitative analysis of the effects of Pax3 and Pax7 on myogenic gene expression.C2C12 were infected with control RV, Pax3 RV, Pax7 RV, Pax3DN RV or Pax7DN RV and analysed for mRNA (72 h later) and protein levels either 72 h or 1 week later. Semi-quantitative RT-PCR showed that constitutive Pax7 expression did not induce endogenous Pax3 mRNA expression, while Pax3DN did not down-regulate endogenous Pax7 mRNA (a – M: DNA ladder; NTC: no template control; amplicon sizes – Pax3: 151 bp; Pax7: 247 bp; Gapdh: 250 bp, representative experiments shown in duplicate). QPCR analysis of Myf5, MyoD and myogenin expression levels 72 h post-infection demonstrated that constitutive Pax3 or Pax7 expression both increased Myf5 levels, whereas the dominant-negative versions had the opposite effects and decreased Myf5 expression (b). Pax3DN or Pax7DN also suppressed both MyoD and myogenin expression (b). Data show mean fold change from control RV-infected cells (mean±SEM, from duplicate measurements from 2 independent experiments n = 2+2). Hypothesis testing performed using REST analysis where an asterisk denotes significant difference at p<0.05, while two asterisks denotes significant difference at p<0.01. Western blot analysis of infected cells showed that constitutive Pax3 or Pax7 expression increased MyoD protein levels, while the dominant-negative versions both resulted in less MyoD protein being present (c). Western blotting demonstrated that although the infection rates were similar, each construct produced eGFP with differing efficiencies from the IRES-eGFP in the retroviral backbone.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2637421&req=5

pone-0004475-g005: Quantitative analysis of the effects of Pax3 and Pax7 on myogenic gene expression.C2C12 were infected with control RV, Pax3 RV, Pax7 RV, Pax3DN RV or Pax7DN RV and analysed for mRNA (72 h later) and protein levels either 72 h or 1 week later. Semi-quantitative RT-PCR showed that constitutive Pax7 expression did not induce endogenous Pax3 mRNA expression, while Pax3DN did not down-regulate endogenous Pax7 mRNA (a – M: DNA ladder; NTC: no template control; amplicon sizes – Pax3: 151 bp; Pax7: 247 bp; Gapdh: 250 bp, representative experiments shown in duplicate). QPCR analysis of Myf5, MyoD and myogenin expression levels 72 h post-infection demonstrated that constitutive Pax3 or Pax7 expression both increased Myf5 levels, whereas the dominant-negative versions had the opposite effects and decreased Myf5 expression (b). Pax3DN or Pax7DN also suppressed both MyoD and myogenin expression (b). Data show mean fold change from control RV-infected cells (mean±SEM, from duplicate measurements from 2 independent experiments n = 2+2). Hypothesis testing performed using REST analysis where an asterisk denotes significant difference at p<0.05, while two asterisks denotes significant difference at p<0.01. Western blot analysis of infected cells showed that constitutive Pax3 or Pax7 expression increased MyoD protein levels, while the dominant-negative versions both resulted in less MyoD protein being present (c). Western blotting demonstrated that although the infection rates were similar, each construct produced eGFP with differing efficiencies from the IRES-eGFP in the retroviral backbone.
Mentions: In order to better understand the dynamics of myogenic gene expression in response to constitutively expressed Pax3 and Pax7, we also examined the mRNA levels. RNA was isolated from C2C12 cells that had been infected with control RV, Pax3 RV, Pax7 RV, Pax3DN RV or Pax7DN RV, and then cultured for 72 h. The results of semi-quantitative RT-PCR for Pax3 and Pax7 transcripts confirmed our immunocytochemical data. Pax3 mRNA was only present at high levels in cultures infected with the Pax3 RV, whereas Pax7 mRNA was detected in all cultures, but was present at much higher levels in cultures infected with Pax7 RV (Figure 5a). The Pax7 primers also recognised transcript produced from the Pax7DN construct, but Western blotting using the Pax7 antibody confirmed that only cultures infected with Pax7 RV contained high levels of Pax7 protein (Figure 5c). We could not assess Pax3 protein levels by Western blot due to the unsuitability of the antibody for this application.

Bottom Line: We now show that constitutive expression of Pax3 or Pax7 in either satellite cells or C2C12 myoblasts results in an increased proliferative rate and decreased cell size.Similarly to the effects of Pax7, retroviral expression of Pax3 increases levels of Myf5 mRNA and MyoD protein, but does not result in sustained inhibition of myogenic differentiation.Our results show that in muscle progenitor cells, Pax3 and Pax7 function to maintain expression of myogenic regulatory factors, and promote population expansion, but are also required for myogenic differentiation to proceed.

View Article: PubMed Central - PubMed

Affiliation: Dubowitz Neuromuscular Centre, UCL Institute of Child Health, London, United Kingdom.

ABSTRACT
Pax3 and Pax7 are paired-box transcription factors with roles in developmental and adult regenerative myogenesis. Pax3 and Pax7 are expressed by postnatal satellite cells or their progeny but are down regulated during myogenic differentiation. We now show that constitutive expression of Pax3 or Pax7 in either satellite cells or C2C12 myoblasts results in an increased proliferative rate and decreased cell size. Conversely, expression of dominant-negative constructs leads to slowing of cell division, a dramatic increase in cell size and altered morphology. Similarly to the effects of Pax7, retroviral expression of Pax3 increases levels of Myf5 mRNA and MyoD protein, but does not result in sustained inhibition of myogenic differentiation. However, expression of Pax3 or Pax7 dominant-negative constructs inhibits expression of Myf5, MyoD and myogenin, and prevents differentiation from proceeding. In fibroblasts, expression of Pax3 or Pax7, or dominant-negative inhibition of these factors, reproduce the effects on cell size, morphology and proliferation seen in myoblasts. Our results show that in muscle progenitor cells, Pax3 and Pax7 function to maintain expression of myogenic regulatory factors, and promote population expansion, but are also required for myogenic differentiation to proceed.

Show MeSH
Related in: MedlinePlus