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NDRG2 promotes myoblast proliferation and caspase 3/7 activities during differentiation, and attenuates hydrogen peroxide - But not palmitate-induced toxicity.

Anderson KJ, Russell AP, Foletta VC - FEBS Open Bio (2015)

Bottom Line: NDRG2 also attenuated apoptosis by reducing cleavage of PARP and caspase 3 and expression of pro-apoptotic Bax while enhancing the pro-survival Bcl-2 and Bcl-xL levels.In contrast, Mcl-1 was not altered, and NDRG2 did not protect against palmitate-induced lipotoxicity.Furthermore, NDRG2 attenuates H2O2-induced oxidative stress and specific serine and threonine amino acid residues appear to contribute to its function in muscle cells.

View Article: PubMed Central - PubMed

Affiliation: Centre for Physical Activity and Nutrition Research (C-PAN), School of Exercise and Nutrition Sciences, Faculty of Health, Deakin University, Melbourne, Australia.

ABSTRACT
The function of the stress-responsive N-myc downstream-regulated gene 2 (NDRG2) in the control of myoblast growth, and the amino acids contributing to its function, are not well characterized. Here, we investigated the effect of increased NDRG2 levels on the proliferation, differentiation and apoptosis in skeletal muscle cells under basal and stress conditions. NDRG2 overexpression increased C2C12 myoblast proliferation and the expression of positive cell cycle regulators, cdk2, cyclin B and cyclin D, and phosphorylation of Rb, while the serine/threonine-deficient NDRG2, 3A-NDRG2, had less effect. The onset of differentiation was enhanced by NDRG2 as determined through the myogenic regulatory factor expression profiles and myocyte fusion index. However, the overall level of differentiation in myotubes was not different. While NDRG2 up-regulated caspase 3/7 activities during differentiation, no increase in apoptosis was measured by TUNEL assay or through cleavage of caspase 3 and PARP proteins. During H2O2 treatment to induce oxidative stress, NDRG2 helped protect against the loss of proliferation and ER stress as measured by GRP78 expression with 3A-NDRG2 displaying less protection. NDRG2 also attenuated apoptosis by reducing cleavage of PARP and caspase 3 and expression of pro-apoptotic Bax while enhancing the pro-survival Bcl-2 and Bcl-xL levels. In contrast, Mcl-1 was not altered, and NDRG2 did not protect against palmitate-induced lipotoxicity. Our findings show that NDRG2 overexpression increases myoblast proliferation and caspase 3/7 activities without increasing overall differentiation. Furthermore, NDRG2 attenuates H2O2-induced oxidative stress and specific serine and threonine amino acid residues appear to contribute to its function in muscle cells.

No MeSH data available.


Related in: MedlinePlus

NDRG2 and 3A-NDRG2 overexpression effect on MRFs expression levels. (A) Myf5, (B) MyoD and (C) Myogenin mRNA (upper panels) and protein (lower panels) expression levels with representative blots indicated in C2C12 cells at P3, D0, D1 and up to D6 for myogenin. Myoblasts are infected with vector alone (black bars), NDRG2 (white bars) and 3A-NDRG2 (gray bars). All data are averages of three independent experiments (n = 3 per condition). ***P < 0.001, **P < 0.01 and *P < 0.05 compared to vector; ##P < 0.01, #P < 0.05 compared to NDRG2.
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f0020: NDRG2 and 3A-NDRG2 overexpression effect on MRFs expression levels. (A) Myf5, (B) MyoD and (C) Myogenin mRNA (upper panels) and protein (lower panels) expression levels with representative blots indicated in C2C12 cells at P3, D0, D1 and up to D6 for myogenin. Myoblasts are infected with vector alone (black bars), NDRG2 (white bars) and 3A-NDRG2 (gray bars). All data are averages of three independent experiments (n = 3 per condition). ***P < 0.001, **P < 0.01 and *P < 0.05 compared to vector; ##P < 0.01, #P < 0.05 compared to NDRG2.

Mentions: The effects of NDRG2 and 3A-NDRG2 overexpression during the early stages of differentiation were next investigated through the expression analyses of the MRFs, Myf5, MyoD and myogenin. NDRG2 overexpression increased Myf5 mRNA and protein expression by 1.5- and 1.3-fold at P3 and D0, respectively, while mRNA levels remained significantly elevated by 1.2-fold at D1 (P < 0.05) while 3A-NDRG2 increased Myf5 protein levels at P3 (P < 0.05; Fig. 4A). For MyoD expression levels, NDRG2 overexpression increased mRNA expression by 1.2-fold at P3 and D0, and protein levels by 1.7-fold at D0 (P < 0.05; Fig. 4B). No difference in MyoD mRNA or protein expression was observed at D1 and no effect by 3A-NDRG2 was found. Finally, myogenin levels were examined over the course of differentiation and NDRG2 overexpression increased myogenin mRNA and protein expression by 4.4-fold at P3 (P < 0.05) as well as increasing myogenin protein expression by 1.5-fold at D0 (P < 0.01) and both mRNA and protein levels at D2 when maximal myogenin expression was reached (P < 0.05; Fig. 4C). No difference in myogenin mRNA or protein expression was observed at D1 or with 3A-NDRG2 overexpression.


NDRG2 promotes myoblast proliferation and caspase 3/7 activities during differentiation, and attenuates hydrogen peroxide - But not palmitate-induced toxicity.

Anderson KJ, Russell AP, Foletta VC - FEBS Open Bio (2015)

NDRG2 and 3A-NDRG2 overexpression effect on MRFs expression levels. (A) Myf5, (B) MyoD and (C) Myogenin mRNA (upper panels) and protein (lower panels) expression levels with representative blots indicated in C2C12 cells at P3, D0, D1 and up to D6 for myogenin. Myoblasts are infected with vector alone (black bars), NDRG2 (white bars) and 3A-NDRG2 (gray bars). All data are averages of three independent experiments (n = 3 per condition). ***P < 0.001, **P < 0.01 and *P < 0.05 compared to vector; ##P < 0.01, #P < 0.05 compared to NDRG2.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4556729&req=5

f0020: NDRG2 and 3A-NDRG2 overexpression effect on MRFs expression levels. (A) Myf5, (B) MyoD and (C) Myogenin mRNA (upper panels) and protein (lower panels) expression levels with representative blots indicated in C2C12 cells at P3, D0, D1 and up to D6 for myogenin. Myoblasts are infected with vector alone (black bars), NDRG2 (white bars) and 3A-NDRG2 (gray bars). All data are averages of three independent experiments (n = 3 per condition). ***P < 0.001, **P < 0.01 and *P < 0.05 compared to vector; ##P < 0.01, #P < 0.05 compared to NDRG2.
Mentions: The effects of NDRG2 and 3A-NDRG2 overexpression during the early stages of differentiation were next investigated through the expression analyses of the MRFs, Myf5, MyoD and myogenin. NDRG2 overexpression increased Myf5 mRNA and protein expression by 1.5- and 1.3-fold at P3 and D0, respectively, while mRNA levels remained significantly elevated by 1.2-fold at D1 (P < 0.05) while 3A-NDRG2 increased Myf5 protein levels at P3 (P < 0.05; Fig. 4A). For MyoD expression levels, NDRG2 overexpression increased mRNA expression by 1.2-fold at P3 and D0, and protein levels by 1.7-fold at D0 (P < 0.05; Fig. 4B). No difference in MyoD mRNA or protein expression was observed at D1 and no effect by 3A-NDRG2 was found. Finally, myogenin levels were examined over the course of differentiation and NDRG2 overexpression increased myogenin mRNA and protein expression by 4.4-fold at P3 (P < 0.05) as well as increasing myogenin protein expression by 1.5-fold at D0 (P < 0.01) and both mRNA and protein levels at D2 when maximal myogenin expression was reached (P < 0.05; Fig. 4C). No difference in myogenin mRNA or protein expression was observed at D1 or with 3A-NDRG2 overexpression.

Bottom Line: NDRG2 also attenuated apoptosis by reducing cleavage of PARP and caspase 3 and expression of pro-apoptotic Bax while enhancing the pro-survival Bcl-2 and Bcl-xL levels.In contrast, Mcl-1 was not altered, and NDRG2 did not protect against palmitate-induced lipotoxicity.Furthermore, NDRG2 attenuates H2O2-induced oxidative stress and specific serine and threonine amino acid residues appear to contribute to its function in muscle cells.

View Article: PubMed Central - PubMed

Affiliation: Centre for Physical Activity and Nutrition Research (C-PAN), School of Exercise and Nutrition Sciences, Faculty of Health, Deakin University, Melbourne, Australia.

ABSTRACT
The function of the stress-responsive N-myc downstream-regulated gene 2 (NDRG2) in the control of myoblast growth, and the amino acids contributing to its function, are not well characterized. Here, we investigated the effect of increased NDRG2 levels on the proliferation, differentiation and apoptosis in skeletal muscle cells under basal and stress conditions. NDRG2 overexpression increased C2C12 myoblast proliferation and the expression of positive cell cycle regulators, cdk2, cyclin B and cyclin D, and phosphorylation of Rb, while the serine/threonine-deficient NDRG2, 3A-NDRG2, had less effect. The onset of differentiation was enhanced by NDRG2 as determined through the myogenic regulatory factor expression profiles and myocyte fusion index. However, the overall level of differentiation in myotubes was not different. While NDRG2 up-regulated caspase 3/7 activities during differentiation, no increase in apoptosis was measured by TUNEL assay or through cleavage of caspase 3 and PARP proteins. During H2O2 treatment to induce oxidative stress, NDRG2 helped protect against the loss of proliferation and ER stress as measured by GRP78 expression with 3A-NDRG2 displaying less protection. NDRG2 also attenuated apoptosis by reducing cleavage of PARP and caspase 3 and expression of pro-apoptotic Bax while enhancing the pro-survival Bcl-2 and Bcl-xL levels. In contrast, Mcl-1 was not altered, and NDRG2 did not protect against palmitate-induced lipotoxicity. Our findings show that NDRG2 overexpression increases myoblast proliferation and caspase 3/7 activities without increasing overall differentiation. Furthermore, NDRG2 attenuates H2O2-induced oxidative stress and specific serine and threonine amino acid residues appear to contribute to its function in muscle cells.

No MeSH data available.


Related in: MedlinePlus