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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

The effect of NDRG2 and 3A-NDRG2 overexpression on C2C12 myoblast proliferation and expression of positive cell cycle regulators at day 2 (P2) or day 3 (P3) of proliferation. (A) Proliferation rate of C2C12 myoblasts following infection with vector (black bars), NDRG2 (white bars) or 3A-NDRG2 (gray bars) (n = 6 per treatment) at P2 and P3. (B) Protein expression of cell cycle regulators cyclin E, Cdk4 and Cdk6. (C) mRNA (upper panel) and protein (lower panel) expression of Cdk2, cyclin B, cyclin D, and (D), Rb phosphorylation levels following vector, NDRG2 and 3A-NDRG2 overexpression at P3. Data are mean of three independent experiments (n = 3 per treatment). HSP90 or β-actin expression indicates protein levels loaded. ***P < 0.001, **P < 0.01, *P < 0.05 compared to vector; ###P < 0.001, ##P < 0.01 and #P < 0.05 compared to NDRG2.
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f0010: The effect of NDRG2 and 3A-NDRG2 overexpression on C2C12 myoblast proliferation and expression of positive cell cycle regulators at day 2 (P2) or day 3 (P3) of proliferation. (A) Proliferation rate of C2C12 myoblasts following infection with vector (black bars), NDRG2 (white bars) or 3A-NDRG2 (gray bars) (n = 6 per treatment) at P2 and P3. (B) Protein expression of cell cycle regulators cyclin E, Cdk4 and Cdk6. (C) mRNA (upper panel) and protein (lower panel) expression of Cdk2, cyclin B, cyclin D, and (D), Rb phosphorylation levels following vector, NDRG2 and 3A-NDRG2 overexpression at P3. Data are mean of three independent experiments (n = 3 per treatment). HSP90 or β-actin expression indicates protein levels loaded. ***P < 0.001, **P < 0.01, *P < 0.05 compared to vector; ###P < 0.001, ##P < 0.01 and #P < 0.05 compared to NDRG2.

Mentions: Having established comparable overexpression levels between NDRG2 and 3A-NDRG2, C2C12 myoblast proliferation rates were assessed at P2 and P3. To note, in this study, our aim was not to compare between time-points, but rather to focus on the effect of the NDRG2 proteins at each given time-point. At P2, NDRG2 increased proliferation 1.25-fold (P < 0.001) when compared with the vector. There was no effect of 3A-NDRG2 on proliferation. At P3, both NDRG2 and 3A-NDRG2 increased proliferation by 1.4-fold (P < 0.001) and 1.2-fold (P < 0.05), respectively (Fig. 2A). The increase in proliferation with NDRG2 overexpression remained higher than the effect of 3A-NDRG2 (P < 0.05). NDRG2 or 3A-NDRG2 did not affect cyclin E, Cdk4 or Cdk6 protein levels (Fig. 2B) at P3. However, NDRG2 increased both the mRNA and protein expression of the positive cell cycle regulators Cdk2 (P < 0.001), cyclin B (P < 0.01) and cyclin D (P < 0.05; Fig. 2C). Similarly, 3A-NDRG2 increased the levels of Cdk2 mRNA and protein, and cyclin B mRNA (P < 0.05). The phosphorylated levels of Rb also increased significantly following NDRG2 overexpression above both vector and 3A-NDRG2 (P < 0.05; Fig. 2D) indicating increased inhibition of Rb and enhanced cell cycle progression.


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)

The effect of NDRG2 and 3A-NDRG2 overexpression on C2C12 myoblast proliferation and expression of positive cell cycle regulators at day 2 (P2) or day 3 (P3) of proliferation. (A) Proliferation rate of C2C12 myoblasts following infection with vector (black bars), NDRG2 (white bars) or 3A-NDRG2 (gray bars) (n = 6 per treatment) at P2 and P3. (B) Protein expression of cell cycle regulators cyclin E, Cdk4 and Cdk6. (C) mRNA (upper panel) and protein (lower panel) expression of Cdk2, cyclin B, cyclin D, and (D), Rb phosphorylation levels following vector, NDRG2 and 3A-NDRG2 overexpression at P3. Data are mean of three independent experiments (n = 3 per treatment). HSP90 or β-actin expression indicates protein levels loaded. ***P < 0.001, **P < 0.01, *P < 0.05 compared to vector; ###P < 0.001, ##P < 0.01 and #P < 0.05 compared to NDRG2.
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f0010: The effect of NDRG2 and 3A-NDRG2 overexpression on C2C12 myoblast proliferation and expression of positive cell cycle regulators at day 2 (P2) or day 3 (P3) of proliferation. (A) Proliferation rate of C2C12 myoblasts following infection with vector (black bars), NDRG2 (white bars) or 3A-NDRG2 (gray bars) (n = 6 per treatment) at P2 and P3. (B) Protein expression of cell cycle regulators cyclin E, Cdk4 and Cdk6. (C) mRNA (upper panel) and protein (lower panel) expression of Cdk2, cyclin B, cyclin D, and (D), Rb phosphorylation levels following vector, NDRG2 and 3A-NDRG2 overexpression at P3. Data are mean of three independent experiments (n = 3 per treatment). HSP90 or β-actin expression indicates protein levels loaded. ***P < 0.001, **P < 0.01, *P < 0.05 compared to vector; ###P < 0.001, ##P < 0.01 and #P < 0.05 compared to NDRG2.
Mentions: Having established comparable overexpression levels between NDRG2 and 3A-NDRG2, C2C12 myoblast proliferation rates were assessed at P2 and P3. To note, in this study, our aim was not to compare between time-points, but rather to focus on the effect of the NDRG2 proteins at each given time-point. At P2, NDRG2 increased proliferation 1.25-fold (P < 0.001) when compared with the vector. There was no effect of 3A-NDRG2 on proliferation. At P3, both NDRG2 and 3A-NDRG2 increased proliferation by 1.4-fold (P < 0.001) and 1.2-fold (P < 0.05), respectively (Fig. 2A). The increase in proliferation with NDRG2 overexpression remained higher than the effect of 3A-NDRG2 (P < 0.05). NDRG2 or 3A-NDRG2 did not affect cyclin E, Cdk4 or Cdk6 protein levels (Fig. 2B) at P3. However, NDRG2 increased both the mRNA and protein expression of the positive cell cycle regulators Cdk2 (P < 0.001), cyclin B (P < 0.01) and cyclin D (P < 0.05; Fig. 2C). Similarly, 3A-NDRG2 increased the levels of Cdk2 mRNA and protein, and cyclin B mRNA (P < 0.05). The phosphorylated levels of Rb also increased significantly following NDRG2 overexpression above both vector and 3A-NDRG2 (P < 0.05; Fig. 2D) indicating increased inhibition of Rb and enhanced cell cycle progression.

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