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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

Protein expression levels of NDRG2, apoptotic and ER stress markers following 0, 8 and 16 h palmitate (PA) treatment of vector, NDRG2 and 3A-NDRG2-infected C2C12 myoblasts at P3. Protein loading is indicated by the α-Tubulin immunoblots.
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f0035: Protein expression levels of NDRG2, apoptotic and ER stress markers following 0, 8 and 16 h palmitate (PA) treatment of vector, NDRG2 and 3A-NDRG2-infected C2C12 myoblasts at P3. Protein loading is indicated by the α-Tubulin immunoblots.

Mentions: We next assessed the role for NDRG2 and 3A-NDRG2 under different stress treatments during myoblast proliferation. Both PA and H2O2 treatments cause significant apoptosis with oxidative and ER stress observed in C2C12 muscle cells [21–23,35]. Accordingly, we determined that 0.5 mM PA and 0.5 mM H2O2 treatments reduced myoblasts proliferation by nearly 50% after 16 and 8 h treatments, respectively (P < 0.001), while shorter exposure times also significantly decreased proliferation (P < 0.05; Table 2). Both NDRG2 and 3A-NDRG2 overexpression were unable to alter the proliferation rates of the myoblasts in the presence of PA treatment (Table 2). Nor did they alter the protein levels of apoptosis or ER stress markers including PARP and caspase 3 cleavage, Bcl-2 family members Bcl-2, Bcl-xL, Bax and Mcl-1, and glucose-regulated protein 78 (GRP78) (Fig. 7). In contrast, NDRG2 overexpression inhibited the decrease in cell proliferation following 4 h H2O2 treatment and attenuated the effect of H2O2 at 8 h with only a 17% proliferation decrease measured (P < 0.05) when compared to the 48% decrease with the vector alone (P < 0.001; Table 2). Moreover, NDRG2 reduced the amount of PARP and caspase 3 cleavage at 8 h (Fig. 8A and B) and reduced the loss of Bcl-2 and Bcl-xL (Fig. 8C and D) but Mcl-1 expression did not change (Fig. 8E). The induction of both Bax and GRP78 expression levels by H2O2 treatment was blocked by NDRG2 at 4 and 8 h time-points (Fig. 8F and G). 3A-NDRG2 only had moderate effects in preventing the loss in cell proliferation following H2O2 treatment (Table 2) and in regulating the expression of the apoptotic and ER stress protein markers (Fig. 8). Interestingly, both endogenous and overexpressed NDRG2 protein levels were significantly reduced by 8 h of H2O2 exposure with 80%, 50% and 60% decrease in NDRG2 expression measured in vector, NDRG2- and 3A-NDRG2-treated myoblasts, respectively (Fig. 8H).


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)

Protein expression levels of NDRG2, apoptotic and ER stress markers following 0, 8 and 16 h palmitate (PA) treatment of vector, NDRG2 and 3A-NDRG2-infected C2C12 myoblasts at P3. Protein loading is indicated by the α-Tubulin immunoblots.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0035: Protein expression levels of NDRG2, apoptotic and ER stress markers following 0, 8 and 16 h palmitate (PA) treatment of vector, NDRG2 and 3A-NDRG2-infected C2C12 myoblasts at P3. Protein loading is indicated by the α-Tubulin immunoblots.
Mentions: We next assessed the role for NDRG2 and 3A-NDRG2 under different stress treatments during myoblast proliferation. Both PA and H2O2 treatments cause significant apoptosis with oxidative and ER stress observed in C2C12 muscle cells [21–23,35]. Accordingly, we determined that 0.5 mM PA and 0.5 mM H2O2 treatments reduced myoblasts proliferation by nearly 50% after 16 and 8 h treatments, respectively (P < 0.001), while shorter exposure times also significantly decreased proliferation (P < 0.05; Table 2). Both NDRG2 and 3A-NDRG2 overexpression were unable to alter the proliferation rates of the myoblasts in the presence of PA treatment (Table 2). Nor did they alter the protein levels of apoptosis or ER stress markers including PARP and caspase 3 cleavage, Bcl-2 family members Bcl-2, Bcl-xL, Bax and Mcl-1, and glucose-regulated protein 78 (GRP78) (Fig. 7). In contrast, NDRG2 overexpression inhibited the decrease in cell proliferation following 4 h H2O2 treatment and attenuated the effect of H2O2 at 8 h with only a 17% proliferation decrease measured (P < 0.05) when compared to the 48% decrease with the vector alone (P < 0.001; Table 2). Moreover, NDRG2 reduced the amount of PARP and caspase 3 cleavage at 8 h (Fig. 8A and B) and reduced the loss of Bcl-2 and Bcl-xL (Fig. 8C and D) but Mcl-1 expression did not change (Fig. 8E). The induction of both Bax and GRP78 expression levels by H2O2 treatment was blocked by NDRG2 at 4 and 8 h time-points (Fig. 8F and G). 3A-NDRG2 only had moderate effects in preventing the loss in cell proliferation following H2O2 treatment (Table 2) and in regulating the expression of the apoptotic and ER stress protein markers (Fig. 8). Interestingly, both endogenous and overexpressed NDRG2 protein levels were significantly reduced by 8 h of H2O2 exposure with 80%, 50% and 60% decrease in NDRG2 expression measured in vector, NDRG2- and 3A-NDRG2-treated myoblasts, respectively (Fig. 8H).

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