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Myostatin negatively regulates satellite cell activation and self-renewal.

McCroskery S, Thomas M, Maxwell L, Sharma M, Kambadur R - J. Cell Biol. (2003)

Bottom Line: BrdU labeling in vivo revealed that, among the Myostatin-deficient satellite cells, higher numbers of satellite cells are activated as compared with wild type.Cell cycle analysis confirms that Myostatin up-regulated p21, a Cdk inhibitor, and decreased the levels and activity of Cdk2 protein in satellite cells.Taken together, these results suggest that Myostatin is a potent negative regulator of satellite cell activation and thus signals the quiescence of satellite cells.

View Article: PubMed Central - PubMed

Affiliation: Animal Genomics, AgResearch, Hamilton 2015, New Zealand.

ABSTRACT
Satellite cells are quiescent muscle stem cells that promote postnatal muscle growth and repair. Here we show that myostatin, a TGF-beta member, signals satellite cell quiescence and also negatively regulates satellite cell self-renewal. BrdU labeling in vivo revealed that, among the Myostatin-deficient satellite cells, higher numbers of satellite cells are activated as compared with wild type. In contrast, addition of Myostatin to myofiber explant cultures inhibits satellite cell activation. Cell cycle analysis confirms that Myostatin up-regulated p21, a Cdk inhibitor, and decreased the levels and activity of Cdk2 protein in satellite cells. Hence, Myostatin negatively regulates the G1 to S progression and thus maintains the quiescent status of satellite cells. Immunohistochemical analysis with CD34 antibodies indicates that there is an increased number of satellite cells per unit length of freshly isolated Mstn-/- muscle fibers. Determination of proliferation rate suggests that this elevation in satellite cell number could be due to increased self-renewal and delayed expression of the differentiation gene (myogenin) in Mstn-/- adult myoblasts. Taken together, these results suggest that Myostatin is a potent negative regulator of satellite cell activation and thus signals the quiescence of satellite cells.

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Delayed cell cycle withdrawal and differentiation of myostatin- adult myoblasts. Isolated myoblasts from C57BL/10 (Mstn+/+) or myostatin- mice (Mstn−/−) were switched to DM, and the cells were pulsed with BrdU before fixing at 0-, 6-, 12-, 24-, or 48-h time points. Percentage of cells that incorporate BrdU are shown for every time point in panel A. *, P < 0.05; **, P < 0.01 (as compared with wild type). Over 1,000 cells were counted in each of three replicates. The data presented are an average of three animals (A). Freshly isolated myoblasts from C57BL/10 (WT) or myostatin- mice (KO) were switched to DM, and proteins were extracted at indicated time points. Time 0 indicates freshly isolated quiescent satellite cells. Western analysis (n = 3) with MyoD (B) and Myogenin (C) antibodies on protein extracts from differentiating myoblasts is shown. Maximum expression was termed 100%, and relative expression at various time points was plotted. Anti–α-tubulin Western indicates equal loading.
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fig7: Delayed cell cycle withdrawal and differentiation of myostatin- adult myoblasts. Isolated myoblasts from C57BL/10 (Mstn+/+) or myostatin- mice (Mstn−/−) were switched to DM, and the cells were pulsed with BrdU before fixing at 0-, 6-, 12-, 24-, or 48-h time points. Percentage of cells that incorporate BrdU are shown for every time point in panel A. *, P < 0.05; **, P < 0.01 (as compared with wild type). Over 1,000 cells were counted in each of three replicates. The data presented are an average of three animals (A). Freshly isolated myoblasts from C57BL/10 (WT) or myostatin- mice (KO) were switched to DM, and proteins were extracted at indicated time points. Time 0 indicates freshly isolated quiescent satellite cells. Western analysis (n = 3) with MyoD (B) and Myogenin (C) antibodies on protein extracts from differentiating myoblasts is shown. Maximum expression was termed 100%, and relative expression at various time points was plotted. Anti–α-tubulin Western indicates equal loading.

Mentions: Myogenic differentiation of myoblasts occurs upon withdrawal of actively growing myoblasts from cell cycle into G0 and subsequent expression of myogenic markers. As the adult myoblasts derived from the Mstn−/− muscle fibers have deregulated S phase entry, we next evaluated the ability of Mstn−/− myoblasts to withdraw from the cell cycle upon mitogen withdrawal. Adult myoblasts derived from both wild-type and myostatin knockout mice were switched to differentiation low serum medium and pulsed with BrdU to identify the total number of myoblasts that are still cycling through the cell cycle in differentiation medium (DM). As shown in Fig. 7 A, nearly 100% of cells incorporated BrdU when the myoblasts were in GM. The majority of wild-type myoblasts (67%) stopped proliferating and withdrew from the cell cycle at 6 h after switching to DM. However, at this time point, 50% of Mstn−/− myoblasts were still in S phase, indicating that these cells were still cycling through the S phase and thus had not withdrawn to the G0 stage to differentiate. Even at the 12-h time point, significantly more Mstn−/− myoblasts were in S phase as compared with wild-type myoblasts (Fig. 7 A). Although delayed, eventually, the majority of Mstn−/− myoblasts do withdraw from the cell cycle and differentiate within 48 h.


Myostatin negatively regulates satellite cell activation and self-renewal.

McCroskery S, Thomas M, Maxwell L, Sharma M, Kambadur R - J. Cell Biol. (2003)

Delayed cell cycle withdrawal and differentiation of myostatin- adult myoblasts. Isolated myoblasts from C57BL/10 (Mstn+/+) or myostatin- mice (Mstn−/−) were switched to DM, and the cells were pulsed with BrdU before fixing at 0-, 6-, 12-, 24-, or 48-h time points. Percentage of cells that incorporate BrdU are shown for every time point in panel A. *, P < 0.05; **, P < 0.01 (as compared with wild type). Over 1,000 cells were counted in each of three replicates. The data presented are an average of three animals (A). Freshly isolated myoblasts from C57BL/10 (WT) or myostatin- mice (KO) were switched to DM, and proteins were extracted at indicated time points. Time 0 indicates freshly isolated quiescent satellite cells. Western analysis (n = 3) with MyoD (B) and Myogenin (C) antibodies on protein extracts from differentiating myoblasts is shown. Maximum expression was termed 100%, and relative expression at various time points was plotted. Anti–α-tubulin Western indicates equal loading.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172861&req=5

fig7: Delayed cell cycle withdrawal and differentiation of myostatin- adult myoblasts. Isolated myoblasts from C57BL/10 (Mstn+/+) or myostatin- mice (Mstn−/−) were switched to DM, and the cells were pulsed with BrdU before fixing at 0-, 6-, 12-, 24-, or 48-h time points. Percentage of cells that incorporate BrdU are shown for every time point in panel A. *, P < 0.05; **, P < 0.01 (as compared with wild type). Over 1,000 cells were counted in each of three replicates. The data presented are an average of three animals (A). Freshly isolated myoblasts from C57BL/10 (WT) or myostatin- mice (KO) were switched to DM, and proteins were extracted at indicated time points. Time 0 indicates freshly isolated quiescent satellite cells. Western analysis (n = 3) with MyoD (B) and Myogenin (C) antibodies on protein extracts from differentiating myoblasts is shown. Maximum expression was termed 100%, and relative expression at various time points was plotted. Anti–α-tubulin Western indicates equal loading.
Mentions: Myogenic differentiation of myoblasts occurs upon withdrawal of actively growing myoblasts from cell cycle into G0 and subsequent expression of myogenic markers. As the adult myoblasts derived from the Mstn−/− muscle fibers have deregulated S phase entry, we next evaluated the ability of Mstn−/− myoblasts to withdraw from the cell cycle upon mitogen withdrawal. Adult myoblasts derived from both wild-type and myostatin knockout mice were switched to differentiation low serum medium and pulsed with BrdU to identify the total number of myoblasts that are still cycling through the cell cycle in differentiation medium (DM). As shown in Fig. 7 A, nearly 100% of cells incorporated BrdU when the myoblasts were in GM. The majority of wild-type myoblasts (67%) stopped proliferating and withdrew from the cell cycle at 6 h after switching to DM. However, at this time point, 50% of Mstn−/− myoblasts were still in S phase, indicating that these cells were still cycling through the S phase and thus had not withdrawn to the G0 stage to differentiate. Even at the 12-h time point, significantly more Mstn−/− myoblasts were in S phase as compared with wild-type myoblasts (Fig. 7 A). Although delayed, eventually, the majority of Mstn−/− myoblasts do withdraw from the cell cycle and differentiate within 48 h.

Bottom Line: BrdU labeling in vivo revealed that, among the Myostatin-deficient satellite cells, higher numbers of satellite cells are activated as compared with wild type.Cell cycle analysis confirms that Myostatin up-regulated p21, a Cdk inhibitor, and decreased the levels and activity of Cdk2 protein in satellite cells.Taken together, these results suggest that Myostatin is a potent negative regulator of satellite cell activation and thus signals the quiescence of satellite cells.

View Article: PubMed Central - PubMed

Affiliation: Animal Genomics, AgResearch, Hamilton 2015, New Zealand.

ABSTRACT
Satellite cells are quiescent muscle stem cells that promote postnatal muscle growth and repair. Here we show that myostatin, a TGF-beta member, signals satellite cell quiescence and also negatively regulates satellite cell self-renewal. BrdU labeling in vivo revealed that, among the Myostatin-deficient satellite cells, higher numbers of satellite cells are activated as compared with wild type. In contrast, addition of Myostatin to myofiber explant cultures inhibits satellite cell activation. Cell cycle analysis confirms that Myostatin up-regulated p21, a Cdk inhibitor, and decreased the levels and activity of Cdk2 protein in satellite cells. Hence, Myostatin negatively regulates the G1 to S progression and thus maintains the quiescent status of satellite cells. Immunohistochemical analysis with CD34 antibodies indicates that there is an increased number of satellite cells per unit length of freshly isolated Mstn-/- muscle fibers. Determination of proliferation rate suggests that this elevation in satellite cell number could be due to increased self-renewal and delayed expression of the differentiation gene (myogenin) in Mstn-/- adult myoblasts. Taken together, these results suggest that Myostatin is a potent negative regulator of satellite cell activation and thus signals the quiescence of satellite cells.

Show MeSH
Related in: MedlinePlus