Kishioka Yasuhiro, Thomas Mark, Wakamatsu Jun-Ichi, Hattori Akihito, Sharma Mridula, Kambadur Ravi, Nishimura Takanori
Meat Science Laboratory, Division of Bioresources and Bioproduction, Graduate School of Agriculture, Hokkaido University, Sapporo, Japan.
J Cell Physiol. 2008 Jun;215(3):856-67. doi: 10.1002/jcp.21371.
Decorin, a small leucine-rich proteoglycan, plays an important role in the regulation of cell growth. Our recent study has shown that immobilized decorin in the collagen matrix sequesters myostatin into the extracellular matrix and prevents its inhibitory action to myoblast proliferation in vitro. However, it still remains unclear whether free decorin could affect the proliferation and differentiation of myogenic cells by regulating myostatin activity. In the present study, we generated stable clonal C2C12 myoblasts that were over-expressing decorin, and showed that decorin over-expressing cells had an increased rate of proliferation as compared to control cells. Decorin over-expressing cells formed multi-giant hypertrophic myotubes with an elongated morphology and larger size as compared to control cells, although the initiation of differentiation in decorin over-expressing cells was somewhat delayed as compared to control cells. Western blot analysis demonstrated that MyoD expression in decorin over-expressing cells was lower than that in control cells until 12 h after induction to differentiate. At 48-h differentiation, the expressions of MyoD, p21 and myogenin were dramatically increased in cells that over-expressed decorin. Furthermore, we revealed that over-expression of decorin suppressed the activity of myostatin endogenously synthesized in C2C12 myoblasts and attenuated the signaling of exogenous myostatin. Consistent with these results, knock-down of decorin impairs C2C12 myoblast growth by increasing the sensitivity to exogenous myostatin. These results clearly show that decorin enhances the proliferation and differentiation of C2C12 myoblasts through suppressing myostatin activity.
核心蛋白聚糖是一种富含亮氨酸的小分子蛋白聚糖，在细胞生长调节中起重要作用。我们最近的研究表明，固定在胶原基质中的核心蛋白聚糖将肌生成抑制素隔离到细胞外基质中，并在体外阻止其对成肌细胞增殖的抑制作用。然而，游离的核心蛋白聚糖是否能通过调节肌生成抑制素活性来影响成肌细胞的增殖和分化仍不清楚。在本研究中，我们构建了稳定克隆的过表达核心蛋白聚糖的C2C12成肌细胞，并表明与对照细胞相比，过表达核心蛋白聚糖的细胞增殖速率增加。与对照细胞相比，过表达核心蛋白聚糖的细胞形成了多巨型肥大肌管，其形态细长且尺寸更大，尽管与对照细胞相比，过表达核心蛋白聚糖的细胞分化起始有所延迟。蛋白质印迹分析表明，在诱导分化后12小时之前，过表达核心蛋白聚糖的细胞中MyoD的表达低于对照细胞。在分化48小时时，过表达核心蛋白聚糖的细胞中MyoD、p21和肌细胞生成素的表达显著增加。此外，我们发现核心蛋白聚糖的过表达抑制了C2C12成肌细胞内源性合成的肌生成抑制素的活性，并减弱了外源性肌生成抑制素的信号传导。与这些结果一致，敲低核心蛋白聚糖会通过增加对外源性肌生成抑制素的敏感性来损害C2C12成肌细胞的生长。这些结果清楚地表明，核心蛋白聚糖通过抑制肌生成抑制素活性来增强C2C12成肌细胞的增殖和分化。
