Department of Biochemistry, Dokkyo Medical University School of Medicine, Shimotsuga-gun, Tochigi, Japan.
Department of Integrative Physiology, Graduate School of Medicine, Gunma University, Showa, Maebashi, Gunma, Japan.
PLoS One. 2022 Dec 1;17(12):e0277830. doi: 10.1371/journal.pone.0277830. eCollection 2022.
Silencing Mediator of Retinoid and Thyroid hormone receptors (SMRT; NCoR2) is a transcriptional corepressor (CoR) which has been recognized as an important player in the regulation of hepatic lipogenesis and in somatic development in mouse embryo. SMRT protein is also widely expressed in mouse connective tissues, for example adipocytes and muscle. We recently reported that mice with global deletion of SMRT develop significant obesity and muscle wasting which are independent from thyroid hormone (TH) signaling and thermogenesis. However, the tissue specific role of SMRT in skeletal muscle is still not clear.
To clarify role of SMRT in muscle differentiation, we made myogenic C2C12 clones which lack SMRT protein (C2C12-SKO) by using CRISPR-Cas9. Wild-type C2C12 (C2C12-WT) and C2C12-SKO cells were cultured in differentiation medium, and the resulting gene and protein profiles were compared between the two cell lines both before and after differentiation. We also analyzed muscle tissues which were dissected from whole body SMRT knockout (KO) mice and their controls.
We found significant up-regulation of muscle specific β-oxidation markers; Peroxisome proliferator-activated receptor δ (PPARδ) and PPARγ coactivator-1α (PGC-1α) in the C2C12-SKO cells, suggesting that the cells had a similar gene profile to what is found in exercised rodent skeletal muscle. On the other hand, confocal microscopic analysis showed the significant loss of myotubes in C2C12-SKO cells similar to the morphology found in immature myoblasts. Proteomics analysis also confirmed that the C2C12-SKO cells had higher expression of markers of fibrosis (ex. Collagen1A1; COL1A1 and Fibroblast growth factor-2; FGF-2), indicating the up-regulation of Transforming growth factor-β (TGF-β) receptor signaling. Consistent with this, treatment with a specific TGF-β receptor inhibitor ameliorated both the defects in myotube differentiation and fibrosis.
Taken together, we demonstrate that SMRT functions as a pivotal transcriptional mediator for both β-oxidation and the prevention for the fibrosis via TGF-β receptor signaling in the differentiation of C2C12 myoblasts. In contrast to the results from C2C12 cells, SMRT does not appear to play a role in adult skeletal muscle of whole body SMRT KO mice. Thus, SMRT plays a significant role in the differentiation of myoblasts.
视黄酸和甲状腺激素受体的沉默介体（SMRT；NCoR2）是一种转录核心抑制因子（CoR），已被认为在肝脂肪生成和小鼠胚胎体发育的调节中起着重要作用。SMRT 蛋白在小鼠结缔组织中也广泛表达，例如脂肪细胞和肌肉。我们最近报道称，SMRT 全身性缺失的小鼠会发展出明显的肥胖和肌肉减少症，这与甲状腺激素（TH）信号和产热无关。然而，SMRT 在骨骼肌中的组织特异性作用仍不清楚。
为了阐明 SMRT 在肌肉分化中的作用，我们使用 CRISPR-Cas9 构建了缺乏 SMRT 蛋白的肌源性 C2C12 克隆（C2C12-SKO）。野生型 C2C12（C2C12-WT）和 C2C12-SKO 细胞在分化培养基中培养，并在分化前后比较这两种细胞系的基因和蛋白谱。我们还分析了从全身 SMRT 敲除（KO）小鼠及其对照中分离的肌肉组织。
我们发现 C2C12-SKO 细胞中肌肉特异性β-氧化标志物的表达显著上调；过氧化物酶体增殖物激活受体δ（PPARδ）和过氧化物酶体增殖物激活受体γ共激活因子 1α（PGC-1α），表明这些细胞具有类似于运动后啮齿动物骨骼肌的基因谱。另一方面，共聚焦显微镜分析显示 C2C12-SKO 细胞中的肌管明显丢失，类似于未成熟成肌细胞的形态。蛋白质组学分析还证实，C2C12-SKO 细胞中纤维化标志物（例如胶原蛋白 1A1；COL1A1 和成纤维细胞生长因子 2；FGF-2）的表达更高，表明转化生长因子-β（TGF-β）受体信号的上调。与此一致的是，使用特定的 TGF-β 受体抑制剂治疗可改善肌管分化和纤维化缺陷。
总之，我们证明 SMRT 作为一种关键的转录介体，通过 TGF-β 受体信号在 C2C12 成肌细胞的分化中发挥作用，既促进β-氧化，又防止纤维化。与 C2C12 细胞的结果相反，SMRT 似乎在全身 SMRT KO 小鼠的成年骨骼肌中不起作用。因此，SMRT 在成肌细胞的分化中发挥重要作用。
