Regulation of Myogenesis by a Na/K-ATPase α1 Caveolin-Binding Motif.
Regulation of Myogenesis by a Na/K-ATPase α1 Caveolin-Binding Motif.
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DOI：
10.1093/stmcls/sxab012
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发表时间：
2022-03-16
期刊：
影响因子：
--
通讯作者：
Cai L
中科院分区：
文献类型：
--
作者：
Huang M;Wang X;Banerjee M;Mukherji ST;Kutz LC;Zhao A;Sepanski M;Fan CM;Zhu GZ;Tian J;Wang DZ;Zhu H;Xie ZJ;Pierre SV;Cai L
Na/K-ATP酶（NKA）α1亚基N端小窝蛋白结合基序（CBM）在动物细胞信号转导和体节发生中起重要作用。为了进一步研究分子机制，我们通过CRISPR/Cas9基因组编辑产生了CBM突变体人诱导多能干细胞（iPSC），并检查了它们分化为骨骼肌（Skm）细胞的能力。与亲本野生型人iPSC相比，CBM突变体细胞失去了它们的Skm分化能力，这通过在最终分化的iSkm（诱导的Skm）细胞中缺乏自发细胞收缩、标记基因表达和亚细胞肌纤维带结构来证明。另一个NKA功能突变体A420 P缺乏NKA/Src信号传导功能，没有产生类似的缺陷。事实上，A420 P突变体iPSC保留了完整的多能性和Skm分化能力。机制上，生肌转录因子MYOD被CBM突变极大地抑制。通过慢病毒递送过表达小鼠Myod cDNA恢复了CBM突变细胞分化为Skm的能力。在MYOD上游，TOPFlash测定证明Wnt信号传导具有类似的抑制作用。通过Wnt 3a配体或GSK 3抑制剂/Wnt途径激活剂CHIR对前体中胚层标记基因BRACHYURY（T）和MESOGENIN 1（MSGN 1）的缺陷诱导，在功能上进一步证实了这种对Wnt活性的影响。通过免疫荧光成像和细胞分级分离的进一步研究揭示了CBM突变体iPSC中β-连环蛋白的膜定位转移，揭示了NKA-Wnt调控的新分子组分。这项研究揭示了通过NKA的CBM和Wnt/β-catenin信号转导的控制对肌肉发生的遗传调控。Na/K-ATP酶中小窝蛋白结合基序的两个氨基酸突变（F97 A; F100 A）改变了β-catenin的定位，阻断了Wnt信号传导和人诱导多能干细胞向骨骼肌细胞的诱导。膜蛋白Na/K-ATP酶作为一个允许门来控制细胞信号和肌肉细胞谱系特化。
The N-terminal caveolin binding motif (CBM) in Na/K-ATPase (NKA) α1 subunit is essential for cell signaling and somitogenesis in animals. To further investigate the molecular mechanism, we have generated CBM mutant human induced pluripotent stem cells (iPSCs) through CRISPR/Cas9 genome editing and examined their ability to differentiate into skeletal muscle (Skm) cells. Compared to the parental wild type human iPSCs, the CBM mutant cells lost their ability of Skm differentiation, which was evidenced by the absence of spontaneous cell contraction, marker gene expression, and subcellular myofiber banding structures in the final differentiated iSkm (induced Skm) cells. Another NKA functional mutant, A420P, which lacks NKA/Src signaling function, did not produce a similar defect. Indeed, A420P mutant iPSCs retained intact pluripotency and ability of Skm differentiation. Mechanistically, the myogenic transcription factor MYOD was greatly suppressed by the CBM mutation. Overexpression of a mouse Myod cDNA through lentiviral delivery restored the CBM mutant cells’ ability to differentiate into Skm. Upstream of MYOD, Wnt signaling was demonstrated from the TOPFlash assay to have a similar inhibition. This effect on Wnt activity was further confirmed functionally by defective induction of the presomitic mesoderm marker genes BRACHYURY (T) and MESOGENIN1 (MSGN1) by Wnt3a ligand or the GSK3 inhibitor/Wnt pathway activator CHIR. Further investigation through immunofluorescence imaging and cell fractionation revealed a shifted membrane localization of β-catenin in CBM mutant iPSCs, revealing a novel molecular component of NKA-Wnt regulation. This study sheds light on a genetic regulation of myogenesis through the CBM of NKA and control of Wnt/β-catenin signaling. Two amino acid mutation (F97A;F100A) of the caveolin-binding motif in Na/K-ATPase shifted β-catenin localization, blocked Wnt signaling and induction of human induced pluripotent stem cells into skeletal muscle cells. The membrane protein Na/K-ATPase serves as a permissive gate to control cell signaling and muscle cell lineage specification.
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