Podocalyxin-like Protein 1透過膽固醇生合成路徑促進人類富潛能幹細胞的自我更新能力

Abstract

維持延展性富潛能幹細胞潛能的訊息傳遞機制與膽固醇在潛能幹細胞中的功能還不是被研究得很清楚。本次研究報導了抑制Podocalyxin-like protein 1 (PODXL) 表現會抑制富潛能幹細胞與延展性富潛能幹細胞自我更新的能力，進而造成c-MYC 與端粒酶的蛋白表現量下降，並且抑制逆編程誘導性富潛能幹細胞與逆編程延展性富潛能幹細胞的細胞聚落產生的數量。反之，若增強Podocalyxin-like protein 1 (PODXL) 表現，則會促進富潛能幹細胞與延展性富潛能幹細胞自我更新的能力，進而造成c-MYC 與端粒酶的蛋白表現量上升，並且增加逆編程誘導性富潛能幹細胞與逆編程延展性富潛能幹細胞的細胞聚落產生的數量。我們也發現PODXL 可以調控hydroxymethylglutaryl-CoA reductase (HMGCR) 與sterol regulatory element-binding proteins 1/2 (SREBP1/2) 的表現量去調控細胞內膽固醇的生合成。更重要地，富潛能幹細胞相對於成體幹細胞 (例如: 神經性成體幹細胞，骨間質幹細胞) 與已經分化的體細胞 (例如: 纖維母細胞)，對於膽固醇抑制劑處理後的反應更為敏感，結果造成自我更新能力下降。 然而，補充膽固醇可以彌補因為抑制PODXL表現，所造成的富潛能幹細胞自我更新能力的下降。我們更進一步地發現PODXL 可以透過調控脂筏(lipid rafts)的形成，並且透過控制F-actin骨架蛋白纖維形成與膽固醇合成，進而促進新生脂筏形成; 並調節脂筏性蛋白分子integrin α2 (ITGA2) 表現。抑制ITGA2 後的確也會抑制富潛能幹細胞自我更新能力的下降。總結，本研究揭露PODXL在富潛能幹細胞與延展性富潛能幹細胞中扮演了透過調控膽固醇細胞代謝維持自我更新能力的重要地位。

The signaling mechanisms of extended pluripotency and cholesterol in human pluripotent stem cells (hPSCs) have rarely been investigated. Here, we report that downregulation of Podocalyxin-like protein 1 (PODXL) in undifferentiated hPSCs significantly blocks self-renewal, leads to decreased protein expression of c-MYC and telomerase, and inhibits the formation of induced pluripotent stem cell (iPSC) and extended pluripotent stem cell (EPSC) colonies. Accordingly, overexpression of PODXL promotes hPSC self-renewal, c-MYC and telomerase expression, and iPSC/EPSC formation. PODXL also regulates hydroxymethylglutaryl-CoA reductase (HMGCR) and sterol regulatory element-binding proteins 1/2 (SREBP1/2) expression. Importantly, hPSCs are more sensitive to cholesterol depletion than neural stem cells, bone marrow stem cells and fibroblasts, resulting in pluripotency loss, whereas cholesterol can fully restore PODXL knockdown-mediated pluripotency loss. Moreover, PODXL regulates lipid raft formation possibly by serving as a nucleating factor to activate actin polymerization and coupled with cholesterol biosynthesis and transmits signals by controlling integrin α2 (ITGA2) in lipid rafts. Finally, loss of ITGA2 blocks hPSC renewal. Our data highlight the important roles of PODXL in controlling cholesterol metabolism to achieve hPSC self-renewal.