Rationale

Forkhead box M1 (FoxM1) is a transcription factor that promotes cell proliferation by regulating a broad spectrum of genes that participate in cell cycle regulation, such as Cyclin B, CDC25B, and Aurora B Kinase. We have shown that hypoxia, a well-known stimulus for pulmonary hypertension (PH), induces FoxM1 in pulmonary artery smooth muscle cells (PASMC) in a HIF-dependent pathway, resulting in PASMC proliferation, while the suppression of FoxM1 prevents hypoxia-induced PASMC proliferation. However, the implications of FoxM1 in the development of PH remain less known.

Methods

We determined FoxM1 levels in the lung samples of idiopathic PAH (pulmonary arterial hypertension) (IPAH) patients and hypoxia-induced PH mice. We generated constitutive and inducible smooth muscle cell (SMC)-specific FoxM1 knockdown or knockout mice as well as FoxM1 transgenic mice which overexpress FoxM1, and exposed them to hypoxia (10% O₂, 90% N₂) or normoxia (Room air, 21% oxygen) for four weeks, and measured PH indices. We also isolated mouse PASMC (mPASMC) and mouse embryonic fibroblasts (MEF) from these mice to examine the cell proliferation and expression levels of SMC contractile proteins.

Results

We showed that in hypertensive human lungs or mouse lungs, FoxM1 levels were elevated. Constitutive knockout of FoxM1 in mouse SMC caused early lethality, whereas constitutive knockdown of FoxM1 in mouse SMC prevented hypoxia-induced PH and PASMC proliferation. Inducible knockout of FoxM1 in SMC reversed hypoxia-induced pulmonary artery wall remodeling in existing PH. Overexpression of FoxM1 enhanced hypoxia-induced pulmonary artery wall remodeling and right ventricular hypertrophy in mice. Alteration of FoxM1 status did not affect hypoxia-induced hypoxia-inducible factor (HIF) activity in mice. Knockout of FoxM1 decreased PASMC proliferation and induced expression of SMC contractile proteins and TGF-β/Smad3 signaling.

Conclusions

Our studies provide clear evidence that altered FoxM1 expression in PASMC contributes to PH and uncover a correlation between Smad3-dependent signaling in FoxM1-mediated proliferation and de-differentiation of PASMC.

中文翻译：

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平滑肌细胞特异性FoxM1控制缺氧引起的肺动脉高压

基本原理

叉头盒M1（FoxM1）是一种转录因子，可通过调控参与细胞周期调控的广泛基因（如细胞周期蛋白B，CDC25B和Aurora B激酶）来促进细胞增殖。我们已经表明，缺氧是一种众所周知的肺动脉高压（PH）刺激，它以HIF依赖性途径在肺动脉平滑肌细胞（PASMC）中诱导FoxM1，从而导致PASMC增殖，而抑制FoxM1可以防止缺氧引起的PASMC增殖。但是，FoxM1在PH的发展中的影响仍然鲜为人知。

方法

我们确定了特发性PAH（肺动脉高压）（IPAH）患者和低氧诱导的PH小鼠的肺样品中的FoxM1水平。我们产生组成型和诱导平滑肌细胞（SMC）特异性敲低FOXM1或敲除小鼠以及FOXM1的转基因小鼠过表达FOXM1，并将它们暴露于缺氧（10％氧气₂，90％N ₂）或含氧量正常（室内空气，氧气（21％氧气）持续4周，并测量PH指数。我们还从这些小鼠中分离了小鼠PASMC（mPASMC）和小鼠胚胎成纤维细胞（ MEF），以检查SMC收缩蛋白的细胞增殖和表达水平。

结果

我们显示在高血压的人肺或小鼠肺中，FoxM1水平升高。小鼠SMC中FoxM1的本构性敲除导致早期致死性，而小鼠SMC中FoxM1的本构性敲除阻止了低氧诱导的PH和PASMC增殖。SMC中FoxM1的诱导性敲除逆转了现有PH中低氧诱导的肺动脉壁重塑。FoxM1的过表达增强缺氧诱导的小鼠肺动脉壁重塑和右心室肥大。FoxM1状态的改变不会影响小鼠中的缺氧诱导的缺氧诱导因子（HIF）活性。FoxM1的敲除降低了PASMC的增殖，并诱导了SMC收缩蛋白和TGF-β/ Smad3信号的表达。

结论

我们的研究提供了明确的证据，表明PASMC中FoxM1表达的改变有助于PH值，并揭示FoxM1介导的PASMC增殖和去分化中Smad3依赖性信号传导之间的相关性。