Department of Pulmonary and Critical Care Medicine, The Affiliated Hospital of Inner Mongolia Medical University, Hohhot, Inner Mongolia Autonomous Region, China 010059.
Health Management Center, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China 510700.
Biomed Res Int. 2021 Apr 8;2021:5551504. doi: 10.1155/2021/5551504. eCollection 2021.
Previous studies have demonstrated the ubiquitin-proteasome inhibitor bortezomib (BTZ) can effectively alleviate hypoxia-induced pulmonary hypertension (HPH) by suppressing the intracellular calcium homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Further evaluation showed that the antiproliferation roles of BTZ are mainly mediated by inhibition of the intracellular calcium homeostasis. Caveolin-1 belongs to one of the key regulators of the intracellular calcium homeostasis in PASMCs, which can regulate the store-operated calcium entry (SOCE). However, the effects of BTZ on Caveolin-1 remain unclear.
Primarily cultured human PASMCs were used as the cell model. CCK-8 assay was performed to assess the PASMCs proliferation. Western blotting and real-time qPCR were used to detect the mRNA and protein expressions. Fura-2-based fluorescence imaging experiments were used to determine the intracellular calcium concentration ([Ca]). The protein synthesis inhibitor cycloheximide (CHX) was utilized to determine the protein degradation process.
Firstly, in cultured human PASMCs, treatment of BTZ for 24 or 60 hours significantly downregulates Caveolin-1 at both mRNA and protein levels. Secondly, in the presence CHX, BTZ treatment also leads to downregulated protein expression and fastened protein degradation of Caveolin-1, indicating that BTZ can promote the Caveolin-1 protein degradation, other than the BTZ on Caveolin-1 mRNA transcription. Then, BTZ significantly attenuates the hypoxia-elevated baseline [Ca], SOCE, and cell proliferation.
We firstly observed that the ubiquitin-proteasome inhibitor BTZ can inhibit the Caveolin-1 expression at both mRNA transcription and protein degradation processes, providing new mechanistic basis of BTZ on PASMC proliferation.
先前的研究表明，蛋白酶体抑制剂硼替佐米（BTZ）可以通过抑制肺动脉平滑肌细胞（PASMC）内的钙稳态来有效缓解低氧诱导的肺动脉高压（HPH）。进一步的评估表明，BTZ 的抗增殖作用主要是通过抑制细胞内钙稳态来介导的。窖蛋白-1（Caveolin-1）属于 PASMC 内细胞钙稳态的关键调节因子之一，可调节储存操纵钙内流（SOCE）。然而，BTZ 对 Caveolin-1 的影响尚不清楚。
原代培养的人 PASMC 作为细胞模型。用 CCK-8 法检测 PASMC 增殖。用 Western blot 和实时 qPCR 检测 mRNA 和蛋白表达。用 Fura-2 荧光成像实验测定细胞内钙浓度([Ca])。用蛋白合成抑制剂环己酰亚胺（CHX）测定蛋白降解过程。
首先，在培养的人 PASMC 中，BTZ 处理 24 或 60 小时可显著下调 Caveolin-1 的 mRNA 和蛋白水平。其次，在 CHX 存在的情况下，BTZ 处理也导致 Caveolin-1 蛋白表达下调和蛋白降解加快，表明 BTZ 可促进 Caveolin-1 蛋白降解，而非 BTZ 对 Caveolin-1 mRNA 转录的影响。然后，BTZ 显著减弱了低氧引起的基础[Ca]、SOCE 和细胞增殖。
我们首次观察到泛素蛋白酶体抑制剂 BTZ 可在 mRNA 转录和蛋白降解过程中抑制 Caveolin-1 的表达，为 BTZ 抑制 PASMC 增殖提供了新的机制基础。
