Department of Periodontology, Osaka University Graduate School of Dentistry, Osaka, Japan.
Department of Periodontology and Endodontology, Tohoku University Graduate School of Dentistry, Sendai, Japan.
J Periodontal Res. 2022 Jun;57(3):470-478. doi: 10.1111/jre.12976. Epub 2022 Feb 9.
To investigate the mutual regulation of hypoxia-inducible factor (HIF)-1α activity and periodontal ligament-associated protein-1 (PLAP-1) expression in human periodontal ligament cells (HPDLs).
Cellular responses to hypoxia regulate various biological events (e.g., inflammation and tissue regeneration) through activation of HIF-1α. PLAP-1, an extracellular matrix protein preferentially expressed in the periodontal ligament, plays important roles in the functions of HPDLs. Although PLAP-1 expression has been demonstrated in hypoxic regions, the involvement of PLAP-1 in responses to hypoxia has not been revealed.
HPDLs were cultured under normoxic (20% O ) or hypoxic (1% O ) conditions with or without deferoxamine mesylate (chemical hypoxia inducer) or chetomin (HIF signaling inhibitor). Expression levels of PLAP-1 and HIF-1α were examined by real-time reverse transcription-polymerase chain reaction and western blot analysis. Luciferase reporter assays of HIF-1α activity were performed using 293T cells stably transfected with a hypoxia response element (HRE)-containing luciferase vector in the presence or absence of recombinant PLAP-1 or PLAP-1 gene transfection.
Cultivation under hypoxic conditions elevated the gene and protein expression levels of PLAP-1 in HPDLs. Deferoxamine mesylate treatment also enhanced PLAP-1 expression in HPDLs. Hypoxia-induced PLAP-1 expression was significantly suppressed in the presence of chetomin. PLAP-1-suppressed HPDLs showed increased HIF-1α accumulation in the nucleus during culture under hypoxic conditions, but not in the presence of recombinant PLAP-1. In the presence of recombinant PLAP-1, hypoxia-induced HRE activity of 293T cells was significantly suppressed in a dose-dependent manner. Transfection of the PLAP-1 gene resulted in a significant reduction of HRE activity during culture under hypoxic conditions.
PLAP-1 expression is upregulated under hypoxic conditions through HIF-1α activation. Moreover, hypoxia-induced PLAP-1 expression regulates HIF-1α signaling.
探讨缺氧诱导因子（HIF）-1α活性与牙周韧带相关蛋白-1（PLAP-1）在人牙周膜细胞（HPDL）中的相互调节作用。
细胞对缺氧的反应通过激活 HIF-1α调节各种生物学事件（如炎症和组织再生）。PLAP-1 是一种优先在牙周韧带中表达的细胞外基质蛋白，在 HPDL 的功能中发挥重要作用。尽管已经在缺氧区域中检测到了 PLAP-1 的表达，但尚未揭示 PLAP-1 在缺氧反应中的参与情况。
将 HPDL 在常氧（20% O ）或缺氧（1% O ）条件下培养，有或没有甲磺酸去铁胺（化学缺氧诱导剂）或 chetomin（HIF 信号抑制剂）。通过实时逆转录聚合酶链反应和 Western blot 分析检测 PLAP-1 和 HIF-1α 的表达水平。使用 293T 细胞进行 HIF-1α 活性的荧光素酶报告基因分析，该细胞稳定转染了含有缺氧反应元件（HRE）的荧光素酶载体，存在或不存在重组 PLAP-1 或 PLAP-1 基因转染。
在缺氧条件下培养会提高 HPDL 中 PLAP-1 的基因和蛋白表达水平。甲磺酸去铁胺处理也增强了 HPDL 中的 PLAP-1 表达。在 chetomin 存在的情况下，缺氧诱导的 PLAP-1 表达显著受到抑制。在缺氧条件下培养时，PLAP-1 抑制的 HPDL 显示核内 HIF-1α积累增加，但不存在重组 PLAP-1。在存在重组 PLAP-1 的情况下，293T 细胞的 HRE 活性在缺氧诱导下呈剂量依赖性显著抑制。PLAP-1 基因转染可显著降低缺氧条件下 HRE 活性。
PLAP-1 表达在缺氧条件下通过 HIF-1α 激活而上调。此外，缺氧诱导的 PLAP-1 表达调节 HIF-1α 信号。
