Department of Periodontology, Osaka University Graduate School of Dentistry, Osaka, Japan.
J Periodontol. 2021 Nov;92(11):1635-1645. doi: 10.1002/JPER.20-0670. Epub 2021 Mar 19.
Cellular responses to hypoxia regulate various biological events, including angiogenesis and extracellular matrix metabolism. Collagen is a major component of the extracellular matrix in periodontal tissues and its coordinated production is essential for tissue homeostasis. In this study, we investigated the effects of hypoxia on collagen production in human gingival fibroblasts (HGFs) and human periodontal ligament cells (HPDLs).
HGFs and HPDLs were cultured under either normoxic (20% O ) or hypoxic (1% O ) conditions. Nuclear expression of hypoxia-inducible factor-1α (HIF-1α) was determined by western blotting. Peri-cellular expression of type I collagen was examined by immunocytochemistry analysis. Synthesis of type I collagen was evaluated by measuring the concentration of procollagen type I C-peptide (PIP) in culture supernatant using enzyme-linked immunosorbent assay. Expression of collagen hydroxylase enzymes prolyl 4-hydroxylase alpha polypeptide 1 (P4HA1) and 2-oxoglutarate 5-dioxygenase 2 (PLOD2) was determined by RT-qPCR and western blotting. The roles of these enzymes were analyzed using siRNA transfection.
Cultivation under hypoxic conditions stimulated type I collagen production via HIF-1α in both cell types. Interestingly, hypoxic conditions did not affect collagen 1a1 or 1a2 gene expression but upregulated that of P4HA1 and PLOD2. Additionally, suppressing P4HA1 significantly decreased the levels of hypoxia-induced procollagen type I C-peptide, a product of stable triple helical collagen, in the supernatant. In contrast, PLOD2 suppression decreased cross-linked collagen expression in the pericellular region.
Our results suggest that hypoxia activates collagen synthesis in HGFs and HPDLs by upregulating hydroxylases P4HA1 and PLOD2 in an HIF-1α-dependent manner.
细胞对缺氧的反应调节着各种生物事件，包括血管生成和细胞外基质代谢。胶原蛋白是牙周组织细胞外基质的主要成分，其协调产生对于组织稳态至关重要。在这项研究中，我们研究了缺氧对人牙龈成纤维细胞（HGFs）和人牙周韧带细胞（HPDLs）胶原产生的影响。
将 HGFs 和 HPDLs 在常氧（20% O ）或缺氧（1% O ）条件下培养。通过 Western blot 测定缺氧诱导因子-1α（HIF-1α）的核表达。通过免疫细胞化学分析检测细胞周围 I 型胶原蛋白的表达。通过酶联免疫吸附试验测量培养上清中 I 型前胶原 C 肽（PIP）的浓度来评估 I 型胶原的合成。通过 RT-qPCR 和 Western blot 测定胶原羟化酶脯氨酰 4-羟化酶 α 多肽 1（P4HA1）和 2-氧戊二酸 5-双加氧酶 2（PLOD2）的表达。使用 siRNA 转染分析这些酶的作用。
在两种细胞类型中，缺氧培养条件通过 HIF-1α刺激 I 型胶原蛋白的产生。有趣的是，缺氧条件不影响胶原 1a1 或 1a2 基因表达，但上调了 P4HA1 和 PLOD2 的表达。此外，抑制 P4HA1 可显著降低上清液中缺氧诱导的稳定三螺旋胶原的前胶原 I C 肽水平。相比之下，抑制 PLOD2 可降低细胞周围区域交联胶原蛋白的表达。
我们的结果表明，缺氧通过 HIF-1α 依赖性方式上调羟化酶 P4HA1 和 PLOD2 激活 HGFs 和 HPDLs 中的胶原合成。
