Hypoxia stimulates collagen hydroxylation in gingival fibroblasts and periodontal ligament cells
Hypoxia stimulates collagen hydroxylation in gingival fibroblasts and periodontal ligament cells
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DOI：
10.1002/jper.20-0670
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发表时间：
2021-03-19
影响因子：
4.3
通讯作者：
Murakami, Shinya
中科院分区：
文献类型：
--
作者：
Morimoto, Chiaki;Takedachi, Masahide;Murakami, Shinya
背景细胞对缺氧的反应调节各种生物学事件，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，其协调产生对组织稳态至关重要。本研究旨在探讨低氧对人牙龈成纤维细胞（HGFs）和人牙周膜细胞（HPDLs）胶原合成的影响。缺氧诱导因子-1 α（HIF-1 α）的核表达通过蛋白质印迹法测定。免疫细胞化学分析检测细胞周围I型胶原的表达。I型胶原的合成通过使用酶联免疫吸附测定法测量培养上清液中I型前胶原C肽（PIP）的浓度来评价。通过RT-qPCR和蛋白质印迹法测定胶原羟化酶脯氨酰4-羟化酶α多肽1（P4 HA 1）和2-酮戊二酸5-双加氧酶2（PLOD 2）的表达。这些酶的作用进行了分析，使用siRNA transfection.Results缺氧条件下的培养刺激I型胶原蛋白的生产，通过HIF-1 α在两种细胞类型。有趣的是，低氧条件下不影响胶原1a 1或1a 2基因的表达，但上调P4 HA 1和PLOD 2。此外，抑制P4 HA 1显着降低缺氧诱导的前胶原I型C肽，稳定的三螺旋胶原蛋白的产品，在上清液中的水平。相反，PLOD 2抑制降低交联胶原蛋白的表达在pericellular region.Conclusion我们的研究结果表明，缺氧激活胶原蛋白的合成在HGF和HPDL通过上调羟化酶P4 HA 1和PLOD 2在HIF-1 α依赖的方式。
Background 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).Methods HGFs and HPDLs were cultured under either normoxic (20% O-2) or hypoxic (1% O-2) conditions. Nuclear expression of hypoxia-inducible factor-1 alpha (HIF-1 alpha) 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.Results Cultivation under hypoxic conditions stimulated type I collagen production via HIF-1 alpha 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.Conclusion Our results suggest that hypoxia activates collagen synthesis in HGFs and HPDLs by upregulating hydroxylases P4HA1 and PLOD2 in an HIF-1 alpha-dependent manner.