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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-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 Cpeptide (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-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 crosslinked 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-1α-dependent manner.

Objective 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). Background 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. Methods HPDLs were cultured under normoxic (20% O2) or hypoxic (1% O2) 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. Results 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. Conclusion PLAP‐1 expression is upregulated under hypoxic conditions through HIF‐1α activation. Moreover, hypoxia‐induced PLAP‐1 expression regulates HIF‐1α signaling.

BackgroundNew cementum forms from existing cementum during periodontal tissue regeneration, indicating that cementoblasts may interact with progenitor cells in the periodontal ligament to enhance cementogenesis. However, the molecular mechanisms of this process are currently unknown. This study aims to clarify the role of cell‐cell interactions between cementoblasts and periodontal ligament cells in differentiation into cementoblasts.MethodsTo analyze the role of human cementoblast‐like cells (HCEMs) on human periodontal ligament cells (HPDLs), we mixed cell suspensions of enhanced green fluorescent protein‐tagged HPDLs and HCEMs, and then seeded and cultured them in single wells (direct co‐cultures). We sorted co‐cultured HPDLs and analyzed their characteristics, including the expression of cementum‐related genes. In addition, we cultured HPDLs and HCEMs in a non‐contact environment using a culture system composed of an upper insert and a lower well separated by a semi‐permeable membrane (indirect co‐cultures), and similar analysis was performed. Gene expression of integrin‐binding sialoprotein (IBSP) in cementoblasts was confirmed in mouse periodontal tissues. We also investigated the effect of Wingless‐type (Wnt) signaling on the differentiation of HPDLs into cementoblasts.ResultsDirect co‐culture of HPDLs with HCEMs significantly upregulated the expression of cementoblast‐related genes in HPDLs, whereas indirect co‐culture exerted no effect. Wnt3A stimulation significantly upregulated IBSP expression in HPDLs, whereas inhibition of canonical Wnt signaling suppressed the effects of co‐culture.ConclusionOur results suggest that direct cell interactions with cementoblasts promote periodontal ligament cell differentiation into cementoblasts. Juxtacrine signaling via the canonical Wnt pathway plays a role in this interaction.