Hypoxia Regulates PLAP-1 Expression in Periodontal Ligament Cells

Objectives: Cellular responses to hypoxia regulate various biological events including inflammation and tissue regeneration via activation of hypoxia-inducible factor (HIF)-1a and several hypoxic responses of PDLCs have also been reported. PLAP-1 (Periodontal Ligament Associated Protein-1), an extracellular matrix specifically expressed in the periodontal ligament, plays important roles in cellular functions of periodontal ligament cells (PDLCs). However, the involvement of PLAP-1 in the hypoxic responses has not been examined yet. In this study, we investigated the mutual regulation between the hypoxic responses and the PLAP-1 expression in PDLCs.

Methods: We cultured PDLCs in normoxic (20%O₂) or hypoxic condition (1%O₂). In some experiments, PDLCs were cultured with or without deferoxamine (DFO, inhibitor of HIF-1a degradation), or chetomin (HIF inhibitor). PLAP-1 expression was examined by RT-qPCR and western blotting. Chromatin immunoprecipitation (ChIP) analysis was performed to elucidate whether HIF-1a would increase PLAP-1 mRNA expression under hypoxic condition. We also examined whether PLAP-1 would modulate HIF-1a signaling in hypoxic response of PDLCs by treating PDLCs with PLAP-1 siRNA.

Results: RT-qPCR and western blotting revealed that both hypoxia and DFO treatments upregulated PLAP-1 expression, and chetomin and HIF-1a siRNA transfection suppressed the hypoxia-induced PLAP-1 expression in PDLCs. ChIP analysis demonstrated that HIF-1a bound to the PLAP-1 promoter region. Furthermore, PLAP-1 siRNA transfection suppressed the expression of HIF-1-targeted genes such as vascular endothelial growth factor (VEGF) under hypoxia.
Conclusions: Our results demonstrated that the expression of PLAP-1 is upregulated in hypoxic condition through HIF-1a activation and that hypoxia-induced PLAP-1 could modulate HIF-1a signaling of PDLCs.