Reciprocal role of PLAP-1 in HIF-1 alpha-mediated responses to hypoxia

Objective To investigate the mutual regulation of hypoxia-inducible factor (HIF)-1 alpha 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-1 alpha. 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% O-2) or hypoxic (1% O-2) conditions with or without deferoxamine mesylate (chemical hypoxia inducer) or chetomin (HIF signaling inhibitor). Expression levels of PLAP-1 and HIF-1 alpha were examined by real-time reverse transcription-polymerase chain reaction and western blot analysis. Luciferase reporter assays of HIF-1 alpha 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-1 alpha 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-1 alpha activation. Moreover, hypoxia-induced PLAP-1 expression regulates HIF-1 alpha signaling.