Calcium Signal Facilitates the Immediate Induction of HIF-1α by Lipopolysaccharide

Objectives: hypoxia-inducible factor 1 alpha (HIF-1α) is an oxygen-sensitive nuclear transcription factor, which responds to hypoxia or non-hypoxia stimuli by transcribing dozens of downstream genes and participates profoundly in cellular physiology. The regulation of HIF-1α is typically through oxygen-sensitive prolyl hydroxylases (PHDs), which hydroxylize HIF-1α to facilitate its degradation. Other mechanisms include the regulation of the transcription and translation of HIF-1α. We previously reported that lipopolysaccharide (LPS) induces HIF-1α in human primary gingival fibroblasts (HGF). The present study aimed to explore the underlying mechanism via looking into Ca²⁺/Calmodulin-dependent protein kinase II (CaMKII) and calcineurin pathway. Methods: Human gingival biopsies were harvested from teeth scheduled for extraction because of orthodontic reasons. HGFs were cultured and subjected to LPS treatment only or in combination with actinomycin D (transcription inhibitor), dimethyloxalylglycine (DMOG, PHDs inhibitor), KN-93 (CaMKII inhibitor) or cyclosporin A (calcineurin inhibitor). Realtime RT-PCR, immunoprecipitation and western blotting were performed to detect the transcript and peptide of HIF-1α. Results: Although HIF-1α accumulation could be detected as early as 3 hours after LPS challenge, the transcription level of HIF-1α gene did not increase until after 6 hours. Moreover, when transcription was prohibited by actinomycin D, LPS still induced HIF-1α. Similarly, LPS enhanced the HIF-1α accumulation in DMOG-treated cells, indicating that the LPS effect on HIF-1α may not be through actions of PHDs. However, cyclosporin A and KN-93 dramatically attenuated the LPS-induced HIF-1α accumulation. Conclusions: Ca²⁺-CaMKII-calcineurin pathway may be essential to the immediate accumulation of HIF-1α in HGFs as a response to LPS challenge.