HIF‐2 Inhibition Supresses Inflammatory Responses and Osteoclastic Differentiation in Human Periodontal Ligament Cells

Abstract

Recent reports suggest that hypoxia inducible factor-2α (HIF-2α) is a key regulator of osteoarthritis cartilage destruction. However, the precise role of HIF-2α in the inflammatory response and osteoclast differentiation remains unclear. The purpose of this study was to investigate the effect of HIF-2α on inflammatory cytokines, extracellular matrix (ECM) destruction enzymes, and osteoclastic differentiation in nicotine and lipopolysaccharide (LPS)-stimulated human periodontal ligament cells (PDLCs). HIF-2α was upregulated in chronically inflamed PDLCs of periodontitis patients, and in nicotine- and LPS-exposed PDLC in dose- and time-dependent manners. HIF-2α inhibitor and HIF-2α siRNA attenuated the nicotine- and LPS- induced production of NO and PGE2 , upregulation of iNOS, COX-2, pro-inflammatory cytokines (IL-1β, TNF-α, IL-1β, IL-6, IL-8, IL-10, IL-11, and IL-17), and matrix metalloproteinases (MMPs; MMP-1, -8, -13, -2 and -9), and reversed the effect on TIMPs (TIMP-1 and -2) in PDLCs. The conditioned medium produced by nicotine and LPS-treated PDLCs increased the number of TRAP-stained osteoclasts, TRAP activity and osteoclast-specific genes, which has been blocked by HIF-2α inhibition and silencing. HIF-2α inhibitor and HIF-2α siRNA inhibited the effects of nicotine and LPS on the activation of Akt, JAK2 and STAT3, ERK and JNK MAPK, nuclear factor-κB, c-Jun, and c-Fos. Taken together, this study is the first to demonstrate that HIF-2α inhibition exhibits anti-inflammatory activity through the inhibition of inflammatory cytokines and impairment of ECM destruction, as well as blocking of osteoclastic differentiation in a nicotine- and periodontopathogen-stimulated PDLCs model. Thus, HIF-2α inhibition may be a novel molecular target for therapeutic approaches in periodontitis.

Expression of Phospholipase D in Periodontitis and Its Role in the Inflammatory and Osteoclastic Response by Nicotine‐ and Lipopolysaccharide‐Stimulated Human Periodontal Ligament Cells

“…In this study, CM obtained from nicotine‐ and LPS‐treated HPDLCs increases the number of TRAP‐positive multinucleated BMM cells compared with a treatment of only RANKL and M‐CSF. These results are consistent with the results of previous studies that used CM from nicotine‐ and LPS‐treated osteoblasts 9 and HPDLCs 8 , 15 . PLD1 is induced by RANKL early in the culture of mouse BMMs 46 .…”

“…Therefore, P. gingivalis LPS is a specific TLR2 agonist, and the data may not be applicable to TLR4‐stimulating LPS. LPS was used at a concentration of 1 μg/mL, because previously this concentration has been shown to upregulate COX‐2 and iNOS expression and the production of NO and PGE 2 in HPDLCs 12–15 …”

“…Furthermore, it was demonstrated previously that nicotine and LPS synergistically induced the production of nitric oxide (NO) and PGE 2 and increased inducible NO synthase (iNOS) and COX‐2 expression via heme oxygenase‐1, 11 , 12 hypoxia‐inducible factor (HIF)‐1α, 13 and resistin 14 regulation in HPDLCs. In addition, it was shown that HIF‐2α inhibition 15 and peptidyl‐prolyl cis‐trans isomerase NIMA‐interacting 1 inhibition 8 suppressed nicotine‐ and LPS‐induced inflammatory mediators and osteoclastic differentiation in HPDLCs.…”

Hypoxia‐inducible transcription factors, HIF1A and HIF2A, increase in aging mucosal tissues

“…Similarly, HIF‐2 α , a key regulator of cartilage destruction, was up‐regulated in periodontal ligament cells from diseased tissues. In vitro , HIF‐2 α appeared to contribute to nicotine and lipopolysaccharide induction of inflammatory mediators through multiple transcription factors . We also observed altered ceruloplasmin mRNA levels.…”

“…In vitro, HIF-2a appeared to contribute to nicotine and lipopolysaccharide induction of inflammatory mediators through multiple transcription factors. 80 We also observed altered ceruloplasmin mRNA levels. As ceruloplasmin is induced by hypoxia and inflammation, Iwata et al 81 noted that polymorphonuclear cells from patients with aggressive periodontitis were primed for production of ceruloplasmin that would enhance hypoxia-mediated O 2 generation and increased oxidative stress.…”