Hypoxia andP. gingivalisSynergistically Induce HIF-1 and NF-κB Activation in PDL Cells and Periodontal Diseases

Abstract

Periodontitis is characterized by deep periodontal pockets favoring the proliferation of anaerobic bacteria like Porphyromonas gingivalis (P. gingivalis), a periodontal pathogen frequently observed in patients suffering from periodontal inflammation. Therefore, the aim of the present study was to investigate the signaling pathways activated by lipopolysaccharide (LPS) of P. gingivalis (LPS-PG) and hypoxia in periodontal ligament (PDL) cells. The relevant transcription factors nuclear factor-kappa B (NF-κB) and hypoxia inducible factor-1 (HIF-1) were determined. In addition, we analyzed the expression of interleukin- (IL-) 1β, matrix metalloproteinase-1 (MMP-1), and vascular endothelial growth factor (VEGF) in PDL cells on mRNA and protein level. This was accomplished by immunohistochemistry of healthy and inflamed periodontal tissues. We detected time-dependent additive effects of LPS-PG and hypoxia on NF-κB and HIF-1α activation in PDL cells followed by an upregulation of IL-1β, MMP-1, and VEGF expression. Immunohistochemistry performed on tissue samples of gingivitis and periodontitis displayed an increase of NF-κB, HIF-1, and VEGF immunoreactivity in accordance with disease progression validating the importance of the in vitro results. To conclude, the present study underlines the significance of NF-κB and HIF-1α and their target genes VEGF, IL-1β, and MMP-1 in P. gingivalis and hypoxia induced periodontal inflammatory processes.

Regulation of p53 under hypoxic and inflammatory conditions in periodontium

“…In these inflamed tissues, p53 was not restricted to the nucleus; in fact, we detected this transcription factor also in the cytoplasm. As we have demonstrated recently for HIF-1α and NF-κB, transcription factors are regular components of the cytoplasm [29]. The cytoplasmic effects of p53 include involvement in biological processes such as autophagy and oxidative stress response [33].…”

“…Using explant technique, PDL cells were cultured in Dulbecco's minimal essential medium (DMEM, Invitrogen, Karlsruhe, Germany) supplemented with 10 % fetal bovine serum (FBS, Invitrogen) 100 units penicillin and 100 mg/ml streptomycin (Biochrom, Berlin, Germany) at 37°C in a humidified atmosphere of 5 % CO 2 . Cells from 9 donors (mean age 17.1 ± 1.6 years, min-max 15-19 years; gender 4 male/5 female) were phenotyped by specific PDL markers as has been described before [10,29,30]. After phenotyping, the cells were pooled in groups of three in dependence of their characterization results and used at the 4th or 5th passage.…”

“…Cells were lysed with 4 M guanidinthiocyanat (GTC) (Roth, Karlsruhe, Germany), and total RNA was extracted using the acid guanidinium thiocyanate/phenol/chloroform extraction method as has been described before [10,29]. A total of 1 μg of RNA was reverse transcribed into cDNA by use of 200 U of Moloney murine leukemia virus (MMLV) reverse transcriptase (Bio-Rad, Munich, Germany).…”

“…are lipopolysaccharides (LPS) as components of their Gram-negative bacteria cell wall. The inflammatory reactions and mechanical stress induced by chewing, grinding, or orthodontic therapy favor ischemia or hypoxia in the PDL leading to the induction of transcription factors like HIF-1α and nuclear factor kappa B (NF-κB) [28,29]. Both, inflammation and hypoxia, lead to an increase in p53 protein due to production or protein stabilization [17,6].…”

Role of the Hypoxia-Inducible Factor in Periodontal Inflammation

“…Recent studies reported that a hypoxic environment may upregulate proinlammatory cytokines and MMPs' expression from host cells during periodontal disease [34]. The idea was that hypoxia further encourages lipopolysaccharide (LPS)-induced TNF-α, interleukin-1β, and interleukin 6 (IL-6) expressions via LPS toll-like receptor (TLR) interaction that, in turn, activates the nuclear factor kappa B (NF-κB) pathway in human PDL cells upon exposure to the aforementioned Gram-negative bacterial surface component [35][36][37].…”