Hypoxia and P. gingivalis synergistically 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.

Figures

Figure 1

Induction of IL-1β mRNA in PDL cells. PDL cells were cultured under normoxic or hypoxic condition and stimulated with LPS-PG (1 μg/mL). Cells cultured in normoxia without LPS stimulation served as control. IL-1β mRNA expression was analyzed by real-time PCR after 2, 4, 8, 24, and 48 h. Statistical differences were analyzed by one-way ANOVA and Dunnett's test as well as Tukey's multiple comparison test; *P < 0.05 indicates a significant difference between the groups (means ± SD; n = 9).

Figure 2

Expression of MMP-1 in PDL cells. (a) PDL cells were cultured under normoxic or hypoxic condition and stimulated with LPS-PG (1 μg/mL). Untreated cells cultured in normoxia served as control. MMP-1 mRNA expression was quantitated by real-time PCR after 2, 4, 8, 24, and 48 h. Statistical differences were analyzed by one-way ANOVA followed by different post hoc tests (Dunnett's and Tukey's multiple comparison test); a P value < 0.05 was assumed as significant. #P < 0.05 depicts a significant difference with respect to the time-matched control, *P < 0.05 indicates a significant difference between groups (means ± SD; n = 9). (b) MMP-1 protein secretion by PDL fibroblasts was determined using the Quantikine enzyme-linked immunoabsorbent assay (ELISA) system according to the manufacturer's instruction. Supernatants of PDL cells stimulated with or without LPS-PG (1 μg/mL) under normoxic or hypoxic condition were collected after varying time points (2, 4, 8, 24, and 48 h). Statistical differences were analyzed by one-way ANOVA followed by different post hoc tests (Dunnett's and Tukey's multiple comparison test); a P value < 0.05 was assumed as significant. *P < 0.05 indicates a significant difference between groups (means ± SD; n = 9).

Figure 3

Activation of NF-κB. (a) NF-κB was visualized by immunofluorescence staining. PDL cells were cultured under normoxic (control) or hypoxic condition (Hox) and stimulated with LPS-PG (1 μg/mL). NF-κB activation was visible as enhanced nuclear staining. Representative pictures of control cells and a 1 hour LPS-PG treatment under hypoxic conditions are shown. The scale bars indicate 100 μm. (b) Density of NF-κB nuclear staining after 1, 2, and 4 h treatment was determined in relation to total cell area using the freely available image-processing software ImageJ 1.43 (http://rsb.info.nih.gov/ij). Statistical analysis of the processed immunofluorescence data were performed by one-way ANOVA followed by Dunnett's test and Tukey's multiple comparison test; a P value < 0.05 was assumed as significant. #P < 0.05 depicts a significant difference with respect to time-matched control, *P < 0.05 indicates a significant difference between groups (means ± SD; n = 6). (c) Tissue samples from healthy gingiva (I), gingivitis (II), healthy periodontal ligament (III), and periodontitis (IV) were obtained after approval of the Ethics Committee of the University of Bonn and parental as well as patients' written consent (n = 3). Polyclonal primary antibody raised against the p65 subunit of NF-κB was used in a concentration of 1 : 100 for immunohistochemistry. We observed an increase in NF-κB immunostaining in accordance to the progression of periodontal inflammation. Arrows indicate the nuclear staining of NF-κB in endothelial cells as well as immune cells in tissue samples of periodontitis. The scale bars indicate 50, 100, or 200 μm in dependence of the magnifications.

Figure 4

Activation of HIF-1α. (a) PDL cells were incubated for the indicated time periods with LPS-PG under normoxic or hypoxic conditions. HIF-1α mRNA expression was quantitated by real-time PCR, and expression levels were calculated as n-fold induction of the respective time matched untreated normoxic control. Statistical differences were analyzed by one-way ANOVA followed by different post hoc tests (Dunnett's and Tukey's multiple comparison test); #P < 0.05 depicts a significant difference with respect to time-matched control, *P < 0.05 indicates a significant difference between groups (means ± SD; n = 6). (b) PDL cells were cultured under normoxic (control) or hypoxic conditions (Hox) and stimulated with LPS-PG (1 μg/mL). HIF-1α protein was visualized by immunofluorescence staining. Representative pictures of cells under normoxic control conditions and after a 2-hour LPS-PG treatment under hypoxic conditions are shown. The scale bars indicate 100 μm. (c) PDL cells were incubated for 1, 2, and 4 hours. HIF-1α protein was detected by immunofluorescence. Density of nuclear HIF-1α staining was determined as in relation to total cell area using the freely available image-processing software ImageJ 1.43 (http://rsb.info.nih.gov/ij). Statistical analysis of the processed data were performed by one-way ANOVA followed by Tukey's multiple comparison test; *P < 0.05 indicates a significant difference between groups (means ± SD; n = 6). (d) Tissue samples from healthy gingiva (I), gingivitis (II), healthy periodontal ligament (III), and periodontitis (IV) were obtained after approval of the Ethics Committee of the University of Bonn and parental as well as patients' written consent (n = 3). Polyclonal primary antibody raised against HIF-1 was used in a concentration of 1 : 100 for immunohistochemistry. We observed an increase of HIF-1α immunostaining in accordance to the progression of periodontal inflammation. The scale bars indicate 50, 100, or 200 μm in dependence of the magnifications.

Figure 5

VEGF protein expression. (a) VEGF protein expression in PDL cells was measured using ELISA. Supernatants of PDL cells, stimulated with or without LPS-PG (1 μg/mL) under normoxic or hypoxic condition, were collected after varying time points (2, 4, 8, 24, and 48 h). Statistical differences were analyzed by one-way ANOVA and the post hoc Dunnett's and Tukey's multiple comparison test; #P < 0.05 depicts a significant difference with respect to the time-matched control, *P < 0.05 indicates a significant difference between groups (means ± SD; n = 9). (b) Healthy gingiva (I), gingivitis (II), healthy periodontal ligament (III), and periodontitis (IV) were obtained after the approval of the Ethics Committee of the University of Bonn and parental as well as patients' written consent (n = 3). A polyclonal primary antibody against VEGF was used in a concentration of 1 : 100 for immunohistochemistry. The scale bars indicate 50, 100, or 200 μm in dependence of the magnifications.