Pleckstrin Levels Are Increased in Patients with Chronic Periodontitis and Regulated via the MAP Kinase-p38α Signaling Pathway in Gingival Fibroblasts

Abstract

Chronic periodontitis (CP) is a bacteria-driven inflammatory disease characterized by the breakdown of gingival tissue, the periodontal ligament, and alveolar bone, leading ultimately to tooth loss. We previously reported the pleckstrin gene (PLEK) to be highly upregulated in gingival tissue of patients with CP and the only gene concurrently upregulated in other inflammatory diseases including rheumatoid arthritis and cardiovascular diseases. Using saliva from 169 individuals diagnosed with CP and healthy controls, we investigated whether pleckstrin could serve as a novel biomarker of periodontitis. Additionally, we explored signal pathways involved in the regulation of PLEK using human gingival fibroblasts (HGFs). Pleckstrin levels were significantly higher (p < 0.001) in the saliva samples of patients with CP compared to controls and closely associated with CP severity. Immunohistochemical analysis revealed the expression of pleckstrin in inflammatory cells and gingival fibroblasts of CP patients. To explore the signal pathways involved in pleckstrin regulation, we stimulated HGFs with either interleukin-1β (IL-1β) or lipopolysaccharides (LPS) alone, or in combination with inhibitors targeting c-Jun N-terminal kinase, tyrosine kinase, protein kinase C, or p38 MAP kinase. Results showed that IL-1β and LPS significantly increased PLEK mRNA and pleckstrin protein levels. VX-745, the p38 MAP kinase inhibitor significantly decreased IL-1β- and LPS-induced pleckstrin levels at both the mRNA and the protein level. Together, these findings show that pleckstrin could serve as a salivary biomarker for the chronic inflammatory disease periodontitis and a regulator of inflammation via the p38 MAP kinase pathway.

Keywords: MAP kinase pathway; PLEK; chronic periodontitis; gingival fibroblasts; inflammation; mPGES-1; pleckstrin.

Figure 1

Pleckstrin levels in saliva samples of patients with CP and controls. Bar plot showing salivary pleckstrin concentrations (ng/ml) of patients with CP compared with healthy controls. Data are presented as the mean values ± SEM. Significance was calculated following Mann Whitney test. ***p-value < 0.001. CP, Chronic periodontitis.

Figure 2

Immunohistochemical staining of Pleckstrin expression in gingival tissue biopsies. (A) Representative histological staining of gingival tissue of patients with CP stained with DAB showing the detection of pleckstrin in gingival inflammatory cells and fibroblasts (black arrows in the highlighted area). (B) Immunofluorescence staining of pleckstrin in human gingival biopsy. The confocal images show the localization of pleckstrin (green) with a DAPI (blue) staining. DAPI was used as a nuclear staining of tissue cells. The square areas were highlighted below the images with inflammatory cells (IC) and fibroblasts (F) for pleckstrin positive staining (white arrows). Scale bars: 20 μm and 50 μm. CP, Chronic periodontitis.

Figure 3

Bar graphs of relative mRNA levels (fold change) of PLEK and mPGES-1 by qPCR. (A) PLEK mRNA levels in HGFs (0.3x10⁶ cells/ml) stimulated with IL-1β (500 pg/ml) for 24 h alone or in combination with SP (20 μM) or PD (2 μM). (B) PLEK mRNA levels in gingival fibroblasts stimulated with P. gingivalis LPS (5μg/ml) alone or in combination with SP or PD. (C) PLEK mRNA levels in HGFs stimulated with IL-1β or LPS alone or in combination with BIS. (D) Gingival fibroblast cells were stimulated with IL-1β and/or LPS alone or in combination with VX-745 (1.0 µM) for 24 h, followed by measurement of relative mRNA levels (fold change) of PLEK. (E) mPGES-1 mRNA level after IL-1β and LPS treatment alone or in combination with VX-745. The relative mRNA levels were normalized to the levels of the housekeeping gene GAPDH. Data are presented as mean values ± SD from at least three independent experiments. Significance was calculated with one-way ANOVA with Tukey’s multiple comparisons test where p-values were set as *p ≤ 0.05 and **p ≤ 0.01. SP, SP600125 (inhibitor of c-Jun N-terminal kinase JNK); PD, PD 153035 hydrochloride (inhibitor of the epidermal growth factor receptor tyrosine kinase EGFR); BIS, Bisindolylmaleimide I, Hydrochloride (Protein kinase C inhibitor); VX-745 , Neflamapimod (selective inhibitor p38α MAPK inhibitor).

Figure 4

Intracellular pleckstrin detection in HGFs analyzed by Flow cytometry. (A) Overlay histogram profile and corresponding bar graph plot (shown below) showing intracellular pleckstrin fold change expression in response to IL-1β treatment alone or in combination with SP in gingival fibroblasts. (B) Overlay histogram profile and corresponding bar graph plot (below) showing intracellular pleckstrin fold change expression after IL-1β treatment alone or in combination with PD in gingival fibroblasts. (C) Overlay histogram profile and corresponding bar graph plot (below) showing intracellular pleckstrin fold change expression after IL-1β treatment alone or in combination with VX-745 in gingival fibroblasts. (D) Overlay histogram profile and corresponding bar graph plot (below) showing intracellular pleckstrin fold change expression after LPS treatment in the absence or presence of VX-745 in gingival fibroblasts. Data are presented as the mean values ± SD from at least three independent experiments. Significance was calculated with one-way ANOVA with Tukey’s multiple comparisons test where p-values were set as *p ≤ 0.05. SP SP600125 (inhibitor of c-Jun N-terminal kinase JNK); PD, PD 153035 hydrochloride (inhibitor of the epidermal growth factor receptor tyrosine kinase EGFR); VX-745, Neflamapimod (selective inhibitor p38α MAPK inhibitor). Raw data images and gating strategies can be found in Supplementary Figure 3 .

Figure 5

Extracellular pleckstrin detection in HGFs using ELISA. (A) Bar plot demonstrating extracellular pleckstrin levels in HGFs stimulated with IL-1β and/or LPS treatment alone or in combination with signal transduction pathway inhibitors SP and PD. (B) Bar plot showing extracellular pleckstrin production after IL-1β or LPS treatment alone or in combination with p38α MAPK inhibitor VX-745 in gingival fibroblasts. (C) Immunofluorescence staining of pleckstrin and mPGES-1 expression after IL-1β treatment for 24 h alone or in combination with p38α MAPK inhibitor VX-745 in gingival fibroblast cells. The confocal images depict the localization of pleckstrin (green) and mPGES-1 (red) with DAPI (blue) staining which was used as a nuclear marker. Isotype or unstained controls for pleckstrin and mPGES-1 staining are used for revealing the specificity of the staining (data not shown here). Scale bars: 20 μm. Data are presented as the mean values ± SD from at least three independent experiments. Significance was calculated with one-way ANOVA with Tukey’s multiple comparisons test where p-values were set as *p ≤ 0.05 and **p ≤ 0.01. SP, SP600125 (inhibitor of c-Jun N-terminal kinase JNK); PD, PD 153035 hydrochloride (inhibitor of the epidermal growth factor receptor tyrosine kinase EGFR); VX-745, Neflamapimod (selective inhibitor p38α MAPK inhibitor).

Figure 6

Visualization and co-localization of pleckstrin protein with mPGES-1 in unstimulated control and IL-1β or LPS stimulated HGFs using confocal microscopy. Immunofluorescence staining of fibroblast cells showing co-localization of pleckstrin (green) with mPGES-1 (red), an inflammatory marker, and DAPI (blue), a nuclear marker. Scale bars: 10 μm and 5μm.