Fig 1.
ANGPTL2 protein levels are increased in GCF from CP patients.
ANGPTL2 protein levels were compared in GCF from CP patients and healthy individuals using the Student’s t-test. Data are expressed as means ± SD. *p < 0.05 vs control.
Fig 2.
ANGPTL2 is upregulated by P. gingivalis LPS in Ca9-22 cells and HGECs.
ANGPTL2 is upregulated by P. gingivalis LPS in Ca9-22 cells and HGECs. (A, B) ANGPTL2 mRNA expression was measured in primed Ca9-22 cells stimulated with (A) 0, 10, 30, 100, 300, or 1000 ng/ml P. gingivalis LPS for 24 h or (B) 30 ng/ml P. gingivalis LPS for 0–48 h. (C) ANGPTL2 mRNA expression was measured in primed HGECs stimulated with 30 ng/ml P. gingivalis LPS for 0–48 h. Differences among groups were analyzed using one-way ANOVA. Data are expressed as means ± SD (n = 3). *p < 0.05, **p < 0.01, *** p < 0.001 vs. control. (D) Western blot showing ANGPTL2 (60 kDa) levels after stimulation with 30 ng/ml P. gingivalis LPS for 24 and 48 h. (E) ANGPTL2 protein levels in Ca9-22 cells culture supernatant after 24 and 48 h stimulation with 30 ng/ml P. gingivalis LPS. (F) ANGPTL2 protein levels in HGEC culture supernatants at 24 and 48 h after stimulation with 30 ng/ml P. gingivalis LPS. Differences among groups were analyzed using one-way ANOVA. Data are expressed as means ± SD (n = 4). *p < 0.05, **p < 0.01 vs. control.
Fig 3.
ANGPTL2 expression is suppressed by TLR2 or TLR4 siRNA in Ca9-22 cells.
(A, B) TLR2 (A) and TLR4 (B) protein levels Ca9-22 cells transfected with TLR2 and TLR4 siRNA (red line), respectively, and control siRNA-transfected cells (black line). (C, D) ANGPTL2 mRNA levels in Ca9-22 cells transfected with TLR2 and TLR4 siRNA after stimulation with 30 ng/ml P. gingivalis LPS for 24 h. Differences among groups were analyzed with the Student’s t-test. Data are expressed as means ± SD (n = 3). *p < 0.05 vs control. (E, F) Western blot analysis of ANGPTL2 protein levels in Ca9-22 cells transfected with TLR2 and TLR4 siRNA after stimulation with 30 ng/ml P. gingivalis LPS for 24 h.
Fig 4.
Inflammatory cytokines are upregulated by rhANGPTL2 in Ca9-22 cells.
(A–C) mRNA levels of inflammatory cytokines were measured in Ca9-22 cells after stimulation with 0–100 ng/ml rhANGPTL2 for 1 h. (D–F) Protein levels of inflammatory cytokines were measured in Ca9-22 cells after stimulation with 10 ng/ml rhANGPTL2 for 3,6,9 and 12 h. Differences among groups were analyzed by one-way ANOVA. Data are expressed as means ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs. control. (G–I) Protein levels of inflammatory cytokines after anti-integrin α5β1 IgG pretreatment followed by stimulation with 10 ng/ml rhANGPTL2 for 9 h. Data are expressed as means ± SD (n = 4). *p < 0.05, **p < 0.01 vs. control. ND: not detectable.
Fig 5.
ANGPTL2 siRNA downregulates the inflammatory cytokine response to P. gingivalis LPS.
(A) ANGPTL2 protein levels in Ca9-22 cells transfected with ANGPTL2 or control siRNA. (B–D) Inflammatory cytokine mRNA levels in Ca9-22 cells transfected with ANGPTL2 or control siRNA after stimulation with P. gingivalis LPS for 24 h. (E–G) Inflammatory cytokine protein levels in Ca9-22 cells transfected with ANGPTL2 or control siRNA after stimulation with P. gingivalis LPS for 24 h. Differences among groups were analyzed using the Student’s t-test. Data are expressed as means ± SD (n = 4). *p < 0.05, **p < 0.01 vs. control siRNA.
Fig 6.
Inflammatory cytokines are downregulated by blocking integrin α5β1.
(A) IL-1β, (B) IL-8, and (C) TNF-α protein levels in Ca9-22 cells pretreated with anti-integrin α5β1 IgG and then stimulated with P. gingivalis LPS for 24 h. Differences among groups were analyzed using one-way ANOVA. Data are expressed as means ± SD (n = 4). *p < 0.05 vs. control. ND: not detectable.
Fig 7.
Diagram summarizing the possible mechanisms responsible for ANGPTL2 function in human gingival epithelial cells.
We identified a novel signaling cascade comprising sequential P. gingivalis LPS stimulation → ANGPTL2 induction → integrin α5β1 → inflammatory cytokine induction, which results in potent periodontal disorganization activity in gingival epithelial cells. The bold line is generally considered as the main pathway mediating inflammatory cytokine production by periodontal pathogens such as P. gingivalis LPS. Non-bold line is shown as a novel signaling pathway via ANGPTL2 in P. gingivalis LPS stimulation. The dashed line is based on a hypothesis that ANGPTL2 is one of key mediator between periodontal disease and systemic disease.