Pannexin3 inhibits TNF-α-induced inflammatory response by suppressing NF-κB signalling pathway in human dental pulp cells

Abstract

Human dental pulp cells (HDPCs) play a crucial role in dental pulp inflammation. Pannexin 3 (Panx3), a member of Panxs (Pannexins), has been recently found to be involved in inflammation. However, the mechanism of Panx3 in human dental pulp inflammation remains unclear. In this study, the role of Panx3 in inflammatory response was firstly explored, and its potential mechanism was proposed. Immunohistochemical staining showed that Panx3 levels were diminished in inflamed human and rat dental pulp tissues. In vitro, Panx3 expression was significantly down-regulated in HDPCs following a TNF-α challenge in a concentration-dependent way, which reached the lowest level at 10 ng/ml of TNF-α. Such decrease could be reversed by MG132, a proteasome inhibitor. Unlike MG132, BAY 11-7082, a NF-κB inhibitor, even reinforced the inhibitory effect of TNF-α. Quantitative real-time PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to investigate the role of Panx3 in inflammatory response of HDPCs. TNF-α-induced pro-inflammatory cytokines, interleukin (IL)-1β and IL-6, were significantly lessened when Panx3 was overexpressed in HDPCs. Conversely, Panx3 knockdown exacerbated the expression of pro-inflammatory cytokines. Moreover, Western blot, dual-luciferase reporter assay, immunofluorescence staining, qRT-PCR and ELISA results showed that Panx3 participated in dental pulp inflammation in a NF-κB-dependent manner. These findings suggested that Panx3 has a defensive role in dental pulp inflammation, serving as a potential target to be exploited for the intervention of human dental pulp inflammation.

Keywords: NF-κB; Pannexin3; TNF-α; human dental pulp cells; proteasome; pulpitis.

Figure 1

The expression of Panx3 was decreased in inflamed human (A) and rat dental pulpitis tissue (B). The dental pulp tissue sections were stained with HE, anti‐Panx3 antibody or anti‐TNF‐α antibody. HE‐stained sections of normal and inflamed dental pulp tissue verified the progression of inflammation. Immunohistochemistry performed on dental pulp tissues showed the presence of Panx3 in normal tissue, but decreased in the inflamed pulp tissue. In contrast to Panx3 expression, TNF‐α was increased in inflamed dental pulp tissue. Cell nuclei were visualized with haematoxylin. Scale bars are stamped in the images, respectively.

Figure 2

TNF‐α repressed Panx3 expression at the mRNA and protein levels in a concentration‐dependent manner in HDPCs. The mRNA and protein expression levels of Panx3 in HDPCs stimulated with TNF‐α (0–10 ng/ml) for 24 hrs were detected by qRT‐PCR (A) and Western blot (B). GAPDH served as an internal control. (C) Immunofluorescence of Panx3 in HDPCs with or without TNF‐α (10 ng/ml) treatment for 24 hrs. Scale bar = 200 μm. Data were expressed as mean ± SEM. *P < 0.05 compared to the group treated without TNF‐α.

Figure 3

MG132 not BAY 11‐7082 rescued the TNF‐α‐induced down‐regulation of Panx3. Cells were pre‐treated with 2 μM BAY 11‐7082, 1 μM MG132 or DMSO for 30 min. before TNF‐α treatment. qRT‐PCR (A, C) and Western blot analysis (B, D) were then performed to determine the Panx3 expression. *P < 0.05 compared to the group without stimulation. ^# P < 0.05 compared to the group treated with TNF‐α alone.

Figure 4

Lentiviral‐mediated expression of Panx3 in HDPCs. (A, B) Cell images of HDPCs/Panx3, HDPC/Plvx, HDPC/shRNA and HCPD/Ctrl groups were photographed in normal light (lower panels) and under a fluorescence microscope (upper panels). Protein and mRNA expression of Panx3 were determined by Western blot (C, D) and qRT‐PCR (E, F) analysis. *P < 0.05 HDPC/Panx3 versus HDPC/Plvx, and HDPC/shRNA versus HDPC/Ctrl. Scale bar: 200 μm.

Figure 5

Panx3 regulates TNF‐α‐induced inflammatory cytokine expression in HDPCs. qRT‐PCR (A, B) and ELISA (C) analysis of IL‐1β and IL‐6 expression in HDPC/Panx3 and HDPC/Plvx cells upon TNF‐α stimulation for 24 hrs. qRT‐PCR (D, E) and ELISA (F) analysis of IL‐1β and IL‐6 expression in HDPC/shRNA and HDPC/Ctrl cells upon TNF‐α stimulation for 24 hrs. *P < 0.05 HDPC/Panx3 versus HDPC/Plvx, and HDPC/shRNA versus HDPC/Ctrl.

Figure 6

Panx3 regulates NF‐κB signalling pathway. (A, B) NF‐κB transcriptional activities in HDPC/Panx3, HDPC/Plvx, HDPC/shRNA and HDPC/Ctrl groups were measured using dual‐luciferase reporter system. *P < 0.05 HDPC/Panx3 versus HDPC/Plvx, HDPC/shRNA versus HDPC/Ctrl. (C, D) Phosphorylated‐p65 and IκBα protein expression were verified by Western blot in HDPC/Panx3, HDPC/Plvx, HDPC/shRNA, HDPC/Ctrl groups with or without TNF‐α stimulation for 30 min. GAPDH was used as the internal control. (E) Elevated nucleus translocation of NF‐κB‐p65 in TNF‐α‐treated HDPCs from the Panx3 silenced group (HDPC/shRNA) compared with HDPC/Ctrl, detected by immunofluorescence. *P < 0.05 HDPC/Panx3 versus HDPC/Plvx, and HDPC/shRNA versus HDPC/Ctrl. Scale bar = 100 μm. (F–H) Inflammatory cytokines expression were quantified by qRT‐PCR and ELISA in HDPC/shRNA, HDPC/Ctrl and HDPC/shRNA‐BAY 11‐7082 groups. ^# P < 0.05 HDPC/shRNA‐ BAY 11‐7082 versus HDPC/shRNA.

Figure 7

Schematic illustration of the role of Panx3 in dental pulp inflammation. TNF‐α could down‐regulate the expression of Panx3 via activating proteasome pathway, meanwhile the NF‐κB might balance the effect. In addition, Panx3 could suppress NF‐κB signalling pathway, leading to inhibition of TNF‐α‐induced inflammatory response. The black lines indicate that the interactions have been clearly established in previous studies. The purple lines indicate the connection established in this study. MG132, a proteasome inhibitor; BAY 11‐7082, a NF‐κB inhibitor.