Integrin Beta-1 Mediated Osteoblast Apoptosis Induced by Gingipains

Objectives: Gingipains, which are trypsin-like protease from P. gingivalis, degrade cell adhesion molecules (CAMs) and cause cells to undergo apoptosis. Our previous study showed that gingipains induced apoptosis of osteoblasts, but the mechanisms are not completely understood. This study aims to investigate the role of integrin beta-1 (Itgb1) in osteoblast apoptosis induced by gingipains.
Methods: Human Calvarial Osteoblasts (HCOs) and mouse preosteoblasts MC3T3-E1 were treated with gingipains. Apoptosis was detected with Annexin V and PI or TUNEL staining. Real-time PCR or Western blotting was used to detect the mRNA or protein expression. Knocking down Itgb1 by siRNA or over-expressing Itgb1 gene by lentiviral transfection were used to confirm the role of Itgb1 in mediating osteoblast apoptosis induced by gingipains.
Results: After exposure to gingipains, HCOs and MC3T3-E1 cells shrank and detached from the culture surface time-dependently. Flow cytometry analysis of Annexin V and PI staining showed that HCOs and MC3T3-E1 cells underwent apoptosis time-dependently in the presence of gingipains, which was consistent with the morphological alterations. Western blotting showed that Itgb1 protein level of HCOs and MC3T3-E1 cells also decreased in a time dependent manner with the lowest level at 48 h and 24 h, respectively. Membrane protein of MC3T3-E1 cells treated with gingipains showed that gingipains degraded Itgb1 completely in 15 min. After blocking the activity of gingipains with TLCK, gingipains barely had impact on morphology, apoptosis, or Itgb1 expression of HCOs and MC3T3-E1 cells. TLCK also inhibited the degradation of Itgb1 in the membrane protein. Knocking down Itgb1 resulted in apoptosis of MC3T3-E1 cells and decrease of p-ERK and p-AKT. Over-expressing Itgb1 gene partially prevented apoptosis of MC3T3-E1 cells from gingipains, which was accompanied by elevation of p-ERK and p-AKT.
Conclusions: Integrin beta-1 plays a critical role in mediating osteoblast apoptosis induced by gingipains of P. gingivalis.