Gingipains disrupt F-actin and cause osteoblast apoptosis via integrin ß1.

ABSTRACT

BACKGROUND AND OBJECTIVE: The aim of this study was to explore the cellular mechanisms underlying gingipain-caused changes in cell morphology and apoptosis of osteoblasts. MATERIAL AND METHODS: Human calvarial osteoblasts and mouse osteoblasts MC3T3-E1 were treated with gingipain extracts from Porphyromonas gingivalis stain W83. Apoptosis was detected with annexin V and propidium iodide flow cytometry analysis or terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling staining. F-actin was determined by immunostaining. Western blotting was used to detect protein expression. Knocking down and overexpressing approaches were used to determine the role of integrin ß1. RESULTS: Osteoblasts exposed to gingipain extracts displayed increased apoptosis, accompanied by loss of F-actin integrity and cell shrinkage. The effects of gingipain extracts were abolished by the cysteine protease inhibitor N-tosyl-l-lysyl chloromethyl-ketone. Notably, gingipain extracts resulted in reduction of integrin ß1, accompanied by diminished active RhoA whereas without effect on the total RhoA. Knockdown of integrin ß1 resembled those seen in gingipain-treated osteoblasts. By contrast, the effects of gingipain extracts were abrogated by either overexpression of integrin ß1 or presence of RhoA agonist CN03. CONCLUSION: Gingipain-induced F-actin disruption and apoptosis are mediated by the degradation of integrin ß1 and inhibition of RhoA activity, which account for osteoblast apoptosis.