α3β1 Integrin-mediated MC3T3-E1 Osteoblasts Adherence Induced by Interleukin-1α

Objective: Osteoblasts play a central role in bone formation during healing phase of periapical periodontitis. Adhesion of osteoblasts to extracellular matrix proteins initiates bone formation at periapical lesion. Integrins compose a superfamily of cell surface receptors involved in cell-cell and cell-matrix adhesion. We have previously reported that interleukin (IL)-1α increased during periapical healing phase in a rat experimental periapical lesion (Arias et al, J Endod, 2007). However, biological roles of IL-1α on periapical healing are still unknown. In the present study, we investigated the IL-1α-induced integrin expression and adherence in MC3T3-E1 osteoblasts to clarify the possible mechanisms of osteoblasts adhesion-mediated periapical healing.

Methods: Mouse osteoblastic cell line, MC3T3-E1 (Riken), was treated with IL-1α (0-10 ng/ml; R&D) for 24 hours, and the cells (1 × 10⁶ cells) were seeded in a 6-well tissue culture plate. Cell morphology was observed by phase-contrast microscopy. Non-adherent cells were removed by washing 3 times with phosphate-buffered saline solution, and adherent cells were counted using hemocytometer. Next, cells were treated with IL-1α for 24 hours, and the total cell lysates were collected for detecting integrin subunits α3 and β1 using Western blotting. In addition, to investigate the effects of p38 MAPK pathway on integrin expression, the specific chemical inhibitor SB203580 (10 μM; Calbiochem) was used.

Results: The attachment of MC3T3-E1 osteoblasts to the culture plates after stimulation with IL-1α increased significantly compared to that of non-stimulated cells (Student's t-test, p＜0.05). Furthermore, IL-1α enhanced the expression of integrin subunits α3, but not β1, in MC3T3-E1 osteoblasts, and SB203580 dramatically suppressed IL-1α-induced expression of integrin subunit α3.

Conclusions: Our findings suggest that the enhancement of α3 integrin expression via p38 MAPK pathway by IL-1α may represent an important mechanism for adherence of MC3T3-E1 osteoblasts in periapical healing.