In vitro Evaluation of the Biological Behavior of SOFAT in Osteoblasts

Objectives: A novel T cell–secreted cytokine, known as secreted osteoclastogenic factor of activated T cells (SOFAT), which induces osteoclastic bone resorption in a RANKL-independent manner, has recently been described. In inflammatory conditions, such as periodontitis, osteoblasts are induced to stimulate RANKL and other mediators, which in turn, activate osteoclastic bone resorption. Thus, the aim of the present study was to evaluate the role of SOFAT on osteoblasts.
Methods: Cell proliferation and viability were assessed using the Trypan exclusion method and MTT test, respectively, after 1, 2 and 3 days, in the presence of different doses of SOFAT (0, 100, 250 and 500 ng/ml). In addition, an ELISA array containing 12 different cytokines was evaluated. The expression of up-regulated cytokines in the ELISA array was confirmed by RT-qPCR. The ability of SOFAT to increase RANKL expression was also evaluated, using RT-qPCR and western blotting. Furthermore, based on previous data, 84 different genes of the IL6/STAT3 signaling pathway were also assessed.
Results: None of the three SOFAT concentrations promoted statistically significant alterations in cell proliferation and viability. IL-6, IL-10 and GM-CSF expression were up-regulated (p<0.05) in the presence of SOFAT. These data were confirmed by gene expression. RANKL expression, however, did not differ significantly in either gene or protein levels (p>0.05). Interestingly, the Bcl2, IL-10, IL-22, IL-2ra, IL-6 and Pias3 genes were up-regulated (at least 4 times higher), whilst the IL-2, IL-21 and Tnf genes were down-regulated (at least 4 times lower) in the presence of SOFAT.
Conclusions: In conclusion, SOFAT exerts a possible role over osteoblastic cells, exacerbating the inflammatory environment.