Methods: Fibroblasts were isolated from keratocystic odontogenic tumor specimens biopsied from patients admitted to Kyushu University Hospital under institutionally approved protocols after obtaining informed consent. The expression of BMP-2 mRNA and protein was measured by quantitative real-time PCR and western immunoblotting or ELISA, respectively.
Results: Elevated levels of [Ca2+]o increased the expression of BMP-2 mRNA in the fibroblasts in a concentration-dependent manner, and a CaSR agonist, neomycin, also stimulated the expression of BMP-2 mRNA. Elevated levels of [Ca2+]o and neomycin increased the expression of BMP-2. Furthermore, a CaSR agonist, R568, increased the secretion of BMP-2, and a CaSR antagonist, NPS2143, inhibited the Ca2+-induced secretion of BMP-2. CaSR mRNA was expressed in the fibroblasts and CaSR protein was detected in the cell surface proteins. CaSR gene silencing with small interfering RNA (siRNA) decreased the expressions of CaSR mRNA and protein, and attenuated the Ca2+-induced expression of BMP-2 mRNA. Elevated levels of [Ca2+]o increased the phosphorylation of p38 MAPK, ERK1/2, and JNK, and induced the translocation of NF-κB p65 to the nucleus in the fibroblasts. Furthermore, inhibitors for p38 MAPK (SB203580), ERK1/2 (PD98059), JNK (SP600125), and NF-κB (PDTC) attenuated the Ca2+-induced expression of BMP-2 mRNA in the cells.
Conclusion: In fibroblasts isolated from keratocystic odontogenic tumors, a high concentration of Ca2+ may stimulate the expression of BMP-2 via activation of the p38, ERK1/2, and JNK signaling pathways as well as the NF-κB cascade through CaSRs.