Calcium-mediated increased expression of fibroblast growth factor-2 acts through NF-κB and PGE₂/EP4 receptor signaling pathways in cementoblasts

We reported previously that cementoblasts are provided with sensing mechanisms for extracellular Ca²⁺ and that elevated extracellular Ca²⁺ increases fibroblast growth factor-2 (FGF-2) gene and protein expression levels via a cyclic AMP/protein kinase A (PKA) dependent pathway. In the present study, we found that stimulation of murine cementoblasts with 10mM CaCl₂ induced cyclooxygenase-2 (COX-2) gene expression and prostaglandin E₂ (PGE₂) biosynthesis. NS-398, a COX-2 inhibitor, significantly reduced CaCl₂-induced increase in Fgf-2 gene expression, indicating that PGE₂ synthesized by COX-2 may be involved in FGF-2 induction. The inhibitory effect of NS-398 was restored completely by the addition of PGE₂ receptor 4 (E-prostanoid receptor 4, called EP4) agonist, but not agonists for EP1, EP2, and EP3. Furthermore, EP4 antagonist significantly reduced CaCl₂-induced Fgf-2 induction, suggesting that it is mediated by EP4 activation. However, stimulation with EP4 agonist alone in the absence of CaCl₂ had no effect on the Fgf-2 induction, indicating that EP4 signaling alone is not sufficient. CaCl₂ also upregulated gene expression levels of Ep4 and Cox-2, as well as Fgf-2 and induction of these genes was abolished by pretreatment with BMS-345541, a nuclear factor-κB (NF-κB) inhibitor, indicating that NF-κB signaling triggered by CaCl₂ is indispensable for FGF-2 induction. Furthermore, CaCl₂-induced Fgf-2 induction was synergistically enhanced by the addition of EP4 agonist. This indicates that the signaling triggered via CaCl₂ and its combination with EP4 agonist may be useful as a novel strategy for periodontal regeneration.