Moreover an increase of extracellular calcium stimulates BMP

Moreover, an increase of extracellular calcium stimulates BMP-2 expression of pulp mdm2 p53 via L-type calcium channel and ERK signaling, which can be attenuated by PD98059 and nifedipine . Dental pulp cells expressed little calcium-sensing receptors . immunohistochemistry staining also shows the strong nucleus expression of CREB Ser133 phosphorylation in odontoblasts and cementoblasts as well as pulp stroma cells of human molar teeth, suggesting the involvement of CREB in mediating various signaling and pulpal functions . Furthermore, PGE may increase intracellular calcium levels of pulp cells . Therefore, it is intriguing to clarify whether PGE may stimulate PLC, intracellular calcium release, and extracellular calcium influx to activate calcium calmodulin and adenosine monophosphate–activated protein kinase (AMPK) to affect cAMP production of pulp cells. Various prostanoids are important in the pulpal inflammation and repair. In this study, to clarify the role of PGE in the pathogenesis of pulpal inflammation and repair, we studied the effect of PGE on adenylate cyclase activity and cAMP production of pulp cells and its upstream signal transduction pathways in dental pulp cells. The results of this study can be helpful to control and treat pulpal inflammation in the future. Materials and Methods
Results
Discussion PGE2 plays a crucial role in pulpal inflammation and repair. Understanding the signal transduction pathways responsible for PGE2-induced events in the dental pulp is useful for future control and treatment of pulpal inflammation. PGE2 has been shown to stimulate cAMP production of rat clonal odontoblastlike RDP4-1 cells and bovine dental pulp cells 22, 23, but a decrease in the cAMP level of dental papilla cells in response to PGE2 is also reported (24). However, the signal transduction for PGE2-induced cAMP production of dental pulp cells is still not clear. To further clarify this question, we studied cAMP production of pulp cells in response to PGE2 and various EP receptor agonists. The EP2 receptor is a G protein–coupled receptor linked to adenylate cyclase activation and cAMP production 9, 10, 11. In the present study, exposure of human dental pulp cells to PGE2 and 19R-OH PGE2 (an EP2 receptor agonist) stimulate cAMP production of pulp cells with a maximal stimulation after 10 to 20 minutes of exposure. However, sulprostone, an EP1/EP3 receptor agonist, shows little effect on cAMP production. This indicates that activation of the EP2 receptor by PGE2 is responsible for adenylate cyclase activation and cAMP production of human dental pulp cells. Accordingly, SQ22536 inhibited the PGE2-induced cAMP production, suggesting the involvement of adenylate cyclase activation. Stimulation of glycosaminoglycan synthesis and alkaline phosphatase activity of pulp cells by PGE2 and cAMP has been reported 25, 26. Because cAMP has been detected in healthy dental pulp as analyzed by immunohistochemistry (27) and is an important second messenger responsible for the neurotransmitter release and painful pulpal and periapical lesions (28), future control of COX expression, PGE2, and cAMP production may be useful for the treatment of painful pulpal and periapical lesions. cAMP production of pulp cells has been reported to be stimulated by PGE2 and parathyroid hormone (23). The majority of cellular effects of cAMP are mediated by cAMP-dependent PKA (29). The inhibition of PKA by H89 enhanced cAMP production, further indicating that PKA is a downstream molecule of cAMP. Dorsomorphin as an AMPK inhibitor is not able to prevent the PGE2-induced PGE2 production of pulp cells, excluding the involvement of AMPK in these events. Activation of the EP receptors may potentially stimulate downstream signaling such as PLC. We interestingly found that the inhibition of PLC by U73122 suppressed PGE2-induced cAMP production, suggesting the involvement of PLC in this event. PLC may stimulate IP3 production and calcium mobilization via the activation of intracellular IP3 receptors of intracellular organelles such as mitochondria or rough endoplasmic reticulum to induce intracellular calcium release or via cell membrane IP3 receptors to induce extracellular calcium influx (30). In addition to PLC, PKA and calcium calmodulin kinase may also regulate IP3 receptors and therefore calcium mobilization (30). PGE2 is shown to stimulate the cytoplasmic-free calcium level and phosphoinositide hydrolysis of rat clonal odontoblastlike RDP4-1 cells (22) and calcium mobilization in dental pulp cells (5), possibly via EP3 receptors. Interestingly, thapsigargin (an inhibitor of intracellular sarcoplasmic reticulum calcium release) can attenuate PGE2-induced cAMP production, indicating the role of intracellular release of calcium is crucial in this event. However, verapamil and EGTA are not able to prevent PGE2-induced cAMP production, excluding the involvement of extracellular calcium and L-type calcium channel in this event. Similarly, PGE2 has been shown to stimulate the EP2 receptor, cAMP production, and intracellular release of calcium stores in astrocytes (31). The inhibitory effect of W7 on PGE2-induced cAMP production further suggests the role of downstream calcium calmodulin in mediating cAMP production of pulp cells by PGE2. Accordingly, in the 9 isoforms of adenylate cyclase, at least types I, III, and VIII adenylate cyclase can be activated by Ca2+/calmodulin 13, 14. Type III adenylate cyclase is present in a number of tissues including the heart, kidney, and liver, whereas type VIII adenylate cyclase is present mainly in the brain 13, 14. Simultaneous activation of protein kinase C and cAMP signaling may induce neurogenic differentiation of pulp cells (32), suggesting the potential application for tissue engineering by stimulation of cAMP/PKA signal transduction pathways.