BMP-7 activates Smad dependent and Smad independent pathways during dentinogenesis

Objective: BMP7 is secreted by differentiated odontoblasts during dentin and pre-dentin formation that regulates the biological activity of these cells through an autocrine mode of action. However, the mechanism of BMP7 signaling during dentinogenesis has not been explored. Here, we investigate the activation of downstream signaling molecules in response to exogenous recombinant BMP7 protein in dental pulp cells. Method: Using immortalized mouse dental pulp cell line, MDPC-23, we employed both western blot and quantitive real time PCR to show protein and mRNA expression respectively. We also used both dominant negative Smad4 retrovirus and small synthetic chemical inhibitor to block Smad and Smad-independent pathways to evaluate the regulation of downstream transcription factors; Runx2 and MSX1 as well as genes involve in dentinogenesis, such as dentin-sialo-phopho-protein (DSPP) and Osteopontin (OPN). Results: Our results demonstrated that in response to BMP7, both protein and mRNA expression of Smad 1, 4, 5 and 8 are increased in time dependent fashion from 0-72 hours post treatment. In addition, partners of Smad-independent pathway, such MEK1/2 and AKT are also phosphorylated within 15 minutes. These pathways induce the activation of Runx2 and MSX1 transcription factors. Finally, DSPP and OPN, two specific marker of dentin formation, are also upregulated in response to BMP7. Upon termination of these pathways by specific inhibitors, all these markers cease to be activated. Conclusion: These findings suggest that BMP7 signaling activates both Smad dependent and Smad-independent pathways to regulate Runx2 and MSX1 transcription factors that induce DSPP and OPN genes during dentinogenesis. Our results might shed light into the future studies to investigate the therapeutic implication of these molecules in dentin repair and formation. This study was supported by RR018759 for the Nebraska Center for Cellular Signaling from the NIH and Veterans Affairs grant (A4290I) to Prof. Beatty.