Abstract
Osteoblastic differentiation is an important landmark for bone formation, bone repair and regeneration; however, it is a very complex process controlled by different signalling mechanisms. Several groups have reported that the cyclic adenosine monophosphate (cAMP) signalling system is responsible for regulating osteoblast cell differentiation. Nonetheless, to date, the principle role of the cAMP molecules related to this process remains controversial. Moreover, the underlying cAMP-dependent signalling cascade governing the osteoblastic differentiation has not been clarified. In this study we investigated the roles of the cAMP-dependent protein kinase A (PKA) signalling in proliferation, differentiation and mineralization of osteoblast-like MC3T3-E1 cells, using the PKA-specific small molecule cAMP analogue, 6-Bnz-cAMP, at 100 µM. Alkaline phosphatase (ALP) activity, runt transcription factor 2 (Runx2), osteopontin (OPN) and osteocalcin (OCN) protein expressions were used as osteoblast-specific markers to demonstrate osteoblastic differentiation. Further, calcium measurement of the extracellular matrix was employed as the hallmark of matrix mineralization or calcification. We report here that activation of PKA by the small molecule 6-Bnz-cAMP induces osteoblastic differentiation and matrix mineralization of osteoblast-like MC3T3-E1 cells. Moreover, 6-Bnz-cAMP does not induce cytotoxicity to the cells, as revealed by our cell proliferation studies. Therefore, based on these findings, we propose that the PKA-specific small molecule 6-Bnz-cAMP may serve as a novel bone-inducing growth factor for repairing and regenerating bone tissues during bone-regenerative engineering.