The small molecule PKA-specific cyclic AMP analogue as an inducer of osteoblast-like cells differentiation and mineralization

Abstract

Osteoblastic differentiation is an important landmark for bone formation, bone repair and regeneration, however it is a very complex process controlled by different signaling mechanisms. Several groups have reported that the cyclic adenosine monophosphate (cAMP) signaling 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 signaling cascade governing the osteoblastic differentiation has not been clarified. In this report, we investigated the roles of the cAMP-dependent protein kinase A (PKA) signaling 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.

Electrospinning of polymer nanofibers for tissue regeneration

“…For instance, a small molecule could be identified to activate signaling pathways that stimulate osteoblast-associated gene expression and matrix mineralization, which are processes associated with bone formation [199]. In fact, data from our group and others demonstrated that small molecule cyclic adenosine monophosphate (cAMP) analogs were able to induce in vitro and in vivo bone formation through the Protein Kinase A (PKA)/cAMP response element-binding protein (CREB) signaling mechanism [200][201][202][203]. It is also worth noting that the advances in high throughput screening technologies have yielded a large number of other osteoinductive small molecules in the past decade [176].…”

Poly (lactic acid)-based biomaterials for orthopaedic regenerative engineering

“…Via a similar mechanism, utilizing a small molecule phenamil, our group recently reported in vitro bone formation through the downregulation of Smurf1 in sintered PLGA microsphere scaffolds [232]. In addition to BMP/smad pathway, several other pathways such as protein kinase A (PKA) simulated by 4-Bnz-cAMP,Wnt, Hh, and BMP/MAPK signaling cascades have also been observed to induce osteogenesis [233–236]. Other small molecules that have been observed to induce bone formation by these pathways include rapamycin, FK-506, and tilorone [237].…”

Culture-expanded human dermal stem cells exhibit donor to donor differences in cAMP generation

“…Growth factors stimulate cell expansion and maintain cell-stemness, whereas stimulated cAMP signalling stops cells dividing and stimulates spontaneous cell differentiation. The cAMP/PKA pathway acts as a regulator of neurite outgrowth/regeneration and osteoblast cell differentiation in several experimental systems (Tegenge et al 2011;Lo et al 2011). The differences in the responses of the cell lines to the db-cAMP stimuli here suggest that isolated adult progenitor cells possess diverse differentiation potential.…”