Functional Interaction between PKA and BMP Signaling during Osteogenic Differentiation of Human Mesenchymal Stem Cells

We approach cell based bone tissue engineering by in vitro expansion and osteogenic differentiation of pluripotent human mesenchymal stem cells (hMSCs) isolated from bone marrow and subsequent seeding onto calcium phosphate ceramics. To optimise the osteogenic differentiation process we want to understand the molecular signalling pathways underlying differentiation of hMSCs into active osteoblast in vitro and in vivo. G protein coupled receptors (GPCR) are involved in bone formation. Therefore we investigated the role of GPCR signalling in hMSC osteogenesis. Short term PKA activation using cAMP or cholera toxin induced expression of the osteogenic markers alkaline phosphatase, osteocalcin, collagen type1 and it enhanced in vitro mineralization. Inhibition of PKA signalling with H89 reversed the cAMP and cholera toxin induced effects. Mcro array studies on 6 hours cAMP treated hMSCs showed an up regulation of BMP target genes Id1, Id2, Id4 and Smad6, which was further confirmed by quantitative PCR. The PKA induced expression of BMP target genes was not inhibited by cycloheximide treatment indicating direct activation of BMP target genes via cyclic AMP response element binding protein (CREB). Mcro array studies further show that hMSCs treated with cAMP for 7 days displayed a strong expression of a number of growth factors and cytokines with known osteogenic activity, including BMP2. Since we observed induction of BMP2 upon PKA activation, we tested autocrine induction of BMP target genes using noggin (an inhibitor of BMP signalling). Noggin partially inhibited PKA induced expression of BMP target genes, but reduced the BMP2 induced expression to the basal level confirming the autocrine effect. A similar effect was observed in MG63, a human osteosarcoma cell line. We conclude that PKA activation in hMSCs not only directs the cells into the osteogenic lineage by direct induction of BMP target genes but also induces the expression of BMPs and other bone inducing cytokines. We currently investigate whether PKA activated hMSCs can stimulate uncommitted progenitor cells into the osteogenic lineage using in vitro and in vivo models. The functional interaction between PKA and BMP signalling pathways can be further explored to augment current bone tissue engineering protocols.