Bone Abstracts

Identifying novel molecules that enhance human skeletal (mesenchymal) stem cells (hMSC) differentiation into osteoblastic bone forming cells (OB), may lead to development of new bone anabolic drugs. We have identified Kix, a small molecule kinase inhibitor that enhanced ex vivo OB differentiation and reduced apoptosis of hMSC. We found that Kix targeted undifferentiated hMSC populations and not their differentiated progeny. In addition, Kix increased in vivo heterotopic bone formation and hMSC survival in an in vivo bone regeneration model of mouse calvarial defect. In order to determine molecular mechanisms, we carried out DNA microarray analysis which revealed that Kix treatment up-regulated gene expression of different components of TGFβ and BMP signaling pathways, e.g. BMP2/4/6, TGFBR1/2/3 and their down- stream target genes e.g. IGFBP3/5, p21, and BHLHB2. Furthermore, Kix treatment of hMSC for 15 minutes enhanced canonical TGFβ signaling, as shown by approximately twofold upregulation of p-smad2 as well as enhancing TGFβ3 effects on p-Smad2. Treatment of hMSC with SB-431542; an inhibitor of TGFβ signaling abolished Kix-mediated increase in alkaline phosphatase (ALP) activity and ex vivo matrix mineralization. Active-site-directed competition kinase binding assay (DiscoveRx KINOMEscan) revealed that Kix inhibited protein kinase A, C, G, and D, known inhibitors of TGFβ receptor signaling and thus resulted in increased TGFβ signaling. From a clinical perspective, Kix may represent an attractive molecule for further development for in vivo targeting of hMSC to increase bone formation.