THE INTRACELLULAR SIGNALING PATHWAYS OF DEMINERLIZED BONE MATRIX

摘要

Bone morphogenetic proteins (BMPs) are a group of structurally related proteins in the transforming growth factor-beta (TGF-beta) family which have been shown to stimulate bone formation in vivo. Since these proteins are concentrated in the organic matrix of bone and would be released after a fracture or during bone resorption, they are likely to have a profound effect on bone healing. There is a lack in the understanding of the intracellular signaling pathways that these proteins and growth factors utilize for healing and remodeling. The current understanding is that the BMPs utilize the SMAD signaling pathway within the cell to regulate target gene expression and thus control cellular events such as proliferation, differentiation, migration, apoptosis, and matrix remodeling. There are, however, numerous other growth factors within the bone matrix which utilize a vast number of intracellular signaling pathways to exert an effect on the cell. The hypothesis of this study was that osteoblast-like cells (OBs) treated with demineralized bone matrix (DBM) would utilize a variety of intracellular signaling pathways, not isolated to the SMAD signaling pathway. Specifically, the aims of this study were to evaluate osteoblast-like cells (MG-63 cell line) for relative levels of phosphorylation of 6 kinase phophorylation sites (three known factors in the JAK STAT pathway and three factors known in the MAPK pathway) after treatment with DBM compared to control, at 30 minutes of incubation. An array of capture and control antibodies to these 6 specific sites were incubated with cellular extracts and detected utilizing secondary antibody and chemiluminescent reagents. Results were evaluated by x-ray and image analysis software for relative pixel density. The levels of phosphorylated SMAD 1/5/8 complex and phosphorylated MAPK were then determined using standard Western blot technique. The results clearly demonstrated that DBM utilizes a variety of signaling mechanisms as seen in the relative levels of phosphorylated intracellular signaling factors. This information is important for understanding the balance of signaling pathways that are necessary for normal bone healing and remodeling.