Emerging Role of Dentin Matrix Protein-1 in Osteolysis

Objectives: To determine the role of Dentin Matrix Protein-1(DMP1) in the activating osteolytic cycle.
Methods: Mouse calvarial preosteoblast MC3T3-E1 cells, Metastatic MDA MB 231 human breast cancer cells and RAW 264.7 monocytes cells were grown in their respective growth media supplemented with FBS and antibiotic. MC3T3-E1 cells were stimulated with DMP1 for 24 hours and Western blot was performed using anti-RANKL antibody. Overexpression and knockout of DMP1 were performed using pcDNA 3.1 DMP1 plasmid (OE cells) or shRNA (Sh-DMP1 cells) for DMP1 MDA MB231using Fugene HD. MDA-MB231 cells transfected with pcDNA3.1 (mock) or scrambled shRNA were used as controls. Total proteins were extracted from these cells stimulated with or without TGF-β/SB20358, and Western Blotting was done to determine the role of MAPK kinase and Smad pathways. Invasion assay, cell proliferation assay and migration were performed on the OE cells and Sh-DMP1 cells. Using a co-culture model, MDA MB231 cells or OE cells with MC3T3-E1 cells, conditioned media was obtained to stimulate the differentiation of RAW 264.7 cells for 7 days. TRAP staining and Western blotting were performed for MAP Kinase pathway and anti-TRAP antibodies.
Results: Results from our studies have shown that osteoblast cells upon differentiation secrete RANKL. Western blot results confirmed DMP1 mediated p38 MAP kinase signaling pathway activation in metastatic breast cancer cells. Results from the co-culture model, showed that OE cells can trigger osteoclast activation. Furthermore, results from TRAP staining shows that DMP1 secreted by OE cells into the extracellular environment can accelerate the differentiation of monocyte, RAW264.7 cells to osteoclast cells.
Conclusions: Overall, the results obtained in this study identifies a new role for DMP1 in the differentiation of breast cancer tumor cells. Supported by NIHDE11657.