The establishment of inducible osteocyte ablation mouse model to elucidate the mechanisms of maxillo-mandibular dysmorphia.

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In maxillo-mandibular growth and development, osteocytes absorb mechanical stress generated by muscle contraction and deform bone structure. Thus, osteocytes are thought to be mechanosensory cells that respond to mechanical stress by sending signals to other bone cells to initiate bone remodeling. However, it has been difficult to show that osteocytic lineage are involved in bone remodeling in vivo. In present study, to define the role of osteocytes in the control of bone remodeling, we have tried to generate an inducible osteocyte ablation mouse model. Precisely, transgenic mice have been generated with mouse dentin matrix protein-1 (dmp-l) promoter, which is used to drive expression of Fas fused to mutant estrogen receptor (Fas-ER^t). Dmp-1 is an osteocyte-specific gene, and is expressed only in differentiated osteocytes but not in osteoblasts. Cells expressing (Fas-ER^t) die of tamoxifen treatment, expression of Fas-ER^t in osteocytes should allow inducible ablation of differentiated osteocytes in vivo without affecting the precursor cells, osteoblasts. First, we generated fusion genes composed of various length of the mouse dmp-1promoter fused to GFP reporter gene. The dmp-1-GFP fusion genes were transfected into osteoblastic MC3T3-E1 cells and some stable transformants were obtained. The transformants were cultured for 30 days and GFP activity was measured every days. Osteocyte-specific enhancer activity was observed in region between -1200 and -800bp. Next, we constructed Flag-tagged Fas-ER^t fusion gene and transiently transfected the gene into MC3T3-E1 cells. We tried to induce apoptosis in Flag-Fas-ER^t expressing cells by addition of anti-Flag antibody and tamoxifen, but we were able to induce cell death in only 30% population of these cells. We have examined another system that is more effective than Fas-ER^t. We have considered to use diphtheria-toxin receptor or herpes simplex virus thymidine kinase for inducible cell ablation system.