Suppression of Sprouty2 Induces Periodontal Tissue Regeneration

Objective: Sprouty was identified as an inhibitor of fibroblast growth factor (FGF) receptor in Drosophila. In mammals, four Sproutys have been identified, and specifically Sprouty2 functions as a negative regulator of receptor tyrosine kinases (RTKs) signaling. The purpose of this study was to investigate whether Sprouty2 could be new therapeutic targets for periodontal tissue regeneration. Methods: MC3T3-E1 osteoblastic cells and GE1 gingival epithelial cells were used for the following experiments. Cells were transfected with a control plasmid, or a dominant negative mutant plasmid of Sprouty2. For immunoblot analysis, cells were extracted after growth factor stimulation and proteins were detected by various antibodies. Using MTT assay, growth factor-induced cell proliferation was measured in transfected osteoblastic and epithelial cells. In order to evaluate calcium-rich deposits in culture, alizarin red S staining assay was performed using Sprouty2 dominant negative mutant MC3T3-E1 cells. Results: After confirming that Sprouty2 was induced by growth factor stimulation and it diminished ERK activation, we transfected with a dominant negative mutant of Sprouty2 in MC3T3-E1 or GE1 cells, respectively. Transfection of Sprouty2 mutation enhanced bFGF-induced ERK activation in MC3T3-E1 cells. By contrast, under the same conditions, it decreased EGF-induced ERK activation in GE1 cells. In addition, when osteoblastic cells were stimulated with bFGF, cell proliferation assay revealed that Sprouty2 mutant cells proliferated faster than control cells. Consistent with this, alizarin red S staining assay showed the ability of mineralization in Sprouty2-mutated MC3T3-E1 cells were partly higher compared with control cells. Conclusions: Suppression of Sprouty2 induced cell proliferation and bone mineralization of osteoblastic cells, while it diminished cell proliferation of gingival epithelial cells. In other words, inhibition of Sprouty2 may effectively allow alveolar bone to grow, with blocking the ingrowth of gingival epithelial cells toward bony defects, as well as an effect of guided tissue regeneration (GTR) methods.