The Effects of Spry2 in Osteoblasts and Gingival Epithelial Cells

Objectives: Sprouty was identified as an inhibitor of the fibroblast growth factor (FGF) receptor, and Sprouty2 (Spry2) functions as a negative regulator of receptor tyrosine kinase signaling. In this study, we investigated how inhibition of Spry2 affects osteoblasts and gingival epithelial cells in periodontal tissue regeneration in vitro.
Methods: MC3T3-E1 and GE1cells were transfected with a dominant-negative mutant of Spry2 (Y55A-Spry2). Using WST-8 assay and Ki-67 staining, growth factor-induced cell proliferation was investigated. In order to evaluate osteoblastic differentiation, ALP staining and RT-PCR detecting genes associated with calcification in MC3T3-1 cells were performed. EGFR downregulation in GE1 cells was confirmed by fluorescence microscope.
Results: Transduction of Y55A-Spry2 enhanced basic fibroblast growth factor (bFGF)- and epidermal growth factor (EGF)-induced ERK activation in MC3T3-E1 osteoblastic cells. In contrast, it decreased their activation in GE1 cells. Consistent with these observations, Y55A-Spry2 increased osteoblast proliferation with bFGF and EGF stimulation, whereas the proliferation of Y55A-Spry2-introduced GE1 cells was decreased via the ubiquitination and degradation of EGF receptors (EGFRs). In addition, Y55A-Spry2 caused upregulation of Runx2 expression and downregulation of Twist, a negative regulator of Runx2, with treatment of bFGF and EGF, resulting in enhanced osteoblastogenesis accompanied by alkaline phosphatase activation and osteocalcin expression in MC3T3-E1 cells.
Conclusions: These data suggest that suppression of Spry2 expression induces proliferation and differentiation of osteoblastic cells after the addition of a bFGF and EGF cocktail but inhibits proliferation in gingival epithelial cells. These in vitro experiments may provide a molecular basis for novel therapeutic approaches in periodontal tissue regeneration. Taken together, our study proposes that combined application of an inhibitor for tyrosine 55 of Spry2, bFGF, and EGF may effectively allow alveolar bone growth and block the ingrowth of gingival epithelial cells toward bony defects, biologically mimicking a barrier effect in guided tissue regeneration, with in vivo investigation in the future.