Fibroblast Growth Factor‐2 Is an Immediate‐Early Gene Induced by Mechanical Stress in Osteogenic Cells

Fifteen minutes of physiological MS induces FGF‐2 in osteogenic cells. Here, we show that MS induced proliferation in both MC3T3‐E1 and BMOp cells isolated from Fgf2+/+ mice; Fgf2−/− BMOp cells required exogenous FGF‐2 for a normal proliferation response. The induction of fgf‐2 is mediated by PKA and ERK pathways. Introduction: Mechanical stress (MS) induces gene expression and proliferation of osteogenic MC3T3‐E1 cells. We have previously shown that physiological levels of MS in MC3T3‐E1 cells causes extracellular signal–regulated kinase (ERK)1/2 phosphorylation. Here we evaluate the induction and importance of fibroblast growth factor‐2 (FGF‐2) for MS‐induced proliferation. Materials and Methods: We characterized the MS induction of fgf‐2 using a 15‐minute pulse of 120 μstrain and studied the stability of fgf‐2 message using actinomycin D. Bone marrow stromal cells (BMOp) isolated from Fgf2−/− and Fgf2+/+ mice were used to study the importance of FGF‐2 in MS‐induced proliferation. Results: We found that the induction of fgf‐2 by MS is dependent on both protein kinase A (PKA) and ERK pathways. MS transiently induces fgf‐2 within 30 minutes. FGF‐2 receptor (FGFR2) was also significantly increased within 1 h. All three isoforms of FGF‐2 (24, 22, and 18 kDa) were significantly increased by MS. The MS‐mediated increase of fgf‐2 mRNA was caused by new synthesis and not stabilization. Pretreatment of MC3T3‐E1 cells with cycloheximide showed that the induction of fgf‐2 did not require new protein synthesis. Pretreating MC3T3‐E1 cells with the mitogen‐activated protein kinase (MAPK)/ERK kinase 1/2 (MEK1/2) inhibitor, U0126, or H‐89, a PKA inhibitor, significantly inhibited the induction of fgf‐2, showing that mechanical induction of fgf‐2 is dependent on ERK and PKA signaling pathways. The downstream consequence of a single 15‐minute stress pulse was a 3.5‐fold increase in cell number in MC3T3‐E1 compared with growth in nonstressed control cells. In studies using bone marrow osteoprogenitor cells (BMOp) isolated from Fgf2+/+and Fgf2−/− mice, we found that FGF‐2 was necessary for a full proliferative response to MS. Conclusions: These studies show that FGF‐2 is an immediate‐early gene induced by MS, and its expression is mediated by both the PKA and MAPK signal transduction pathways. FGF‐2 was required for a full proliferative response.