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.


BACKGROUND
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.


METHODS
We characterized the MS induction of fgf-2 using a 15-minute pulse of 120 mustrain 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.
Li
Chai-Fei
CF
Laboratory of Cell Growth, Northern California Institute for Research and Education, San Francisco, California, USA.
Hughes-Fulford
Millie
M
D000818:Animals; D049109:Cell Proliferation; D002478:Cells, Cultured; D051546:Cyclooxygenase 2 / Q000378:metabolism; D051766:Early Growth Response Protein 1 / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism*; D017781:Genes, Immediate-Early / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D010012:Osteogenesis / Q000502:physiology*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D051497:Receptor, Fibroblast Growth Factor, Type 2 / Q000378:metabolism; D013314:Stress, Mechanical; D015854:Up-Regulation
D051766:Early Growth Response Protein 1; D020033:Protein Isoforms; D012333:RNA, Messenger; D016222:Fibroblast Growth Factor 2; D051546:Cyclooxygenase 2; D051497:Receptor, Fibroblast Growth Factor, Type 2