The Effects of Fibroblast Growth Factor-2 on Human Neonatal Calvaria Osteoblastic Cells Are Differentiation Stage Specific

Abstract

Fibroblast growth factors (FGFs) appear to play an important role in human cranial osteogenesis. We therefore investigated the effects of recombinant human FGF-2 (rhFGF-2) on human calvaria (HC) osteoblastic cells. Immunocytochemical analysis showed that confluent HC cells express both FGF receptors -1 and -2. In short-term culture, rhFGF-2 (0.1-100 ng/ml, 2-5 days) increased HC cell growth and decreased alkaline phosphatase (ALP) activity and type I collagen (ColI) synthesis, as evaluated by P1CP levels. When HC cells were induced to differentiate in long-term culture in the presence of 50 g/ml ascorbic acid and 3 mM phosphate, HC cells initially proliferated, then ALP activity and ColI synthesis decreased and calcium content in the extracellular matrix increased. Continuous treatment with rhFGF-2 (50 ng/ml) for 1-28 days, or a transient rhFGF-2 treatment for 1-7 days, slightly increased DNA synthesis at 7 days, whereas a late treatment for 8 -28 days had no effect on cell growth. The continuous and transient treatments with rhFGF-2 decreased ALP activity, ColI synthesis, and matrix mineralization. This was associated with a transient fall in osteocalcin (OC) production at 7 days. In contrast, the late rhFGF-2 treatment for 8 -28 days only slightly inhibited ALP activity and increased matrix mineralization. In addition, both continuous and late treatments with rhFGF-2 increased OC production in more mature cells at 3-4 weeks of culture. We also found that the early and late treatments with rhFGF-2 had opposite effects on transforming growth factor ␤2 production in proliferating cells and more mature cells. The results show that rhFGF-2 slightly stimulates cell growth and reduces the expression of osteoblast markers in less mature cells, whereas it induces OC production and matrix mineralization in more mature cells, indicating that the effects of FGF-2 are differentiation stage specific and that FGF-2 may modulate HC osteogenesis by acting at distinct stages of cell maturation. (J Bone Miner Res 1998;13:645-654)

Fibroblast growth factor signaling in skeletal development and disease

“…While it is well accepted that FGF/FGFR signaling plays an important role in osteoblastogenesis, it is now clear that the effects in osteogenic cells are complex, as they depend on the type of FGFs and FGFRs expressed, the stage of cell maturation, and the microenvironment (proteoglycans and interacting proteins) that may either enhance or attenuate FGF/FGFR signaling in bone cells. The collective data suggest that FGF2 signaling increases cell proliferation in immature osteoblasts and thereby expands the pool of osteoblast precursor cells that then fully differentiate in the bone environment (Debiais et al 1998;Shimoaka et al 2002;Ignelzi et al 2003;Fakhry et al 2005). Accordingly, blocking FGF2 biological activity reduces osteogenesis in vivo (Moore et al 2002).…”

“…In addition to controlling osteoblast proliferation and differentiation, FGF signaling regulates osteoblast apoptosis. In osteoblast precursor cells, FGF2 induces osteoblast survival through activation of PI3K/AKT signaling (Debiais et al 1998) and FGFR1-mediated increased Bcl2/Bax ratio . In more differentiated osteoblasts, FGF treatment or overexpression of FGF2 in transgenic mice induces apoptosis in mouse calvaria (Mansukhani et al 2000;Ignelzi et al 2003), which limits the increase in the osteoblast population.…”

FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease

“…In vitro studies show that FGF1, FGF2, FGF4, or FGF18 can potentiate growth of fetal or neonatal calvarial osteoblasts (Canalis et al 1988;Tang et al 1996;Hurley et al 2001;Shimoaka et al 2001) but not mature osteoblasts (Debiais et al 1998;Mansukhani et al 2000). In vivo, FGF2 increases the number of osteogenic cells and promotes calvarial osteogenesis (Mundy et al 1999).…”

“…In vitro, FGFs inhibit type I collagen expression and bone nodule formation of rat calvarial cells (Tang et al 1996;Hurley et al 2001). However, prolonged treatment with FGF2 increases calvarial osteoblast differentiation (Debiais et al 1998). FGF2 promotes osteocalcin transcription in rodent and human calvarial osteoblasts (Schedlich et al 1994;Boudreaux and Towler 1996;Newberry et al 1996Newberry et al , 1998Debiais et al 1998).…”

“…However, prolonged treatment with FGF2 increases calvarial osteoblast differentiation (Debiais et al 1998). FGF2 promotes osteocalcin transcription in rodent and human calvarial osteoblasts (Schedlich et al 1994;Boudreaux and Towler 1996;Newberry et al 1996Newberry et al , 1998Debiais et al 1998). This effect, together with the reported increase in sodium-dependent phosphate transport (Suzuki et al 2000), may contribute to the regulation of intramembranous calcification.…”