Basic Fibroblast Growth Factor is Synthesized and Released by Isolated Ovine Fetal Growth Plate Chondrocytes: Potential Role as an Autocrine Mitogen

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Abstract
Basic fibroblast growth factor (basic FGF) is a mitogen for isolated epiphyseal growth plate chondrocytes. To determine whether basic FGF might function as an autocrine stimulus to longitudinal skeletal growth in utero, we investigated the synthesis and release of basic FGF by isolated growth plate chondrocytes from the ovine fetus, the expression of mRNA for a high affinity basic FGF receptor by these cells, and the contribution of endogenous basic FGF to the DNA synthetic rate of the cells in vitro. Chondrocytes were isolated from the proximal tibial growth plate of the lamb fetuses between 35 and 132 days' gestation using collagenase, and were cultured in monolayer before use between passages 3 and 6. Viability was confirmed over the duration of the experiments by the exclusion of trypan blue, and an absence of lactate dehydrogenase accumulation in conditioned medium. Immunocytochemistry of chondrocyte monolayers showed immunoreactive basic FGF to be present in the cytoplasm of approximately 80% of sub-confluent cells which was accompanied by pronounced nuclear staining in approximately 30% of cells. Serum-free, conditioned culture medium, extracellular matrix and chondrocyte cytoplasm contained 52±2 pM/μg DNA, 66±2 pM/μg DNA and 22±3 pM/μg DNA basic FGF, respectively (mean±S.E.M., n=8 fetuses), for cells obtained from animals of 35-40 days' gestation when assessed by radioimmunoassay. Chondrocyte-conditioned medium increased endothelial cell proliferation in vitro (a specific bio-assay for basic FGF and related peptides); and the mitogenic activity was removed from conditioned medium by incubation with heparin-Sepharose demonstrating that this was due to heparin-binding protein(s). Western blot analysis of conditioned medium using a specific basic FGF antibody revealed a single immunoreactive protein of approximately 18 kDa molecular size. The appearance of radiommunoassayable basic FGF in conditioned medium, extracellular matrix, and chondrocyte cytoplasm observed during culture was blocked by co-incubation with cycloheximide. The levels of immunoreactive basic FGF present in each compartment decreased with gestational age as did basal DNA synthetic rate assessed by the incorporation of PH] thymidine. Incubation of chondrocytes with transforming growth factor β, resulted in a significant increase while exposure to insulin-like growth factors or insulin caused a decrease, in the content and release of basic FGF. Basic FGF presence was unaltered when medium was supplemented with varying amounts of glucose (2.7-16.7 mM). In situ hybridization on cell monolayers using a cRNA probe encoding the high affinity flg receptor for FGFs showed an abundant expression of mRNA for the receptor. When chondrocytes were cultured in the presence of exogenous basic FGF both DNA synthetic rate and cell number were increased, the half-maximal effective dose of basic FGF being 0.2 nM. Co-culture with an anti-basic FGF antibody caused a significant decrease of 23% in DNA synthetic rate under basal conditions, and completely blocked the effects of exogenous basic FGF. We conclude that isolated ovine fetal growth plate chondrocytes synthesize and release basic FGF, a proportion of which is in soluble form, which makes a significant contribution to basal DNA synthetic rate, possibly through the expression of the flg receptor. This suggests an autocrine action of basic FGF in the epiphyseal growth plate during fetal life.