Cell Cycle-Dependent Localization of Irnmunoreactive Basic Fibroblast Growth Factor to Cytoplasm and Nucleus of Isolated Ovine Fetal Growth Plate Chondrocytes

Basic fibroblast growth factor (basic FGF) is a potent mitogen for chondrocytes in vitro and is present in developing cartilage in vivo. Studies of intracellular basic FGF localization in other cell types have revealed a transient nuclear presence. We have examined ovine fetal growth plate chondrocytes for the presence of intracellular basic FGF by immunocytochemistry. Chondrocytes were isolated from the proximal tibial growth plate of lamb fetuses between 75 and 80 days' gestation using collagenase, and were cultured in monolayer before use between passages 3 and 6. In non-synchronized cell cultures 58 +/- 6% of cells (mean +/- s.d., n=3) demonstrated cytoplasmic staining for immunoreactive basic FGF. Of these cells, 18 +/- 3% also exhibited strong nuclear staining. Chondrocytes were growth-restricted and restarted into the cell cycle with 2% (vol/vol) fetal calf serum. The timing of S phase was followed by nuclear labelling of nuclei with [H-3] thymidine followed by autoradiogaphy, or by the incorporation of [H-3] thymidine into trichloroacetic acid-precipitable DNA in parallel cultures. A cytoplasmic presence of immunoreactive basic FGF did not appear, following immunocytochemistry, until the second half of G(1) with 97% of cells immunopositive 2 hr prior to S phase. Nuclear staining for basic FGF appeared 2 hr before peak nuclear labelling index, and 56% of cell nuclei were immunopositive. Following entry into S phase cytoplasmic and nuclear basic FGF immunostaining rapidly disappeared. When these experiments were repeated with or without the presence of anti-basic FGF antibody or heparin, the presence of the antibody significantly reduced peak [H-3] thymidine incorporation into DNA during S phase while exposure to heparin increased this. However, the proportion of cells demonstrating cytoplasmic or nuclear staining for immunoreactive basic FGF, and the time of onset of staining, were unaltered. Incubation of cells with suramin blocked subsequent DNA synthesis and no intracellular basic FGF was visualized. Cell-conditioned culture medium, extracellular matrix and cytoplasm from synchronized cultures of chondrocytes were taken at time points throughout the cell cycle and assessed for basic FGF content by radioimmunoassay. Basic FGF was detectable in each compartment and steadily rose throughout the second half of G(1) to reach maximum values around the S phase. The accumulation of basic FGF in medium, matrix and cytoplasm was blocked by the presence of cycloheximide. When cells were labelled with [H-3] leucine and newly-synthesized basic FGF immunoprecipitated from cytoplasmic or nuclear fractions, peak values of peptide were seen in the cytoplasm 2 hr prior to its transient appearance in the nucleus which was immediately before entry into S phase. The amount of immunoprecipitable, tritiated basic FGF recovered from cytoplasm and nucleus was unaffected by incubation with antibody against basic FGF. Separation of immunoprecipitated, tritiated basic FGF by gel electrophoresis revealed two major forms with molecular sizes 18 kDa and 23 kDa in both the cytoplasm and nucleus. Only the former was present in conditioned culture medium. The results suggest that chondrocytes synthesize and release basic FGF during the second half of G(1) in response to hormonal signals contained in fetal calf serum. This basic FGF contributes to the subsequent DNA synthetic rate of the cells. During late G(1) a brief translocation of basic FGF from cytoplasm to nucleus occurs, but this is not directly related to alterations in DNA synthesis.