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

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/micrograms DNA, 66 +/- 2 pM/micrograms DNA and 22 +/- 3 pM/micrograms 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 [3H] thymidine. Incubation of chondrocytes with transforming growth factor beta, 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Cloning and molecular characterization of a novel gene strongly induced by the adenovirus E1A gene in rat thyroid cells

“…One may gain insight into the stimuli and the mechanism whereby cells produce the secreted or intracellular form of CL2 by looking at already characterized proteins that have the same distribution as CL2. A number of peptide growth factors (bFGF (Bouche et al, 1987;Baldin et al, 1990;Hill et al, 1992), aFGF (Imamura et al, 1990), FGF3 (Antoine et al, 1997), PDGF (Maher et al, 1989), angiogenin (Moroianu andRiordan, 1994) and PTHrP (Henderson et al, 1995)) are known to modulate cellular function by a dual mode of action: first, by activating classical signal transduction pathways (secreted form) and, second, by activities within the nucleus (increted form). Furthermore, the localization and, thus, the function of one form can counteract the function of the other.…”

The nucleus: A target site for parathyroid hormone-related peptide (PTHrP) action

“…A number of peptide growth factors (bFGF [Bouche et al, 1987;Baldin et al, 1990;Hill et al, 1992], aFGF [Imamura et al, 1990], FGF3 [Kiefer et al, 1994], PDGF [Maher et al, 1989], and angiogenin [Moroianu and Riordan, 1994]) are known to modulate cellular function in a dual mode of action: first, by activation of classical signal transduction pathways, and, second, by nuclear actions that critically rely on an endocytosis-dependent pathway. It would appear that, after binding to cell surface receptors, the ligand-receptor complex is internalized and the ligand translocates to the nucleus and/or nucleolus of the target cells (Fig.…”

Coordination of chondrogenesis and osteogenesis by fibroblast growth factor 18

“…Fgf4, Fgf9, and Fgf17 are also expressed in the developing limb (Martin 1998;Colvin et al 1999). Fgf8 and Fgf17 are expressed in some skeletal elements (Xu et al 1999), and Fgf2 is abundantly expressed in chondrocytes (Hill et al 1992;Twal et al 1994;Luan et al 1996). However, gene targeting experiments have not identified a role for these ligands, individually, in either limb or skeletal development (Dono et al 1998;Ortega et al 1998;Zhou et al 1998;Sun et al 2000;Xu et al 2000;Colvin et al 2001a,b).…”