Insulin acts directly as a mitogen on isolated embryonic tissues but has been shown to be inactive at physiological concentrations on several fetal cell types. Since insulin availability is obligatory for optimal fetal growth, we have investigated its mitogenic actions on chondrocytes isolated from the epiphyseal growth plates of fetal lambs. Chondrocytes were isolated from the proximal tibial growth plate of lamb fetuses between 40 and 130 days gestation using collagenase and were cultured in monolayer before use between passages 2 and 6. The synthesis of DNA was assessed from the incorporation of [3H]thymidine after incubation in medium supplemented with glucose (0.7 mM-25 mM) with or without insulin (0.08 nM-167 nM). Increase in cell number was assessed after incubation with test medium for up to 8 days. Insulin substantially increased both DNA synthesis, and cell number, compared to control incubations with a biphasic dose response; an initial 3- to 5-fold increase in DNA synthesis occurring at approximately 1 nM insulin with a second response seen at approximately 50 nM. Within the physiological range of concentrations insulin was only 50% as active as insulin-like growth factor I (IGF I), but was 15 times more active than IGF II. Similar effects of insulin were observed throughout the fetal age range, although the DNA synthetic rate in basal medium declined with both fetal age and cell passage number. The mitogenic actions of insulin were glucose-dependent and were maximal in the presence of 2.7 mM glucose. Insulin did not cause any change in chondrocyte cell cycle duration. Chondrocytes released immunoreactive IGF II but no detectable IGF I. While exposure to insulin concentrations of approximately 50 nM or greater resulted in a statistically significant increase in IGF II release from chondrocytes, no change in IGF II release was seen in response to physiological insulin concentrations. However, exposure of cells to a blocking monoclonal antibody against human IGF I, McAb SM 1.2, which also negates the mitogenic actions of IGF II, consistently reduced insulin-stimulated DNA synthesis suggesting that IGF II presence may be necessary for optimal insulin action. Combination experiments using maximal concentrations of IGF I (13.3 nM) and increasing amounts of insulin (0.16 nM-1.67 nM) showed additive effects on DNA synthesis, suggesting that each hormone was acting through distinct receptor populations. We conclude that insulin, at physiological concentrations, may exert direct growth-promoting actions at the epiphyseal growth plates of the fetal lamb throughout gestation.

Chondrogenesis is thought to be controlled by interactions between circulating anabolic hormones and locally produced peptide growth factors, and involves ordered changes in matrix composition which ultimately allow endochondral calcification. We have used a model of isolated ovine fetal growth-plate chondrocytes to examine the actions and interactions of basic fibroblast growth factor (basic FGF), insulin-like growth factors-I and -II (IGF-I and -II), insulin and transforming growth factor-beta 1 (TGF-beta 1) on total protein, collagen or non-collagenous protein and sulphated glycosaminoglycan synthesis. These parameters were determined by assessment of the incorporation by monolayer cultures of early passage chondrocytes of [3H]leucine, [14C]proline and [35S]sulphate respectively, followed by partial molecular characterization. Basic FGF enhanced total protein synthesis with a half-maximal effective concentration of 270 +/- 60 pmol/l (mean +/- S.E.M., four animals) and was sixfold more active on a molar basis than IGF-I or insulin, and 28-fold more active that IGF-II which is the endogenously synthesized IGF. The actions of basic FGF were additive to those of IGF-I or insulin. More detailed analysis of extracellular-matrix component synthesis showed that basic FGF, IGF-I and insulin each caused significant increases in the synthesis of collagen and sulphated glycosaminoglycans. TGF-beta 1 had no effect on total protein synthesis by chondrocytes when present alone at concentrations of 200 pmol/l or less, but was inhibitory at 400 pmol/l. However, the use of this parameter masked a stimulatory action of 50 or 100 pmol TGF-beta 1 on sulphated glycosaminoglycan synthesis and a relative shift in the ratio of collagen: non-collagenous protein synthesis in favour of the former. A synergistic interaction existed between TGF-beta 1 (20-100 pmol/l) and basic FGF which potentiated total protein and collagen synthesis, and their actions on sulphated glycosaminoglycan production were additive. The same concentrations of TGF-beta 1 inhibited the ability of IGF-I or insulin to stimulate total protein or collagen synthesis, but were additive to their stimulatory effects on sulphated glycosaminoglycan synthesis. The results suggest that matrix-molecule composition and the anabolic status of the epiphyseal growth-plate may be modulated in utero by multiple interactions between peptide growth factors produced locally, such as basic FGF, IGF-II and TGF-beta 1, and circulating hormones such as insulin and IGF-I.

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)