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The IR-IGF1 production by rabbit epiphyseal chondrocytes cultured in serum-free medium was analyzed. Cell proliferation was induced by the addition of 10 ng/ml basic fibroblast growth factor (bFGF) without or with 100 ng/ml recombinant human growth hormone (hGH). GH alone induced no cell multiplication. Chondrocytes treated with bFGF alone secreted an IR-IGF1 activity proportional to the mitotic activity of the cells. A specific positive IGF1 immunostaining was localized in the Golgi of control and hGH-treated cells. The IR-IGF1 activity recovered into culture medium was mainly composed of three fractions of apparent MW 6-8 kDa, 9-14 kDa, and 16-18 kDa. [35S]Methionine pulse-chase experiments indicated that the radiolabeled 16-18 kDa IR-IGF1 fraction was partly converted into the 9-14 kDa and 6-8 kDa fractions. At equilibrium, 70% of the chondrocyte IR-IGF1 activity was recovered as 9- to 18-kDa forms which contained high IR-proIGF1A activity. The 6-8 kDa fraction had biochemical characteristics similar to those of the mature IGF1 peptide. Similar results were observed when 4% fetal calf serum was added to the culture. The addition of 100 ng/ml of hGH significantly and specifically increased IGF1 precursor material, which thus represented 90% of total IR-IGF1 activity. On Day 16 of the culture, when cells stopped dividing, the amount of chondrocyte IR-IGF1 was significantly lower than during cell proliferation, and hGH had no effect on this production. These data indicate that cultured chondrocytes produce more IGF1 precursors than mature IGF1 and that GH specifically stimulates biosynthesis of IGF1 precursors but not IGF1 per se. A GH-dependent biological function of IGF1 proforms in chondrocytes remains to be demonstrated.

This study concerns the immunoreactive somatomedin C secretion by prepubertal rabbit epiphyseal chondrocytes cultured in a defined serum-free medium. In such culture conditions, chondrocytes mainly synthesized Type II collagen (80% of total collagen) during 10 days. A small amount of Type I collagen was also found with a significant (p less than 0.05) higher level during the period of cell multiplication (6.4 +/- 1.5%) than when cells reached confluency (0.9 +/- 0.2%). During the 10 days of culture without serum and without hormone added, a Sm-C/IGF1 activity was measured by RIA at a mean level of 30 +/- 5 mU/ml/10 micrograms DNA. This value was significantly higher (p less than 0.001) than in the medium not incubated with the cells (1.7 +/- 0.9 mU/ml). When hGH was added to the culture medium during the period of cell division, the level of Sm-C/IGF1 activity was significantly elevated at 39 +/- 4 mU/ml/10 micrograms DNA (p less than 0.05) and at 55 +/- 3 mU/ml/10 micrograms DNA (p less than 0.001) with 50 ng/ml and 100 ng/ml hGH concentrations respectively. On the contrary, no difference was observed at confluency in treated and non treated cells.

To determine whether growth hormone has a direct effect on skeletal tissues not mediated by somatomedins, and to better define the role of somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) in skeletal development, bovine growth plate, and rabbit articular and growth plate chondrocytes in primary culture were evaluated under a variety of experimental conditions designed to elicit growth hormone and Sm-C/IGF-I stimulation. Under none of these conditions did bovine growth plate chondrocytes respond to either homologous bovine growth hormone or heterologous hGH. Under the same conditions, these cells were highly responsive to human Sm-C/IGF-I with respect to both [3H]thymidine and [35S]sulfate incorporation, indices of mitotic and differentiated cell functions, respectively. Similarly, both rabbit articular and growth plate chondrocytes showed enhanced incorporation of [3H] thymidine and [35S]sulfate in the presence of Sm-C/IGF-I, but did not respond to either native or recombinant hGH. Cells at different stages of maturation within the bovine growth plate differed in their reaction to Sm-C/IGF-I with proliferative zone cells manifesting a greater response to the peptide than cells of the reserve zone. These results suggest that the action of Sm-C/IGF-I on growth plate and articular chondrocytes is direct and that the effect of GH on these cells is indirect. The data further suggest that within the growth plate, the transition from reserve to proliferative status is associated with an increased Sm-C/IGF-I responsiveness, a change which may contribute to the functional differences in these cells.