Stimulation of DNA Synthesis in Rat A10 Vascular Smooth Muscle Cells by Threonine-59 Insulin-like Growth Factor I

The clonal smooth muscle cell line A10, derived from fetal rat aorta, binds I-lnsulin-like growth factor I at a Type 1 insulin-like growth factor receptor. Threonine-59 insulin-like growth factor I, multiplication stimulating activity, and insulin inhibit the binding with IC50 = 10 nM, 84 nM, and 500 nM, respectively. Insulin in high concentrations (>5 μM) completely inhibits I-insulin-like growth factor I binding to A10 cells. Threonine-59 insulin-like growth factor I and insulin stimulate [H]thymidine incorporation into DNA in A10 cells that had been growth arrested by incubation in serum-free media (DMEM/0.1% BSA) for 24–36 hours. The stimulation produced by the peptides is 50–60% of the stimulation produced by 10% fetal calf serum. Low levels of serum (0.1 and 0.5%) also stimulate DNA synthesis, and the effects of Threonine-59 insulin-like growth factor I and low serum are additive. The ED50 for the effects of Threonine-59 insulin-like growth factor I, multiplication stimulating activity, and insulin are 6.8 % 0.3 nM, 36 % 2.5 nM, and 360 % 242 nM, respectively. Incubation of A10 cells for 24 hours with Threonine-59 insulin-like growth factor I or serum increases the protein content per culture dish by 85 % 21 and 183 % 26%, respectively (mean % SEM). Thus, both protein levels and DNA synthesis are increased by incubation with peptides. However, Threonine-59 insulinlike growth factor I does not increase the number of cells in serum starved cultures, although 10% fetal calf serum does. Platelet-derived growth factor also stimulates DNA synthesis in A10 cells, but epidermal growth factor and acidic fibroblast growth factor do not. The effects of platelet-derived growth factor and Threonine-59 insulin-like growth factor I are additive. DNA synthesis begins 12 hours after the addition of Threonine-59 insulin-like growth factor I or serum to growth-arrested A10 cells. Threonine-59 insulin-like growth factor I can be removed from the cells after 8 hours, and maximal stimulation of DNA synthesis still occurs. Thus, a minimum 8-hour exposure of A10 cells to Threonine-59 insulin-like growth factor I is necessary to initiate the events required for the cells to progress from G1 to S phase. These data suggest that insulin-like growth factor I stimulates DNA synthesis in the A10 rat vascular smooth muscle cell line in the absence of serum and other exogenously added growth factors. However, insulin-like growth factor I alone apparently is not sufficient for cells