Mitogenic activity of epidermal growth factor on newborn rat astroglia: interaction with insulin-like growth factors

Newborn rat astroglia cells possess epidermal growth factor (EGF) and insulin-like growth factor (IGF) receptors, which suggests that these growth factors regulate their growth and development. To determine the relative roles and interactions between the two growth factors on astroglial growth, primary cultures of astroglial cells from newborn rats (1 day postnatal) were treated with pure peptides, singly or in combination in various concentrations, and the growth response was determined by DNA synthesis ([3H]thymidine incorporation). EGF, IGF-I, and IGF-II, as single peptides, stimulated DNA synthesis, with half-maximal stimulatory concentrations of 0.25 ng/ml for EGF, 2.0 ng/ml for IGF-I, and 25 ng/ml for IGF-II, respectively. These findings indicate that astroglial cells are responsive to these growth factors in physiological concentrations, with the relative sensitivity of EGF greater than IGF greater than IGF-II. When EGF and IGF-I were added in combination, the growth stimulatory effect was greater than the additive effects of each growth factor added alone, indicating that the two growth factors act in synergism with each other. In particular, addition of increasing concentrations of EGF from 0.25-10 ng/ml to a constant concentration of 50 ng/ml IGF-I resulted in significant potentiation of [3H]thymidine incorporation of astroglial cells. To determine if the synergistic effect was due to a local synthesis of IGF-I by astroglia, a specific monoclonal antibody against IGF-I (Sm 1.2) was added to the peptides. Sm 1.2 decreased not only IGF-I-stimulated DNA synthesis, but also EGF-stimulated DNA synthesis, suggesting that the effects of EGF were contributed to in part by the local synthesis of IGF-I by astroglial cells. Analysis of conditioned medium from cells treated with EGF revealed a significant increase (approximately 2-fold) in IGF-I (from 4.5 to 8.8 ng/ml), but not IGF-II. To determine if the EGF effect on IGF synthesis was at the level of IGF-I mRNA transcription, stable IGF-I mRNA levels were determined in the astroglial cells before and after stimulation with EGF, using Northern analysis and quantification by densitometry. Astroglia expressed four IGF-I mRNA transcripts as in the adult and fetal liver, but only one (3.6 kilobases) IGF-II mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)