Characterization and estrogen regulation of uterine growth factor activity

Abstract

Estradiol-17B has been shown to stimulate glucose transport, measured by phosphorylation of 2-deoxyglucose, and stimulate DNA synthesis, as measured by 3H-thymidine incorporation, In rat uterus in vivo. Attempts to demonstrate these responses in uterine cells in primary culture and in uterine tumor cells In culture have been unsuccessful. These observations led to the hypothesis that some responses to estradiol are mediated through the local action of peptide factors. Acid extracts of rat, bovine and rabbit uterus stimulated glucose transport and DNA synthesis in uterine tumor cells and in primary cultures of rat uterine cells. The stimulation of glucose transport was of the same magnitude (1.5-to 3.0-fold) and followed the same time course (maximum stimulation at 2-3 h) as estradiol stimulation in vivo. Uteri from estradiol treated rats contained 4 times more glucose transport-stimulating activity as did control rat uteri. The activity was acid and heat stable, was inactivated by trypsin, but not removed by dextran-coated charcoal treatment. The activity eluted in 6-12 kDa range on Sephadex G-50. DNA synthetic activity in rat uterine homogenates was elevated 3-fold within 18-24 h after estradiol injection and remained elevated with subsequent Injections. The growth-promoting activity was acid and heat stable, was reduced by trypsin but not reduced by treatment with dextran-coated charcoal. Gel filtration showed molecular weight heterogeneity with activity eluting at MW 10,000-30,000. The effect of purified growth factors on DNA synthesis in primary cultures of rat uterine cells was examined. Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and transforming growth factor-B (TGFB) had no effect on 3H-thymidine incorporation under optimal conditions of incorporation. Relaxin and multiplication stimulating activity (MSA) demonstrated a stimulatory effect only at high concentrations, 207% of control at 100 |ag/ml and 175% of control at 100 ng/ml, respectively. Insulin stimulated incorporation 350% at 100 ng/ml, insulinlike growth factor-l (IGF-I) stimulated incorporation 300-400% at 10-100 g/ml, and platelet-derived growth factor (PDGF)stimulated incorporation 450% at 3 units/ml. When the positive effectors (insulin, IGF-I, MSA, and PDGF) were analyzed either combined or individually in the presence of uterine extract, the level of stimulation was greater than the maximum stimulation bserved with extract alone and approached that seen with 10% serum. Uterine extracts from estradiol treated and control rats were analyzed for IGF-I by radioimmunoassay. IGF-I was elevated 500-1000% in estradiol treated extracts relative to control levels. In summary, estradiol increases the growthpromoting activity and glucose transport-stimulating activity of uterine extracts. IGF-I was a positive stimulator of DNA synthesis in primary uterine cultures and was elevated in uterine extracts. This growth factor may be involved in the stimulation of DNA synthesis In rat uterus by estradiol.