Insulin-like growth factor binding protein-3 mediates IGF-I action in a bovine mammary epithelial cell line independent of an IGF interaction

IGF-I is mitogenic for the bovine mammary epithelial cell line MAC-T. in ad dition, IGF-I specifically upregulates IGFBP-3 synthesis in these cells. To investigate this effect on cell growth and IGF-I responsiveness, cell line s were developed that constitutively express IGFBP-3. MAC-T cells transfect ed with IGFBP-3 (+BP3) or vector alone (Mock) grew similarly over 7 days in 10 or 1% fetal calf serum. Basal DNA synthesis was lower (70%) in +BP3 cel ls compared to Mock cells. However, DNA synthesis was increased by IGF-I (1 -50 ng/ml) relative to untreated controls to a greater extent in +BP3 cells compared to Mock cells. ICF-I (20 ng/ml) increased DNA synthesis 11- and t hreefold in +BP3 and Mock cells, respectively. Additionally, +BP3 cells wer e more sensitive to the lower concentrations of ICF-I (1-5 ng/ml). In contr ast, preincubation of Mock cells with exogenous IGFBP-3 did not enhance res ponsiveness or sensitivity to IGF-I. Basal DNA synthesis was unaffected by either an IGF neutralizing antibody or exogenous IGFBP3, indicating the dif ferences observed between +BP3 and Mock cells were not attributable to sequ estration of endogenous ICF-I by IGFBP-3. There were no differences between +BP3 and Mock cells in IGF-I receptor number or affinity. DNA synthesis wa s also increased in +BP3 cells, compared to controls, in response to 5 mu g /ml insulin and 2.5 ng/ml Long R(3)IGF-I, indicating that the potentiated r esponse did not require an interaction with IGFBP-3. These results suggest that IGF-I regulation of IGFBP-3 represents a regulatory loop, the function of which is to increase IGF-I bioactivity, using a mechanism that does req uire an IGF-I-IGFBP-3 interaction. (C) 2000 Wiley-Liss, Inc.