Regulation of IGF binding protein synthesis by a bovine mammary epithelial cell line.
IGF-I has been proposed as a key regulator of mammary epithelial cell (MEC) growth and differentiation. As IGF-I bioactivity is modulated by specific, high-affinity binding proteins (IGFBP), the forms of IGFBP that are secreted by the bovine MEC line, MAC-T, were identified. Media conditioned by MAC-T cells contained four forms of IGFBP that were identified, by western blotting with specific antibodies, as IGFBP-2, -3, -4 and -6. The amounts of IGFBP-3 in conditioned media were relatively low under basal conditions when analyzed by ligand blotting with 125I-IGF-II, but were increased dramatically relative to serum-free controls by exposure to IGF-I (100 ng/ml) or IGF-II (100 ng/ml) for 24 h. These increases in IGFBP-3 protein corresponded with dose-dependent increases in IGFBP-3 mRNA, with IGF-II eliciting a smaller response than was elicited by IGF-I at each concentration. Leu-IGF-I, which has reduced affinity for the IGF-I receptor but normal affinity for IGFBPs, failed to increase IGFBP-3 protein and mRNA levels, whereas B-chain IGF-I (normal affinity for the receptor but reduced affinity for IGFBPs) elicited the response, thus implying an IGF-I receptor-mediated event. Time-course studies indicated that IGFBP-3 mRNA was increased fourfold by 3 h of IGF-I treatment, with maximal increases of eightfold above serum-free controls observed between 8 and 13 h of treatment. By 24 h of treatment, IGFBP-3 mRNA levels had declined and were approximately threefold above controls in cells exposed to IGF-I. Amounts of messenger RNA of IGFBP-6 and IGFBP-2 were not increased by IGF treatment. However, retinoic acid (10(-6) M) stimulated both IGFBP-2 and IGFBP-6 protein and mRNA levels, but it decreased IGFBP-3 mRNA levels relative to controls. The combination of retinoic acid plus IGF-I had no additional effect on IGFBP-6 or -2 above that observed with retinoic acid alone, whereas IGF-I together with retinoic acid attenuated the decrease in IGFBP-3 observed with retinoic acid alone. Protein kinase A-mediated pathways were also shown to alter IGFBP synthesis. Forskolin, which increases cAMP, increased IGFBP-3 protein and mRNA levels. The combination of IGF-I plus forskolin resulted in greater increases in both protein and mRNA than were observed with either treatment alone. In contrast, forskolin decreased IGFBP-6 mRNA relative to controls, but had no effect on IGFBP-2. The decrease in IGFBP-6 was less marked when cells were treated with a combination of IGF-I and forskolin. Forskolin had no effect on IGFBP-2 mRNA levels. In summary, the ability of IGF-I specifically to regulate IGFBP-3 synthesis represents a mechanism whereby IGF-I may regulate its own bioactivity. In addition, the differential regulation of IGFBP-2, -3 and -6 by retinoic acid (which inhibits proliferation) and IGF-I (which stimulates proliferation) suggests that these forms of IGFBP have different roles in regulating mammary epithelial cell physiology.
Cohick
W S
WS
Department of Animal Sciences, Rutgers University, New Brunswick, New Jersey, USA.
Turner
J D
JD
D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D005260:Female; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D018974:Insulin-Like Growth Factor Binding Protein 4 / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D018976:Insulin-Like Growth Factor Binding Protein 6 / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D018969:Insulin-Like Growth Factor Binding Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D007334:Insulin-Like Growth Factor I / Q000378:metabolism / Q000494:pharmacology*; D008321:Mammary Glands, Animal / Q000187:drug effects / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis; D014212:Tretinoin / Q000494:pharmacology
D018971:Insulin-Like Growth Factor Binding Protein 2; D018972:Insulin-Like Growth Factor Binding Protein 3; D018974:Insulin-Like Growth Factor Binding Protein 4; D018976:Insulin-Like Growth Factor Binding Protein 6; D018969:Insulin-Like Growth Factor Binding Proteins; D012333:RNA, Messenger; D005576:Colforsin; D014212:Tretinoin; D007334:Insulin-Like Growth Factor I; D000242:Cyclic AMP