Microsome factions prepared from the mammary glands of non-pregnant, pregnant and lactating sheep have been used to study binding of 125I-labelled transforming growth factor-alpha (TGF-alpha). Binding was dependent on microsomal protein concentration, time and temperature. It showed the characteristics of an epidermal growth factor (EGF) receptor, being displaced by TGF-alpha and EGF, but not by insulin or IGF-I. The non-linear curve fitting program LIGAND was used to determine affinity and number of binding sites. A single class of high-affinity binding sites was found. The apparent dissociation constant (Kd) was similar in all physiological states (2.43 +/- 0.27 mol/l x 10(-10), n = 23). Numbers of binding sites were lower in late-pregnant (20 weeks) and lactating sheep (14.07 +/- 2.45 fmol/mg protein, n = 10) than in non-pregnant, 10- or 15-week pregnant sheep (43.04 +/- 5.93 fmol/mg protein, n = 13). DNA synthesis by mammary alveolar epithelial cells cultured on collagen gels was increased twofold by TGF-alpha (maximum response at 10 micrograms/l; 1.8 nmol/l) but not by EGF. Cells derived from 15- to 20-week pregnant sheep responded significantly to TGF-alpha on day 3 of culture, but the response was delayed to day 4-5 of culture in cells from other physiological states. Dose-response was not significantly affected. TGF-alpha and IGF-I produced an additive effect on DNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
Amphiregulin is a heparin-binding member of the epidermal growth factor (EGF) family, which we have recently shown to be expressed in sheep mammary gland. Uniquely among known EGF-like growth factors, its mitogenic activity is inhibited by soluble heparin, but heparin-like molecules on the cell surface and/or in extracellular matrix appear to be necessary for amphiregulin to exert its biological effect. In primary cultures of sheep mammary alveolar epithelial cells, heparin (1-20 mg/l) inhibited DNA synthesis in a dose-dependent manner. The extent of the inhibition was influenced by physiological state, being greater (P<0·05) in mammary cell cultures derived from 5-to 10-week pregnant sheep (63·1 8·2%, mean ..., n=8) than in cultures derived from sheep which were non-pregnant (35·8 8·3% inhibition, n=6) or late, 20-week, pregnant (39·8 5·6%, n=6). Both EGF and transforming growth factor-(TGF-) significantly (P<0·001) increased DNA synthesis in the presence of heparin. The effect of TGF-was dose-related, wholly reversing the inhibitory effect of heparin in cell cultures from non-pregnant and 20-week pregnant sheep. DNA synthesis was stimulated by amphiregulin and TGF-increased the maximum response. The heparin antagonist, hexadimethrine, inhibited DNA synthesis, but, in the presence of amphiregulin, approximately equivalent concentrations of heparin overcame this inhibitory effect. In the presence of heparin, TGF-showed synergistic interactions with insulin or IGF-I. The results indicate interactive effects of EGF and IGF growth factor families in sheep mammary growth.
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