DNA-SYNTHESIS BY OVINE MAMMARY ALVEOLAR EPITHELIAL-CELLS - EFFECTS OFHEPARIN, EPIDERMAL GROWTH-FACTOR RELATED PEPTIDES AND INTERACTION WITH STAGE OF PREGNANCY

Amphiregulin is a heparin-binding member of the epidermal growth facto r (EGF) family, which we have recently shown to be expressed in sheep mammary gland. Uniquely among known EGF-like growth factors, its mitog enic activity is inhibited by soluble heparin, but heparin-like molecu les on the cell surface and/or in extracellular matrix appear to be ne cessary for amphiregulin to exert its biological effect. In primary cu ltures 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 shee p (63.1 +/- 8.2%, mean +/- S.E.M., 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 gro wth factor-alpha (TGF-alpha) significantly (P<0.001) increased DNA syn thesis in the presence of heparin. The effect of TGF-alpha was dose-re lated, wholly reversing the inhibitory effect of heparin in cell cultu res from non-pregnant and 20-week pregnant sheep. DNA synthesis was st imulated by amphiregulin and TGF-alpha increased the maximum response. The heparin antagonist, hexadimethrine, inhibited DNA synthesis, but, in the presence of amphiregulin, approximately equivalent concentrati ons of heparin overcame this inhibitory effect. In the presence of hep arin, TGF-alpha showed synergistic interactions with insulin or IGF-I. The results indicate interactive effects of EGF and IGF growth factor families in sheep mammary growth.