Abstract

We investigated the effects of transforming growth factor α (TGF-α) on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes and examined the influence of α and β adrenoceptor agonists on the TGF-α-induced responses. TGF-α (1.0 ng/ml) produced a 4.1-fold elevation of DNA synthesis during 3 h of culture and a 1.2-fold increase in the nucleus number (proliferation) during 4 h of culture at a cell density of 3.3 × 10⁴ cells/cm². The TGF-α-induced hepatocyte DNA synthesis and proliferation were dose-dependent at EC₅₀ values of 0.36 ng/ml and 0.45 ng/ml, respectively. Hepatocyte DNA synthesis and proliferation induced by 1.0 ng/ml TGF-α did not reduce even at higher initial plating densities (5.0 × 10⁴ and 1.0 × 10⁵ cells/cm²). Increasing concentrations of the β₂ adrenoceptor agonist metaproterenol (10⁻⁷–10⁻⁶ M) markedly reduced the proliferative effects of TGF-α, whereas those of the α₂ adrenoceptor agonist 5-bromo-6-[2-imidazolin-2-yl-amino]-quinoxaline (UK-14304; 10⁻⁶–10⁻⁵ M) and the α₁adrenoceptor agonist phenylephrine (10⁻⁷–10⁻⁶ M) significantly potentiated the TGF-α action. The proliferative effects of TGF-α (1.0 ng/ml) were not affected significantly by a monoclonal antiepidermal growth factor receptor antibody (1–100 ng/ml) and were almost completely blocked by specific inhibitors of signal transducers such as genistein (10⁻⁵ M), 1–6[[17β-3methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrol2,5-dione (U-73122; 0⁻⁵ M), wortmannin (5 × 10⁻⁷M), sphingosine (5 × 10⁻⁶ M), 2′-amino-3′-methoxyflavone (PD98059; 5 × 10⁻⁵ M), and rapamycin (10 ng/ml). These results suggest that among the elements that link signals of cell surface receptor to the nucleus, the proliferative action of TGF-α is mediated, at least, by tyrosine kinase, phospholipase C, phosphatidylinositol 3-kinase, protein kinase C, mitogen-activated protein kinase kinase, and ribosomal protein p70 S6 kinase.