Stimulation by Transforming Growth Factor-a of DNA Synthesis and Proliferation of Adult Rat Hepatocytes in Primary Cultures: Modulation by a- and b-Adrenoceptor Agonists

We investigated the effects of transforming growth factor a (TGF-a) on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes and examined the influence of a and b adrenoceptor agonists on the TGF-a-induced responses. TGF-a (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 3 10 cells/cm. The TGF-a-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-a did not reduce even at higher initial plating densities (5.0 3 10 and 1.0 3 10 cells/cm). Increasing concentrations of the b2 adrenoceptor agonist metaproterenol (10–10 M) markedly reduced the proliferative effects of TGF-a, whereas those of the a2 adrenoceptor agonist 5-bromo-6-[2-imidazolin-2-yl-amino]-quinoxaline (UK-14304; 10–10 M) and the a1 adrenoceptor agonist phenylephrine (10–10 M) significantly potentiated the TGF-a action. The proliferative effects of TGF-a (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 M), 1– 6[[17b-3methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrol2,5-dione (U-73122; 0 M), wortmannin (5 3 10 M), sphingosine (5 3 10 M), 29-amino-39-methoxyflavone (PD98059; 5 3 10 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-a 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. Mature rat liver in its normal state is quiescent and thus does not proliferate (Michalopoulos and DeFrances, 1997). However, the liver does have a tremendous capacity to regenerate. For example, after extensive hepatic resection (involving ;70% of the liver mass), the remaining hepatocytes proliferate to restore the mass of the organ within 2 weeks. The key to understanding the process may lie in a knowledge of the action and interactions of the various growth factors and growth modulators that have stimulatory or inhibitory activities (Diehl and Rai, 1996; Michalopoulos and DeFrances, 1997). Studies searching for a trigger substance have implicated humoral factors, portal-derived hepatotrophic factors, and liver-derived growth factors. However, despite extensive analysis of this precisely regulated process, the mechanisms that initiate, maintain, and terminate this intrinsic regenerative process are not well understood. This is probably because such studies are complicated by the fact that so many factors are involved in the hepatic regenerative