Vol. 291, Issue 1, 171-180, October 1999

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

Mitsutoshi Kimura and Masahiko Ogihara

Department of Pharmacology and Therapeutics, Faculty of Pharmaceutical Sciences, Josai University, Saitama, Japan

We investigated the effects of transforming growth factor  (TGF-) on DNA synthesis and proliferation in primary cultures of adult rat hepatocytes and examined the influence of  and  adrenoceptor agonists on the TGF--induced responses. TGF- (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--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- did not reduce even at higher initial plating densities (5.0 × 10⁴ and 1.0 × 10⁵ cells/cm²). Increasing concentrations of the ₂ adrenoceptor agonist metaproterenol (10⁷-10⁶ M) markedly reduced the proliferative effects of TGF-, whereas those of the ₂ adrenoceptor agonist 5-bromo-6-[2-imidazolin-2-yl-amino]-quinoxaline (UK-14304; 10⁶-10⁵ M) and the ₁ adrenoceptor agonist phenylephrine (10⁷-10⁶ M) significantly potentiated the TGF- action. The proliferative effects of TGF- (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 (10⁵ M), 1-6[[17-3methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrol2,5-dione (U-73122; 0⁵ M), wortmannin (5 × 10⁷ M), sphingosine (5 × 10⁶ M), 2'-amino-3'-methoxyflavone (PD98059; 5 × 10⁵ 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- 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.