ALPHA-ADRENERGIC INHIBITION OF PROLIFERATION IN HEPG2 CELLS STABLY TRANSFECTED WITH THE ALPHA(1B)-ADRENERGIC RECEPTOR THROUGH A P42(MAPKINASE) P21(CIP1/WAF1)-DEPENDENT PATHWAY/

Activation of alpha(1B) adrenergic receptors (alpha(1B)AR) promotes DN A synthesis in primary cultures of hepatocytes, yet expression of alph a(1B)AR in hepatocytes rapidly declines during proliferative events, H epG2 human hepatoma cells, which do not express alpha(1B)AR, were stab ly transfected with a rat alpha(1B)AR cDNA (TFG2 cells), in order to s tudy the effects of maintained alpha(1B)AR expression on hepatoma cell proliferation. TFG2 cells had a decreased rate of growth compared to mock transfected HepG2 cells as revealed by a decrease in [H-3]thymidi ne incorporation into DNA, Stimulation of alpha(1B)AR with phenylephri ne caused a further large reduction in TFG2 cell growth, whereas no ef fect on growth was observed in mock transfected cells, Reduced cell gr owth correlated,vith increased percentages of cells found in G(0)/G(1) and G(2)/M phases of the cell cycle. In TFG2 cells, phenylephrine inc reased p42(MAPkinase) activity by 1.5- to 2.0-fold for up to 24 h and increased expression of the cyclin dependent kinase inhibitor protein p21(Cip1/WAF1). Treatment of TFG2 cells with the specific MEK1 inhibit or PD98059, or infection with a -/- MEK1 recombinant adenovirus permit ted phenylephrine to increase rather than decrease [H-3]thymidine inco rporation. In addition, inhibition of MAP kinase signaling by PD98059 or MEK1 -/- blunted the ability of phenylephrine to increase p21(Cip1/ WAF1) expression. In agreement with a role for increased p21(Cip1/WAF1 ) expression in causing growth arrest, infection of TFG2 cells with a recombinant adenovirus to express antisense p21(Cip1/WAF1) mRNA blocke d the ability of phenylephrine to increase p21(Cip1/WAF1) expression a nd to inhibit DNA synthesis. Antisense p21(Cip1/WAF1) permitted phenyl ephrine to stimulate DNA synthesis in TFG2 cells, and abrogated growth arrest. These results suggest that transformed hepatocytes may turn o ff the expression of alpha(1B)ARs in order to prevent the activation o f a growth inhibitory pathway. Activation of this inhibitory pathway v ia alpha(1B)AR appears to be p42(MAPkinase) and p21(Cip1/WAF1) depende nt. (C) 1998 Federation of European Biochemical Societies.