Role of the I kappa B kinase complex in oncogenic Ras- and Raf-mediated transformation of rat liver epithelial cells

NF-kappa B/Rel factors have been implicated in the regulation of liver cell death during development, after partial hepatectomy, and in hepatocytes in culture. Rat liver epithelial cells (RLEs) display many biochemical and ul trastructural characteristics of oval cells, which are multipotent cells th at can differentiate into mature hepatocytes. While untransformed RLEs unde rgo growth arrest and apoptosis in response to transforming growth factor b eta 1 (TGF-beta 1) treatment, oncogenic Ras- or Raf-transformed RLEs are in sensitive to TGF-beta 1-mediated growth arrest. Here me have tested the hyp othesis that Ras- or Raf-transformed RLEs have altered NF-kappa B regulatio n, leading to this resistance to TGF-beta 1. We show that classical NF-kapp a B is aberrantly activated in Ras- or Raf-transformed RLEs, due to increas ed phosphorylation and degradation of I kappa B-alpha protein. Inhibition o f NF-kappa B activity with a dominant negative form of I kappa B-alpha rest ored TGF-beta 1-mediated cell killing of transformed RLEs. IKK activity med iates this hyperphosphorylation of I kappa B-alpha protein. As judged by ki nase assays and transfection of dominant negative IKK-1 and IKK-2 expressio n vectors, NF-kappa B activation by Ras appeared to be mediated by both IKK -1 and IKK-2, while Raf-induced NF-kappa B activation was mediated by IKK-2 . NF-kappa B activation in the Ras-transformed cells was mediated by both t he Raf and phosphatidylinositol 3-kinase pathways, while in the Raf-transfo rmed cells, NF-kappa B induction was mediated by the mitogen-activated prot ein kinase cascade. Last, inhibition of either IKK-1 or IKK-2 reduced focus -forming activity in Ras-transformed RLEs. Overall, these studies elucidate a mechanism that contributes to the process of transformation of liver cel ls by oncogene Ras and Raf through the I kappa B kinase complex leading to constitutive activation of NF-kappa B.