Regulation of Cell Cycle Progression and Cellular Survival in Primary Rat Hepatocytes

In liver disease, irregular proliferation of hepatocytes is an important factor favoring hepatocarcinogenesis. The aim of this cell biological thesis has been to study the impact of epidermal growth factor (EGF) and cellular stress on the cell cycle- and cell survival machinery, mainly focusing on the MAP kinase pathway and PI3 kinase-Akt signalling. EGF-induced proliferation is partly driven by the cyclic formation of cyclin-CDK complexes. By using a combination of immunological methods, it was demonstrated that PI3 kinase activity was essential for activation of CDK4. CDK4 in turn was crucial for CDK2 activity, which is an important event in the G1 phase of hepatocyte cell cycle progression. Growth factor-induced PI3 kinase activity also mediated p53 induction, and the induction of this tumor suppressor protein played a key role in cell cycle progression by regulating CDK4 and CDK2 activity, via p21Cip1. EGF stimulation of hepatocytes activates the small GTP-protein Ras that signals through both ERK- and PI3 kinase pathways. Transient transfection of active- and inactive mutants of the Ras isoforms H-Ras and K-Ras, revealed different selectivity for the downstream mediators ERK and PI3 kinase. This distinction had impact on the iso-specific roles of H-Ras and K-Ras in survival and proliferation. It was further demonstrated that redox-activated ERK mediated survival from the cytoplasm and played an important role in adapting hepatocytes to a stressful environment

Inhibitors of AKT kinase increase LDL receptor mRNA expression by two different mechanisms.

Protein kinase B (AKT) is a serine/threonine kinase that functions as an important downstream effector of phosphoinositide 3-kinase. We have recently shown that MK-2206 and triciribine, two highly selective AKT inhibitors increase the level of low density lipoprotein receptor (LDLR) mRNA which leads to increased amount of cell-surface LDLRs. However, whereas MK-2206 induces transcription of the LDLR gene, triciribine stabilizes LDLR mRNA, raising the possibility that the two inhibitors may actually affect other kinases than AKT. In this study, we aimed to ascertain the role of AKT in regulation of LDLR mRNA expression by examining the effect of five additional AKT inhibitors on LDLR mRNA levels. Here we show that in cultured HepG2 cells, AKT inhibitors ARQ-092, AKT inhibitor VIII, perifosine, AT7867 and CCT128930 increase LDLR mRNA levels by inducing the activity of LDLR promoter. CCT128930 also increased the stability of LDLR mRNA. To study the role of AKT isoforms on LDLR mRNA levels, we examined the effect of siRNA-mediated knockdown of AKT1 or AKT2 on LDLR promoter activity and LDLR mRNA stability. Whereas knockdown of either AKT1 or AKT2 led to upregulation of LDLR promoter activity, only knockdown of AKT2 had a stabilizing effect on LDLR mRNA. Taken together, these results provide strong evidence for involvement of AKT in regulation of LDLR mRNA expression, and point towards the AKT isoform specificity for upregulation of LDLR mRNA expression

EGF Activates Autocrine TGFα to Induce Prolonged EGF Receptor Signaling and Hepatocyte Proliferation

Background/Aims: EGF receptor is a main participant in the regulation of liver regeneration. In primary hepatocyte cultures, EGF or TGFα binding to EGF receptor activates Erk1/2 and PI3K pathways, induces cyclin D1 and thus initiates DNA synthesis. We have explored mechanisms by which prolonged EGF receptor activation induces hepatocyte proliferation. Methods: EGF receptor activation, as well as Erk1/2 and PI3K signaling were explored in EGF-stimulated primary hepatocyte cultures by Western blotting and immunocytochemistry. TGFα release to the medium was quantified by ELISA. Effects of a neutralizing antibody to TGFα on EGF receptor signaling and proliferation were explored. Results: Inhibitors of PI3K or Erk1/2 inhibited cyclin D1 expression and G1 progression when added 12 hours after EGF stimulation, whereas depletion of EGF from the medium at this time point did not. ELISA demonstrated that EGF induced TGFα release to the medium. Cyclin D1 induction and cellular proliferation were efficiently inhibited when a neutralizing antibody to TGFα was added to the medium. This also occurred when the antibody was added 12 hours after EGF stimulation. Conclusion: Sustained EGF receptor activity and signaling through both Erk1/2 and PI3K pathways were necessary for proliferation. This was achieved by EGF activation of autocrine TGFα