c-Fos overexpression increases the proliferation of human hepatocytes by stabilizing nuclear Cyclin D1.
OBJECTIVE
To investigate the effect of stable c-Fos overexpression on immortalized human hepatocyte (IHH) proliferation.


METHODS
IHHs stably transfected with c-Fos (IHH-Fos) or an empty vector (IHH-C) were grown in medium supplemented with 1% serum or stimulated with 10% serum. Cell proliferation was assessed by cell counts, 3H-thymidine uptake and flow cytometry analyses. The levels of cell cycle regulatory proteins (Cyclin D1, E, A) cyclin dependent kinases (cdk) cdk2, cdk4, cdk6, and their inhibitors p15, p16, p21, p27, total and phosphorylated GSK-3beta and epidermal growth factor receptor (EGF-R) were assayed by Western blotting. Analysis of Cyclin D1 mRNA levels was performed by reverse transcription-polymerase chain reaction and real-time polymerase chain reaction (PCR) analysis. Stability of Cyclin D1 was studied by cycloheximide blockade experiments.


RESULTS
Stable c-Fos overexpression increased cell proliferation under low serum conditions and resulted in a two-fold increase in [3H]-thymidine incorporation following serum addition. Cell cycle analysis by flow cytometry showed that c-Fos accelerated the cell cycle kinetics. Following serum stimulation, Cyclin D1 was more abundantly expressed in c-Fos overexpressing cells. Cyclin D1 accumulation did not result from increased transcriptional activation, but from nuclear stabilization. Overexpression of c-Fos correlated with higher nuclear levels of inactive phosphorylated GSK-3beta, a kinase involved in Cyclin D1 degradation and higher levels of EGF-R mRNA, and EGF-R protein compared to IHH-C both in serum starved, and in serum stimulated cells. Abrogation of EGF-R signalling in IHH-Fos by treatment with AG1478, a specific EGF-R tyrosine kinase inhibitor, prevented the phosphorylation of GSK-3beta induced by serum stimulation and decreased Cyclin D1 stability in the nucleus.


CONCLUSIONS
Our results clearly indicate a positive role for c-Fos in cell cycle regulation in hepatocytes. Importantly, we delineate a new mechanism by which c-Fos could contribute to hepatocarcinogenesis through stabilization of Cyclin D1 within the nucleus, evoking a new feature to c-Fos implication in hepatocellular carcinoma.
Güller
Meryem
M
Institut National de la Sante et de la Recherche Medicale U773, Hôpital Saint-Louis, and Université Paris 7 Denis Diderot, Centre de Recherche Bichat Beaujon CRB3, BP 416, Paris F-75018, France.
Toualbi-Abed
Kahina
K
Legrand
Agnès
A
Michel
Laurence
L
Mauviel
Alain
A
Bernuau
Dominique
D
Daniel
Fanny
F
D002453:Cell Cycle; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D002471:Cell Transformation, Neoplastic / Q000378:metabolism; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000378:metabolism*; D050756:Cyclin-Dependent Kinase Inhibitor Proteins / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D055550:Protein Stability; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism*; D011799:Quinazolines; D012333:RNA, Messenger / Q000378:metabolism; D014162:Transfection; D020032:Tyrphostins / Q000494:pharmacology; D015854:Up-Regulation
C530104:CCND1 protein, human; D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D047428:Protein Kinase Inhibitors; D011500:Protein Synthesis Inhibitors; D016760:Proto-Oncogene Proteins c-fos; D011799:Quinazolines; D012333:RNA, Messenger; D020032:Tyrphostins; D019938:Cyclin D1; C101044:RTKI cpd; D003513:Cycloheximide; C512478:EGFR protein, human; D066246:ErbB Receptors; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D018844:Cyclin-Dependent Kinases; D038362:Glycogen Synthase Kinase 3