Proteomic analysis reveals a major role for contact inhibition in the terminal differentiation of hepatocytes.
OBJECTIVE
Hepatocytes are considered an exception of the paradigmatic inverse correlation between cell proliferation and terminal differentiation. In fact, hepatic vital functions are guaranteed by proliferating parenchymal cells during liver regeneration. However, a fine molecular characterization of the relationship between proliferation and differentiation in hepatocytes has been hampered by the lack of reliable in vivo or in vitro models.


METHODS
The hepatocyte terminal differentiation program was characterized in the immortalized, untransformed and differentiated hepatocytic cell line MMH, using several techniques. Particularly, two-dimensional difference gel electrophoresis combined to tandem mass spectrometry proteomic approach was used. Cell cycle and cell adhesion properties of MMH have been altered using either myc-overexpression and MEK1/2 inhibition or a constitutive active beta-catenin mutant, respectively.


RESULTS
The hepatocyte terminal differentiation program is stimulated by the exit from the cell cycle induced by cell-cell contact. Comparative proteomic analysis of proliferating versus quiescent hepatocytes validated the importance of contact inhibition, identifying 68 differently expressed gene products, representing 49 unique proteins. Notably, enzymes involved in important liver functions such as detoxification processes, lipid metabolism, iron and vitamin A storage and secretion, anti-inflammatory response and exocytosis were found significantly up-regulated in quiescent hepatocytes. Finally, we found that: (i) cell cycle arrest induced by MEK1/2 inhibition is not sufficient to induce hepatic product expression; (ii) constitutive activation of beta-catenin counteracts the contact inhibition-induced terminal differentiation.


CONCLUSIONS
The hepatocyte terminal differentiation program requires a quiescent state maintained by cell-cell contact through the E-cadherin/beta-catenin pathway, rather than the inhibition of proliferation.
Mancone
Carmine
C
Laboratory of Gene Expression, National Institute for Infectious Diseases L. Spallanzani IRCCS, Via Portuense 292, Rome, Italy.
Conti
Beatrice
B
Amicone
Laura
L
Bordoni
Veronica
V
Cicchini
Carla
C
Calvo
Ludovica
L
Perdomo
Ariel Basulto
AB
Fimia
Gian Maria
GM
Tripodi
Marco
M
Alonzi
Tonino
T
D000818:Animals; D050260:Carbohydrate Metabolism; D002453:Cell Cycle; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D003260:Contact Inhibition / Q000502:physiology*; D005089:Exocytosis; D022781:Hepatocytes / Q000166:cytology* / Q000502:physiology*; D050356:Lipid Metabolism; D051379:Mice; D040081:Protein Array Analysis; D014176:Protein Biosynthesis; D040901:Proteomics; D011994:Recombinant Proteins / Q000378:metabolism; D013312:Stress, Physiological; D015854:Up-Regulation; D051176:beta Catenin / Q000378:metabolism
C495267:CTNNB1 protein, mouse; D011994:Recombinant Proteins; D051176:beta Catenin