Convergence of Wnt signaling on the HNF4alpha-driven transcription in controlling liver zonation.
OBJECTIVE
In each hepatocyte, the specific repertoire of gene expression is influenced by its exact location along the portocentrovenular axis of the hepatic lobule and provides a reason for the liver functions compartmentalization defined "metabolic zonation." So far, few molecular players controlling genetic programs of periportal (PP) and perivenular (PV) hepatocytes have been identified; the elucidation of zonation mechanisms remains a challenge for experimental hepatology. Recently, a key role in induction and maintenance of the hepatocyte heterogeneity has been ascribed to Wnt/beta-catenin pathway. We sought to clarify how this wide-ranging stimulus integrates with hepatocyte specificity.


METHODS
Reverse transcriptase polymerase chain reaction (RT-PCR) allowed the transcriptional profiling of hepatocytes derived from in vitro differentiation of liver stem cells. The GSK3beta inhibitor 6-bromoindirubin-3'-oxime (BIO) was used for beta-catenin stabilization. Co-immunoprecipitations were used to study biochemical protein interactions while ChIP assays allowed the in vivo inspection of PV and PP genes regulatory regions.


RESULTS
We found that spontaneous differentiation of liver stem cells gives rise to PP hepatocytes that, after Wnt pathway activation, switch into PV hepatocytes. Next, we showed that the Wnt downstream player LEF1 interacts with the liver-enriched transcriptional factor HNF4alpha. Finally, we unveiled that the BIO induced activation of PV genes correlates with LEF1 binding to both its own and HNF4alpha consensus, and the repression of PP genes correlates with HNF4alpha displacement from its own consensus.


CONCLUSIONS
Our data show a direct and hitherto unknown convergence of the canonical Wnt signaling on the HNF4alpha-driven transcription providing evidences of a mechanism controlling liver zonated gene expression.
Colletti
Marta
M
Department of Cellular Biotechnologies and Haematology, Istituto Pasteur-Fondazione Cenci Bolognetti, University Sapienza of Rome, Italy.
Cicchini
Carla
C
Conigliaro
Alice
A
Santangelo
Laura
L
Alonzi
Tonino
T
Pasquini
Emiliano
E
Tripodi
Marco
M
Amicone
Laura
L
D000818:Animals; D015153:Blotting, Western; D002454:Cell Differentiation / Q000235:genetics*; D049109:Cell Proliferation; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D051557:Hepatocyte Nuclear Factor 4 / Q000235:genetics / Q000378:metabolism*; D022781:Hepatocytes / Q000166:cytology* / Q000502:physiology; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D015398:Signal Transduction / Q000235:genetics* / Q000502:physiology; D014162:Transfection; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism*; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism*
C496592:HNF4A protein, human; D051557:Hepatocyte Nuclear Factor 4; D051153:Wnt Proteins; D051176:beta Catenin