An MLCK-dependent window in late G1 controls S phase entry of proliferating rodent hepatocytes via ERK-p70S6K pathway.
We show that MLCK (myosin light chain kinase) plays a key role in cell cycle progression of hepatocytes: either chemical inhibitor ML7 or RNA interference led to blockade of cyclin D1 expression and DNA replication, providing evidence that MLCK regulated S phase entry. Conversely, inhibition of RhoK by specific inhibitor Y27632 or RhoK dominant-negative vector did not influence progression in late G1 and S phase entry. Inhibition of either MLCK or RhoK did not block ERK1/2 phosphorylation, whereas MLCK regulated ERK2-dependent p70S6K activation. In addition, DNA synthesis was reduced in hepatocytes treated with p70S6K siRNA, demonstrating the key role played by the kinase in S phase entry. Interestingly, after the G1/S transition, DNA replication in S phase was no longer dependent on MLCK activity. We strengthened this result by ex vivo experiments and evidenced an MLCK-dependent window in late G1 phase of regenerating liver after two-thirds partial hepatectomy. In conclusion, our results underline an MLCK-dependent restriction point in G1/S transition, occurring downstream of ERK2 through the regulation of p70S6K activation, and highlighting a new signaling pathway critical for hepatocyte proliferation.
Bessard
Anne
A
INSERM U522, Unité de Recherches Hépatologiques; IFR 140; Université de Rennes1, Rennes, France.
Coutant
Alexandre
A
Rescan
Claude
C
Ezan
Frédéric
F
Frémin
Christophe
C
Courselaud
Brice
B
Ilyin
Gennady
G
Baffet
Georges
G
D000577:Amides / Q000494:pharmacology; D000818:Animals; D001381:Azepines / Q000494:pharmacology; D015152:Blotting, Northern; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000187:drug effects / Q000235:genetics; D004261:DNA Replication / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051606:G-Protein-Coupled Receptor Kinase 1 / Q000187:drug effects; D016193:G1 Phase; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D008856:Microscopy, Fluorescence; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009281:Naphthalenes / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D012313:RNA / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000235:genetics / Q000378:metabolism*; D016196:S Phase
D000577:Amides; D001381:Azepines; D004791:Enzyme Inhibitors; D009281:Naphthalenes; D011725:Pyridines; C070571:ML 7; D019938:Cyclin D1; C108830:Y 27632; D012313:RNA; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D051606:G-Protein-Coupled Receptor Kinase 1; C496765:Grk1 protein, rat; D009219:Myosin-Light-Chain Kinase