An MLCK-dependent window in late G1 controls S phase entry of proliferating rodent hepatocytes via ERK-p70S6K pathway

Abstract

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. T he G1 phase is a preparative step where cells temporally integrate complex signals from the microenvironment. At this time, the cells are able to switch between proliferation, differentiation or apoptosis, depending on extracellular matrix composition, cytokines, and cell-cell contacts. In the hepatocyte, a highly differentiated cell, cell cycle progression in vivo during liver regeneration is regulated by a sequential acquisition of complex signals depending on transduction pathway activation. 1-3 Primary cultures of hepatocytes are a powerful model in studying the precise sequence of events that are necessary for cell progression from a G0-like state to S phase. The model mimics the physiological process of hepatic regeneration after liver injury or partial hepatectomy. In this context, we have already demonstrated that epidermal growth factor (EGF) possesses two distinct and complementary effects on G1 phase hepatocytes. 4 First, the growth factor is a morphogen in early G1 phase by inducing controlled spreading of hepatocytes via integrin ␤1 regulation. Second, a mitogenic effect occurs in midlate G1 phase and allows hepatocytes to progress through a restriction point located two thirds of the way through G1 phase. [5][6][7] In addition, EGF promotes cell progression up to late G1. 7 In hepatocytes, EGF activates specific transduction pathways. MEK and PI3K signaling cascades are essential for progression past the G1/S checkpoint and hepatocyte progression to S phase. 8 They both control expression of cyclin D1, a key cell cycle protein that is upregulated in the pre-replicative phase of liver regeneration and in primary culture hepatocytes. 4,9,10 MLCK and RhoK, two kinases linked to the contractile apparatus, are known to be involved in the regulation of a wide range of cellular functions, including prol...

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Hepatocyte-specific deletion of Cdc42 results in delayed liver regeneration after partial hepatectomy in mice

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