The Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Cascade Activation Is a Key Signalling Pathway Involved in the Regulation of G₁ Phase Progression in Proliferating Hepatocytes

Abstract

In this study, activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signalling pathway was analyzed in proliferating rat hepatocytes both in vivo after partial hepatectomy and in vitro following epidermal growth factor (EGF)-pyruvate stimulation. First, a biphasic MEK/ERK activation was evidenced in G 1 phase of hepatocytes from regenerating liver but not from shamoperated control animals. One occurred in early G 1 (30 min to 4 h), and the other occurred in mid-late G 1 , peaking at around 10.5 h. Interestingly, the mid-late G 1 activation peak was located just before cyclin D1 induction in both in vivo and in vitro models. Second, the biological role of the MEK/ERK cascade activation in hepatocyte progression through the G 1 /S transition was assessed by adding a MEK inhibitor (PD 98059) to EGF-pyruvate-stimulated hepatocytes in primary culture. In the presence of MEK inhibitor, cyclin D1 mRNA accumulation was inhibited, DNA replication was totally abolished, and the MEK1 isoform was preferentially targeted by this inhibition. This effect was dose dependent and completely reversed by removing the MEK inhibitor. Furthermore, transient transfection of hepatocytes with activated MEK1 construct resulted in increased cyclin D1 mRNA accumulation. Third, a correlation between the mid-late G 1 MEK/ERK activation in hepatocytes in vivo after partial hepatectomy and the mitogen-independent proliferation capacity of these cells in vitro was established. Among hepatocytes isolated either 5, 7, 9, 12 or 15 h after partial hepatectomy, only those isolated from 12-and 15-h regenerating livers were able to replicate DNA without additional growth stimulation in vitro. In addition, PD 98059 intravenous administration in vivo, before MEK activation, was able to inhibit DNA replication in hepatocytes from regenerating livers. Taken together, these results show that (i) early induction of the MEK/ERK cascade is restricted to hepatocytes from hepatectomized animals, allowing an early distinction of primed hepatocytes from those returning to quiescence, and (ii) mid-late G 1 MEK/ERK activation is mainly associated with cyclin D1 accumulation which leads to mitogen-independent progression of hepatocytes to S phase. These results allow us to point to a growth factor dependency in mid-late G 1 phase of proliferating hepatocytes in vivo as observed in vitro in proliferating hepatocytes and argue for a crucial role of the MEK/ERK cascade signalling pathway.

RNAi-mediated ERK2 knockdown inhibits growth of tumor cells in vitro and in vivo

“…Our results confirm the importance of the MAPK ERK1/2 pathway in the regulation of proliferation of the two transformed hepatic cell lines analysed, similar to that previously described in normal rat and mouse hepatocytes (Talarmin et al, 1999;Fremin et al, 2007). Moreover, this inhibition of proliferation and the blockade in G 1 /S phase are reversible and not toxic in agreement with a nonapoptotic engagement of MEK-inhibited cells as measured by terminal caspases activity.…”

Steatosis does not impair liver regeneration after partial hepatectomy

“…Cell-cycle progression and regenerative mechanisms are controlled by Erk activity via suppressing apoptosis and regulation of DNA replication. 42,43 Our findings demonstrate elevated serum leptin and increased Hgf expression, associated with an altered Erk1/2 phosphorylation pattern in WD-fed mice after PHx. Whereas SD-fed mice showed strong Erk1/2 phosphorylation preoperatively diminishing on PHx þ 1 and PHx þ 2, phosphorylated Erk1/2 was only marginally present in WD mice before surgery and increased after surgery.…”

EGF receptor‐mediated, c‐Src‐dependent, activation of Stat5b is downregulated in mitogenically responsive hepatocytes

“…At the time of seeding, most of the hepatocytes are in the early G 1 phase of the cell cycle (Christoffersen et al, 2000). During culturing, the hepatocytes progress to a restriction point in mid/ late-G 1 where they become highly responsive to mitogenic stimulation by EGF (Sand and Christoffersen, 1987;Wollenberg et al, 1989;Guren et al, 1996;Loyer et al, 1996;Talarmin et al, 1999;Christoffersen et al, 2000). In these cells, the EGF-induced activation of Stat5b was completely downregulated, but EGF readily elicited tyrosine phosphorylation of the EGF receptor and of the adapter protein Shc and induced activation of ERK.…”