The mitogen-activated protein kinase kinase extracellular signal-regulatedkinase cascade activation is a key signalling pathway involved in the regulation of G(1) phase progression in proliferating hepatocytes

In this study, activation of the mitogen-activated protein kinase kinase (M EK)/extracellular signal-regulated kinase (ERK) signalling pathway was anal yzed in proliferating rat hepatocytes both in vivo after partial hepatectom y and in vitro following epidermal growth factor (EGF)-pyruvate stimulation . First, a biphasic MEK/ERK activation was evidenced in G(1) phase of hepat ocytes from regenerating liver but not from sham-operated control animals. One occurred in early G(1) (30 min to 4 h), and the other occurred in mid-l ate G(1), peaking at around 10.5 h. Interestingly, the mid-late G(1) activa tion peak was located just before cyclin D1 induction in both in vivo and i n vitro models. Second, the biological role of the MEK/ERK cascade activati on 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 accumula tion was inhibited, DNA replication was totally abolished, and the MEK1 iso form was preferentially targeted by this inhibition. This effect was dose d ependent and completely reversed by removing the MEK inhibitor. Furthermore , transient transfection of hepatocytes with activated MEK1 construct resul ted in increased cyclin D1 mRNA accumulation. Third, a correlation between the mid-late G(1) MEK/ERK activation in hepatocytes in vivo after partial h epatectomy and the mitogen-independent proliferation capacity of these cell s in vitro was established. Among hepatocytes isolated either 5, 7, 9, 12 o r 15 h after partial hepatectomy, only those isolated from 12 and 15-h rege nerating livers were able to replicate DNA without additional growth stimul ation in vitro. In addition, PD 98059 intravenous administration in vivo, b efore MEK activation, was able to inhibit DNA replication in hepatocytes fr om regenerating livers. Taken together, these results show that (i) early i nduction of the MEK/ERK cascade is restricted to hepatocytes from hepatecto mized animals, allowing an early distinction of primed hepatocytes from, th ose returning to quiescence, and (ii) mid-late G(1) MEK/ERK activation is m ainly associated with cyclin D1 accumulation which leads to mitogen-indepen dent 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 hepa tocytes in vivo as observed in vitro in proliferating hepatocytes and argue for a crucial role of the MEK/ERK cascade signalling pathway.