Multiple division cycles and long-term survival of hepatocytes are distinctly regulated by extracellular signal-regulated kinases ERK1 and ERK2

Abstract

We investigated the specific role of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1 (ERK1)/ERK2 pathway in the regulation of multiple cell cycles and long-term survival of normal hepatocytes. An early and sustained epidermal growth factor (EGF)-dependent MAPK activation greatly improved the potential of cell proliferation. In this condition, almost 100% of the hepatocytes proliferated, and targeting ERK1 or ERK2 via RNA interference revealed the specific involvement of ERK2 in this regulation. However, once their first cell cycle was performed, hepatocytes failed to undergo a second round of replication and stayed blocked in G1 phase. We demonstrated that sustained EGF-dependent activation of the MAPK/ERK kinase (MEK)/ERK pathway was involved in this blockage as specific transient inhibition of the cascade repotentiated hepatocytes to perform a new wave of replication and multiple cell cycles. We identified this mechanism by showing that this blockage was in part supported by ERK2-dependent p21 expression. Moreover, continuous MEK inhibition was associated with a lower apoptotic engagement, leading to an improvement of survival up to 3 weeks. Using RNA interference and ERK1 knockout mice, we extended these results by showing that this improved survival was due to the specific inhibition of ERK1 expression/phosphorylation and did not involve ERK2. Conclusion: Our results emphasize that transient MAPK inhibition allows multiple cell cycles in primary cultures of hepatocytes and that ERK2 has a key role in the regulation of S phase entry. Moreover, we revealed a major and distinct role of ERK1 in the regulation of hepatocyte survival. Taken together, our results represent an important advance in understanding long-term survival and cell cycle regulation of hepatocytes. (HEPATOLOGY 2009;49:930-939.) D ue to the difficulties of in vivo studies, primary cultures of hepatocytes are widely studied in vitro to examine the expression of liver-specific functions, notably regulation and activity of detoxifying pathways; this is a good method of predicting the hepatic elimination of xenobiotics. Nevertheless, the main obstacle in developing culture conditions that allow these investigations is the maintenance of cell survival and liverspecific functions that rapidly and markedly decrease after hepatocyte seeding. 1,2 Primary culture also represents an interesting tool for cell cycle studies. A growth factor restriction point has been located at two-thirds of G1 phase, 3 and hepatocytes usually undergo only one round of division in conventional primary cultures. Many studies have shown that hepatocyte growth factor/scatter factor, epidermal growth factor (EGF), and transforming growth factor ␣ are the primary mitogens for hepatocytes. In addition to their well-known proliferating functions, growth factors are also characterized by pleiotropic activ-

P2Y2 purinergic receptor activation is essential for efficient hepatocyte proliferation in response to partial hepatectomy

“…Recent studies have defined the functional significance of calcium signaling for efficient hepatocyte cell cycle progression from G 0 to G 1 and S phases in regenerating livers (15,38,52). Among the many intracellular mitogenic targets of calcium signaling, p44/42 ERK MAPKs are well-known mediators of hepatocyte cell cycle progression at the G 1 phase and S phase entry (9,24,57,65). In this study, we provide evidence that both ERK MAPK activation in response to 70% PH in vivo and ATP-treatment of isolated hepatocytes in vitro were dependent on intact P2Y2 purinergic receptor expression in hepatocytes.…”

ERK2 but not ERK1 mediates HGF-induced motility in non small cell lung carcinoma cell lines

“…In contrast to such a redundant function of both ERK isoforms, there are recent studies that have identified some isoform specific functions. Thus, ERK1 and ERK2 have been demonstrated to fulfil different roles in Ras-dependent signalling (Vantaggiato et al, 2006) and long-term survival of hepatocytes (Frémin et al, 2009). While some differences in ERK isoform signalling have been attributed to differences in the N-terminal domain of both isoforms (Marchi et al, 2008), other studies have shown that differences might be due to the expression level of each isoform (Lefloch et al, 2008;Lefloch et al, 2009).…”

The Activation of MEK/ERK Signaling Pathway by Bone Morphogenetic Protein 4 to Increase Hepatocellular Carcinoma Cell Proliferation and Migration

“…On the basis of these studies, we found that further upregulation of the expression of cyclin B1 and CDK1 could result from Smad4 deficiency in HepG2 cells, whereas inhibition of the MEK/ERK signaling pathway efficiently attenuates BMP4-induced upregulation and maintained basal levels of cyclin B1 and CDK1 in HepG2 cells. MEK/ERK signaling is believed to play an important role in the regulation of cell proliferation and its signaling pathway is activated by different growth factors, including BMP4 cytokines (41,42). Growth factor-induced MEK/ ERK cascade is considered to be an inducer for the regulation of multiple phosphorylating proteins and their target proteins and to promote the interaction between retinoblastoma (Rb) protein and E2F-1 and to affect transcriptional regulation of target genes involved in cell-cycle control (43)(44)(45).…”