ERK2 but not ERK1 plays a key role in hepatocyte replication: An RNAi-mediated ERK2 knockdown approach in wild-type and ERK1 null hepatocytes

Abstract

The mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 have been implicated in various physiological events, and specific targeting of these MAPKs could affect cell proliferation in many cell types. First, to evaluate the potential specific roles of these two MAPKs, we analyzed the mitogenic response in regenerating liver after partial hepatectomy (PH) and in primary culture of hepatocytes isolated from ERK1-deficient mice. We show that ERK1 knockout and wild-type (wt) cells replicate with the same kinetics after PH in liver, in vivo, and in primary cultures of hepatocytes, in vitro. Indeed, Cyclin D1 and Cdk1 appear to be expressed concomitantly in knockout and wt cells, highlighting that hepatocytes progress in the cell cycle independently of the presence of ERK1. Second, we specifically abolished ERK2 expression by RNA interference in mouse and rat hepatocytes. We investigated whether small interfering RNA (siRNA) targeting ERK2 could specifically inhibit its expression and interfere with the process of replication. In ERK1-deficient hepatocytes, silencing ERK2 expression by RNA interference and ERK2 activation by U0126 clearly demonstrate that DNA replication is regulated by an ERK2-dependent mechanism. Furthermore, in rat wt hepatocytes, whereas ERK2 targeting inhibits late G 1 and S phase progression, ERK1 silencing is devoid of any effect on cell proliferation, indicating that ERK1 cannot rescue ERK2 deficiency. Conclusion: Our results emphasize the importance of the MAPK cascade in hepatocyte replication and allow us to conclude that ERK2 is the key form involved in this regulation, in vivo and in vitro. T he Ras-dependent mitogen-activated protein kinase (MAPK) signaling cascade participates in control of cell fate, proliferation, and survival in various mammalian organs, including the liver. In adult rodent hepatocytes and regenerating liver, the MAPK MEK/ERK cascade plays a key role in regulating G 1 phase progression and consequently proliferation. 1,2 The ability to control precisely the order and timing of events is determinant for cell cycle regulation. 3,4 According to our previous data, the first part of G 1 is devoted to growth factor-dependent MEK/ERK-morphogenic events, whereas the mitogenic signal occurs in mid-G 1 phase. We showed that the sequential control mechanism of hepatocyte morphology and S phase entry by growth factor could involve successive activation of MEK2-ERK2 and then MEK1/MEK2-ERK1/ERK2 isoforms. 3 The process in early G 1 in relation to cytoskeletal reorganization induces hepatocyte spreading, making them permissive to DNA replication. In late G 1 phase, MEK1/MEK2-ERK1/ERK2 activation is associated with accumulation of Cyclin D1 and mitogen-dependent progression of hepatocytes to S phase. The central role of ERK2 in regulation was highlighted by the observation that ERK2 was preferentially activated in early and midlate G 1 phase. However, although ERK1 was slightly expressed and phosphorylated in early G 1 , a gradual

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.…”

Regulator of G Signaling 16 Is a Marker for the Distinct Endoplasmic Reticulum Stress State Associated with Aggregated Mutant α1-Antitrypsin Z in the Classical Form of α1-Antitrypsin Deficiency

“…Furthermore, network analysis suggests that ERK2, caspase 3, apoptotic peptidase activating factor 1, cyclin-dependent kinase inhibitor 2a, p53, Fos, and Myc functional activity is activated. Activation of ERK2 has recently been shown to play a key role in hepatocyte replication after partial hepatectomy (30). Effects on expression of genes that alter cell proliferation and carcinogenesis are completely consistent with our recent observations that accumulation of ATZ in hepatocytes has a profound effect on cell proliferation and cell death and engenders a cancerprone proliferative state on hepatocytes with lesser accumulation of ATZ in the liver in AT deficiency (31,32).…”

MiR‐483–5p suppresses the proliferation of glioma cells via directly targeting ERK1

“…A recent study in ischemic heart disease suggested an additional role of miR-483-5p in angiogenesis inhibition mediated by its target SRF [23], suggesting that by controlling different processes, miR-483-5p might inhibit tumorigenesis through various functions. Addtionally, Although ERK2 is the structurally related isoform of ERK1 and also contributes to cell proliferation [24,25], we observed no significant change in ERK2 expression after miR-483-5p manipulation.…”