Mechanism in the Sequential Control of Cell Morphology and S Phase Entry by Epidermal Growth Factor Involves Distinct MEK/ERK Activations

Abstract

Cell shape plays a role in cell growth, differentiation, and death. Herein, we used the hepatocyte, a normal, highly differentiated cell characterized by a long G1 phase, to understand the mechanisms that link cell shape to growth. First, evidence was provided that the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) cascade is a key transduction pathway controlling the hepatocyte morphology. MEK2/ERK2 activation in early G1 phase did not lead to cell proliferation but induced cell shape spreading and demonstration was provided that this MAPK-dependent spreading was required for reaching G1/S transition and DNA replication. Moreover, epidermal growth factor (EGF) was found to control this morphogenic signal in addition to its mitogenic effect. Thus, blockade of cell spreading by cytochalasin D or PD98059 treatment resulted in inhibition of EGF-dependent DNA replication. Our data led us to assess the first third of G1, is exclusively devoted to the growth factor-dependent morphogenic events, whereas the mitogenic signal occurred at only approximately mid-G1 phase. Moreover, these two growth factor-related sequential signaling events involved successively activation of MEK2-ERK2 and then MEK1/2-ERK1/2 isoforms. In addition, we demonstrated that inhibition of extracellular matrix receptor, such as integrin beta1 subunit, leads to cell arrest in G1, whereas EGF was found to up-regulated integrin beta1 and fibronectin in a MEK-ERK-dependent manner. This process in relation to cytoskeletal reorganization could induce hepatocyte spreading, making them permissive for DNA replication. Our results provide new insight into the mechanisms by which a growth factor can temporally control dual morphogenic and mitogenic signals during the G1 phase.

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

“…No significant differences could be noticed between the two chemically modified sequences and no changes with all control mutated sequences. In parallel, we looked at cell cycle markers expressions of cyclin D1 (Figure 5a) and Cdk1 (Figure 5b), two markers which are expressed, respectively, at late G 1 and S phase in normal hepatocytes and in HCC (Albrecht et al, 1993(Albrecht et al, , 1995Ito et al, 1998;Rescan et al, 2001). Our results showed that cyclin D1 inhibition was partially inhibited in transfected cells.…”

“…In primary culture of normal hepatocytes, expression of Cdk1, one marker of G 1 /S phase transition, is regulated by the MEK/ERK pathway (Albrecht et al, 1993;Rescan et al, 2001;Huynh et al, 2003). Therefore, we looked at the regulation of this protein in transformed FAO cells and showed that Cdk1 expression was significantly blocked when cells were treated with U0126 ( Figure 1b).…”

“…Total protein (30 mg) was resolved on a 10% polyacrylamide gel and transferred to nitrocellulose membranes for 1 h as previously described (Rescan et al, 2001). Membranes were incubated overnight at 4 1C with the following primary antibody in Tris-buffered saline (pH 7,4): a-phospho-ERK1/ 2 and a-phospho-p38 (Thr180/Tyr182) (Cell Signaling, Saint Quentin en Yvelines, France); a-pan ERK1 (sc-94) and ERK2 (sc-154), a-cyclin E (sc-481) and a-HSC 70 (sc-7298) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); a-PCNA (Dako, Trappes, France); a-cyclin D1 (Neomarkers, Westinghouse, CA, USA).…”

Dysregulation of growth factor signaling in human hepatocellular carcinoma

“…TGFa and EGF stimulate hepatocellular DNA synthesis and act as potent mitogens for hepatocytes (Mead and Fausto, 1989;Rescan et al, 2001). Overexpression of TGFa is frequently observed in human HCCs and patients suffering from HCC may have elevated urinary TGFa levels (Yeh et al, 1987;Chuang et al, 1991).…”

Epidermal growth factor-induced hepatocellular carcinoma: gene expression profiles in precursor lesions, early stage and solitary tumours

“…EGF and TGFa are potent mitogens for hepatocytes (Wang et al, 1999;Rescan et al, 2001). Signalling of these mitogens is through binding to members of the EGF-receptor family.…”