Suppression of Epidermal Growth Factor-Induced Phospholipase C Activation Associated With Actin Rearrangement in Rat Hepatocytes in Primary Culture

Abstract

Hepatocytes maintained in primary culture for periods of 1 to 24 hours exhibited a rapid decline in epidermal growth factor (EGF)-induced activation of phospholipase C (PLC), as was evident in a loss of EGF-induced inositol 1,4,5-trisphosphate (IP 3 ) formation and mobilization of Ca 2؉ from intracellular Ca 2؉ stores. The loss of PLC activation was not the result of a decrease in EGF receptor or phospholipase C-␥1 (PLC␥1) protein levels, nor the result of a loss of tyrosine phosphorylation of these proteins, but was associated with a decrease in EGF-induced translocation of PLC␥1 to the Triton-insoluble fraction, presumably reflecting binding to the actin cytoskeleton. Disruption of F-actin by treatment of cultured hepatocytes with cytochalasin D recovered the EGF-induced IP 3 formation and Ca 2؉ mobilization to the same level and with the same dose-response relationship as was obtained in freshly isolated cells. Analysis of PLC␥1 colocalization with F-actin by confocal microscopy showed that PLC␥1 was mostly distributed diffusely in the cytosol, both in freshly plated cells and in cells in culture for 24 hours, despite marked differences in actin structures. EGF stimulation caused a modest redistribution of PLC␥1 and a detectable increase in colocalization with cortical actin structures in freshly plated cells or in cytochalasin D-treated cells, but in cells that had been maintained and spread in culture only a limited PLC␥1 relocation was detected to specific actin-structure associated with lamellipodia and membrane ruffles. We conclude that actin cytoskeletal structures can exert negative control over PLC␥1 activity in hepatocytes and the interaction of the enzyme with specific actin structures dissociates PLC␥1 tyrosine phosphorylation from activation of its enzymatic activity. (HEPATOLOGY 2000;32:947-957.)Binding of the epidermal growth factor (EGF) to its receptor (EGFR) initiates dimerization of the receptor protein and activation of its intrinsic tyrosine kinase, resulting in autophosphorylation of multiple tyrosine residues on the cytoplasmic domain of the receptor. 1-3 These phosphotyrosine residues act as specific docking sites for target proteins that contain characteristic protein domains, such as the src homology-2 (SH2) domain. 4,5 Activation of different target proteins initiates a branching network of signaling reactions in the cell, resulting in the appropriate biological response patterns. 6 One of the intracellular signaling pathways activated by EGF involves phospholipase C-␥1 (PLC␥1), a member of the phosphoinositide-specific phospholipase C family of proteins, which, in addition to the catalytic domain and Ca 2ϩ -binding domains shared with other PLC isoforms, contains several tyrosine phosphorylation sites (Tyr771, Tyr783, and Tyr 1254) that are targets for the tyrosine kinase activity of growth-factor receptors, two SH2 domains that bind to autophosphorylation sites on the receptor, an SH3 domain that interacts with proline-rich regions in proteins, and a pleckstrin homology (PH) domain...

The diacylglycerol and protein kinase C pathways are not involved in insulin signalling in primary rat hepatocytes

“…A similar controversy also exists concerning the mechanism of action of EGF in hepatocytes. A review of the literature shows that EGF‐dependent phospholipase activation, and increases in DAG, PA, inositoltrisphosphate and cytosolic calcium, were detected to various degrees when hepatocyte suspensions or glucocorticoid‐free hepatocyte cultures were used [36,39,41,46,47]. In contrast, in our dexamethasone‐treated cultures, EGF had no effect on DAG levels, which is in agreement with the results of Dajani et al .…”

“…Data represent mean values ± SD from three different hepatocyte preparations. increases in DAG, PA, inositoltrisphosphate and cytosolic calcium, were detected to various degrees when hepatocyte suspensions or glucocorticoid-free hepatocyte cultures were used [36,39,41,46,47]. In contrast, in our dexamethasonetreated cultures, EGF had no effect on DAG levels, which is in agreement with the results of Dajani et al [38] who used similar culture conditions.…”

An Electrostatic Engine Model for Autoinhibition and Activation of the Epidermal Growth Factor Receptor (EGFR/ErbB) Family

“…Thus we postulate that Ca/CaM drives a positive feedback mechanism that produces maximal activation of ErbB. The mechanism functions only when the local [Ca 2ϩ ] is sufficiently elevated to provide a source of Ca/CaM; intracellular [Ca 2ϩ ] measurements indicate this occurs only for times Ͻ15 min after exposure to EGF (Hughes et al, 1991;Nojiri and Hoek, 2000). This electrostatic engine model predicts that CaM inhibitors (or agents that increase the intracellular Ca 2ϩ buffering capacity) will diminish the transient increase in trans autophosphorylation observed within 15 min of EGF stimulation, but have little effect on the steady-state level of activity measured at later times.…”

“…For example, Hughes et al (1991) reported the intracellular [Ca 2ϩ ] in A431 cells increases to 600 nM in 5 min, but falls to 150 nM by 15 min. Nojiri and Hoek (2000) show the intracellular [Ca 2ϩ ] in hepatocytes increases to 450 nM in 1-2 min, and then falls to 150 nM in the next 2-3 min. Fig.…”

Tumor Necrosis Factor-α Promotes Survival of Opossum Kidney Cells via Cdc42-induced Phospholipase C-γ1 Activation and Actin Filament Redistribution

“…PLC-␥1 is a signaling molecule that is involved in regulation of cytoskeletal organization (Yu et al, 1998;Nojiri and Hoek 2000;Papakonstanti et al, 2000). Accordingly, we examined whether PLC-␥1 was phosphorylated and/or activated in our system.…”