Activation of epidermal growth factor receptor by metal-ligand complexes decreases levels of extracellular amyloid beta peptide
Introduction
Receptor tyrosine kinases (RTKs) are cell surface receptors consisting of a single transmembrane domain separating an intracellular kinase domain and an extracellular ligand-binding domain (Sefton & Hunter, 1984; Singh & Harris, 2005; Thompson & Gill, 1985). The prototypal example of RTKs is the epidermal growth factor receptor (EGFR). Activation of RTKs induces kinase activity directed against tyrosine residues located both within the receptor itself (auto-phosphorylation) and on target downstream molecules (Schlessinger, 2002; Ullrich & Schlessinger, 1990). The pleiotropic cell responses from ligand binding include cell proliferation, migration, and differentiation, as well as homeostatic functioning (Alroy & Yarden, 1997; Brown, 1995; Singh & Harris, 2005). In normal tissue, the EGFR is activated by a variety of receptor-specific ligands including EGF, transforming growth factor-a (TGF-a) and heparin-binding EGF-like growth factor (HB-EGF) (Bogdan & Klambt, 2001; Harris, Chung, & Coffey, 2003). The activation of the EGFR has marked effects on phospholipid metabolism and can also activate both the ras/raf-mitogen activated protein (MAP) kinase and phosphoinositol-3-kinase (PI3K) pathways and the c-src family of tyrosine kinases.
In addition to its cognate ligands, the EGFR can be ‘trans-activated’ by non-physiologic agents including metals such as zinc (Zn) and copper (Cu) (Wu et al., 2004). Evidence increasingly supports a central role for Cu and Zn in many diseases. Recent studies reveal that relatively high levels of Zn and Cu as metal salts can trigger EGFR signalling in different cell-types including murine fibroblasts (B82L), human epidermoid carcinoma cells and human bronchial epithelial cells (A431) (Samet, Dewar, Wu, & Graves, 2003; Wu et al., 1999; Wu, Graves, Gill, Parsons, & Samet, 2002; Wu et al., 2004). The activation of EGFR by metal leads to subsequent activation of a number of downstream signalling pathways including ras/raf-MAP kinase (Samet et al., 2003, Wu et al., 2004) and PI3K-Akt-p70 S6 kinase pathways (Samet et al., 2003). Although the mechanism by which metals activate EGFR is not fully understood, it may involve activation of c-src kinases, inhibition of protein tyrosine kinases or up-regulated release of EGFR soluble ligands (Tal et al., 2006; Wu et al., 2004).
While these studies have demonstrated that simple metal salts added to the media at high concentration can activate EGFR, little is known about how cell permeable metal complexes modulate receptor expression and activity. The importance of this is highlighted by the rapidly increasing development of metal ligands and metal complexes as potential therapeutic agents in the treatment of neurodegenerative disorders and cancer (White et al., 2006). Recently, the 8-hydroxyquinoline derivative, 5-chloro-7-iodo-8-hydroxyquinoline or clioquinol (CQ), induced a marked reduction in amyloid plaque formation in the brain and improved cognitive performance in treated AD transgenic animals (Cherny et al., 2001). Similar protective effects have been observed in animal models of AD treated with alternative metal ligands such as DP-109 and pyrrolidine dithiocarbamate (PDTC) (Lee, Freidman, Angel, Kozak, & Koh, 2004; Malm et al., 2007). CQ has also revealed protective effects in animal models of Parkinson's disease and Huntington's disease (Kaur et al., 2003; Nguyen, Hamby, & Massa, 2005). Additionally, metal ligands and metal complexes such as tetrathiomolybdate, d-penicillamine and bis(thiosemicarbazones) have also been investigated for treatment of human metal storage disorders, arthritis and cancer (Chen et al., 2007; Cowley et al., 2005).
The consequence of altering biometal metabolism through the use of metal complexes is still largely unknown. Importantly, metal-complexing agents such as CQ and PDTC have the potential to transport metals into cells, thereby substantially altering cellular metal homeostasis (Malm et al., 2007; Treiber et al., 2004; White et al., 2006). Recently, our laboratory demonstrated that treatment of epithelial or neuronal cells with low levels of 8-hydroxyquinoline (8HQ) or phenanthroline complexes (Caragounis et al., 2007; White et al., 2006) stimulates PI3K-Akt resulting in downstream activation of c-Jun N-terminal kinase (JNK) and extracellular signal regulated kinase (ERK), up-regulation of matrix metalloproteases (MMP) and degradation of Aß. Due to the potential for metals to activate these pathways through stimulation of membrane receptor kinases, we have now investigated whether the complexes induced activation of EGFR. In this study, we found that low concentrations of CQ or other metal complexes were able to stimulate EGFR activation, resulting in marked downstream ERK phosphorylation and increased degradation of extracellular Aß1-40. These effects were mediated through cognate ligand-independent activation of src kinase by metal-complexes. These findings demonstrate that metal-ligand complexes can modulate activity of the EGFR and potentially other RTKs and may have important implications for the development of metal complex-based drugs.
Section snippets
Materials
5-Chloro-7-iodo-8-hydroxyquinoline (CQ), 8-hydroxyquinoline (8HQ), neocuproine (NC), pyrrolidine dithiocarbamate (PDTC), puromycin, dimethyl sulfoxide (DMSO), poly-d-lysine (PDL), fibronectin, cis diamminedichloroplatinum (cisplatin), PD153035 and PD98059 were purchased from Sigma–Aldrich (Sydney, Australia). GM6001, SB203580, PP2 and MMP Inhibitor-I (broad spectrum MMP inhibitor) were obtained from Merck Biosciences (Victoria, Australia). Antibodies to total or phospho-specific forms of
CQ-Cu complexes altered expression of EGFR in APP-CHO cells
In a previous study, we demonstrated that APP-CHO cells treated with CQ-Cu complexes revealed activation of PI3K and downstream phosphorylation of Akt, glycogen synthase kinase 3 and MAP kinases (White et al., 2006). In this study, we investigated how CQ-Cu may induce activation of protein kinases in these cells. To achieve this, we used antibody microarray analysis of CQ-Cu treated cells to identify differential protein expression. After treatment of APP-CHO cells with 10 µM CQ-Cu for 6 h, cell
Discussion
This study has shown for the first time that CQ-Cu and other Cu-complexes can induce potent activation of EGFR in multiple cell-types. CQ-Cu mediated activation of EGFR resulted in activation of ERK most likely through stimulation of the ras/raf kinase pathway. In contrast, PI3K and JNK activation by CQ-Cu were not induced through EGFR phosphorylation. The up-regulation of ERK activity resulted in a significant reduction of extracellular Aß levels in cells expressing APP and when synthetic Aß
Conflict of interest
Professor Colin L. Masters is a co-founding scientist of Prana Biotechnologies, which is currently developing compounds related to clioquinol as therapeutic agents for treatment of AD. Until recently, he was a member of its Board of Directors, and is currently a major shareholder and serves on its Research and Development Advisory Board. Prana Biotechnologies has established a major research collaboration with the Mental Health Research Institute, which is currently directed by Professor
Acknowledgments
This work was funded by the National Health and Medical Research Council of Australia and the Australian Research Council. ARW is an NH & MRC R.D. Wright Fellow. KJB is an NH & MRC Senior Research Fellow.
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