Epidermal Growth Factor Receptor Is a Common Mediator of Quinone-induced Signaling Leading to Phosphorylation of Connexin-43

Abstract

Rat liver epithelial cells were exposed to three quinones with different properties: menadione (2-methyl-1,4-naphthoquinone, vitamin K 3 ), an alkylating as well as redox-cycling quinone, the strongly alkylating p-benzoquinone (BQ), and the non-arylating redox-cycler, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). All three quinones induced the activation of extracellular signalregulated kinase (ERK) 1 and ERK 2 via the activation of epidermal growth factor receptor (EGFR) and MAPK/ ERK kinases (MEK) 1/2. ERK activation resulted in phosphorylation at Ser-279 and Ser-282 of the gap junctional protein, connexin-43, known to result in the loss of gap junctional intercellular communication. Another EGFRdependent pathway was stimulated, leading to the activation of the antiapoptotic kinase Akt via phosphoinositide 3-kinase. The activation of EGFR-dependent signaling by these quinones was by different mechanisms: (i) menadione, but not BQ or DMNQ, inhibited a protein-tyrosine phosphatase regulating the EGFR, as concluded from an EGFR dephosphorylation assay; (ii) although menadione-induced activation of ERK was unimpaired by pretreatment of cells with N-acetyl cysteine, activation by BQ and DMNQ was prevented; (iii) cellular glutathione (GSH) levels were strongly depleted by BQ. The mere depletion of GSH by application of diethyl maleate EGFR-dependently activated ERK and Akt, thus mimicking BQ effects. GSH levels were only moderately decreased by menadione and not affected by DMNQ. In summary, EGFR-dependent signaling was mediated by protein-tyrosine phosphatase inactivation (menadione), GSH depletion (BQ), and redox-cycling (DMNQ), funneling into the same signaling pathway.

The Phosphatase Inhibitor Menadione (Vitamin K3) Protects Cells from EGFR Inhibition by Erlotinib and Cetuximab

“…We sought to extend the previously reported data indicating that menadione induces EGFR phosphorylation in rat liver epithelial cells to human skin keratinocyte cells (48). HaCaT cells were exposed to different concentrations of menadione or with the same volume of medium as control for 1 hour at 37 C and then EGFR phosphorylation was detected by immunohistochemical staining with monoclonal anti-EGFR antibody and polyclonal anti-p-EGFR antibody as described in Materials and Methods.…”

“…[45][46][47]. Menadione has been shown to phosphorylate EGFR in rat hepatocytes (48). It has been used in the clinic as a topical treatment of vascular disorders in the skin to enhance vascular regeneration with an acceptable toxicity profile, although topical side effects have been observed in some patients (49,50).…”

Effects of 2-Methyl-1,4-naphtoquinone (Menadione) on Cellular Signaling in RBL-2H3 Cells

“…RBL cells were pretreated with different concentrations of menadione for 15 min and stimulated with antigen (DNP 7 -BSA) for 20 min. 23) As shown in Fig. 2, antigen-induced degranulation was inhibited by menadione dose dependently (white column), and degranulation decreased to about 75% of the control value at 400 mM.…”

Connexins as Substrates for Protein Kinases and Phosphoprotein Phosphatases

“…Leykauf et al used a specific antibody against PSer279-PSer282 of Cx43 to demonstrate that different phosphorylated forms of Cx43 coexist at the plasma membrane (Leykauf et al, 2003). Two antibodies recognizing the same phosphosites were used to show that EGF and activation of its receptor with quinones induce phosphorylation of Cx43 in these serine residues (Abdelmohsen et al, 2003;Leykauf et al, 2003). Using an antibody that specifically recognizes Cx43 phosphorylated at serines 325, 328 and/or 330 (PS325/328/330), Lampe and colleagues showed that while Cx43 relocalizes to the lateral edges in ischemic hearts, Cx43 phosphorylated at these residues remained mostly at the intercalated disk (Lampe et al, 2006).…”