Nitric oxide reversibly inhibits the epidermal growth factor receptor tyrosine kinase

Abstract

Although it has been demonstrated that NO inhibits the proliferation of different cell types, the mechanisms of its anti-mitotic action are not well understood. In this work we have studied the possible interaction of NO with the epidermal growth factor receptor (EGFR), using transfected fibroblasts which overexpress the human EGFR. The NO donors S-nitroso-N-acetylpenicillamine (SNAP), 1,1-diethyl-2-hydroxy-2-nitrosohydrazine (DEA-NO) and N-{4-[1-(3-aminopropyl)-2-hydroxy-2-nitrosohydrazino]butyl}propane -1, 3-diamine (DETA-NO) inhibited DNA synthesis of fibroblasts growing in the presence of fetal calf serum, epidermal growth factor (EGF) or EGF plus insulin, as assessed by [methyl-3H]thymidine incorporation. Neither 8-bromo-cGMP nor the cGMP-phosphodiesterase inhibitor zaprinast mimicked this effect, suggesting that NO is unlikely to inhibit cell proliferation via a cGMP-dependent pathway. SNAP, DEA-NO and DETA-NO also inhibited the transphosphorylation of the EGFR and its tyrosine kinase activity toward the exogenous substrate poly-l-(Glu-Tyr), as measured in permeabilized cells using [gamma-32P]ATP as phosphate donor. In contrast, 3-[morpholinosydnonimine hydrochloride] (SIN-1), a peroxynitrite-forming compound, did not significantly inhibit either DNA synthesis or the EGFR tyrosine kinase activity. The inhibitory action of DEA-NO on the EGFR tyrosine kinase was prevented by haemoglobin, an NO scavenger, but not by superoxide dismutase, and was reversed by dithiothreitol. The binding of EGF to its receptor was unaffected by DEA-NO. The inhibitory action of DEA-NO on the EGF-dependent transphosphorylation of the receptor was also demonstrated in intact cells by immunoblot analysis using an anti-phosphotyrosine antibody. Taken together, these results suggest that NO, but not peroxynitrite, inhibits in a reversible manner the EGFR tyrosine kinase activity by S-nitrosylation of the receptor.

Nitric Oxide Is a Physiological Inhibitor of Neurogenesis in the Adult Mouse Subventricular Zone and Olfactory Bulb

“…Because in vitro studies demonstrate that NO is primarily a direct cytostatic agent in many cell types including neuroblasts (Garg and Hassid, 1990; Peunova and Enikolopov, 1995; Estrada et al, 1997;Sciorati et al, 1997;Nakaya et al, 2000;Murillo-Carretero et al, 2002), general increases in cerebral NO concentration, such as those derived from NO donor injections or iNOS induction, may promote neurogenesis by indirect mechanisms. In this sense, it is interesting that after stroke, NO stimulates the secretion of vascular endothelial growth factor (Zhang et al, 2003), which has been shown to induce neurogenesis both in vivo and in vitro (Jin et al, 2002).…”

“…A variety of molecules are involved in the antiproliferative action of NO in different cell types (Lepoivre et al, 1994;Estrada et al, 1997;Sciorati et al, 1997;Nakaya et al, 2000). Among them, EGFR is a good candidate to be mediating the NO effects in the SVZ.…”

“…First, a role for NO in embryonic brain development is well established on the basis of the variable anatomical distribution of nitrergic cells during ontogenesis (Bredt and Snyder, 1994;Keilhoff et al, 1996;Santacana et al, 1998) as well as on the consequences of preventing NO formation on brain development (Kuzin et al, 1996;Peunova et al, 2001). Second, NO is antiproliferative for several cell types in vitro Hassid, 1989, 1990;Cornwell et al, 1994;Estrada et al, 1997), including tumoral cells of neuronal lineage (Peunova and Enikolopov, 1995;Murillo-Carretero et al, 2002). Finally, one of the mechanisms involved in the antiproliferative action of NO is the inhibition of the epidermal growth factor receptor (EGFR) (Estrada et al, 1997;MurilloCarretero et al, 2002), and it is known that EGFR activation is required for the proliferation of SVZ neuronal precursors both in vitro (Reynolds and Weiss, 1992;Morshead et al, 1994;Gritti et al, 1999) and in vivo (Craig et al, 1996;Kuhn et al, 1997;Tropepe et al, 1997).…”

“…Second, NO is antiproliferative for several cell types in vitro Hassid, 1989, 1990;Cornwell et al, 1994;Estrada et al, 1997), including tumoral cells of neuronal lineage (Peunova and Enikolopov, 1995;Murillo-Carretero et al, 2002). Finally, one of the mechanisms involved in the antiproliferative action of NO is the inhibition of the epidermal growth factor receptor (EGFR) (Estrada et al, 1997;MurilloCarretero et al, 2002), and it is known that EGFR activation is required for the proliferation of SVZ neuronal precursors both in vitro (Reynolds and Weiss, 1992;Morshead et al, 1994;Gritti et al, 1999) and in vivo (Craig et al, 1996;Kuhn et al, 1997;Tropepe et al, 1997).…”

S-nitrosylation: Physiological regulation of NF-κB

“…Other proven or likely targets for nitrosylation in the NF-B pathway include the epidermal growth factor and src tyrosine kinases (24,25), which activate NF-B through p21 ras ; tyrosine phosphatases (26); and NADPH oxidase (27), known to activate NF-B through IKK (presumably by means of the oxidative inactivation of phosphatase). Given the multiple loci of S-nitrosylation in NF-Brelated pathways, the effects of S-nitrosylation may be considered analogous to those of phosphorylation, with a large number of regulatory permutations that combine ultimately to optimize cellular responses.…”