Role of intracellular Ca2+ and calmodulin/MAP kinase kinase/extracellular signal-regulated protein kinase signalling pathway in the mitogenic and antimitogenic effect of nitric oxide in glia- and neurone-derived cell lines.
To elucidate the mechanism of cell growth regulation by nitric oxide (NO) and the role played in it by Ca2+, we studied the relationship among intracellular Ca2+ concentration ([Ca2+]i), mitogen-activated protein kinases [extracellular signal-regulated protein kinase (ERK)] and proliferation in cell lines exposed to different levels of NO. Data showed that NO released by low [(z)-1-[2-aminiethyl]-N-[2-ammonioethyl]amino]diazen-1-ium-1,2diolate (DETA/NO) concentrations (10 microm) determined a gradual, moderate elevation in [Ca2+]i (46.8 +/- 7.2% over controls) which paralleled activation of ERK and potentiation of cell division. Functionally blocking Ca2+ or inhibiting calmodulin or MAP kinase kinase activities prevented ERK activation and antagonized the mitogenic effect of NO. Experimental conditions favouring Ca2+ entry into cells led to increased [Ca2+]i (189.5 +/- 4.8%), ERK activation and cell division. NO potentiated the Ca2+ elevation (358 +/- 16.8%) and ERK activation leading to expression of p21Cip1 and inhibition of cell proliferation. Furthermore, functionally blocking Ca2+ down-regulated ERK activation and reversed the antiproliferative effect of NO. Both the mitogenic and antimitogenic responses induced by NO were mimicked by a cGMP analogue whereas they were completely antagonized by selective cGMP inhibitors. These results demonstrate for the first time that regulation of cell proliferation by low NO levels is cGMP dependent and occurs via the Ca2+/calmodulin/MAP kinase kinase/ERK pathway. In this effect the amplitude of Ca2+ signalling determines the specificity of the proliferative response to NO possibly by modulating the strength of ERK activation. In contrast to the low level, the high levels (50-300 microm) of DETA/NO negatively regulated cell proliferation via a Ca2+-independent mechanism.
Meini
Antonella
A
Dipartimento di Scienze Biomediche, Universita di Siena, via A. Moro 2, 53100 Siena, Italy.
Garcia
Julian Blanco
JB
Pessina
Gian Paolo
GP
Aldinucci
Carlo
C
Frosini
Maria
M
Palmi
Mitri
M
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D002147:Calmodulin / Q000502:physiology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D006152:Cyclic GMP / Q000502:physiology; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000096:biosynthesis; D004247:DNA / Q000096:biosynthesis; D048049:Extracellular Signal-Regulated MAP Kinases / Q000502:physiology*; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000096:biosynthesis; D048052:Mitogen-Activated Protein Kinase 3 / Q000096:biosynthesis; D020929:Mitogen-Activated Protein Kinase Kinases / Q000502:physiology*; D008938:Mitosis; D009457:Neuroglia / Q000166:cytology*; D009474:Neurons / Q000166:cytology*; D009569:Nitric Oxide / Q000502:physiology*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D009603:Nitroso Compounds / Q000494:pharmacology; D051381:Rats; D015398:Signal Transduction
C493956:CDKN1A protein, human; D002147:Calmodulin; D050759:Cyclin-Dependent Kinase Inhibitor p21; D020030:Nitric Oxide Donors; D009603:Nitroso Compounds; C094210:2,2'-(hydroxynitrosohydrazono)bis-ethanamine; D009569:Nitric Oxide; D004247:DNA; D048049:Extracellular Signal-Regulated MAP Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases; D006152:Cyclic GMP; D002118:Calcium