A nitric oxide/Ca2+/Calmodulin/ERK1/2 mitogen-activated protein kinase pathway is involved in the mitogenic effect of IL-1beta in human astrocytoma cells

Background and purpose: Evidence is accumulating to support a role for interleukin-1b (IL-1b) in astrocyte proliferation. However, the mechanism by which this cytokine modulates this process is not fully elucidated. Experimental approach: In this study we used human astrocytoma U-373MG cells to investigate the role of nitric oxide (NO), intracellular Ca2þ concentration ([Ca2þ]i), and extracellular signal-regulated protein kinase (ERK) in the signalling pathway mediating IL-1b-induced astrocyte proliferation. Key results: Low IL-1b concentrations induced dose-dependent ERK activation which paralleled upregulation of cell division, whereas higher concentrations gradually reversed both these responses by promoting apoptosis. Pretreatment with the nonspecific NOS inhibitor, N-o-nitro-l-arginine methyl ester (L-NAME) or the selective iNOS inhibitor, N-[[3-(aminomethyl)- phenyl]methyl]-ethanimidamide dihydrochloride (1400W), antagonized ERK activation and cell proliferation induced by IL-1b. Inhibition of cGMP formation by the guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), partially inhibited ERK activation and cell division. Functionally blocking Ca2þ release from endoplasmic reticulum with ryanodine or 2-aminoethoxydiphenylborane (2-APB), inhibiting calmodulin (CaM) activity with N-(6-aminohexyl)-5-chloro-1- naphthalenesulphonamide hydrochloride (W7) or MAPK kinase activity with 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthiol] butadiene (U0126) downregulated IL-1b-induced ERK activation as well as cell proliferation. The cytokine induced a transient and time-dependent increase in intracellular NO levels which preceded elevation in [Ca2þ]i. Conclusions and implications: These data identified the NO/Ca2þ/CaM/ERK signalling pathway as a novel mechanism mediating the mitogenic effect of IL-1b in human astrocytes. As astrocyte proliferation is a hallmark of reactive astrogliosis, our results reveal a new potential target for therapeutic intervention in neuroinflammatory disorders.