Elsevier

Neuroscience

Volume 120, Issue 4, 15 September 2003, Pages 941-950
Neuroscience

Nuclear factor κb activation by muscarinic receptors in astroglial cells: effect of ethanol

https://doi.org/10.1016/S0306-4522(03)00401-9Get rights and content

Abstract

Activation of muscarinic receptors leads to proliferation of astroglial cells and this effect is inhibited by ethanol. Among the intracellular pathways involved in the mitogenic action of muscarinic agonists, activation of the atypical protein kinase C ζ (PKC ζ) appears to be of most importance, and is also affected by low ethanol concentrations. PKC ζ has been reported to activate nuclear factor κB (NF-κB), a transcription factor that has been shown to play an important role in cell proliferation. The aim of this study was, therefore, to determine whether muscarinic receptors would activate NF-κB in astroglial cells, whether such activation would play a role in the mitogenic action of muscarinic agonists, and whether it would represent a possible target for ethanol. Carbachol activated NF-κB in human 1321N1 astrocytoma cells, as evidenced by translocation of the p65 subunit of NF-κB to the nucleus, phosphorylation and degradation of IκBα in the cytosol, and increase NF-κB binding to DNA. Carbachol also induced translocation of p65 to the nucleus in primary rat astrocytes. Carbachol-induced NF-κB activation was mediated by the M3 subtype of muscarinic receptors and appeared to involve Ca2+ mobilization and activation of PKC ϵ and PKC ζ, but not PI3-kinase and mitogen-activated protein kinase. The NF-κB peptide inhibitor SN50, but not the inactive peptide SN50M, strongly inhibited carbachol-induced astrocytoma cells proliferation and p65 translocation to the nucleus. Increased DNA synthesis was also antagonized by the IκBα kinase inhibitor BAY 11–7082. Ethanol (25–100 mM) inhibited the translocation of p65 and the binding of NF-κB to DNA in both 1321N1 astrocytoma cells and primary rat cortical astrocytes. Together, these results suggest that activation of NF-κB by muscarinic receptors in astroglial cells is important for carbachol-induced DNA synthesis and that ethanol-mediated inhibition of cell proliferation may be due in part to inhibition of NF-κB activation.

Section snippets

Cell culture

The human astrocytoma cell line 1321N1 was maintained in Dulbecco's Modified Eagle medium (DMEM) low glucose, supplemented with 5% of fetal bovine serum, 1 U/ml penicillin G and 1 mg/ml streptomycin, all purchased from Invitrogen (Carlsbad, CA, USA), under a humidified atmosphere of 5% CO2/95% air at 37 °C, as previously described (Guizzetti et al., 1996). For NF-κB experiments, cells were plated in 100 mm dishes (2×106 cells/dish); for [3H]thymidine experiments, cells were plated in 24-well

Results

NF-κB activation was measured after incubating 1321N1 astrocytoma cells for 15 min, 0.5, 1 and 2 h with 1 mM carbachol. Carbachol induced phosphorylation (not shown) and degradation of IκBα (Fig. 1a), translocation of p65 from the cytosol to the nucleus (Fig. 1b), and increased the binding of NF-κB to DNA (Fig. 1c) with maximal activation observed after 1 h. Carbachol also induced translocation of p65 in primary rat astrocytes, with maximal activation after 1 h (Fig. 1d). Fig. 2 shows that

Discussion

A first novel finding of this study is that the cholinergic agonist carbachol activates NF-κB in astroglial cells in a time- and concentration-dependent manner (Fig. 1, Fig. 2). NF-κB activation was assessed in human astrocytoma cells by measuring four different parameters: phosphorylation and degradation of the NF-κB inhibitor IκBα (which binds and anchors NF-κB in the cytoplasm preventing its transfer to the nucleus); translocation of p65 NF-κB to the nucleus; and binding of NF-κB to DNA

Acknowledgements

This work was supported in part by grants AA-08154 and ES-07033 from the National Institutes of Health. We thank Ms Khoi Dao for her assistance in some of the experiments.

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