Mechanism of glial activation by S100B: involvement of the transcription factor NFκB
Introduction
The chronic activation of glial cells (astrocytes and microglia) is believed to play a central role in the development and propagation of neuronal dysfunction and degeneration in disorders such as Alzheimer’s disease (AD) [4]. A number of different endogenous and exogenous stimuli can induce or increase the susceptibility of glial activation, including cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), lipopolysaccharide (LPS) and β-amyloid peptide 1-42 (Aβ). The resulting neurotoxic glial products can lead to further glial activation, suggesting that long-term exposure to products derived from activated glia contributes to the development and progression of chronic neurodegenerative diseases [4], [24]. Therefore, dissection of the molecular mechanisms involved in the amplification and maintenance of glial activation may yield new targets upon which to focus drug discovery efforts aimed at reducing the incidence, or slowing the progression of neuroinflammatory-linked diseases such as AD.
The S100 proteins are calcium binding proteins belonging to the EF-hand protein family [see 19 for review]. There are more than 15 members of the S100 family, of which S100B (formerly called S100β) is one of the most extensively characterized. S100B is a glial-derived cytokine and is present in the highest concentration in the vertebrate nervous system [19]. The gene for human S100B maps to the Down’s Syndrome region of chromosome 21 [1], and the protein has been found to be overexpressed in AD [26], [49], Down’s Syndrome [26], and epilepsy [25]. Moreover, elevated S100B levels in AD coincide with regions of high neuropathology, such as regions around amyloid plaques [7], [47], [49], and mice that overexpress S100B have been shown to have defects in brain function [21], [51], [52]. These lines of evidence support the view that elevated S100B levels may have potentially detrimental consequences in the brain. S100B is expressed primarily by astrocytes and has intra- and extracellular activities [19]. A secreted disulfide-linked form of S100B is known to have a beneficial role in the developing brain, stimulating neurite outgrowth at low nM concentrations [12], [38], [39]. However, higher concentrations (high nM to low μM range) of extracellular S100B have been shown to stimulate production of inducible nitric oxide synthase (iNOS) in astrocytes [32] and enhance Aβ -induced astrocyte activation [30], suggesting that the detrimental effects of S100B are through its ability to induce pro-inflammatory cytokines and oxidative stress enzymes in glia, and to enhance other stimuli to activate glia. In support of this, it has been previously shown that S100B can induce neuronal cell death through iNOS production and subsequent release of NO from activated astrocytes [31].
The aim of the present study was to determine the molecular mechanisms by which S100B leads to glial activation and iNOS expression. One possible pathway centers on the transcription factor NFκB. NFκB plays a pivotal role in the regulation of expression of many genes involved in a variety of physiological responses such as control of cell proliferation and apoptosis, neural development, and immune and inflammatory responses, as well as in the organism’s response to injury and infection [for reviews, see [8], [22], [44]]. Because this transcription factor is important in diverse cellular processes, NFκB has been found to be activated in many cell types in response to a broad range of conditions and stimuli. Some examples of NFκB activating stimuli include inflammatory cytokines such as IL-1β and TNFα, Aβ, mitogens, viral and bacterial products, and intracellular and extracellular stresses.
In unstimulated cells, NFκB exists in the cytoplasm as an inactive dimer bound to an inhibitor protein, IκB. There are currently five well-defined NFκB subunits in mammals: p65 (RelA), p50, p52, c-Rel and RelB that can form homo- and hetero-dimers [22], [42], [48]. In addition, there are a number of different mammalian forms of IκB, including IκBα, IκBβ, IκBγ (the p105 precursor of p50), IκBδ (the p100 precursor of p52), IκBϵ, and Bcl-3 [see [8], [16] for review]. This diversity provides one means by which NFκB activation can be fine-tuned in a given cell by regulation of which NFκB and IκB forms are present in the complex. Another means for regulation of NFκB is through the signal transduction pathways that lead to NFκB activation. The most well-characterized mechanism involves the p65/p50 NFκB dimer and its association with IκBα. Upon stimulation of a cell with an NFκB activator, IκBα is phosphorylated on two serine residues (S32 and S36), which targets the inhibitor protein for ubiquitination and degradation. The released NFκB dimer can then translocate to the nucleus where it binds to specific DNA response elements in a variety of target genes and stimulates transcription. More recently, alternative pathways to NFκB activation have been described, including regulation of processing of the p50 precursor, p105, that leads to increased levels of p50/p65 dimer formation [13]; a tyrosine kinase-dependent phosphorylation of IκBα that leads to NFκB activation [14], [34], or a direct activation of NFκB by H2O2 or other oxidants [16].
In the CNS, NFκB has been found to be important for transcriptional regulation of genes in both neurons and glia [44], and NFκB activation has been reported in neurodegenerative disorders like AD [15]. In many cell types, NFκB activation is involved in iNOS expression, and we recently reported [3] that Aβ stimulates NO production in astrocytes through an NFκB-dependent mechanism. Finally, S100B has been reported to stimulate NFκB activity in neuronal and endothelial cells [5], [29]. Therefore, it was logical to address whether S100B could stimulate NFκB in astrocytes, and whether the NFκB activation was important for iNOS induction. We report here that S100B activates NFκB in rat cortical astrocyte cultures, and that the subsequent iNOS expression and NO production are dependent on NFκB activation.
Section snippets
Cell culture
Primary rat cortical astrocytes were prepared and maintained as described elsewhere [32]. Briefly, cells were maintained in αMEM supplemented with 10% fetal bovine serum (FBS) (HyClone) and antibiotics [100 units/ml penicillin/100 μg/ml streptomycin (GIBCO/BRL)]. Prior to experiments, astrocytes were washed twice with PBS, and maintained for 24 h in serum-free αMEM containing N2 media supplement (GIBCO/BRL). Cells were then treated with S100B, PBS (negative control), or lipopolysaccharide (LPS,
S100B stimulates NFκB activation in astrocytes
In order to determine if S100B treatment of astrocytes led to a stimulation of NFκB activation, we first determined if S100B induced NFκB translocation into the nucleus. We have previously reported [3] that all five NFκB family members can be detected by Western blots in our rat astrocyte cultures, but that only p65 and p50 participate in NFκB activation in response to a glial activating stimulus. Therefore, astrocyte cultures were treated for 6 h with either vehicle control (PBS), LPS positive
Discussion
Through the use of multiple experimental approaches, we determined that S100B stimulates glial iNOS via a signal transduction pathway that involves the transcription factor NFκB. We found that NFκB was activated in S100B-treated rat glial cells by nuclear translocation of the p65 NFκB subunit, stimulation of NFκB specific DNA binding activity and stimulation of NFκB dependent transcriptional activity. Furthermore, S100B-induced iNOS promoter activation was abrogated upon mutation of the NFκB
Acknowledgements
This work was supported by a grant from the Alzheimer’s Association (to DMW), NIH grant AG13939 (to LVE), and NIH training grants AG00260 (to TK), GM08061 (to KTA), and GM08152 (to JMC). We thank Dr. Thomas Lukas for assistance with the amino acid analyses.
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Current address: Rockefeller University, New York, NY USA.