S100β Stimulates Inducible Nitric Oxide Synthase Activity and mRNA Levels in Rat Cortical Astrocytes

Abstract

The glia-derived, neurotrophic protein S100␤ has been implicated in development and maintenance of the nervous system. However, S100␤ has also been postulated to play a role in mechanisms of neuropathology, because of its specific localization and selective overexpression in Alzheimer's disease. To begin to address the question of whether S100␤ can induce potentially toxic signaling pathways, we examined the effects of the protein on nitric oxide synthase (NOS) activity in cultures of rat cortical astrocytes. S100␤ treatment of astrocytes induced a time-and dose-dependent increase in accumulation of the NO metabolite, nitrite, in the conditioned medium. The S100␤-stimulated nitrite production was blocked by cycloheximide and by the NOS inhibitor N-nitro-L-arginine methylester, but not by the inactive D-isomer of the inhibitor. Direct measurement of NOS enzymatic activity in cell extracts and analysis of NOS mRNA levels showed that the NOS activated by S100␤ addition is the calcium-independent, inducible isoform. Furthermore, the specificity of the effects of S100␤ on activation of NOS was demonstrated by the inability of S100␣ and calmodulin to induce an increase in nitrite levels. Our data indicate that S100␤ can induce a potent activation of inducible NOS in astrocytes, an observation that might have relevance to the role of S100␤ in neuropathology.The normal development and maintenance of the brain involves the temporal and spatial coordination and proper functioning of a number of intracellular and cell-cell signaling events, and the contribution of glial cells to these signaling processes is becoming more widely appreciated. The classical concept of the role of glia in brain function is rapidly changing with newer evidence of the crucial nature of these cells in controlling neurotransmitter levels, maintaining calcium homeostasis, and synthesizing and releasing neurotrophic and growth factors (for review, see Ref. 1). One such glia-derived factor is S100␤, a protein that promotes neuritic outgrowth of specific neuronal populations (e.g. cortical (2, 3), dorsal root ganglia (4), serotonergic (5, 6), and motoneurons (7)) and enhances survival of neurons during development (7,8) and after insult (9). S100␤ is also a glial mitogen, inducing phosphoinositide hydrolysis, increases in intracellular calcium, and protooncogene expression (10, 11). These trophic functions require nanomolar concentrations of a disulfide-linked S100␤ dimer (see Ref. 12). Thus, S100␤ may be beneficial during development of the nervous system, and increased S100␤ expression and secretion following acute glial activation in response to central nervous system injury may be one mechanism the brain uses in attempts to repair injured neurons.However, S100␤ may also reach concentrations that are deleterious, e.g. in neurodegenerative diseases like Alzheimer's disease and Down syndrome where chronic glial activation occurs (13). It has been found that S100␤ levels in severely affected brain regions of Alzheimer's disease patients are severalfol...

S100B Protein Stimulates Microglia Migration via RAGE-dependent Up-regulation of Chemokine Expression and Release

“…Because of these observations, the concept has emerged that S100B, when present in the brain extracellular milieu in relatively high amounts, might act as an unconventional cytokine and/or a damage-associated molecular pattern molecule playing a role in the pathophysiology of neurodegenerative disorders and inflammatory brain diseases (1,(5)(6)(7)(8)(9)(10)(11)(12)(13). Indeed, treatment of astrocytes with S100B results in up-regulation of expression of inducible NOS, stimulation of inducible NOS activity, NO release, and NO-dependent killing of astrocytes and co-cultured neurons (14,15) and in stimulation of release of the inflammatory cytokine IL-1␤ (16). Neurons and microglia also were shown to be targets of extracellular S100B.…”

“…Neurons and microglia also were shown to be targets of extracellular S100B. Specifically, S100B was shown to cause neuronal death by increasing reactive oxygen species production in neurons (17,18), to up-regulate inducible NOS expression in and stimulate NO release by microglia in the presence of bacterial endotoxin or interferon-␥ (19,20), to up-regulate the expression in and the release of IL-1␤ and TNF-␣ by microglia (14,(21)(22)(23), to up-regulate the expression of the proinflammatory enzyme, COX-2, in micro-glia (22), and to synergize with IL-1␤ and TNF-␣ to up-regulate COX-2 expression in microglia (23). Most of these effects were observed at relatively high doses of S100B, pointing to the need of accumulation of the protein in the brain extracellular space for it to act as an inflammatory cytokine/damage-associated molecular pattern factor and/or a neurotoxic factor.…”

Estudo da distribuição da proteína S100b em encéfalo de ratos.

“…As concentrações µM em células em cultura, induzem efeitos tóxicos, onde se observou apoptose de neurônios e astrócitos. O aumento da proteína induz uma resposta inflamatória mediada por NFkB (Fator de transcrição nuclear) em astrócitos, dessa forma iNOS (enzima de síntese de óxido nítrico) passa a produzir o NO (óxido nítrico), que consequentemente provoca morte celular com características definidas por apoptose (Hu et al, 1996(Hu et al, e 1997 Esses efeitos produzem danos neurodegenerativos através da indução do RNA (ácidoribonucleico) mensageiro e expressão da proteína precursora da β-amilóide (LI et al, 1998). Da mesma forma ocorre com os níveis de cálcio intracelular, não somente alterando a função de proteínas ligantes de cálcio, como também a expressão, como documentado em doenças humanas neurodegenerativas (Fano et al, 1995).…”

“…Ainda com relação à associação de condições patológicas e S100β, mesmo que indiretamente, a elevação nos níveis de S100Β induz, a aumento na liberação de óxido nítrico no cérebro de ratos (Hu et al, 1996), alterações estas que podem induzir á apoptose e morte de células nervosas (Hu et al, 1997).…”

“…Essa situação somente aparece em disfunções do SNC, onde seu conteúdo aumentado é verificado no soro ou no líquor (Aurell et al, 1991;Hu et al, 1996Hu et al, , 1997Murphy, 2000). De fato, S100β não apresenta as mesmas propriedades de GFAP, como marcadora, mas desempenha ação direta sobre diferentes proteínas do citoesqueleto, respectivamente, filamentos intermediários, microtúbulos e GFAP, no interir das glias (Bianchi et al, 1993;Donato et al, 1999;Whitaker-Azmitia, 2001;Donato, 2003).…”