Heat Shock Protein 70 Suppresses Astroglial-inducible Nitric-oxide Synthase Expression by Decreasing NFκB Activation

Abstract

In brain glial cells, expression of calcium independent nitric-oxide synthase (NOS-2) is induced following stimulation with bacterial endotoxin (lipopolysaccharide (LPS)) and/or pro-inflammatory cytokines. We have investigated the effects of heat shock (HS), which can reduce inflammatory responses in several cell types, on the induction of glial NOS-2 expression. Preincubation of cells for 20 -60 min at 43°C decreased subsequent levels of NOS-2 induction, with a maximal 80% reduction after 60 min of HS. Following HS, cells were refractory to NOS inducers for up to 4 h, after which time little or no suppression was observed. HS reduced cytosolic NOS-2 enzymatic activity (3-fold), steady state mRNA levels (2-3-fold), and gene promoter activity (by 50%). HS also reduced LPS-induced nuclear accumulation of transcription factor NFB p65 subunit, suggesting perturbation of NFB activation.A role for HS protein (HSP) 70 in NOS-2 suppression by HS is supported by the demonstration that 1) transfection with human HSP70 cDNA partially replicated HS effects; 2) antisense, but not sense, oligonucleotides directed against rat HSP70 partially blocked HS effects; and 3) rat fibroblasts stably expressing human HSP70 did not express NOS-2 in response to LPS plus cytokines. As with heat-shocked cells, HSP70-expressing cells also exhibited decreased NFB p65 subunit nuclear accumulation. These results demonstrate that in glial cells, as well as other cell types, NOS-2 induction can be modulated by the HS response, mediated at least in part by HSP70 expression.

Heat Shock Protein 70 Is a Potential Virulence Factor in MurineToxoplasmaInfection Via Immunomodulation of Host NF-κB and Nitric Oxide

“…A number of precedents exist for HSP70 modulation of NF-B uptake to the nucleus; however, the mechanism by which HSP70 could interfere with the activation of NF-B nuclear uptake is not yet fully elucidated. In cases involving HSP70-mediated inhibition of NF-B, Feinstein et al (18) proposed that stress proteins physically interact with I B. The association of inhibitory I B with NF-B subunits occurs via interaction with I B ankyrin domains with nuclear localization sites present in the p50 and p65 proteins.…”

“…Other possibilities are that HSP70, which is capable of migrating to the nucleus upon induction of the stress response, impedes NF-B nuclear translocation by competing for access to the same nuclear pore complexes through which NF-B must be transported to the nucleus (18). HSP70 could potentially inhibit NF-B activation by direct interaction with one or more of the NF-B constituents.…”

“…Thus, HSP70s are associated with protection against NO-induced cellular pathology and inhibition of NF-B activity in a range of eukaryotic cells. Kim et al (16) demonstrated that HSP70 antisense oligonucleotides abrogate NO-induced protection against TNF in rat hepatocytes, Bellmann et al (17) showed that transfection of the human gene encoding HSP70 into rat insulinoma cells is protective against NO-induced cell lysis, and Feinstein et al (18) found that stress-induced HSP70 reduces NF-B nuclear uptake and subsequent downstream iNOS expression and activity in astroglial cells. Thus, in this study we tested the hypotheses that the HSP70 of T. gondii inhibits iNOS expression, NO production, and NF-B activity, and that such effects are confined to virulent strains of the parasite.…”

Microglial Activation in Stroke: Therapeutic Targets

“…193,194 Glial cells exposed to heat shock or transfected with HSP-70 showed less nuclear NF B translocation and less iNOS expression (NF B-regulated protein) when treated with bacterial lipopolysaccharide. 195 Similarly, in brain inflammation elicited by bacterial lipopolysaccharide and cytokine injection, prior heat stress led to less microglial activation and less NF B activity. 196 Pyruvate.…”

The Oct DNA motif participates in the alcohol inhibition of the inducible nitric oxide synthase gene promoter in rat C6 glioma cells

“…The co-precipitation of calcium phosphate and DNA method was used to prepare stable transfectants following standard protocols for adherent cells incorporating minor modifications for C6 cells (Feinstein, et al, 1996). The C6 cells were cotransfected with pBK-CMV plasmid to allow selection by growth in 0.6 or 0.3 mg/mL geneticin.…”