HEAT-SHOCK PROTEIN-70 SUPPRESSES ASTROGLIAL-INDUCIBLE NITRIC-OXIDE SYNTHASE EXPRESSION BY DECREASING NF-KAPPA-B ACTIVATION

In brain glial cells, expression of calcium independent nitric oxide s ynthase (NOS-2) is induced following stimulation with bacterial endoto xin (lipopolysaccharide (LPS)) and/or pro-inflammatory cytokines. We h ave investigated the effects of heat shock (HS), which can re duce inf lammatory responses in several cell types, on the induction of glial N OS-2 expression. Preincubation of cells for 20-60 min at 43 degrees C decreased subsequent levels of NOS-2 induction, with a maximal 80% red uction 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), stea dy state mRNA levels (2-3-fold), and gene promoter activity (by 50%). HS also reduced LPS induced nuclear accumulation of transcription fact or NF kappa B p65 subunit, suggesting perturbation of NF kappa B activ ation. A role for HS protein (HSP) 70 in NOS-2 suppression by HS is su pported by the demonstration that 1) transfection with human HSP70 cDN A partially replicated HS effects; 2) antisense, but not sense, oligon ucleotides directed against rat HSP70 partially blocked HS effects; an d 3) rat fibroblasts stably expressing human HSP70 did not express NOS -2 in response to LPS plus cytokines. As with heat-shocked cells, HSP7 0 expressing cells also exhibited decreased NF kappa B p65 subunit nuc lear accumulation. These results demonstrate that in glial cells, as w ell as other cell types, NOS-2 induction can be modulated by the HS re sponse, mediated at least in part by HSP70 expression.