Suppression of Glial Nitric Oxide Synthase Induction by Heat Shock: Effects on Proteolytic Degradation of IκB-α

Abstract

Rat C6 glioma cells were stably transfected with a human cDNA encoding heat shock protein (HSP) 70. Immunostaining revealed the presence of largely cytosolic HSP70 in C6-hsp70 cells, but not in control (vector transfected) C6-pTK cells. Induction of nitric oxide synthase (NOS-2) expression in C6-hsp70 cells, assessed by nitrite accumulation, was significantly reduced compared to control C6-pTK cells (25 ± 8% of control cell induction,P< 0.005), when induced with a maximally stimulatory combination of bacterial endotoxin lipopolysaccharide (LPS) plus a mixture of three cytokines (“CM:” TNF-α, IL1-β, and IFN-γ). Immunostaining for the transcription factor NFκB p65 subunit revealed decreased cytokine-dependent nuclear uptake in HSP70 expressing cells compared to control cells. Activation of C6 cell NFκB by LPS plus CM required IκB degradation by the 20S proteasome, since NOS-2 expression was blocked by a selective proteasome inhibitor. In parental C6 cells, the presence of LPS plus CM caused a rapid (within 30 min) decrease in inhibitory IκB-α protein levels, and this loss was abolished by prior heat shock of the cells. In contrast, IκB-α levels in transfected cells were not modified by the expression of HSP70. These results demonstrate that constitutive HSP70 expression in glial cells can reduce NOS-2 induction, presumably due to inhibition of NFκB nuclear uptake. Furthermore, whereas prevention of decreases in IκB-α can account for the suppressive effects of heat shock, the results suggest that HSP70 blocks NOS-2 induction by interfering at a later step in the NFκB activation pathway.