Suppression of glial nitric oxide synthase induction by heat shock: effects on proteolytic degradation of IkappaB-alpha.
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-alpha, IL1-beta, and IFN-gamma). Immunostaining for the transcription factor NFkappaB p65 subunit revealed decreased cytokine-dependent nuclear uptake in HSP70 expressing cells compared to control cells. Activation of C6 cell NFkappaB by LPS plus CM required IkappaB 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 IkappaB-alpha protein levels, and this loss was abolished by prior heat shock of the cells. In contrast, IkappaB-alpha 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 NFkappaB nuclear uptake. Furthermore, whereas prevention of decreases in IkappaB-alpha 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 NFkappaB activation pathway.
Department of Neurology and Neuroscience, Cornell University Medical College, New York, New York 10021, USA. dlfeins@med.cornell.edu
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D000818:Animals; D004790:Enzyme Induction; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D018869:Heat-Shock Response; D006801:Humans; D006868:Hydrolysis; D051550:I-kappa B Kinase; D007150:Immunohistochemistry; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D009457:Neuroglia / Q000201:enzymology* / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D014162:Transfection; D014407:Tumor Cells, Cultured
D018840:HSP70 Heat-Shock Proteins; D016328:NF-kappa B; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D017346:Protein Serine-Threonine Kinases; C496559:CHUK protein, human; D051550:I-kappa B Kinase; C496560:IKBKB protein, human; C496565:IKBKE protein, human