Feinstein D L, Galea E, Reis D J
Department of Neurology and Neuroscience, Cornell University Medical College, New York, New York 10021, USA.
Nitric Oxide. 1997 Apr;1(2):167-76. doi: 10.1006/niox.1997.0117.
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.
用编码热休克蛋白（HSP）70的人cDNA稳定转染大鼠C6胶质瘤细胞。免疫染色显示，C6 - hsp70细胞中主要存在于胞质中的HSP70，但在对照（载体转染）C6 - pTK细胞中不存在。与对照C6 - pTK细胞相比，用细菌内毒素脂多糖（LPS）加三种细胞因子混合物（“CM”：TNF -α、IL1 -β和IFN -γ）的最大刺激组合诱导时，通过亚硝酸盐积累评估的C6 - hsp70细胞中一氧化氮合酶（NOS - 2）表达的诱导显著降低（为对照细胞诱导的25±8%，P < 0.005）。转录因子NFκB p65亚基的免疫染色显示，与对照细胞相比，HSP70表达细胞中细胞因子依赖性核摄取减少。LPS加CM对C6细胞NFκB的激活需要20S蛋白酶体降解IκB，因为NOS - 2表达被选择性蛋白酶体抑制剂阻断。在亲代C6细胞中，LPS加CM的存在导致抑制性IκB -α蛋白水平迅速（30分钟内）下降，而细胞预先热休克可消除这种下降。相反，转染细胞中的IκB -α水平不受HSP70表达的影响。这些结果表明，胶质细胞中组成型HSP70表达可降低NOS - 2诱导，推测是由于抑制了NFκB核摄取。此外，虽然防止IκB -α下降可以解释热休克的抑制作用，但结果表明HSP70通过在NFκB激活途径的后期步骤进行干扰来阻断NOS - 2诱导。
