Suppression of glial nitric oxide synthase induction by heat shock:: Effects on proteolytic degradation of IκB-α
Suppression of glial nitric oxide synthase induction by heat shock:: Effects on proteolytic degradation of IκB-α
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DOI：
10.1006/niox.1997.0117
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发表时间：
1997-04-01
影响因子：
3.9
通讯作者：
Reis, DJ
中科院分区：
文献类型：
--
作者：
Feinstein, DL;Galea, E;Reis, DJ
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。免疫组化显示C6-hsp 70细胞中存在大量的胞质HSP 70，但在对照（载体转染）C6-pTK细胞中不存在。当用细菌内毒素脂多糖（LPS）加三种细胞因子（“CM”：TNF-α、IL 1-β和IFN-γ）的混合物的最大刺激组合诱导时，通过亚硝酸盐积累评估的C6-hsp 70细胞中一氧化氮合酶（NOS-2）表达的诱导与对照C6-pTK细胞相比显著降低（对照细胞诱导的25 +/-8%，P < 0.005）。转录因子NF κ B B p65亚基的免疫染色显示，与对照细胞相比，表达HSP 70的细胞中的精氨酸依赖性核摄取减少。LPS加CM激活C6细胞NF κ B需要205蛋白酶体降解I κ B，因为选择性蛋白酶体抑制剂阻断了NOS-2的表达。在亲本C6细胞中，LPS加CM的存在引起抑制性I κ B-α蛋白水平的快速（30分钟内）降低，并且这种损失通过细胞的先前热休克而消除。相反，在转染细胞中的I κ B-α水平没有被HSP 70的表达所改变。这些结果表明，在胶质细胞中的组成型HSP 70表达可以减少NOS-2的诱导，推测是由于抑制NF κ B核摄取。此外，虽然预防I κ B-α的减少可以解释热休克的抑制作用，但结果表明HSP 70通过在NF κ B活化途径的后续步骤中干扰来阻断NOS-2诱导。(C)北京：科学出版社.
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 NF kappa B p65 subunit revealed decreased cytokine-dependent nuclear uptake in HSP70 expressing cells compared to control cells. Activation of C6 cell NF kappa B by LPS plus CM required I kappa B degradation by the 205 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 I kappa B-alpha protein levels, and this loss was abolished by prior heat shock of the cells. In contrast, I kappa B-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 NF kappa B nuclear uptake. Furthermore, whereas prevention of decreases in I kappa B-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 NF kappa B activation pathway. (C) 1997 Academic Press.