Gupta A K, Diaz R A, Higham S, Kone B C
Departments of Internal Medicine and of Integrative Biology, Pharmacology and Physiology, The University of Texas Health Science Center at Houston, Houston, Texas 77030, USA.
Kidney Int. 2000 Jun;57(6):2239-48. doi: 10.1046/j.1523-1755.2000.00084.x.
alpha-Melanocyte-stimulating hormone (alpha-MSH) is an endogenous tridecapeptide that exerts anti-inflammatory actions and abrogates postischemic renal injury in rodents. alpha-MSH inhibits lipopolysaccharide (LPS)-induced gene expression of several cytokines, chemokines, and nitric oxide synthase-2 (NOS2), but the molecular mechanisms underlying these effects have not been clearly defined. To test the hypothesis that alpha-MSH inhibits the expression of inducible trans-activating factors involved in NOS2 regulation, we used RAW 264.7 macrophage cells to examine the effects of alpha-MSH on the activation of nuclear factor-kappaB (NF-kappaB) and CCAAT/enhancer binding protein-beta (C/EBPbeta), trans-acting factors known to be involved in LPS + interferon (IFN)-gamma induction of the NOS2 gene.
Gel shift assays were performed to identify NF-kappaB and C/EBP DNA binding activities in LPS + IFN-gamma-treated RAW 264.7 cells in the presence and absence of alpha-MSH. NOS2 promoter assays were conducted to identify the effects of alpha-MSH on LPS + IFN-gamma-mediated induction of NOS2 transcription.
Gel shift assays demonstrated LPS + IFN-gamma induction of NF-kappaB and C/EBP family protein-DNA complexes in nuclei harvested from the cells. Supershift assays revealed that the C/EBP complexes were comprised of C/EBPbeta, but not C/EBPalpha, C/EBPdelta, or C/EBPepsilon. alpha-MSH (100 nmol/L) inhibited the LPS + IFN-gamma-mediated induction of nuclear DNA binding activity of C/EBPbeta, but not that of NF-kappaB (in contrast to reports in other cell types), as well as the activity of a murine NOS2 promoter-luciferase construct. In contrast, alpha-MSH (100 nmol/L) had no effect on the induction of NOS2 promoter-luciferase genes harboring deletion or mutation of the C/EBP box.
These data indicate that alpha-MSH inhibits the induction of C/EBPbeta DNA binding activity and that this effect is a major mechanism by which alpha-MSH inhibits the transcription of the NOS2 gene. The inability of alpha-MSH to inhibit LPS + IFN-gamma induction of NF-kappaB in murine macrophage cells, which contrasts with inhibitory effects of the neuropeptide in other cell types, suggests that cell-type-specific mechanisms are involved.
α-黑素细胞刺激素（α-MSH）是一种内源性十三肽，具有抗炎作用，并可减轻啮齿动物缺血后肾损伤。α-MSH可抑制脂多糖（LPS）诱导的多种细胞因子、趋化因子及一氧化氮合酶2（NOS2）的基因表达，但其作用的分子机制尚未明确。为验证α-MSH抑制参与NOS2调控的诱导性反式激活因子表达这一假说，我们利用RAW 264.7巨噬细胞检测α-MSH对核因子-κB（NF-κB）及CCAAT/增强子结合蛋白-β（C/EBPβ）激活的影响，这两种反式作用因子参与LPS +干扰素（IFN）-γ诱导NOS2基因的表达。
采用凝胶迁移试验鉴定在有或无α-MSH存在情况下，LPS + IFN-γ处理的RAW 264.7细胞中NF-κB及C/EBP的DNA结合活性。进行NOS2启动子试验以确定α-MSH对LPS + IFN-γ介导的NOS2转录诱导的影响。
凝胶迁移试验表明，LPS + IFN-γ可诱导从细胞收获的细胞核中NF-κB及C/EBP家族蛋白-DNA复合物的形成。超迁移试验显示，C/EBP复合物由C/EBPβ组成，而非C/EBPα、C/EBPδ或C/EBPε。α-MSH（100 nmol/L）可抑制LPS + IFN-γ介导的C/EBPβ核DNA结合活性的诱导，但对NF-κB无此作用（与其他细胞类型的报道相反），同时也抑制小鼠NOS2启动子-荧光素酶构建体的活性。相比之下，α-MSH（100 nmol/L）对含有C/EBP框缺失或突变的NOS2启动子-荧光素酶基因的诱导无影响。
这些数据表明，α-MSH可抑制C/EBPβ DNA结合活性的诱导，且此效应是α-MSH抑制NOS2基因转录的主要机制。α-MSH不能抑制小鼠巨噬细胞中LPS + IFN-γ诱导的NF-κB，这与该神经肽在其他细胞类型中的抑制作用相反，提示存在细胞类型特异性机制。
