Sakatsume M, Narita I, Yamazaki H, Saito A, Nakagawa Y, Kuriyama H, Kuwano R, Gejyo F, Arakawa M
Department of Medicine (II) and Research Laboratory for Molecular Genetics, Niigata University School of Medicine, Niigata, Japan.
Kidney Int. 2000 Feb;57(2):455-63. doi: 10.1046/j.1523-1755.2000.00865.x.
Interferon-gamma (IFN-gamma) is secreted by T lymphocytes and natural killer (NK) cells in the cellular immunity-mediated inflammatory lesion, including endocapillary or extracapillary proliferative glomerulonephritis. It induces and/or enhances multiple histocompatibility complex (MHC) class I and II, intercellular adhesion molecule-1 (ICAM-1), inducible nitric oxide synthase (iNOS), and Fc receptor expression in renal resident cells, resulting in the initiation and promotion of inflammation. Recently, the signaling mechanism of IFN-gamma has been investigated, and it appears that Stat1alpha is essential for signaling. We investigated Stat1alpha activation by IFN-gamma in mesangial cells and attempted to regulate the signal transduction by gene transfer.
Western blot with anti-Stat1 and antiphosphotyrosine after immunoprecipitation of Stat1 and Northern blot for detection of Stat1 mRNA were performed. The dominant negative form of Flag-tagged Stat1 was expressed in cultured rat mesangial cells. Flag was immunostained in transfectants, and luciferase reporter assay was carried out to measure the transcriptional activity of Stat1alpha. The expression of IFN-gamma-inducible genes such as MHC class II (Ia-Aalpha) and MHC class II transactivator (CIITA) was detected by reverse transcriptase-polymerase chain reaction (RT-PCR).
Stat1alpha was tyrosine phosphorylated and activated by IFN-gamma in mesangial cells, and the mRNA and protein level of Stat1alpha increased upon stimulation by IFN-gamma. Overexpression of Stat1-mutant lacking 35 C-terminal amino acids strongly suppressed the IFN-gamma-induced signal transduction and inhibited the expression of MHC class II and CIITA genes in mesangial cells.
Stat1alpha is a critical molecule in the signal transduction of IFN-gamma in mesangial cells. The inhibition of an endogenous function of Stat1alpha by gene transfer of the Stat1 mutant may be a new method to reduce the inflammatory effects of IFN-gamma in localized inflammation of the kidney.
在细胞免疫介导的炎症病变中，包括毛细血管内或毛细血管外增生性肾小球肾炎，γ干扰素（IFN-γ）由T淋巴细胞和自然杀伤（NK）细胞分泌。它诱导和/或增强肾固有细胞中多种主要组织相容性复合体（MHC）Ⅰ类和Ⅱ类分子、细胞间黏附分子-1（ICAM-1）、诱导型一氧化氮合酶（iNOS）以及Fc受体的表达，从而引发并促进炎症。最近，对IFN-γ的信号传导机制进行了研究，似乎Stat1α对信号传导至关重要。我们研究了系膜细胞中IFN-γ对Stat1α的激活作用，并试图通过基因转移来调节信号转导。
进行Stat1免疫沉淀后用抗Stat1和抗磷酸酪氨酸的蛋白质印迹法以及用于检测Stat1 mRNA的Northern印迹法。带有Flag标签的Stat1的显性负性形式在培养的大鼠系膜细胞中表达。对转染细胞中的Flag进行免疫染色，并进行荧光素酶报告基因测定以测量Stat1α的转录活性。通过逆转录聚合酶链反应（RT-PCR）检测IFN-γ诱导基因如MHCⅡ类分子（Ia-Aα）和MHCⅡ类反式激活因子（CIITA）的表达。
在系膜细胞中，Stat1α被IFN-γ酪氨酸磷酸化并激活，且在IFN-γ刺激后Stat1α的mRNA和蛋白质水平增加。缺乏35个C末端氨基酸的Stat1突变体的过表达强烈抑制了IFN-γ诱导的信号转导，并抑制了系膜细胞中MHCⅡ类分子和CIITA基因的表达。
Stat1α是系膜细胞中IFN-γ信号转导的关键分子。通过Stat1突变体的基因转移抑制Stat1α的内源性功能可能是减少IFN-γ在肾脏局部炎症中炎症效应的一种新方法。
