Nakashima O, Terada Y, Hanada S, Yamamoto K, Kuwahara M, Sasaki S, Marumo F
Second Department of Internal Medicine and First Department of Internal Medicine, Tokyo Medical and Dental University, Tokyo, Japan.
Kidney Int. 2000 Jun;57(6):2249-57. doi: 10.1046/j.1523-1755.2000.00085.x.
JAK-STAT signaling has been shown to promote development and proliferation in lymphopoietic and hematopoietic lineages. We investigated the effect of activated STAT1 on mesangial cell proliferation.
Rat mesangial cells of primary culture (rMCs) were used in the following experiments: (1) Whole cell lysates were immunoblotted against JAK1 and JAK2. (2) Whole cell lysates and nuclear proteins were extracted from rMCs with or without treatment with interferon-gamma, and immunoblotting was performed against both STAT1 and tyrosine (701)-phosphorylated STAT1. (3) rMCs and rMCs electroporated with either wild-type STAT1, mutated STAT1, or antibody against STAT1 were incubated with interferon-gamma for 20 hours, followed by a further incubation with [3H]-thymidine for four hours.
JAK1, JAK2, and STAT1 were detected in whole cell lysates, suggesting that JAK-STAT signaling could be activated by interferon-gamma (INF-gamma). Using an antibody specific for tyrosine-phosphorylated STAT1, we detected signal in the INF-gamma-treated nuclear extracts, which showed translocation of phosphorylated STAT1 to the nucleus. [3H]-thymidine incorporation in the presence of INF-gamma was significantly lower than that of control in a dose-dependent manner. The introduction of wild-type STAT1 enhanced the effect of interferon-gamma and decreased [3H]-thymidine incorporation, whereas tyrosine-mutated (Y701F) STAT1 and SH2 domain (R602T)-mutated STAT1 reversed INF-gamma-induced suppression of [3H]-thymidine incorporation. Electroinjected antibody against STAT1 increased [3H]-thymidine incorporation upon stimulation with INF-gamma.
STAT1 activated by interferon-gamma suppresses mesangial cell proliferation.
JAK-STAT信号通路已被证明可促进淋巴细胞和造血细胞系的发育和增殖。我们研究了活化的STAT1对系膜细胞增殖的影响。
原代培养的大鼠系膜细胞（rMCs）用于以下实验：（1）全细胞裂解物用抗JAK1和JAK2抗体进行免疫印迹。（2）用或不用干扰素-γ处理rMCs，提取全细胞裂解物和核蛋白，并用抗STAT1和酪氨酸（701）磷酸化的STAT1抗体进行免疫印迹。（3）将rMCs与野生型STAT1、突变型STAT1或抗STAT1抗体电穿孔处理后，与干扰素-γ孵育20小时，然后再与[3H]-胸腺嘧啶孵育4小时。
在全细胞裂解物中检测到JAK1、JAK2和STAT1，表明JAK-STAT信号通路可被干扰素-γ（INF-γ）激活。使用酪氨酸磷酸化的STAT1特异性抗体，我们在INF-γ处理的核提取物中检测到信号，这表明磷酸化的STAT1易位至细胞核。在INF-γ存在下，[3H]-胸腺嘧啶掺入量以剂量依赖方式显著低于对照组。野生型STAT1的导入增强了干扰素-γ的作用并降低了[3H]-胸腺嘧啶掺入量，而酪氨酸突变（Y701F）的STAT1和SH2结构域（R602T）突变的STAT1逆转了INF-γ诱导的[3H]-胸腺嘧啶掺入抑制。电注射抗STAT1抗体在INF-γ刺激后增加了[3H]-胸腺嘧啶掺入量。
干扰素-γ激活的STAT1抑制系膜细胞增殖。
