Down-regulation of interferon-gamma signaling by gene transfer of Stat1 mutant in mesangial cells

Background. Interferon-gamma (IFN-gamma) is secreted by T lymphocytes and n atural killer (NK) cells in the cellular immunity-mediated inflammatory les ion, including endocapillary or extracapillary proliferative glomerulonephr itis. It induces and/or enhances multiple histocompatibility complex (MHC) class I and II, intercellular adhesion molecule-1 (ICAM-1), inducible nitri c 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 Stat1 alpha is essential for signaling. We investigated Stat1 alpha activa tion by IFN-gamma in mesangial cells and attempted to regulate the signal t ransduction by gene transfer. Methods. Western blot with anti-Stat1 and antiphosphotyrosine after immunop recipitation of Stat1 and Northern blot for detection of Stat1 mRNA were pe rformed. The dominant negative form of Flag-tagged Stat1 was expressed in c ultured rat mesangial cells. Flag was immunostained in transfectants, and l uciferase reporter assay was carried out to measure the transcriptional act ivity of Stat1 alpha. The expression of IFN-gamma-inducible genes such as M HC class II (Ia-A alpha) and MHC class II transactivator (CIITA) was detect ed by reverse transcriptase-polymerase chain reaction (RT-PCR). Results. Stat1 alpha was tyrosine phosphorylated and activated by IFN-gamma in mesangial cells, and the mRNA and protein level of Stat1 alpha increase d upon stimulation by IFN-gamma. Overexpression of Stat1-mutant lacking 35 C-terminal amino acids strongly suppressed the IFN-gamma-induced signal tra nsduction and inhibited the expression of MHC class II and CIITA genes in m esangial cells. Conclusions. Stat1 alpha is a critical molecule in the signal transduction of IFN-gamma in mesangial cells. The inhibition of an endogenous function o f Stat1 alpha by gene transfer of the Stat1 mutant may be a new method to r educe the inflammatory effects of IFN-gamma in localized inflammation of th e kidney.