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 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.


METHODS
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).


RESULTS
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.


CONCLUSIONS
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.
Sakatsume
M
M
Department of Medicine (II) and Research Laboratory for Molecular Genetics, Niigata University School of Medicine, Niigata, Japan. sakatsum@med.niigata-u.ac.jp
Narita
I
I
Yamazaki
H
H
Saito
A
A
Nakagawa
Y
Y
Kuriyama
H
H
Kuwano
R
R
Gejyo
F
F
Arakawa
M
M
D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology; D018014:Gene Transfer Techniques; D005920:Glomerular Mesangium / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D005921:Glomerulonephritis / Q000276:immunology; D007371:Interferon-gamma / Q000494:pharmacology*; D008297:Male; D016296:Mutagenesis / Q000276:immunology; D010766:Phosphorylation; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D050794:STAT1 Transcription Factor; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics* / Q000276:immunology; D015534:Trans-Activators / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects / Q000276:immunology; D014443:Tyrosine / Q000378:metabolism
D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D050794:STAT1 Transcription Factor; C494081:Stat1 protein, rat; D015534:Trans-Activators; D014443:Tyrosine; D007371:Interferon-gamma