Targeted inhibition of interferon-gamma-dependent intercellular adhesion molecule-1 (ICAM-1) expression using dominant-negative Stat1

A subset of epithelial immune-response genes (including intercellular adhesion molecule-1 (ICAM-1)) depends on an IFN-gamma signal transduction pathway with the Stat1 transcription factor as a critical intermediate, Excessive local activation of this pathway may lead to airway inflammation, so we sought to selectively downregulate the pathway using a dominant-negative strategy for inhibition of epithelial Stat1 in a primary culture airway epithelial cell model. Using a Stat1-deficient cell line, we demonstrated that transfection of wild-type Stat1 expression plasmid restored appropriate Stat1 expression and IFN-gamma-dependent phosphorylation as well as consequent lFN-gamma activation of cotransfected ICAM-1 promoter constructs and endogenous ICAM-1 gene expression. However, mutations of Stat1 at Tyr-701 (JAR kinase phosphorylation site), Glu-428/429 (putative DNA-binding site), His-713 (splice site resulting in Stat1 beta formation), or Ser-727 (MAP kinase phosphorylation site) all decreased Stat1 capacity to activate the ICAM-1 promoter, The Tyr-701 mutant (followed by the His-713 mutant) were most effective in disabling Stat1 function and in overcoming the activating effect of cotransfected wild-type Stat1 in this cell system thereby highlighting the effectiveness of blocking Stat1 homo-and hetero-dimerization. In experiments using primary culture human tracheobronchial epithelial cells (hTBECs) and each of the four Stat1 mutant plasmids, transfection with the Tyr-701 and His-713 mutants again most effectively inhibited IFN-gamma activation of an ICAM-1 gene promoter construct, Then by transfecting hTBECs with wild-type or mutant Stat1 tagged with a Flag reporter sequence, we used dual immunofluorescence to show that hTBECs expressing the Tyr-701 or His-713 mutants were prevented from expressing endogenous ICAM-1 in response to IFN-gamma treatment. The capacity of a specific Stat1 mutations to exert a potent dominant-negative effect on IFN-gamma signal transduction provides for further definition of Statl structure function and a means for natural or engineered expression of mutant Stat1 to selectively down-regulate activity of this pathway in a cell type-or tissue-specific manner during immune and/or inflammatory responses.