At the cell surface, lignai transducers and activators of transcription (STAT) proteins specifically interact with cytokine-receptors and receptor-associated kinases, but the nature of subsequent STAT interactions in the nucleus is less certain. To further investigate the basis for Stall-dependent transcription, we have studied the requirements for IFN-y-responsiveness of the intercellular adhesion molecule-1 (ICAM-1) gene using a human tracheobronchial epithelial cell (hTBEC) model. Transfection experiments with constructs containing ICAM-1 gene sequences upstream to endogenous or heterologous promoters driving a reporter gene demonstrated that IFN-y-induced activation of ICAM-1 gene transcription is dependent on the presence of an II bp inverted-repeat (IR) at -116 to -106 and GC box at -99 to -94. Electrophoretic mobility shift assays using transcription factor-specific antibodies demonstrated that Stall binds at the IR while specificity protein 1 (Spl) binds at the GC box. Expression of a Gal4/Spl fusion protein restored full responsiveness to a relatively inactive transfection construct containing ICAM-1 5'-flanking sequence with a Gal4- in place of the Spl-binding site. The capacity of Spl to restore full responsiveness was not shared with the transactivating domains of several other transcription factors (including p6S/RelA, Fos, or Ela), even though these factors were capable of increasing basal promoter activity to a similar level. Coimmunoprecipitation experiments using protein extracts from hTBEC indicated direct Spl/Statl interaction that was not dependent on Stall Tyr-701 or Ser-727 phosphorylation state. hTBECs also contain Stat2, 3, 5, and 6, however, only Stal3 appeared to directly interact with Spl. The results indicate that both IRStatl and GC box-Spl binding are required for maximal activation of the ICAM-1 gene by IFN-γ. Evidence of direct and selective Statl/Spl interaction further suggests that Statl/Spl synergy confers an element of specificity in the pathway leading to cytotine-activated transcription.
|State||Published - Dec 1 1996|