Dampened SOCS: An altered balance between STAT activating and SOCS inhibitory signals regulate interferon-γ expression in LPMC

Abstract

Activated T cells and T cell-associated cytokines, in particular IFN-% are implicated in the pathogenesis of inflammatory bowel disease. Mucosal lamina propria mononuclear cells (LPMC) exhibit enhanced IFN-.,/secretion, compared to PBL, through activation of the CD2 pathway. In PBL, cell surface receptor activation of the Jak/STAT pathway tacil~]tes cytokinemediated gene expression. STATs are latent transcription factors, which bind to the activated receptor complex, are phosphorylated and translocated to the nucleus, leading to transcriptional activation. Activation of the Jak/STAT pathway is short-lived with SOCS family proteins downregulating STAT activity in a classic negative feedback loop. Although studies have identified STAT binding regions within the IFN-x promoter and first intron, the role of STATs and SOCS in regulating IFN--y expression has not been clearly defined. In this study we show that CD2 activation of LPMC results in transactivation of the IFN-~/promoter region and concomitant phosphorylation and binding of STAT1 and STAT4 heterodimers to a STAT-binding enhancer, within the first intron, within 15 minutes. Mutation of the STAT element within the first intron attenuates enhancer activity. In contrast, the STAT binding element located upstream at -236 bp, exhibited binding of a nuclear protein complex in unstimulatad cells which remained unchanged following CD2 signaling. CD2 signaling leads to upregulation of SOCS1, SOCS 2, SOCS 3 and CIS expression in both PBL and LPL. Since SOCS is believed to function in a classic feedback loop, the kinetics of expression lags behind STAT activation as expected, with upregulation seen within 60 to 120 minutes following CD2 activation. However, although the kinetics of CIS expression are similar in both PBL and LPL, the magnitude of CIS expression was muted in LPL in comparison to PBL. In contrast to PBL, overexpression of SOCS/CIS protein in LPL leads to down regulation of transfected IFN--y promoter constructs. These studies represent the first reports of CD2-mediated activation linked to the STAT and SOCS pathways. They provide evidence for the participation of dual positive and negative regulatory mechanism involved in IFN-~/gene expression in LPMC compared to PBL: enhanced transactivation via the STAT pathway as well as dampened negative regulatory signals via the SOCS pathway. Targeted alterations in the levels of STAT and SOCS expression may provide novel approaches to regulate IFN--y secretion in the gut.