Stimulation of signal transducer and activator of transcription-1 (STAT1)-dependent gene transcription by lipopolysaccharide and interferon-γ is regulated by mammalian target of rapamycin

Mammalian target of rapamycin ( mTOR) and phosphatidylinositol 3-kinase (PI3K) regulate cell growth, protein synthesis, and apoptosis in response to nutrients and mitogens. As an important source of nitric oxide during inflammation, human inducible nitric oxide synthase also plays a role in the regulation of cytokine-driven cell proliferation and apoptosis. The role of mTOR and PI3K in the activation of human inducible nitric oxide synthase transcription by cytokines and lipopolysaccharide (LPS) was investigated in lung epithelial adenocarcinoma (A549) cells. LY294002, a dual mTOR and PI3K inhibitor, blocked human inducible nitric oxide synthase (hiNOS) promoter activation and mRNA induction by cytokines and LPS in a PI3K-independent fashion. On gene expression analysis, LY294002 selectively blocked the induction of a subset of 14 LPS/interferon-gamma (IFN-gamma)-induced genes, previously characterized as signal transducer and activator of transcription-1 (STAT1)-dependent. LY294002, but not wortmannin, inhibited LPS/IFN-gamma-dependent STAT1 phosphorylation at Ser-727 and STAT1 activity. Consistent with dual inhibition of mTOR and PI3K by LY294002, dominant-negative mTOR, anti-mTOR small interfering RNA, or rapamycin each inhibited phosphorylation of STAT1 only in the presence of wortmannin. LPS/IFN-gamma led to the formation of a macromolecular complex containing mTOR, STAT1, as well as protein kinase Cdelta, a known STAT1alpha kinase. Thus, LPS and IFN-gamma activate the PI3K and mTOR pathways, which converge to regulate STAT1-dependent transcription of pro-apoptotic and pro-inflammatory genes in a rapamycin-insensitive manner.