Abstract
Interferon (IFN)-gamma actions on the vessel wall play an important role in the pathogenesis of arteriosclerosis, yet the contribution of different IFN-gamma signaling pathways to the phenotypic modulation of vascular smooth muscle cells (VSMCs) are poorly understood. We investigated the effects of IFN-gamma on VSMCs and arteries through interactions involving signal transducer and activator of transcription (STAT) proteins. In addition to STAT1 activation, IFN-gamma consistently phosphorylated STAT3 in human VSMCs but weakly or not at all in human endothelial cells or mouse VSMCs. STAT3 activation resulted in nuclear translocation of this transcription factor. By selectively inhibiting STAT3 and not STAT1 signaling, we identified a number of candidate IFN-gamma-inducible, STAT3-dependent gene products by microarray analysis. Results for selected genes, including the pro-apoptotic molecules X-linked inhibitor of apoptosis associated factor-1 (XAF1) and Noxa, were verified by real time quantitative reverse transcription-PCR and immunoblot analyses. IFN-gamma-induced STAT3 and STAT1 signaling in VSMCs demonstrated reciprocal inhibition. STAT3 activation by IFN-gamma sensitized VSMCs to apoptosis triggered by both death receptor- and mitochondrial-mediated pathways. Knock down of XAF1 and Noxa expression inhibited the priming of VSMCs to apoptotic stimuli by IFN-gamma. Finally, we confirmed the in vivo relevance of our observations using a chimeric animal model of immunodeficient mice bearing human coronary artery grafts in which the expression of XAF1 and Noxa as well as the pro-apoptotic effects induced by IFN-gamma were dependent on STAT3. The data suggest STAT1-independent signaling by IFN-gamma via STAT3 that promotes the death of human VSMCs.
Highlights
Interferon (IFN ␥),4 the prototypical TH1 cytokine, plays a key effector role in the pathogenesis of inflammatory arteriosclerotic diseases, such as atherosclerosis and graft arteriosclerosis, through its actions on vascular cells [1]
We report that the IFN-␥ induces expression of X-linked inhibitor of apoptosis associated factor-1 (XAF1) and Noxa in human vascular smooth muscle cells (VSMCs) through STAT3 activation and that these proteins sensitize VSMCs in vitro and in vivo to apoptosis triggered via death receptor or mitochondrial mechanisms
IFN-␥-activated STAT3 in VSMCs—To determine whether STAT1-independent signaling by IFN-␥ occurred in VSMCs, we first analyzed IFN-␥-treated cultured human VSMCs for activation of other signal transducer and activator of transcription (STAT) proteins
Summary
VSMCs were transduced with retroviral vectors in the presence of Polybrene at 8 g/ml for 6 h and cultured in 20% fetal bovine serum-supplemented media. Microarray Analysis—Total RNA was isolated from cultured VSMCs under various conditions using RNeasy mini kits (Qiagen) with on-column DNase treatment. VSMCs were fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin, and labeled with rabbit antibody to human cleaved caspase 3 (Cell Signaling Technology) or control IgG followed by fluorescein isothiocyanate-conjugated goat anti-rabbit IgG. Graft Analysis—Immunohistochemistry was performed as previously described [25] using primary antibodies to human major histocompatibility complex class I antigens (Dako, Carpinteria, CA), STAT3 (BD Biosciences), Noxa (Oncogene Research Product), and XAF1 (Santa Cruz Biotechnology) as well as the 2E1 antibody [26] developed against ASO 13920 (ISIS Pharmaceuticals). Differences with p Ͻ 0.05 were considered to indicate statistical significance
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