Interferon-γ-mediated Inhibition of Serum Response Factor-dependent Smooth Muscle-specific Gene Expression

Abstract

IFN␥ exerts multiple biological effects on effector cells by regulating many downstream genes, including smooth muscle-specific genes. However, the molecular mechanisms underlying IFN␥-induced inhibition of smooth muscle-specific gene expression remain unclear. In this study, we have shown that serum response factor (SRF), a common transcriptional factor important in cell proliferation, migration, and differentiation, is targeted by IFN␥ in a STAT1-dependent manner. We show that the molecular mechanism by which IFN␥ regulates SRF is via activation of the 2-5A-RNase L system, which triggers SRF mRNA decay and reduced SRF expression. As a result, decreased SRF expression reduces expression of SRF target genes such as smooth muscle ␣-actin and smooth muscle myosin heavy chain. Additionally, IFN␥ reduced p300 and acetylated histone-3 binding in both smooth muscle ␣-actin and SRF promoters, epigenetically decreasing smooth muscle ␣-actin and SRF transcriptional activation. Our data reveal that SRF is a novel IFN␥-regulated gene and further elucidate the molecular pathway between IFN␥, IFN␥-regulated genes, and SRF and its target genes.IFN␥, a pleiotropic cytokine that is primarily produced by T cells and NK cells, plays a complex and central role in antiviral, antiproliferative, antifibrogenesis, antitumor, and immunomodulatory activities (reviewed in Refs. 1 and 2). The complexity of such a variety of IFN␥ effects appears to be achieved by its signaling to over 200 genes, leading to a highly networked pattern of cell-specific gene regulation (3). In the canonical pathway, through binding to cognate IFN type II receptors, IFN␥ initiates a cascade that includes JAK family kinases and the STAT1 family of transcriptional factors to induce STAT1-dependent gene expression, which largely mediates the actions of IFN␥ (reviewed in Ref. 4). Among IFN␥-regulated genes, smooth muscle ␣-actin, a cytoskeleton protein that is critical in smooth muscle cell differentiation (reviewed in Ref. 5), myofibroblast activation (reviewed in Ref. 6), and epithelial-mesenchymal transition (7), is negatively regulated by IFN␥ (8, 9). Although the observation that IFN␥ regulates smooth muscle ␣-actin is well established, the molecular mechanisms underlying IFN␥-induced inhibition of smooth muscle ␣-actin expression remain unclear.Regulation of smooth muscle ␣-actin expression is complex, having been studied extensively in cardiovascular and vascular diseases, particularly in smooth muscle cells (reviewed in Ref. 5). It has been well demonstrated that most muscle-specific genes, including smooth muscle ␣-actin, are SRF 2 target genes. SRF binds to the CArG boxes (CC(A/T) 6 GG) of the smooth muscle ␣-actin gene promoter and activates smooth muscle ␣-actin transcription (reviewed in Ref. 10). Extracellular factors can exert their effects on smooth muscle gene expression through regulating SRF or/and SRF cofactors (11). TGF␤, a well characterized cytokine important in smooth muscle cell differentiation and epithelial-mesenchymal transition, up-reg...

RNase L Attenuates Mitogen-stimulated Gene Expression via Transcriptional and Post-transcriptional Mechanisms to Limit the Proliferative Response

“…This result demonstrated the RNase L-dependent down-regulation of TTP transcription (81,82) in mitogenstimulated MEFs and suggested that RNase L directly targets a transcription factor that mediates TTP induction by mitogens. Consistent with this model, SRF is a master transcriptional regulator of serum-induced genes that was previously reported to be regulated by RNase L in IFN␥-stimulated smooth muscle cells (66). Furthermore, analysis of the ZFP36/TTP gene promoter revealed a serum-response element, the site at which SRF binds to induce transcription of mitogen-induced genes, at positions Ϫ281 to Ͼ264 relative to the ZFP36/TTP transcription start site (83).…”

“…A binding site for the serum-response factor (SRF), a master transcriptional regulator of serum-induced genes, was identified in the ZFP36 gene promoter. Furthermore, SRF mRNA was previously reported to be down-regulated by RNase L in IFN␥-stimulated mouse embryo fibroblasts (MEFs) (66). Together, these observations suggested that SRF mRNA is a direct target of RNase L and that this regulation may account for the RNase L-dependent regulation of TTP in mitogen-stimulated cells.…”

“…SRF is a master transcriptional regulator of serum-induced genes (81,82,91), and RNase L was previously reported to regulate SRF in IFN␥-treated smooth muscle cells (66). Furthermore, an SRF-binding site was identified in the ZFP36 gene promoter (84,85).…”

Vascular pathobiology in chronic liver disease and cirrhosis – Current status and future directions

“…For example, transforming growth factor beta-1 (TGF-β1) the most profibrogenic cytokine for stellate cells, may also regulate smooth muscle α actin gene expression [37]. Recent evidence indicates that smooth muscle α actin expression in stellate cells is controlled transcriptionally by canonical muscle specific transcription factors, including serum response factor [38]. Further, it appears that there may be interplay between the smooth muscle α actin cytoskeleton and various stellate cell functions, including cell motility and contractility [39].…”

Stellate Cells and Portal Hypertension

“…The molecular mechanism of upregulation of these smooth muscle contractile proteins appears to be via a robust smooth muscle transcriptional program, including upregulation of a family of transcription factors important in smooth muscle cell biology [13]. Although controversy exists regarding the magnitude of contraction of normal stellate cells, available data emphasize that stellate cells in the injured liver exhibit enhanced contractility and moreover, the degree of contraction appears to be proportional to the degree of liver injury [14,15].…”