Abstract
ROR family of nuclear receptor transcription factors forms nodes connecting metabolic and inflammatory signaling pathways. The RORα members of the family have intrinsic transcriptional activity and they are involved in both activation and repression of a wide range of genes. The role of RORα in control of inflammation has been extensively studied using animal models but its function in human cells is not as well understood. To address this shortcoming, we analyzed how RORα is shaping the inflammatory state of human macrophages. Using CRISPR-Cas9 system, we deleted RORA in THP-1 human monocytic cell line. In mutant cells we observed a dramatic increase in basal expression of a subset of NF-κB regulated genes, including TNF, IL-1β and IL-6, at both transcriptional and translational levels. Furthermore, RORA-deletion cells produced notable amounts of pro-IL-1β even in the absence of LPS stimulation. Subsequent LPS stimulation induced cleavage of pro-IL-1β to mature form. Our RNAseq analysis further confirmed the key role of RORA in setting the inflammatory state of macrophages and defined the set of differentially regulated genes. Overall, our data provides evidence supporting the anti-inflammatory function of RORα in human macrophages.
Highlights
RAR-related Orphan Receptors (RORs) are members of nuclear receptor (NR) transcription factor family that tie together metabolic and inflammatory signaling pathways
To test which of the observations made in the model system translate to human cells, we chose to determine the role of RORα in control of inflammatory signaling in human macrophages
When compared to control cell lines carrying irrelevant renilla guide RNA, we found that deletion of RORA in THP-1 cells made a substantial impact on their inflammatory state (Fig 1)
Summary
RAR-related Orphan Receptors (RORs) are members of nuclear receptor (NR) transcription factor family that tie together metabolic and inflammatory signaling pathways. There are three major isotypes of RORs: α, β, and γ. All three of these isotypes are further diversified by alternative splicing and different promoter usage producing numerous cell-type specific isoforms differing at their N-terminus [1,2,3,4]. Recent studies suggested that oxysterols, cholesterol, 7-dehydroxycholeterol, hydroxyderivatives of vitamin D and lumisterol derivatives can act as RORα ligands while all-trans retinoic acid have been shown to stimulate RORβ activity [5,6,7,8,9,10]. Simvastatin, a cholesterol synthesis inhibitor, has been shown to decrease viability of macrophages and this process can be reversed by SR1001, a synthetic ligand of RORα [11]
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