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RORα Regulates Cholesterol Metabolism of CD8+ T Cells for Anticancer Immunity - PubMed

  • ️Wed Jan 01 2020

RORα Regulates Cholesterol Metabolism of CD8+ T Cells for Anticancer Immunity

In Kyu Lee et al. Cancers (Basel). 2020.

Abstract

Retinoic acid-related orphan receptor α (RORα) functions as a transcription factor for various biological processes, including circadian rhythm, inflammation, cancer, and lipid metabolism. Here, we demonstrate that RORα is crucial for maintaining cholesterol homeostasis in CD8+ T cells by attenuating NF-kB transcriptional activity. Cholesterol sulfate, the established natural agonist of RORα, exhibits cellular cytotoxicity on, and increased effector responses in, CD8+ T cells. Transcript analysis reveals that the suppression of RORα leads to the upregulation of NF-kB target genes in T cells. Chromatin immunoprecipitation analysis was used to determine the corecruitment of RORα and histone deacetylase (HDAC) on NF-kB target promoters and the subsequent dismissal of coactivators for transcriptional repression. We demonstrate that RORα/HDAC-mediated attenuation of NF-kB signaling controls the balance of cholesterol metabolism in CD8+ T cells, and that therapeutic strategies targeting this epigenetic regulation could be beneficial to the treatment of solid tumors including colon cancers.

Keywords: CD8+ T cell; Cholesterol; NF-kB; RORα.

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Conflict of interest statement

The authors declare that they have no competing financial interests.

Figures

Figure 1
Figure 1

Increased RORα activity is crucial for maintaining cholesterol levels in T cells. (A) Transcriptional levels of RORα, RORγ, LXRα, LXRβ, and G6P in stimulated Jurkat cells by activation times (+: 24 hours). Protein levels of RORα were detected by immunoblot analysis in stimulated Jurkat cells. The p-value was calculated by a t-test (*** p < 0.001). (B) Transcriptional levels of Abcg1 and Acat1 mRNAs in Jurkat cells treated with CS. The p-value was calculated by a t-test (** p < 0.01, *** p < 0.001). (C) Transcriptional levels of LXRα, LXRβ, ChREBP, and G6P in Jurkat cells treated with SR1078 or SR3335. The p-value was calculated by a t-test (** p < 0.01, *** p < 0.001). (D) Transcriptional levels of Abca1, Acat1, and Acat2 in Jurkat cells treated with SR1078 or SR3335. The p-value was calculated by a t-test (*** p < 0.001). (E) Transcriptional levels of cholesterol synthesis genes DHCR24, HMGCR, and SREBP2 in Jurkat cells treated with CS. The p-value was calculated by a t-test (* p < 0.05, *** p < 0.001).

Figure 2
Figure 2

RORα potentiates T cell cholesterol synthesis via NF-κB inhibition. (A) NF-κB-luciferase reporter-expressed Jurkat cells were treated with SR1078 or SR3335 at doses of 1, 5, 10, and 20 μM for 1 day. Luciferase was reacted by the Luciferase Assay System and measured using a microplate reader. The p-value was calculated by a t-test (* p < 0.05, ** p < 0.01, *** p < 0.001). (B) Co-immunoprecipitation assays between RORα and p50, NF-κB component (left). The binding affinity of RORα WT or C90A with p50 was assessed in 293T cells (middle). The binding between RORα and p50 was assessed with SR3335 treatment in 293T cells (right) (C) The introduction of RORαC90A decreased the transcriptional activation of the 5XRORE luciferase reporter (left). Compared to RORE, the introduction of RORα WT, C90A, and ΔAF2 mutants had the same effect of transrepression on the 3XNFκB luciferase reporter (middle and right). The p-value was calculated by a t-test (** p < 0.01, *** p < 0.001). (D) Effects of RORα knockdown on 3XNFκB luciferase reporter in 293T cells treated with LPS (shRORα, +: 0.5μg/μL, ++: 2μg/μL). (E) The illustration represents the location of NF-κB elements on Acat1 and Abca1 promoter. Proposed model of RORα recruitment with HDAC3 and binding on the NF-κB target promoters (left). ChIP assays on Acat1 or Abca1 promoters in WT, ROR KO (sg), or p50 KO MEFs (right). The p-value was calculated by a t-test (*** p < 0.001).

Figure 3
Figure 3

RORα potentiates CD8+ T cells through cholesterol esterification. (A) Dot plot analysis for the proliferation of CD8+ T cells treated with CS at doses of 5, 10, and 20 μM, or SR3335 at doses of 5, 10, and 20 μM. (B) Quantification of dot plat analysis in Figure 3A. The p-value was calculated by a t-test (*** p < 0.001). (C,E) IFN-γ production of CD8+ cells treated with 0, 10, and 20 μM of SR3335 (C) or 0 and 10 μM of CS (E). (D,F) Quantification of IFN-γ production of CD8+ cells treated with SR3335 (D) in Figure 3C or CS (F) in Figure 3E. The p-value was calculated by a t-test (* p < 0.05, *** p < 0.001). (G) Transcriptional level of Acat1 in CD8+ cells treated with CS. The p-value was calculated by a t-test (*** p < 0.001). (H–J) Jurkat cells were transfected with either siRNA for control (siCTL) or siRNA of RORα, and the cells were activated at the indicated times. Transcriptional levels of Acat1 (H), RORα (I), and Acat2/Abcg1 (J) were measured. The p-value was calculated by a t-test (*** p < 0.001).

Figure 4
Figure 4

Enhanced cholesterol level by RORα may potentiate the antitumor activity of CD8+ T cells. (A,B) Cholesterol quantification of CD8+ T cells (A) or Jurkat (B) cells by Filipin III staining. CD8+ T cells or Jurkat cells were activated in the presence of CS 20 μM. The p-value was calculated by a t-test (*** p < 0.001). (C,D, and E) Quantification of cell death in colon cancer. HCT116 cells were cocultured with CD8+ T cells pretreated with SR3335 for three days. The p-value was calculated by a t-test (* p < 0.05, ** p < 0.01, *** p < 0.001). (F–H) Quantification of cell death in cancer. HCT116 cells were cocultured with CD8+ T cells pretreated with CS for three days. The p-value was calculated by a t-test (* p < 0.05, ** p < 0.01, *** p < 0.001).

Figure 5
Figure 5

Schematic models show RORα agonist effect as inducing cholesterol metabolism in cytotoxic T cells. The models illustrate the role of RORα in CD8+ cytotoxic T cells. RORα agonist attenuates NF-κB signaling via HDAC recruitment on the Acat1/2 and Abca1 promoters for changing the status of T cells from dormant (left) to cytotoxic (right). These increased levels of cholesterol in the cell membrane of cytotoxic T cells induce a clustering of TCRs that interact with the antigens presented on the membrane of the cancer cells. Increased levels of cholesterol in cytotoxic T cells also trigger phosphorylation (P) of the linker of activated T cells (LAT), which, with TCRs, functions to stimulate the formation of immunological synapses.

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References

    1. Jetten A.M., Joo J.H. Retinoid-Related Orphan Receptors (Rors): Roles in Cellular Differentiation and Development. Adv. Dev. Biol. 2006;16:313–355. doi: 10.1016/s1574-3349(06)16010-x. - DOI - PMC - PubMed
    1. Hamilton B.A., Frankel W.N., Kerrebrock A.W., Hawkins T.L., FitzHugh W., Kusumi K., Russell L.B., Mueller K.L., van Berkel V., Birren B.W., et al. Disruption of the Nuclear Hormone Receptor Roralpha in Staggerer Mice. Nature. 1996;379:736–739. doi: 10.1038/379736a0. - DOI - PubMed
    1. Oh S.K., Kim D., Kim K., Boo K., Yu Y.S., Kim I.S., Jeon Y., Im S.K., Lee S.H., Lee J.M., et al. Roralpha Is Crucial for Attenuated Inflammatory Response to Maintain Intestinal Homeostasis. Proc. Natl. Acad. Sci. USA. 2019;116:21140–21149. doi: 10.1073/pnas.1907595116. - DOI - PMC - PubMed
    1. Kim K., Lee J.M., Yu Y.S., Kim H., Nam H.J., Moon H.G., Noh D.Y., Kim K.I., Fang S., Baek S.H. Roralpha2 Requires Lsd1 to Enhance Tumor Progression in Breast Cancer. Sci. Rep. 2017;7:11994. doi: 10.1038/s41598-017-12344-0. - DOI - PMC - PubMed
    1. Shin D., Kim I.S., Lee J.M., Shin S.Y., Lee J.H., Baek S.H., Cho K.H. The Hidden Switches Underlying Roralpha-Mediated Circuits That Critically Regulate Uncontrolled Cell Proliferation. J. Mol. Cell. Biol. 2014;6:338–348. doi: 10.1093/jmcb/mju023. - DOI - PubMed