SRC-3 Functions As a Coactivator of T-Bet by Regulating The

SRC-3 Functions As a Coactivator of T-Bet by Regulating The

Author Manuscript Published OnlineFirst on June 19, 2020; DOI: 10.1158/2326-6066.CIR-20-0181 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. SRC-3 functions as a coactivator of T-bet by regulating the maturation and antitumor activity of natural killer cells Mengjia Hu1, Yukai Lu1, Yan Qi1, Zihao Zhang1, Song Wang1, Yang Xu1, Fang Chen1, Yong Tang1, Shilei Chen1, Mo Chen1, Changhong Du1, Mingqiang Shen1, Fengchao Wang1, Yongping Su1, Youcai Deng2, Junping Wang1. 1State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, Chongqing Engineering Research Center for Nanomedicine, College of Preventive Medicine, Third Military Medical University, Chongqing 400038, China. 2Institute of Materia Medica, College of Pharmacy, Third Military Medical University, Chongqing 400038, China. Running title: SRC-3 regulates NK cell maturation and antitumor activity Key Words: natural killer cell, SRC-3, T-bet, coactivator, tumor surveillance Correspondence author: Dr. & Prof. Junping Wang, State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, College of Preventive Medicine, Third Military Medical University, Gaotanyan Street 30 Chongqing 400038, China. Tel: +86-023-68771515; Fax: +86-023-68752009; Email: [email protected] Conflict of interest: The authors declare no potential conflicts of interest. Financial support: This work was supported by grants from the National Natural Science Foundation of China (No. 81725019, 81930090, 81573084, 81500087), and the Scientific Research Project of PLA (AWS16J014). 1 Downloaded from cancerimmunolres.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 19, 2020; DOI: 10.1158/2326-6066.CIR-20-0181 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract word count: 191 Main text word count: 3198 Number of figures: 6 Number of supplementary files: 2 2 Downloaded from cancerimmunolres.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 19, 2020; DOI: 10.1158/2326-6066.CIR-20-0181 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Natural killer (NK) cell development and maturation is a well-organized process. The steroid receptor coactivator 3 (SRC-3) is a regulator of the hematopoietic and immune systems; however, its role in NK cells is poorly understood. Here, SRC-3 displayed increased nuclear translocation in NK cells during terminal differentiation and upon inflammatory cytokine stimulation. Targeted deletion of SRC-3 altered normal NK cell distribution and compromised NK cell maturation. SRC-3 deficiency led to significantly impaired NK cell functions, especially their antitumor activity. The expression of several critical T-bet target genes, including Zeb2, Prdm1 and S1pr5, but not T-bet itself, was markedly decreased in NK cells in the absence of SRC-3. There was a physiological interaction between SRC-3 and T-bet proteins, where SRC- 3 was recruited by T-bet to regulate the transcription of the aforementioned genes. Collectively, our findings unmask a previously unrecognized role of SRC-3 as a coactivator of T-bet in NK cell biology and indicate that targeting SRC-3 may be a promising strategy to increase the tumor surveillance function of NK cells. 3 Downloaded from cancerimmunolres.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 19, 2020; DOI: 10.1158/2326-6066.CIR-20-0181 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Natural killer (NK) cells, innate lymphocytes (ILCs) with cytotoxic functions, are indispensable for early immunosurveillance of tumors and elimination of infected cells (1,2). NK cells play an important role in the regulation of the adaptive immune response by secreting cytokines (3). Unraveling the antitumor properties of NK cells is crucial (4,5), thus a deep understanding of the mechanisms underlying NK cell differentiation, maturation and function may advance the development of NK cell- based immunotherapy. NK cells are derived from hematopoietic stem cells (HSCs) (6). After development in the bone marrow (BM), NK cells traffic to peripheral organs (7). The commitment of NK cell specific differentiation is characterized by the gradual acquisition of CD122, NK1.1 and DX5 expression (8). As NK cells fully mature, CD27 expression gradually decreases and CD11b expression gradually increases (9). NK cell development, maturation and function are controlled by a series of intrinsic and extrinsic factors, but these mechanisms are not fully understood (10). T-box transcription factor T-bet plays a critical role in this transcriptional regulatory network (11,12) with many factors, such as Ets1, Foxo1, Tox2, Gata3 and mTOR, modulating NK cell biology by at least partially affecting T-bet (13-17). However, little is known about how T-bet regulates downstream transcription factors or effector molecules and whether it needs interacting partners in NK cells. Steroid receptor coactivator 3 (SRC-3), also called NCOA3/ACTR/AIB1/pCIP, is a coactivator of nuclear receptors (18). Similar to other p160 family members, SRC-3 can be recruited by nuclear receptors to modulate the transcription of target genes (19). SRC-3 can regulate the activity of several nonnuclear receptor proteins, including p53, AP-1, and E2F1, indicating a nuclear receptor-independent function (18). SRC-3 has 4 Downloaded from cancerimmunolres.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 19, 2020; DOI: 10.1158/2326-6066.CIR-20-0181 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. attracted considerable attention due to its oncogenic property, whereas its physiological function is largely overlooked (20). SRC-3 is involved in many important physiological processes, such as somatic growth, hematopoiesis, immunoregulation and energy metabolism (21-23). In this study, we evaluated the role of SRC-3 in NK cells. Based on the observations that SRC-3 displayed increased nuclear translocation during NK cell terminal differentiation and upon inflammatory cytokine stimulation, we generated mice with hematopoietic and NK-specific SRC-3 deletion. SRC-3 was required to maintain the maturation and effector function of NK cells via modulation of several critical T-bet-dependent genes. Collectively, our study provides new insight into the regulatory mechanism of NK cell maturation and antitumor activity via SRC-3. Materials and methods Animals. Normal wild-type (WT) C57BL/6J mice were purchased from the Institute of Zoology (Chinese Academy of Sciences, Beijing, China). SRC-3flox/+ (SRC-3fl/+) mice were generated at the Shanghai Model Organisms Center (Shanghai, China). Ncr1-Cre mice were obtained from Beijing Biocytogen Co, Ltd (Beijing, China) (24). Vav1-Cre and T-bet-/- mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). SRC-3fl/fl/Ncr1-Cre (or Vav1-Cre) mice were generated by crossing SRC- 3fl/fl mice with Ncr1-Cre or Vav1-Cre mice. All mice used in the experiment are 6-8 weeks old. All animal experiments were approved by the Animal Care Committee of The Third Military Medical University. Cell culture. NK-92, Yac-1 and B16F10 cells were purchased from BeNa Culture Collection (Beijing, China, December 2017). NK-92 cells were grown in complete medium containing 75% DMEM Alpha (GIBCO, Carlsbad, CA, USA; supplemented 5 Downloaded from cancerimmunolres.aacrjournals.org on September 24, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on June 19, 2020; DOI: 10.1158/2326-6066.CIR-20-0181 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. with 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid and 100-200 U/ml recombinant IL-2), 12.5% horse serum (GIBCO) and 12.5% fetal bovine serum (FBS; GIBCO). Yac-1 cells and B16F10 cells were cultured in 90% RPMI (HyClone, Logan, UT, USA) containing 10% FBS. All cell lines were passaged up to 10 times, authenticated by flow cytometry (last authentication, November 2019) and tested for mycoplasma contamination using a MycoFluor™ Mycoplasma Detection Kit (Invitrogen, Carlsbad, CA, USA; last test, November 2019). Flow cytometry. Single-cell suspensions from the BM, spleen, peripheral lymph nodes (pLNs), lungs and liver of mice were obtained as previously described (14,22). The following flow cytometric antibodies were used: anti-CD3e (145-2C11, #100353, 100321, dilution 1:100), anti-Gr-1 (RB6-8C5, #108417, dilution 1:100), anti-Ter119 (TER-119, #116215, dilution 1:100), anti-B220 (RA3-6B2, #103225, dilution 1:100), anti-CD19 (6D5, #115546, 115521, dilution 1:100), anti-NK1.1 (PK136, #108718, 108732, dilution 1:100), anti-Nkp46 (29A1.4, #137608, dilution 1:100), anti-CD146 (ME-9F1, #134704, dilution 1:100), and anti-CD107a (1D4B, #1216124, dilution 1:50) from Biolegend (San Diego, CA, USA); anti-c-Kit (2B8, #14-1171-82, dilution 1:100), anti-CD244 (eBio244F4, #25-2441-82, dilution 1:100), anti-CD127 (A7R34, #45-1271-82, dilution 1:100), anti-CD135 (A2F10, #17-1351-82, dilution 1:50), anti- CD122 (TM-beta1, #48-1222-82, dilution 1:100), anti-CD27 (LG.7F9, #12-0271-82, dilution 1:100), anti-CD11b (M1/70, #11-0112-82, 45-0112-82, dilution 1:200), anti- KLRG1 (2F1, #12-5893-8, dilution 1:100), anti-Granzyme B (NGZB, #12-8898-82, dilution 1:50), anti-Perforin (eBioOMAK-D, #12-9392-82, dilution 1:50), anti-T-bet (4B10, #12-5825-82, dilution 1:50), anti-Ki67 (SolA15, #25-5698-82, dilution 1:200), anti-p-STAT3Tyr705 (LUVNKLA, #12-9033-42, dilution 1:50), anti-p-STAT5Tyr694 (SRBCZX, #12-9010-42, 1:50), and anti-p-STAT4Tyr693 (4LURPIE, #17-9044-42, 6 Downloaded from cancerimmunolres.aacrjournals.org on September 24, 2021.

View Full Text

Details

  • File Type
    pdf
  • Upload Time
    -
  • Content Languages
    English
  • Upload User
    Anonymous/Not logged-in
  • File Pages
    37 Page
  • File Size
    -

Download

Channel Download Status
Express Download Enable

Copyright

We respect the copyrights and intellectual property rights of all users. All uploaded documents are either original works of the uploader or authorized works of the rightful owners.

  • Not to be reproduced or distributed without explicit permission.
  • Not used for commercial purposes outside of approved use cases.
  • Not used to infringe on the rights of the original creators.
  • If you believe any content infringes your copyright, please contact us immediately.

Support

For help with questions, suggestions, or problems, please contact us