Skip to main content

Differential function and regulation of orphan nuclear receptor TR3 isoforms in endothelial cells

Tumor Biology volume 37, pages 3307–3320 (2016)Cite this article

Abstract

TR3 has been reported to be an excellent target for angiogenesis therapies. We reported three TR3 transcript variant messenger RNAs (mRNAs) are expressed in human umbilical vein endothelial cell (HUVEC) and are differentially regulated by vascular endothelial growth factor (VEGF). TR3 transcript variant 1 (TR3-TV1) and variant 2 (TR3-TV2) encoding the same TR3 isoform 1 protein (TR3-iso1) that was named TR3 has been extensively studied. However, the function of TR3 isoform 2 protein (TR3-iso2) encoded by TR3 transcript variant 3 (TR3-TV3) is still not known. Here, we clone and express the novel TR3-iso2 protein and find that expression of TR3-iso2, in contrast to TR3-iso1, inhibits endothelial cell proliferation induced by VEGF-A, histamine, and phorbol-12-myristate-13-acetate (PMA). The differential function of TR3-iso2 correlates with the down-regulation of cyclin D1. However, TR3-iso2 plays similar roles in endothelial cell migration and monolayer permeability as TR3-iso1. We further demonstrate that several intracellular signaling pathways are involved in histamine-induced TR3 transcript variants, including histamine receptor H1-mediated phospholipase C (PLC)/calcium /calcineurin/protein kinase C (PKC)/protein kinase D (PKD) pathway and ERK pathway, as well as histamine receptor H3-mediated PKC-ERK pathway. Further, expressions of TR3-TV1, TR3-TV2, and TR3-TV3 by VEGF and histamine are regulated by different promoters, but not by their mRNA stability.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Abbreviations

VEGF:

Vascular endothelial growth factor

HUVEC:

Human umbilical vein endothelial cells

TR3-TV1:

TR3 transcript variant 1 mRNA

TR3-TV2:

TR3 transcript variant 2 mRNA

TR3-TV3:

TR3 transcript variant 3 mRNA

TR3-iso1:

TR3 isoform 1 protein

TR3-iso2:

TR3 isoform 2 protein

IGF-1R:

Insulin-like growth factor-1 receptor

VEGFR:

VEGF receptor

shRNA :

Short hairpin RNA

References

  1. Shibuya M. VEGF-VEGFR signals in health and disease. Biomol Ther (Seoul). 2014;22:1–9.

    CAS  Article  Google Scholar 

  2. Hayman SR, Leung N, Grande JP, Garovic VD. VEGF inhibition, hypertension, and renal toxicity. Curr Oncol Rep. 2012;14:285–94.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. Bautch VL. Cancer: tumour stem cells switch sides. Nature. 2010;468:770–1.

    CAS  Article  PubMed  Google Scholar 

  4. Ebos JM, Kerbel RS. Antiangiogenic therapy: impact on invasion, disease progression, and metastasis. Nat Rev Clin Oncol. 2011;8:210–21.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Ebos JM, Lee CR, Christensen JG, Mutsaers AJ, Kerbel RS. Multiple circulating proangiogenic factors induced by sunitinib malate are tumor-independent and correlate with antitumor efficacy. Proc Natl Acad Sci U S A. 2007;104:17069–74.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Kienast Y, von Baumgarten L, Fuhrmann M, Klinkert WE, Goldbrunner R, Herms J, et al. Real-time imaging reveals the single steps of brain metastasis formation. Nat Med. 2010;16:116–22.

    CAS  Article  PubMed  Google Scholar 

  7. Li JL, Sainson RC, Oon CE, Turley H, Leek R, Sheldon H, et al. DLL4-Notch signaling mediates tumor resistance to anti-VEGF therapy in vivo. Cancer Res. 2011;71:6073–83.

    CAS  Article  PubMed  Google Scholar 

  8. Loges S, Schmidt T, Carmeliet P. Mechanisms of resistance to anti-angiogenic therapy and development of third-generation anti-angiogenic drug candidates. Genes Cancer. 2010;1:12–25.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. Ricci-Vitiani L, Pallini R, Biffoni M, Todaro M, Invernici G, Cenci T, et al. Tumour vascularization via endothelial differentiation of glioblastoma stem-like cells. Nature. 2010;468:824–8.

    CAS  Article  PubMed  Google Scholar 

  10. Shojaei F. Anti-angiogenesis therapy in cancer: current challenges and future perspectives. Cancer Lett. 2012;320:130–7.

    CAS  Article  PubMed  Google Scholar 

  11. Shojaei F, Wu X, Qu X, Kowanetz M, Yu L, Tan M, et al. G-CSF-initiated myeloid cell mobilization and angiogenesis mediate tumor refractoriness to anti-VEGF therapy in mouse models. Proc Natl Acad Sci U S A. 2009;106:6742–7.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. Wang R, Chadalavada K, Wilshire J, Kowalik U, Hovinga KE, Geber A, et al. Glioblastoma stem-like cells give rise to tumour endothelium. Nature. 2010;468:829–33.

    CAS  Article  PubMed  Google Scholar 

  13. Helfrich I, Scheffrahn I, Bartling S, Weis J, von Felbert V, Middleton M, et al. Resistance to antiangiogenic therapy is directed by vascular phenotype, vessel stabilization, and maturation in malignant melanoma. J Exp Med. 2010;207:491–503.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  14. Shojaei F, Wu X, Malik AK, Zhong C, Baldwin ME, Schanz S, et al. Tumor refractoriness to anti-VEGF treatment is mediated by CD11b+Gr1+ myeloid cells. Nat Biotechnol. 2007;25:911–20.

    CAS  Article  PubMed  Google Scholar 

  15. Qin L, Zhao D, Xu J, Ren X, Terwilliger EF, Parangi S, et al. The vascular permeabilizing factors histamine and serotonin induce angiogenesis through TR3/Nur77 and subsequently truncate it through thrombospondin-1. Blood. 2013;121:2154–64.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  16. Zeng H, Qin L, Zhao D, Tan X, Manseau EJ, Van Hoang M, et al. Orphan nuclear receptor TR3/Nur77 regulates VEGF-A-induced angiogenesis through its transcriptional activity. J Exp Med. 2006;203:719–29.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  17. Zhao D, Qin L, Bourbon PM, James L, Dvorak HF, Zeng H. Orphan nuclear transcription factor TR3/Nur77 regulates microvessel permeability by targeting endothelial nitric oxide synthase and destabilizing endothelial junctions. Proc Natl Acad Sci U S A. 2011;108:12066–71.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Flaig R, Greschik H, Peluso-Iltis C, Moras D. Structural basis for the cell-specific activities of the NGFI-B and the Nurr1 ligand-binding domain. J Biol Chem. 2005;280:19250–8.

    CAS  Article  PubMed  Google Scholar 

  19. Cho SD, Lee SO, Chintharlapalli S, Abdelrahim M, Khan S, Yoon K, et al. Activation of nerve growth factor-induced B alpha by methylene-substituted diindolylmethanes in bladder cancer cells induces apoptosis and inhibits tumor growth. Mol Pharmacol. 2010;77:396–404.

    CAS  Article  PubMed  Google Scholar 

  20. Zhan Y, Du X, Chen H, Liu J, Zhao B, Huang D, et al. Cytosporone B is an agonist for nuclear orphan receptor Nur77. Nat Chem Biol. 2008;4:548–56.

    CAS  Article  PubMed  Google Scholar 

  21. Lee SL, Wesselschmidt RL, Linette GP, Kanagawa O, Russell JH, Milbrandt J. Unimpaired thymic and peripheral T cell death in mice lacking the nuclear receptor NGFI-B (Nur77). Science. 1995;269:532–5.

    CAS  Article  PubMed  Google Scholar 

  22. Maxwell MA, Cleasby ME, Harding A, Stark A, Cooney GJ, Muscat GE. Nur77 regulates lipolysis in skeletal muscle cells. Evidence for cross-talk between the beta-adrenergic and an orphan nuclear hormone receptor pathway. J Biol Chem. 2005;280:12573–84.

    CAS  Article  PubMed  Google Scholar 

  23. Kanzleiter T, Schneider T, Walter I, Bolze F, Eickhorst C, Heldmaier G, et al. Evidence for Nr4a1 as a cold-induced effector of brown fat thermogenesis. Physiol Genomics. 2005;24:37–44.

    CAS  Article  PubMed  Google Scholar 

  24. Hsu HC, Zhou T, Mountz JD. Nur77 family of nuclear hormone receptors. Curr Drug Targets Inflamm Allergy. 2004;3:413–23.

    CAS  Article  PubMed  Google Scholar 

  25. Campos-Melo D, Galleguillos D, Sanchez N, Gysling K, Andres ME. Nur transcription factors in stress and addiction. Front Mol Neurosci. 2013;6:44.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. McMorrow JP, Murphy EP. Inflammation: a role for NR4A orphan nuclear receptors? Biochem Soc Trans. 2011;39:688–93.

    CAS  Article  PubMed  Google Scholar 

  27. Mohan HM, Aherne CM, Rogers AC, Baird AW, Winter DC, Murphy EP. Molecular pathways: the role of NR4A orphan nuclear receptors in cancer. Clin Cancer Res. 2012;18:3223–8.

    CAS  Article  PubMed  Google Scholar 

  28. Pearen MA, Muscat GE. Minireview: nuclear hormone receptor 4A signaling: implications for metabolic disease. Mol Endocrinol. 2010;24:1891–903.

    CAS  Article  PubMed  Google Scholar 

  29. Niu G, Ye T, Qin L, Bourbon PM, Chang C, Zhao S, et al. Orphan nuclear receptor TR3/Nur77 improves wound healing by upregulating the expression of integrin beta4. Faseb J. 2014.

  30. Zhao S, Zhou L, Niu G, Li Y, Zhao D, Zeng H. Differential regulation of orphan nuclear receptor TR3 transcript variants by novel vascular growth factor signaling pathways. Faseb J. 2014;28:4524–33.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. Bohula EA, Salisbury AJ, Sohail M, Playford MP, Riedemann J, Southern EM, et al. The efficacy of small interfering RNAs targeted to the type 1 insulin-like growth factor receptor (IGF1R) is influenced by secondary structure in the IGF1R transcript. J Biol Chem. 2003;278:15991–7.

    CAS  Article  PubMed  Google Scholar 

  32. Hazel TG, Nathans D, Lau LF. A gene inducible by serum growth factors encodes a member of the steroid and thyroid hormone receptor superfamily. Proc Natl Acad Sci U S A. 1988;85:8444–8.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Ryseck RP, Macdonald-Bravo H, Mattei MG, Ruppert S, Bravo R. Structure, mapping and expression of a growth factor inducible gene encoding a putative nuclear hormonal binding receptor. Embo J. 1989;8:3327–35.

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Milbrandt J. Nerve growth factor induces a gene homologous to the glucocorticoid receptor gene. Neuron. 1988;1:183–8.

    CAS  Article  PubMed  Google Scholar 

  35. Yoon JK, Lau LF. Transcriptional activation of the inducible nuclear receptor gene nur77 by nerve growth factor and membrane depolarization in PC12 cells. J Biol Chem. 1993;268:9148–55.

    CAS  PubMed  Google Scholar 

  36. Kolluri SK, Bruey-Sedano N, Cao X, Lin B, Lin F, Han YH, et al. Mitogenic effect of orphan receptor TR3 and its regulation by MEKK1 in lung cancer cells. Mol Cell Biol. 2003;23:8651–67.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. Liu ZG, Smith SW, McLaughlin KA, Schwartz LM, Osborne BA. Apoptotic signals delivered through the T-cell receptor of a T-cell hybrid require the immediate-early gene nur77. Nature. 1994;367:281–4.

    CAS  Article  PubMed  Google Scholar 

  38. Woronicz JD, Calnan B, Ngo V, Winoto A. Requirement for the orphan steroid receptor Nur77 in apoptosis of T-cell hybridomas. Nature. 1994;367:277–81.

    CAS  Article  PubMed  Google Scholar 

  39. Wu H, Lin Y, Li W, Sun Z, Gao W, Zhang H, et al. Regulation of Nur77 expression by beta-catenin and its mitogenic effect in colon cancer cells. Faseb J. 2010;25:192–205.

    Article  PubMed  Google Scholar 

  40. Cheng LE, Chan FK, Cado D, Winoto A. Functional redundancy of the Nur77 and Nor-1 orphan steroid receptors in T-cell apoptosis. Embo J. 1997;16:1865–75.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. Azmitia EC. Modern views on an ancient chemical: serotonin effects on cell proliferation, maturation, and apoptosis. Brain Res Bull. 2001;56:413–24.

    CAS  Article  PubMed  Google Scholar 

  42. Beaven M. Histamine Its role in physiological and pathological process. 1978.

  43. Faerber L, Drechsler S, Ladenburger S, Gschaidmeier H, Fischer W. The neuronal 5-HT3 receptor network after 20 years of research—evolving concepts in management of pain and inflammation. Eur J Pharmacol. 2007;560:1–8.

    CAS  Article  PubMed  Google Scholar 

  44. Haas HL, Sergeeva OA, Selbach O. Histamine in the nervous system. Physiol Rev. 2008;88:1183–241.

    CAS  Article  PubMed  Google Scholar 

  45. Thurmond RL, Gelfand EW, Dunford PJ. The role of histamine H1 and H4 receptors in allergic inflammation: the search for new antihistamines. Nat Rev Drug Discov. 2008;7:41–53.

    CAS  Article  PubMed  Google Scholar 

  46. Nguyen T, Shapiro DA, George SR, Setola V, Lee DK, Cheng R, et al. Discovery of a novel member of the histamine receptor family. Mol Pharmacol. 2001;59:427–33.

    CAS  PubMed  Google Scholar 

  47. Repka-Ramirez MS. New concepts of histamine receptors and actions. Curr Allergy Asthma Rep. 2003;3:227–31.

    Article  PubMed  Google Scholar 

  48. Ghosh AK, Hirasawa N, Ohtsu H, Watanabe T, Ohuchi K. Defective angiogenesis in the inflammatory granulation tissue in histidine decarboxylase-deficient mice but not in mast cell-deficient mice. J Exp Med. 2002;195:973–82.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  49. Dang L, Seale JP, Qu X. High glucose-induced human umbilical vein endothelial cell hyperpermeability is dependent on protein kinase C activation and independent of the Ca2+-nitric oxide signalling pathway. Clin Exp Pharmacol Physiol. 2005;32:771–6.

    CAS  Article  PubMed  Google Scholar 

  50. Yuan SY. Signal transduction pathways in enhanced microvascular permeability. Microcirculation. 2000;7:395–403.

    CAS  Article  PubMed  Google Scholar 

  51. Limbourg A, Limbourg F, Drexler H. Cell-based therapies for ischemic heart disease—“trick and treat”. Circ J. 2009;73:2179–82.

    Article  PubMed  Google Scholar 

  52. Hao F, Wu DD, Xu X, Cui MZ. Histamine induces activation of protein kinase D that mediates tissue factor expression and activity in human aortic smooth muscle cells. Am J Physiol Heart Circ Physiol. 2012;303:H1344–52.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  53. Hao F, Tan M, Xu X, Cui MZ. Histamine induces Egr-1 expression in human aortic endothelial cells via the H1 receptor-mediated protein kinase Cdelta-dependent ERK activation pathway. J Biol Chem. 2008;283:26928–36.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. Matsubara M, Tamura T, Ohmori K, Hasegawa K. Histamine H1 receptor antagonist blocks histamine-induced proinflammatory cytokine production through inhibition of Ca2+-dependent protein kinase C, Raf/MEK/ERK and IKK/I kappa B/NF-kappa B signal cascades. Biochem Pharmacol. 2005;69:433–49.

    CAS  Article  PubMed  Google Scholar 

  55. Francis H, Onori P, Gaudio E, Franchitto A, DeMorrow S, Venter J, et al. H3 histamine receptor-mediated activation of protein kinase Calpha inhibits the growth of cholangiocarcinoma in vitro and in vivo. Mol Cancer Res. 2009;7:1704–13.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  56. Dequiedt F, Kasler H, Fischle W, Kiermer V, Weinstein M, Herndier BG, et al. HDAC7, a thymus-specific class II histone deacetylase, regulates Nur77 transcription and TCR-mediated apoptosis. Immunity. 2003;18:687–98.

    CAS  Article  PubMed  Google Scholar 

  57. Youn HD, Liu JO. Cabin1 represses MEF2-dependent Nur77 expression and T cell apoptosis by controlling association of histone deacetylases and acetylases with MEF2. Immunity. 2000;13:85–94.

    CAS  Article  PubMed  Google Scholar 

  58. Chen HZ, Zhao BX, Zhao WX, Li L, Zhang B, Wu Q. Akt phosphorylates the TR3 orphan receptor and blocks its targeting to the mitochondria. Carcinogenesis. 2008;29:2078–88.

    CAS  Article  PubMed  Google Scholar 

  59. Chen HZ, Liu QF, Li L, Wang WJ, Yao LM, Yang M, et al. The orphan receptor TR3 suppresses intestinal tumorigenesis in mice by downregulating Wnt signalling. Gut. 2011;61:714–24.

    Article  CAS  PubMed  Google Scholar 

  60. Wang A, Rud J, Olson Jr CM, Anguita J, Osborne BA. Phosphorylation of Nur77 by the MEK-ERK-RSK cascade induces mitochondrial translocation and apoptosis in T cells. J Immunol. 2009;183:3268–77.

    CAS  Article  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Institutes of Health [grant numbers R01CA133235 to H. Z., R01DK095873 and R21DK080970 to D. Z.] by the American Cancer Society [grant numbers RSG CSM 113297 to D.Z.], by the China Scholarship Council [Scholarships to Y.L., J.P., L.Z., S.Z., G.N., P.C.], and by Renji Hospital, P.R. China [Scholarships to T.Y.].

Author information

Affiliations

  1. Center for Vascular Biology Research and Division of Molecular and Vascular Medicine, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, 02215, USA

    Lei Zhou, Pengfei Cui, Shengqiang Zhao, Taiyang Ye, Yan Li, Jin Peng, Gengming Niu, Dezheng Zhao & Huiyan Zeng

  2. Department of Hepatobiliary Surgery, The First Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, China

    Lei Zhou

  3. Department of General Surgery, The First Affiliated Hospital, School of Medicine, Xi’an Jiaotong University, Xi’an, China

    Lei Zhou

  4. Pancreatic Disease Institute, Department of General Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, China

    Pengfei Cui

  5. Department of Gastroenterology, Shandong Provincial Hospital Affiliated to Shandong University, Ji-nan, China

    Shengqiang Zhao & Yan Li

  6. Department of Obstetrics & Gynecology, Renji Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai, China

    Taiyang Ye

  7. Department of Medical Oncology & Radiation Oncology, Zhongnan Hospital of Wuhan University, Wuhan, China

    Jin Peng

  8. Department of General Surgery, Huashan Hospital, Fudan University, Shanghai, China

    Gengming Niu

  9. Division of Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, 02215, USA

    Dezheng Zhao

  10. Division of Molecular and Vascular Medicine, Department of Medicine, Beth Israel Deaconess Medical Center, and Harvard Medical School, 99 Brookline Ave. RN 270F, Boston, MA, 02215, USA

    Huiyan Zeng

Authors
  1. Lei Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Pengfei Cui
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shengqiang Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Taiyang Ye
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yan Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jin Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Gengming Niu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Dezheng Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Huiyan Zeng
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Huiyan Zeng.

Ethics declarations

Conflicts of interest

None

Informed consent

Consent was obtained from all individual participants included in the study.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhou, L., Cui, P., Zhao, S. et al. Differential function and regulation of orphan nuclear receptor TR3 isoforms in endothelial cells. Tumor Biol. 37, 3307–3320 (2016). https://doi.org/10.1007/s13277-015-4157-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13277-015-4157-9

Keywords

  • Angiogenesis
  • Cell signaling
  • Cell proliferation
  • Migration
  • TR3/Nur77 transcription variants