Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

The JNK-EGR1 signaling axis promotes TNF-α-induced endothelial differentiation of human mesenchymal stem cells via VEGFR2 expression

Abstract

Mesenchymal stem cells (MSCs) can differentiate into endothelial cells; however, the mechanisms underlying this process in the tumor microenvironment (TME) remain elusive. This study shows that tumor necrosis factor alpha (TNF-α), a key cytokine present in the TME, promotes the endothelial differentiation of MSCs by inducing vascular endothelial growth factor receptor 2 (VEGFR2) gene expression. EGR1 is a member of the zinc-finger transcription factor family induced by TNF-α. Our findings indicate that EGR1 directly binds to the VEGFR2 promoter and transactivates VEGFR2 expression. We also demonstrate that EGR1 forms a complex with c-JUN activated by c-JUN N-terminal kinase (JNK) to promote VEGFR2 transcription and endothelial differentiation in MSCs in response to TNF-α stimulation. The shRNA-mediated silencing of EGR1 or c-JUN abrogates TNF-α-induced VEGFR2 transcription and the endothelial differentiation of MSCs. To further evaluated the role of EGR1 in the endothelial differentiation of BM-MSCs, we used a syngenic tumor implantation model. 4T1 mouse mammary tumor cells were injected subcutaneously into BALB/c mice with primary mBM-MSCs isolated from wild-type (Egr1+/+) or Egr1-null (Egr1−/−) mice. CD31-positive cells were predominantly observed at the border of the tumor in the 4T1 plus wild-type MSC group, while staining less in the 4T1 alone or 4T1 plus Egr1-null MSC group. Collectively, these findings demonstrate that the JNK-EGR1 signaling axis plays a crucial role in the TNF-α-induced endothelial differentiation of MSCs in the TME, which could be a potential therapeutic target for solid tumors vasculatures.

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

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: Effects of TNF-α stimulation on the endothelial differentiation of BM-MSCs.
Fig. 2: VEGFR2 expression is required for the TNF-α-induced endothelial differentiation of BM-MSCs.
Fig. 3: Identification of the EBS in the TNF-α-inducible element of the VEGFR2 promoter.
Fig. 4: EGR1 regulates TNF-α-induced VEGFR2 expression and the endothelial differentiation of BM-MSCs.
Fig. 5: The JNK pathway regulates the TNF-α-induced endothelial differentiation of BM-MSCs through EGR1 induction.
Fig. 6: EGR1 and c-JUN synergistically regulate the endothelial differentiation of BM-MSCs.
Fig. 7: Interaction between EGR1 and c-JUN on the TNF-α-inducible promoter region of the VEGFR2.
Fig. 8: Egr1 deficiency limits the endothelial differentiation of BM-MSCs in vivo.

Data availability

The data sets generated during the current study are available from the corresponding author upon reasonable request. The microarray data have been deposited in NCBI Gene Expression Omnibus (Accession number: GSE214834).

References

  1. Forster JC, Harriss-phillips WM, Douglass MJ, Bezak E. A review of the development of tumor vasculature and its effects on the tumor microenvironment. Hypoxia. 2017;5:21–32.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Teleanu RI, Chircov C, Grumezescu AM, Teleanu DM. Tumor angiogenesis and anti-angiogenic strategies for cancer treatment. J Clin Med. 2020;9:84.

    Article  CAS  Google Scholar 

  3. Ruoslahti E. Specialization of tumour vasculature. Nat Rev Cancer. 2002;2:83–90.

    Article  PubMed  Google Scholar 

  4. Lugano R, Ramachandran M, Dimberg A. Tumor angiogenesis: causes, consequences, challenges and opportunities. Cell Mol Life Sci. 2020;77:1745–70.

    Article  CAS  PubMed  Google Scholar 

  5. Rohde E, Malischnik C, Thaler D, Maierhofer T, Linkesch W, Lanzer G, et al. Blood monocytes mimic endothelial progenitor cells. Stem Cells. 2006;24:357–67.

    Article  PubMed  Google Scholar 

  6. Coukos G, Benencia F, Buckanovich RJ, Conejo-Garcia JR. The role of dendritic cell precursors in tumour vasculogenesis. Br J Cancer. 2005;92:1182–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Pittenger MF, Discher DE, Péault BM, Phinney DG, Hare JM, Caplan AI. Mesenchymal stem cell perspective: cell biology to clinical progress. NPJ Regen Med. 2019;4:22.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Satija NK, Singh VK, Verma YK, Gupta P, Sharma S, Afrin F, et al. Mesenchymal stem cell-based therapy: a new paradigm in regenerative medicine. J Cell Mol Med. 2009;13:4385–402.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Torsvik A, Bjerkvig R. Mesenchymal stem cell signaling in cancer progression. Cancer Treat Rev. 2013;39:180–8.

    Article  CAS  PubMed  Google Scholar 

  10. Hsu HS, Lin JH, Hsu TW, Su K, Wang CW, Yang KY, et al. Mesenchymal stem cells enhance lung cancer initiation through activation of IL-6/JAK2/STAT3 pathway. Lung Cancer. 2012;75:167–77.

    Article  PubMed  Google Scholar 

  11. Tsai KS, Yang SH, Lei YP, Tsai CC, Chen HW, Hsu CY, et al. Mesenchymal stem cells promote formation of colorectal tumors in mice. Gastroenterology. 2011;141:1046–56.

    Article  CAS  PubMed  Google Scholar 

  12. Mishra PJ, Mishra PJ, Humeniuk R, Medina DJ, Alexe G, Mesirov JP, et al. Carcinoma-associated fibroblast-like differentiation of human mesenchymal stem cells. Cancer Res. 2008;68:4331–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Yang KQ, Liu Y, Huang QH, Mo N, Zhang QY, Meng QG, et al. Bone marrow-derived mesenchymal stem cells induced by inflammatory cytokines produce angiogenetic factors and promote prostate cancer growth. BMC Cancer. 2017;17:1–10.

    Article  Google Scholar 

  14. Yuan L, Sakamoto N, Song G, Sato M. High-level shear stress stimulates endothelial differentiation and VEGF secretion by human mesenchymal stem cells. Cell Mol Bioeng. 2013;6:220–9.

    Article  CAS  Google Scholar 

  15. Lozito TP, Kuo CK, Taboas JM, Tuan RS. Human mesenchymal stem cells express vascular cell phenotypes upon interaction with endothelial cell matrix. J Cell Biochem. 2009;107:714–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Oswald J, Boxberger S, Jørgensen B, Bornhaeuser M, Ehninger G, Werner C. Mesenchymal stem cells can be differentiated into endothelial cells in vitro. Stem Cells. 2004;22:377–84.

    Article  PubMed  Google Scholar 

  17. Batlle R, Andrés E, Gonzalez L, Llonch E, Igea A, Gutierrez-Prat N, et al. Regulation of tumor angiogenesis and mesenchymal–endothelial transition by p38α through TGF-β and JNK signaling. Nat Commun. 2019;10:1–18.

    Article  CAS  Google Scholar 

  18. Balkwill F. Tumour necrosis factor and cancer. Nat Rev Cancer. 2009;9:361–71.

    Article  CAS  PubMed  Google Scholar 

  19. Sethi G, Sung B, Aggarwal BB. TNF: a master switch for inflammation to cancer. Front Biosci. 2008;13:5094–107.

    Article  CAS  PubMed  Google Scholar 

  20. Kwon YW, Heo SC, Jeong GO, Yoon JW, Mo WM, Lee MJ, et al. Tumor necrosis factor-α-activated mesenchymal stem cells promote endothelial progenitor cell homing and angiogenesis. Biochim Biophys Acta Mol Basis Dis. 2013;1832:2136–44.

    Article  CAS  Google Scholar 

  21. Huang H, Zhao N, Xu X, Xu Y, Li S, Zhang J, et al. Dose-specific effects of tumor necrosis factor alpha on osteogenic differentiation of mesenchymal stem cells. Cell Prolif. 2011;44:420–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Tetzlaff F, Fischer A. Human endothelial cell spheroid-based sprouting angiogenesis assay in collagen. Bio Protoc. 2018;8:e2995.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Xie Z, Bailey A, Kuleshov MV, Clarke DJB, Evangelista JE, Jenkins SL, et al. Gene set knowledge discovery with enrichr. Curr Protoc. 2021;1:e90.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Mootha VK, Lindgren CM, Eriksson K-F, Subramanian A, Sihag S, Lehar J, et al. PGC-1α-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat Genet. 2003;34:267–73.

    Article  CAS  PubMed  Google Scholar 

  25. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA. 2005;102:15545–50.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Stauffer W, Sheng H, Lim HN. EzColocalization: an ImageJ plugin for visualizing and measuring colocalization in cells and organisms. Sci Rep. 2018;8:15764.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Herrmann C, Avgousti D, Weitzman M. Differential salt fractionation of nuclei to analyze chromatin-associated proteins from cultured mammalian cells. Bio Protoc. 2017;7:1–13.

    Article  CAS  Google Scholar 

  28. Yeo H, Ahn SS, Lee YH, Shin SY. Regulation of pro-opiomelanocortin (POMC) gene transcription by interleukin-31 via early growth response 1 (EGR-1) in HaCaT keratinocytes. Mol Biol Rep. 2020;47:5953–62.

    Article  CAS  PubMed  Google Scholar 

  29. Lee SL, Wang Y, Milbrandt J. Unimpaired macrophage differentiation and activation in mice lacking the zinc finger transcription factor NGFI-A (EGR1). Mol Cell Biol. 1996;16:4566–72.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Huang S, Xu L, Sun Y, Wu T, Wang K, Li G. An improved protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow. J Orthop Transl. 2015;3:26–33.

    Google Scholar 

  31. Bouïs D, Hospers GAP, Meijer C, Molema G, Mulder NH. Endothelium in vitro: a review of human vascular endothelial cell lines for blood vessel-related research. Angiogenesis. 2001;4:91–102.

    Article  PubMed  Google Scholar 

  32. Janeczek Portalska K, Leferink A, Groen N, Fernandes H, Moroni L, van Blitterswijk C, et al. Endothelial differentiation of mesenchymal stromal cells. PLoS One. 2012;7:e46842.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Grimbacher B, Aicher WK, Peter HH, Eibel H. TNF-α induces the transcription factor Egr-1, pro-inflammatory cytokines and cell proliferation in human skin fibroblasts and synovial lining cells. Rheumatol Int. 1998;17:185–92.

    Article  CAS  PubMed  Google Scholar 

  34. Son SW, Min BW, Lim Y, Lee YH, Shin SY. Regulatory mechanism of TNFα autoregulation in HaCaT cells: The role of the transcription factor EGR-1. Biochem Biophys Res Commun. 2008;374:777–82.

    Article  CAS  PubMed  Google Scholar 

  35. Chaudhary LR, Cheng SL, Avioli LV. Induction of early growth response-1 gene by interleukin-1β and tumor necrosis factor-α in normal human bone marrow stromal and osteoblastic cells: regulation by a protein kinase C inhibitor. Mol Cell Biochem. 1996;156:69–77.

    Article  CAS  PubMed  Google Scholar 

  36. Wang B, Guo H, Yu H, Chen Y, Xu H, Zhao G. The role of the transcription factor EGR1 in cancer. Front Oncol. 2021;11:1–10.

    Google Scholar 

  37. Kim JH, Jung E, Choi J, Min DY, Lee YH, Shin SY. Leptin is a direct transcriptional target of EGR1 in human breast cancer cells. Mol Biol Rep. 2019;46:317–24.

    Article  CAS  PubMed  Google Scholar 

  38. Ip YT, Davis RJ. Signal transduction by the c-Jun N-terminal kinase (JNK) - From inflammation to development. Curr Opin Cell Biol. 1998;10:205–19.

    Article  CAS  PubMed  Google Scholar 

  39. Vleugel MM, Greijer AE, Bos R, van der Wall E, van Diest PJ. c-Jun activation is associated with proliferation and angiogenesis in invasive breast cancer. Hum Pathol. 2006;37:668–74.

    Article  CAS  PubMed  Google Scholar 

  40. Ma J, Zhang L, Han W, Shen T, Ma C, Liu Y, et al. Activation of JNK/c-Jun is required for the proliferation, survival, and angiogenesis induced by EET in pulmonary artery endothelial cells. J Lipid Res. 2012;53:1093–105.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Lee SP, Jun G, Yoon EJ, Park S, Yang CH. Inhibitory effect of methyl caffeate on Fos-Jun-DNA complex formation and suppression of cancer cell growth. Bull Korean Chem Soc. 2001;22:1131–5.

    CAS  Google Scholar 

  42. Nakashima A, Ota A, Sabban EL. Interactions between Egr1 and AP1 factors in regulation of tyrosine hydroxylase transcription. Brain Res Mol Brain Res. 2003;112:61–9.

    Article  CAS  PubMed  Google Scholar 

  43. Goel HL, Mercurio AM. VEGF targets the tumour cell. Nat Rev Cancer. 2013;13:871–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Beckermann BM, Kallifatidis G, Groth A, Frommhold D, Apel A, Mattern J, et al. VEGF expression by mesenchymal stem cells contributes to angiogenesis in pancreatic carcinoma. Br J Cancer. 2008;99:622–31.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Marcelo KL, Goldie LC, Hirschi KK. Regulation of endothelial cell differentiation and specification. Circ Res. 2013;112:1272–87.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Al-Lamki RS, Mayadas TN. TNF receptors: signaling pathways and contribution to renal dysfunction. Kidney Int. 2015;87:281–96.

    Article  CAS  PubMed  Google Scholar 

  47. Guo D, Dunbar JD, Yang CH, Pfeffer LM, Donner DB. Induction of Jak/STAT signaling by activation of the type 1 TNF receptor. J Immunol. 1998;160:2742–50.

    CAS  PubMed  Google Scholar 

  48. Gashler A, Sukhatme VP. Early Growth Response Protein 1 (Egr-1): Prototype of a Zinc-finger family of transcription factors. Prog Nucleic Acid Res Mol Biol. 1995;50:191–224.

    Article  CAS  PubMed  Google Scholar 

  49. Li L, Ameri AH, Wang S, Jansson KH, Casey OM, Yang Q, et al. EGR1 regulates angiogenic and osteoclastogenic factors in prostate cancer and promotes metastasis. Oncogene. 2019;38:6241–55.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Wu SY, Rupaimoole R, Shen F, Pradeep S, Pecot CV, Ivan C, et al. A miR-192-EGR1-HOXB9 regulatory network controls the angiogenic switch in cancer. Nat Commun. 2016;7:1–14.

    CAS  Google Scholar 

  51. Seong Y, Moon J, Kim J. Egr1 mediated the neuronal differentiation induced by extremely low-frequency electromagnetic fields. Life Sci. 2014;102:16–27.

    Article  CAS  PubMed  Google Scholar 

  52. Guerquin MJ, Charvet B, Nourissat G, Havis E, Ronsin O, Bonnin MA, et al. Transcription factor EGR1 directs tendon differentiation and promotes tendon repair. J Clin Invest. 2013;123:3564–76.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Hess J, Angel P, Schorpp-Kistner M. AP-1 subunits: Quarrel and harmony among siblings. J Cell Sci. 2004;117:5965–73.

    Article  CAS  PubMed  Google Scholar 

  54. Vinson C, Myakishev M, Acharya A, Mir AA, Moll JR, Bonovich M. Classification of Human B-ZIP proteins based on dimerization properties. Mol Cell Biol. 2002;22:6321–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Hamik A, Wang B, Jain MK. Transcriptional regulators of angiogenesis. Arterioscler Thromb Vasc Biol. 2006;26:1936–47.

    Article  CAS  PubMed  Google Scholar 

  56. Morgunova E, Taipale J. Structural perspective of cooperative transcription factor binding. Curr Opin Struct Biol. 2017;47:1–8.

    Article  CAS  PubMed  Google Scholar 

  57. Chen L, Glover JN, Hogan PG, Rao A, Harrison SC. Structure of the DNA-binding domains from NFAT, Fos and Jun bound specifically to DNA. Nature. 1998;392:42–8.

    Article  CAS  PubMed  Google Scholar 

  58. Wei X, Guo J, Li Q, Jia Q, Jing Q, Li Y, et al. Bach1 regulates self-renewal and impedes mesendodermal differentiation of human embryonic stem cells. Sci Adv. 2019;5:eaau7887.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Belew MS, Bhatia S, Keyvani Chahi A, Rentas S, Draper JS, Hope KJ. PLAG1 and USF2 co-regulate expression of Musashi-2 in human hematopoietic stem and progenitor cells. Stem Cell Rep. 2018;10:1384–97.

    Article  CAS  Google Scholar 

  60. Hernandez N. TBP, a universal eukaryotic transcription factor? Genes Dev. 1993;7:1291–308.

    Article  CAS  PubMed  Google Scholar 

  61. Franklin CC, McCulloch AV, Kraft AS. In vitro association between the Jun protein family and the general transcription factors, TBP and TFIIB. Biochem J. 1995;305:967–74.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Wang B, Chen J, Santiago FS, Janes M, Kavurma MM, Chong BH, et al. Phosphorylation and acetylation of histone H3 and autoregulation by early growth response 1 mediate interleukin 1β induction of early growth response 1 transcription. Arterioscler Thromb Vasc Biol. 2010;30:536–45.

    Article  CAS  PubMed  Google Scholar 

  63. Silverman ES, Du J, Williams AJ, Wadgaonkar R, Drazen JM, Collins T. cAMP-response-element-binding-protein-binding protein (CBP) and p300 are transcriptional co-activators of early growth response factor-1 (Egr-1). Biochem J. 1998;336:183–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Kim BK, Im JY, Han G, Lee WJ, Won KJ, Chung KS, et al. P300 cooperates with c-Jun and PARP-1 at the p300 binding site to activate RhoB transcription in NSC126188-mediated apoptosis. Biochim Biophys Acta Gene Regul Mech. 2014;1839:364–73.

    Article  CAS  Google Scholar 

  65. Liu L, Guan H, Li Y, Ying Z, Wu J, Zhu X, et al. Astrocyte elevated gene 1 interacts with acetyltransferase p300 and c-Jun to promote tumor aggressiveness. Mol Cell Biol. 2017;37:e00456–16.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This paper was supported by the KU Research Professor Program of Konkuk University.

Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (2018R1A2B2004653 (YHL) and 2020R1A2C1005845 (SYS).

Author information

Authors and Affiliations

Authors

Contributions

Conceptualization, YHL and SYS; Methodology, EJ, SO, SSA, and HY; Investigation, EJ, SO, SSA, and HY; Formal Analysis, EJ, SSA, and HY; Writing – Original Draft, EJ; Writing – Review & Editing, YHL and SYS; Funding Acquisition, YHL and SYS; Project Administration and Supervision, SYS. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Soon Young Shin.

Ethics declarations

Competing interests

The authors declare no competing interests.

Ethics statement

All animal experiments were approved by the Konkuk University Institutional Animal Care and Use Committee (IACUC) (approval number KU22041).

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Edited by JP Medema

Supplementary information

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Jung, E., Ou, S., Ahn, S.S. et al. The JNK-EGR1 signaling axis promotes TNF-α-induced endothelial differentiation of human mesenchymal stem cells via VEGFR2 expression. Cell Death Differ (2022). https://doi.org/10.1038/s41418-022-01088-8

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41418-022-01088-8

Search

Quick links