Skip to main content

Advertisement

SpringerLink
Log in

2-Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine is an anti-inflammatory TLR-2, -4 and -5 response mediator in human monocytes

  • Original Research Paper
  • Published:
Inflammation Research Aims and scope Submit manuscript

Abstract

Objective and design

To elucidate the influence of 2-amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine (AMBMP), a canonical Wnt/β-catenin pathway activator, on the inflammatory response of TLR-engaged innate cells in vitro.

Material or subject

Primary human monocytes.

Treatment

AMPMB (0–10 μM), LPS (0–1.0 μg/ml), Pam3CSK4, FSL-1, or S. typhimurium flagellin (0–0.25 μg/ml).

Methods

TLR-induced cytokine release (TNF, IL-6, IL-12 p40) was monitored by ELISA while Wnt-related signals (GSK3β, p65, IκB, β-catenin) were assessed by Western blot, pharmaceutical inhibition and gene silencing.

Results

AMBMP induced the rapid phosphorylation of NFκB p65 at Ser536 and abrogated total IκB, accompanied by a subsequent increase in pro-inflammatory cytokine production (TNF, IL-6, IL-12 p40) in otherwise naive monocytes. However, in TLR2, -4 and -5-engaged monocytes, AMBMP-suppressed cytokine production. In the context of LPS stimulation, this occurred concomitant with the phosphorylative inactivation of GSK3β at Ser9, β-catenin accumulation and abrogation of NFκB p65 phosphorylation. AMBMP-mediated suppression of the TLR4 -induced inflammatory response was reversed by two pharmaceutical Wnt/β-catenin pathway inhibitors, IWP-2 and PNU-74654 and by Wnt3a silencing.

Conclusions

Herein, we show that AMBMP induces canonical Wnt signaling events and acts as a suppressor of inflammation in surface TLR-engaged primary human monocytes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

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

Similar content being viewed by others

References

  1. Lamont RJ. Mike martin, a tribute. Mol Oral Microbiol. 2011;26:174.

    Article  Google Scholar 

  2. Wang H, Kumar A, Lamont RJ, Scott DA. GSK3beta and the control of infectious bacterial diseases. Trends Microbiol. 2014;22:208–17.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  3. Rehani K, Scott DA, Renaud D, Hamza H, Williams LR, Wang H, et al. Cotinine-induced convergence of the cholinergic and PI3 kinase-dependent anti-inflammatory pathways in innate immune cells. Biochim Biophys Acta. 2008;1783:375–82.

    Article  PubMed  CAS  Google Scholar 

  4. Zhou H, Gao S, Duan X, Liang S, Scott DA, Lamont RJ, et al. Inhibition of serum- and glucocorticoid-inducible kinase 1 enhances TLR-mediated inflammation and promotes endotoxin-driven organ failure. FASEB J. 2015.

  5. Di Liddo R, Bertalot T, Schuster A, Schrenk S, Tasso A, Zanusso I, et al. Anti-inflammatory activity of Wnt signaling in enteric nervous system: in vitro preliminary evidences in rat primary cultures. J Neuroinflamm. 2015;12:23.

    Article  Google Scholar 

  6. Wang H, Liao H, Ochani M, Justiniani M, Lin X, Yang L, et al. Cholinergic agonists inhibit HMGB1 release and improve survival in experimental sepsis. Nat Med. 2004;10:1216–21.

    Article  PubMed  CAS  Google Scholar 

  7. Denicol AC, Dobbs KB, McLean KM, Carambula SF, Loureiro B, Hansen PJ. Canonical WNT signaling regulates development of bovine embryos to the blastocyst stage. Sci Rep. 2013;3:1266.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Lim JC, Kania KD, Wijesuriya H, Chawla S, Sethi JK, Pulaski L, et al. Activation of beta-catenin signalling by GSK-3 inhibition increases p-glycoprotein expression in brain endothelial cells. J Neurochem. 2008;106:1855–65.

    PubMed  CAS  PubMed Central  Google Scholar 

  9. Liu J, Wu X, Mitchell B, Kintner C, Ding S, Schultz PG. A small-molecule agonist of the Wnt signaling pathway. Angew Chem Int Ed Engl. 2005;44:1987–90.

    Article  PubMed  CAS  Google Scholar 

  10. Kuncewitch M, Yang WL, Molmenti E, Nicastro J, Coppa GF, Wang P. Wnt agonist attenuates liver injury and improves survival after hepatic ischemia/reperfusion. Shock. 2013;39:3–10.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  11. Kuncewitch M, Yang WL, Jacob A, Khader A, Giangola M, Nicastro J, et al. Stimulation of Wnt/beta-catenin signaling pathway with Wnt agonist reduces organ injury after hemorrhagic shock. J Trauma Acute Care Surg. 2015;78:793–800.

    Article  PubMed  CAS  Google Scholar 

  12. Ma Y, Lv X, He J, Liu T, Wen S, Wang L. Wnt agonist stimulates liver regeneration after small-for-size liver transplantation in rats. Hepatol Res 2015.

  13. Schaale K, Neumann J, Schneider D, Ehlers S, Reiling N. Wnt signaling in macrophages: augmenting and inhibiting mycobacteria-induced inflammatory responses. Eur J Cell Biol. 2011;90:553–9.

    Article  PubMed  CAS  Google Scholar 

  14. Angers S, Moon RT. Proximal events in Wnt signal transduction. Nat Rev Mol Cell Biol. 2009;10:468–77.

    Article  PubMed  CAS  Google Scholar 

  15. Wada A. GSK-3 inhibitors and insulin receptor signaling in health, disease, and therapeutics. Front Biosci. 2009;14:1558–70.

    Article  CAS  Google Scholar 

  16. Willert K, Jones KA. Wnt signaling: is the party in the nucleus? Genes Dev. 2006;20:1394–404.

    Article  PubMed  CAS  Google Scholar 

  17. Bradley EW, Drissi MH. WNT5A regulates chondrocyte differentiation through differential use of the CaN/NFAT and IKK/NF-kappaB pathways. Mol Endocrinol. 2010;24:1581–93.

    Article  PubMed  CAS  Google Scholar 

  18. Duan Y, Liao AP, Kuppireddi S, Ye Z, Ciancio MJ, Sun J. beta-Catenin activity negatively regulates bacteria-induced inflammation. Lab Invest. 2007;87:613–24.

    Article  PubMed  CAS  Google Scholar 

  19. Kuhl M, Sheldahl LC, Park M, Miller JR, Moon RT. The Wnt/Ca2+ pathway: a new vertebrate Wnt signaling pathway takes shape. Trends Genet. 2000;16:279–83.

    Article  PubMed  CAS  Google Scholar 

  20. Dejmek J, Safholm A. Kamp Nielsen C, Andersson T, Leandersson K. Wnt-5a/Ca2+ -induced NFAT activity is counteracted by Wnt-5a/Yes-Cdc42-casein kinase 1alpha signaling in human mammary epithelial cells. Mol Cell Biol. 2006;26:6024–36.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  21. Lai SL, Chien AJ, Moon RT. Wnt/Fz signaling and the cytoskeleton: potential roles in tumorigenesis. Cell Res. 2009;19:532–45.

    Article  PubMed  CAS  Google Scholar 

  22. Pukrop T, Klemm F, Hagemann T, Gradl D, Schulz M, Siemes S, et al. Wnt 5a signaling is critical for macrophage-induced invasion of breast cancer cell lines. Proc Natl Acad Sci USA. 2006;103:5454–9.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  23. Blumenthal A, Ehlers S, Lauber J, Buer J, Lange C, Goldmann T, et al. The Wingless homolog WNT5A and its receptor Frizzled-5 regulate inflammatory responses of human mononuclear cells induced by microbial stimulation. Blood. 2006;108:965–73.

    Article  PubMed  CAS  Google Scholar 

  24. Neumann J, Schaale K, Farhat K, Endermann T, Ulmer AJ, Ehlers S, et al. Frizzled1 is a marker of inflammatory macrophages, and its ligand Wnt3a is involved in reprogramming Mycobacterium tuberculosis-infected macrophages. FASEB J. 2010;24:4599–612.

    Article  PubMed  CAS  Google Scholar 

  25. Martin M, Rehani K, Jope RS, Michalek SM. Toll-like receptor-mediated cytokine production is differentially regulated by glycogen synthase kinase 3. Nat Immunol. 2005;6:777–84.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  26. Martin M, Schifferle RE, Cuesta N, Vogel SN, Katz J, Michalek SM. Role of the phosphatidylinositol 3 kinase-Akt pathway in the regulation of IL-10 and IL-12 by Porphyromonas gingivalis lipopolysaccharide. J Immunol. 2003;171:717–25.

    Article  PubMed  CAS  Google Scholar 

  27. Bagaitkar J, Zeller I, Renaud DE, Scott DA. Cotinine inhibits the pro-inflammatory response initiated by multiple cell surface Toll-like receptors in monocytic THP cells. Tob Induc Dis. 2012;10:18.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  28. Lee H, Bae S, Choi BW, Yoon Y. WNT/beta-catenin pathway is modulated in asthma patients and LPS-stimulated RAW264.7 macrophage cell line. Immunopharmacol Immunotoxicol. 2012;34:56–65.

    Article  PubMed  CAS  Google Scholar 

  29. Manicassamy S, Reizis B, Ravindran R, Nakaya H, Salazar-Gonzalez RM, Wang YC, et al. Activation of beta-catenin in dendritic cells regulates immunity versus tolerance in the intestine. Science. 2010;329:849–53.

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  30. Chen Y, Guan Y, Liu H, Wu X, Yu L, Wang S, et al. Activation of the Wnt/beta-catenin signaling pathway is associated with glial proliferation in the adult spinal cord of ALS transgenic mice. Biochem Biophys Res Commun. 2012.

  31. Wei J, Fang F, Lam AP, Sargent JL, Hamburg E, Hinchcliff ME, et al. Wnt/beta-catenin signaling is hyperactivated in systemic sclerosis and induces Smad-dependent fibrotic responses in mesenchymal cells. Arthritis Rheum 2012.

  32. Chua AW, Gan SU, Ting Y, Fu Z, Lim CK, Song C, et al. Keloid fibroblasts are more sensitive to Wnt3a treatment in terms of elevated cellular growth and fibronectin expression. J Dermatol Sci. 2011;64:199–209.

    Article  PubMed  CAS  Google Scholar 

  33. You J, Nguyen AV, Albers CG, Lin F, Holcombe RF. Wnt pathway-related gene expression in inflammatory bowel disease. Dig Dis Sci. 2008;53:1013–9.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

Michael Martin, a leading light in oral and systemic inflammation research, sadly passed away on February 22nd 2011, aged 37 years [1]. Mike initiated this project. The paper is dedicated to his family—Jayme, Jack and Clara. This work was supported by the US Department of Health and Human Services (National Institute for Dental and Craniofacial Research grants DE023633 (HW) DE017921 (RJL) and DE017680 (DAS).

Author information

Authors and Affiliations

  1. Department of Oral Immunology and Infectious Diseases, University of Louisville, 501 South Preston Street, Louisville, KY, 40292, USA

    Huizhi Wang, Mark W. Graves II, Huaxin Zhou, Zhen Gu, Richard J. Lamont & David A. Scott

Authors
  1. Huizhi Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mark W. Graves II
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Huaxin Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhen Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Richard J. Lamont
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. David A. Scott
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to David A. Scott.

Ethics declarations

Conflict of interests

The author(s) declare(s) that they have no competing interests.

Additional information

Responsible Editor: John Di Battista.

Electronic supplementary material

Below is the link to the electronic supplementary material.

11_2015_891_MOESM1_ESM.pdf

Supplemental Figure 1: AMBMP reduces p65 phosphorylation in response to LPS. (A) LPS (100 ng/ml) alone induced a rapid increase in the phosphorylated p65 (Ser536) signal, as measured by Western blot in cell lysates (20 μg protein). AMBMP clearly suppressed the LPS-induced phosphorylation of p65. (B) The densitometric ratio of phospho-p65 to β-actin is shown. Typical data are presented (PDF 87 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, H., Graves, M.W., Zhou, H. et al. 2-Amino-4-(3,4-(methylenedioxy)benzylamino)-6-(3-methoxyphenyl)pyrimidine is an anti-inflammatory TLR-2, -4 and -5 response mediator in human monocytes. Inflamm. Res. 65, 61–69 (2016). https://doi.org/10.1007/s00011-015-0891-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00011-015-0891-0

Keywords

Search

Navigation