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Glycoconjugate Journal

, Volume 27, Issue 6, pp 583–600 | Cite as

Thymoquinone-induced Neu4 sialidase activates NFκB in macrophage cells and pro-inflammatory cytokines in vivo

  • Trisha M. Finlay
  • Samar Abdulkhalek
  • Alanna Gilmour
  • Christina Guzzo
  • Preethi Jayanth
  • Schammim Ray Amith
  • Katrina Gee
  • Rudi Beyaert
  • Myron R. SzewczukEmail author
Article

Abstract

Thymoquinone (TQ) derived from the nutraceutical black cumin oil has been reported to be a novel agonist of Neu4 sialidase activity in live cells (Glycoconj J DOI 10.1007/s10719-010-9281-6). The activation of Neu4 sialidase on the cell surface by TQ was found to involve GPCR-signaling via membrane targeting of Gαi subunit proteins and matrix metalloproteinase-9 activation. Contrary to other reports, TQ had no anti-inflammatory effects in vitro. Here, we show that MyD88/TLR4 complex formation and subsequent NFκB activation are induced by the Neu4 activity associated with TQ-stimulated live primary bone marrow (BM) macrophage cells from WT and Neu1-deficient mice, HEK-TLR4/MD2 cells and BMC-2 macrophage cell line but not with primary macrophage cells from Neu4-knockout mice. Tamiflu (oseltamivir phosphate), pertussis toxin (PTX), a specific inhibitor of Gαi proteins of G-protein coupled receptor (GPCR) and the broad range inhibitor of matrix metalloproteinase (MMP) galardin applied to live primary BM macrophage cells completely block TQ-induced MyD88/TLR4 complex formation. Using immunocytochemistry and western blot analyses, Tamiflu, galardin and PTX inhibit NFκB activation induced by Neu4 activity associated with TQ-stimulated BMC-2 cells, HEK-TLR4/MD2 cells and primary BM macrophages from WT mice. EMSA analyses on HEK-TLR4/MD2 nuclear cell extracts confirm the nuclear localization and DNA binding of TQ-induced NFκB activation in a biphasic manner within 30 min. Co-immunoprecipitation experiments reveal for the first time that MMP-9 may be an important intermediate link in the TQ-induced Neu4 activity circuitously targeting TLR4 receptors. Central to this process is that Neu4 forms a complex with MMP-9, which is already bound to TLR4 receptors. Fluorescence spectrophotometer analyses of live CD14-THP1 cells treated with TQ show Neu4 sialidase activity over 5 min. Using flow cytometry analyses, CD14-THP1 cells treated with TQ express stable protein levels of Neu4, TLR4 and MMP9 on the cell surface over 30 min except for a marked diminution of MMP9 at 15 min. Using cytokine array profiling analyses of serum, Neu4-knockout mice respond poorly to TQ in producing pro-inflammatory cytokines and chemokines after 5-h treatment compared to the wild-type or hypomorphic cathepsin A mice with a secondary 90% Neu1 deficient mice. Our findings establish an unprecedented signaling paradigm for TQ-induced Neu4 sialidase activity. It signifies that MMP-9 forms an important molecular signaling platform in complex with TLR4 receptors at the ectodomain and acts as the intermediate link for TQ-induced Neu4 sialidase in generating a functional receptor with subsequent NFκB activation and pro-inflammatory cytokine production in vivo.

Keywords

Thymoquinone Cell signaling Receptor activation TOLL-like receptor 4 Neu4 sialidase Cellular sialidase NFκB Cytokines 

Abbreviations

TQ

thymoquinone

oseltamivir phosphate

Tamiflu

LPS

lipopolysaccharide

BM

bone marrow

M-CSF

monocyte colony-stimulating factor

4-MUNANA

2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid

PVDF

polyvinylidene fluoride

HEK293

human embryonic kidney 293 cells

Neu1 KD

hypomorphic cathepsin A mice with the secondary ~90% reduction of the Neu1 activity

Neu4 KO

Neu4 knockout

EMSA

electrophoretic mobility shift assay

Notes

Acknowledgements

These studies are partially supported by grants to MRS from Natural Sciences and Engineering Research Council of Canada (NSERC). T.M.F. was a recipient of the Queen’s University Research Award. A.G. and S.A. are the recipients of the Queen’s Graduate Awards. P.J. is a recipient of the Queen’s Graduate Award and the Robert J. Wilson Fellowship. S.R.A. was a recipient of the Queen’s University Research Award, the Robert J. Wilson Fellowship and the Ontario Graduate Scholarship. Research work on the TLR transfected cell lines was supported by grants to R.B. from the ‘Interuniversitaire Attractiepolen’ (IAP6/18), the ‘Fonds voor Wetenschappelijk Onderzoek-Vlaanderen’ (FWO; grant 3G010505), and the ‘Geconcerteerde Onderzoeksacties’ of the Ghent University (GOA; grant 01G06B6).

We acknowledge Prof. Alexey V. Pshezhetsky and Dr. Volkan Seyrantepe from the Departments of Pediatrics and Biochemistry, Montreal University, Service de Genetique, Ste-Justine Hospital, 3175 Cote-Ste-Catherine, H3T1C5, Montreal, QC, Canada for provided us the Neu1-deficient and Neu4 knockout mice. The authors acknowledge Merry Guo in generating some of the graphic figures in this report.

Authors Contributions

M.R.S. and T.M.F. wrote the paper, designed and performed experiments; P.J., A.G. and S.A. performed the TLR4 and Neu4 IP WB; K.G. assisted with the cytokine profiling; R.B. generated HEK-TLR4/MD2 cells; S.R.A. initially helped with the experiments; M.R.S. supervised the research design and the writing of the paper. All authors read and commented on the manuscript, and declare no competing financial interests.

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Trisha M. Finlay
    • 1
    • 4
  • Samar Abdulkhalek
    • 1
  • Alanna Gilmour
    • 1
  • Christina Guzzo
    • 1
  • Preethi Jayanth
    • 1
  • Schammim Ray Amith
    • 1
    • 5
  • Katrina Gee
    • 1
  • Rudi Beyaert
    • 2
    • 3
  • Myron R. Szewczuk
    • 1
    Email author
  1. 1.Department of Microbiology & ImmunologyQueen’s UniversityKingstonCanada
  2. 2.Department for Molecular Biomedical Research, VIBUnit for Molecular Signal Transduction in InflammationZwijnaardeBelgium
  3. 3.Department of Biomedical Molecular BiologyGhent UniversityGhentBelgium
  4. 4.Hotchkiss Brain InstituteUniversity of CalgaryCalgaryCanada
  5. 5.Conway InstituteUniversity College DublinBelfieldIreland

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