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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 UGent and Myron R Szewczuk (2010) GLYCOCONJUGATE JOURNAL. 27(6). p.583-600
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 G alpha i 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 NF kappa B 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 G alpha i 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 NF kappa B 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 NF kappa B 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 NF kappa B activation and pro-inflammatory cytokine production in vivo.
Please use this url to cite or link to this publication:
author
organization
alternative title
Thymoquinone-induced Neu4 sialidase activates NF kappa B in macrophage cells and pro-inflammatory cytokines in vivo
year
type
journalArticle (original)
publication status
published
subject
keyword
Thymoquinone, Cell signaling, Receptor activation, TOLL-like receptor 4, Neu4 sialidase, Cellular sialidase, NF kappa B, Cytokines, INTERLEUKIN-1 RECEPTOR ANTAGONIST, GROWTH-FACTOR RECEPTOR, MOUSE MODEL, AIRWAY INFLAMMATION, BLACK CUMIN, ALPHA, TRANSACTIVATION, EXPRESSION, PATHWAY, SIGNAL
journal title
GLYCOCONJUGATE JOURNAL
Glycoconjugate J.
volume
27
issue
6
pages
583 - 600
Web of Science type
Article
Web of Science id
000282098400003
JCR category
BIOCHEMISTRY & MOLECULAR BIOLOGY
JCR impact factor
2.7 (2010)
JCR rank
148/284 (2010)
JCR quartile
3 (2010)
ISSN
0282-0080
DOI
10.1007/s10719-010-9302-5
language
English
UGent publication?
yes
classification
A1
copyright statement
I have transferred the copyright for this publication to the publisher
id
1057092
handle
http://hdl.handle.net/1854/LU-1057092
date created
2010-10-11 10:38:02
date last changed
2016-12-19 15:46:27
@article{1057092,
  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 G alpha i 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 NF kappa B 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 G alpha i 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 NF kappa B 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 NF kappa B 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 NF kappa B activation and pro-inflammatory cytokine production in vivo.},
  author       = {Finlay, Trisha M and Abdulkhalek, Samar and Gilmour, Alanna and Guzzo, Christina and Jayanth, Preethi and Amith, Schammim Ray and Gee, Katrina and Beyaert, Rudi and Szewczuk, Myron R},
  issn         = {0282-0080},
  journal      = {GLYCOCONJUGATE JOURNAL},
  keyword      = {Thymoquinone,Cell signaling,Receptor activation,TOLL-like receptor 4,Neu4 sialidase,Cellular sialidase,NF kappa B,Cytokines,INTERLEUKIN-1 RECEPTOR ANTAGONIST,GROWTH-FACTOR RECEPTOR,MOUSE MODEL,AIRWAY INFLAMMATION,BLACK CUMIN,ALPHA,TRANSACTIVATION,EXPRESSION,PATHWAY,SIGNAL},
  language     = {eng},
  number       = {6},
  pages        = {583--600},
  title        = {Thymoquinone-induced Neu4 sialidase activates NF\ensuremath{\kappa}B in macrophage cells and pro-inflammatory cytokines in vivo},
  url          = {http://dx.doi.org/10.1007/s10719-010-9302-5},
  volume       = {27},
  year         = {2010},
}

Chicago
Finlay, Trisha M, Samar Abdulkhalek, Alanna Gilmour, Christina Guzzo, Preethi Jayanth, Schammim Ray Amith, Katrina Gee, Rudi Beyaert, and Myron R Szewczuk. 2010. “Thymoquinone-induced Neu4 Sialidase Activates NFκB in Macrophage Cells and Pro-inflammatory Cytokines in Vivo.” Glycoconjugate Journal 27 (6): 583–600.
APA
Finlay, T. M., Abdulkhalek, S., Gilmour, A., Guzzo, C., Jayanth, P., Amith, S. R., Gee, K., et al. (2010). Thymoquinone-induced Neu4 sialidase activates NFκB in macrophage cells and pro-inflammatory cytokines in vivo. GLYCOCONJUGATE JOURNAL, 27(6), 583–600.
Vancouver
1.
Finlay TM, Abdulkhalek S, Gilmour A, Guzzo C, Jayanth P, Amith SR, et al. Thymoquinone-induced Neu4 sialidase activates NFκB in macrophage cells and pro-inflammatory cytokines in vivo. GLYCOCONJUGATE JOURNAL. 2010;27(6):583–600.
MLA
Finlay, Trisha M, Samar Abdulkhalek, Alanna Gilmour, et al. “Thymoquinone-induced Neu4 Sialidase Activates NFκB in Macrophage Cells and Pro-inflammatory Cytokines in Vivo.” GLYCOCONJUGATE JOURNAL 27.6 (2010): 583–600. Print.