Nucleotide oligomerization domain 1 is a dominant pathway for NOS2 induction in vascular smooth muscle cells: comparison with Toll-like receptor 4 responses in macrophages.
OBJECTIVE
Gram-negative bacteria contain ligands for Toll-like receptor (TLR) 4 and nucleotide oligomerization domain (NOD) 1 receptors. Lipopolysaccharide (LPS) activates TLR4, while peptidoglycan products activate NOD1. Activation of NOD1 by the specific agonist FK565 results in a profound vascular dysfunction and experimental shock in vivo.


METHODS
Here, we have analysed a number of pharmacological inhibitors to characterize the role of key signalling pathways in the induction of NOS2 following TLR4 or NOD1 activation.


RESULTS
Vascular smooth muscle (VSM) cells expressed NOD1 mRNA and protein, and, after challenge with Escherichia coli or FK565, NOS2 protein and activity were induced. Macrophages had negligible levels of NOD1 and were unaffected by FK565, but responded to E. coli and LPS by releasing increased NO and expression of NOS2 protein. Classic pharmacological inhibitors for NF-kappaB (SC-514) and mitogen-activated protein kinase (SB203580, PD98059) signalling pathways inhibited responses in both cell types regardless of agonist. While TLR4-mediated responses in macrophages were specifically inhibited by the pan-caspase inhibitor z-VAD-fmk and the PKC inhibitor Gö6976, NOD1-mediated responses in VSM cells were inhibited by the Rip2 inhibitor PP2.


CONCLUSIONS
Our findings suggest a selective role for NOD1 in VSM cells, and highlight NOD1 as a potential novel therapeutic target for the treatment of vascular inflammation.
Moreno
L
L
Cardiothoracic Pharmacology, National Heart and Lung Institute, Imperial College, London, UK. laura.moreno@imperial.ac.uk
McMaster
S K
SK
Gatheral
T
T
Bailey
L K
LK
Harrington
L S
LS
Cartwright
N
N
Armstrong
P C J
PC
Warner
T D
TD
Paul-Clark
M
M
Mitchell
J A
JA
D000818:Animals; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004790:Enzyme Induction; D018836:Inflammation Mediators / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D017737:Macrophages, Peritoneal / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000201:enzymology*; D016328:NF-kappa B / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis*; D053474:Nod1 Signaling Adaptor Protein / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011921:Rats, Inbred WKY; D053475:Receptor-Interacting Protein Serine-Threonine Kinase 2; D053422:Receptor-Interacting Protein Serine-Threonine Kinases / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D051197:Toll-Like Receptor 4 / Q000819:agonists / Q000378:metabolism*
D018836:Inflammation Mediators; D008070:Lipopolysaccharides; D016328:NF-kappa B; D053474:Nod1 Signaling Adaptor Protein; C507873:Nod1 protein, mouse; D009842:Oligopeptides; D012333:RNA, Messenger; C493487:Tlr4 protein, mouse; C495347:Tlr4 protein, rat; D051197:Toll-Like Receptor 4; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; C496318:Nos2 protein, rat; D053475:Receptor-Interacting Protein Serine-Threonine Kinase 2; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; C506255:Ripk2 protein, mouse; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; C039537:heptanoyl-gamma-D-glutamyl-L-meso-diaminopimelyl-D-alanine