LPS-Induced NF-kappaB activation and TNF-alpha release in human monocytes are protein tyrosine kinase dependent and protein kinase C independent.
BACKGROUND
Tumor necrosis factor alpha (TNF-alpha) is an important mediator of septic shock. Endotoxin (LPS) signal transduction in human monocytes leads to activation of nuclear factor-kappa B (NF-kappaB) and TNF-alpha release. Previous studies have implicated activation of both protein kinase C (PKC) and protein tyrosine kinases (PTK) in LPS-induced NF-kappaB activation and TNF-alpha production. We hypothesized that inhibition of either PKC or PTK would decrease LPS-induced NF-kappaB DNA binding and TNF-alpha release in human monocytes.


METHODS
Human monocytes were stimulated with PMA (50 ng/ml) alone or LPS (100 ng/ml) with and without a nonspecific serine/threonine protein kinase inhibitor staurosporine (Stauro), a specific pan-PKC inhibitor bisindolylmaleimide (Bis), or an inhibitor of PTK genistein (Gen). TNF-alpha release in culture supernatants was measured by an ELISA. NF-kappaB DNA binding was evaluated by electrophoretic mobility shift assay.


RESULTS
LPS increased NF-kappaB DNA binding and TNF-alpha release in human monocytes. Nonspecific protein kinase inhibition inhibited NF-kappaB activation and TNF-alpha release, while specific PKC inhibition with Bis had no effect on LPS-induced NF-kappaB DNA binding or TNF-alpha release. PTK inhibition with Gen attenuated both LPS-induced NF-kappaB DNA binding and TNF-alpha production in human monocytes. Direct activation of PKC with PMA induced both NF-kappaB activation and TNF-alpha production by human monocytes.


CONCLUSIONS
These results suggest that LPS-induced NF-kappaB activation and TNF-alpha release in human monocytes are independent of PKC activity. Furthermore, our results provide evidence that PTK plays a role in LPS-induced NF-kappaB activation and TNF-alpha release in human monocytes and thus could be a potential therapeutic target in inflammatory states.
Shames
B D
BD
Department of Surgery, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA. brian.shames@uchsc.edu
Selzman
C H
CH
Pulido
E J
EJ
Meng
X
X
Meldrum
D R
DR
McIntyre
R C
RC
Harken
A H
AH
Banerjee
A
A
D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019833:Genistein / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008301:Maleimides / Q000494:pharmacology; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D004791:Enzyme Inhibitors; D007211:Indoles; D008070:Lipopolysaccharides; D008301:Maleimides; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; D004247:DNA; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D019311:Staurosporine; C088060:bisindolylmaleimide; D013755:Tetradecanoylphorbol Acetate