Involvement of tyrosine kinase in the induction of cyclo‐oxygenase and nitric oxide synthase by endotoxin in cultured cells

Abstract

1 Cyclo-oxygenase (COX) and nitric oxide synthase (NOS) are two enzymes which have distinct cytokine-inducible isoforms (COX-2 and iNOS). Many cytokine receptors have an intracellular tyrosine kinase domain. Here we have used the tyrosine kinase inhibitors, erbstatin and genistein, to investigate the potential role of tyrosine kinase activation in the induction on COX-2 and iNOS caused by endotoxin (lipopolysaccharide; LPS) in bovine aortic endothelial cells (BAEC) and J774.2 macrophages. 2 The main COX metabolites, 6-oxo-prostaglandin Fl, (6-oxo-PGFI,) (for BAEC) and PGF2, (for 774.2 macrophages) were measured by radioimmunossay: (i) accumulation of COX metabolites from endogenous arachidonic acid was measured at 24 h after addition of LPS (1 jig ml-'); (ii) in experiments designed to measure 'COX activity', COX metabolites generated by BAEC or J774.2 macrophages activated with LPS were assayed (at 12 h after LPS administration) after incubation of the washed cells with exogenous arachidonic acid (30 tiM for 15 min). Western blot analysis with a specific antibody to COX-2 was used to determine the expression of COX-2 protein caused by LPS in cell extracts. Accumulation of nitrite (measured by the Griess reaction) was used as an indicator of NO formation and, hence, iNOS activity. 3 Erbstatin (0.05 to 5 jg ml-') or genistein (0.5 to 50 pg ml-') caused a dose-dependent inhibition of the accumulation of COX metabolites in the supernatant of BAEC or J774.2 macrophages activated with LPS. Erbstatin or genistein also caused a dose-dependent inhibition of 'COX activity' in both cell types. Western blot analysis showed that erbstatin (5 ig ml1') or genistein (50gg ml-') inhibited the expression of COX-2 protein in BAEC and J774.2 macrophages activated with LPS (lLgml-' for 24 h). 4 Erbstatin or genistein also caused a dose-dependent inhibition of nitrite accumulation in J774.2 macrophages activated with LPS (1 sg ml-' for 24 h). In contrast to J774.2 macrophages, BAEC stimulated with LPS (1 pg ml-' for 24 h) did not produce detectable amounts (<1PiM) of nitrite. 5These results suggest that tyrosine phosphorylation is part of the signal transduction mechanism that mediates (i) the induction of COX-2 and iNOS elicited by LPS in J774.2 macrophages, and (ii) the induction of COX-2 by LPS in BAEC.

Upregulation of COX-2 During Cardiac Allograft Rejection

“…27 The co-induction of COX-2 and iNOS has also been observed in studies in vitro of rat vascular smooth muscle cells, 28,29 glomerular mesangial cells, 7 murine macrophages, 30 rat islets of Langerhans, 31 human endothelial cells, 32 articular chondrocytes, 33 and rabbit hepatocytes 34 incubated with endotoxin and/or cytokines but not in similar studies of human fetal cell fibroblasts 6 or bovine aortic endothelial cells. 35 Proinflammatory cytokines known to be synthesized and released by T lymphocytes and macrophages during cardiac allograft rejection are probably responsible for induction of COX-2 in this situation. 36 In studies of porcine endothelial cells, exposed to xenoreactive antibodies and complement, IL-1␤ mediated the upregulation of COX-2 and synthesis of PGE 2 and TXA 2 .…”

Induction of NOS in rat blood PMN in vivo and in vitro: modulation by tyrosine kinase and involvement in bactericidal activity

“…The induction of NO synthase (iNOS) in mononuclear phagocytes, in vivo and in vitro, is dependent on the activation by cytokines, such as interferons (IFN-␣, -␤, or -␥) and tumor necrosis factor ␣ (TNF-␣), acting synergistically with bacterial products, promoting the expression of iNOS gene [6,7]. In macrophages, the molecular mechanism implicated in the activation of NOS after cytokine or lipopolysaccharides (LPS) stimulation apparently involves a protein tyrosine kinase pathway [8,9]. The stimulation of blood neutrophils (PMN) in vitro with LPS, at doses that usually induce NOS in other cells [10], failed to induce NO production [11].…”

“…After incubation of rat blood PMN with IFN-␥ (10 U/mL) for different times, cells were washed twice in PBS buffer and lysed in ice-cold buffer [8]. Protein samples (50 µg) were determined in the lysates and subjected to gel electrophoresis and immunoblotting.…”

Relaxin Activates the l -Arginine–Nitric Oxide Pathway in Vascular Smooth Muscle Cells in Culture

“…40,43 Of note, NF-B activation in response to cytokines has been shown to be under the control of different signal transduction effectors, including tyrosine kinase 44 and PC-PLC, 45 both of which are also known to upregulate iNOS expression. 46,47 Moreover, cytokine-stimulated cells produce reactive oxygen radicals 40 that are able to activate NF-B. 40,48 Through all of the aforementioned pathways, cells are prompted to express iNOS protein and to generate NO.…”