Effect of acetylsalicylic acid on endogenous I kappa B kinase activity in lung epithelial cells

The anti-inflammatory effect of acetylsalicylic acid (ASA) has been thought to be secondary to the inhibition of prostaglandin synthesis. Because doses of ASA necessary to treat chronic inflammatory diseases are much higher than those needed to inhibit prostaglandin synthesis, a prostaglandin-independent pathway has been emerging as the new anti-inflammatory mechanism of ASA. Here, we examined the effect of ASA on the interleukin (IL)-1 beta- and tumor necrosis factor (TNF)-alpha-induced proinflammatory cytokine expression and evaluated whether this effect is closely linked to the nuclear factor (NF)-kappa B/I kappa B-alpha pathway. A high dose of ASA blocked IL-1 beta- and TNF-alpha-induced TNF-alpha and IL-8 expression, respectively. ASA inhibited TNF-alpha-induced activation of NF-kappa B by preventing phosphorylation and subsequent degradation of I kappa B-alpha in a prostanoid-independent manner. TNF-alpha-induced activation of I kappa B kinase was also suppressed by ASA pretreatment. These observations suggest that the anti-inflammatory effect of ASA in lung epithelial cells may be due to suppression of I kappa B kinase activity, which thereby inhibits subsequent phosphorylation and degradation of I kappa B-alpha, activation of NF-kappa B, and proinflammatory cytokine expression in lung epithelial cells.     

A functional role of I kappa B-epsilon in endothelial cell activation

The NF-kappa B inhibitor I kappa B-epsilon is a new member of the I kappa B protein family, but its functional role in regulating NF-kappa B-mediated induction of adhesion molecule expression is unknown. In vascular endothelial cells, I kappa B-epsilon associates predominantly with the NF-kappa B subunit Rel A and to a lesser extent with c-Rel, whereas I kappa B-alpha and I kappa B-beta associate with Rel A only. Following stimulation with TNF-alpha, pyrrolidine dithiocarbamate (PDTC), N-acetylcysteine, and dexamethasone prevented I kappa B kinase-induced I kappa B-alpha, but not I kappa B-beta or I kappa B-epsilon phosphorylation and degradation. Since the activation of NF-kappa B is required for the induction of adhesion molecule expression, we examined the role of I kappa B-epsilon in the transactivation of promoters from VCAM-1, ICAM-1, and E-selectin. Using reporter gene constructs of adhesion molecule promoters, PDTC inhibited VCAM-1 and E-selectin, but to a lesser extent, ICAM-1 promoter activity. Subcloning of kappa B cis-acting elements of VCAM-1, E-selectin, and ICAM-1 into a heterologous promoter construct revealed that PDTC inhibited VCAM-1 and E-selectin, but to a lesser extent, ICAM-1 kappa B promoter activity. By electrophoretic mobility shift assay, NF-kappa B heterodimers containing c-Rel specifically bind to the kappa B motif in the ICAM-1, but not VCAM-1 or E-selectin promoter. Indeed, overexpression of c-Rel induced ICAM-1 kappa B promoter activity to a greater extent than that of E-selectin and overexpression of I kappa B-epsilon inhibited ICAM-1 and VCAM-1 promoter activity in endothelial cells. These findings indicate that c-Rel-associated I kappa B-epsilon is involved in the induction of ICAM-1 expression.     

Inducible nuclear expression of NF-kappa B in primary B cells stimulated through the surface Ig receptor

Constitutive expression of NF-kappa B has been associated with developmental maturity in B cells on the basis of studies using continuously growing cell lines and plasmacytomas; however, little is known about the behavior of NF-kappa B in primary, mature B cells. In the present work, the regulation of NF-kappa B expression was studied by analyzing subcellular fractions of adult murine splenic B cells with the electrophoretic mobility shift assay using a kappa B-containing oligonucleotide. Although nuclear extracts from resting B cells contained kappa B-binding activity, additional kappa B-binding activity was present in cytosolic fractions in a form that became apparent after treatment with detergent. Competition analysis indicated that the DNA binding activity detected by electrophoretic gel mobility shift assay was specific for the kappa B motif, and UV photo-cross-linking showed the molecular size of kappa B-binding protein to be similar to that of the DNA binding subunit of NF-kappa B. Nuclear expression of kappa B-binding activity was markedly induced by treatment of B cells with phorbol ester or with LPS. Most notably, kappa B-binding activity was induced after surface IgR cross-linking, and the mechanism of this induction involved PKC. Further, anti-Ig-induced activity was superinduced in the presence of cycloheximide. These results indicate that nuclear NF-kappa B is rapidly induced as a result of B cell stimulation, and further suggest that NF-kappa B may play a specific role in mature B cells after ligand binding to surface Ig distinct from its postulated developmental role as a stage-specific factor involved in kappa-enhancer function.     

Cyclooxygenase (COX)-2 inhibitor celecoxib abrogates TNF-induced NF-kappa B activation through inhibition of activation of I kappa B alpha kinase and Akt in human non-small cell lung carcinoma: correlation with suppression of COX-2 synthesis

The cyclooxygenase 2 (COX-2) inhibitor celecoxib (also called celebrex), approved for the treatment of colon carcinogenesis, rheumatoid arthritis, and other inflammatory diseases, has been shown to induce apoptosis and inhibit angiogenesis. Because NF-kappa B plays a major role in regulation of apoptosis, angiogenesis, carcinogenesis, and inflammation, we postulated that celecoxib modulates NF-kappa B. In the present study, we investigated the effect of this drug on the activation of NF-kappa B by a wide variety of agents. We found that celecoxib suppressed NF-kappa B activation induced by various carcinogens, including TNF, phorbol ester, okadaic acid, LPS, and IL-1 beta. Celecoxib inhibited TNF-induced I kappa B alpha kinase activation, leading to suppression of I kappa B alpha phosphorylation and degradation. Celecoxib suppressed both inducible and constitutive NF-kappa B without cell type specificity. Celecoxib also suppressed p65 phosphorylation and nuclear translocation. Akt activation, which is required for TNF-induced NF-kappa B activation, was also suppressed by this drug. Celecoxib also inhibited the TNF-induced interaction of Akt with I kappa B alpha kinase (IKK). Celecoxib abrogated the NF-kappa B-dependent reporter gene expression activated by TNF, TNF receptor, TNF receptor-associated death domain, TNF receptor-associated factor 2, NF-kappa B-inducing kinase, and IKK, but not that activated by p65. The COX-2 promoter, which is regulated by NF-kappa B, was also inhibited by celecoxib, and this inhibition correlated with suppression of TNF-induced COX-2 expression. Besides NF-kappa B, celecoxib also suppressed TNF-induced JNK, p38 MAPK, and ERK activation. Thus, overall, our results indicate that celecoxib inhibits NF-kappa B activation through inhibition of IKK and Akt activation, leading to down-regulation of synthesis of COX-2 and other genes needed for inflammation, proliferation, and carcinogenesis.