Nuclear factor-kappa B (NF-B) is a transcription factor that regulates the expression of many genes that play a key role in inflammation and inflammatory diseases such as sepsis. NF-B activity is tightly controlled by the I B kinase (IKK) complex, which consists of at least three subunits, IKK, IKKß and IKK (Zandi et al., 1997; Yanaoka et al., 1998) and, these proteins may represent potentially novel targets for anti-inflammatory drugs. One additional target may be the recently described inducible isoform of IKK, termed IKKi. As IKKi displays sequence homology to IKK/ß and has also been shown to activate NF-B (Shimada et al., 1999), we sought to determine the molecular mechanisms regulating the stimulated expression of IKKi. This includes the roles of the mitogen- activated protein (MAP) kinases and NFB itself.

RAW 264.7 macrophages were used in this study. Induction of IKKi protein was examined by immunoprecipitation followed by Western blotting. In parallel IKKi RNA was extracted from cells and subjected to RT-PCR analysis. NF-B-DNA binding was assayed by EMSA. IKK activity was measured by an immunocomplex kinase assay performed in vitro.

Lipopolysaccharide (LPS) (1 µg/ml) stimulated a significant increase in protein levels for IKKi in murine macrophages, which was evident at 4 hours (LPS, 4hr =8.8 ± 1.6 fold, n=3), maximal by 6 hours (LPS, 6hr =15.1 ± 2.0 fold, p<0.05, n=3) before returning to basal values by 24h. The kinetics of IKKi mRNA levels closely paralleled protein expression. The Toll-like receptor (TLR) 2 specific ligands PGN (10 µg/ml), LTA (10 µg/ml) and PAM3CSK4 (300ng/ml), the TLR3 specific ligand POLY(I:C) (10 µg/ml), and also highly purified LPS (1 µg/ml) specific for TLR4 also increased IKKi protein levels as did interferon- (IFN-, 6hr =11.6 ± 4.9 fold, p <0.05, n=3). However in contrast, protein expression was not augmented by the protein kinase C activator phorbol myristate acetate (PMA). Again protein levels closely correlated with IKKi mRNA expression. In RAW 264.7 cells, LPS also stimulated a substantial increase in p42/44 MAP kinase, p38 MAP kinase and JNK activities. Never-the-less, pre-treatment of cells with pharmacological inhibitors PD 098059, SB 203580 and SP 60012 which inhibit p42/p44 MAP kinase, p38 MAP kinase and JNK pathways respectively, were without significant effect on LPS-stimulated IKKi induction. However, the IKK inhibitor compound BAY-11-7082 (50 µM) significantly decreased IKKi protein levels (LPS =7.5 ± 2.8, LPS+BAY-11-7082 =1.3 ± 0.86 fold, p<0.05, n=3). This was also true for IKKi mRNA.

Taken together these results indicate that IKKi is an inducible IB kinase that is induced by LPS in a NF- B dependent and a MAP kinase-independent manner. It remains to be determined what contribution the pathway makes to LPS-stimulated NF-B activation and induction of pro-inflammatory genes.

Shimada, T., et al. (1999) Int. Immunol. 11: 1357-62
Zandi, E., et al. (1997) Cell 91: 243-252
Yanaoka, S., et al. (1998) Cell 93: 1231-40

This work was supported by a British Heart Foundation grant to AP and RP. GM is a recipient of a Carnegie Studentship award