Validation of the Anti-Inflammatory Properties of Small- Molecule I B Kinase (IKK)-2 Inhibitors by Comparison with Adenoviral-Mediated Delivery of Dominant-Negative IKK1 and IKK2 in Human Airways Smooth Muscle

Asthma and chronic obstructive pulmonary disease (COPD) are characterized by chronic airway inflammation. However, because patients with COPD and certain patients with asthma show little or no therapeutic benefit from existing corticosteroid therapies, there is an urgent need for novel anti-inflammatory strategies. The transcription factor nuclear factorB (NFB) is central to inflammation and is necessary for the expression of numerous inflammatory genes. Proinflammatory cytokines, including interleukin (IL)-1 and tumor necrosis factor (TNF), activate the I B kinase complex (IKK) to promote the degradation of inhibitory I B proteins and activate NFB. This pathway and, in particular, the main I B kinase, IKK2, are now considered prime targets for novel anti-inflammatory drugs. Therefore, we have used adenoviral overexpression to demonstrate NFB and IKK2 dependence of key inflammatory genes, including intercellular adhesion molecule (ICAM)-1, cyclooxygenase-2, IL-6, IL-8, granulocyte macrophage–colony-stimulating factor (GM-CSF), regulated on activation normal T cell expressed and secreted (RANTES), monocyte chemotactic protein-1 (MCP-1), growth-regulated oncogene(GRO ), neutrophil-activating protein-2 (NAP-2), and epithelial neutrophil activating peptide 78 (ENA-78) in primary human airways smooth muscle cells. Because this cell type is central to the pathogenesis of airway inflammatory diseases, these data predict a beneficial effect of IKK2 inhibition. These validated outputs were therefore used to evaluate the novel IKK inhibitors N-(6-chloro9H-carbolin-8-yl) nicotinamide (PS-1145) and N-(6-chloro-7methoxy-9H-carbolin-8-yl)-2-methyl-nicotinamide (ML120B) on IL-1 and TNF -induced expression, and this was compared with the corticosteroid dexamethasone. As observed above, ICAM-1, IL-6, IL-8, GM-CSF, RANTES, MCP-1, GRO , NAP-2, and ENA-78 expression was reduced by the IKK inhibitors. Furthermore, this inhibition was either as effective, or for ICAM-1, MCP-1, GRO , and NAP-2, more effective, than a maximally effective concentration of dexamethasone. We therefore suggest that IKK inhibitors may be of considerable benefit in inflammatory airways diseases, particularly in COPD or severe asthma, in which corticosteroids are ineffective. Asthma and chronic obstructive pulmonary disease (COPD) are inflammatory diseases of the lung that are associated with both chronic inflammation of the airways and, in the case of COPD, a progressive nonreversible decline in lung function (Barnes, 2004; Barnes and Hansel, 2004). Although in the majority of asthma cases, inflammation and disease severity can be controlled by inhaled or oral corticosteroids, This study was supported by a grant from Millennium Pharmaceuticals (to R.N. and P.J.B.). R.N. is a Canadian Institutes of Health Research New Investigator and an Alberta Heritage Foundation for Medical Research Scholar. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.106.023150. □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. ABBREVIATIONS: COPD, chronic obstructive pulmonary disease; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; GFP, green fluorescent protein; HASM, human airway smooth muscle; IKK, I B kinase complex; MOI, multiplicity of infection; MTT, 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyltetrazolium bromide; RANTES, regulated on activation normal T cell expressed and secreted; GM-CSF, granulocyte macrophage–colony-stimulating factor; GRO , growth-regulated oncogene; NAP-2, neutrophil-activating protein-2; DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immunosorbent assay; HA, hemagglutinin; COX-2, cyclooxygenase-2; NFB, nuclear factor B; IL, interleukin; ICAM, intercellular adhesion molecule; TNF , tumor necrosis factor ; MCP-1, monocyte chemotactic protein-1; PS-1145, N-(6-chloro-9H-carbolin8-yl) nicotinamide; ML120B, N-(6-chloro-7-methoxy-9H-carbolin-8-yl)-2-methyl-nicotinamide. 0026-895X/06/7002-697–705$20.00 MOLECULAR PHARMACOLOGY Vol. 70, No. 2 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 23150/3127452 Mol Pharmacol 70:697–705, 2006 Printed in U.S.A. 697 http://molpharm.aspetjournals.org/content/suppl/2006/05/11/mol.106.023150.DC1 Supplemental material to this article can be found at: at A PE T Jornals on Jne 6, 2017 m oharm .aspeurnals.org D ow nladed from there remains a group of patients whose asthma remains poorly controlled because of disease severity or to insensitivity to corticosteroid treatment (Adcock and Ito, 2004; Barnes, 2004). In contrast, COPD is generally unresponsive to corticosteroid treatment, and apart from smoking cessation, which halts the accelerated decline in lung function, the only genuinely effective treatment is lung transplantation (Barnes and Hansel, 2004; Wouters, 2004). Given the highly invasive nature of this therapy and the shortage of donor tissue, effective treatments such as the development of more specific and potent anti-inflammatory agents that target the inflammation associated with COPD and severe and steroid insensitive asthma are desperately needed (Adcock and Ito, 2004; Barnes, 2004; Barnes and Hansel, 2004; Wouters, 2004). NFB is a ubiquitously expressed transcription factor that consists of heteroor homodimers of the Rel family of proteins and regulates the expression of many genes involved in immune and inflammatory responses (Barnes and Karin, 1997; Li and Verma, 2002). Thus, the NFB signaling pathway is considered to be a potential target for novel antiinflammatory compounds, and indeed, a number of pharmaceutical companies are developing compounds that target this pathway (Barnes and Karin, 1997; Li and Verma, 2002; Karin et al., 2004). A number of these have now been shown to retain anti-inflammatory effects in animal models and could therefore be useful in the therapeutic treatment of inflammatory airways disease (Castro et al., 2003; Karin et al., 2004). In unstimulated or resting cells, NFB is localized to the cytoplasm and is associated with a members of a family of inhibitory proteins known as I B (inhibitor of B) (Hayden and Ghosh, 2004). NFB activation is initiated in response to a wide range of stimuli, including the pro-inflammatory cytokines, IL-1 and TNF, chemokines, and bacterial and viral products (Barnes and Karin, 1997; Li and Verma, 2002; Hayden and Ghosh, 2004). These lead to phosphorylation of I B by the I B kinase (IKK) complex, which in turn leads to I B polyubiquitination and subsequent degradation by the 26S proteosome (Li and Verma, 2002; Hayden and Ghosh, 2004). As a result, NFB proteins are liberated from I B, usually I B , and translocate to the nucleus, where they bind to the promoter regions of NFB responsive genes and initiate gene transcription (Li and Verma, 2002; Hayden and Ghosh, 2004; Karin et al., 2004). A critical step in the NFB pathway is the phosphorylation of I B by the IKK complex (Li and Verma, 2002). This complex consists of at least two catalytic subunits, IKK1 (IKK ) and IKK2 (IKK ) and a regulatory subunit, IKK /NEMO, which has no catalytic function, but whose structural role is absolutely required for IKK activation. Although IKK1 and IKK2 are structurally similar, studies in knockout mice and derived mouse embryonic fibroblasts suggest that IKK2 is the predominant kinase involved in I B phosphorylation and hence NFB activation (Li and Verma, 2002; Hayden and Ghosh, 2004). Airway smooth muscle cells are a potent source of many cytokines, chemokines, and other mediators and have been implicated in the local amplification of airway inflammatory responses (Howarth et al., 2004). To help evaluate the therapeutic potential of inhibiting the NFB pathway in this cell type, we have used primary human airway smooth muscle (HASM) cells and a combination of adenoviral delivery of dominant-negative IKK1, IKK2, and dominant I B (I B N), as well as novel small-molecule IKK inhibitors. Taken together, the data presented in these studies strongly suggest that therapeutic strategies to inhibit NFB may be beneficial in airway inflammatory diseases. Materials and Methods Reagents. IL-1 and TNF were from R&D systems (Abingdon, UK). The IKK-selective inhibitor PS-1145 has been described previously, and the structure has been published elsewhere (Hideshima et al., 2002; Castro et al., 2003). ML120B is a novel ATP binding site IKK2-selective (IC50 at 50 M ATP) inhibitor that does not inhibit either IKK1 (EC50 100 M) or IKK (EC50 100 M) or a panel of 28 other kinases at EC50 values of up to 50 M (Nagashima et al., 2006; Wen et al., 2006). Both PS-1145 and ML120B were supplied as free bases by Millennium Pharmaceuticals (Cambridge, MA) and were dissolved in dimethyl sulfoxide (DMSO) before dilution in tissue culture medium. All other reagents were from Sigma (Poole, Dorset, UK) unless otherwise stated. Cell Culture and Adenovirus Infection. Human bronchial tissue was obtained from patients undergoing lung transplantation or surgical resection for carcinoma. The isolation and culture of HASM cells from these tissues has been described previously (Sukkar et al., 2004). The null, green fluorescent protein (Ad5-GFP), dominant I B N, and dominant-negative IKK1(KM), IKK2(KA)-expressing viruses have all been described previously (Krappmann et al., 1996; Zandi et al., 1997; Catley et al., 2003, 2005). All viruses were titered by endpoint dilution and plaque assay to determine plaque-forming units. Viruses were diluted in Dulbecco’s modified Eagle’s medium (Sigma) to a multiplicity of infection (MOI) of 30 before infection of HASM cells. This dose of virus showed no significant effect on cell viability as determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) cell viability assay (Fig. 1). Cells were then incubated for 24 h and serum-starved for an additional 24 h before microscopic inspection to confirm normal morphology. The cells were then treated with cytokines or prepared for experimental procedures. Cells grown on glass coverslips were incubated with various MOIs of Ad5-GFP virus before staining with 4 ,6 -diamidino-2-phenylindole dihydrochloric hydrate and analysis of GFP fluorescence as described previously (Catley et al., 2003). For NFB reporter assays, HASM cells were infected with MOI 30 of an adenovirus carrying an NFB-dependent reporter (Ad-NFB-luc). This construct was obtained by inserting the NFB enhancer [five copies of the classic NFB motif (underlined) 5 -TGG GGA CTT TCC GC-3 ], TATA box, and luciferase gene from pNFB-luc (Stratagene, La Jolla, CA) into the Ad5 parent vector. After incubation for 24 h in Dulbecco’s modified Eagle’s medium, the cells were then changed to fresh serum-free medium and stimulated with IL-1 or TNF for 8 h before harvesting for luciferase assay. IKK Kinase Assay. Kinase assays were performed as described previously with the following modifications (Nasuhara et al., 1999; Catley et al., 2004). Confluent six-well plates were placed in serumfree medium for 24 h before stimulation with IL-1 or TNF for 5 min. Cells were immediately put on ice and washed twice with ice-cold Hanks’ balanced salt solution. Triplicate wells were then scraped and pooled into one tube before centrifugation and subsequent lysis. Cell lysates were precleared with agarose-conjugated normal rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h before immunoprecipitation of the IKK complex with an agaroseconjugated IKK -specific antibody (Santa Cruz Biotechnology). Antibody target complexes were then collected by centrifugation and washed before resuspension in kinase buffer (20 mM HEPES, pH 7.9, 2 mM MgCl, 2 mM MnCl, 10 mM -glycerophosphate, 10 mM NaF, 10 mM 4-nitrophenyl phosphate, 0.5 mM Na3VO4, 1 mM benzamidine, 0.5 mM phenylmethylsulfonyl fluoride, aprotinin 25 g/ ml, 10 g/ml leupeptin, 2 m/ml pepstatin, and 1 mM dithiothreitol). Kinase reactions were performed in kinase buffer using a commer698 Catley et al. at A PE T Jornals on Jne 6, 2017 m oharm .aspeurnals.org D ow nladed from cially available I B substrate peptide (Upstate Biotechnology, Lake Placid, NY). For concentration-response experiments, ML120B and PS-1145 were diluted in kinase buffer to the required concentration. The whole reaction mix was run on a 4 to 12% NuPage SDS gel, which was cut at the 22-kDa marker. The bottom portion of each gel was dried for autoradiography, whereas the top portion was subject to Western analysis for IKK . Electromobility Shift Assays and Luciferase Assay. Electromobility shift assays (EMSAs) were performed as described previously (Nasuhara et al., 1999; Catley et al., 2004). Cells for luciferase assay were harvested in 1 reporter lysis buffer (Promega) before luciferase assay according to the manufacturers’ instructions (Promega) (Nasuhara et al., 1999; Catley et al., 2004). Western Blotting and Cytokine Release Measurements. Detection of proteins by Western blotting was carried out as described previously (Nasuhara et al., 1999; Holden et al., 2004). Measurement of cytokine release was determined with the SearchLight Proteome Array sandwich ELISAs using the Pierce custom service (Perbio,