Modulating the Tumor Necrosis Factor-a Induced Inflammatory Response in Human Colonic Epithelial Cells by Inhibiting NFkB SignalingJ. Rivard, A. James, G. Wegner, K. Reagan │ Array Group, R&D Systems Inc., 614 McKinley PL. NE, Minneapolis, MN, 55413

AbstractIn colonic epithelia, tumor necrosis factor alpha (TNF-a) secreted by inflammatory cells is a key mediator of inflammatory bowel disease. TNF-α is a well-characterized activator of the NFkB signaling pathway in intestinal epithelial cells and is known to up-regulate the expression of a subset of chemokines and cytokines. In this study, we used antibody arrays and ELISAs to profile the chemokines secreted upon activation of the human colon carcinoma cell line HT-29. Elevated CXCL1, CXCL7, CXCL8, CXCL10, and CCL20 protein levels were observed in conditioned media after TNF-a treatment of HT-29 cells. To better understand the contributions of the classical NFkB signaling pathway to TNF-a activation of HT-29 cells we used TPCA-1, an inhibitor with high selectivity for IKK2 over IKK1. In addition, both classical and the alternative NFkB signaling pathways were inhibited downstream of IKK1 and IKK2 by using the NEDD8 activating enzyme (NAE)-inhibitor MLN4924. TPCA-1 and MLN4924 both attenuated TNF-a activated secretion of these chemokines in a dose dependent manner in HT-29 cells. Confirmation of these array data by quantitative ELISAs supports the use of arrays for analysis of inhibitor biomarkers.

IntroductionChemokines play critical roles in inflammatory diseases and cancer progression. Nuclear factor kB (NFkB) upstream signaling pathways have emerged as potential therapeutic targets for the treatment of inflammatory diseases.1 TPCA-1 is a potent, selective inhibitor of IkB kinase 2 (IKK2) that displays 22-fold selectivity over IKK1. Both classical and alternative NFkB pathways are regulated downstream of IKK1 and IKK2 by the NEDD8 activating enzyme (NAE). Degradation of IkB-a following its phosphorylation by IKK2, and NFkB2/p100 processing in response to its phosphorylation by IKK1, both require ubiquitination by a cullin-RING subtype ubiquitin ligase complex of the SKP1-cullin 1-F-box family. The activity of this E3 ubiquitin ligase is dependent on neddylation of the Cul-1 component by NAE, which is inhibited by the potent and selective small molecule inhibitor MLN4924.

ConclusionThe small molecule inhibitors TPCA-1 and MLN4924 inhibited TNF-a-induced CXCL8,2 CXCL1, CXCL10, and CCL20 secretion in HT-29 cells in a dose dependent manner by targeting the upstream regulation of NFkB transcription factors. The chemokine secretion profiles generated using the Proteome Profiler Human Chemokine Array revealed that the effect of TPCA-1 and MLN4924 on secreted chemokines was not substantially different. Subsequent measurement of CXCL8, CXCL1, CXCL10, and CCL20 protein levels by using Quantikine ELISA Kits confirmed these array data. This report demonstrates the effective use of antibody arrays combined with ELISA kits for the detection and measurement of chemokine biomarkers expressed during inflammation.

References

1. Yamamoto, Y. and R.B. Gaynor (2001) J. Clin. Invest. 107:135.2. Rauert-Wunderlich et al. (2013) PLOS ONE, 8:e59292.

Arrays are composed of capture and control antibodies spotted in duplicate on nitrocellulose membranes. Cell culture supernate or extracts were diluted, mixed with a cocktail of biotinylated detection antibodies, and incubated overnight with the Proteome Profiler Human Chemokine Antibody Array (Catalog #ARY017). Streptavidin-HRP and chemiluminescent detection reagents were applied to the arrays, and the signal produced at each capture spot corresponded to the amount of protein bound.

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CCL21/6CkineCCL28CXCL16ChemerinCXCL5/ENA-78CCL26/Eotaxin-3CX3CL1/FractalkineCXCL1/GROaCCL14/HCC-1CCL1/I-309CXCL8/IL-8

IL-16CXCL10/IP-10CXCL11/I-TACXCL1/LymphotactinCCL2/MCP-1CCL7/MCP-3CCL22/MDCMidkineCXCL9/MIGCCL3/CCL4/MIP-1a/bCCL15/MIP-1d

CCL20/MIP-3aCCL19/MIP-/3CXCL7/NAP-2CCL18/PARCCXCL4/PF4CCL5/RANTESCXCL12/SDF-1CCL17/TARCCXCL17/VCC-1

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Figure 1. TNF-a induced chemokine secretion is inhibited by TPCA-1 and MLN4924 Figure 2. Confirmation of Antibody Array Data Using ELISAs

Figure 3. TPCA-1 targets the classical NFkB pathway, while MLN4924 targets both classical and alternative NFkB pathways

CXCL1, CXCL8, CXCL10, and CCL20 secretion by Human TNF-a treated HT-29 cells is inhibited by TPCA-1 and MLN4924 in a dose dependent manner. (A) HT-29 cells were untreated or treated with 50 ng/mL Recombinant Human TNF-a (Catalog # 210-TA) for 6 hours. No inhibitor pretreatment was used. Cell culture supernates (500 µL per array) were tested using the Proteome Profiler Human Chemokine Antibody Array Kit (Catalog # ARY017). One minute exposures to X-ray film are shown. (B) HT-29 cells were untreated or pretreated with the indicated concentrations of TPCA-1 (Catalog # 2559/10) or MLN4924 (Catalog # I-502-01M) for 1 hour followed by treatment with 50 ng/mL Recombinant Human TNF-a (Catalog # 210-TA) for 6 hours.

ELISA analysis confirms antibody array data showing TPCA-1 and MLN4924 dose dependent inhibition of chemokine secretion by TNF-a treated HT-29 cells. Antibody array images were analyzed using image analysis software. The Human CXCL1/GROa Quantikine® ELISA Kit (Catalog # DGR00), Human CXCL8/IL-8 Quantikine ELISA Kit (Catalog # D8000C), Human CXCL10/IP-10 Quantikine ELISA Kit (Catalog # DIP100), and Human CCL20/MIP-3a Quantikine ELISA Kit (Catalog # DM3A00) were run to confirm antibody array data. ELISA measurement of highly expressed chemokines CXCL1 and CXCL8 show dose dependent inhibition more clearly than antibody arrays. TPCA-1 and MLN4924 did not differ substantially in their inhibition of secreted chemokines.

TPCA-1 blocks TNF-a-induced phosphorylation and degradation of IkB-a, without affecting TWEAK-induced p100 processing, while MLN4924 blocks TNF-a induced degradation of phospho-IkB-a and p100 processing. MLN4924 blocks downstream of IKK1 and IKK2, resulting in accumulation of phospho-IkB-a and inhibition of p100 cleavage. (A) HT-29 cells were pretreated with TPCA-1 or MLN4924 for 1 hour followed by treatment with 50 ng/mL Recombinant Human TNF-a (Catalog # 210-TA) for 3 and 10 minutes. Western blot analysis of whole cell lysates was done using Rabbit Anti-Human Phospho-IkB-a (S32/S36) Antibody (Catalog # AF4809) followed by Goat Anti-Rabbit IgG-HRP Antibody (Catalog # HAF008); or Mouse Anti-Human IkB-a Antibody (Catalog # MAB4299) followed by Goat Anti-Mouse IgG-HRP Antibody (Catalog # HAF007); or Goat Anti-Human GAPDH Antibody (Catalog # AF5718) followed by Donkey Anti-Goat IgG-HRP Antibody (Catalog # HAF109). (B) HT-29 cells were pretreated with TPCA-1 or MLN4924 for 1 hour followed by treatment with 200 ng/mL Recombinant Human TWEAK (Catalog # 1090-TW) for 18 hours. Western blot analysis of whole cell lysates was done using Mouse Anti-Human NFkB2 Antibody (Catalog # MAB28881) followed by Goat Anti-Mouse IgG-HRP Antibody (Catalog # HAF007); or Goat Anti-Human GAPDH Antibody (Catalog # AF5718) followed by Donkey Anti-Goat IgG-HRP Antibody (Catalog # HAF109).