IKK-α Axis Activation of the Canonical NF-κB Pathway

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TNF-a Modulation of Intestinal Tight Junction Permeability Is Mediated by NIK/IKK-a Axis Activation of the Canonical NF-kB Pathway Q18

Rana Al-Sadi, Shuhong Guo, Dongmei Ye, Manmeet Rawat, and Thomas Y. Ma From the Department of Internal Medicine, University of New Mexico School of Medicine and Albuquerque Veterans Affairs Medical Center, Albuquerque, New Mexico Accepted for publication December 22, 2015. Address correspondence to Thomas Y. Ma, M.D., Ph.D., Internal MedicineGastroenterology, MSC10 5550, University of New Mexico, Albuquerque, NM 87131-0001. E-mail: tma@ salud.unm.edu.

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Tumor necrosis factor (TNF)-a, a key mediator of intestinal inflammation, causes an increase in intestinal epithelial tight junction (TJ) permeability by activating myosin light chain kinase (MLCK; official name MYLK3) gene. However, the precise signaling cascades that mediate the TNF-aeinduced activation of MLCK gene and increase in TJ permeability remain unclear. Our aims were to delineate the upstream signaling mechanisms that regulate the TNF-a modulation of intestinal TJ barrier function with the use of in vitro and in vivo intestinal epithelial model systems. TNF-a caused a rapid activation of both canonical and noncanonical NF-kB pathway. NF-kBeinducing kinase (NIK) and mitogen-activated protein kinase kinase-1 (MEKK-1) were activated in response to TNF-a. NIK mediated the TNF-a activation of inhibitory kB kinase (IKK)-a, and MEKK1 mediated the activation of IKK complex, including IKK-b. NIK/IKK-a axis regulated the activation of both NF-kB p50/p65 and RelB/p52 pathways. Surprisingly, the siRNA induced knockdown of NIK, but not MEKK-1, prevented the TNF-a activation of both NF-kB p50/p65 and RelB/p52 and the increase in intestinal TJ permeability. Moreover, NIK/IKK-a/NF-kB p50/p65 axis mediated the TNF-aeinduced MLCK gene activation and the subsequent MLCK increase in intestinal TJ permeability. In conclusion, our data show that NIK/IKK-a/regulates the activation of NF-kB p50/p65 and plays an integral role in the TNF-aeinduced activation of MLCK gene and increase in intestinal TJ permeability. (Am J Pathol 2016, -: 1e15; http://dx.doi.org/10.1016/j.ajpath.2015.12.016)

It is well established that in intestinal permeability disorders, the defective intestinal tight junction (TJ) barrier allows paracellular permeation of luminal antigens, which can induce or propagate inflammatory response.1,2 Previous studies from our laboratory and others have shown that proinflammatory cytokines, including tumor necrosis factor (TNF)-a, (IL-1b, IL-6, and interferon-g, cause an increase in intestinal TJ permeability and contribute to the inflammatory process by allowing luminal antigenic penetration.3e6 Conversely, antiinflammatory cytokine IL-10 was shown to promote intestinal TJ barrier function.7,8 In IL-10
/
mice, the development of intestinal inflammation was preceded by an increase in intestinal permeability,7,9 and enhancement of intestinal TJ barrier with a TJ barrier enhancing agent (AT-1001) prevented the development of intestinal inflammation.10,11 TNF-a is a multifunctional proinflammatory cytokine that was shown to play a central role in intestinal inflammation

of Crohn disease (CD).2,12,13 Patients with CD have marked increase in TNF-a levels in their intestinal tissues, sera, and stool. Treatment with antieTNF-a antibodies was shown to be an effective therapeutic strategy in CD.14,15 Previous studies have shown that TNF-a causes an increase in intestinal TJ permeability in vitro and in vivo and that the TNF-aeinduced increase in intestinal permeability contributes to the development of intestinal inflammation by allowing increased antigenic penetration.16e19 Clinical studies have also shown that antieTNF-a therapy leads to a rapid re-tightening of the intestinal barrier and early resolution of active CD.20e22 Supported by a Veterans Affairs (VA) Merit Review grant from the VA Research Service and National Institute of Diabetes and Digestive and Kidney Diseases grants RO 1-DK-64165 and RO 1-DK-81429. Q2 Disclosures: None declared.

Published by Elsevier Inc. on behalf of the American Society for Investigative Pathology. http://dx.doi.org/10.1016/j.ajpath.2015.12.016

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Al-Sadi et al Previous studies have shown that NF-kB signaling plays an important role in TNF-a modulation of intestinal epithelial TJ barrier by targeting myosin light chain kinase (MLCK; official name MYLK3) gene activation.5,23 These studies indicated that TNF-a causes a rapid activation of NF-kB p50/ p65 dimer. The inhibition of NF-kB p50/p65 activation by pharmacologic inhibitors or siRNA silencing of p65 subunit completely inhibited the TNF-aeinduced activation of MLCK gene and increase in intestinal TJ permeability.23 The upstream signaling pathways that mediate the TNF-a modulation in NF-kB p50/p65 activation, MLCK gene activity, or increase in intestinal epithelial TJ permeability remain unknown. Mitogen-activated protein kinase kinase kinases (MAP3 kinases) are recruited by TNF-a receptor complex after TNF-a binding and play a crucial regulatory role in a variety of biological activities in intestinal epithelial cells.24e26 The MAP3 kinases mitogen-activated protein kinase kinase kinase-1 (MEKK-1) and NF-kBeinducing kinase (NIK), are important regulators of NF-kB pathways.24,25,27 Two distinct pathways were described that lead to activation of NF-kB dimers: the canonical (or classic) and the noncanonical (or alternative) pathways.28e32 In the canonical pathway, TNF-a binding to the cell surface receptor leads to the TNF-a receptor complex recruitment of membrane shuttle kinases that ultimately lead to the phosphorylation and degradation of inhibitory kB (IkB)-a and activation of NF-kB dimer p50/p65.33,34 The TNF-ae induced activation of the noncanonical pathway results in the phosphorylation of p100 subunit, leading to the generation and activation of RelB/p52 dimer.30,35 The role of MAP3 kinases in TNF-a modulation of intestinal TJ barrier remains unknown. Our aim was to determine the regulatory role of MAP3 kinases MEKK-1 and NIK in TNF-aeinduced increase in intestinal epithelial TJ permeability, using filter-grown Caco-2 intestinal epithelial monolayers and recycling mouse intestinal perfusion as in vitro and in vivo model systems, respectively. Herein, we show for the first time that NIK mediates the TNF-ae induced activation of MLCK gene and increase in intestinal TJ permeability by regulating the activation of the canonical (NF-kB p50/p65) pathway. Our results also show for the first time that NIK, a MAP kinase known to regulate the noncanonical pathway (NF-kB p50/p52), regulates the activation of the canonical pathway via IkB kinase (IKK)-ae induced activation of NF-kB p50/p65. This is in direct contrast to what we previously described with IL-1b where the canonical pathway of NF-kB and MLCK gene activity were regulated by MEKK1 activation of IKK-b (a part of the IKK complex).36

Materials and Methods Q4

Chemicals

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Cell culture media (Dulbecco’s modified Eagle’s medium), trypsin, fetal bovine serum, glutamine, penicillin, streptomycin,

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and phosphate-buffered saline (PBS) were purchased from GIBCO-BRL (Grand Island, NY). AntieMEKK-1, NIK, IKK-a, IKK-b, IkB-a, MLCK, and antieb-actin antibodies were obtained from Sigma-Aldrich (St. Louis, MO). Antiephospho-MEKK1, phospho-NIK, phosphoeIKK-a/b antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). AntieNF-kB p65 and p100/p52 antibodies were purchased from Abcam (Cambridge, MA). Horseradish peroxidase-conjugated secondary antibodies for Western blot analysis were purchased from Invitrogen (San Francisco, CA). siRNA of MEKK-1, NIK, IKK-a, IKK-b, p65, and p100 and transfection reagents were obtained from Dharmacon (Lafayette, CO). All other chemicals were purchased from Sigma-Aldrich, VWR (West Chester, PA), or Fisher Scientific (Pittsburgh, PA).

Cell Cultures Caco-2 cells (passage 20) were purchased from the ATCC (Rockville, MD) and maintained at 37 C in a culture medium composed of Dulbecco’s modified Eagle’s medium with 4.5 mg/mL glucose, 50 U/mL penicillin, 50 U/mL streptomycin, 4 mmol/L glutamine, 25 mmol/L HEPES, and 10% fetal bovine serum. The cells were kept at 37 C in a 5% CO2 environment. Culture medium was changed every 2 days. Caco-2 cells were subcultured after partial digestion with 0.25% trypsin and 0.9 mmol/L EDTA in Ca2þ- and Mg2þ-free PBS.4,37

Determination of Epithelial Monolayer Resistance and Paracellular Permeability An epithelial voltohmeter (World Precision Instruments, Sarasota, FL) was used for measurements of the transepithelial electrical resistance (TER) of the filter-grown Caco-2 intestinal monolayers as previously reported.17,37 The effect of TNF-a on Caco-2 paracellular permeability was determined with an established paracellular marker inulin (mol. wt. Z 5000 g/mol).38 For determination of mucosal-to-serosal flux rates of inulin, Caco-2eplated filters with epithelial resistance of 400 to 500 U$cm2 were used. Known concentrations of inulin (2 mmol/L) and its radio- Q6 active tracer were added to the apical solution.

Assessment of Protein Expression by Western Blot Analysis Caco-2 monolayers were treated with 10 ng/mL TNF-a for varying time periods. At the end of the experimental period, Q7 Caco-2 monolayers were immediately rinsed with ice-cold PBS, and cells were lyzed with lysis buffer (50 mmol/L Tris$HCl, pH 7.5, 150 mmol/L NaCl, 500 mmol/L NaF, 2 mmol/L EDTA, 100 mmol/L vanadate, 100 mmol/L phenylmethylsulfonyl fluoride, 1 mg/mL leupeptin, 1 mg/mL pepstatin A, 40 mmol/L paranitrophenyl phosphate, 1 mg/mL aprotinin, and 1% Triton X-100) and scraped, and the cell

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Mechanism of TNF-a Regulation of Intestinal Barrier Function Q1 lysates were placed in Microfuge tubes. Cell lysates were centrifuged to yield a clear lysate. Supernatant fluid was collected, and protein measurement was performed with the Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hercules, CA). Laemmli gel loading buffer was added to the lysate that contained 10 to 20 mg of protein and boiled for 7 minutes, after which time proteins were separated on SDS-PAGE gel. Proteins from the gel were transferred to the membrane (Trans-Blot Transfer Medium, Nitrocellulose Membrane; Bio-Rad Laboratories) overnight. The membrane was incubated for 2 hours in blocking solution (5% dry milk in trisbuffered salineeTween 20 buffer). The membrane was incubated with appropriate primary antibodies in blocking solution. After being washed in tris-buffered salinee1% Tween buffer, the membrane was incubated in appropriate secondary antibodies and developed with the Santa Cruz Western Blotting Luminol Reagents (Santa Cruz Biotechnology) on the Kodak BioMax MS film (Fisher Scientific, Pittsburgh, PA).

method. The NF-kB p65 and p52 DNA-binding assay was performed with Trans-AM enzyme-linked immunosorbent assay (ELISA)-based kits from Active Motif (Carlsbad, CA) according to the manufacturer’s protocol. In brief, the binding reactions contained 1 pmol/L biotinylated probe (Integrated DNA Technologies, Coralville, IA) and 5 mg of nuclear extract in complete binding buffer with a total volume of 50 mL. After 30 minutes of incubation, the solution was transferred to an individual well on a 96-well plate and incubated for 1 hour. Appropriate antibody (2 mg/mL) was added to the well to bind the target protein in nuclear extract. After incubation for 1 hour, the antibody was removed, and 100 mL of horseradish peroxidase-conjugated secondary antibody was added to the well and incubated for 1 hour. Subsequently, 100 mL of developing solution was added for 2 to 10 minutes, and 100 mL of stop solution were added. The absorbance at 450 nm was determined with the SpectraMax 190 (Molecular Devices, Sunnyvale, CA).

RNA Isolation and Reverse Transcription siRNA of MEKK-1, NIK, IKK-a, IKK-b, p65, and p52 Targeted siRNAs were obtained from Dharmacon, Inc. (Chicago, IL). Caco-2 monolayers were transiently transfected with DharmaFect transfection reagent (Dharmacon).4 Briefly, 5  105 cells per filter were seeded into a 12-well transwell plate and grown to confluency. Caco-2 monolayers were then washed with PBS twice, and 1.0 mL OptiMEM medium was added to the apical compartment of each filter, and 1.5 mL was added to the basolateral compartment of each filter. Five nanograms of the siRNA of interest and 2 mL of DharmaFect reagent were preincubated in OptiMEM. After 5 minutes of incubation, two solutions were mixed and incubated for another 20 minutes, and the mixture was added to the apical compartment of each filter. The TNF-a experiments were performed 96 hours after transfection. The efficiency of silencing was confirmed by Western blot analysis.

Nuclear Extracts and ELISA for Transcription Factor Activation

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Filter-grown Caco-2 monolayers were treated with 10 ng/mL TNF-a for 30 minutes. Caco-2 monolayers were washed with ice-cold PBS, scraped, collected, and centrifuged at 14,000 rpm for 30 seconds. The cell pellets were resuspended in 200 mL of buffer A (in millimoles: 10 HEPES-KOH, 1.5 MgCl2, 10 KCl, 0.5 dithiothreitol, and 0.2 phenylmethylsulfonyl fluoride; pH 7.9), and incubated on ice for 15 minutes. After centrifugation at 14,000 rpm for 30 seconds, pelleted nuclei were resuspended in 30 mL of buffer C [in millimoles: 20 HEPES-KOH (25% glycerol), 420 NaCl, 1.5 MgCl2, 0.2 EDTA, 0.5 dithiothreitol, and 0.2 phenylmethylsulfonyl fluoride; pH 7.9]. After incubation on ice for 20 minutes, the lysates were centrifuged at 14,000 rpm for 20 minutes. Protein concentrations were determined with the Bradford

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Caco-2 cells (5  105 per filter) were seeded into -well transwell permeable inserts and grown to confluence. Filtergrown Caco-2 cells were then treated with appropriate experimental reagents for desired time periods. At the end of the experimental period, cells were washed twice with icecold PBS. Total RNA was isolated with Qiagen RNeasy Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Total RNA concentration was determined by absorbance at 260/280 nm with the use of SpectrraMax 190 (Molecular Devices). The reverse transcription was Q9 outperformed with the GeneAmp Gold RNA PCR core kit (Applied Biosystems, Foster city, CA). Two micrograms of total RNA from each sample were reverse transcribed into cDNA in a 40-mL reaction that contained 1 RT-PCR buffer, 2.5 mmol/L MgCl2, 250 mmol/L of each dNTP, 20 U RNase inhibitor, 10 mmol/L dithiothreitol, 1.25 mmol/L random hexamer, and 30 U multiscribe reverse transcriptase. The reverse transcription reactions were performed in a thermocycler (MyCycler; Bio-Rad Laboratories) at 25 C for 10 minutes, 42 C for 30 minutes, and 95 C for 5 minutes.

Quantification of Gene Expression Using Real-Time PCR The real-time PCRs were performed with ABI prism 7900 sequence detection system and TaqMan universal PCR master mix kit (Applied Biosystems, Branchburg, NJ) as previously described.23 Each real-time PCR reaction contained 10 mL reverse transcription reaction mix, 25 mL 2 TaqMan universal PCR master mix, 0.2 mmol/L probe, and 0.6 mmol/L primers. Primer and probe design for the realtime PCR was made with Primer Express version 2 from Applied Biosystems. [The primers used in this study are as follows: MLCK-specific primer pairs consisted of 50 -AGGAAGGCAGCATTGAGGTTT-30 (forward), 50 -GCTTTCAGCAGGCAGAGGTAA-30 (reverse); probe specific for

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Al-Sadi et al MLCK consisted of FAM 50 -TGAAGATGCTGGCTCC-30 TAMRA; the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific primer pairs consisted of 50 CCACCCATGGCAAATTCC-30 (forward), 50 -TGGGATTTCCATTGATGACCAG-30 (reverse); probe specific for GAPDH consisted of JOE 50 -TGGCACCGTCAAGGCTGAGAACG-30 TAMRA.] All runs were performed according to the default PCR protocol (50 C for 2 minutes, 95 C for 10 minutes, 40 cycles of 95 C for 15 seconds, and 60 C for 1 minute). For each sample, real-time PCR reactions were performed in triplicate, and the average threshold cycle (Ct) was calculated. A standard curve was generated to convert the Ct to copy numbers. Expression of MLCK mRNA was normalized with GAPDH mRNA expression. The average copy number of MLCK mRNA expression in control samples was set to 1.0. The relative expression of MLCK mRNA in treated samples was determined as a fold increase compared with control samples.

Transfection of MLCK DNA and Measurement of Promoter Activity The MLCK promoter region was cloned with GenomeWalker system (Clontech Laboratories, Inc., Mountain View, CA). A 2091-bp DNA fragment (
2109 to
18) was amplified by PCR.23 The amplification condition was 1 cycle at 94 C for 2 minutes, followed by 43 cycles at 94 C for 1 minute, 50 C for 1 minute, and 72 C for 2 minutes and 1 cycle at 72 C for 5 minutes. The resultant PCR product was digested with HindIII and KpnI and inserted into pGL3basic luciferase reporter vector (Promega, Madison, WI). The sequence was confirmed by DNA services at the University of New Mexico. MLCK promoter was transiently transfected into Caco-2 cells with the use of transfection reagent lipofectamine 2000 (Life Technologies, Carlsbad, CA). Renilla luciferase vector (pRL-TK; Promega) was cotransfected with each plasmid construct as an internal control. Cells (5  105 per filter) were seeded into a 6-well transwell plate and grown to confluence. Caco-2 monolayers were then washed with PBS twice, 1.0 mL Opti-MEM medium was added to the apical compartment of each filter, and 1.5 mL was added to the basolateral compartment of each filter. One microgram of each plasmid construct and 0.25 mg pRL-TK or 2 mL lipofectamine 2000 was preincubated in 250 mL Opti-MEM, respectively. After 5 minutes of incubation, two solutions were mixed and incubated for another 20 minutes, and the mixture was added to the apical compartment of each filter. After incubation for 3 hours at 37 C, 500 mL Dulbecco’s modified Eagle’s medium that contained 10% fetal bovine serum was added to both sides of the filter to reach a 2.5% final concentration of fetal bovine serum. Subsequently, media were replaced with normal Caco-2 growth media 16 hours after transfection. Specific experiments were performed 48 hours after transfection. At the completion of specific experimental treatments, Caco-2 cells were washed twice with

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1 mL ice-cold PBS, followed by the addition of 400 mL 1 passive lysis buffer, incubated at room temperature for 15 minutes, scraped, transferred into an Eppendorf tube, and centrifuged for 15 seconds at 13,000  rpm in a micro- Q10 centrifuge. Luciferase activity was determined with the dual luciferase assay kit (Promega). Twenty microliters of the supernatant fluid was used for each assay. Luciferase values were determined by Lumat LB 9507 (EG&G Berthold, Oak Ridge, TN). The value of reporter luciferase activities were then divided by that of renilla luciferase activities to normalize for differences in transfection efficiencies. The average activity value of the control samples was set to 1.0. The luciferase activity of MLCK promoter in treated samples was determined relative to the control samples.

In Vivo Mouse Intestinal Permeability Measurements The Laboratory Animal Care and Use Committee at the University of New Mexico approved all experimental protocols. The mouse intestinal permeability was measured by recycling small intestinal perfusion as previously described.39e41 After the experimental period, mice were anesthetized with isoflurane. After midline incision of the abdomen, 5 cm of intestine segment was isolated and cannulated at the proximal and distal ends with 0.76-mm internal diameter polyethylene tubing. Flushing solution (140 mmol/L NaCl, 10 mmol/L HEPES, pH 7.4) warmed to 37 C was first perfused through the intestine at 1 mL per minute for 20 minutes, followed by air flush to remove residual contents with the use of an external pump (Bio-Rad Laboratories). This was followed by perfusion of 5 mL perfusate solution (85 mmol/L NaCl, 10 mmol/L HEPES, 20 mmol/L sodium ferrocyanide, 5 mmol/L KCl, 5 mmol/L CaCl2, pH 7.4.) that contained Texas Red-labeled dextran (10 kDa) in a recirculating manner at 0.75 mL per minute for 2 hours. The abdominal cavity was covered with moistened gauze, body temperature was measured via rectal thermometer, and temperature was maintained at 37.5 C  0.5 C with the use of a heating lamp. One-milliliter aliquots of test solution were removed at the beginning and end of the perfusion. After perfusion, the animal was sacrificed, and the perfused intestine segment was excised and the length was measured. The excised intestinal tissue was then snap-frozen in optimal cutting temperature compound or used for protein and RNA analyses. Ferrocyanide concentration in the perfusate was measured with the colorimetric assay. Texas Red-labeled dextran 10 kDa Q11 concentration was measured with an excitation wavelength of 595 nm and an emission wavelength of 615 nm in a microplate reader. Probe clearance was calculated as Cprobe Z (CiVi
CfVf)/(CavgTL). In the equation, Ci represents the measured initial probe concentration; Cf represents the measured final probe concentration; Vi represents the measured initial perfusate volume; Vf was calculated as Vi([ferrocyanide]i/ [ferrocyanide]f); Cavg was calculated as (Ci
Cf)/ln(Ci/Cf). T represents hours of perfusion, and L represents the length of the perfused intestine section in centimeters.

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Animal Surgery and in Vivo Transfection of NF-kB p65, MEKK-1, and NIK siRNA Mice were deprived of food for 24 hours before the surgery. Mice were anesthetized with isoflurane (4% for surgical induction, 1% for maintenance) with the use of oxygen as carrier during surgical procedures. Surgical procedures were performed with sterile technique. The abdomen was opened by a midline incision, and 6 cm of intestine segment was isolated at the proximal and distal ends and tied with sutures. siRNA transfection solution (0.5 mL; containing Accell medium; 2.5 nmol p38 or ATF-2 siRNA and 50 mL transfecting agent lipofectamine) was introduced into the isolated intestine segment (surface area 6 cm2) for 1-hour transfection period. Control animals underwent shamoperation, where the siRNA transfection solution contained Accell medium; 2.5 nmol nontarget siRNA, and 50 mL transfecting agent lipofectamine. The abdominal cavity was covered with moistened gauze. Body temperature was monitored continuously with a rectal probe and maintained at 37.5 C  0.5 C with the use of a heating pad. After 1-hour transfection period, each end of the intestinal segment was untied, the intestine was placed back in the abdominal cavity, and the abdomen was closed. Three days after transfection, functional studies of intestinal epithelial barrier were performed. The surgery and the in vivo transfection procedures had no effect on the food intake and the body weight of the animals during the experimental period. The average animal weight averaged between 23 and 25 g during the experimental period.

Statistical Analysis Statistical significance of differences between mean values was assessed with Student’s t-tests for unpaired data and analysis of variance analysis whenever required. All

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559 560 561 562 563 564 Figure 1 Time course effect of TNF-a on Caco565 2 NIK and MEKK-1 activation. A: TNF-a (10 ng/mL) 566 causes a time-dependent increase in Caco-2 NIK 567 P-MEKK-1 phosphorylation (total NIK was used for equal 568 Total NIK protein loading). B: TNF-a causes a time569 dependent increase in MEKK-1 phosphorylation 570 (total MEKK-1 was used for equal protein loading). 571 MEKK-1, mitogen-activated protein kinase kinase kinase-1; NIK, NF-kB-inducing kinase; TNF, tumor 572 Q17 necrosis factor. 573 574 575 576 577 Time (min) 578 579 580 reported significance levels represent two-tailed P values. 581 P < 0.05 was used to indicate statistical significance. All 582 583 in vitro experiments that used Caco-2 monolayers, including 584 assessment of TJ barrier, biochemical and molecular studies, 585 and kinase activity measurements, were performed in trip586 licates or quadruplicates and were repeated at a minimum of 587 three times for reproductivity. The immunoblot analysis and 588 cell imaging studies were repeated three to four times. The 589 animal studies were performed individually, and each 590 experimental group consisted of three to six animals. 591 592 593 Results 594 595 Role of MAP3 Kinase Pathways in TNF-aeInduced 596 Increase in Caco-2 TJ Permeability 597 598 The role of MAP3 kinases NIK and MEKK-1 in mediating 599 the TNF-aeinduced increase in Caco-2 TJ permeability was 600 investigated. TNF-a caused a time-dependent increase in 601 NIK phosphorylation (Thr 559) in Caco-2 cells, starting at 602 about 10 minutes and continuing up to 60 minutes as 603 determined by phospho-NIK immunoblotting (Figure 1A). ½F1 604 605 TNF-a treatment (10 ng/mL) also caused a rapid activation 606 of MEKK-1 as assessed by phospho-MEKK-1 (Thr 1381) 607 immunoblotting (Figure 1B). These results suggested that 608 TNF-a causes rapid activation of both NIK and MEKK-1 in 609 filter-grown Caco-2 monolayers. 610 The requirement of NIK and/or MEKK-1 in TNF-ae 611 induced increase in Caco-2 TJ permeability was examined 612 by targeted knockdown of NIK or MEKK-1 by siRNA 613 transfection of filter-grown Caco-2 monolayers. Two 614 different controls were used in these experiments; a control 615 group exposed to normal media and a control group trans616 617 fected with nontargeted siRNA. The NIK siRNA trans618 fection resulted in a near-complete depletion of NIK expression in Caco-2 cells (Figure 2A); NIK silencing by ½F2 619 620

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Mechanism of TNF-a Regulation of Intestinal Barrier Function

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Figure 2

Effect of siRNA-induced NIK and MEKK-1 knockdown on TNF-aeinduced increase in Caco-2 TJ permeability. A: NIK siRNA transfection results in a near complete depletion in NIK protein expression. B: NIK silencing prevents the TNF-aeinduced drop in Caco-2 TER. C: NIK silencing by siRNA transfection prevents the TNF-aeinduced increase in inulin flux. D: MEKK-1 siRNA transfection results in a near complete depletion in MEKK-1 protein expression. E: siRNAinduced knockdown of MEKK-1 does not prevent the TNF-aeinduced drop in Caco-2 TER. F: siRNA-induced knockdown of MEKK-1 does not prevent the TNFaeinduced increase in inulin flux. G: NIK inhibitor (4H-isoquinoline-1,3-dione; 50 mmol/L) prevents the TNF-phosphorylation of NIK. H: NIK inhibitor prevents the TNF-aeinduced drop in Caco-2 TER. Data are expressed as means  SEM. n Z 4. **P < 0.005 versus control; yyP < 0.005 versus TNF-a treatment. C, no siRNA controls; inh, inhibitor; MEKK-1, mitogen-activated protein kinase kinase kinase-1; NIK, NF-kB-inducing kinase; NT, non-targeted siRNA; TER, transepithelial electrical resistance; TJ, tight junction; TNF, tumor necrosis factor.

siRNA transfection prevented the TNF-aeinduced drop in Caco-2 TER and increase in apical-to-serosal flux of paracellular marker inulin (Figure 2, B and C). In contrast, the siRNAeinduced knockdown of MEKK-1 (Figure 2D) did not affect the TNF-aeinduced drop in Caco-2 TER (Figure 2E) or the increase in inulin flux (Figure 2F). The treatment of Caco-2 monolayers with pharmacologic NIK inhibitor 4H-isoquinolinee1,3-dione (50 mmol/L) also inhibited the TNF-aeinduced drop in Caco-2 TER (Figure 2, G and H). These data suggested that NIK activation, but not MEKK-1, was required for the TNF-ae induced increase in Caco-2 TJ permeability.

Involvement of Canonical versus NonCanonical NF-kB Pathways The role of noncanonical pathway (NF-kB p52/Rel B) or canonical pathway (NF-kB p50/p65) in mediating the TNF-a modulation of Caco-2 TJ permeability was

6

examined. In the following studies, the TNF-a effect on canonical pathway was determined by assessing the degradation of IkB-a and activation of NF-kB p50/p65 dimer, and the effect on noncanonical pathway was determined by the activation of p100/p52 subunit with the use of ELISAbased assay.5,37 TNF-a caused a rapid degradation of IkB-a in Caco-2 cells (Figure 3A). Interestingly, TNF-a caused ½F3 activation of both p50/p65 and p100/p52 dimers as evidenced by the binding of the active dimers to the respective kB binding site on the oligonucleotide probe as determined by ELISA binding assay (Figure 3, B and C), indicating that both canonical and noncanonical NF-kB pathways were activated in Caco-2 cells. The siRNA-induced silencing of p65 resulted in a complete inhibition of TNF-aeinduced drop in Caco-2 TER and increase in inulin flux (Figure 3, D and E). In contrast, siRNA-induced knockdown of p52 did not affect the TNF-a drop in Caco-2 TER or increase in inulin flux (Figure 3, F and G). Collectively, these data suggested that TNF-a activation of the canonical pathway,

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807 808 **** 809 6 15 **** Time (min) 0 10 30 60 120 810 **** **** **** 4 10 811 **** **** Iκ κB-α 812 **** 2 5 β-actin 813 814 0 0 0 10 30 60 120 0 10 30 60 120 815 Time (min) Time (min) 816 817 6 120 G D 120 E 6 ** F ** ** ** 818 ** †† †† †† †† †† 819 100 100 4 4 820 ** ** †† ** 80 80 821 ** ** 2 2 †† †† 822 †† †† 60 60 823 0 0 824 C TNF siNT siNT sip52 sip52 control TNF siNT siNT sip52 sip52 C TNF siNT siNT sip65 sip65 control TNF siNT siNT sip65 sip65 +TNF +TNF 825 +TNF +TNF +TNF +TNF +TNF +TNF 826 Figure 3 Effect of 10 ng/mL TNF-a on Caco-2 NF-kB pathway (p65 and p52) activation. A: TNF-a causes a time-dependent degradation in IkB-a 827 expression. B: TNF-a treatment causes a significant increase in Caco-2 NF-kB p65 binding to the DNA probe as assayed by ELISA-based DNA binding assay of 828 NF-kB p65. C: TNF-a treatment causes a significant increase in Caco-2 NF-kB p52 binding to the DNA probe as assayed by ELISA-based DNA binding assay of 829 NF-kB p52. D: NF-kB p65 siRNA transfection prevents the TNF-aeinduced drop in Caco-2 TER. E: NF-kB p65 siRNA transfection prevents the TNF-aeinduced 830 increase in inulin flux. F: NF-kB p52 siRNA transfection does not prevent the TNF-aeinduced drop in Caco-2 TER. G: NF-kB p52 induced silencing by siRNA 831 transfection does not prevent the TNF-aeinduced increase in inulin flux. Data are expressed as means  SEM. n Z 4. **P < 0.005, ****P < 0.0001 versus control; yyP < 0.005 versus TNF-a treatment. C, no siRNA controls; ELISA, enzyme-linked immunosorbent assay; IkB, inhibitory kB; NT, non-targeted siRNA; 832 TER, transepithelial electrical resistance; TNF, tumor necrosis factor. 833 834 but not the noncanonical pathway, was required for the degradation of IkB-a and activation of p65 subunit 835 TNF-aeinduced increase in intestinal TJ permeability. (Figure 6). These were unexpected findings and suggested ½F6 836 Next, the regulatory role of NIK or MEKK-1 on TNFthat IKK-a, and not IKK-b (as part of IKK complex), 837 aeinduced activation of p50/p65 dimer was examined. The was the catalytic subunit responsible for mediating the 838 839 siRNA-induced knockdown of NIK prevented the TNF840 A B aeinduced degradation of IkB-a and activation of p50/p65 841 ½F4 in Caco-2 cells (Figure 4, A and B). However, the siRNA **** **** C siNIK TNF siNIK 842 knockdown of MEKK-1 did not affect the degradation of +TNF 843 Iκ κB-α IkB-a or the activation of p50/p65 (Figure 4, C and D). †† 844 These data suggested that NIK (but not MEKK1) mediated β-actin †††† †††† 845 the TNF-a activation of the canonical pathway. 846 847 Role of IKK Catalytic Subunits in TNF-aeinduced 848 C D 20 Increase in Caco-2 TJ Permeability 849 **** **** 15 **** C siMEKK-1 TNF siMEKK-1 850 +TNF 10 The role of IKKs in mediating the TNF-aeinduced increase 851 IκB-α 5 in intestinal TJ permeability was examined. In these studies, 852 †††† †††† β-actin 853 0 the involvement of IKK catalytic subunits, IKK-a and IKKC TNF siNT siNT siMEKK1siMEKK1 854 +TNF +TNF b, was examined. TNF-a (10 ng/mL) caused a rapid acti855 vation of both IKK-a and IKK-b subunits as assessed by Figure 4 Effect of siRNA-induced MAP3 kinase knockdown on TNF-a 856 Q12 ½F5 subunit phosphorylation (Ser176/177) (Figure 5A). To activation of NF-kB p65. A: NIK siRNA transfection prevents the TNF857 aeinduced degradation of IkB-a as assessed by Western blot analysis. B: NIK identify which IKK subunit mediated the TNF-a effect on 858 silencing inhibits the TNF-aeinduced binding of p65 to its binding site on Caco-2 TJ permeability, the expression of IKK-a or IKK-b 859 DNA probe as measured by DNA ELISA-binding assay. C: siRNA-induced was selectively silenced via siRNA transfection. The IKK-a knockdown of MEKK-1 does not prevent the TNF-aeinduced degradation of 860 knockdown, but not IKK-b knockdown, prevented both the IkB-a. D: siRNA-induced knockdown of MEKK-1 does not prevent the TNF861 aeinduced binding of p65 to its binding site on DNA probe as measured TNF-aeinduced drop in Caco-2 TER and increase in par862 by DNA ELISA-binding assay. Data are expressed as means  SEM. acellular permeability (Figure 5), suggesting that IKK-a but 863 yy yyyy ****P < 0.0001 versus control; P < 0.005, P < 0.0001 versus TNF-a not IKK-b was required for the increase in Caco-2 TJ 864 treatment. C, no siRNA controls; ELISA, enzyme-linked immunosorbent assay; 865 permeability. Next, the involvement of IKK subunits in the IkB, inhibitory kB; MAP3 kinase, mitogen-activated protein kinase, kinase, 866 TNF-a activation of NF-kB p65 was also examined. IKK-a, kinases; MEKK-1, mitogen-activated protein kinase kinase kinase-1; NIK, NF867 kB-inducing kinase; NT, non-targeted siRNA; TNF, tumor necrosis factor. but not IKK-b, silencing inhibited the TNF-aeinduced 868

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Mechanism of TNF-a Regulation of Intestinal Barrier Function

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869 A B C C siIKK-α 870 IKK-α 871 872 120 8 β-actin 873 Time (min) 0 10 30 60 120 †† 874 †† ** P-IKK-α 100 †† 6 875 ** P-IKK-β β 876 80 4 β-actin 877 ** ** †† 878 †† 60 †† 2 879 880 40 0 control TNF siNT siNT siIKK-α siIKK-α 881 C TNF siNT siNT siIKK-α siIKK-α +TNF +TNF 882 +TNF +TNF 883 C siIKK-β D E 884 IKK-β 885 120 8 β-actin 886 887 †† ** †† 100 6 888 ** ** 889 80 4 890 ** 891 ** ** †† †† 892 2 60 893 894 0 40 control TNF siNT siNT siIKK-β siIKK-β C TNF siNT siNT siIKK-β siIKK-β 895 +TNF +TNF +TNF +TNF 896 897 Figure 5 Time course effect of TNF-a on Caco-2 IKK catalytic subunit activation. A: TNF-a (10 ng/mL) causes a time-dependent increase in Caco-2 IKK-a and IKK-b phosphorylation. B: IKK-a siRNA transfection completely prevents the TNF-aeinduced drop in Caco-2 TER. C: siRNA-induced knockdown of IKK-a 898 prevented the TNF-aeinduced increase in inulin flux. D: IKK-b siRNA transfection does not prevent the TNF-aeinduced drop in Caco-2 TER. E: siRNA-induced 899 knockdown of IKK-b does not inhibit the TNF-aeinduced increase in inulin flux. Data are expressed as means  SEM. n Z 4. **P < 0.005 versus control; 900 yy P < 0.005 versus TNF-a treatment. C, no siRNA controls; IKK, inhibitory kB kinase; NT, non-targeted siRNA; TER, transepithelial electrical resistance; 901 TNF, tumor necrosis factor. 902 903 904 TNF-aeinduced degradation of IkB-a and activation of in MLCK promoter activity, MLCK mRNA expression, and 905 NF-kB p65 and the subsequent increase in TJ permeability. MLCK protein expression (Figure 7, CeE).23,42 The siRNA 906 Next, the MAP3 kinase responsible for the TNF-a actiknockdown of NIK prevented the TNF-aeinduced increase 907 vation of IKK-a was determined. The siRNA knockdown of in MLCK promoter activity, MLCK mRNA expression, and 908 NIK inhibited the TNF-aeinduced phosphorylation of IKKMLCK protein expression (Figure 7, CeE). The siRNA 909 a; however, MEKK-1 knockdown did not have any effect silencing of MEKK-1 did not have any effect (Figure 7, 910 FeH). These data indicated that the TNF-aeinduced in911 ½F7 (Figure 7, A and B). Interestingly, the siRNA depletion of crease in Caco-2 TJ permeability was regulated by NIK MEKK-1, but not NIK, inhibited the phosphorylation of 912 pathway activation of MLCK gene. IKK-b (data not shown). These results suggested that 913 NIK was responsible for the TNF-aeinduced activation 914 915 of IKK-a and MEKK-1 mediated the phosphorylation Potential Crosstalk between NIK and MAP Kinases 916 of IKK-b. ERK1/2 and p38 Kinase 917 918 Previous studies from our laboratory showed that the Role of NIK in TNF-a Regulation of MLCK Gene Activity 919 TNF-aeinduced increase in intestinal TJ permeability was 920 mediated in part by extracellular signal-regulated kinase Previous studies have shown that the TNF-aeinduced 921 increase in Caco-2 TJ permeability was regulated by the (ERK)1/2 signaling pathway16 and that IL-1b increase in 922 42 activation of MLCK gene. In the following studies, we intestinal TJ permeability involved p38 kinase pathway.40 923 examined the involvement of NIK in MLCK gene regulaBecause, TNF-a is known to activate MAP kinases, the 924 tion by assessing MLCK promoter activity and MLCK possible crosstalk between MAP3 kinase NIK and MAP 925 mRNA transcription. To assess the MLCK promoter activkinases ERK1/2 and p38 kinase in TNF-aeinduced acti926 927 ity, Caco-2 cells were transfected with plasmid vector vation of the canonical pathway was also examined. TNF-a 928 encoding the MLCK promoter region and the luciferase caused an activation of both ERK1/2 and p38 kinase, as 929 reporter gene. The TNF-a treatment resulted in an increase evidenced by the phosphorylation of ERK1/2 and p38 930

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Effect of siRNA IKK subunit knockdown on TNF-aeinduced activation of NF-kB p65. A: siRNA-induced knockdown of IKK-a completely abolishes the TNF-aeinduced degradation of IkB-a. B: IKK-a siRNA transfection inhibits the TNF-aeinduced binding of p65 to its binding site on DNA probe as measured by DNA ELISA-binding assay. C: IKK-b siRNA transfection does not prevent the TNF-aeinduced degradation of IkB-a. D: siRNA-induced knockdown of IKK-b does not inhibit the TNF-aeinduced binding of p65 to its binding site on DNA probe as measured by DNA ELISAbinding assay. ****P < 0.0001 versus control; yyyyP < 0.0001 versus TNF-a treatment. C, no siRNA controls; ELISA, enzyme-linked immunosorbent assay; IkB, inhibitory kB; IKK, inhibitory kB kinase; NT, non-targeted siRNA; TNF, tumor necrosis factor.

kinase (Figure 7, I and J). To determine the possible regulatory role of NIK in the MAP kinase activation, the effect of siRNA knockdown of NIK on TNF-aeinduced activation of ERK1/2 and p38 kinase was examined. The siRNA knockdown of NIK did not affect the TNF-aeinduced phosphorylation of ERK1/2 but inhibited the p38 kinase phosphorylation (Figure 7, I and J), indicating that p38 kinase activation depended on NIK. Note that MEKK1 knockdown completely prevented the TNF-aeinduced phosphorylation of ERK1/2 (data not shown). Interestingly, the siRNA-induced knockdown of p38 kinase did not affect the TNF-aeinduced degradation of IkB-a (Figure 7K). In combination, our results suggested that the TNF-aeinduced activation of p38 kinase is regulated by NIK, but p38 kinase is not involved in TNF-aeinduced degradation of IkB-a. Thus, it appears that the NIK pathway activation of NF-kB p50/p65 is independent of MAP kinases ERK1/2 and p38 kinase.

Role of NIK in TNF-aeInduced Increase in Intestinal Permeability in Vivo The above-mentioned studies indicated that NIK/IKK-a/ p50/p65 axis mediated the TNF-aeinduced activation of MLCK gene and Caco-2 permeability. However, whether similar kinase axis also plays a role in in vivo animal system remains unclear. In the following studies, we examined the role of NIK signaling cascade in the TNF-aeinduced

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1055 increase in mouse intestinal permeability by in vivo recy39 1056 cling intestinal perfusion of mouse small intestine. The 1057 intraperitoneal injection of TNF-a (5 mg) caused a threefold 1058 increase in mouse intestinal permeability to paracellular 1059 marker dextran 10 kDa (Figure 8A). TNF-a treatment also ½F8 1060 resulted in a time-dependent degradation of IkB-a in mouse 1061 intestinal tissue (Figure 8B), consistent with the activation 1062 of NF-kB p50/p65. The TNF-a administration also caused a 1063 time-dependent phosphorylation of NIK (Thr 559) and 1064 1065 MEKK-1 (Thr 1381) in mouse intestinal tissue (Figure 8C), 1066 confirming the activation of the upstream MAP3 kinases in 1067 response to the TNF-a treatment. TNF-a also caused an 1068 increase in intestinal tissue MLCK mRNA and protein expression in mouse small intestine (Figure 9, A and B). To ½F9 1069 1070 determine the requirement of NIK or MEKK-1 in TNF-a 1071 regulation of MLCK expression and intestinal permeability 1072 in vivo, intestinal tissue NIK or MEKK-1 expression was 1073 selectively silenced with the in vivo siRNA transfection 1074 method.39e41 Briefly, 6 cm of mouse small intestine was 1075 isolated with sutures, and the mucosal surface was exposed 1076 1077 to siRNA transfection solution that contained NIK or 1078 MEKK-1 siRNA for 1 hour. The sutures were removed, the 1079 intestinal segment was reinserted into the original location 1080 in the abdomen, and the abdominal cavity was closed with 1081 sutures. After 2 days, mice were treated with TNF-a, and 1082 intestinal permeability studies were performed on day 3. 1083 The in vivo siRNA knockdown of intestinal tissue NIK, but 1084 not MEKK-1 (Figure 9, C and F), prevented the TNF1085 aeinduced increase in intestinal MLCK expression 1086 (Figure 9, D and G) and intestinal permeability (Figure 9, E 1087 and H), demonstrating the requirement of NIK in TNF1088 1089 aeinduced increase in MLCK expression and intestinal 1090 permeability in vivo. Next, the effect of siRNAeinduced 1091 silencing of NIK or MEKK-1 on IkB-a degradation was 1092 examined. The siRNA-induced knockdown of NIK but not 1093 MEKK-1 prevented the TNF-aeinduced degradation of 1094 IkB-a in mouse intestine (Figure 10, A and B), suggesting ½F10: 1095 that NIK mediated the TNF-aeinduced degradation of 1096 IkB-a and activation of NF-kB p50/p65 in mouse intestine. 1097 The in vivo siRNA knockdown of intestinal NF-kB p65 1098 prevented the TNF-aeinduced increase in intestinal tissue 1099 expression of MLCK and increase in mouse intestinal 1100 1101 permeability (Figure 10, C and D). Together, these data 1102 suggested that the TNF-aeinduced increase in mouse in1103 testinal permeability was also regulated by NIK/NF-kB p50/ 1104 p65 axis activation of MLCK gene and MLCK-dependent 1105 increase in mouse intestinal permeability.

Discussion Previous studies have shown that TNF-a causes an increase in intestinal TJ permeability.5,17 The TNF-aeinduced increase in intestinal TJ permeability is an important factor that contributes to the observed increase in intestinal permeability in inflammatory bowel disease patients.13,43 In Q14

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Effect of siRNA-induced MEKK-1 and NIK knockdown on TNF-a activation of IKK-a. A: siRNA-induced knockdown of NIK prevents the TNFaeinduced phosphorylation of IKK-a as assessed by Western blot analysis. B: siRNA-induced knockdown of MEKK-1 does not prevent the TNF-aeinduced phosphorylation of IKK-a. Effect of siRNA induced knockdown of NIK and MEKK-1 on TNF-aeinduced increase in MLCK gene activity and protein expression. C: siRNA-induced knockdown of NIK results in a complete inhibition of TNF-aeinduced increase in MLCK promoter activity. D: siRNA-induced knockdown of NIK prevents the TNF-aeinduced increase in MLCK mRNA levels. E: NIK silencing by siRNA transfection prevents the TNF-aeinduced increase in MLCK protein expression. F: siRNA-induced knockdown of MEKK-1 does not prevent the TNF-aeinduced increase in MLCK promoter activity. G: Knocking-down MEKK-1 by siRNA does not prevent the TNF-aeinduced increase in MLCK mRNA levels. H: Knocking-down MEKK-1 by siRNA does not affect the TNF-aeinduced increase in MLCK protein expression. I: Knocking-down NIK by siRNA prevents the TNF-aeinduced phosphorylation of p38 kinase (siNIK: siRNA NIK transfection). J: Knocking-down NIK by siRNA does not inhibit the TNF-aeinduced phosphorylation of ERK1/2. K: Knocking-down p38 kinase by siRNA does not inhibit the TNF-aeinduced degradation of IkB-a. *P < 0.05, **P < 0.005 versus control; yyP < 0.005, yyyP < 0.001 versus TNF-a treatment. C, no siRNA controls; ERK, extracellular signal-related kinase; IKK, inhibitory kB kinase; MEKK-1, mitogen-activated protein kinase kinase kinase-1; MLCK, myosin light chain kinase; NIK, NF-kB-inducing kinase; NT, nontargeted siRNA; siNIK: siRNA NIK transfection; sip38: siRNA p38 kinase transfection; T, TNF-a for 30 minutes; TNF, tumor necrosis factor.

this regard, treatment of inflammatory bowel disease patients with antieTNF-a antibody therapy resulted in rapid retightening of the intestinal barrier and improvement of the disease.20,22,44,45 Previous studies from our laboratory have shown that NF-kB p50/p65 plays a central role in the TNFaeinduced increase in intestinal TJ permeability by targeting MLCK gene activation.17,23,42 However, the signaling pathways and the intracellular mechanisms that mediate TNF-aeinduced activation of NF-kB p50/p65 and MLCK gene in intestinal epithelial cells remain unknown. Herein, we investigated the involvement of canonical and

10

noncanonical NF-kB pathways and the specific protein kinases that mediate the TNF-aeinduced activation of NFkB and MLCK gene and increase in intestinal TJ permeability. Our results demonstrate a new signaling pathway activation of NF-kB p50/p65 or canonical pathway in which NIK, a MAP3 kinase shown to be involved primarily in the activation of the noncanonical pathway, activates the canonical pathway via IKK-a degradation of IkB-a. The MAP3 kinases, including NIK and MEKK-1, are important upstream regulators of NF-kB activation.26,46 The NF-kB dimers are formed by the individual Rel subunits,

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Figure 8 Effect of TNF-a activation of NF-kB pathway in mouse intestinal permeability. A: TNF-a (5 mg) causes an increase in mouse intestinal mucosal-to-serosal flux of dextran 10 kDa. B: TNF-a causes a time-dependent increase in IkB-a degradation in mouse intestinal tissue, starting at 2 hours and continuing up to 24 hours as assessed by Western blot analysis. C: TNF-a caused a time-dependent increase in phosphorylation of NIK and MEKK-1 in mouse intestinal tissue as assessed by Western blot analysis. **P < 0.01. IkB, inhibitory kB; MEKK-1, mitogen-activated protein kinase kinase kinase-1; NIK, NF-kB-inducing kinase; TNF, tumor necrosis factor.

pathways.30,49 In the canonical pathway, the ligand binding to the membrane receptor leads to the phosphorylation and degradation of IkB protein (most common subtype being the IkB-a) and activation of NF-kB dimer p50/p65.49e52

including p65 (often referred to as RelA), RelB, c-Rel, p50, and p52.47,48 Two distinct NF-kB signaling pathways have been identified that regulate the activation of the canonical (or classic) and the noncanonical (or alternative) 2.5

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Effect of TNF-a on MLCK expression in vivo. A: TNF-a causes an increase in mouse intestinal tissue MLCK mRNA transcript as assessed by real-time PCR; TNF-a 24 hours of treatment. B: TNF-a causes a time-dependent increase in mouse intestinal tissue MLCK protein expression as assessed by Western blot analysis. C: NIK siRNA transfection in vivo results in a near-complete knockdown of NIK expression in mouse intestinal tissue. D: NIK siRNA transfection in vivo prevents the TNF-aeinduced increase in mouse intestinal MLCK protein expression as assessed by Western blot analysis. E: NIK siRNA transfection in vivo prevents the TNF-aeinduced increase in mouse intestinal mucosal-to-serosal flux of dextran 10 kDa. F: MEKK-1 siRNA transfection in vivo results in a near-complete knockdown of MEKK-1 expression in mouse intestinal tissue. G: siRNA-induced knockdown of MEKK-1 in vivo does not prevent the TNF-aeinduced increase in mouse intestinal MLCK protein expression as assessed by Western blot analysis. H: MEKK-1 siRNA transfection in vivo does not prevent the TNF-aeinduced increase in mouse intestinal mucosal-to-serosal flux of dextran 10 kDa. **P < 0.01, ***P < 0.001 versus control; yyP < 0.005 versus TNF-a treatment. MEKK-1, mitogenactivated protein kinase kinase kinase-1; MLCK, myosin light chain kinase; NIK, NF-kB-inducing kinase; TNF, tumor necrosis factor.

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Effect of siRNA-induced silencing of NIK and MEKK-1 on NF-kB signaling pathway and mouse intestinal permeability. A: NIK siRNA transfection in vivo prevents the TNF-aeinduced degradation of IkB-a in mouse intestinal tissues. B: MEKK-1 siRNA transfection in vivo does not inhibit the TNFaeinduced degradation of IkB-a in mouse intestinal tissues. C: NF-kB p65 siRNA transfection in vivo prevents the TNFeaeinduced increase in mouse intestinal MLCK protein expression as assessed by Western blot analysis. D: NF-kB p65 siRNA transfection in vivo prevents the TNF-aeinduced increase in mouse intestinal mucosal-to-serosal flux of dextran 10 kDa. ***P < 0.001 versus control; yyP < 0.005 versus TNF-a treatment. IkB, inhibitory kB; MEKK-1, mitogenactivated protein kinase kinase kinase-1; MLCK, myosin light chain kinase; NIK, NF-kB-inducing kinase; TNF, tumor necrosis factor.

NF-kB p50/p65 is the dominant NF-kB dimer and makes up >90% of the NF-kB dimers present in the cells. In the noncanonical pathway, ligand binding leads to the activation of NIK, which, in turn, leads to the phosphorylation and processing of p100 subunit into p52 and generation of activated RelB/p52 dimer.29,53 To date, NIK is the only MAP3 kinase that has been shown to signal RelB/p52 activation or the noncanonical pathway.54,55 Previous studies have shown MEKK-1 to be an important regulator of the canonical pathway; MEKK-1 induces the activation of the trimeric IKK complex, consisting of the catalytic IKK subunits, IKK-a and IKK-b, and the regulatory subunit IKK-g/NEMO.36,56e58 IKK-g does not have catalytic activity but plays a critical role in the IKK complex formation.59,60 The activation of MEKK-1 leads to the activation of IKK complex with phosphorylation of IKK-a and IKK-b.56 The final outcome of the canonical pathway signaling is the phosphorylation and degradation of IkB-a and activation of p50/p65 dimer, and the noncanonical pathway is the phosphorylation and processing of p100 and the generation of RelB/p52 dimer.26,35,51,56,61 Herein, we show that TNF-a causes activation of both canonical and noncanonical pathways in Caco-2 monolayers as evidenced by the activation of p50/p65 and RelB/p52 dimers. Our data suggested that NIK was responsible for activating both the canonical and the noncanonical pathways, because siRNA-induced silencing of NIK inhibited the TNF-a activation of p50/p65 and RelB/p52 dimers. Surprisingly, MEKK-1 depletion did not affect the TNF-a activation of p50/p65. These data suggested that NIK but not MEKK-1 mediated the TNF-aeinduced activation of both noncanonical (RelB/p52) and canonical (p50/p65) pathways. It had been previously shown that in the noncanonical pathway, NIK phosphorylates IKK-a at Ser-176 and Ser-180 without affecting the trimeric IKK complex. Whereas, in the canonical pathway, MAP3 kinases, including MEKK-1, activate the IKK complex, leading to the phosphorylation of serine residues within the activation loop of the catalytic subunits IKK-a (Ser-176 and Ser-180) and IKK-b (Ser-177 and Ser-181).46,56 The activated IKK catalytic subunits then phosphorylate IkB-a protein at Ser-32 and Ser-36, leading to

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the ubiquitination and proteasome-induced degradation and nuclear translocation of p50/p65 dimer.62e65 Although both IKK-b and IKK-a can catalyze IkB-a phosphorylation, IKK-b is the dominant catalytic subunit that catalyzes the IkB-a phosphorylation.65e67 NIK is known to phosphorylate and activate IKK-a, but it can also directly phosphorylate p100.68,69 Our results indicate that TNF-a caused the activation of both canonical and noncanonical pathways, but only the siRNA depletion of p65 but not p52 inhibited the TNFaeinduced activation of MLCK gene and increase in Caco-2 TJ permeability, confirming that p50/p65 but not RelB/p52 dimer mediated the TNF-a modulation of MLCK gene activity and increases in Caco-2 TJ permeability. As described previously, the increase in MLCK expression leads to MLCKdependent opening of the TJ barrier.70,71 It has been shown that MLCK catalyzes the phosphorylation of MLC; which in turn activates Mg2þemyosin ATPase, leading to the contraction of perijunctional acto-myosin filaments and mechanical tension-induced opening of the TJ barrier.72,73 MLCK expression has been shown to be markedly increased in intestinal tissue of patients with inflammatory bowel disease.74 MLCK is also essential to the permeability of intestinal epithelial barrier function in vitro and in vivo; MLCK gene is activated in response to proinflammatory cytokine, including TNF and IL-1b in the inflamed intestinal tissues.5,36,75 In addition, several recent studies have found the pathogenic role of MLCK in both intestinal barrier dysfunction and intestinal inflammation in animal models of inflammatory bowel disease.39,76 In addition, other studies found that interferon-g induces endocytosis of TJ transmembrane proteins and increase in intestinal epithelial TJ permeability in T-84 monolayers by triggering selective vacuolarization of the apical plasma membrane that was mediated by myosin IIedriven contraction.77 An important novel finding of our study is the demonstration that the TNF-aeinduced activation of NF-kB p50/ p65 in Caco-2 monolayers is mediated by what is traditionally considered to be a noncanonical pathway NIK/IKKa axis degradation of IkB-a. Although both IKK-b and IKK-a were activated in response to TNF-a treatment, only IKK-a depletion inhibited the TNF-a degradation of IkB-a

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and subsequent MLCK gene activation. Neither MEKK-1 (which catalyzed the IKK-b phosphorylation and activation) nor IKK-b depletion affected the IkB-a degradation, p50/ p65 activation or MLCK gene activation. This is in contrast to previous published reports that show IKK-b (as part of the IKK complex) to be the kinase mainly responsible for IkB-a phosphorylation in various cell types.78e80 Although there are a number of reports showing MEKK-1/IKK-b to be the upstream kinases that regulate NF-kB p50/p65 activation,56,57,81 there are also a few reports showing that NIK can also mediate p50/p65 activation.82,83 In this regard, previous studies with peptidoglycan and IL-1a showed that IKK-a activation was associated with the canonical NF-kB pathway activation in HeLa and fibroblast cells.84e86 Herein, we show for the first time that NIK/IKK-a axis, independent of MEKK-1 and IKK-b, regulates the TNFaeinduced activation of NF-kBp50/p65 in intestinal epithelial cells and, thereby, also mediates MLCK gene activation and increase in intestinal epithelial TJ permeability. These present findings with TNF-a are quite distinct from our previous studies showing that IL-1beinduced activation of MLCK gene was mediated by MEKK1/IKK-beinduced degradation of IkB-a and NF-kB p50/p65 activation. Thus, our results demonstrate that the TNF-a and IL-1b activation of NF-kB p50/p65 is mediated by distinct MAP3 kinases and IKK catalytic subunits. The in vivo mouse intestinal perfusion studies were also performed to determine the involvement of the NIK in TNFaeinduced increase in mouse intestinal permeability. Similar to the cell culture studies with Caco-2 monolayers, the in vivo mouse studies also showed that TNF-a causes activation of both NIK and MEKK-1 in mouse small intestine. The siRNA depletion of NIK prevented the TNFaeinduced increase in mouse intestinal expression of MLCK and increase in intestinal permeability, whereas MEKK-1 knockdown did not have any effect. These data suggested that intestinal tissue activation of NIK was required for the TNF-aeinduced increase in mouse intestinal MLCK expression and intestinal permeability. Our in vivo studies also showed that NIK was a key regulator of TNF-aeinduced IkB-a degradation and p50/p65 activation. Thus, in vivo data indicated that NIK also plays an integral role in mediating the TNF-aeinduced activation of the canonical pathway, increase in MLCK gene expression and increase in mouse intestinal permeability.

Conclusion In conclusion, the results of this study provide important novel insight into the signaling processes that mediate TNFa modulation of intestinal epithelial TJ permeability in vitro and in vivo. Our data show that TNF-aeinduced increase in intestinal TJ permeability is mediated by NIK/IKK-a/ NF-kB p50/p65 axis activation of MLCK gene. Although TNF-a also caused the activation of MEKK-1/IKK-b axis,

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the traditional pathway known to regulate the canonical pathway, this axis was not involved in the TNF-aeinduced activation of NF-kB p50/p65 or the increase in intestinal TJ permeability. Thus, our results identify an important new pathway activation of NF-kB p50/p65 in the intestinal epithelial cells and provide novel insight into the mechanism of intestinal TJ barrier regulation.

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