Inhibition of Interleukin-1-stimulated NF-κB RelA/p65 Phosphorylation by Mesalamine Is Accompanied by Decreased Transcriptional Activity*

Nuclear factor κB (NF-κB) is an inducible transcription factor that regulates genes important in immunity and inflammation. The activity of NF-κB is highly regulated: transcriptionally active NF-κB proteins are sequestered in the cytoplasm by inhibitory proteins, IκB. A variety of extracellular signals, including interleukin-1 (IL-1), activate NF-κB by inducing phosphorylation and degradation of IκB, allowing nuclear translocation and DNA binding of NF-κB. Many of the stimuli that activate NF-κB by inducing IκB degradation also cause phosphorylation of the NF-κB RelA (p65) polypeptide. The transactivating capacity of RelA is positively regulated by phosphorylation, suggesting that in addition to cytosolic sequestration by IκB, phosphorylation represents another mechanism for control of NF-κB activity. In this report, we demonstrate that mesalamine, an anti-inflammatory aminosalicylate, dose-dependently inhibits IL-1-stimulated NF-κB-dependent transcription without preventing IκB degradation or nuclear translocation and DNA binding of the transcriptionally active NF-κB proteins, RelA, c-Rel, or RelB. Mesalamine was found to inhibit IL-1-stimulated RelA phosphorylation. These data suggest that pharmacologic modulation of the phosphorylation status of RelA regulates the transcriptional activity of NF-κB, independent of nuclear translocation and DNA binding. These findings highlight the importance of inducible phosphorylation of RelA in the control of NF-κB activity.

The NF-B 1 family of proteins are important transcriptional regulators of genes involved in immunity and inflammation (1,2). The activity of NF-B is induced by a wide variety of stimuli, including pro-inflammatory cytokines such as IL-1 and TNF-␣, oxidant stress, bacterial endotoxin, and viral infection. Elevated NF-B activity has been observed in a number of inflammatory disease states, including the gut mucosa in inflammatory bowel disease (3,4), the inflamed synovium in rheumatoid arthritis (5), and reactive airways in asthma (6). For these reasons, the NF-B system is an attractive target for therapeutic inhibition in chronic inflammatory conditions (7).
The transcriptionally active NF-B proteins, RelA (p65), c-Rel, and RelB are characterized by a conserved region known as the Rel homology domain, after homology to the viral oncogene v-Rel. The Rel homology domain contains motifs essential for dimerization, nuclear localization, and DNA binding of NF-B. The activity of NF-B is highly regulated. In the basal unstimulated state, NF-B resides in the cytoplasm as homoor heterodimers bound to a family of inhibitory molecules termed IB, which mask the nuclear localization signal. Stimuli that activate NF-B initiate a cascade of signaling events that culminate in phosphorylation, ubiquination, and subsequent proteasomal degradation of IB (8). Consequently, NF-B is freed to translocate to the nucleus and initiate transcription of a variety of genes, including a gene encoding one of its cytosolic inhibitors, IB␣.
Recently it has been demonstrated that the transcriptional activity of NF-B can be regulated by mechanisms other than cytosolic sequestration by IB. The NF-B protein RelA can be inducibly phosphorylated by the same stimuli that cause degradation of IB. Several reports have demonstrated that the DNA binding (9,10) and transactivating capacity of NF-B (11)(12)(13) are up-regulated by inducible phosphorylation of RelA. Thus, pathways that regulate the phosphorylation status of RelA and possibly other transcriptionally active NF-B proteins constitute another potential mechanism for control of transcriptional activity.
Salicylate drugs are in widespread use as inhibitors of inflammation in a variety of disease states. Sodium salicylate and acetyl salicylic acid inhibit NF-B by preventing inducible degradation of IB, and this action may underlie the in vivo anti-inflammatory effects of these agents (14). Aminosalicylates such as sulfasalazine (an azo-conjugated aminosalicylate) and mesalamine (5-aminosalicylic acid, a free aminosalicylate) inhibit gut inflammation in inflammatory bowel disease (15) (Fig. 1). Like conventional salicylates, sulfasalazine has been reported to inhibit NF-B activity by preventing inducible IB␣ degradation (16). In this report we show that mesalamine is an inhibitor of inducible NF-B-dependent transcription in intestinal epithelial cells and T lymphocytes. However, unlike conventional salicylates and sulfasalazine, mesalamine does not prevent IL-1-induced IB␣ or ␤ degradation. The transcriptionally active NF-B proteins RelA, RelB, and c-Rel translocate to the nucleus and bind to B sites on DNA but are unable to initiate transcription in the presence of mesalamine. Further experiments demonstrated that mesalamine inhibits IL-1-induced phosphorylation of RelA. This suggests that mesalamine regulates NF-B activity by modulating the phosphorylation of one of its transcriptionally active proteins and that the path-ways leading to inducible RelA phosphorylation constitute an independent mechanism for the regulation of NF-B activity.
Transient Transfections and NF-B Reporter Gene Assay-An NF-B reporter construct, consisting of the firefly luciferase gene under control of three copies of the consensus NF-B site from the IgG promoter was used to quantify NF-B transcriptional activity (18). A Renilla luciferase reporter under control of the herpes simplex virus thymidine kinase promoter, pRLTK (Promega, Madison, WI), was used to normalize the NF-B reporter gene activity, to prevent nonspecific drug effects such as cytotoxicity confounding the results. Caco-2 cells were first brought into suspension by trypsinization. Caco-2 or Ju.1 cells (10 7 ) were mixed with 10 g NF-B reporter gene, 20 ng of pRLTK and 20 g of filler DNA and pulsed once with 325 V for 10 ms using a square wave electroporater (BTX, San Diego, CA). For experiments, 10 7 transfected Caco-2 cells were distributed into 30 wells of a 48-well tissue culture plate, and 10 7 transfected Jurkat cells were distributed into 6 wells of a 6-well tissue culture plate. Experiments were performed 12-48 h after transfection. For luciferase assays, cell lysates were prepared, and luciferase activities were read according to the manufacturer's instructions (Dual luciferase, Promega), using a model LB 9501/16 Lumat luminometer (Berthold Systems, Aliquippa, PA). Results shown are representative of at least three independent experiments.
Analysis of Protein Synthesis-Caco-2 cells (2.5 ϫ 10 5 ) were plated in 6-well tissue culture plates and allowed to adhere overnight. Cells were depleted of leucine by replacing the medium with leucine free RPMI supplemented with 2% dialyzed fetal calf serum for 4 h, which was changed once. After addition of mesalamine or control, 25 Ci of [ 3 H]leucine (L-4,5-[ 3 H]leucine, 136 Ci/mmol, Amersham Pharmacia Biotech) was added to each well. After various durations, [ 3 H]leucine incorporation into precipitable protein was quantified. Medium was aspirated, and the monolayers were washed twice with calcium and magnesium-free phosphate-buffered saline containing 1 mM EDTA. Cells were scraped, transferred to microtubes, and centrifuged, and the supernatant was discarded. Protein was precipitated by addition of 500 l of 10% trichloroacetic acid to the cell pellet, which was then vortexed and centrifuged at 12,000 rpm for 10 min. The supernatant was discarded, and the protein precipitation step was repeated once. The precipitated protein was resolubilized by addition of 500 l of 0.3 N NaOH in 1% SDS for 30 min with vortexing. [ 3 H]Leucine incorporation was determined by liquid scintillation counting of aliquots of the resolubilized protein.
Quantitative Immunoblot Analysis of Cytosolic IB-Caco-2 cells (6 ϫ 10 6 ) were plated in 60-mm tissue culture dishes and allowed to adhere overnight. After various experimental procedures, cytosolic lysates were prepared as follows: medium was aspirated, and the Caco-2 monolayers were washed twice with ice-cold phosphate-buffered saline. 1 ml of lysis buffer consisting of 50 mM Tris, 300 mM NaCl, 5 mM EDTA, 10 mM iodoacetamide, 1 mM Na 2 VO 4 , 0.5% Triton X-100, pH 7.6, supplemented immediately before use with 0.4 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, and 10 g/ml aprotinin, was added to each monolayer and allowed to lyse the cells for at least 30 min at 4°C. Ju.1 cells in suspension culture were pelleted by centrifugation and lysed in the same buffer. Lysates were then transferred to sialized microtubes and cleared by centrifugation at 12,000 rpm for 20 min at 4°C. IB␣ was immunoprecipitated from cytosolic preparations using antibody to IB␣ conjugated to protein A-Sepharose beads as described previously, under conditions of antibody excess (18). Immunoprecipitated proteins were separated by 8.75% SDS-PAGE and transferred to Immobilon-P membrane (Millipore, Bedford, MA). Blots were probed using antibody to IB␣ as described previously (18), and immunoreactive protein was detected using enhanced chemiluminescence reagents (Amersham Pharmacia Biotech).
Quantitative Immunoblot Analysis of Nuclear RelA, RelB, and c-Rel-Caco-2 cells (6 ϫ 10 6 ) were plated in 60-mm tissue culture dishes and allowed to adhere overnight. After various experimental procedures, nuclear lysates were prepared as follows: medium was aspirated, and the Caco-2 monolayers were washed twice with ice-cold calciumand magnesium-free phosphate-buffered saline containing 1 mM EDTA. Cells were disrupted by incubating the monolayer for 4 min in 1 ml of ice-cold buffer consisting of 10 mM HEPES, pH 7.9, 1.5 mM MgCl 2 , and 10 mM NaCl, supplemented immediately before use with 0.4 mM phenylmethylsulfonyl fluoride, 2 mM dithiothreitol, 50 g/ml leupeptin, 10 g/ml aprotinin, 150 M spermine, 500 M spermidine, and 0.4% Nonidet P-40. The disrupted monolayer was mixed in a pipette, transferred to microtubes, and centrifuged at 1500 rpm for 5 min at 4°C, and the cell pellet was washed with buffer lacking Nonidet P-40 to remove contaminating cytosolic proteins. Nuclear extracts were prepared by vortexing the cell pellets for 30 min at 4°C in 50 l of ice-cold buffer consisting of 20 mM HEPES, pH 7.9, 20% v/v glycerol, 0.4 M NaCl, 1.5 mM MgCl 2 , and 0.2 mM EDTA supplemented immediately before use with 200 M phenylmethylsulfonyl fluoride, 2 mM dithiothreitol, 50 g/ml leupeptin, 10 g/ml aprotinin, 150 M spermine, and 500 M spermidine. After centrifugation at 10,000 rpm for 10 min at 4°C, c-Rel, RelB, and RelA were sequentially immunoprecipitated from the supernatant using the appropriate antibodies conjugated to protein A-Sepharose beads under conditions of antibody excess, as described previously (18,19). SDS-PAGE and Western blotting were carried out as described above for cytosolic lysates.
Electrophoretic Mobility Shift Analysis-Nuclear lysates were prepared as for immunoblots, and the protein concentration was determined using the Coomassie Plus kit (Pierce). Mobility shift reactions were carried out using 6 g of protein from each nuclear extract incubated with 32 P-labeled IL-2 NF-B oligonucleotide probe as described previously (18). The DNA sequence of the probe used in this study is 5Ј-CCCGACCAAGAGGGATTTCACCTAAATCCATT-3Ј (coding strand only).
Phosphoprotein Analysis-Ju.1 and Caco-2 cells were labeled with 32 P i (ICN Radiochemicals, Irvine, CA), as described previously (18). Labeled cells were divided into aliquots (10 7 /sample) and, after various experimental conditions, were washed with ice-cold phosphate-buffered saline containing 400 M Na 3 VO 4 , 5 mM EDTA, and 10 mM NaF, pH 7.4. After centrifugation, the cells were lysed and immunoprecipitated for c-Rel, RelB, and RelA as described above. The precipitated phosphoproteins were separated by 10% SDS-PAGE and analyzed by autoradiography at Ϫ70°C. ulated NF-B transcription with a maximal effect at 40 mM and a half-maximal effect at 16 mM (Fig. 2a). At these concentrations, mesalamine did not inhibit transcription from the control reporter (data not shown). Consistent with previous reports (16), sulfasalazine also inhibited NF-B reporter gene activity, with a maximal effect at 10 mmol/liter and a half-maximal effect at 1.4 mmol/l (Fig. 2a). To determine whether mesalamine inhibited NF-B reporter gene activity in cell types other than epithelial cells, parallel NF-B reporter gene experiments were performed in Ju.1 lymphoid cells. Mesalamine also inhibited NF-B reporter gene activity in Ju.1 T lymphocytes, demonstrating that the effect is not cell type-specific (Fig. 2b).

Mesalamine Inhibits the Transcriptional Activity of NF-
To ensure that the mesalamine-mediated inhibition of NF-B reporter gene activity was not simply a nonspecific blockade of translation, total protein synthesis in Caco-2 cells was estimated by the rates of incorporation of [ 3 H]leucine into precipitable protein in the presence and absence of the drug. Mesalamine 40 mM, or vehicle were added to leucine depleted Caco-2 cells 5 min before addition of [ 3 H]leucine tracer. After 1, 2, and 4 h of incubation, means of 17, 21, and 25%, respectively, of the added tracer was incorporated in the control cells, compared with 16, 18, and 27% in the mesalamine-treated cells (p Ͼ 0.05 at each time point; Fig. 3). Thus, mesalamine had no detectable effect on translation, at a concentration that produced maximum inhibition of reporter gene activity.
Mesalamine Does Not Inhibit Inducible IB␣ or IB␤ Degradation-Acetyl salicylic acid, sodium salicylate, and sulfasalazine inhibit NF-B by preventing degradation of its cytosolic inhibitor, IB␣ (14,16). To determine whether mesalamine inhibited IB␣ degradation in intestinal epithelial cells, Caco-2 cells were stimulated with IL-1 in the presence and absence mesalamine or sulfasalazine. After stimulation, the kinetics of IB␣ degradation were determined by analysis of quantitative immunoblots of cytosolic lysates. Consistent with previous reports (20), stimulation of control cells with IL-1 or phorbol myristate acetate caused rapid depletion of cytosolic IB␣ pools, followed by its gradual reappearance after 1 h (Fig. 4). Mesalamine treatment of Caco-2 cells did not prevent IL-1induced IB␣ degradation. However, reappearance of IB␣ in the cytosol was delayed by over 2 h in mesalamine-treated cells but had reached base-line levels within 4 h. In contrast to mesalamine, sulfasalazine completely inhibited IL-1-stimulated degradation of IB␣. Mesalamine also did not prevent IL-1-stimulated degradation of IB␤ (data not shown).
Mesalamine Does Not Inhibit Nuclear Translocation of Transcriptionally Active NF-B Proteins-Because mesalamine inhibited NF-B transactivation but did not prevent degradation of its cytosolic inhibitor, we hypothesized that this drug might be inhibiting nuclear translocation of transcriptionally active NF-B proteins. To test this hypothesis, we assessed the effect of mesalamine on the kinetics of IL-1-inducible RelA, c-Rel, and RelB nuclear translocation in Caco-2 cells by quantitative immunoblotting from nuclear extracts. Consistent with previous reports, IL-1 caused rapid but transient nuclear translocation of RelA, c-Rel and RelB, followed later by a decrease in abundance of these proteins in the nuclear extracts (Fig. 5). Mesalamine did not prevent IL-1-induced nuclear localization of RelA, c-Rel, or RelB in Caco-2 cells. Interestingly, nuclear extracts of Caco-2 cells treated with only mesalamine contained RelB, whereas untreated cells displayed no detectable amounts of nuclear RelB.
Mesalamine Does Not Prevent NF-B DNA Binding-Mesalamine inhibits NF-B transcriptional activity without preventing degradation of IB␣ or nuclear translocation of the transcriptionally active NF-B proteins. To determine whether mesalamine interferes with the ability of NF-B to bind to DNA B sites, electrophoretic mobility shift analysis was performed. Caco-2 cells were treated with mesalamine or vehicle and then stimulated with IL-1 for varying durations. Analysis of DNA binding activity in Caco-2 nuclear extracts revealed that IL-1 stimulated NF-B DNA binding in the presence and absence of mesalamine (Fig. 6). Identical results were obtained using the IL-2 and HIV NF-B oligonucleotide probes (data not shown). Because mesalamine is absent from the nuclear extracts at the time of incubation with NF-B oligonucleotide probes, a direct Mesalamine Inhibits the Inducible Phosphorylation of RelA-NF-B transcriptional activity depends on the inducible degradation its cytosolic inhibitor IB, followed by nuclear translocation and DNA binding of the transcriptionally active proteins RelA, c-Rel, and RelB. Mesalamine inhibits NF-B-dependent transactivation without any detectable interference in this classic mechanism of regulation. This suggests that mesalamine is interfering with an alternative pathway for regulation of NF-B. To determine whether mesalamine directly affects the post-translational modification of NF-B proteins, we examined the inducible phosphorylation of RelA, c-Rel, and RelB in the presence and absence of this drug. In Caco-2 cells, RelA was found to be basally phosphorylated by 32 P labeling. IL-1 induced further phosphorylation of RelA in control cells, but the IL-1-induced phosphorylation was inhibited by pre-treatment with mesalamine (Fig. 7). Comparable results were obtained in Ju.1 cells (data not shown). Low levels of IL-1induced phosphorylation of c-Rel precluded definitive identification of mesalamine effects on c-Rel phosphorylation. RelB is not inducibly phosphorylated by IL-1 in either Caco-2 or Ju.1 cells. These data demonstrate that mesalamine inhibits a pathway that directly regulates the inducible phosphorylation of the RelA NF-B protein. DISCUSSION The nuclear localization and transcription regulation activity of NF-B is stimulated by the pro-inflammatory cytokine, IL-1. After exposure to IL-1, cells rapidly degrade cytosolic IB, allowing NF-B to translocate to the nucleus and initiate specific gene transcription. Inhibition of the signaling pathways that lead to IB degradation prevents NF-B-dependent gene transcription, for example by the oxygen radical scavenger pyrolidine dithiocarbamate and by anti-inflammatory drugs, such as salicylates (7). In this report, we describe a novel pharmacologic mechanism for control of NF-B activity. Pretreatment of cells with mesalamine, an anti-inflammatory aminosalicylate, prevented IL-1-stimulated NF-B-dependent transcription without affecting IB degradation, NF-B nuclear translocation, or DNA binding. IL-1 stimulation of colonic epithelial cells and T lymphocytes inducibly phosphorylated RelA. Mesalamine-mediated inhibition of NF-B transcriptional activity was accompanied by inhibition of IL-1-stimulated RelA phosphorylation. This suggests that the inducible phosphorylation of RelA positively regulates gene transcription by NF-B and that RelA phosphorylation constitutes an independent mechanism for the control of NF-B activity.
The activity of transcription factors is highly regulated to control the timely and co-ordinated expression of different genes. Increased transcriptional activity of NF-B has been identified in a number of different disease states, including chronic inflammatory disorders (2), cancers (21), and the antiviral state (1). Because of the need for fine control of transcription factor activity, multiple levels of regulation have evolved. Regulation can result from subcellular localization or posttranslational modifications by phosphorylation on tyrosine, threonine, or serine residues. The control of the Rel/NF-B family of transcription factors has been extensively studied, notably with regard to the cytoplasmic sequestration of NF-B proteins by their natural inhibitors, IB. Like many other transcription factors, including AP-1 and CREB, NF-B activity can also be regulated by the phosphorylation state of its transcriptionally active components, especially RelA. Using a variety of experimental conditions and stimuli, it has been demonstrated that the DNA binding of NF-B (9, 10) and NF-B-dependent transcription (11)(12)(13) are positively regulated by RelA phosphorylation.
Identification of the sites of inducible RelA phosphorylation and the responsible signaling pathways and kinases are currently active fields of investigation. Zhong et al. (12) showed that lipopolysaccharide treatment of cells stimulated protein kinase A-dependent phosphorylation of RelA on serine 276. In a cell-free system, it appeared that protein kinase A could directly phosphorylate RelA. Phosphorylation of this serine residue did not affect DNA binding but greatly increased RelA transcriptional activity by promoting interaction with the coactivator, cAMP response element binding protein/p300 (22). Wang and Baldwin demonstrated that TNF-␣ stimulates phosphorylation of RelA on serine 529 (13). Inducible NF-B transcription but not DNA binding was dependent upon this phosphorylation. It has recently been shown that IKK ␣ and ␤, in addition to phosphorylating IB proteins, can directly phosphorylate RelA (23). Thus, different NF-B-inducing stimuli appear to cause phosphorylation of RelA at unique sites, at least two distinct kinases can directly phosphorylate RelA, and phosphorylation of RelA appears to promote transactivation. Our experimental results are consistent with the hypothesis that phosphorylation of RelA can positively regulate NF-B-dependent transcription, because inhibition of this process with mesalamine prevented transactivation without affecting DNA binding.
What is the likely mechanism for the mesalamine-mediated inhibition of IL-1-stimulated RelA phosphorylation? The pathways that transduce the signal from the activated IL-1 receptor that culminate in phosphorylation and degradation of IB␣ are incompletely characterized. After ligand binding, the activated IL-1 receptor associates with the transmembrane protein, IL-1 receptor-accessory protein. The IL-1 receptor-accessory protein complex recruits IL-1 receptor-activated kinase (24), a serine threonine kinase, and TRAF-6 (25). TRAF-6 transduces the activation signal to a complex consisting of a mitogen-activated protein kinase kinase kinase called NIK. TRAF-6-mediated activation of NIK initiates the phosphorylation and activation of IKK␣ and ␤. Once activated, the IKK ␣/␤ heterodimer directly phosphorylates IB␣ and IB␤ on N-terminal serine residues. The kinases responsible for IL-1-stimulated phosphorylation of RelA, and the target for inhibition of this process by mesalamine are not known. However, because mesalamine inhibits IL-1-stimulated RelA phosphorylation without preventing IB degradation, IL-1 receptor-activated kinase, TRAF-6, NIK, and IKK should not be inhibited by mesalamine. Rather, a kinase with specificity for RelA and not IB␣/␤ is implicated. It has been shown that aspirin and sodium salicylate inhibit IKK␤ but not IKK␣ by competing with ATP for enzyme binding (26). Similarly, recent studies from IKK␣ Ϫ/Ϫ and IKK␤ Ϫ/Ϫ mice show that TNF-␣ and IL-1 activate NF-B through IKK␤ and not IKK␣ (27)(28)(29). Interestingly, although IKK␤ Ϫ/Ϫ mice did not respond to TNF-␣ by degrading IB proteins and activating NF-B, the IL-1-initiated NF-B response was only partially inhibited (29). Thus, IL-1 may activate an IKK␣/␤ independent kinase that phosphorylates IB polypeptides. Such redundant IL-1-inducible kinases could exhibit overlapping but preferential interactions with IB and RelA substrates. Mesalamine may selectively inhibit a kinase that preferentially interacts with RelA without inhibiting another kinase that preferentially interacts with IB polypeptides. Mesalamine should be a valuable reagent to further characterize IL-1-inducible signaling events that regulate NF-B transcriptional activity.
Uncontrolled overactivity of NF-B may be prevented by positive transcriptional regulation of the IB␣ gene by NF-B proteins. Newly synthesized IB␣ has been reported to downregulate NFB activity by directly interacting with transcriptionally active NF-B proteins in the nucleus and cytosol (30). Although mesalamine completely inhibited NF-B reporter gene activity, the late (4 h post stimulation) cytosolic reappearance of IB␣ was not blocked (Fig. 4). This suggests that not all NF-B-regulated gene transcription is blocked by mesalamine. RelB is not inducibly phosphorylated, so mesalamine may be unable to prevent activation of genes responsive to this transcription factor. In contrast to its effects on RelA and c-Rel, mesalamine caused nuclear translocation of RelB in the absence of stimulation (Fig. 5), further supporting a differential sensitivity to mesalamine among the NF-B family members. Alternatively, it is also possible that the late reappearance of IB␣ in mesalamine-treated cells is due to activity of a transcription factor other than NF-B. The IB␣ promoter is known to contain Sp-1 binding sequences (31), and this ubiquitous transcription factor may be responsible for late appearing IB␣ in mesalamine-treated cells.
These data demonstrate that aminosalicylates can inhibit NF-B activity by two different mechanisms. Sulfasalazine, the azo-conjugated aminosalicylate studied here, is considered a pro-drug, which is activated when bacterial azo-reductase enzymes in the colon split the molecule to release the active component mesalamine and sulfapyridine (32). If inhibition of NF-B is the true anti-inflammatory mechanism of action of aminosalicylates in vivo, sulfasalazine may in fact have dual anti-NF-B actions, as a pro-drug of mesalamine and as an active compound itself. It has recently been demonstrated that acetyl salicylic acid inhibits IKK␤ but not IKK␣ (26). Currently it is unknown whether any of the aminosalicylates inhibit IKK␣ or ␤ or other NF-B regulatory kinases. A more detailed understanding of the biochemical interactions of the structurally related aminosalicylates with the proteins of the NF-B system is necessary.
These findings suggest that a novel, pharmacologically manipulable mechanism for regulation of NF-B activity exists. Identification of the IL-1-stimulated signaling pathway responsible for the selective phosphorylation of RelA that is inhibited by mesalamine will be an important extension of this work. This pathway is an attractive potential target for therapeutic inhibition in inflammatory diseases.