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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA

Thionamides Inhibit the Transcription Factor Nuclear Factor-B by Suppression of Rac1 and Inhibitor of B Kinase

Center for Clinical Research, Department of Anesthesiology, University Hospital Freiburg, Freiburg, Germany (M.H., H.D., P.S., U.G., M.R., R.S., C.I.S., T.L., K.K.G., H.L.P.); Department of Medicine IV and Kidney Research Center Cologne, University of Cologne, Cologne, Germany (N.A.); and Department of Anesthesiology, University Hospital Duesseldorf, Duesseldorf, Germany (B.H.J.P.)

Received October 1, 2007; accepted November 29, 2007.

	Abstract

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Thionamides, inhibitors of the thyroid peroxidase-mediated iodination, are clinically used in the treatment of hyperthyroidism. However, the use of antithyroid drugs is associated with immunomodulatory effects, and recent studies with thionamide-related heterocyclic thioderivates demonstrated direct anti-inflammatory and immunosuppressive properties. Using primary human T-lymphocytes, we show that the heterocyclic thionamides carbimazole and propylthiouracil inhibit synthesis of the proinflammatory cytokines tumor necrosis factor (TNF) and interferon (IFN). In addition, DNA binding of nuclear factor (NF)-B, a proinflammatory transcription factor that regulates both TNF and IFN synthesis, and NF-B-dependent reporter gene expression were reduced. Abrogation of NF-B activity was accompanied by reduced phosphorylation and proteolytic degradation of inhibitor of B (IB), the inhibitory subunit of the NF-B complex. Carbimazole inhibited NF-B via the small GTPase Rac-1, whereas propylthiouracil inhibited the phosphorylation of IB by its kinase inhibitor of B kinase . Methimazole had no effect on NF-B induction, demonstrating that drug potency correlated with the chemical reactivity of the thionamide-associated sulfur group. Taken together, our data demonstrate that thioureylenes with a common, heterocyclic structure inhibit inflammation and immune function via the NF-B pathway. Our results may explain the observed remission of proinflammatory diseases upon antithyroid therapy in hyperthyroid patients. The use of related thioureylenes may provide a new therapeutic basis for the development and application of anti-inflammatory compounds.

The aberrant activation of the transcription factor nuclear factor (NF)-B is involved in chronic and pathological inflammation, autoimmunity, sepsis, and heart disease (Kumar et al., 2004), arguing that this transcription factor might participate in thionamide-mediated side effects. In most cell types, NF-B is sequestered in the cytoplasm by the IB family of proteins (Karin and Delhase, 2000). After Rac-1 and IB-kinase (IKK) activation, IB proteins become phosphorylated, ubiquitinilated, and degraded by the proteasome (Karin and Delhase, 2000; Marinari et al., 2002; Piccolella et al., 2003). In subsequent experiments, NF-B proteins translocate to the nucleus to bind their cognate DNA sequences and to initiate transcription of proinflammatory cytokines, chemokines, adhesion molecules, enzymes, and antimicrobial peptides.

We recently described thiobarbiturates as potent inhibitors of IKKs, NF-B, and proinflammatory cytokine production (Loop et al., 2002, 2003). Although thiobarbiturates and thionamides have different clinical utilities, both are thioureylenes with a related structure and overlapping cellular activities. Thus, thiobarbiturates reduce thyroid function (Farwell and Braverman, 1996) and inhibit adaptive and cellular immune responses (Corrêa-Sales et al., 1997; Nishina et al., 1998).

In the present study, we investigated whether thionamides, comparable with the structurally related barbiturates, impede immune functions by a general, structure-dependent mechanism. In this study, we demonstrated that thionamides inhibited the activation of the transcription factor NF-B, which might be responsible for the remission of some immune-related diseases observed during antithyroid treatment of patients. Comparable with thiobarbiturates, we found that propylthiouracil inhibited the IKK-dependent phosphorylation of IB, whereas carbimazole repressed the proximal GTPase Rac-1. The biological effects were mediated by redox-active sulfur of thionamides. Because thioureylenes affect ubiquitously expressed proteins that regulate important cellular responses in health and disease, the development of new pharmaceutical compounds on a mononuclear heterocyclic thioureylene basis as key building blocks in medicinal chemistry may evolve.

	Materials and Methods

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Isolation and Treatment of Human Primary T-Lymphocytes. Peripheral blood mononuclear cells were purified by Ficoll-Paque PLUS (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK) centrifugation of whole blood. T-cells were enriched by immunomagnetic cell sorting with anti-CD3 microbeads using LS⁺ selection columns (Miltenyi Biotech, Auburn, CA). Isolated cells were suspended in RPMI 1640, supplemented with 10 mM HEPES, pH 7.3, 50 µM β-mercaptoethanol, and 2 mM L-glutamine. Cell suspensions were pretreated with methimazole (Sigma-Aldrich, St. Louis, MO), propylthiouracil (Sigma-Aldrich), or carbimazole (LKT Laboratories, St. Paul, MN) at indicated concentrations. T-lymphocytes were activated by CD3/CD28 cross-linking with T cell Expander Dynabeads (Dynal Biotech, Lake Success, NY) or 15 ng/ml phorbol 12-myristate 13-acetate (Sigma-Aldrich).

Cytokine ELISA. Cytokine concentrations in supernatants of 2 x 10⁶ primary human T-lymphocytes were analyzed using the human TNF- or IFN Quantikine Immunoassay (R&D Systems, Minneapolis, MN). Cells were seeded at 10⁷ T-cells/ml and preincubated with various concentrations (0.1–5 mM) of thionamides in different wells of a 96-well round bottom plate (Greiner Bio-One GmbH, Frickenhausen, Germany). T-cells were induced by 10⁶ T-cell Expander Dynabeads 2 h after the onset of thionamide treatment. After 15 h, supernatants were collected, diluted 1:5 in Assay Diluent, and the TNF- or IFN concentration was determined according to the manufacturer's instructions.

NF-B-Dependent Luciferase Reporter Gene Expression. Jurkat cells were transiently transfected with 2 µg of pNF-B-Luc (Clontech, Mountain View, CA) using Lipofectamine2000 (Invitrogen, Carlsbad, CA) according to the manufacturer's recommendations. Transfection reactions were pooled, redistributed at 10⁵ cells/well, and incubated with thionamides and 15 ng/ml phorbol 12-myristate 13-acetate for 15 h. Luciferase reporter gene expression was measured by harvesting cells in 100 µl of luciferase reporter lysis buffer (Promega, Madison, WI) and assaying in a Microluminat Plus LB 96P luminometer (Berthold Technologies, Bad Wildbach, Germany). Protein levels were normalized by the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA).

ELISA-Based NF-B Transcription Factor Activity Assay. Nuclear extracts were analyzed by the TransAM NF-B family transcription factor activation assay according to the manufacturer's recommendations (Active Motif, Carlsbad, CA). NF-B complexes were captured by binding to a consensus 5'-GGGACTTTCC-3' oligonucleotide immobilized on a 96-well plate. The contend of bound NF-B was determined by different primary antibodies, detecting p50 or p65 proteins followed by a secondary horseradish peroxidase-conjugated goat anti-rabbit IgG for spectrophotometric detection at OD₄₅₀ nm. Data were expressed as a percentage of NF-B/DNA binding compared with activated cells (=100%).

Nuclear Protein Extraction and Electrophoretic Mobility Shift Assays. Preparation of nuclear cell extracts and electrophoretic mobility shift assays was performed as described previously (Loop et al., 2002). For DNA binding, the NF-B motif 5'-AGTTGAGGGGACTTTCCCAGGC-3' was used as a probe. Binding reactions were carried out at room temperature for 30 min in a volume of 20 µl containing 20 µg of nuclear cell extract, 22 mM HEPES, pH 7.9, 70 mM KCl, 50 µM EDTA, 2.2 mM dithiothreitol, 2% glycerol, 4% Ficoll, 0.1% Nonidet P40, 30 µM phenylmethylsulfonyl fluoride, 20 µg of bovine serum albumin, 2 µg of poly(dI-dC), and 1.75 pmol ³²P end-labeled oligonucleotides (5 µCi/pmol). The resulting DNA complexes were displayed by electrophoresis on 4% nondenaturating polyacrylamide gels and subsequent autoradiography.

IB: Phosphorylation and Degradation. Primary human CD3⁺ T-lymphocytes were preincubated at 2 x 10⁶ T-cells/well with various concentrations (0.1–5 mM) of thionamides for 4 h. In addition, lymphocytes were exposed to 10 µM MG132 (Calbiochem, San Diego, CA) for measurement of IB phosphorylation and 5 µg/ml actinomycin D (Sigma-Aldrich) for measurement of IB degradation. CD3/CD28 receptor stimulation was induced by 10⁶ T-cell Expander Dynabeads for the last 20 min (IB phosphorylation) or 10 min (IB degradation) of the experiment. Reactions were terminated by the addition of 30 µl of 3x SDS sample buffer and boiling. Proteins were separated by 10% SDS-PAGE gels. Immunoblots were analyzed with 0.1 µg/ml of a phosphospecific IB(Ser32) antibody (New England Biolabs, Ipswich, MA) or 0.1 µg/ml of an IB(total) antibody (New England Biolabs). β-Actin was recognized by 20 ng/ml of a β-actin antibody (New England Biolabs). Specific bands were visualized using secondary horseradish peroxidase-conjugated antibodies and enhanced chemiluminescence reagents (GE Healthcare).

In Vitro Kinase Assay. T-lymphocytes were lysed for 20 min at 4°C in a volume of 600 µl of kinase buffer (20 mM HEPES, 2 mM dithiothreitol, 10 mM NaF, 1 mM Na₃VO₄, 10 mM β-glycerophosphate, 0.5% Nonidet P-40, 20 µg/ml aprotinin, 20 µg/ml leupeptin, 1 µg/ml pepstatin A, and 0.2 µg/ml pefabloc). For the kinase reaction, 100 µl of cell lysate, 2 µg of GST-IB-Sepharose beads (Santa Cruz Biotechnology, Santa Cruz, CA), and 0.5 µmol ATP were incubated for 2 h at 30°C. In subsequent experiments, GST-IB-Sepharose beads were repeatedly washed by kinase buffer and analyzed for phospho-IB (Ser32) or total IB by immunoblotting.

In alternative experiments, IKK proteins were immunoprecipitated from cell lysates with 2 µg of anti-IKK antibody-Sepharose beads (Santa Cruz Biotechnology) for 1 h at 4°C under constant rotation. The precipitates were washed four times in kinase buffer. Kinase reactions were performed for 20 min at 22°C in 20 µlof kinase buffer containing 10 µM ATP, 5 µCi of [-³²P]ATP, and 4 µg of GST-IB (Santa Cruz Biotechnology) for each immunoprecipitate. Reactions were terminated by addition of 5x SDS loading buffer and separated by 10% SDS-PAGE. Gels were fixed, dried, and analyzed by autoradiography.

Analysis of G-Proteins. Rac-1 activity was determined by the Rac activation assay kit (Cytoskeleton, Denver, CO). In brief, 2 x 10⁷ T-cells were harvested and lysed in 300 µl of ice-cold extraction buffer (50 mM Tris, 100 mM NaCl, 2 mM MgCl₂, 1 mM phenylmethylsulfonyl fluoride, 1% Nonidet P-40, 10% glycerol, 1 µg/ml leupeptin, and 2 µg/ml aprotinin). Where indicated, cell lysate supernatants were induced by 100 µM GTPS plus 1 mM EDTA on a rocker platform for 30 min at room temperature. Reactions were stopped by the addition of 50 mM MgCl₂. For Rac-1 precipitation, cell lysate supernatants were incubated with 20 µl of GST-PAK PBD protein beads for 40 min at 4°C under constant rotation. Protein beads were repeatedly washed with 1 ml of extraction buffer, then resuspended and boiled in 40 µl of 2x SDS sample buffer. Proteins were separated by 12% SDS-PAGE and analyzed for Rac-1 precipitation by immunoblotting using 0.25 µg/ml anti-Rac-1 (clone 102; BD Biosciences, San Jose, CA). Total cell extracts were analyzed by immunoblotting using an anti-Rac-1 antibody (Cytoskeleton) and an anti-phosphotyrosine antibody (clone 4G10; Millipore Corporation, Billerica, MA).

Immunoblotting. Proteins from cell lysates were separated by SDS-PAGE and electroblotted to a polyvinylidene difluoride membrane (Millipore Corporation). For immunodetection antibodies were used according to manufacturer's recommendations. Antibodies were directed against IB(Ser32) (New England Biolabs), IB (New England Biolabs), β-actin (New England Biolabs), IB(Ser32/36) (New England Biolabs), GST (Rockland Immunochemicals, Gilbertsville, PA), IKK (Santa Cruz Biotechnology), phosphotyrosines (PharMingen, San Diego, CA), ZAP 70(Tyr318)/Syk(Tyr352) (New England Biolabs), VAV-1(Tyr160) (Sigma-Aldrich), Rac-1(Ser71) (New England Biolabs), Rac-1 (Cytoskeleton), ZAP-70 (New England Biolabs), and VAV (New England Biolabs).

Statistical Analysis. Data are shown as the median ± S.E.M. Statistical analysis was performed using a one-way analysis of variance followed by a Holm Sidak post-hoc test. P values less than 0.05 were considered significant.

	Results

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Thionamides Inhibited the Proinflammatory Cytokine Synthesis. Because clinical observations suggest anti-inflammatory properties, the heterocyclic thioureylenes methimazole, propylthiouracil, and carbimazole were analyzed for their ability to inhibit cytokine production (Fig. 1). Secretion of the proinflammatory cytokines IL-1, TNF, and IFN was measured in supernatants of primary human T-lymphocytes by ELISA. CD3/CD28 receptor cross-linking induced TNF and IFN secretion (Fig. 2, A and B). In contrast, IL-1, a cytokine primarily secreted by mononuclear phagocytes, could not be detected in human T lymphocyte supernatants (data not shown). Preincubation with either propylthiouracil or carbimazole significantly reduced TNF or IFN synthesis, whereas methimazole showed no effect on cytokine production (Fig. 2, A and B).

View larger version (16K): [in this window] [in a new window]   Fig. 1. Structure of heterocyclic thioderivates in clinical use. The thioureylenes used in this study include the antithyroid drugs methimazole, propylthiouracil, and carbimazole.

View larger version (28K): [in this window] [in a new window]   Fig. 2. Thionamides inhibit TNF and IFN secretion. Cytokine ELISAs of supernatants from peripheral human T-lymphocytes are shown. T cells were either untreated or preincubated with increasing doses of propylthiouracil, carbimazole, methimazole, MG132, or lactacystin for 15 h. Cytokine synthesis was induced by 106 CD3/CD28 T cell Expander Dynabeads (0.5 beads/cell) for the final 13 h of culture. A, propylthiouracil significantly inhibited TNF synthesis at 1 mM and carbimazole at 100 µM, whereas methimazole treatment showed no effect. B, carbimazole inhibited IFN production stronger than propylthiouracil, and methimazole showed no effect. C, NF-B inhibitors MG132 and lactacystin repress synthesis of TNF and IFN demonstrating NF-B-dependent transcription. Error bars, median ± S.E.M. of four independent experiments. *, P < 0.05 versus positive controls was considered as significant.

Several transcription factors participate in the transcription of TNF and IFN. To demonstrate that the central mediator of proinflammatory cytokine synthesis is NF-B, specific inhibitors of the proinflammatory NF-B pathway were used (Fig. 2C). The proteasome inhibitors MG132 and lactacystin, which block IB degradation, significantly suppressed cytokine synthesis in a dose-dependent manner (Fig. 2C). In addition, 25 mM of the antioxidant N-acetylcysteine, an inhibitor of the IB kinases, repressed cytokine synthesis but was associated with cytotoxicity (data not shown). We observed that NF-B inhibitors were more potent inhibitors of TNF than of IFN production. In addition, propylthiouracil repressed TNF secretion at lower doses than IFN (Fig. 2, A and B).

Thionamides Inhibited the Activation of the Transcription Factor NF-B. In a previous study, we described that structurally related thiobarbiturates inhibit NF-B (Loop et al., 2002). To evaluate whether thionamides influence the proinflammatory transcription factor NF-B by a similar mechanism, NF-B-dependent reporter gene expression studies (Fig. 3) and NF-B DNA-binding assays were performed (Fig. 4).

View larger version (16K): [in this window] [in a new window]   Fig. 3. Propylthiouracil and carbimazole inhibit NF-B-dependent reporter gene expression. Jurkat cells were transfected with 2 µg of pNF-B-Luc and incubated with 0 to 5 mM methimazole, propylthiouracil, or carbimazole for 15 h. Cells were stimulated with 15 ng/ml phorbol 12-myristate 13-acetate to induce NF-B for the final 13 h of culture. Lysates were analyzed for luciferase reporter gene activity, and the results were normalized to protein levels. Results are displayed as a percentage of relative light units compared with stimulated, transfected cells in the absence of thionamides. Statistics represent the median ± S.E.M. of four independent experiments. *, P < 0.05 versus positive control (stimulation in the absence of thionamides) was considered as significant.

View larger version (31K): [in this window] [in a new window]   Fig. 4. Carbimazole and propylthiouracil inhibit DNA binding of NF-B. CD3+ T cells were treated for 3 h with thionamides at the indicated concentrations and subsequently stimulated by CD3/CD28 T-cell receptor cross-linking for 30 min (107 T-cell Expander Dynabeads/assay). In A, nuclear extracts were prepared and analyzed for DNA binding of p50 or p65(RelA) by an electrophoretic mobility shift assay. NF-B DNA complexes, the nonspecific binding activity of the probe (*), and unbound oligonucleotides (>) are indicated. In B, for quantification of DNA-bound NF-B, a plate-immobilized oligonucleotide containing a 5'-GGGACTTTTTCC-3' NF-B binding site was used. NF-B members were recognized by specific anti-p50 or anti-p65(RelA) antibodies and secondary horseradish peroxidase-immune complexes for colorimetric readout. Spectrophotometric data were expressed as a percentage of induction by absorbance compared with activated cells (=100%). Error bars, median ± S.E.M. of six independent experiments. *, P < 0.05 versus positive controls was considered as significant. Propylthiouracil and carbimazole inhibited binding of p50 and p65(RelA) NF-B subunits.

NF-B is composed of homo- and heterodimeric complexes of various NF-B family members. However, the p50/p65 heterodimers are the most common dimers found in the NF-B signaling pathways. Therefore, DNA binding of p50 and p65(RelA) was analyzed by electrophoretic mobility shift assay using nuclear extracts of CD3/CD28-activated T-cells (Fig. 4A). The position and specificity of p50 and p65(RelA) was identified by supershift and competition experiments on independent blots (data not shown). The DNA-binding activity of NF-B was significantly impaired by preincubation with either propylthiouracil or carbimazole. In contrast, methimazole did not affect p50 or p65(RelA) binding to its corresponding DNA consensus sequence.

For quantification of p50 or p65(RelA) DNA binding, an ELISA-based DNA-binding assay was used. Results in Fig. 4B displayed a minor DNA-binding activity of p50 and p65-(RelA) to a (5'-GGGACTTTCG-3') DNA consensus sequence in resting T-cells. This was induced two to four times upon CD3/CD28 T-cell receptor stimulation. The presence of methimazole did not significantly alter DNA binding. In contrast, preincubation with either propylthiouracil or carbimazole dose-dependently inhibited binding of p50 or p65 to its correspondent DNA consensus sequence.

Repression of NF-B Was Due to Inhibition of IB Phosphorylation and Proteolytic Degradation by Reduced IB-Kinase Activity. The reason for reduced NF-B/DNA binding was analyzed. In most cell types, NF-B exists in an inactive form in the cytoplasm, bound to inhibitory IB proteins. Phosphorylation and proteolytic cleavage of IB result in the release and nuclear translocation of NF-B and in specific gene activation (Li and Verma, 2002). Therefore, we investigated whether thionamides impair the phosphorylation (Fig. 5A) and degradation (Fig. 5B) of IB proteins. Because the phosphorylated form of IB is highly transient and difficult to detect due to rapid proteasomal degradation, the proteasome inhibitor MG132 was added (Fig. 5A). CD3/CD28 receptor stimulation of T-lymphocytes induced the phosphorylation of IB proteins as determined by immunoblotting (Fig. 5A). Preincubation with propylthiouracil or carbimazole prevented the CD3/CD28-induced phosphorylation of IB, whereas methimazole had no effect. Reduced levels of IB phosphorylation were not attributed to proteolysis because MG132 efficiently blocked proteasomal degradation. Equal amounts of total IB proteins could be detected in each reaction (Fig. 5A, bottom blots).

View larger version (58K): [in this window] [in a new window]   Fig. 5. Carbimazole and propylthiouracil inhibit the phosphorylation and degradation of IB. CD3+ T cells were treated for 3 h with thionamides at the indicated concentrations. CD3/CD28 T-cell receptor cross-linking was induced for 20 min to detect phosphorylation or 10 min to detect IB degradation (106 T-cell Expander Dynabeads/assay). Total cellular lysates were analyzed by immunoblotting. In A, both, propylthiouracil and carbimazole inhibited IB phosphorylation in a dose-dependent manner, whereas methimazole had no effect. Analysis of total IB protein demonstrated equal amounts of protein in each lane because IB degradation was inhibited during thionamide treatment and T-cell activation by addition of 10 µM of the proteasome inhibitor MG132. In B, effects of thionamides on IB degradation are demonstrated (top blot). To inhibit protein synthesis, thionamide treatment and CD3/CD28 T-cell receptor stimulation were performed in the presence of 5 µg/ml actinomycin D but in the absence of MG132. Total cell lysate supernatants were analyzed for β-actin to demonstrate that equal amounts of protein were included (bottom blot). Representatives of three independent experiments are shown.

In previous experiments, we have shown that thiobarbiturates inhibit the phosphorylation and the proteasomal degradation of IB by a repression of IKKs (Loop et al., 2003). To analyze whether thionamides mediate similar effects due to their related structure, IKK activity was examined in the presence of methimazole, propylthiouracil, and carbimazole. T-cells contain multiple different IKK complexes (Kupfer and Scheinman, 2002). Therefore, the overall IKK activity was determined by a kinase activity assay using total cytosolic extracts of activated T cells and immobilized recombinant GST-IB as a substrate. IKK-specific phosphorylation was measured by an IB (Ser32/Ser36) phosphospecific antibody and by immunoblotting (Fig. 6A). A small amount of phospho-IB was detected when lysates, derived from quiescent T-lymphocytes, were used. However, CD3/CD28 activation resulted in a marked increase in phosphorylated GST-IB. Pretreatment of T-cells with propylthiouracil or carbimazole prevented IB Ser32 and Ser36 phosphorylation, indicating a reduced IKK activity, whereas methimazole treatment had no effect. Carbimazole was more potent in inhibiting IKKs than propylthiouracil (Fig. 6A).

View larger version (37K): [in this window] [in a new window]   Fig. 6. Carbimazole and propylthiouracil inhibit the IB-kinase activity. Kinase activity assays are shown. Primary human T-lymphocytes (2 x 107) were pretreated with thionamides for 2 h and stimulated with 0.5 CD3/CD28 T-cell Expander Dynabeads/cell for 10 min. In A, total cell lysates were prepared and incubated with 2 µg of GST-IB-Sepharose beads plus 0.5 µmol ATP to perform an IB kinase reaction at 30°C for 2 h. Phosphorylation of IB at Ser32 and Ser36 were visualized by immunoblotting (top blot). Reprobed blots using an anti-GST-specific antibody demonstrated equal amounts of GST-IB substrate in each reaction (input control, middle blot). Total cell lysates contained equal amounts of IKK (bottom blot). In B, IKK-specific IB phosphorylation is demonstrated. IKK was immunoprecipitated from total cell lysates by 2 µg of antibody-Sepharose beads. Immunoprecipitates were incubated with 4 µg of GST-IB kinase substrate, 10 µM ATP, and 5 µCi [-32P]ATP for 20 min at 22°C. Carbimazole- or propylthiouracil-treated cells demonstrated a dose-dependent decline of IKK-specific kinase activity (top blots). Western blot analysis of the immunoprecipitates demonstrated equal amounts of IKK in each kinase reaction (input control, bottom blots).

Carbimazole- but Not Propylthiouracil-Mediated Repression of IKK Activity Was Due to Inhibition of the Small G-Protein Rac-1. T-cell receptor-mediated T-cell activation is induced by a supramolecular activation complex referred to as the immunological synapse (Schmitz et al., 2003). Because activation of the immunological synapse leads to the induction of NF-B, a suppression of signal transmission via the T-cell receptor might explain the inhibition of IKKs. However, antiphosphotyrosine immunoblots of total cell lysates displayed no fundamental decrease in activating tyrosine phosphorylation after CD3/CD28 receptor stimulation and thionamide treatment (Fig. 7A). In fact, carbimazole induced a marked increase in tyrosine phosphorylation of proteins at 70 kDa (Fig. 7A, arrows). Subsequent experiments revealed that ZAP-70/Syk protein tyrosine kinase was a target of carbimazole-mediated tyrosine phosphorylation. Increased phosphorylation of ZAP-70/Syk was accompanied by an increased kinase activity. VAV-1, a direct substrate of ZAP-70, was also hyperphosphorylated at tyrosine 160 (Fig. 7B) in the presence of carbimazole.

View larger version (33K): [in this window] [in a new window]   Fig. 7. Carbimazole induces phosphorylation of ZAP70/Syk and VAV-1 but inhibits activation of the small GTPase Rac-1. Immunoblots are shown. T cells were pretreated with thionamides for 3 h and stimulated with 0.5 CD3/CD28 T-cell Expander Dynabeads/cell for 10 min as indicated. In A, protein phosphorylation was detected by an antiphosphotyrosine (4G10) horseradish peroxidase-conjugated antibody using whole-cell lysates. <, position of increased tyrosine phosphorylation in the presence of carbimazole. In alternative experiments, tyrosine phosphorylation of ZAP-70/Syk, Rac-1, and VAV-1 was analyzed by protein-specific antibodies (B). As a loading control, antibodies directed against total Rac-1, ZAP-70, and VAV-1 were used to demonstrate equal amounts of protein in each lane. In C, the effect of thionamides on Rac-1 activity was determined by a PAK pull-down assay. Active Rac was pulled down from whole cellular lysates with GST-PAK PRB fusion proteins. Precipitated proteins (top panels) show the active fraction of the GTPase, whereas the bottom panels show the total amount of Rac-1 in each cell lysate fraction. GTPS was used for direct activation of Rac-1 in cellular lysates, in the absence of CD3/CD28 T-cell receptor coactivation (final blots). Representatives of three independent experiments are shown.

The carbimazole-mediated inhibition of Rac-1 was more closely investigated. Activation of Rac-1 by GTPS is independent of regulatory events by associated signaling cascades. As a result, inhibition of Rac-1/Pak binding must be direct. Indeed, GTPS-activated cell lysates showed a direct repression of Rac-1/Pak binding upon carbimazole treatment (Fig. 7C, bottom blot). In contrast, phosphorylation of Rac-1 at Ser71 that both inhibits GTP-binding and activation of the small G protein (Kwon et al., 2000) was not responsible for the inhibitory potential of carbimazole (Fig. 7B). In addition, isoprenylation that is essential for membrane association and biological function of Rac-1 (Didsbury et al., 1990) was not involved in carbimazole-mediated effects because GTPS-dependent Rac-1 activation does not require a specific subcellular protein localization (Fig. 7C, bottom blot). In contrast, both methimazole and propylthiouracil did not influence the GTPS-dependent activation of Rac-1 as observed in pull-down experiments before, using CD3/CD28-activated T-cells (Fig. 7C). In summary, carbimazole directly inhibits Rac-1 and thus demonstrates another inhibitory profile than heterocyclic thioderivates with a pyrimidine-like nucleus (Fig. 8).

View larger version (23K): [in this window] [in a new window]   Fig. 8. Effects of heterocyclic thioderivates on T-cell receptor-dependent NF-B activation are shown. T-cell activation involves a supramolecular activation complex (immunological synapse) and is mediated by tyrosine phosphorylation and protein-protein interactions. VAV-1, a direct substrate of ZAP-70 and a GDP/GTP exchange factor of the small GTPase Rac-1, is induced by tyrosine phosphorylation, leading to the activation of the transcription factor NF-B by a Rac-1/MLK-3/IKK or a Rac-1/MEKK1/IKK-dependent pathway. Carbimazole, a sulfur derivate of imidazole, directly inhibits the small G-protein Rac-1, whereas thioderivates with a pyrimidine-like nucleus inhibit the IB-kinase activity.

	Discussion

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NF-B serves as an ubiquitous regulator of the host immune and inflammatory response, and its participation in apoptosis, proliferation, and differentiation has recently been demonstrated (Karin, 1998, 2006; Li and Verma, 2002). However, excessive activation or deregulation of NF-B is thought to play a fundamental role in the pathogenesis of several immunologic and inflammatory disorders such as morbus Alzheimer, atherosclerosis, adult respiratory distress syndrome, asthma, rheumatoid arthritis, cardiovascular disease, inflammatory bowel disease, cystic fibrosis, and multiple sclerosis (Wright and Christman, 2003; Kumar et al., 2004).

It is interesting to note that single case studies with hyperthyroid patients suffering from inflammatory myopathy described a complete clinical and pathologic resolution upon antithyroid treatment with carbimazole (Hardiman et al., 1997). Thionamide therapy is also beneficial to some chronic inflammatory disorders such as psoriasis (Elias, 2004). In addition, it protects from autoimmune diseases in experimental animal models (Singer et al., 1994; Mozes et al., 1998) or diminishes aberrant leukocyte adhesion as observed in pathological inflammation (Dagia et al., 2004). These observations may now be explained by the thionamide-mediated inhibition of NF-B.

We observed that the mechanism of thionamide-mediated immunosuppression depended on the chemical structure of the individual reagent. Thioureylenes with a pyrimidine-like nucleus such as the antithyroid drug propylthiouracil (presented data) or the barbiturates thiopental or thiamylal (Loop et al., 2002, 2003) showed high resemblance in dose response, and both inhibited the IB-kinase complex. In contrast, carbimazole, a sulfur-containing imidazole derivate, repressed the proximal activation of Rac-1, necessary for MLK3- or MEKK1-dependent activation of IKKβ.

Redox-active sulfur was central for the NF-B-inhibitory potential of carbimazole, propylthiouracil, and thiobarbiturates as demonstrated by comparison with methimazole or the oxy-analogs of barbiturates. Thiobarbiturates inhibited NF-B, but their oxy-analogs showed only marginal effects on their activation (Loop et al., 2002, 2003). Likewise, carbimazole and methimazole demonstrated diametric properties depending on the biochemical activity of the associated sulfur group. Methimazole, due to its stable aromatic structure, shows no anti-inflammatory activity, whereas carbimazole, although it contains an analog imidazole nucleus, is not an aromatic compound but includes redox-active sulfur. Carbohydrate side chains (ethylcarbamate) and components of the heterocycle (thiourea, malonic acid, imidazole) had no immunoregulatory effects because these reagents did not significantly inhibit NF-B-dependent reporter gene expression (M. Humar, unpublished observations).

Several publications confirm that molecular targets of thioureylenes are susceptible to sulfur. The structural analysis of IKK subunits suggests that cysteine residues are present in the activation loop within the kinase domain at sites critical for enzymatic activity (Byun et al., 2006). These sites might serve as molecular targets for heterocyclic thioderivates. In addition, it has been described that thiol-reactive agents block IKK activity and prevent the subsequent activation of NF-B (Jeon et al., 2000; Loop et al., 2003). For Rac-1 activation, a redox-reactive cysteine within the P-loop motif, is crucially involved in the guanine nucleotide exchange (Heo and Campbell, 2005). The X-ray crystal structure indicates that this Cys18 thiol is solvent-accessible (Heo and Campbell, 2005) and thus might represent a direct target for carbimazole. Most probably, active posttranslational modifications do not participate in carbimazole-mediated Rac-1 inhibition because the interaction of Rac-1 and Pak was repressed in cellular lysates despite GTPS stimulation.

NF-B is critically involved in the pathogenesis of diseases independent from inflammation (Kumar et al., 2004). In these cases, heterocyclic thioderivates might also be useful for molecular intervention and medical treatment. Screening methods and molecular modeling indicate that heterocyclic agents containing thionamides and thiourea are potent inhibitors of protein function (Buchholz et al., 2006). To evaluate the application of heterocyclic thioureylenes in NF-B-related diseases, more case observations and clinical studies are necessary. We suggest the analysis of hyperthyroid patients with secondary clinical symptoms associated with NF-B deregulation and ongoing therapy with thionamides. After treatment, differences in the clinical manifestation of NF-B-associated symptoms might be related to our observations. In addition, the inhibitory potential of heterocyclic thioureylenes might be optimized by chemical modifications.

In summary, our work demonstrates a molecular mechanism of immunosuppression by mononuclear heterocyclic thioureylenes. Due to the fact that thionamides inhibit proteins that are involved in numerous aspects of cellular responses and are ubiquitously expressed in most tissues, extensive side effects of treatment might be expected. At the same time, new therapeutic applications may evolve using thioureylenes for the development of new molecules in medical chemistry.

	Footnotes

 
This study was supported by departmental funding and by the Else Kroener-Fresenius-Stiftung (Grant 1087002001), Bad Homburg, Germany.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.132407.

ABBREVIATIONS: IL, interleukin; NF, nuclear factor; IB, inhibitor of B; IKK, inhibitor of B kinase; CD, cluster(s) of differentiation; ELISA, enzyme-linked immunosorbent assay; TNF, tumor necrosis factor; IFN, interferon; MG132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; GTPS, guanosine 5'-3-O-(thio)triphosphate; ZAP, -associated protein; MLK, mixed lineage kinase; MEKK, mitogen-activated protein kinase kinase kinase.

Address correspondence to: Dr. Matjaz Humar, Center for Clinical Research, Breisacher Strasse 66, D-79106 Freiburg, Germany. E-mail: humar{at}ana1.ukl.uni-freiburg.de

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