Norikazu Harii

Phenyl methimazole inhibits TNF-α-induced VCAM-1 expression in an IFN regulatory factor-1-dependent manner and reduces monocytic cell adhesion to endothelial cells

Proinflammatory cytokine (e.g., TNF-α)-induced expression of endothelial cell adhesion molecules (ECAMs) on the lumenal surface of the vascular endothelium and a consequent increase in leukocyte adhesion are key aspects of pathological inflammation. A promising therapeutic approach to diminish aberrant leukocyte adhesion is, therefore, to inhibit cytokine-induced ECAM expression at the transcription level. Several studies suggest that methimazole, a compound used clinically to treat autoimmune diseases, such as Graves’ disease, may also diminish pathological inflammation by suppressing ECAM expression. In this study we probed the hypothesis that a derivative of methimazole, phenyl methimazole (compound 10), can reduce cytokine-induced ECAM expression and consequent leukocyte adhesion. We found that compound 10 1) dramatically inhibits TNF-α-induced VCAM-1 mRNA and protein expression in human aortic endothelial cells (HAEC), has a relatively modest inhibitory effect on TNF-α induced E-selectin expression and has no effect on ICAM-1 expression; 2) significantly reduces TNF-α-induced monocytic (U937) cell adhesion to HAEC under in vitro flow conditions similar to that present in vivo; 3) inhibits TNF-α-induced IFN regulatory factor-1 binding to VCAM-1 promoter; and 4) reduces TNF-α-induced IRF-1 expression in HAEC. Combined, the results indicate that phenyl methimazole can reduce TNF-α-induced VCAM-1 expression in an IFN regulatory factor-1-dependent manner and that this contributes significantly to reduced monocytic cell adhesion to TNF-α-activated HAEC.

Phenylmethimazole Decreases Toll-Like Receptor 3 and Noncanonical Wnt5a Expression in Pancreatic Cancer and Melanoma Together with Tumor Cell Growth and Migration

Purpose: To evaluate whether (a) Wnt5a expression in pancreatic cancer and malignant melanoma cells might be associated with constitutive levels of Toll-like receptor 3 (TLR3) and/or TLR3 signaling; (b) phenylmethimazole (C10), a novel TLR signaling inhibitor, could decrease constitutive Wnt5a and TLR3 levels together with cell growth and migration; and (c) the efficacy of C10 as a potential inhibitor of pancreatic cancer and malignant melanoma cell growth in vivo. Experimental Design: We used a variety of molecular biology techniques including but not limited to PCR, Western blotting, and ELISA to evaluate the presence of constitutively activated TLR3/Wnt5a expression and signaling. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based technology and scratch assays were used to evaluate inhibition of cell growth and migration, respectively. TLR3 regulation of cell growth was confirmed using small interfering RNA technology. Nude and severe combined immunodeficient mice were implanted with human pancreatic cancer and/or melanoma cells and the effects of C10 on tumor growth were evaluated. Results: We show that constitutive TLR3 expression is associated with constitutive Wnt5a in human pancreatic cancer and malignant melanoma cell lines, that C10 can decrease constitutive TLR3/Wnt5a expression and signaling, suggesting that they are interrelated signal systems, and that C10 inhibits growth and migration in both of these cancer cell lines. We also report that C10 is effective at inhibiting human pancreatic cancer and malignant melanoma tumor growth in vivo in nude or severe combined immunodeficient mice and associate this with inhibition of signal transducers and activators of transcription 3 activation. Conclusions: C10 may have potential therapeutic applicability in pancreatic cancer and malignant melanoma.

Thyroglobulin (Tg) induces thyroid cell growth in a concentration-specific manner by a mechanism other than thyrotropin/cAMP stimulation

Thyroglobulin (Tg), a major product of the thyroid gland, serves as a macromolecular precursor of thyroid hormone biosynthesis. In addition, Tg stored in the thyroid follicles is a potent regulator of thyroid-specific gene expression. In conjunction with thyroid stimulating hormone (TSH) and iodide, Tg regulates thyroid follicle function, which is the minimal functional unit of the thyroid gland. In the present study, we show that Tg stimulates growth of FRTL-5 thyroid cells in the absence of TSH, insulin and serum. Unlike TSH, Tg did not increase cellular cyclic AMP (cAMP) levels; rather, the TSH signal counteracted Tg-induced cell growth. A specific inhibitor of A-kinase, H-89, did not modulate the effect of Tg. Tg increased kinase activity of Akt to the same level as TSH, insulin and 5% serum, while LY294002 abolished Tg-induced growth. Interestingly, low Tg concentrations maximized growth-promotion activity and induction of the apical iodide transporter (PDS; SLC26A4), whereas high Tg concentrations suppressed both cell growth and the expression of thyroid-specific genes. These results suggest that a low levels of Tg in the follicular lumen might stimulates cell growth and iodide transport to accelerate the iodide organification process; however, elevated Tg levels in the follicle might then shut down all of these functions.

Pax-8 Is Essential for Regulation of the Thyroglobulin Gene by Transforming Growth Factor-β1

Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine that is thought to play a major role in the regulation of growth and differentiation of thyroid cells. However, little is known of its detailed mechanisms of action in thyrocytes. We have therefore studied the molecular mechanisms of TGF-β1 action on thyroglobulin (TG) gene expression by focusing our attention on TGF-β1 regulation of thyroid-specific transcription factors. TGF-β1 decreased TG messenger RNA (mRNA) expression both in the presence and in the absence of TSH in rat thyroid FRTL-5 cells. Transfected into FRTL-5 cells, the activity of reporter plasmids containing the rat TG promoter ligated to a luciferase gene was significantly suppressed by the addition of TGF-β1. When the nuclear extracts prepared from TGF-β1-treated FRTL-5 cells were used in gel mobility shift assays, the amount of protein-DNA complex formed by Pax-8 was reduced, both in the presence and in the absence of TSH, but protein-DNA complexes formed by thyroid tra...

A TSHR-LH/CGR Chimera that Measures Functional Thyroid-Stimulating Autoantibodies (TSAb) Can Predict Remission or Recurrence in Graves' Patients Undergoing Antithyroid Drug (ATD) Treatment

CONTEXTnA functional thyroid-stimulating autoantibodies (TSAb) assay using a thyroid-stimulating hormone receptor chimera (Mc4) appears to be clinically more useful than the commonly used assay, a binding assay that measures all the antibodies binding to the thyroid-stimulating hormone receptor without functional discrimination, in diagnosing patient with Graves disease (GD).nnnOBJECTIVEnThe objective of the study was to investigate whether an Mc4 assay can predict relapse/remission of hyperthyroidism after antithyroid drug (ATD) treatment in patients with GD.nnnDESIGNnAn Mc4 assay was used to prospectively track TSAb activity in GD patients treated with ATD over a 5-yr period.nnnSETTING AND PATIENTSnGD patients from the Chieti University participated in this study.nnnINTERVENTIONSnInterventions included the assessment of patients sera using the Mc4 assay, the Mc4-derivative assay (Thyretain), and a human monoclonal thyroid-stimulating hormone receptor antibody, M22 assay.nnnMAIN OUTCOME MEASURESnThe Mc4 assay, a sensitive index of remission and recurrence, was used in this study.nnnRESULTSnThe TSAb levels significantly decreased only in the remitting group as evidenced by Mc4 assay values at the end of ATD (0.96 ± 1.47, 10.9 ± 26.6. and 24.7 ± 37.5 arbitrary units for the remitting, relapsing, and unsuspended therapy groups, respectively). Additional prognostic help was obtained by thyroid volume measurements at the end of treatment. Although not statistically significant, the Mc4 assay has a trend toward improved positive predictive value (95.4 vs. 84.2 or 87.5%), specificity (96.4 vs. 86.4 and 90.9%), and accuracy (87.3 vs. 83.3 and 80.9%) comparing the Mc4, Thyretain, and M22 assays, respectively. Thyretain has a trend toward improved negative predictive value (82.6 vs. 81.8 and 76.9%) and sensitivity (80 vs. 77.8 and 70%) comparing Thyretain, Mc4, and M22 assays, respectively.nnnCONCLUSIONnThe Mc4 assay is a clinically useful index of remission and relapse in patients with GD. Larger studies are required to confirm these findings.

Extracellular adenosine increases Na+/I− symporter gene expression in rat thyroid FRTL-5 cells

We studied the effect of extracellular adenosine on iodide (I-) transport in FRTL-5 thyroid cells. I- accumulation increases after a 48 h exposure to adenosine in a concentration-dependent manner, reaching a maximum of 7.9-fold basal levels at 72 h after the addition of 300 microM adenosine. Neither I- efflux nor intracellular cyclic adenosine monophosphate accumulation is affected by the exposure to adenosine. The stimulation of I- transport by adenosine is partly as a result of an increase in Na+/I- symporter (NIS) mRNA and protein levels. Northern blot analysis revealed that adenosine increases NIS mRNA levels at 24 h, reaching a maximum at 36 h. Western blot analysis demonstrated that adenosine increases NIS protein levels at 36 h, reaching a maximum at 72 h, in parallel with the kinetics of adenosine-induced I- transport. Adenosine increased the promoter activity of a full-length NIS promoter-luciferase chimera, suggesting that the effect of adenosine on NIS mRNA levels is transcriptional. The stimulatory effect of adenosine on NIS mRNA levels, is mimicked by N6-(L-2-phenylisopropyl) adenosine (PIA), an A1 adenosine receptor agonist, and inhibited by 1,3-dipropyl-8-cyclopentylxanthine, an A1 adenosine receptor antagonist, suggesting that the effect is mediated via the A1 adenosine receptor stimulation in FRTL-5 cells. Incubating cells with islet-activating protein inhibited the adenosine-induced NIS mRNA levels. In sum, extracellular adenosine increases NIS gene expression and stimulates I- transport via the A1 adenosine receptor-Gi/Go protein signal transduction pathway.

Tumor Necrosis Factor-α Regulation of Thyroid Transcription Factor-1 and Pax-8 in Rat Thyroid FRTL-5 Cells

Tumor necrosis factor-α (TNF-α) is known to modulate the expression of thyroid-specific genes, such as thyroglobulin (TG), contributing to the pathogenesis of autoimmune thyroid disease. In the present study, we show that TNF-α suppresses DNA-binding activity of thyroid transcription factors, Pax-8 and thyroid transcription factor-1 (TTF-1), which is, in part, involved in TNF-α-induced decrease in TG gene expression. Transfected into rat thyroid FRTL-5 cells, the activity of reporter plasmid containing the rat TG promoter ligated to a luciferase gene was significantly suppressed in the presence of TNF-α. In gel mobility shift analyses, protein-DNA complexes formed by TTF-1 and Pax-8 were reduced when the nuclear extracts prepared from TNF-α-treated FRTL-5 cells were used. The suppressive effect of TNF-α on TTF-1-DNA complex formation is, in part, caused by suppression of TTF-1 gene transcription by TNF-α. Expressions of TTF-1 messenger RNA and protein, which were assessed by Northern blot and Western blot...

A TSHr-LH/CGr Chimera that Measures Functional TSAb in Graves' Disease.

CONTEXTnStimulating thyrotropin receptor (TSHr) autoantibodies (TSAb) are the cause of hyperthyroidism in Graves disease. In a patients serum, TSAb can coexist with antagonist TSHr autoantibodies that block TSAb stimulatory activity (TSBAb); both can vary in amount and time.nnnOBJECTIVEnThe objective of the study was to create a functional assay that detects only TSAb, thus having an increased accuracy for diagnosing Graves disease.nnnDESIGNnA TSHr chimera (Mc4) that retains an agonist-sensitive TSAb epitope but replaces a TSBAb epitope was stably transfected in cells to establish the Mc4 assay.nnnSETTINGnThe study was conducted at the Chieti University (Outpatient Endocrine Clinic) and the University of Pisa (the Department of Endocrinology).nnnPATIENTSnThe assay was validated using sera from 170 individuals with Graves disease, Hashimotos thyroiditis, and nonautoimmune hyperthyroidism and normal subjects from Chieti University. A second blinded study evaluated sera from 175 patients with autoimmune thyroid disease (mainly Graves disease) from the University of Pisa.nnnINTERVENTIONSnInterventions included the assessment of patients sera using human wild-type TSHr (WT-TSHr), Mc4 chimera, and binding (TRAb) assays.nnnMAIN OUTCOME MEASURESnThe Mc4 assay has the best accuracy for diagnosing Graves disease.nnnRESULTSnThe Mc4 assay has a better diagnostic accuracy than WT-TSHr and second-generation TRAb assays. Indeed, the sensitivity of the WT-TSHr, TRAb, and Mc4 assays was 97.3, 86.5, and 100%, respectively, whereas the specificity was 93.1, 97, and 98.5%, respectively.nnnCONCLUSIONnThe Mc4 assay is a functional assay with improved sensitivity and specificity for the detection of TSAb and is clinically useful in diagnosing Graves disease.

Phenyl methimazole suppresses dextran sulfate sodium-induced murine colitis

Ulcerative colitis is an autoimmune-inflammatory disease characterized by abnormally increased expression of Toll-like receptor-4 (TLR4) in colonic epithelial cells, increased production of pro-inflammatory cytokines (e.g., TNF-alpha, IL-1beta, IL-6, IL-12), chemokines (e.g., IP-10), and endothelial cell adhesion molecules (e.g., VCAM-1), plus enhanced leukocyte infiltration into colonic interstitium. Previously, we have shown that phenyl methimazole (C10) markedly decreases virally-induced TLR-3 expression and signaling and potently inhibits both TNF-alpha-induced VCAM-1 expression and the resultant leukocyte-endothelial cell adhesion. In this study we probed the hypothesis that C10 is efficacious in a TLR-4- and VCAM-1-associated murine model [the dextran sulfate sodium (DSS) model] of human colitis. C10 was administered intraperitoneally coincident with or after DSS treatment was initiated. Macroscopic colon observations revealed that C10 significantly reversed DSS-induced shortening of the colon (P<0.05) and reduced the presence of blood in the colon. Histological analyses of colonic tissues revealed that C10 distinctly attenuated both DSS-induced edema as well as leukocyte infiltration in the colonic mucosa and resulted in pronounced protection against DSS-induced crypt damage (P<0.001). Northern blot analyses and immunohistochemistry of colonic tissue revealed that C10 markedly diminished DSS-induced expression of pertinent inflammatory mediators: TNF-alpha, IL-1beta, IL-6, IL-12, IP-10, TLR-4 and VCAM-1. Most importantly, C10 significantly improved survival and protected mice against DSS-induced colitic-death: 75% by comparison to 12.5% with identical treatment with DMSO-control (log rank test: P=0.005). These results provide direct evidence that C10 suppresses DSS-induced colitis by inhibiting expression of key inflammatory mediators and leukocyte infiltration, and is a potentially attractive therapeutic for colitis.

Phenylmethimazole inhibits production of proinflammatory mediators and is protective in an experimental model of endotoxic shock

Background:One form of sepsis, or endotoxic shock, is a hyperactivated systemic response caused by excessive expression of proinflammatory mediators, which results from Gram-negative bacterial lipopolysaccharide-stimulated Toll-like receptor-4 signaling. This lipopolysaccharide signaling is known to consist of a MyD88-dependent nuclear factor-&kgr;B-mediated pathway that results in production of proinflammatory mediators (tumor necrosis factor-&agr;, interleukin-6, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, inducible nitric oxide synthase, cyclooxygenase-2) and a MyD88-independent interferon regulatory factor-mediated pathway that regulates production of Type 1 interferon-inducible proteins (interferon &ggr;-induced protein-10, monocyte chemotactic protein-1). In prior studies, phenylmethimazole markedly decreased virally induced Toll-like receptor-3 expression and signaling and significantly suppressed murine colitis in an experimental model wherein lipopolysaccharide is known to play an important role. Objective:In this study, we probed the hypothesis that phenylmethimazole inhibits lipopolysaccharide-mediated Toll-like receptor-4 signaling and is efficacious in attenuating inflammatory changes and improving survival in an in vivo murine model of endotoxic shock. Design:Experimental animal model. Setting:University laboratory. Subjects:Male C57BL/6J mice weighing 18–22 g. Interventions:Phenylmethimazole (1 mg/kg) was administered intraperitoneally to mice before a lethal lipopolysaccharide challenge (25 mg/kg). RAW264.7 mouse macrophage cells were pretreated with phenylmethimazole followed by lipopolysaccharide stimulation. Measurements and Main Results:Macroscopic observations revealed that phenylmethimazole was significantly protective in controlling clinical manifestations of endotoxic shock and death under conditions wherein flunixin of meglumine and prednisolone were marginally effective. A combination of enzyme-linked immunosorbent assay, Northern blot, reverse transcriptase-polymerase chain reaction, immunohistochemistry, and Western blot analyses showed that phenylmethimazole attenuated lipopolysaccharide-induced increases in production of proinflammatory cytokines (tumor necrosis factor-&agr;, interleukin-6, interferon-&ggr;), endothelial cell adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), inducible nitric oxide synthase and cyclooxygenase-2, interferon regulatory factor-1, interferon-inducible proteins (interferon &ggr;-induced protein-10, monocyte chemotactic protein-1), and signal transducer and activator of transcription-1 phosphorylation in multiple tissues in mice. Consistent with these observations, electrophoretic mobility shift assay demonstrated that phenylmethimazole inhibited in vitro lipopolysaccharide-induced nuclear factor-&kgr;B and interferon regulatory factor-1 activation in RAW 264.7 mouse macrophages. Conclusions:Collectively, these results provide direct evidence that phenylmethimazole diminishes lipopolysaccharide-induced MyD88-dependent as well as MyD88-independent signaling pathways and is protective in an experimental model of endotoxic shock.