The function of peroxisome proliferator-activated receptors PPAR-γ and PPAR-δ in Mycobacterium leprae-induced foam cell formation in host macrophages.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae (M. leprae). In lepromatous leprosy (LL), skin macrophages, harboring extensive bacterial multiplication, gain a distinctive foamy appearance due to increased intracellular lipid load. To determine the mechanism by which M. leprae modifies the lipid homeostasis in host cells, an in vitro M. leprae infection system, using human macrophage precursor THP-1 cells and M. leprae prepared from the footpads of nude mice, was employed. RNA extracted from skin smear samples of patients was used to investigate host gene expressions before and after multidrug therapy (MDT). We found that a cluster of peroxisome proliferator-activated receptor (PPAR) target genes associated with adipocyte differentiation were strongly induced in M. leprae-infected THP-1 cells, with increased intracellular lipid accumulation. PPAR-δ and PPAR-γ expressions were induced by M. leprae infection in a bacterial load-dependent manner, and their proteins underwent nuclear translocalization after infection, indicating activation of PPAR signaling in host cells. Either PPAR-δ or PPAR-γ antagonist abolished the effect of M. leprae to modify host gene expressions and inhibited intracellular lipid accumulation in host cells. M. leprae-specific gene expressions were detected in the skin smear samples both before and after MDT, whereas PPAR target gene expressions were dramatically diminished after MDT. These results suggest that M. leprae infection activates host PPAR signaling to induce an array of adipocyte differentiation-associated genes, leading to accumulation of intracellular lipids to accommodate M. leprae parasitization. Certain PPAR target genes in skin lesions may serve as biomarkers for monitoring treatment efficacy.

Inhibitory effects of methimazole and propylthiouracil on iodotyrosine deiodinase 1 in thyrocytes.

Methimazole (MMI) and propylthiouracil (PTU) are commonly used for the treatment of Graves' disease. They share similar inhibitory effects on thyroid hormone biosynthesis by interfering with thyroid peroxidase (TPO)-mediated oxidation and organification of iodine. However, their potential effects on other thyroid functional molecules have not been explored in depth. To identify novel effects of MMI and PTU, DNA microarray analysis, real-time PCR, Western blotting, immunofluorescence staining and confocal laser scanning microscopy were performed using FRTL-5 rat thyroid cells. DNA microarray analysis indicated that both MMI and PTU suppress iodotyrosine deiodinase 1 (Iyd, Dehal1) mRNA levels. Further studies revealed that Dehal1 mRNA levels was stimulated by TSH, insulin and serum, while it was suppressed by iodine and a follicular concentration of thyroglobulin. MMI and PTU significantly suppressed Dehal1 expression induced by TSH, insulin and serum. On the other hand, although MMI suppressed Dehal1 expression in the absence of TSH, PTU only weakly suppressed Dehal1 without TSH. These results suggest that PTU and MMI may use different mechanisms to regulate Dehal1 expression, and TSH may play essential and differential roles in mediating PTU and MMI signals in thyrocytes. The drugs also inhibited re-distribution of Dehal1 protein into newly formed lysosomes following thyroglobulin endocytosis. These findings imply complex and multifaceted regulation of Dehal1 in the thyroid and suggest that MMI and PTU modulate Dehal1 expression and distribution of the protein in thyrocytes to exert their effect.

Follicular thyroglobulin induces cathepsin H expression and activity in thyrocytes.

Thyroglobulin (Tg) stored in thyroid follicles exerts a potent negative-feedback effect on each step of pre-hormone biosynthesis, including Tg gene transcription and iodine uptake and organification, by suppressing the expression of specific transcription factors that regulate these steps. Pre-hormones are stored in the follicular colloid before being reabsorbed. Following lysosomal proteolysis of its precursor, thyroid hormone (TH) is released from thyroid follicles. Although the suppressive effects of follicular Tg on each step of pre-hormone biosynthesis have been extensively characterized, whether follicular Tg accumulation also affects hormone reabsorption, proteolysis, and secretion is unclear. In this study we explored whether follicular Tg can regulate the expression and function of the lysosomal endopeptidases cathepsins. We found that in the rat thyroid cell line FRTL-5 follicular Tg induced cathepsin H mRNA and protein expression, as well as cathepsin H enzyme activity. Double immunofluorescence staining showed that Tg endocytosis promoted cathepsin H translocalization into lysosomes where it co-localized with internalized Tg. These results suggest that cathepsin H is an active participant in lysosome-mediated pre-hormone degradation, and that follicular Tg stimulates mobilization of pre-hormones by activating cathepsin H-associated proteolysis pathways.

Excessive Cytosolic DNA Fragments as a Potential Trigger of Graves' Disease: An Encrypted Message Sent by Animal Models.

Graves' hyperthyroidism is caused by autoantibodies directed against the thyroid-stimulating hormone receptor (TSHR) that mimic the action of TSH. The establishment of Graves' hyperthyroidism in experimental animals has proven to be an important approach to dissect the mechanisms of self-tolerance breakdown that lead to the production of thyroid-stimulating TSHR autoantibodies (TSAbs). "Shimojo's model" was the first successful Graves' animal model, wherein immunization with fibroblasts cells expressing TSHR and a major histocompatibility complex (MHC) class II molecule, but not either alone, induced TSAb production in AKR/N (H-2k) mice. This model highlights the importance of coincident MHC class II expression on TSHR-expressing cells in the development of Graves' hyperthyroidism. These data are also in agreement with the observation that Graves' thyrocytes often aberrantly express MHC class II antigens via mechanisms that remain unclear. Our group demonstrated that cytosolic self-genomic DNA fragments derived from sterile injured cells can induce aberrant MHC class II expression and production of multiple inflammatory cytokines and chemokines in thyrocytes in vitro, suggesting that severe cell injury may initiate immune responses in a way that is relevant to thyroid autoimmunity mediated by cytosolic DNA signaling. Furthermore, more recent successful Graves' animal models were primarily established by immunizing mice with TSHR-expressing plasmids or adenovirus. In these models, double-stranded DNA vaccine contents presumably exert similar immune-activating effect in cells at inoculation sites and thus might pave the way toward successful Graves' animal models. This review focuses on evidence suggesting that cell injury-derived self-DNA fragments could act as Graves' disease triggers.

A Novel Role for Flotillin-Containing Lipid Rafts in Negative-Feedback Regulation of Thyroid-Specific Gene Expression by Thyroglobulin.

BACKGROUND: Thyroglobulin (Tg) stored in thyroid follicles regulates follicular function in thyroid hormone (TH) synthesis by suppressing thyroid-specific gene expression in a concentration-dependent manner. Thus, Tg is an intrinsic negative-feedback regulator that can restrain the effect of thyrotropin (TSH) in the follicle. However, the underlying mechanisms by which Tg exerts its prominent autoregulatory effect following recognition by thyrocytes remains unclear. METHODS: In order to identify potential proteins that recognize and interact with Tg, mass spectrometry was used to analyze immunoprecipitated Tg-bound proteins derived from Tg-treated rat thyroid FRTL-5 cells. RESULTS: Flotillin 1 and flotillin 2, two homologs that are integral membrane proteins in lipid rafts, were identified as novel Tg-binding proteins with high confidence. Further studies revealed that flotillins physically interact with endocytosed Tg, and together these proteins redistribute from the cell membrane to cytoplasmic vesicles. Treatment with the lipid raft disrupter methyl-ß-cyclodextrin abolished both the endocytosis and the negative-feedback effect of Tg on thyroid-specific gene expression. Meanwhile, siRNA-mediated knockdown of flotillin 1 or flotillin 2 also significantly inhibited Tg effects on gene expression. CONCLUSION: Together these results indicate that flotillin-containing lipid rafts are essential for follicular Tg to be recognized by thyrocytes and exert its negative-feedback effects in the thyroid.

Follicular thyroglobulin enhances gene expression necessary for thyroid hormone secretion.

We have previously shown that follicular thyroglobulin (Tg) has an unexpected function as an autocrine negative-feedback regulator of thyroid hormone (TH) biosynthesis. Tg significantly suppressed the expression of genes necessary for iodide transport and TH synthesis by counteracting stimulation by TSH. However, whether follicular Tg also regulates intracellular TH transport and its secretion from thyrocytes is not known. In the present study, we examined the potential effect of follicular Tg on TH transport and secretion by quantifying the expression of two TH transporters: monocarboxylate transporter 8 (MCT8) and µ-crystallin (CRYM). Our results showed that follicular Tg at physiologic concentrations enhanced both the mRNA and protein expression levels of MCT8 and CRYM in a time- and dose-dependent manner in rat thyroid FRTL-5 cells. Although both the sodium/iodide symporter (NIS), an essential transporter of iodide from blood into the thyroid, and MCT8, a transporter of synthesized TH from the gland, were co-localized on the basolateral membrane of rat thyrocytes in vivo, Tg decreased NIS expression and increased the expression of MCT8 by counteracting TSH action. Thus, the effect of Tg on TH secretion opposed its previously described negative-feedback suppression of TH synthesis. Our results indicate that Tg mediates a complex intrinsic regulation of gene expression that is necessary to balance two opposing vectorial transport systems: the inflow of newly synthesized TH and the outflow of TH by external secretion.

Detection of Mycobacterium ulcerans subsp. shinshuense DNA from a water channel in familial Buruli ulcer cases in Japan.

AIM: To determine if Mycobacterium ulcerans subsp. shinshuense is present in the common living environment in a case of familial concurrent occurrence of Buruli ulcer in Japan. MATERIALS & METHODS: Environmental samples were collected primarily from a water channel in the patient's residence. A combination of whole-genome amplification, touchdown PCR and DNA sequencing was used to detect M. ulcerans subsp. shinshuense DNA. RESULTS: M. ulcerans subsp. shinshuense DNA was detected in a crayfish sampled in July, but not in other samples including a crayfish sampled in October. CONCLUSION: These findings support an association between contaminated aquatic environments and Buruli ulcer in Japan. The data also suggest dynamic seasonal appearance of the pathogen in the environment may contribute to the seasonal variation of Buruli ulcer occurrence in Japan.

Rapid preparation of high-purity nuclear proteins from a small number of cultured cells for use in electrophoretic mobility shift assays.

BACKGROUND: Highly purified nuclear protein is required when using an electrophoretic mobility shift assay (EMSA) to study transcription factors, e.g. nuclear factor-κB (NF-κB), a major transcription factor that regulates both innate and adaptive immune responses following infection. Although many protocols have been developed for nuclear protein extraction, they are not necessarily optimized for use in EMSA, often require a large number of cells and long processing times, and do not always result in complete separation of the nuclear and cytoplasmic fractions. RESULTS: We have developed a simple, rapid and cost-effective method to prepare highly purified nuclear proteins from a small number of both suspended and adherent cultured cells that yields nuclear proteins comparable to those prepared by a standard large-scale method. The efficiency of the method was demonstrated by using EMSA to show the successful detection, in multilple concurrent samples, of NF-κB activation upon tetradecanoyl phorbol acetate (TPA) stimulation. CONCLUSIONS: This method requires only a small number of cells and no specialized equipment. The steps have been simplified, resulting in a short processing time, which allows researchers to process multiple samples simultaneously and quickly. This method is especially optimized for use in EMSA, and may be useful for other applications that include proteomic analysis.

Iodine excess as an environmental risk factor for autoimmune thyroid disease.

The global effort to prevent iodine deficiency disorders through iodine supplementation, such as universal salt iodization, has achieved impressive progress during the last few decades. However, iodine excess, due to extensive environmental iodine exposure in addition to poor monitoring, is currently a more frequent occurrence than iodine deficiency. Iodine excess is a precipitating environmental factor in the development of autoimmune thyroid disease. Excessive amounts of iodide have been linked to the development of autoimmune thyroiditis in humans and animals, while intrathyroidal depletion of iodine prevents disease in animal strains susceptible to severe thyroiditis. Although the mechanisms by which iodide induces thyroiditis are still unclear, several mechanisms have been proposed: (1) excess iodine induces the production of cytokines and chemokines that can recruit immunocompetent cells to the thyroid; (2) processing excess iodine in thyroid epithelial cells may result in elevated levels of oxidative stress, leading to harmful lipid oxidation and thyroid tissue injuries; and (3) iodine incorporation in the protein chain of thyroglobulin may augment the antigenicity of this molecule. This review will summarize the current knowledge regarding excess iodide as an environmental toxicant and relate it to the development of autoimmune thyroid disease.

Paleopathological evidence and detection of Mycobacterium leprae DNA from archaeological skeletal remains of Nabe-kaburi (head-covered with iron pots) burials in Japan.

The Nabe-kaburi is a unique burial method, the purpose of which is shrouded in mystery. The burials were performed during the 15(th) to 18(th) centuries in eastern Japan, and involved covering the heads of the deceased with iron pots or mortars. The identification of leprosy-specific osteological lesions among some of the excavated remains has led to the suggestion that Nabe-kaburi burials were a reflection of the social stigma against certain infectious diseases, such as leprosy, tuberculosis or syphilis. However, molecular evidence for the presence of disease has been lacking. The goal of this study was to detect Mycobacterium leprae (M. leprae) DNA in archaeological human skeletal remains from Nabe-kaburi burials. The paleopathological data from three Nabe-kaburi burials were re-evaluated before small samples were taken from affected and control areas. DNA was extracted and used as a template to target the M. leprae-specific DNA using a combination of whole genome amplification, PCR analysis and DNA sequencing. M. leprae DNA fragments were detected in the two sets of skeletal remains that had also shown paleopathological evidence of leprosy. These findings provide definitive evidence that some of the Nabe-kaburi burials were performed for people affected by leprosy. Demonstration of the presence of M. leprae DNA, combined with archeological and anthropological examinations, will aid in solving the mystery of why Nabe-kaburi burials were performed in medieval Japan.

Thyroglobulin increases thyroid cell proliferation via the suppression of specific microRNAs.

Thyroglobulin (Tg), stored in the follicular lumen, has also been shown recently to perform two unexpected roles: as an autocrine negative-feedback suppressor of thyroid function in the presence of TSH and as a potent inducer of thyroid cell growth in the absence of TSH. However, the underlying molecular mechanism(s) remain unclear. To elucidate a molecular pathway linking Tg to increased cell proliferation, we examined the regulation of microRNAs (miRNAs) by Tg using an miRNA microarray. We identified 21 miRNAs whose expression was significantly suppressed by Tg in rat thyroid FRTL-5 cells. Using specific miRNA analogs, we determined that miR-16, miR-24, and miR-195 mediate the induction of thyroid cell growth by Tg. The expression of miR-16 and miR-195 target genes, Mapk8, Ccne1, and Cdc6, which were previously shown to be essential for TSH-stimulated thyroid cell growth, were also induced by Tg. Moreover, the Tg-induced expression of these genes was reduced by overexpression of miR-16 and miR-195. Similarly, the induction of c-Myc by Tg was reduced by miR-24 overexpression. These results suggest that Tg could alter thyroid cell proliferation by increasing the expression of cell division-related genes such as Mapk8, Ccne1, Cdc6, and c-Myc through its suppression of specific microRNAs (miR-16, miR-24, and miR-195). In addition, we identified phosphatidylinositol 3-kinase as a key signaling pathway, linking Tg with cell proliferation. The present data support an important role for miRNAs as effectors for the effect of Tg on cell proliferation and perhaps other functions of Tg in the thyroid cell.

Thyroglobulin suppresses thyroid-specific gene expression in cultures of normal but not neoplastic human thyroid follicular cells.

CONTEXT: It was shown in the rat thyroid that thyroglobulin (Tg) stored in the follicular lumen is a potent regulator of thyroid-specific gene expression to maintain the function of individual follicles. However, the actions of Tg as a regulatory molecule in human thyroid have not been studied. OBJECTIVE: Our objective was to determine the effect of Tg on gene expression in normal and diseased human thyroid and to examine whether the proposed model of negative-feedback autocrine regulation of thyroid function by Tg is applicable in the human as well as the rat. DESIGN: Primary cultures of human thyrocytes were established from normal thyroid, Graves' disease thyroid, adenomatous goiter, follicular adenoma, and papillary carcinoma tissues obtained during surgery. Cells were stimulated with physiologic (ie, follicular) concentrations of Tg, and mRNA and protein expression of genes involved in thyroid hormonogenesis were evaluated. The effects of Tg on thyroid-specific gene expression were also assessed in 2 human papillary carcinoma cell lines. RESULTS: Transcript levels of genes participating in thyroid hormone biosynthesis were significantly reduced by Tg in thyrocyte cultures derived from normal and Graves' thyroid, but not in cultures derived from thyroid neoplasms and adenomatous goiter. CONCLUSION: It was confirmed that Tg acts as a negative-feedback regulator of gene expression in human thyrocytes, suggesting that Tg signaling may constitute a common mechanism for maintaining thyroid homeostasis in species with follicular thyroid morphology. However, certain diseases of intrinsic thyroid overgrowth appear to be associated with an escape from the regulatory mechanism of Tg.

Leprosy as a model of immunity.

 Leprosy displays a spectrum of clinical manifestations, such as lepromatous and tuberculoid leprosy, and type I and II lepra reactions, which are thought to be a reflection of the host's immunological response against Mycobacterium leprae. Therefore, differential recognition of M. leprae, as well as its degraded components, and subsequent activation of cellular immunity will be an important factor for the clinical manifestation of leprosy. Although M. leprae mainly parasitizes tissue macrophages in the dermis and the Schwann cells of peripheral nerves, the presence of M. leprae in other organs, such as the liver, may also play important roles in the further modification of seesaw-like bipolar phenotypes of leprosy. Thus, leprosy is an exciting model for investigating the role of the human immune system in host defense and susceptibility to infection.

Hsp90 prevents interaction between CHIP and HERG proteins to facilitate maturation of wild-type and mutant HERG proteins.

AIMS: We examined the role of Hsp90 in expression and maturation of wild-type (WT) and mutant ether-a-go-go related gene (HERG) proteins by using Hsp90 inhibitors, geldanamycin (GA) and radicicol, and Hsp90 overexpression. METHODS AND RESULTS: The proteins were expressed in HEK293 cells or collected from HL-1 mouse cardiomyocytes, and analysed by western blotting, immunoprecipitation, immunofluorescence, and whole-cell patch-clamp techniques. GA and radicicol suppressed maturation of HERG-FLAG proteins and increased their immature forms. Co-expression of Hsp90 counteracted the effects of Hsp90 inhibitors and suppressed ubiquitination of HERG proteins. Overexpressed Hsp90 also inhibited the binding of endogenous C-terminus of Hsp70-interacting protein (CHIP) to HERG-FLAG proteins. Hsp90-induced increase of functional HERG proteins was verified by their increased expression on the cell surface and enhanced HERG channel currents. CHIP overexpression decreased both mature and immature forms of HERG-FLAG proteins in cells treated with GA. Hsp90 facilitated maturation of endogenous ERG proteins, whereas CHIP decreased both forms of ERG proteins in HL-1 cells. Mutant HERG proteins harbouring disease-causing missense mutations were mainly in the immature form and had a higher binding capacity to CHIP than the WT; Hsp90 overexpression suppressed this association. Overexpressed Hsp90 increased the mature form of HERG(1122fs/147) proteins, reduced its ubiquitinated form, increased its immunoreactivity in the endoplasmic reticulum and on the plasma membrane, and increased the mutant-mediated membrane current. CHIP overexpression decreased the immature form of HERG(1122fs/147) proteins. CONCLUSION: Enhancement of HERG protein expression through Hsp90 inhibition of CHIP binding might be a novel therapeutic strategy for long QT syndrome 2 caused by trafficking abnormalities of HERG proteins.

Demonstration of innate immune responses in the thyroid gland: potential to sense danger and a possible trigger for autoimmune reactions.

BACKGROUND: Autoimmune thyroid disease is an archetypal organ-specific autoimmune disorder that is characterized by the production of thyroid autoantibodies and lymphocytic infiltration into the thyroid. However, the underlying mechanisms by which specific thyroid antibodies are produced are largely unknown. Recent studies have shown that innate immune responses affect both the phenotype and the severity of autoimmune reactions. Moreover, it appears that even non-immune cells, including thyroid cells, have an ability to launch such responses. The aim of this study was to conduct a more detailed analysis of innate immune responses of the thyroid upon stimulation with various "non-self" and "self" factors that might contribute to the initiation of autoimmune reactions. METHODS: We used rat thyroid FRTL-5 cells, human thyroid cells, and mice to investigate the effects of various pathogen-associated molecular patterns (PAMPs), danger-associated molecular patterns (DAMPs), and iodide on gene expression and function that were related to innate immune responses. RESULTS: RT-PCR analysis showed that both rat and human thyroid cells expressed mRNAs for Toll-like receptors (TLRs) that sensed PAMPs. Stimulation of thyrocytes with TLR ligands resulted in activation of the interferon-beta (IFN-ß) promoter and the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB)-dependent promoter. As a result, pro-inflammatory cytokines, chemokines, and type I interferons were produced. Similar activation was observed when thyroid cells were stimulated with double-stranded DNA, one of the typical DAMPs. In addition to these PAMPs and DAMPs, treatment of thyroid cells with high concentrations of iodide increased mRNA expression of various cytokines. CONCLUSION: We show that thyroid cells express functional sensors for exogenous and endogenous dangers, and that they are capable of launching innate immune responses without the assistance of immune cells. Such responses may relate to the development of thyroiditis, which in turn may trigger autoimmune reactions.

Comprehensive analysis of prokaryotes in environmental water using DNA microarray analysis and whole genome amplification.

The microflora in environmental water consists of a high density and diversity of bacterial species that form the foundation of the water ecosystem. Because the majority of these species cannot be cultured in vitro, a different approach is needed to identify prokaryotes in environmental water. A novel DNA microarray was developed as a simplified detection protocol. Multiple DNA probes were designed against each of the 97,927 sequences in the DNA Data Bank of Japan and mounted on a glass chip in duplicate. Evaluation of the microarray was performed using the DNA extracted from one liter of environmental water samples collected from seven sites in Japan. The extracted DNA was uniformly amplified using whole genome amplification (WGA), labeled with Cy3-conjugated 16S rRNA specific primers and hybridized to the microarray. The microarray successfully identified soil bacteria and environment-specific bacteria clusters. The DNA microarray described herein can be a useful tool in evaluating the diversity of prokaryotes and assessing environmental changes such as global warming.

Clofazimine modulates the expression of lipid metabolism proteins in Mycobacterium leprae-infected macrophages.

Mycobacterium leprae (M. leprae) lives and replicates within macrophages in a foamy, lipid-laden phagosome. The lipids provide essential nutrition for the mycobacteria, and M. leprae infection modulates expression of important host proteins related to lipid metabolism. Thus, M. leprae infection increases the expression of adipophilin/adipose differentiation-related protein (ADRP) and decreases hormone-sensitive lipase (HSL), facilitating the accumulation and maintenance of lipid-rich environments suitable for the intracellular survival of M. leprae. HSL levels are not detectable in skin smear specimens taken from leprosy patients, but re-appear shortly after multidrug therapy (MDT). This study examined the effect of MDT components on host lipid metabolism in vitro, and the outcome of rifampicin, dapsone and clofazimine treatment on ADRP and HSL expression in THP-1 cells. Clofazimine attenuated the mRNA and protein levels of ADRP in M. leprae-infected cells, while those of HSL were increased. Rifampicin and dapsone did not show any significant effects on ADRP and HSL expression levels. A transient increase of interferon (IFN)-ß and IFN-γ mRNA was also observed in cells infected with M. leprae and treated with clofazimine. Lipid droplets accumulated by M. leprae-infection were significantly decreased 48 h after clofazimine treatment. Such effects were not evident in cells without M. leprae infection. In clinical samples, ADRP expression was decreased and HSL expression was increased after treatment. These results suggest that clofazimine modulates lipid metabolism in M. leprae-infected macrophages by modulating the expression of ADRP and HSL. It also induces IFN production in M. leprae-infected cells. The resultant decrease in lipid accumulation, increase in lipolysis, and activation of innate immunity may be some of the key actions of clofazimine.

Propylthiouracil increases sodium/iodide symporter gene expression and iodide uptake in rat thyroid cells in the absence of TSH.

BACKGROUND: Propylthiouracil (PTU) and methimazole (MMI) are drugs that are widely used to treat Graves' disease. Although both exert an antithyroid effect primarily by blocking thyroid peroxidase activity, their molecular structure and other actions are different. We hypothesized that PTU and MMI may have differential effects on thyroid-specific gene expression and function. METHODS: The effects of PTU and MMI on thyroid-specific gene expression and function were examined in rat thyroid FRTL-5 cells using DNA microarray, reverse transcriptase (RT)-polymerase chain reaction (PCR), real-time PCR, Western blot, immunohistochemistry, and radioiodine uptake studies. RESULTS: DNA microarray analysis showed a marked increase in sodium/iodide symporter (NIS) gene expression after PTU treatment, whereas MMI had no effect. RT-PCR and real-time PCR analysis revealed that PTU-induced NIS mRNA levels were comparable to those elicited by thyroid-stimulating hormone (TSH). PTU increased 5'-1880-bp and 5'-1052-bp activity of the rat NIS promoter. While PTU treatment also increased NIS protein levels, the size of the induced protein was smaller than that induced by TSH, and the protein localized predominantly in the cytoplasm rather than the plasma membrane. Accumulation of (125)I in FRTL-5 cells was increased by PTU stimulation, but this effect was weaker than that produced by TSH. CONCLUSIONS: We found that PTU induces NIS expression and iodide uptake in rat thyroid FRTL-5 cells in the absence of TSH. Although PTU and MMI share similar antithyroid activity, their effects on other thyroid functions appear to be quite different, which could affect their therapeutic effectiveness.

Regulation of dual oxidase expression and H2O2 production by thyroglobulin.

BACKGROUND: Thyroglobulin (Tg) is a macromolecular precursor in thyroid hormone synthesis to which iodine is stably bound. Tg, which is stored in the follicular space, is also a potent negative feedback regulator of follicular function, and this is achieved by suppressing mRNA levels of thyroid-specific genes such as the sodium/iodide symporter (Slc5a5), Tg, and thyroid peroxidase. Dual oxidase 1 (DUOX1) and DUOX2, originally identified in the thyroid, are nicotinamide adenine dinucleotide phosphate (NADPH) oxidases that are necessary to produce the H2O2 required for thyroid hormone biosynthesis. Since follicular Tg regulates the expression of genes that are essential for thyroid hormone synthesis, we hypothesized that Tg might also regulate DUOX expression and H2O2 production. METHODS: Rat thyroid FRTL-5 cells were treated with Tg, and the mRNA expression of Duox1 and Duox2 and their corresponding maturation factors Duoxa1 and Duoxa2 were evaluated by DNA microarray and real-time PCR. Duox2 promoter activity was examined by luciferase reporter gene assay. Protein levels of DUOX2 were also examined by Western blot analysis. Intracellular H2O2 generation was quantified by a fluorescent dye, 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, and acetyl ester (CM-H2DCFDA). RESULTS: mRNA levels of Duox2 and its activation factor Duoxa2 (but not Duox1 or Duoxa1) were significantly suppressed by Tg in a dose-dependent manner and a time-dependent fashion in rat thyroid FRTL-5 cells. DUOX2 promoter activity was significantly suppressed by Tg in a dose-dependent manner. Protein levels of DUOX2 and H2O2 generation in cells were also reduced by Tg treatment. CONCLUSIONS: We show that physiological concentrations of Tg suppressed the expression and function of DUOX2 in thyroid cells. These results suggest that Tg is a strong suppressor of the expression and the activity of DUOX2/DUOXA2, thereby regulating iodide organification and hormone synthesis in the thyroid. The evidence supports a reported model in which accumulated Tg in thyroid follicles plays important roles in autoregulating the function of individual follicles, which produces the basis of follicular heterogeneity.

Essential role of hormone-sensitive lipase (HSL) in the maintenance of lipid storage in Mycobacterium leprae-infected macrophages.

Mycobacterium leprae (M. leprae), the causative agent of leprosy, parasitizes within the foamy or enlarged phagosome of macrophages where rich lipids accumulate. Although the mechanisms for lipid accumulation in the phagosome have been clarified, it is still unclear how such large amounts of lipids escape degradation. To further explore underlying mechanisms involved in lipid catabolism in M. leprae-infected host cells, we examined the expression of hormone-sensitive lipase (HSL), a key enzyme in fatty acid mobilization and lipolysis, in human macrophage THP-1 cells. We found that infection by live M. leprae significantly suppressed HSL expression levels. This suppression was not observed with dead M. leprae or latex beads. Macrophage activation by peptidoglycan (PGN), the ligand for toll-like receptor 2 (TLR2), increased HSL expression; however, live M. leprae suppressed this increase. HSL expression was abolished in the slit-skin smear specimens from patients with lepromatous and borderline leprosy. In addition, the recovery of HSL expression was observed in patients who experienced a lepra reaction, which is a cell-mediated, delayed-type hypersensitivity immune response, or in patients who were successfully treated with multi-drug therapy. These results suggest that M. leprae suppresses lipid degradation through inhibition of HSL expression, and that the monitoring of HSL mRNA levels in slit-skin smear specimens may be a useful indicator of patient prognosis.