STAT6 blockade ameliorates thyroid function in Graves' disease via downregulation of the Sodium/Iodide Symporter

STAT6 blockade ameliorates thyroid function in Graves' disease via downregulation of the Sodium/Iodide Symporter | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article STAT6 blockade ameliorates thyroid function in Graves' disease via downregulation of the Sodium/Iodide Symporter Qian Yang, Qinnan zhang, Fanfan Pan Pan, Bingbing Zha This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3936891/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Signal Transducer and Activator of Transcription 6 (STAT6) is an important nuclear transcription factor. Previous study demonstrated that blockading STAT6 can ameliorate thyroid function by reducing serum T3 and T4. Sodium/iodide symporter (NIS) is a key protein that mediates active iodine uptake and plays an important role in regulating thyroid function. This study explored the interaction between STAT6 and NIS. Methods Immunohistochemical staining was performed for detecting the expression of NIS in different tissues. Reverse transcription-polymerase chain reaction (RT-PCR) was performed for evaluating the mRNA level of NIS when Nthy-ori 3-1cells were incubated with IL4, TSH (Thyroid stimulating hormone) or monoclonal TSAb (thyroid-specific stimulatory autoantibody) for 24h. Quantitative RT-PCR,Western blot and immunofluorescence analysis were performed for detecting NIS expression after inhibiting STAT6 phosphorylation by AS1517499. Finally, we used Luciferase reporter assays to explore the ability of STAT6 to regulate the promoter activity of the NIS-coding gene. Results NIS was highly expressed in thyroid epithelial cells of EAGD mice or Graves' disease(GD) patients and TSAb increased the expression of NIS. We show that STAT6 phosphorylation inhibitor can attenuate the effect of TSAb on increasing NIS protein and mRNA levels. Finally, we confirm that transcription factor STAT6 can mediate NIS transcription and co-activator P100 protein can enhance STAT6-dependent transcriptional activation. Conclusion In Graves' disease, TSAb induces STAT6 signaling to upregulate NIS expression and STAT6 blockade ameliorates thyroid function via downregulation of the Sodium/Iodide Symporter. Our study furthers understanding of the effects of STAT6 on thyroid function and reveals new avenues for GD treatment. Graves' disease Sodium/Iodide Symporter Signal Transducer and Activator of Transcription 6 TSAb Figures Figure 1 Figure 2 Figure 3 Introduction Graves' disease (GD) 1 , 2 , characterized by thyroid-specific stimulatory autoantibodies (TSAbs) and high radioactive iodine uptake (RAIU), is the most common cause of hyperthyroidism. It is a disease that affects approximately 1% of Chinese people and is closely associated with many disorders including hyperglycemia, abnormal liver function and cardiovascular dysfunction 3 , 4 . It is fast becoming a growing challenge for public health but the pathogenesis of GD remains incompletely understood. Signal Transducer and Activator of Transcription 6 (STAT6) 5 belongs to the STAT family, stimulated by IL-4 and IL-13 that are involved in the pathophysiology of asthma, atopic dermatitis, rheumatoid arthritis and tumor microenvironment regulation 6 – 9 . In the last two decades, Land established that stat6 −/− mice were protected against Graves' disease, implying that STAT6 plays a pivotal role in its development 10 . In our previous study, we established a mouse model of autoimmune Graves' disease (EAGD), induced by repeatedly immunizing mice with an adenovirus vector expressing TSHR-289 11 . First, we found STAT6 phosphorylation was highly expressed in thyroid epithelial cells (TECs) of EAGD mice. Since STAT6 is involved in increasing epithelial cell growth and thyroid TEC hyperplasia is a feature of GD, we established the indispensable role of STAT6 in stimulating TEC hyperplasia in GD by targeting Bcl-xL and cyclin-D1. Bcl-xL and cyclin-D1 inhibit apoptosis and cell proliferation, respectively. We also revealed that stat6 −/− mice injected with Ad-TSHR-289 exhibited lower thyroid function, mainly characterized by lower serum T3 and T4 level. Nonetheless the pathogenesis of STAT6 deficiency and its role in amelioration of thyroid function remain unclear. Previous studies have confirmed that stimulatory autoantibodies result in thyroid hormone overproduction that is uncontrolled by the hypothalamic-pituitary axis. It is well established that a specific gene is involved in thyroid hormone biosynthesis 12 , 13 : thyroperoxidase (TPO), thyroglobulin (Tg), iodide transporters Na + /I − symporter (NIS), pendrin, calcium and NADPH dependent oxidases (DUOX 1/2). Accumulation of I − is the first step in thyroid hormone synthesis and is an active transport process finely regulated by NIS. NIS 14 , 15 , a key plasma membrane glycoprotein with 13 transmembrane segments, and an extracellular amino terminus and an intracellular carboxy terminus, transports two Na + ions per single I − ion into the thyroid. Thyroid stimulating hormone (TSH) 16 , the essential regulator of thyroid cell proliferation, differentiation and function, has been shown to be the primary regulator of NIS in the thyroid 17 . It strongly increases the expression of NIS in mRNA and protein levels by activation of Gɑs-mediated cAMP production. Our study aimed to determine whether STAT6 plays an important role in ameliorating thyroid function by regulating NIS. First, we found that NIS was highly expressed in TECs of patients with GD and mouse model of GD. Since NIS activity is reflected by Iodine uptake, retrospective study indicated a significant and linear correlation of thyrotrophin receptor antibody(TRAb) level with Iodine uptake ratio(RAIU). Further study indicated that STAT6 phosphorylation inhibitor could decrease the effect of TSH and TRAb on increasing NIS protein and mRNA levels. Finally, we confirmed that the TRAb-IL4/STAT6-NIS pathway is crucial for thyroid function. Our results revealed the role of STAT6 in the specific gene involved in thyroid hormone biosynthesis and may offer a novel strategy for treatment of GD. Materials and Methods Study population A retrospective cohort study was performed from 2010 to 2020 at Shanghai Fifth People's Hospital, Fudan University in 1000 patients with newly diagnosed or relapsed GD. Diagnosis of GD was based on signs, symptoms, and ancillary laboratory tests,such as diffuse goiter, a suppressed TSH value, elevated in serum thyroxine (T4),TRAb,TSAb and RAIU. Data on thyroid function and TRAb were extracted from the Hospital's Health Information System. The study protocol was approved by the Medical Ethics Committee of the Fifth People's Hospital of Shanghai, Fudan University (NO.2017-029) Cell culture Nthy-ori 3 − 1 cells (a normal human thyroid follicular epithelial cell line) were obtained from Shanghai Royal Industrial Co., Ltd (Shanghai, China). Nthy-ori 3 − 1 cells were grown on RIPM-1640 medium (Gibco, Portland, OR, USA) supplemented with 10% fetal bovine serum (Gibco, Portland, OR, USA), 2 mmol/L glutamine (Gibco, Portland, OR, USA), 100 IU/mL penicillin and 100 mg/mL streptomycin sulfate. Normal human colon epithelium cells (NCM460) were purchased from ATCC (Manassas, VA) and cultured with RPMI-1640 medium containing 10% fetal bovine serum. Tissues collection EAGD mouse model induction had been described in our previous study 11 . Wild-type (WT) BALB/c mice(n = 6) and EAGD mice(n = 6) were killed by anesthesia, and thyroid gland was removed and analyzed. Thyroid tissue(n = 6) was obtained from GD patients undergoing thyroidectomy and normal thyroid samples (n = 6) were obtained from unaffected glands of patients undergoing parathyroidectomy. Q-PCR Total RNA was isolated by applying RNAiso plus reagent (Takara Biotechnology Co.,Ltd,Dalian,China). Then we measured RNA content by NanoDrop2000 (Thermo Fisher Scientific,Waltham, MA, USA). cDNA was synthesized using the Prime Script RT Master Mix Perfect Real Time kit (Takara Biotechnology Co.,Ltd,Dalian,China), according to the manufacturer's protocol. The expression of NIS mRNA was detected by Q-PCR using SYBR Green premix (Takara Biotechnology Co.,Ltd,Dalian,China). The data were analyzed by the QuantStudio 3 Real-Time PCR System (Applied Biosystems). The expression level of NIS was normalized to the expression level of GAPDH. The fold change in gene expression was calculated according to the 2 −∆∆Ct method. The sequence-specific primers of NIS: NIS primer sense CTGCGACTC TCC CACTGA, NIS primer antisense CGCAGCTCTAGGTACTGGTA. Western blotting analysis Nthy-ori 3 − 1 cells were stimulated with TSH (Sigma-Aldrich, St Louis, MO,USA),M22 (a monoclonal stimulatory TSHR antibody, RSR-TSHR hMAB Cardiff, UK),IL-4(Proteintech Group Inc.,Wuhan,China) and AS1517499(a potent STAT6 phosphorylation inhibitor, HY-100614, MCE, NJ, USA). Cells were harvested and lysed with radio immunoprecipitation assay (RIPA) buffer (CoWin Biosciences,China). Cell lysates were quantified by BCA protein assay (Thermo Fisher Scientific,Waltham, MA, USA) and separated using standard SDS-PAGE. Proteins were then electrotransferred to nitrocellulose membranes and blocked with 5% bovine serum albumin for 1h. Membranes were incubated with primary antibodies and protein bands were developed by incubating with appropriate fluorophore-conjugated secondary antibodies, followed by imaging using the Odyssey laser digital imaging system. Immunohistochemistry Thyroid tissue was fixed in paraformaldehyde, embedded in paraffin, sectioned to 5µm thickness for immunohistochemical analysis. After deparaffinization, sections were incubated with 3% BSA at room temperature for 1h. Sections were stained with rabbit monoclonal antibody against NIS( Proteintech Group Inc.,Wuhan,China) overnight at 4℃. The corresponding secondary antibodies, HRP-conjugated anti-rabbit antibody, was applied for 1 h at room temperature. Sections were washed in PBS, then washed with diaminobenzidine substrate for 1 minute and hematoxylin for 1 minute. Sections were rinsed with water, dehydrated, cleared and finally mounted with neutral balsam. Sections were examined and visualized by Leica Microsystems.Six fields at ×400 magnification were selected randomly to quantify the number of NIS-positive epithelial cells per high-powered field (HPF). Immunofluorescence assay Nthy-ori 3 − 1 cells were cultured and then washed with phosphate-buffered saline (PBS). Cells were incubated in 4% paraformaldehyde (dissolved in PBS, pH 7.4) for 15 min at room temperature. Then cells were permeated with 0.1% Triton X-100, and stained with the primary antibodiesagainst NIS (1:500). After blocking with 5% goat serum for 2h, plates were incubated overnight at 4°C. Then, the corresponding secondary antibodies were incubated for 1 hour at room temperature, and DAPI (Beyotime, Shanghai, China) was used to stain nuclei. Images were captured using a confocal microscope. Plasmid construction and luciferase assay The − 1282 to + 198 bp DNA fragment of the human NIS promoter was amplified from human colon epithelial cell (NCM460) genomic DNA and cloned into a PGL4.17 vector with XhoI and HindIII restriction enzyme sites. Site-directed mutagenesis was performed by PCR with oligonucleotides carrying the desired mutation using Phusion Hot Start II DNA Polymerase (Thermo Fisher Scientific Waltham, MA, USA), followed by template plasmid digestion with DpnI. All constructs were sequenced to confirm the correct sequence. The expression vectors encoding STAT6 and P100 constructs were purchased from Promega Corporation. After 12 h of transfection, NCM460 cells were irradiated and then added to the medium for 36 h. Luciferase activity was analyzed using the Luciferase Assay System (Thermo Fisher Scientific Waltham, MA, USA) according to the manufacturer’s protocol. Statistical analysis Results are presented as the mean ± SE of at least three independent experiments. Statistical tests were performed using Prism 6.0 software (GraphPad Software). Continuous variables were compared using independent sample t test. Spearman rank correlation was used to evaluate the relationship between TRAb level and 3hRAIU or 24hRAIU. The differences were considered significant at p < 0.05 (*p < 0.05; **p < 0.01; ***p < 0.001) Results 1. TRAb increases the expression of genes for thyroid hormone biosynthesis. Activation of thyrotropin-receptor antibodies (TSAb) induces thyroid hormone overproduction. Since NIS is an intrinsic transmembrane protein and the first step in thyroid hormone synthesis, we focused on the effect of TSAb on NIS expression. We first explored NIS expression in TECs from GD patients and a GD mouse model. Immunohistochemical staining revealed that NIS was highly expressed in TECs of both GD patients and mice compared with those of control individuals (Fig. 1 a,b). Iodine uptake rate (RAIU) is widely applied in the clinical diagnosis of thyrotoxicosis since NIS affects absorption of iodine. Since NIS activity is reflected by I- uptake, we performed simple linear regression analyses to determine TRAb level with RAIU. There was a linear correlation of TRAb level with 3hRAIU (r = 0.495,P < 0.05) and 24hRAIU (R = 0.216,P < 0.05)(supplementary Fig. 1a,b). We established that IL-4 enhanced NIS mRNA expression in a dose-dependent manner, with induction evident at 0.5 ng/mL and reaching a peak at 50 ng/mL (Fig. 1 c).To study the correlation of TSAb with thyroid hormone biosynthesis genes, Nthy-ori 3-1cells were exposed to IL-4(50ng/mL), TSH (50ng/mL) or M22 (TSAb 1ug/ml) for 24h and expression of thyroid hormone biosynthesis genes examined. Results revealed that IL4,TSH and TSAb increased the expression of NIS (Fig. 1 d) and other thyroid-specific genes (supplementary Fig. 2). 2. STAT6 regulates the expression of NIS stimulated by TRAb. Thyroid TEC hyperplasia and thyroid hormone overproduction are features of GD. Previous study showed that STAT6 phosphorylation (p-STAT6), activated by IL-4, promotes TEC hyperplasia in GD. To investigate whether p-STAT6 plays an important role for thyroid hormone biosynthesis genes, we analyzed the effect of AS1517499 (STAT6 inhibitor) on NIS expression in Nthy-ori 3 − 1 cells stimulated by TSH or TRAb. Real-time quantitative reverse transcription PCR and Western blot assays were performed to assess NIS expression level following AS1517499 treatment. As shown in the figures(2a,2b), NIS expression induced by TSH or TRAb decreased dramatically after 24 h of AS1517499 treatment. Immunofluorescence analysis indicated that NIS was localized in the plasma membrane and cytoplasm. After stimulation by TRAb for 24h, nuclear staining revealed that NIS expression was increased in the plasma membrane and cytoplasm (Fig. 2 c). Nonetheless NIS protein in the plasma membrane, cytoplasm and nuclear staining (Fig. 2 c) were reduced markedly when Nthy-ori 3 − 1 cells were stimulated by TSH plus AS1517499 or TRAb plus AS1517499. 3. Coactivator P100 protein enhances STAT6 to modulate NIS transcriptional expression. p100 is a key coactivator of STAT6 enhancer that can interact with STAT6 to enhance STAT6-mediated gene transcriptional activation. To determine whether STAT6 binds to NIS promoter, we first analyzed the human NIS promoter region from the JASPAR database to identify candidate motifs for STAT6 binding. Finally, the − 1282 to + 198 bp of the human NIS promoter region was confirmed. The human NIS promoter was then amplified and cloned into a PGL4.17 vector. Nthy-ori 3 − 1 cells were transfected with NIS promoter reporter gene (PGL4-NIS) at different concentrations of pCDNA3.1-STAT6, with or without pCDNA3.1-p100.Then recombinant human IL-4 (50ng/mL) was added and incubated for 48h. Luciferase assay results demonstrated slightly increased activity when stimulated by a high concentration of pCDNA3.1-STAT6 (100ng/mL). Nonetheless there was no obvious enhancement of reporter gene activity when a low concentration of pCDNA3.1-STAT6 was applied (supplementary Fig. 3). This indicated that STAT6 may activate NIS indirectly. As shown in figure (3a), P100 had a slight effect on activity of the NIS reporter. Nonetheless when NIS promoter reporter gene and pCDNA3.1-p100 were co-transfected with different concentrations of pCDNA3.1-STAT6 into Nthy-ori 3 − 1 cells, luciferase activity was enhanced significantly in a dose-dependent manner. To further evaluate the role of coactivator P100 protein on STAT6 regulation of NIS transcriptional expression, we mutated the sequence of -1096 to -1086 bp of the human NIS promoter region AGCTTATGGAGAAGG into AGCTGATAGAGCAGG (Fig. 3 b). Results demonstrated no significant effect on control of the promoter (Fig. 3 c ). Discussion Thyroid hormones (TH) are important metabolic hormones that are synthesized by thyroid follicular epithelial cells and influence almost every tissue and organ system 18 . Inappropriately high synthesis and secretion of thyroid hormones has adverse effects on quality of life. Graves' disease is characterized by excessive thyroid hormone levels and is becoming a growing challenge for public health, affecting 0.5-2% of the world's adult population 4 . TH biosynthesis and production are controlled by a complex mechanism. Proteins, such as NIS, TPO, TG, TSHR, PDS and DUOX2, are important contributors to TH biosynthesis. Among them, active iodide accumulation in the thyroid mediated by the sodium iodide symporter (NIS) is the first step in thyroid hormone biosynthesis, and relies on the functional expression of NIS on the cell membrane 15 . Saito confirmed that NIS protein was increased in the thyroid tissue of patients with GD compared with normal thyroid tissue 19 . In this study, we demonstrated that NIS protein was increased in Graves' disease or EAGD thyroid tissue compared with normal thyroid tissue. Further analysis of the correlation between TRAb level and NIS activity revealed that increased TRAb level was closely associated with RAIU. Moreover, NIS mRNA and protein levels increased following TSH or TRAb stimulation. These data suggest that thyroid NIS expression in GD may be regulated by TRAb. Elevated IL-4/STAT6 signaling was observed in thyroid epithelial cells of GD patients and an EAGD mouse model 11 . STAT6 activation plays an essential role in promoting thyroid epithelial cell growth. Blockade of STAT6 could reduce T3 and T4 level, indicating it may be a potential therapeutic target in Graves' disease. Our study demonstrated that blockade of STAT6 phosphorylation by AS1517499 reduced NIS expression when TECs were stimulated by IL4, TSH or TRAb. This suggests that IL-4/STAT6 signaling plays a vital role in regulating NIS. Previous study has established that transcription factors NKX2.1, FOXE1, Krüppel-like zinc finger transcription factor GLI-similar 3 (GLIS3), nuclear factor-κB (NF-κB), and PAX8, can mediate NIS transcription 20 – 22 . Recently, Di Giusto confirmed that transcription factor CREB3L1 regulates promoter activity of the NIS-coding gene 23 . We hypothesized that transcription factor STAT6 is involved in NIS transcriptional regulation. We analyzed the combination with luciferase promoter assay of the human NIS gene using JASPAR database ( http://jaspar.genereg.net/ ) and identified STAT6 putative binding sites. Since transcriptional activation by STAT6 requires the interaction with coactivators like p100, we performed luciferase assay and revealed that STAT6 could enhance NIS transcriptional activity, especially when TECs were transfected with p100. The limitations of our study are as follows. Firstly, we only performed transfection or co-transfection experiments to explore whether STAT6 regulates promoter activity of the NIS-coding gene. However, experiments on protein-DNA interaction (for example, ChIP or EMSA) were not performed. Moreover, the interaction between STAT6 and p100 should be confirmed using additional methods, for example co-immunoprecipitation. Our findings highlight a new role of IL-4/STAT6 signaling in thyroid hormone biosynthesis. Blockade of STAT6 may serve as a potential therapeutic target in the treatment of GD. Author Contributors: Qian Yang, Qinnan Zhang and Fanfan Pan: Conceptualization, Methodology, Data curation, Writing- Original draft preparation Qian Yang, Bingbing Zha: Writing- Reviewing and Editing Bingbing Zha : Visualization,Supervision Dr. Bingbing Zha is the guarantor of this work and had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis Data Availability: The datasets generated and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request. Declarations Author Contributors: Qian Yang, Qinnan Zhang and Fanfan Pan: Conceptualization, Methodology, Data curation, Writing- Original draft preparation Qian Yang, Bingbing Zha: Writing- Reviewing and Editing Bingbing Zha : Visualization,Supervision Dr. Bingbing Zha is the guarantor of this work and had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis Data Availability: The datasets generated and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request. Acknowledgements We are grateful to S. Aglionby (London, England) for editing the manuscript. References Smith TJ, Hegedus L. Graves' Disease. N Engl J Med. 2016;375:1552–1565. DOI: 10.1056/NEJMra1510030 . Rapoport B, McLachlan SM. TSH Receptor Cleavage Into Subunits and Shedding of the A-Subunit; A Molecular and Clinical Perspective. Endocr Rev. 2016;37:114–134. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3936891","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":271740709,"identity":"35bd1b7f-5fab-4288-929d-b42c71411edf","order_by":0,"name":"Qian Yang","email":"","orcid":"","institution":"Department of Endocrinology, Fifth People's Hospital of Shanghai Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Yang","suffix":""},{"id":271740710,"identity":"bd930e8c-0dab-44f2-94cf-96b6cbe03ec9","order_by":1,"name":"Qinnan zhang","email":"","orcid":"","institution":"Department of Endocrinology, Fifth People's Hospital of Shanghai Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Qinnan","middleName":"","lastName":"zhang","suffix":""},{"id":271740711,"identity":"66287c06-974c-4b8a-86d4-b29152bf9709","order_by":2,"name":"Fanfan Pan Pan","email":"","orcid":"","institution":"Department of Endocrinology, Fifth People's Hospital of Shanghai Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Fanfan","middleName":"Pan","lastName":"Pan","suffix":""},{"id":271740712,"identity":"b198614f-577f-4af6-8603-b13da299afbc","order_by":3,"name":"Bingbing Zha","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCElEQVRIiWNgGAWjYBACA2YGBmYGAyBLgoHxQQWQzQBmE6mF2eAMUVoYGODK2CSI0mLOznv4dUHBHbv5s5ufVRxss5Y3Z2A+eJuHwS4PlxbLZr406xkGz5Ib5xwzu3GwLd1wZwNbsjUPQ3IxTocd5jEz5jE4nMwskWB2+2PbYcYNB3jMpHkYDiQ2ENLCJpH+reBg22H7DQf4vxHSYvwYqMWORyLHjAGoJRFoCxteLZbNPGbMMwwOJ0hI5BRLHDiXnrzhMJux5RyDZJxazPnPGH8u+HPYXn5G+sYPB8qsbTccb354402FHU4tQMAGigUkBeCoMcCtHqTkA5Cwx6tkFIyCUTAKRjYAAGzuVkMFYi1UAAAAAElFTkSuQmCC","orcid":"","institution":"Department of Endocrinology, Fifth People's Hospital of Shanghai Fudan University","correspondingAuthor":true,"prefix":"","firstName":"Bingbing","middleName":"","lastName":"Zha","suffix":""}],"badges":[],"createdAt":"2024-02-07 13:02:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3936891/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3936891/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51020328,"identity":"4acb2daf-b9ae-4607-a0b0-ccb307348072","added_by":"auto","created_at":"2024-02-12 19:44:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":610673,"visible":true,"origin":"","legend":"\u003cp\u003eTRAb increase expression of thyroid hormone biosynthesis genes.NIS was increased in TECs from both GD patients and EAGD mice. a: Immunohistochemical staining was used to detect NIS in 6 normal controls and 6 GD patients. b:Immunohistochemical staining was used to detect NIS in 6 normal controls and 6 GD mouse model. c,d:The mRNA level of NIS were detected after Nthy-ori 3-1cells were incubated with different concentrations of IL4,TSH and TSAb(M22) for 24h. Data are mean ± SE and were analyzed by independent sample t test. Magnification: × 100; × 400. (* p \u0026lt; 0.05,** p \u0026lt; 0.01,*** p \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3936891/v1/7c9811f00df012fc3f909fb1.png"},{"id":51020326,"identity":"823e247d-bd3c-4f5a-aff2-18690dbf909b","added_by":"auto","created_at":"2024-02-12 19:44:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":516433,"visible":true,"origin":"","legend":"\u003cp\u003eSTAT6 regulates the expression of NIS stimulated by TRAb. (a,b) The mRNA and protein level of NIS was detected by QPCR. (c) The expression of NIS was determined by immunoblotting. Immunofluorescence of Nthy-ori 3-1cells. blue indicated for nucleus, green indicated NIS positivity. Data are mean ± SE and were analyzed by independent sample t test. (* p \u0026lt; 0.05,** p \u0026lt; 0.01,*** p \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3936891/v1/5d5a519f122984035ca16242.png"},{"id":51020327,"identity":"9bc94ac6-4baa-4962-88f1-41c0743f77df","added_by":"auto","created_at":"2024-02-12 19:44:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":155023,"visible":true,"origin":"","legend":"\u003cp\u003eSTAT6 modulates NIS promoter activity. (a) Nthy-ori 3-1cells were stimulated with IL-4 50ng/mL and transfected with NIS promoter constructs PGL4-NIS 200ng,pCDNA3.1-p100 100ng and different concentrations of pCDNA3.1-STAT6 for 24h. (b) The sequence -1096 to -1086 bp of the human NIS promoter region were mutated. (c) The relative luciferase activity was detected after transfection with mutant versions. Data are expressed as the mean ± SEM and were analyzed by independent sample t test. (* p \u0026lt; 0.05,** p \u0026lt; 0.01,*** p \u0026lt; 0.001)\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3936891/v1/b8255f12db994dae0ef4a9d9.png"},{"id":51607741,"identity":"78a670f5-7aad-4d7a-a09f-f6e956e63951","added_by":"auto","created_at":"2024-02-25 16:44:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1148937,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3936891/v1/1d5b20fa-21e9-4015-8034-3409993f9cfa.pdf"},{"id":51020329,"identity":"16e23e17-0395-4e4f-b219-0bcf7bb72e34","added_by":"auto","created_at":"2024-02-12 19:44:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":9019546,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigure.docx","url":"https://assets-eu.researchsquare.com/files/rs-3936891/v1/78871073ed1c70e6d95eab4b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"STAT6 blockade ameliorates thyroid function in Graves' disease via downregulation of the Sodium/Iodide Symporter","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGraves' disease (GD)\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, characterized by thyroid-specific stimulatory autoantibodies (TSAbs) and high radioactive iodine uptake (RAIU), is the most common cause of hyperthyroidism. It is a disease that affects approximately 1% of Chinese people and is closely associated with many disorders including hyperglycemia, abnormal liver function and cardiovascular dysfunction\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. It is fast becoming a growing challenge for public health but the pathogenesis of GD remains incompletely understood.\u003c/p\u003e \u003cp\u003eSignal Transducer and Activator of Transcription 6 (STAT6)\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e belongs to the STAT family, stimulated by IL-4 and IL-13 that are involved in the pathophysiology of asthma, atopic dermatitis, rheumatoid arthritis and tumor microenvironment regulation\u003csup\u003e\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In the last two decades, Land established that \u003cem\u003estat6\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice were protected against Graves' disease, implying that STAT6 plays a pivotal role in its development\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In our previous study, we established a mouse model of autoimmune Graves' disease (EAGD), induced by repeatedly immunizing mice with an adenovirus vector expressing TSHR-289\u003csup\u003e11\u003c/sup\u003e. First, we found STAT6 phosphorylation was highly expressed in thyroid epithelial cells (TECs) of EAGD mice. Since STAT6 is involved in increasing epithelial cell growth and thyroid TEC hyperplasia is a feature of GD, we established the indispensable role of STAT6 in stimulating TEC hyperplasia in GD by targeting Bcl-xL and cyclin-D1. Bcl-xL and cyclin-D1 inhibit apoptosis and cell proliferation, respectively. We also revealed that \u003cem\u003estat6\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice injected with Ad-TSHR-289 exhibited lower thyroid function, mainly characterized by lower serum T3 and T4 level. Nonetheless the pathogenesis of STAT6 deficiency and its role in amelioration of thyroid function remain unclear. Previous studies have confirmed that stimulatory autoantibodies result in thyroid hormone overproduction that is uncontrolled by the hypothalamic-pituitary axis. It is well established that a specific gene is involved in thyroid hormone biosynthesis\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e: thyroperoxidase (TPO), thyroglobulin (Tg), iodide transporters Na\u003csup\u003e+\u003c/sup\u003e/I\u003csup\u003e\u0026minus;\u003c/sup\u003e symporter (NIS), pendrin, calcium and NADPH dependent oxidases (DUOX 1/2). Accumulation of I\u003csup\u003e\u0026minus;\u003c/sup\u003e is the first step in thyroid hormone synthesis and is an active transport process finely regulated by NIS. NIS\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, a key plasma membrane glycoprotein with 13 transmembrane segments, and an extracellular amino terminus and an intracellular carboxy terminus, transports two Na\u003csup\u003e+\u003c/sup\u003e ions per single I\u003csup\u003e\u0026minus;\u003c/sup\u003e ion into the thyroid. Thyroid stimulating hormone (TSH)\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, the essential regulator of thyroid cell proliferation, differentiation and function, has been shown to be the primary regulator of NIS in the thyroid\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. It strongly increases the expression of NIS in mRNA and protein levels by activation of Gɑs-mediated cAMP production.\u003c/p\u003e \u003cp\u003eOur study aimed to determine whether STAT6 plays an important role in ameliorating thyroid function by regulating NIS. First, we found that NIS was highly expressed in TECs of patients with GD and mouse model of GD. Since NIS activity is reflected by Iodine uptake, retrospective study indicated a significant and linear correlation of thyrotrophin receptor antibody(TRAb) level with Iodine uptake ratio(RAIU). Further study indicated that STAT6 phosphorylation inhibitor could decrease the effect of TSH and TRAb on increasing NIS protein and mRNA levels. Finally, we confirmed that the TRAb-IL4/STAT6-NIS pathway is crucial for thyroid function. Our results revealed the role of STAT6 in the specific gene involved in thyroid hormone biosynthesis and may offer a novel strategy for treatment of GD.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy population\u003c/h2\u003e \u003cp\u003eA retrospective cohort study was performed from 2010 to 2020 at Shanghai Fifth People's Hospital, Fudan University in 1000 patients with newly diagnosed or relapsed GD. Diagnosis of GD was based on signs, symptoms, and ancillary laboratory tests,such as diffuse goiter, a suppressed TSH value, elevated in serum thyroxine (T4),TRAb,TSAb and RAIU. Data on thyroid function and TRAb were extracted from the Hospital's Health Information System. The study protocol was approved by the Medical Ethics Committee of the Fifth People's Hospital of Shanghai, Fudan University (NO.2017-029)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eNthy-ori 3\u0026thinsp;\u0026minus;\u0026thinsp;1 cells (a normal human thyroid follicular epithelial cell line) were obtained from Shanghai Royal Industrial Co., Ltd (Shanghai, China). Nthy-ori 3\u0026thinsp;\u0026minus;\u0026thinsp;1 cells were grown on RIPM-1640 medium (Gibco, Portland, OR, USA) supplemented with 10% fetal bovine serum (Gibco, Portland, OR, USA), 2 mmol/L glutamine (Gibco, Portland, OR, USA), 100 IU/mL penicillin and 100 mg/mL streptomycin sulfate. Normal human colon epithelium cells (NCM460) were purchased from ATCC (Manassas, VA) and cultured with RPMI-1640 medium containing 10% fetal bovine serum.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eTissues collection\u003c/h2\u003e \u003cp\u003eEAGD mouse model induction had been described in our previous study\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Wild-type (WT) BALB/c mice(n\u0026thinsp;=\u0026thinsp;6) and EAGD mice(n\u0026thinsp;=\u0026thinsp;6) were killed by anesthesia, and thyroid gland was removed and analyzed. Thyroid tissue(n\u0026thinsp;=\u0026thinsp;6) was obtained from GD patients undergoing thyroidectomy and normal thyroid samples (n\u0026thinsp;=\u0026thinsp;6) were obtained from unaffected glands of patients undergoing parathyroidectomy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eQ-PCR\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated by applying RNAiso plus reagent (Takara Biotechnology Co.,Ltd,Dalian,China). Then we measured RNA content by NanoDrop2000 (Thermo Fisher Scientific,Waltham, MA, USA). cDNA was synthesized using the Prime Script RT Master Mix Perfect Real Time kit (Takara Biotechnology Co.,Ltd,Dalian,China), according to the manufacturer's protocol. The expression of NIS mRNA was detected by Q-PCR using SYBR Green premix (Takara Biotechnology Co.,Ltd,Dalian,China). The data were analyzed by the QuantStudio 3 Real-Time PCR System (Applied Biosystems). The expression level of NIS was normalized to the expression level of GAPDH. The fold change in gene expression was calculated according to the 2\u003csup\u003e\u0026minus;∆∆Ct\u003c/sup\u003e method. The sequence-specific primers of NIS: NIS primer sense CTGCGACTC TCC CACTGA, NIS primer antisense CGCAGCTCTAGGTACTGGTA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting analysis\u003c/h2\u003e \u003cp\u003eNthy-ori 3\u0026thinsp;\u0026minus;\u0026thinsp;1 cells were stimulated with TSH (Sigma-Aldrich, St Louis, MO,USA),M22 (a monoclonal stimulatory TSHR antibody, RSR-TSHR hMAB Cardiff, UK),IL-4(Proteintech Group Inc.,Wuhan,China) and AS1517499(a potent STAT6 phosphorylation inhibitor, HY-100614, MCE, NJ, USA). Cells were harvested and lysed with radio immunoprecipitation assay (RIPA) buffer (CoWin Biosciences,China). Cell lysates were quantified by BCA protein assay (Thermo Fisher Scientific,Waltham, MA, USA) and separated using standard SDS-PAGE. Proteins were then electrotransferred to nitrocellulose membranes and blocked with 5% bovine serum albumin for 1h. Membranes were incubated with primary antibodies and protein bands were developed by incubating with appropriate fluorophore-conjugated secondary antibodies, followed by imaging using the Odyssey laser digital imaging system.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry\u003c/h2\u003e \u003cp\u003eThyroid tissue was fixed in paraformaldehyde, embedded in paraffin, sectioned to 5\u0026micro;m thickness for immunohistochemical analysis. After deparaffinization, sections were incubated with 3% BSA at room temperature for 1h. Sections were stained with rabbit monoclonal antibody against NIS( Proteintech Group Inc.,Wuhan,China) overnight at 4℃. The corresponding secondary antibodies, HRP-conjugated anti-rabbit antibody, was applied for 1 h at room temperature. Sections were washed in PBS, then washed with diaminobenzidine substrate for 1 minute and hematoxylin for 1 minute. Sections were rinsed with water, dehydrated, cleared and finally mounted with neutral balsam. Sections were examined and visualized by Leica Microsystems.Six fields at \u0026times;400 magnification were selected randomly to quantify the number of NIS-positive epithelial cells per high-powered field (HPF).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence assay\u003c/h2\u003e \u003cp\u003eNthy-ori 3\u0026thinsp;\u0026minus;\u0026thinsp;1 cells were cultured and then washed with phosphate-buffered saline (PBS). Cells were incubated in 4% paraformaldehyde (dissolved in PBS, pH 7.4) for 15 min at room temperature. Then cells were permeated with 0.1% Triton X-100, and stained with the primary antibodiesagainst NIS (1:500). After blocking with 5% goat serum for 2h, plates were incubated overnight at 4\u0026deg;C. Then, the corresponding secondary antibodies were incubated for 1 hour at room temperature, and DAPI (Beyotime, Shanghai, China) was used to stain nuclei. Images were captured using a confocal microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid construction and luciferase assay\u003c/h2\u003e \u003cp\u003eThe \u0026minus;\u0026thinsp;1282 to +\u0026thinsp;198 bp DNA fragment of the human NIS promoter was amplified from human colon epithelial cell (NCM460) genomic DNA and cloned into a PGL4.17 vector with XhoI and HindIII restriction enzyme sites. Site-directed mutagenesis was performed by PCR with oligonucleotides carrying the desired mutation using Phusion Hot Start II DNA Polymerase (Thermo Fisher Scientific Waltham, MA, USA), followed by template plasmid digestion with DpnI. All constructs were sequenced to confirm the correct sequence. The expression vectors encoding STAT6 and P100 constructs were purchased from Promega Corporation. After 12 h of transfection, NCM460 cells were irradiated and then added to the medium for 36 h. Luciferase activity was analyzed using the Luciferase Assay System (Thermo Fisher Scientific Waltham, MA, USA) according to the manufacturer\u0026rsquo;s protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eResults are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SE of at least three independent experiments. Statistical tests were performed using Prism 6.0 software (GraphPad Software). Continuous variables were compared using independent sample t test. Spearman rank correlation was used to evaluate the relationship between TRAb level and 3hRAIU or 24hRAIU. The differences were considered significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001)\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e1. TRAb increases the expression of genes for thyroid hormone biosynthesis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eActivation of thyrotropin-receptor antibodies (TSAb) induces thyroid hormone overproduction. Since NIS is an intrinsic transmembrane protein and the first step in thyroid hormone synthesis, we focused on the effect of TSAb on NIS expression. We first explored NIS expression in TECs from GD patients and a GD mouse model. Immunohistochemical staining revealed that NIS was highly expressed in TECs of both GD patients and mice compared with those of control individuals (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea,b). Iodine uptake rate (RAIU) is widely applied in the clinical diagnosis of thyrotoxicosis since NIS affects absorption of iodine. Since NIS activity is reflected by I- uptake, we performed simple linear regression analyses to determine TRAb level with RAIU. There was a linear correlation of TRAb level with 3hRAIU (r\u0026thinsp;=\u0026thinsp;0.495,P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and 24hRAIU (R\u0026thinsp;=\u0026thinsp;0.216,P\u0026thinsp;\u0026lt;\u0026thinsp;0.05)(supplementary Fig.\u0026nbsp;1a,b). We established that IL-4 enhanced NIS mRNA expression in a dose-dependent manner, with induction evident at 0.5 ng/mL and reaching a peak at 50 ng/mL (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec).To study the correlation of TSAb with thyroid hormone biosynthesis genes, Nthy-ori 3-1cells were exposed to IL-4(50ng/mL), TSH (50ng/mL) or M22 (TSAb 1ug/ml) for 24h and expression of thyroid hormone biosynthesis genes examined. Results revealed that IL4,TSH and TSAb increased the expression of NIS (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed) and other thyroid-specific genes (supplementary Fig.\u0026nbsp;2).\u003c/p\u003e\n\u003cp\u003e2.\u0026nbsp;\u003cstrong\u003eSTAT6 regulates the expression of NIS stimulated by TRAb.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThyroid TEC hyperplasia and thyroid hormone overproduction are features of GD. Previous study showed that STAT6 phosphorylation (p-STAT6), activated by IL-4, promotes TEC hyperplasia in GD. To investigate whether p-STAT6 plays an important role for thyroid hormone biosynthesis genes, we analyzed the effect of AS1517499 (STAT6 inhibitor) on NIS expression in Nthy-ori 3\u0026thinsp;\u0026minus;\u0026thinsp;1 cells stimulated by TSH or TRAb. Real-time quantitative reverse transcription PCR and Western blot assays were performed to assess NIS expression level following AS1517499 treatment. As shown in the figures(2a,2b), NIS expression induced by TSH or TRAb decreased dramatically after 24 h of AS1517499 treatment. Immunofluorescence analysis indicated that NIS was localized in the plasma membrane and cytoplasm. After stimulation by TRAb for 24h, nuclear staining revealed that NIS expression was increased in the plasma membrane and cytoplasm (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec). Nonetheless NIS protein in the plasma membrane, cytoplasm and nuclear staining (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec) were reduced markedly when Nthy-ori 3\u0026thinsp;\u0026minus;\u0026thinsp;1 cells were stimulated by TSH plus AS1517499 or TRAb plus AS1517499.\u003c/p\u003e\n\u003cp\u003e3.\u0026nbsp;\u003cstrong\u003eCoactivator P100 protein enhances STAT6 to modulate NIS transcriptional expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ep100 is a key coactivator of STAT6 enhancer that can interact with STAT6 to enhance STAT6-mediated gene transcriptional activation. To determine whether STAT6 binds to NIS promoter, we first analyzed the human NIS promoter region from the JASPAR database to identify candidate motifs for STAT6 binding. Finally, the \u0026minus;\u0026thinsp;1282 to +\u0026thinsp;198 bp of the human NIS promoter region was confirmed. The human NIS promoter was then amplified and cloned into a PGL4.17 vector. Nthy-ori 3\u0026thinsp;\u0026minus;\u0026thinsp;1 cells were transfected with NIS promoter reporter gene (PGL4-NIS) at different concentrations of pCDNA3.1-STAT6, with or without pCDNA3.1-p100.Then recombinant human IL-4 (50ng/mL) was added and incubated for 48h. Luciferase assay results demonstrated slightly increased activity when stimulated by a high concentration of pCDNA3.1-STAT6 (100ng/mL). Nonetheless there was no obvious enhancement of reporter gene activity when a low concentration of pCDNA3.1-STAT6 was applied (supplementary Fig.\u0026nbsp;3). This indicated that STAT6 may activate NIS indirectly.\u003c/p\u003e\n\u003cp\u003eAs shown in figure (3a), P100 had a slight effect on activity of the NIS reporter. Nonetheless when NIS promoter reporter gene and pCDNA3.1-p100 were co-transfected with different concentrations of pCDNA3.1-STAT6 into Nthy-ori 3\u0026thinsp;\u0026minus;\u0026thinsp;1 cells, luciferase activity was enhanced significantly in a dose-dependent manner. To further evaluate the role of coactivator P100 protein on STAT6 regulation of NIS transcriptional expression, we mutated the sequence of -1096 to -1086 bp of the human NIS promoter region AGCTTATGGAGAAGG into AGCTGATAGAGCAGG (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb). Results demonstrated no significant effect on control of the promoter (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec ).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThyroid hormones (TH) are important metabolic hormones that are synthesized by thyroid follicular epithelial cells and influence almost every tissue and organ system\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Inappropriately high synthesis and secretion of thyroid hormones has adverse effects on quality of life. Graves' disease is characterized by excessive thyroid hormone levels and is becoming a growing challenge for public health, affecting 0.5-2% of the world's adult population\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTH biosynthesis and production are controlled by a complex mechanism. Proteins, such as NIS, TPO, TG, TSHR, PDS and DUOX2, are important contributors to TH biosynthesis. Among them, active iodide accumulation in the thyroid mediated by the sodium iodide symporter (NIS) is the first step in thyroid hormone biosynthesis, and relies on the functional expression of NIS on the cell membrane\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Saito confirmed that NIS protein was increased in the thyroid tissue of patients with GD compared with normal thyroid tissue\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. In this study, we demonstrated that NIS protein was increased in Graves' disease or EAGD thyroid tissue compared with normal thyroid tissue. Further analysis of the correlation between TRAb level and NIS activity revealed that increased TRAb level was closely associated with RAIU. Moreover, NIS mRNA and protein levels increased following TSH or TRAb stimulation. These data suggest that thyroid NIS expression in GD may be regulated by TRAb.\u003c/p\u003e \u003cp\u003eElevated IL-4/STAT6 signaling was observed in thyroid epithelial cells of GD patients and an EAGD mouse model\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. STAT6 activation plays an essential role in promoting thyroid epithelial cell growth. Blockade of STAT6 could reduce T3 and T4 level, indicating it may be a potential therapeutic target in Graves' disease. Our study demonstrated that blockade of STAT6 phosphorylation by AS1517499 reduced NIS expression when TECs were stimulated by IL4, TSH or TRAb. This suggests that IL-4/STAT6 signaling plays a vital role in regulating NIS. Previous study has established that transcription factors NKX2.1, FOXE1, Kr\u0026uuml;ppel-like zinc finger transcription factor GLI-similar 3 (GLIS3), nuclear factor-κB (NF-κB), and PAX8, can mediate NIS transcription\u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Recently, Di Giusto confirmed that transcription factor CREB3L1 regulates promoter activity of the NIS-coding gene\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. We hypothesized that transcription factor STAT6 is involved in NIS transcriptional regulation. We analyzed the combination with luciferase promoter assay of the human NIS gene using JASPAR database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://jaspar.genereg.net/\u003c/span\u003e\u003cspan address=\"http://jaspar.genereg.net/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and identified STAT6 putative binding sites. Since transcriptional activation by STAT6 requires the interaction with coactivators like p100, we performed luciferase assay and revealed that STAT6 could enhance NIS transcriptional activity, especially when TECs were transfected with p100. The limitations of our study are as follows. Firstly, we only performed transfection or co-transfection experiments to explore whether STAT6 regulates promoter activity of the NIS-coding gene. However, experiments on protein-DNA interaction (for example, ChIP or EMSA) were not performed. Moreover, the interaction between STAT6 and p100 should be confirmed using additional methods, for example co-immunoprecipitation.\u003c/p\u003e \u003cp\u003eOur findings highlight a new role of IL-4/STAT6 signaling in thyroid hormone biosynthesis. Blockade of STAT6 may serve as a potential therapeutic target in the treatment of GD.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAuthor Contributors:\u003c/h2\u003e \u003cp\u003eQian Yang, Qinnan Zhang and Fanfan Pan: Conceptualization, Methodology, Data curation, Writing- Original draft preparation\u003c/p\u003e \u003cp\u003eQian Yang, Bingbing Zha: Writing- Reviewing and Editing\u003c/p\u003e \u003cp\u003eBingbing Zha : Visualization,Supervision\u003c/p\u003e \u003cp\u003eDr. Bingbing Zha is the guarantor of this work and had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eData Availability:\u003c/h2\u003e \u003cp\u003eThe datasets generated and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributors:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQian Yang,\u0026nbsp;Qinnan Zhang and\u0026nbsp;Fanfan Pan: Conceptualization, Methodology, Data curation, Writing- Original draft preparation\u003c/p\u003e\n\u003cp\u003eQian Yang, Bingbing Zha: Writing- Reviewing and Editing\u003c/p\u003e\n\u003cp\u003eBingbing Zha\u0026nbsp;: Visualization,Supervision\u003c/p\u003e\n\u003cp\u003eDr. Bingbing Zha is the guarantor of this work and had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to S. Aglionby (London, England) for editing the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSmith TJ, Hegedus L. Graves' Disease. N Engl J Med. 2016;375:1552\u0026ndash;1565. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1056/NEJMra1510030\u003c/span\u003e\u003cspan address=\"10.1056/NEJMra1510030\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRapoport B, McLachlan SM. TSH Receptor Cleavage Into Subunits and Shedding of the A-Subunit; A Molecular and Clinical Perspective. Endocr Rev. 2016;37:114\u0026ndash;134. 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Transcription Factor CREB3L1 Regulates the Expression of the Sodium/Iodide Symporter (NIS) in Rat Thyroid Follicular Cells. Cells-Basel. 2022;11. DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3390/cells11081314\u003c/span\u003e\u003cspan address=\"10.3390/cells11081314\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Graves' disease, Sodium/Iodide Symporter, Signal Transducer and Activator of Transcription 6, TSAb","lastPublishedDoi":"10.21203/rs.3.rs-3936891/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3936891/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSignal Transducer and Activator of Transcription 6 (STAT6) is an important nuclear transcription factor. Previous study demonstrated that blockading STAT6 can ameliorate thyroid function by reducing serum T3 and T4. Sodium/iodide symporter (NIS) is a key protein that mediates active iodine uptake and plays an important role in regulating thyroid function. This study explored the interaction between STAT6 and NIS.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e \u003cp\u003eImmunohistochemical staining was performed for detecting the expression of NIS in different tissues. Reverse transcription-polymerase chain reaction (RT-PCR) was performed for evaluating the mRNA level of NIS when Nthy-ori 3-1cells were incubated with IL4, TSH (Thyroid stimulating hormone) or monoclonal TSAb (thyroid-specific stimulatory autoantibody) for 24h. Quantitative RT-PCR,Western blot and immunofluorescence analysis were performed for detecting NIS expression after inhibiting STAT6 phosphorylation by AS1517499. Finally, we used Luciferase reporter assays to explore the ability of STAT6 to regulate the promoter activity of the NIS-coding gene.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eNIS was highly expressed in thyroid epithelial cells of EAGD mice or Graves' disease(GD) patients and TSAb increased the expression of NIS. We show that STAT6 phosphorylation inhibitor can attenuate the effect of TSAb on increasing NIS protein and mRNA levels. Finally, we confirm that transcription factor STAT6 can mediate NIS transcription and co-activator P100 protein can enhance STAT6-dependent transcriptional activation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusion\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn Graves' disease, TSAb induces STAT6 signaling to upregulate NIS expression and STAT6 blockade ameliorates thyroid function via downregulation of the Sodium/Iodide Symporter. Our study furthers understanding of the effects of STAT6 on thyroid function and reveals new avenues for GD treatment.\u003c/p\u003e","manuscriptTitle":"STAT6 blockade ameliorates thyroid function in Graves' disease via downregulation of the Sodium/Iodide Symporter","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-12 19:44:47","doi":"10.21203/rs.3.rs-3936891/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5905008f-182b-480a-a682-e0f9ae126660","owner":[],"postedDate":"February 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-25T16:36:27+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-12 19:44:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3936891","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3936891","identity":"rs-3936891","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}