| Background and Objective:Subclinical hypothyroidism(SCH)refers to an abnormal thyroid function in which the serum thyroid stimulating hormone(TSH)is higher than the upper limit of the reference value and the free thyroxine(FT4)is normal.According to the cross-sectional survey conducted by our research group in 2018,the prevalence of SCH in mainland China was 12.93%,ranking first among all kinds of thyroid dysfunction.In particular,the prevalence of SCH increased significantly in patients with more than adequate iodine(urinary iodine 200-299 μg/L)and excess iodine(urinary iodine ≥300 μg/L).Combined with the above evidence and previous epidemiological investigations,it is generally believed that chronic iodine excess is significantly correlated with serum TSH elevation,but its mechanism remains to be further explored.In general,the mechanism of iodine-induced hyperthyrotropinemia can be mainly summarized into two mechanisms:"thyroid hypothesis" and "hypothalamic-pituitary hypothesis".Among them,thyroid hypothesis refers to the significantly increased prevalence or incidence of autoimmune thyroiditis(AIT)caused by iodine overdose.As the destruction of thyroid follicular epithelium is aggravated by iodine,the negative feedback inhibition of thyroid hormone on pituitary TSH is reduced,and serum TSH is increased.This conclusion was confirmed in a five-year cohort study by our group.However,a recent national crosssectional investigation suggested that the prevalence of SCH with positive thyroid autoantibody(thyroid peroxidase antibody,TPOAb;thyroglobulin antibody,TgAb)showed no significant upward trend with the elevation of urinary iodine concentration(UIC).On the contrary,the prevalence of SCH with negative thyroid autoantibody was significantly increased with the increase of UIC.This manuscript suggested that the association between iodine and AIT was similar to iodine-induced hyperthyroidism,namely,the increased prevalence of AIT after iodine supplementation is probably transient.Under the influence of chronic iodine excess,the thyroid and autoimmune system may become tolerant to increased iodine intake.In conclusion,in addition to the"thyroid hypothesis",there may be some non-autoimmune mechanisms of iodineinduced hyperthyrotropinemia remain exploration.Our research group used potassium iodide(KI)at different concentrations to intervene Wistar rats without autoimmune predisposition.The study results confirmed that KI overdose could induce SCH in Wistar rats.This may be related to the intensification of ubiquitination modification and degradation of pituitary type 2 deiodinase(D2),which leads to the decreased activity of D2.D2 is a selenium protease mainly distributed in endoplasmic reticulum(ER)and Golgi membrane and is widely expressed in the central nervous system.The D2 catalyzes the formation of highly active triiodothyronine(T3)from the low bioactivity thyroxine(T4),which can regulate the T4/T3 ratio in TSH cells and the negative feedback efficiency of thyroid hormone on the pituitary.However,the mechanism of chronic iodine excess-pituitary D2 ubiquitination modification-pituitary D2 inactivation and serum TSH elevation may be incomplete for the "hypothalamic-pituitary hypothesis".The aim of this study was to investigate the mechanism of hyperthyrotropinemia induced by iodine excess by focusing on whether iodine leads to the dysregulation of D2 active centers through endoplasmic reticulum stress(ERS)in the pituitary gland.This study supplemented the existing basic research evidence and explained the reason why D2 activity decreased in the high-iodine group from the perspective of its synthesis mechanism,which provided more explanations for the results of some population studies in recent years.Methods:Part One:Wistar rats at four weeks of age were given high iodine(10 times high iodine,10HI;50 times high iodine,50HI)for 24 weeks.The control group(normal iodine,NI)was given normal chow and water.Venous blood and urine samples were collected at baseline,week 6,week 12,week 18,and week 24 by jugular vein sampling and metabolic cage.Inductively coupled plasma mass spectrometry(ICP-MS)was applied to monitor the UIC from baseline to each time point.Serum was collected after venous centrifugation,and enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of TSH,FT4 and FT3 at baseline and each time point.Thyroid hormone sensitivity were calculated,including three central sensitivity parameters of thyroid hormone TFQI,TSHI and TT4RI.At the 24th week,pituitary tissues were collected,and quantitative reverse transcription polymerase chain reaction(qPCR)and western blot(WB)were used to evaluate the expression level of pituitary D2 in each group.The D2 activity of pituitary gland in each group was evaluated by high performance liquid chromatography(HPLC)with radioactive iodine-labeled T4(5’-125I-T4)as the substrate.Illumina NovaSeq 6000 platform was applied for mRNA transcriptome sequencing(RNA-seq)to analyze the differentially expressed genes of NI group and 50HI group.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were conducted,and the potential mechanisms related to pituitary D2 inactivation and serum TSH elevation were screened.Finally,we conducted qPCR and WB to verify and improve the enrichment results of RNA-seq.Part Two:According to the results of part one,the whole-genome distribution characteristics and differentially enriched sites of the marker protein of ERS,as well as a typical transcription factor,C/EBP homologous protein(CHOP or GADD153),were compared using Chromatin Immunoprecipitation and Deep Sequencing(ChIP-seq)in NI and 50HI groups.The biological processes,cell components,molecular functions,and pathways belonging to differentiated peak related genes were identified by GO and KEGG.Based on the results of the enrichment analysis,we used an Integrative Genomics Viewer(IGV)browser to visually browse bigWig files obtained from the ChIP-seq.We compared the enrichment differences of CHOP near the transcription start sites(TSSs)of the indicators involved in the synthesis of D2 active centers,thyroid hormone receptors and thyroid hormone transporter-related indicators.Additionally,the expression differences of these indicators were analyzed by qPCR and WB in NI,10HI and 50HI groups.Finally,lentivirus was used to down-regulate or up-regulate Secisbp2 expression in GH4C1 pituitary tumor cell lines,and the association between Secisbp2 expression and D2 activity in cell lines was verified in vitro.Part Three:Based on the experiments in part one and part two,this part of the experiment firstly verified the conclusions of animal experiments by using GH4C1 cell line.The catalytic activity of D2 in each group was analyzed 72 h after intervention with KI medium with final concentration ranging from 0.01 to 10 mM.After chronic iodine excess intervention,expression levels of markers related to ERS and D2 active centers in each group of cell line were verified by qPCR,WB or immunofluorescence(IF).In addition,KI,4-phenylbutyric acid(4PBA)and tunicamycin(TN)were given to the medium alone or in combination.We also treated GH4C1 cell lines with lentivirus to down-regulate or up-regulate ATF6/CHOP to validate the role of ERS and Bip-ATF6-CHOP pathway in iodine-induced D2 inactivation in GH4C1 cell lines.Results:Part One:After the intervention of 1627.32 μg/L(10HI)and 8859.43μg/L(50HI)KI solution,the UIC level of rats was significantly increased,and gradually increased with the extension of time.In general,the serum TSH of the two intervention groups also increased gradually with the extension of intervention.Serum TSH in the 10HI group was significantly higher than baseline at week 24(p<0.05),and serum TSH in 50HI group was significantly higher than baseline value at weeks 12,18 and 24(p<0.05).Moreover,serum FT4 in the 50HI group was significantly higher than baseline at week 24(p<0.05),the FT4 and FT3 levels of the other two intervention groups at each time point had no statistical difference compared with the baseline.After 24 weeks of intervention,TFQI,TSHI and TT4RI in 50HI group were significantly higher than baseline values(p<0.05).The peripheral sensitivity index of thyroid hormone FT3/FT4 was significantly lower than baseline value(p<0.05).Pituitary D2 activity was significantly reduced in 10HI and 50HI groups(p<0.05),but there were no significant differences in mRNA or protein expression compared with NI group.By RNA-seq analysis,a total of 13878 genes were co-expressed in the NI and 50HI groups,among which 1858 genes had statistically different expression levels,and a total of 1062 genes were significantly up-regulated in the 50HI group.The GO database was used to analyze the biological processes in which the differential expressed genes were located.The results suggested that the response to ERS(GO:0034976)was statistically different between the two groups,and the differential expressed genes could also be significantly enriched in the ER-Golgi membrane system(GO:0005793).qPCR and WB were used to further verify the expression of the ERS pathway indicators in NI,10HI and 50HI groups.The results suggested that the mRNA and protein levels of binding immunoglobulin protein(Bip or Grp78),inositol requiring enzyme 1α(IRE1α),activating transcription factor 6(ATF6)and CHOP were significantly increased in the 50HI group(p<0.05).Additionally,the mRNA and protein expression levels of Bip,ATF6 and CHOP were also significantly increased in 10HI group(p<0.05).Part Two:In addition,three pairs of pituitary tissues via NI and 50HI groups were selected for ChIP-seq analysis,and 24447 peaks were significantly different between the two groups,among which 12546 peaks were significantly enriched and increased in the 50HI group.The gene whose TSS were the closest to the peak were considered as the genes related to the corresponding peak,and there were 12071 differential expressed genes related to peak.KEGG pathway enrichment analysis suggested that the differential enriched genes were mainly related to metabolic pathways,oxidative phosphorylation,amino acid biosynthesis,et al.GO analysis suggested that the biological processes and molecular functions involved in differential genes were mainly related to the morphogenesis or development of organs and the binding ability of transcription factors to DNA.The enrichment analysis of cell components indicated that the top five entries were all located in ribosomes.In view of the specific function of ribosomes and the specificity of the synthesis mechanism of D2 active centers,it is speculated that there may be differentially enriched sites of CHOP in the TSS region of genes related to D2 active centers.The results of IGV visualization indicated that CHOP enrichment was significantly increased in the promoter region of SECIS binding protein 2(SBP2)in the 50HI group.Except for SBP2,no significant difference in enrichment of these genes in TSS was observed by CHOP for other indexes related to D2 active centers.In addition,qPCR and WB indicated that the expression level of SBP2 in the pituitary gland of 10HI and 50HI groups decreased significantly(p<0.05).By comparing CHOP enrichment in TSS region of other genes related to central sensitivity of thyroid hormone(Thra,Thrb,Slc16a2,Slcolcl),no statistical difference was found via IGV visualization.qPCR and WB experiments showed that the expression levels of thyroid transporter related genes(Slc16a2 and Slcolcl)were similar in all groups,but the mRNA and protein expression levels of THRβ decreased significantly in 50HI group(p<0.05).In addition,GH4C1 cell line was used to explore the effect of pituitary SBP2 expression difference on D2 activity.The results showed that with the down-regulation or up-expression of Secisbp2 gene,D2 activity in the corresponding two groups was also significantly lower or higher than that in the control group(p<0.05).Part Three:We added different concentrations of KI to the medium of GH4C1 cell lines and found that the 1000HI group had significantly reduced D2 activity 72 hours after intervention compared with the baseline value and the control group.After 72 h of 1000HI administration,the expression level of D2 was similar to that of NI group.However,the mRNA and protein expression levels of SBP2 were significantly decreased,while the expression levels of Bip,ATF6 and CHOP were significantly increased.When 4PBA was added to the medium to inhibit ERS response(final concentration 1mM),it was found that the ERS activation,SBP2 expression inhibition and D2 inactivation induced by KI were significantly alleviated with the addition of 4PBA.On the contrary,TN was added to the medium to enhance the ERS response(final concentration 2μM),and the above effects were significantly enhanced.ATF6 or CHOP was overexpressed or knocked down by lentivirus,and KI-induced SBP2 expression inhibition and D2 inactivation were also significantly intensified or mitigated.Conclusion:1.Similar to previous findings,chronic iodine excess resulted in SCH in Wistar rats,and the pituitary D2 activity decreased significantly.This study is the first to show that chronic iodine excess induces a significant decrease in both central and peripheral thyroid hormone sensitivity.After RNA-seq and subsequent analysis,iodine excess may lead to the activation of IRE1α and ATF6 pathways in the pituitary gland,suggesting the stimulating effect of chronic iodine excess on the pituitary ERS.2.Under the condition of chronic iodine excess,CHOP,as a marker protein of ERS and an important transcription factor,can significantly enrich at the TSS of Secisbp2 gene and inhibit its transcription.We demonstrated for the first time at the pituitary level that D2 activity significantly decreased or increased with Secisbp2 gene knockdown or overexpression using GH4C1 pituitary tumor cell lines.It is speculated that the overexpression of CHOP caused by iodine overdose can inhibit the expression of SBP2,thereby affecting the formation of D2 active center and leading to a decrease in the central sensitivity of thyroid hormone.3.After intervention with different concentrations of iodine in GH4C1 cell lines,excessive iodine resulted in decreased SBP2 expression and decreased D2 activity.The ERS mediated Bip-ATF6-CHOP pathway may play an important role upstream of D2 inactivation. |
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