Study On The Regulation Of Iodine On Thyroid Function And Its Molecular Mechanisms

The reserve of organic thyroid iodine and its circulation change the metabolism of thyroid and its sensitivity to pituitary TSH, which reflects the effects of iodine on thyroid function and is independent of pituitary TSH. This mechanism is a part of the self-regulation of thyroid function, which is conducive to keeping suitable iodine content and the stability of thyroid function.The synthesis and secretion of thyroid hormone are durative and complicated physiological process which relate to the expressions of thyroid-specific proteins including the sodium/iodide cotransporter (NIS), thyroid peroxidase (TPO), thyroglobulin (Tg) and thyroid stimulating hormone receptor (TSHr) as well as their transcriptional regulators, such as TTF1, TTF-2 and PAX8. The changes of internal and external iodine concentration bring about the alteration of the expressions of thyroid transcriptional factors and thyroid-specific proteins through various complicated mechanisms, which finally results in the self-regulation of thyroid function.The proper experimental model is a key point for the research on the regulation of thyroid function. Research in animals is affected by many factors and is unable to elucidate the detailed functions and mechanisms of some element; most thyroid cells cultured in vitro grow as a monolayer and cannot form the follicle structure, they do not possess the ability of hormonal synthesis, reserve and secretion, which are unsuitable for research on thyroid functional regulation. The recombinant thyroid follicle (RTF) is an ideal experimental model for thyroid functional regulation because it both breaks from the complex regulatory conditions in vivo and possesses the integral functions.We probed the methods of inducing swine recombinant thyroid follicle in vitro, and the RTF model was confirmed to be successful through identifying the morphology and function of thyroid follicle and the expression of thyroid-specific proteins. A series of methods including Western-blotting, realtime RT-PCR, ELISA, RIA, immunofluorescence, laser scanning confocal microscope and the techniques for extraction of thyroid follicular TG were employed to investigate the secretion of thyroid hormone, iodinated extent of follicular TG and the expressions of thyroid-specific proteins and thyroid transcriptional factors under the stress of high or low concentration of iodine. We aimed to elucidate the molecular mechanisms of iodine-regulated thyroid function. The results are as follows:(一)Establishment and identification of swine recombinant thyroid follicle①The methods of establishing swine recombinant thyroid follicle in vitro are briefly introduced as follows: thyroid tissues of swine were digested with 0.1% typsin and vibrated to disperse cells. Cells were maintained in RPMI-1640 medium supplemented with 15% fetal calf serum containing 1 mIU/mlTSH at the density of 1.5-3x106/L and the next day 10% calf serum replaced the fetal calf serum. After cultured 3 days thyroid cells accumulated together to form recombinant thyroid follicle.②Morphological identification of recombinant thyroid follicle Thyroid cells accumulated together to form a circular structure with diameter of 10 to 100 um under the inverted microscope. Observation of phase-contrast microscope revealed the circular structure was the surface of hemisphere follicle closing to the coverslip and the fornix was established with lots of cells. Laser confocal scanning microscope observation revealed that the cell aggregate consisted of thyroid cells and the inner follicle, which could be clearly visualized after immunofluorescence staining.③Functional identification of recombinant thyroid follicleIn the supernate of RTF group FT3 and FT4 were secreted and TG was reserved in the follicle, while all of them were not detected in the supernate of MC group.④Under the stress of high concentration of iodine the expressions of thyroid-specific proteins, NIS, TG, TPO, and TSHr, and the mRNA levels of thyroid transcriptional factors, TTF-1 and PAX8 were all up-regulated in RTF group, whose levels were all down-regulated in MC group.(二)Study the role of iodine on the regulation of thyroid function based on the in vitro experimental model of swine recombinant thyroid follicle①The mRNA levels of NIS, TPO and TG, which were related to the synthesis of thyroid hormone, were all up-regulated in HI group, while were down-regulated in LI group.②The secretion of FT3 and FT4 were enhanced in the supernate of HI group medium, while were significantly decreased in LI group supernate.(三)The molecular mechanism of iodine on the regulation of thyroid function①In group HI, the mRNA levels of TSHr, TTF-1 and PAX8 were all up-regulated as well as their protein synthesis.②The iodinated extent of follicular TG in group HI was relative higher than that in other groups, and it was decreased in group"HI+MMI"; when compared with"MMI+HI"group the iodinated extent of TG in group"HI+MMI"significantly rose.("MMI+HI"group and"HI+MMI"group were dealt with a different procedure; cells in"HI+MMI"group were first treated with high concentration of iodine for 36 h and then MMI while cells in group"MMI+HI"were treated with a reverse sequence.)When follicular TG was highly iodinated, the mRNA levels of TSHr, TTF-1 and PAX8 were markedly improved as well as their protein synthesis.④The mRNA levels of TTF-1 and TSHr in group of H89 (inhibitor of PKA) and LY294002 (inhibitor of IP3) were both significantly enhanced as well as their protein synthesis;⑤The expression of NIS in group of H89 (inhibitor of PKA) was down-regulated while NIS2 (the subtype of NIS) was up-regulated; while in group of LY294002 the expression of NIS and NIS2 were up-regulated and down-regulated, respectively.Conclusions:1. Keeping the integrity of cell membrane, seeding at high density and increasing the opportunities of cell aggregation, adhesion and contact were the key points of successfully establishing recombinant thyroid follicle in vitro; one very important element was avoid using fetal bovine serum which could drive cells forming thyroid follicle to be monolayer growth.2. Recombinant thyroid follicle was an ideal experimental model for the research on the mechanism of regulation of thyroid function, because it both broke away from the complex environment in vivo and had integrated thyroid function. Therefore, the recombinant thyroid follicle could be used for observing the process of the production of thyroid follicle and exploring the regulatory roles of follicular structure and substances on thyroid function.3. The expressions of NIS, TG and TPO, which were all related to the synthesis of thyroid hormone, were overexpressed; the secretions of FT3 and FT4 in the supernate of culture medium were enhanced, indicating the recombinant thyroid follicle was undergoing vibrant hormonal synthesis and secretion. All evidences above proved that iodine promoted the synthesis and secretion of thyroid hormone at follicular level.4. Iodine and MMI affected the iodination of follicular TG and they had no direct effects on the expressions of TSHr and TTF-1; the CAMP/AKP and C/IP3 pathways, which belonged to TSH/TSHr signal, did not up-regulate the expressions of TTF-1 and TSHr, indicating highly iodinated TG directly resulted in the overexpressions of TTF-1 and TSHr.5. Iodine improved the iodinated extent of follicular TG and increased the expression level of TSHr through regulating TTF-1, which finally resulted in promoting the sensitivity of thyroid follicle to central TSH/TSHr signal. This is one of the important pathways for thyroid functional self-regulation.6. When TG was highly iodinated, the expressions of TSHr, NIS, TPO and TG was enhanced as well as the hormone secretion, and follicle was sensitive to central signal, which was one of the reasons for the vibrant thyroid functions.7. Different signal transduction pathways of TSH/TSHr signal played different regulatory roles on the expressions of NIS and NIS2, indicating central signals regulated the expression of different subtype of NIS at the level of mRNA splicing.In summary, we found iodine affected the iodinated extent of follicular TG from the aspect of follicle, and promoted the expression of TSHr through regulating TTF-1, which finally changed the sensitivity of follicle to central TSH/TSHr signal. TG, which was iodinated on different extent, led to the different sensitivity of follicle to central TSH/TSHr signal, this was one of the important pathways for thyroid adapting itself to different concentration of iodine and achieving functional self-regulation. The highly iodinated TG enhanced the expression of TSHr, drove follicle to be sensitive to central signal, and promoted the secretion of thyroid hormone and the expression of NIS, TPO and TG. Taken follicle as a research model, iodine efficiently expedited the synthesis and secretion of thyroid hormone through the mechanisms mentioned above.Over the past 10 years, the incidences of thyroid dysfunction, autoimmune thyroiditis and thyroid cancer that were all high iodine-related diseases obviously increased. At present little is known about how iodine regulates thyroid functions. Our work offered methodology basis and directions for the research on this area and had important theoretical and practical significance for exploring the pathogenesis of iodine-related thyroid diseases and making the scientific strategies for iodine supplement.