INFLUENCE OF ALIMENTARY DEPRIVATION ON MORPHOLOGICAL CHANGES OF RAT'S THYROID GLAND

Introduction: Despite the well-studied effect of alimentary deprivation on the body, the literature data on its effect on functional activity and, in particular, on morphological changes in the thyroid gland are single and often contradictory, which does not allow unambiguous conclusions. All this requires a more detailed study of the role and mechanisms of the impact of restricted nutrition on the thyroid gland. Aim: To investigate the effect of alimentary deprivation on morphological changes in the thyroid gland of young rats. Methods: The study was conducted on 24 male Wistar rats aged 3 months. Rats of all groups were in uniform conditions, on a standard diet. Animals of the experimental group, for 28 days, received a diet reduced by 30 %. Work with rats was carried out in accordance with the principles of the Declaration of Helsinki. Histological preparations were made from the central areas of the thyroid tissue according to the standard method. Using a digital camera, the micropreparations were photographed under a Nikon Eclipse E 100 microscope (Japan). Morphometry was performed using a computer program "Image J". Results: Histological analysis of the rat's thyroid gland affected by alimentary deprivation revealed that it had an unchanged physiological structure. The follicles were mostly of oval shape and of various sizes. Colloid in the follicles of experimental animals is of moderate density and contains numerous resorption vacuoles. Thyroid cells are of prismatic and cubic shape. It was found that in the thyroid gland of experimental rats the area of ​​follicles, colloid, their inner diameter decreases, the height of thyrocytes increases, the stereological resorption index increases and the colloid accumulation index decreases, the number of interfollicular islands increases. Also in experimental animals there was a decrease in the width of the interlobar and interfollicular connective tissue. Conclusion: In rats fed on a reduced diet, morphological signs of increased functional activity of the thyroid gland were found.

Rationale for therapeutic decision-making in locally advanced and metastatic radioactive iodine (RAI)-refractory differentiated thyroid cancer, starting from a clinical case

Iodine uptake and organification are the hallmarks of thyroid cells differentiation. The loss of these characteristics in thyroid cancer leads to radioactive iodine refractoriness, a rare condition that bears a low survival rate and poor prognosis. We present a 52-year-old patient presenting dry cough and dyspnea in the supine position. Imaging examinations revealed a thyroid nodule with a high suspicion of malignancy in the right thyroid lobe, multiple laterocervical and mediastinal lymph nodes, lung, bone, and brain metastases. Fine needle aspiration cytologic features have advocated for papillary thyroid cancer (PTC). The patient underwent total thyroidectomy and selective lymphadenectomy. Subsequently, the patient received suppressive treatment with levothyroxine and four courses of radioactive iodine therapy. In addition, to treat bone and brain metastases, the patient experienced external radiotherapy and glucocorticoid therapy. Despite this rigorous therapeutic management, the patient obtained an incomplete structural and functional response. Although the last two posttherapeutic 131I whole-body scans were negative, the patient had elevated stimulated thyroglobulin levels and loco-regional recurrence by thyroid ultrasound. This aspect would suggest that thyroid cells become unable to uptake 131I, most likely through the emergence of new genetic mutations in the cancer cells. In conclusion, our patient's case suggests a 131I-refractory PTC, requiring the initiation of novel targeted systemic agents such as tyrosine kinase inhibitors, in order to improve structural and functional outcomes of radioactive iodine therapy and to afford prolonged progression-free survival advantage.

Hormonal Regulation of the MHC Class I Gene in Thyroid Cells: Role of the Promoter “Tissue-Specific” Region

In previous studies we have demonstrated that the expression of the Major Histocompatibility Complex (MHC) class I gene in thyrocytes is controlled by several hormones, growth factors, and drugs. These substances mainly act on two regions of the MHC class I promoter a “tissue-specific” region (−800 to −676 bp) and a “hormone/cytokines-sensitive” region (−500 to −68 bp). In a previous study, we have shown that the role of the “tissue-specific” region in the MHC class I gene expression is dominant compared to that of the “hormone/cytokines-sensitive” region. In the present report we further investigate the dominant role of the “tissue-specific” region evaluating the effect of thyroid stimulating hormone (TSH), methimazole (MMI), phenylmethimazole (C10), glucose and thymosin-α1. By performing experiments of electrophoretic mobility shift assays (EMSAs) we show that TSH, MMI and C10, which inhibit MHC class I expression, act on the “tissue-specific” region increasing the formation of a silencer complex. Glucose and thymosin-α1, which stimulate MHC class I expression, act decreasing the formation of this complex. We further show that the silencer complex is formed by two distinct members of the transcription factors families activator protein-1 (AP-1) and nuclear factor-kB (NF-kB), c-jun and p65, respectively. These observations are important in order to understand the regulation of MHC class I gene expression in thyroid cells and its involvement in the development of thyroid autoimmunity.

MAPK Inhibition Requires Active RAC1 Signaling to Effectively Improve Iodide Uptake by Thyroid Follicular Cells

The Sodium/Iodide Symporter (NIS) is responsible for the active transport of iodide into thyroid follicular cells. Differentiated thyroid carcinomas (DTCs) usually preserve the functional expression of NIS, allowing the use of radioactive iodine (RAI) as the treatment of choice for metastatic disease. However, a significant proportion of patients with advanced forms of TC become refractory to RAI therapy and no effective therapeutic alternatives are available. Impaired iodide uptake is mainly caused by the defective functional expression of NIS, and this has been associated with several pathways linked to malignant transformation. MAPK signaling has emerged as one of the main pathways implicated in thyroid tumorigenesis, and its overactivation has been associated with the downregulation of NIS expression. Thus, several strategies have been developed to target the MAPK pathway attempting to increase iodide uptake in refractory DTC. However, MAPK inhibitors have had only partial success in restoring NIS expression and, in most cases, it remained insufficient to allow effective treatment with RAI. In a previous work, we have shown that the activity of the small GTPase RAC1 has a positive impact on TSH-induced NIS expression and iodide uptake in thyroid cells. RAC1 is a downstream effector of NRAS, but not of BRAF. Therefore, we hypothesized that the positive regulation induced by RAC1 on NIS could be a relevant signaling cue in the mechanism underlying the differential response to MEK inhibitors, observed between NRAS- and BRAF-mutant tumors. In the present study, we found that the recovery of NIS expression induced through MAPK pathway inhibition can be enhanced by potentiating RAC1 activity in thyroid cell systems. The negative impact on NIS expression induced by the MAPK-activating alterations, NRAS Q61R and BRAF V600E, was partially reversed by the presence of the MEK 1/2 inhibitors AZD6244 and CH5126766. Notably, the inhibition of RAC1 signaling partially blocked the positive impact of MEK inhibition on NIS expression in NRAS Q61R cells. Conversely, the presence of active RAC1 considerably improved the rescue of NIS expression in BRAF V600E thyroid cells treated with MEK inhibitors. Overall, our data support an important role for RAC1 signaling in enhancing MAPK inhibition in the context of RAI therapy in DTC, opening new opportunities for therapeutic intervention.

Analysis of NIS Plasma Membrane Interactors Discloses Key Regulation by a SRC/RAC1/PAK1/PIP5K/EZRIN Pathway with Potential Implications for Radioiodine Re-Sensitization Therapy in Thyroid Cancer

The functional expression of the sodium–iodide symporter (NIS) at the membrane of differentiated thyroid cancer (DTC) cells is the cornerstone for the use of radioiodine (RAI) therapy in these malignancies. However, NIS gene expression is frequently downregulated in malignant thyroid tissue, and 30% to 50% of metastatic DTCs become refractory to RAI treatment, which dramatically decreases patient survival. Several strategies have been attempted to increase the NIS mRNA levels in refractory DTC cells, so as to re-sensitize refractory tumors to RAI. However, there are many RAI-refractory DTCs in which the NIS mRNA and protein levels are relatively abundant but only reduced levels of iodide uptake are detected, suggesting a posttranslational failure in the delivery of NIS to the plasma membrane (PM), or an impaired residency at the PM. Because little is known about the molecules and pathways regulating NIS delivery to, and residency at, the PM of thyroid cells, we here employed an intact-cell labeling/immunoprecipitation methodology to selectively purify NIS-containing macromolecular complexes from the PM. Using mass spectrometry, we characterized and compared the composition of NIS PM complexes to that of NIS complexes isolated from whole cell (WC) lysates. Applying gene ontology analysis to the obtained MS data, we found that while both the PM-NIS and WC-NIS datasets had in common a considerable number of proteins involved in vesicle transport and protein trafficking, the NIS PM complexes were particularly enriched in proteins associated with the regulation of the actin cytoskeleton. Through a systematic validation of the detected interactions by co-immunoprecipitation and Western blot, followed by the biochemical and functional characterization of the contribution of each interactor to NIS PM residency and iodide uptake, we were able to identify a pathway by which the PM localization and function of NIS depends on its binding to SRC kinase, which leads to the recruitment and activation of the small GTPase RAC1. RAC1 signals through PAK1 and PIP5K to promote ARP2/3-mediated actin polymerization, and the recruitment and binding of the actin anchoring protein EZRIN to NIS, promoting its residency and function at the PM of normal and TC cells. Besides providing novel insights into the regulation of NIS localization and function at the PM of TC cells, our results open new venues for therapeutic intervention in TC, namely the possibility of modulating abnormal SRC signaling in refractory TC from a proliferative/invasive effect to the re-sensitization of these tumors to RAI therapy by inducing NIS retention at the PM.

Potential targets for the hepatitis C virus-mediated autoimmune thyroiditis

Introduction: The mechanisms by which hepatitis C virus (HCV) infection induces autoimmune thyroiditis (AIT) have been studied, and it was suggested that inflammatory cytokines during HCV infection would change the thyroperoxidase (TPO) signaling cascade and thyroglobulin (Tg) determining autoimmune thyroid disease.Objective: To show the signaling pathway, of TPO and Tg, and their potential targets mediated HCV in individuals with hepatitis C.Methods: The mapping of the signaling pathway was based on a review study approach and performed using the automatic annotation server of the Kyoto and Genome Encyclopedia (KEGG). PathVisio is free software for analysis and design of open source routes, and was used for the graphic representation of the signaling pathway.Results: The contigs were extracted from the KEGG database and their mapped transcription represents the signaling pathway of the main biomolecules that triggers the AIT. The action of HCV, or its treatment can trigger AIT that is characterized by the presence of autoantibodies against TPO and Tg. In AIT, autoreactive CD4 + T lymphocytes recruit B cells and CD8 + T cells in the thyroid. The progression of the disease leads to the death of thyroid cellsand hypothyroidism.Conclusion: HCV or its treatment activates several signaling pathways with thyroid cells damage resulting in AIT and secondary hypothyroidism to cellular apoptosis.

Senescent Thyrocytes, Similarly to Thyroid Tumor Cells, Elicit M2-like Macrophage Polarization In Vivo

Inflammation plays a critical role in thyroid cancer onset and progression. We previously characterized the in vitro interplay between macrophages and senescent human thyrocytes and thyroid tumor-derived cell lines, modeling the early and the late thyroid tumor phases, respectively. We reported that both models are able to induce pro-tumoral M2-like macrophage polarization, through the activation of the COX2-PGE2 axis. Here, we investigated the presence of macrophage infiltrating cells in mouse xenografts derived from the above described cells models. We showed that subcutaneous injection in immunodeficient mice of both senescent human thyrocytes and thyroid tumor-derived cell lines elicits macrophage recruitment. Furthermore, considering the type of macrophage infiltrate, we observed a stronger infiltration of Arginase I positive cells (M2-like). Overall, these results demonstrate the in vivo capability of senescent and tumor thyroid cells to recruit and polarize macrophages, suggesting that the promotion of a pro-tumoral microenvironment through tumor associated macrophages may occurs in late as well as in early thyroid tumor stages, favoring tumor onset and progression.

Prescription of Sageretia hamosa Brongn Relieved Goiter through Promoted Apoptosis of Thyroid Cells via miR-511-3p and PTEN/PI3K/Akt Pathway

Goiter is thyroid enlargement, in China, Sageretia hamosa Brongn (SHB) can be used to treat goiter, but it has not been reported. Therefore, data analytics of SHB prescription on thyroid were explored in this study to provide a theoretical support for SHB in the treatment of goiter. In this study, rat in goiter model was constructed by using propylthiouracil (PTU) and treated with SHB prescription. Thyroid function about the triiodothyronine (T3), free thyroxine (T4), free triiodothyronine (FT3), free thyroxine (FT4), and thyroid-stimulating hormone (TSH) were measured by ELISA; thyroid coefficient was calculated after weighed thyroid; and HE staining was applied to assess the morphology of thyroid tissue. miRNA microarrays were employed to detect miRNA expression in thyroid tissue of rats. Expression of miR-511-3p was measured by RT-qPCR; expression of proteins (PTEN and apoptosis-related proteins) was tested by western blotting; relationship between miR-511-3p and PTEN was investigated by dual luciferase reporter gene assay; cell viability rate was determined by CCK-8; and cell cycle distribution and apoptosis rate were detected by flow cytometry. The results showed that SHB prescription ameliorated goiter and downregulated miR-511-3p. miR-511-3p targeted PTEN in thyroid cells and PTEN negatively regulated the activation of PI3K/Akt pathway. Furthermore, the inhibition of apoptosis in thyroid cells caused by the overexpression of miR-511-3p or the activation of PI3K/Akt pathway was reversed by treatment of SHB prescription, inhibition of miR-511-3p, or overexpression of PTEN. In conclusion, SHB prescription promoted apoptosis of thyroid through decreased miR-511-3p and regulated PTEN/PI3K/Akt pathway, it might suggest possible medical applications.

Crosstalk Between Abnormal TSHR Signaling Activation and PTEN/PI3K in the Dedifferentiation of Thyroid Cancer Cells

Iodide uptake and the metabolism of thyroid cells are regulated by thyrotropin (TSH)-TSH receptor (TSHR) signaling. Thus, it is necessary to elevate serum TSH levels by T4 withdraw or rTSH administration to facilitate radioiodide (131I) therapy for differentiated thyroid cancer (DTC). However, non-iodide-avid metastases of DTC which is dedifferentiated do not respond to stimulation by high levels of TSH, suggesting abnormal TSH-TSHR signal transduction in cancer cells. In addition, PI3K/AKT/mTOR signaling activation has been shown to be associated with the dedifferentiated phenotype of thyroid cancer, but the mechanism remains elusive. Therefore, in this study, we aimed to explore the role of abnormal TSH-TSHR signaling activation in regulating iodide uptake and cell mobility in thyroid cancer and its relationship with PI3K/AKT/mTOR signaling. We found that in thyroid cancer cells, TSH binds TSHR coupled to the Gα12/13 protein and then activates RhoA through interacting with leukemia associated RhoA guanine exchange factor (LARG). This results in a promigration tumorigenic phenotype independent of canonical TSHR-GαS signaling that regulates the expression of molecules involved in iodine uptake and metabolism. We observed that signaling pathways downstream of Gα12/13 signaling were increased, while that of Gαs signaling was decreased in thyroid cancer cells undergoing dedifferentiation compared to control cells following stimulation with different levels of TSH. PI3K/AKT/mTOR signaling activation enhanced Gα12/13 signaling through increasing LARG levels but also inhibited the expression of molecules downstream of Gαs signaling, including thyroid-specific molecules, and iodide uptake. In summary, our results demonstrate the noncanonical activation of TSH-TSHR signaling and its role in increasing the cell mobility and dedifferentiation of thyroid cancer through crosstalk with PI3K/AKT/mTOR signaling.