Vitamin D3 Treatment Alters Thyroid Functional Morphology in Orchidectomized Rat Model of Osteoporosis.

Vitamin D plays an essential role in prevention and treatment of osteoporosis. Thyroid hormones, in addition to vitamin D, significantly contribute to regulation of bone remodeling cycle and health. There is currently no data about a possible connection between vitamin D treatment and the thyroid in the context of osteoporosis. Middle-aged Wistar rats were divided into: sham operated (SO), orchidectomized (Orx), and cholecalciferol-treated orchidectomized (Orx + Vit. D3; 5 µg/kg b.m./day during three weeks) groups (n = 6/group). Concentration of 25(OH)D in serum of the Orx + Vit. D3 group increased 4 and 3.2 times (p < 0.0001) respectively, compared to Orx and SO group. T4, TSH, and calcitonin in serum remained unaltered. Vit. D3 treatment induced changes in thyroid functional morphology that indicate increased utilization of stored colloid and release of thyroid hormones in comparison with hormone synthesis, to maintain hormonal balance. Increased expression of nuclear VDR (p < 0.05) points to direct, TSH independent action of Vit. D on thyrocytes. Strong CYP24A1 immunostaining in C cells suggests its prominent expression in response to Vit. D in this cell subpopulation in orchidectomized rat model of osteoporosis. The indirect effect of Vit. D on bone, through fine regulation of thyroid function, is small.

Effects of chronic exposure to nonylphenol at environmental concentration on thyroid function and thyroid hyperplasia disease in male rats.

The aim of this work was to determine whether chronic exposure to nonylphenol (NP), a representative substance of environmental endocrine disruptors (EEDs), at environmental concentration would have toxic effects on thyroid function and thyroid hyperplasia disease. Two hundred SPF Sprague-Dawley rats were divided into five groups (n = 40 per group): blank control group (corn oil), low-dose NP exposure group (0.4 mg/kg/d), medium-dose NP exposure group (4 mg/kg/d), high-dose NP exposure group (40 mg/kg/d), and estradiol control group (E2: 30 µg/kg/d). The rats were treated by gavage for 34 weeks, which were sampled twice (17 weeks and 34 weeks respectively). NP accumulation in the thyroid tissue (F = 52.93, P < 0.001) and serum (F = 5.54, P = 0.00) continuously increased in a significant dose-effect relationship. After NP exposure, the serum FT3 levels exhibited a dose-dependent increasing trend (F = 4.68, P = 0.01), while the serum FT4 level showed an opposite trend (F = 3.93, P= 0.01). Compared with the control group, hyperechoic areas (i.e., calcification points) were observed in the high-dose group. Follicular epithelial stratification was extremely severe, the monolayer cubic epithelial cells became flat, and the area of single follicles was even smaller in the high-dose group. In the high-dose NP group, there were numerous mitochondria that were severely swollen. The rough endoplasmic reticulum was abundant, with obvious expansion and vesiculation. The relative expression of ERα (F = 5.29, P = 0.00), ERß (F = 10.17, P = 0.00), TRα (F = 7.71, P = 0.00), TRß (F = 3.52.17, P = 0.02) and HMGB1 (F = 10.16, P = 0.01) proteins in the thyroid tissue in each NP exposure group was increased compared with the control group, and the relative expression of proteins increased if the exposure time was prolonged under the same exposure dose. Chronic exposure to NP at environmental concentration could have toxic effects on thyroid function, and induce thyroid hyperplasia disease in male rats.

Stromal interaction molecule 1 (STIM1) knock down attenuates invasion and proliferation and enhances the expression of thyroid-specific proteins in human follicular thyroid cancer cells.

Stromal interaction molecule 1 (STIM1) and the ORAI1 calcium channel mediate store-operated calcium entry (SOCE) and regulate a multitude of cellular functions. The identity and function of these proteins in thyroid cancer remain elusive. We show that STIM1 and ORAI1 expression is elevated in thyroid cancer cell lines, compared to primary thyroid cells. Knock-down of STIM1 or ORAI1 attenuated SOCE, reduced invasion, and the expression of promigratory sphingosine 1-phosphate and vascular endothelial growth factor-2 receptors in thyroid cancer ML-1 cells. Cell proliferation was attenuated in these knock-down cells due to increased G1 phase of the cell cycle and enhanced expression of cyclin-dependent kinase inhibitory proteins p21 and p27. STIM1 protein was upregulated in thyroid cancer tissue, compared to normal tissue. Downregulation of STIM1 restored expression of thyroid stimulating hormone receptor, thyroid specific proteins and increased iodine uptake. STIM1 knockdown ML-1 cells were more susceptible to chemotherapeutic drugs, and significantly reduced tumor growth in Zebrafish. Furthermore, STIM1-siRNA-loaded mesoporous polydopamine nanoparticles attenuated invasion and proliferation of ML-1 cells. Taken together, our data suggest that STIM1 is a potential diagnostic and therapeutic target for treatment of thyroid cancer.

Resveratrol Alleviates the Inhibitory Effect of Tunicamycin-Induced Endoplasmic Reticulum Stress on Expression of Genes Involved in Thyroid Hormone Synthesis in FRTL-5 Thyrocytes.

Recently, ER stress induced by tunicamycin (TM) was reported to inhibit the expression of key genes involved in thyroid hormone synthesis, such as sodium/iodide symporter (NIS), thyroid peroxidase (TPO) and thyroglobulin (TG), and their regulators such as thyrotropin receptor (TSHR), thyroid transcription factor-1 (TTF-1), thyroid transcription factor-2 (TTF-2) and paired box gene 8 (PAX-8), in FRTL-5 thyrocytes. The present study tested the hypothesis that resveratrol (RSV) alleviates this effect of TM in FRTL-5 cells. While treatment of FRTL-5 cells with TM alone (0.1 µg/mL) for 48 h strongly induced the ER stress-sensitive genes heat shock protein family A member 5 (HSPA5) and DNA damage inducible transcript 3 (DDIT3) and repressed NIS, TPO, TG, TSHR, TTF-1, TTF-2 and PAX-8, combined treatment with TM (0.1 µg/mL) and RSV (10 µM) for 48 h attenuated this effect of TM. In conclusion, RSV alleviates TM-induced ER stress and attenuates the strong impairment of expression of genes involved in thyroid hormone synthesis and their regulators in FRTL-5 thyrocytes exposed to TM-induced ER stress. Thus, RSV may be useful for the treatment of specific thyroid disorders, provided that strategies with improved oral bioavailability of RSV are applied.

The Association of Myo-Inositol and Selenium Contrasts Cadmium-Induced Thyroid C Cell Hyperplasia and Hypertrophy in Mice.

Previous studies have demonstrated that, in addition to inducing structural changes in thyroid follicles, cadmium (Cd) increased the number of C cells. We examined the effects of myo-inositol (MI), seleno-L-methionine (Se), MI + Se, and resveratrol on C cells of mice exposed to cadmium chloride (Cd Cl2), as no data are currently available on the possible protective effects of these molecules. In contrast, we have previously shown this protective effect against CdCl2 on the thyroid follicles of mice. Ninety-eight C57 BL/6J adult male mice were divided into 14 groups of seven mice each: (i) 0.9% NaCl (vehicle; 1 ml/kg/day i.p.); (ii) Se (0.2 mg/kg/day per os); (iii) Se (0.4 mg/kg/day per os); (iv) MI (360 mg/kg/day per os); (v) Se (0.2 mg/kg/day) + MI; (vi) Se (0.4 mg/kg/day) + MI; (vii) resveratrol (20 mg/kg); (viii) CdCl2 (2 mg/kg/day i.p.) + vehicle; (ix) CdCl2 + Se (0.2 mg/kg/day); (x) CdCl2 + Se (0.4 mg/kg/day); (xi) CdCl2 + MI; (xii) CdCl2 + Se (0.2 mg/kg/day) + MI; (xiii) CdCl2 + Se (0.4 mg/kg/day) + MI; (xiv) CdCl2 + resveratrol (20 mg/kg). After 14 days, thyroids were processed for histological, immunohistochemical, and morphometric evaluation. Compared to vehicle, Cd significantly decreased follicle mean diameter, increased CT-positive cells number, area and cytoplasmic density, and caused the disappearance of TUNEL-positive C cells, namely, the disappearance of C cells undergoing apoptosis. Se at either 0.2 or 0.4 mg/kg/day failed to significantly increase follicular mean diameter, mildly decreased CT-positive cells number, area and cytoplasmic density, and was ineffective on TUNEL-positive C cells. Instead, MI alone increased significantly follicular mean diameter and TUNEL-positive cells number, and decreased significantly CT-positive cells number, area and cytoplasmic density. MI + Se 0.2 mg/kg/day or MI + Se 0.4 mg/kg/day administration improved all five indices more markedly. Indeed, follicular mean diameter and TUNEL-positive cells number increased significantly, while CT-positive cells number, area and cytoplasmic density decreased significantly. Thus, all five indices overlapped those observed in vehicle-treated mice. Resveratrol improved significantly all the considered parameters, with a magnitude comparable to that of MI alone. In conclusion, the association Myo + Se is effective in protecting the mouse thyroid from the Cd-induced hyperplasia and hypertrophy of C cells. This benefit adds to that exerted by Myo + Se on thyrocytes and testis.

Pathological thyroid findings in amiodarone-induced thyrotoxicosis.

Amiodarone (AMD) is a class III antiarrhythmic drug whose chronic or high dosage administration alters the tests of thyroid function. AMD is also associated with hypothyroidism or thyrotoxicosis. Total thyroidectomy is an efficient treatment of AMD-induced thyrotoxicosis in cases resistant to medical therapy, worsening of cardiac function and/or severe thyrotoxicosis. Although AMD is a widely used drug, its pathological consequences are not well known. We describe the pathological findings in the thyroid gland of a patient who underwent total thyroidectomy due to AMD-induced thyrotoxicosis. The surgical specimen was macroscopically normal, but histologically showed multiple follicles totally or partially invaded by clear vacuolated (foamy) histiocytes, sometimes multinucleated. Loss of thyrocytes, breaks in the follicular basal membrane and stromal fibrosis could also be appreciated but no lymphocytic infiltrates were found. An awareness of these histopathological features is particularly important for surgical pathologists, especially as there are very few published reports describing these alterations.

Novel Correctors and Potentiators Enhance Translational Readthrough in CFTR Nonsense Mutations.

Premature-termination codons (PTCs) in CFTR (cystic fibrosis [CF] transmembrane conductance regulator) result in nonfunctional CFTR protein and are the proximate cause of ∼11% of CF-causing alleles, for which no treatments exist. The CFTR corrector lumacaftor and the potentiator ivacaftor improve CFTR function with terminal PTC mutations and enhance the effect of readthrough agents. Novel correctors GLPG2222 (corrector 1 [C1]), GLPG3221 (corrector 2 [C2]), and potentiator GLPG1837 compare favorably with lumacaftor and ivacaftor in vitro. Here, we evaluated the effect of correctors C1a and C2a (derivatives of C1 and C2) and GLPG1837 alone or in combination with the readthrough compound G418 on CFTR function using heterologous Fischer rat thyroid (FRT) cells, the genetically engineered human bronchial epithelial (HBE) 16HBE14o- cell lines, and primary human cells with PTC mutations. In FRT lines pretreated with G418, GLPG1837 elicited dose-dependent increases in CFTR activity that exceeded those from ivacaftor in FRT-W1282X and FRT-R1162X cells. A three-mechanism strategy consisting of G418, GLPG1837, and two correctors (C1a + C2a) yielded the greatest functional improvements in FRT and 16HBE14o- PTC variants, noting that correction and potentiation without readthrough was sufficient to stimulate CFTR activity for W1282X cells. GLPG1837 + C1a + C2a restored substantial function in G542X/F508del HBE cells and restored even more function for W1282X/F508del cells, largely because of the corrector/potentiator effect, with no additional benefit from G418. In G542X/R553X or R1162X/R1162X organoids, enhanced forskolin-induced swelling was observed with G418 + GLPG1837 + C1a + C2a, although GLPG1837 + C1a + C2a alone was sufficient to improve forskolin-induced swelling in W1282X/W1282X organoids. Combination of CFTR correctors, potentiators, and readthrough compounds augments the functional repair of CFTR nonsense mutations, indicating the potential for novel correctors and potentiators to restore function to truncated W1282X CFTR.

Regulation of solute carrier family 26 member 7 (Slc26a7) by thyroid stimulating hormone in thyrocytes.

Iodine transportation is an important step in thyroid hormone biosynthesis. Uptake of iodine into the thyroid follicle is mediated mainly by the basolateral sodium-iodide symporter (NIS or solute carrier family 5 member 5: SLC5A5), and iodine efflux across the apical membrane into the follicular lumen is mediated by pendrin (SLC26A4). In addition to these transporters, SLC26A7, which has recently been identified as a causative gene for congenital hypothyroidism, was found to encode a novel apical iodine transporter in the thyroid. Although SLC5A5 and SLC26A4 have been well-characterized, little is known about SLC26A7, including its regulation by TSH, the central hormone regulator of thyroid function. Using rat thyroid FRTL-5 cells, we showed that the mRNA levels of Slc26a7 and Slc26a4, two apical iodine transporters responsible for iodine efflux, were suppressed by TSH, whereas the mRNA level of Slc5a5 was induced. Forskolin and dibutyryl cAMP (dbcAMP) had the same effect as that of TSH on the mRNA levels of these transporters. TSH, forskolin and dbcAMP also had suppressive effects on SLC26A7 promoter activity, as assessed by luciferase reporter gene assays, and protein levels, as determined by Western blot analysis. TSH, forskolin and dbcAMP also induced strong localization of Slc26a7 to the cell membrane according to immunofluorescence staining and confocal laser scanning microscopy. Together, these results suggest that TSH suppresses the expression level of Slc26a7 but induces its accumulation at the cell membrane, where it functions as an iodine transporter.

Possible mechanism of bamboo shoots (Bambusa balcooa) induced thyroid disruption - An in vitro study.

Endemic goitre and associated iodine deficiency disorders (IDDs) are a major concern in public health even in the period of post salt iodization in many regions. Among others the consumption of cyanogenic plants found responsible for the persistence of such diseases. Bamboo shoots (BS) is one such cyanogenic plant food that caused disruption of certain thyroid hormone synthesizing regulatory element as has already been reported in our earlier study. In this investigation the possible mechanism of thyrocytes disruption along with interruption of thyroid hormone biosynthesis by BS has been worked out. Commonly consumed BS, Bambusa Balcooa Roxb (BBR) water extract was analysed by GC MS; three doses below IC50 were administered to thyrocytes in culture with and without iodine. Expressions of thyroglobulin (Tg), pendrin (PDS) and monocarboxylate transporter 8 (MCT8) were evaluated in thyrocytes with cell cycle analysis, reactive oxygen species (ROS) generation, DNA oxidation and apoptotic regulation through Bax, Bcl-2 and p53. Phytochemical analysis of BBR extract revealed the presence of precursors and metabolic end products of cyanogenic glycosides. Dose dependent decrease in expression of Tg and PDS with concomitant decrease in gene expression of these with MCT8 were observed. Increased ROS, DNA oxidation and associated imbalance were found through increased Bax and p53 with decreased Bcl-2 that perturbed thyrocytes cell cycle. Cyanogenic constituents of BBR generates ROS associated oxidative changes in thyrocytes with DNA damage and oxidation and cell cycle disruption followed by inhibition of thyroid hormone synthesizing regulatory elements; addition of extra iodine showed partial prevention.

Innate Immune-Modulatory Activity of Prunella vulgaris in Thyrocytes Functions as a Potential Mechanism for Treating Hashimoto's Thyroiditis.

Prunella vulgaris (PV), a perennial herb, has been used to treat thyroid diseases in China for over 2,000 years. In particular, its therapeutic effect has been described for Hashimoto's thyroiditis, including reducing titers autoantibodies against thyroid peroxidase and thyroglobulin of and T helper 17 (Th17) cells. However, the underlying mechanism for how PV exerts such effects has not been investigated. We examined the effects of PV on innate immune activation, which is thought to be one of the triggers for the development of autoimmune diseases, including Hashimoto's thyroiditis. In cultured thyrocytes, PV reduced mRNA levels of inflammatory cytokines that were originally induced as a result of innate immune activation initiated by transfection of double-stranded DNA (dsDNA) or dsRNA. PV suppressed activation of nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF3), and suppressed corresponding promoter activation, which were initially activated by dsDNA or dsRNA. PV also suppressed the mRNA levels of molecules responsible for antigen processing and presentation, and PV protected thyrocytes from apoptosis induced by dsDNA and dsRNA. Additionally, PV suppressed the expression of genes involved in iodide uptake and oxidation. Taken together, these results suggest that PV exerts its protective effect on thyrocytes by suppressing both innate and adaptive immune responses and cell death. PV may also protect cells from iodide-associated oxidative injury. This report is among the first to identify the mechanisms to explain PV's beneficial effects in Hashimoto's thyroiditis.

Hyaluronan oligosaccharides modulate inflammatory response, NIS and thyreoglobulin expression in human thyrocytes.

Autoimmune thyroid diseases, such as Hashimoto's thyroiditis, are characterized by lymphocytic infiltration and altered function of the thyroid. During inflammation, it has been reported a decreased expression in Tg and NIS, accompanied by an increase in HA production that accumulates in the gland. HA fragments produced in different pathological states can modulate gene expression in a variety of cell types and may prime inflammatory response by interacting with the TLR-2, TLR-4 and CD44 that, in turn, induce NF-kB activation finally responsible of inflammatory mediator transcription, such as IL-1ß, TNF-α and IL-6. The aim of this study was to investigate the potential inflammatory effect and the biochemical pathways activated by 6-mer HA oligosaccharides in cultured human thyrocytes. 6-mer HA treatment induced up-regulation of TLR-2, TLR-4, CD44 mRNA and related protein levels, increased HA production and NF-kB activation, that in turn increased IL-1ß and IL-6 concentrations. Instead, we found evidence of an opposite effect on thyroid specific-gene Tg and NIS, that were decreased after 6-mer HA addition. Thyrocytes exposition to specific blocking antibodies for TLR-2, TLR-4 and CD44 abolished up-regulation of NF-κB activation and the consequent pro-inflammatory cytokine production, while restored Tg and NIS levels. A further goal of this study was demonstrate that also other LMW HA have pro inflammatory proprieties. These data suggest that HA fragments, through the involvement of TLR-2, TLR-4 and CD44 signaling cascade, contribute to prime the inflammatory response in thyrocytes and, by reducing the expression of thyroid-specific genes, could promote the loss of function of gland such as in Hashimoto's thyroiditis.

Thyrotropin regulation of differentiated gene transcription in adult human thyrocytes in primary culture.

Thyroid transcription factors (TTFs) - NKX2-1, FOXE1, PAX8 and HHEX - regulate multiple genes involved in thyroid development in mice but little is known about TTF regulation of thyroid-specific genes - thyroglobulin (TG), thyroid peroxidase (TPO), deiodinase type 2 (DIO2), sodium/iodide symporter (NIS) and TSH receptor (TSHR) - in adult, human thyrocytes. Thyrotropin (thyroid-stimulating hormone, TSH) regulation of thyroid-specific gene expression in primary cultures of human thyrocytes is biphasic yielding an inverted U-shaped dose-response curve (IUDRC) with upregulation at low doses and decreases at high doses. Herein we show that NKX2-1, FOXE1 and PAX8 are required for TSH-induced upregulation of the mRNA levels of TG, TPO, DIO2, NIS, and TSHR whereas HHEX has little effect on the levels of these thyroid-specific gene mRNAs. We show that TSH-induced upregulation is mediated by changes in their transcription and not by changes in the degradation of their mRNAs. In contrast to the IUDRC of thyroid-specific genes, TSH effects on the levels of the mRNAs for NKX2-1, FOXE1 and PAX8 exhibit monophasic decreases at high doses of TSH whereas TSH regulation of HHEX mRNA levels exhibits an IUDRC that overlaps the IUDRC of thyroid-specific genes. In contrast to findings during mouse development, TTFs do not have major effects on the levels of other TTF mRNAs in adult, human thyrocytes. Thus, we found similarities and important differences in the regulation of thyroid-specific genes in mouse development and TSH regulation of these genes in adult, human thyrocytes.

Primary 3D Culture of Human Thyroid Follicle-Like Structures in Platelet Lysate-Based Gel.

The results of 3D culturing of human thyroid follicle-like structures in a gel based on platelet lysate at the gel-air interface are presented. During culturing up to 4 months, no new follicle-like structures were formed and none were destroyed. During the first 2 months, most follicle-like structures increased in size; then, their grown decelerated, but they retained viability. Ki-67+ cells were observed in the majority of follicle-like structures. Most of them produced thyroglobulin. Follicle-like structures get closer, the number of contacts between them increased, and cluster appeared. Thus, the developed 3D culturing system in a gel based on platelet lysate is an adequate approach for maintaining structure and functional activity of human follicle-like structures in vitro for at least 2 months.

Perfluorooctane sulfonic acid, a persistent organic pollutant, inhibits iodide accumulation by thyroid follicular cells in vitro.

Poly- and perfluoroalkyl substances (PFAS) are a class of endocrine disrupting chemicals (EDCs) reported to alter thyroid function. Iodide uptake by thyroid follicular cells, an early step in the synthesis of thyroid hormones, is a potential target for thyroid disruption by EDCs. The aim of the present study was to evaluate the acute effects of perfluorooctane sulfonic acid (PFOS) and perfluorooctane carboxylic acid (PFOA), two of the most abundant PFAS in the environment, on iodide transport by thyroid follicular cells in vitro. Dynamic changes in intracellular iodide concentration were monitored by live cell imaging using YFP-H148Q/I152, a genetically encoded fluorescent iodide biosensor. PFOS, but not PFOA, acutely and reversibly inhibited iodide accumulation by FRTL-5 thyrocytes, as well as by HEK-293 cells transiently expressing the Sodium Iodide Symporter (NIS). PFOS prevented NIS-mediated iodide uptake and reduced intracellular iodide concentration in iodide-containing cells, mimicking the effect of the NIS inhibitor perchlorate. PFOS did not affect iodide efflux from thyroid cells. The results of this study suggest that disruption of iodide homeostasis in thyroid cells may be a potential mechanism for anti-thyroid health effects of PFOS. The study also confirms the utility of the YFP-H148Q/I152 cell-based assay to screen environmental PFAS, and other EDCs, for anti-thyroid activity.

Rosmarinic acid alleviates di-2-ethylhexyl phthalate (DEHP) -induced thyroid dysfunction via multiple inflammasomes activation.

DEHP (di-2-ethylhexyl phthalate), an environmental endocrine disruptor, is widely used in industrial products, particularly as plasticizers and softeners which could disrupt the function of the hypothalamic-pituitary-thyroid (HPT) axis. Rosmarinic acid (RA) possesses potential antioxidant and anti-inflammatory capacities in disease models. Nevertheless, evidence on the association between DEHP-induced thyroid dysfunction and inflammation, as well as the molecular mechanism underlying the protective effects of RA-mitigated DEHP-induced thyroid injury remains inconclusive. Male Sprague Dawley (SD) rats were intragastrically administered DEHP (150 mg/kg, 300 mg/kg, 600 mg/kg) once a day for 90 consecutive days. Also, FRTL-5 cells were treated with a wide range of DEHP concentrations (10-8, 10-7, 10-6, 10-5, 10-4, 10-3, 10-2 M) for 24 hr. Subsequently, RA (50 µM) was administered for 24 hr before 10-4 M DEHP challenge. We found that DEHP induced thyroid damage and inflammatory infiltration in vivo. In addition, we showed that DEHP triggered inflammatory cell death, which is mediated by multiple inflammasomes. Moreover, RA, pyroptosis inhibitor (Ac-YVAD-cmk) and antioxidant inhibitor (NAC) treatment significantly alleviated DEHP-induced thyrocyte death, suppressing pro-inflammatory cytokine production, inhibiting multiple inflammasomes activation and attenuating thyrocyte death, respectively. Collectively, our results reveal that a critical role of inflammasomes activation in DEHP-induced thyroid injury, and suggest that RA confers protection against DEHP-induced thyroid inflammation, and facilitating control of the effects of DEHP after given pyroptosis inhibitor or antioxidant inhibitor. These results indicate that it should be possible to provide novel insights into toxicologically and pharmacologically targeting this molecule to DEHP-induced inflammation.

The dual phosphodiesterase 3/4 inhibitor RPL554 stimulates rare class III and IV CFTR mutants.

Over 2,000 mutations have been reported in the cystic fibrosis transmembrane conductance regulator (cftr) gene, many of which cause disease but are rare and have no effective treatment. Thus, there is an unmet need for new, mutation-agnostic therapies for cystic fibrosis (CF). Phosphodiesterase (PDE) inhibitors are one such class of therapeutics that have been shown to elevate intracellular cAMP levels and stimulate CFTR-dependent anion secretion in human airway epithelia; however, the number of people with CF that could be helped by PDE inhibitors remains to be determined. Here we used Fisher rat thyroid (FRT) cells stably transduced with rare human CFTR mutants and studied their responsiveness to the dual phosphodiesterase 3/4 inhibitor RPL554 (Verona Pharma). Through its inhibitory effect on PDE4D, we find that RPL554 can elevate intracellular cAMP leading to a potentiation of forskolin-stimulated current mediated by R334W, T338I, G551D, and S549R mutants of CFTR when used alone or in combination with CFTR modulators. We also were able to reproduce these effects of RPL554 on G551D-CFTR when it was expressed in primary human bronchial epithelial cells, indicating that RPL554 would have stimulatory effects on rare CFTR mutants in human airways and validating FRT cells as a model for PDE inhibitor studies. Furthermore, we provide biochemical evidence that VX-809 causes surprisingly robust correction of several class III and IV CFTR mutants. Together, our findings further support the therapeutic potential of RPL554 for patients with CF with class III/IV mutations and emphasize the potential of PDEs as potential drug targets that could benefit patients with CF.

KLF5 influences cell biological function and chemotherapy sensitivity through the JNK signaling pathway in anaplastic thyroid carcinoma.

We aimed to investigate the effects of Krüppel-like factor 5 (KLF5) on cell biological function and chemotherapy sensitivity of anaplastic thyroid carcinoma (ATC) and explore the underlying mechanism. In this study, we found that KLF5 was expressed higher in ATC cells than that in normal thyroid cells. Knockdown of KLF5 inhibited proliferation, induced apoptosis and restrained invasion and migration abilities of ATC cells. KLF5 overexpression promoted proliferation and inhibited apoptosis of ATC cells in response to doxorubicin (Dox), whereas KLF5 knockdown increased the sensitivity of ATC cells to Dox. Multidrug resistance gene 1/permeability glycoprotein and ATP-binding cassette superfamily G member 2 were heightened in ATC cells with KLF5 overexpression, but the opposite results were found in sh-KLF5-treated cells. Phosphorylation (p)-c-Jun N-terminal kinase (JNK) was upregulated in KLF5 overexpression cells, whereas it was downregulated in the KLF5 knockdown treatment group. Furthermore, KLF5 knockdown inhibited ATC growth and enhanced the Dox sensitivity of ATC by inactivating the JNK signaling pathway. Taken together, our findings concluded that KLF5 knockdown can remarkably inhibit the proliferation, invasion, and migration and induce apoptosis of ATC cells, and increase the chemotherapy sensitivity of ATC, all of which probably through inhibiting the JNK signaling pathway.

Low Doses of Methylmercury Induce the Proliferation of Thyroid Cells In Vitro Through Modulation of ERK Pathway.

Exposure to environmental endocrine disruptors has been associated with an increased frequency of thyroid pathology. In this study, we evaluated the effects of various concentrations of methylmercury (MeHg) on immortalized, non-tumorigenic thyroid cells (Nthy-ori-3-1). Exposure to MeHg at 2.5 and 5 µM for 24 h caused a reduction in cell viability with a decrease of the cell population in sub-G0 phase, as detected by MTT and flow cytometry. Conversely, MeHg at the lower concentration of 0.1 µM increased the cell viability with a rise of G2/M phase. An immunoblot analysis showed higher expression levels of phospho-ERK and not of phospho-Akt. Further enhancement of the cell growth rate was observed after a prolonged exposure of the cells up to 18 days to MeHg 0.1 µM. The present findings demonstrate the toxicity of high concentrations of MeHg on thyroid cells, while showing that treatment with lower doses of Hg, as may occur after prolonged exposure to this environmental contaminant, exerts a promoting effect on thyroid cell proliferation, by acting on the ERK-mediated pro-oncogenic signal transduction pathway.

Thyrocyte-derived exosome-targeted dendritic cells stimulate strong CD4+ T lymphocyte responses.

Exosomes have been intensively studied in autoimmune diseases, and circulating exosomes and microvesicles have also been explored in autoimmune thyroiditis (AITD). However, the role of thyroid cell-derived exosomes in immune responses is unclear. We showed that IFN-γ-treated Nthy-ori 3-1 cell-derived exosomes (IFN-γ-Exo) harbored TPO, HSP60 and MHC-II and activated dendritic cells (DCs) in vitro. Compared with Exo-targeted DCs (DCExo), IFN-γ-Exo-targeted DCs (DCIFN-γ-Exo) promoted the expression and release of proinflammatory cytokines, such as IFN-γ, IL-17A and IL-22, from CD4+ T lymphocytes and inhibited the expression and release of anti-inflammatory cytokines, such as IL-4, IL-10 and TGF-ß1; however, IFN-γ-Exo did not have this effect compared with Nthy-ori 3-1 cell-derived exosomes (Exo). DCIFN-γ-Exo stimulates the expression and release of cytokines from CD4+ T lymphocytes more efficiently than IFN-γ-Exo. Thus, DCIFN-γ-Exo may effectively induce CD4+ T lymphocyte-mediated immune responses and play a role in the occurrence and development of AITD.