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The TSH receptor (TSHR) and its many forms are the primary antigens of Graves' disease as evidenced by the presence of TSHR antibodies of differing biological activity. The TSH holoreceptor undergoes complex post-translational changes including cleavage of its ectodomain and oligomer formation. We have previously shown that the TSHR exists in both monomeric and dimeric structures in the thyroid cell membrane and have demonstrated, by modeling, that the transmembrane domains (TMD) can form stable dimeric structures. Based on these earlier simulations of the TSHR-TMD structure and our most recent model of the full-length TSHR we have now built models of full length TSHR multimers with and without TSH ligand in addition to multimers of the extracellular leucine-rich domain (LRD) - the site of TSH and autoantibody binding. Starting from these models we ran molecular dynamics (MD) simulation of the receptor oligomers solvated with water and counterions; the full-length oligomers also were embedded in a DPPC bilayer. The full length TSHR dimer and trimer models stayed in the same relative orientation and distance during 2000 ns (or longer) MD simulation in keeping with our earlier report of TMD dimerization. Simulations were also performed to model oligomers of the LRD alone; we found a trimeric complex to be even more stable than the dimers. These data provide further evidence that different forms of the TSHR add to the complexity of the immune response to this antigen which in patients with autoimmune thyroid disease generate an autoantibody reactome with multiple types of autoantibody to the TSHR.
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The receptor for thyroid stimulating hormone (TSHR), a GPCR, is the primary antigen in autoimmune hyperthyroidism (Graves' disease) caused by stimulating TSHR antibodies. While we have previously published a full length model of the TSHR, including its leucine rich domain (LRD), linker region (LR) and transmembrane domain (TMD), to date, only a partial LRD (aa 21-261) stabilized with TSHR autoantibodies has been crystallized. Recently, however, cryo-EM structures of the full-length TSHR have been published but they include only an incomplete LR. We have now utilized the cryo-EM models, added disulfide bonds to the LR and performed longer (3000 ns) molecular dynamic (MD) simulations to update our previous model of the entire full-length TSHR, with and without the presence of TSH ligand. As in our earlier work, the new model was embedded in a lipid membrane and was solvated with water and counterions. We found that the 3000 ns Molecular Dynamic simulations showed that the structure of the LRD and TMD were remarkably constant while the LR, known more commonly as the "hinge region", again showed significant flexibility, forming several transient secondary structural elements. Analysis of the new simulations permitted a detailed examination of the effect of TSH binding on the structure of the TSHR. We found a structure-stabilizing effect of TSH, including increased stability of the LR, which was clearly demonstrated by analyzing several intrinsic receptor properties including hydrogen bonding, fluctuation of the LRD orientation, and radius of gyration. In conclusion, we were able to quantify the flexibility of the TSHR and show its increased stability after TSH binding. These data indicated the important role of ligands in directing the signaling structure of a receptor.
Assuntos
Receptores da Tireotropina , Tireotropina , Receptores da Tireotropina/química , Receptores da Tireotropina/metabolismo , Ligantes , Tireotropina/química , Tireotropina/metabolismo , LeucinaRESUMO
The thyroid-stimulating hormone receptor (TSHR) transmembrane domain (TMD) is found in the plasma membrane and consists of lipids and water molecules. To understand the role of TSHR-associated water molecules, we used molecular dynamic simulations of the TMD and identified a network of putative receptor-associated transmembrane water channels. This result was confirmed with extended simulations of the full-length TSHR with and without TSH ligand binding. While the transport time observed in the simulations via the TSHR protein was slower than via the lipid bilayer itself, we found that significantly more water traversed via the TSHR than via the lipid bilayer, which more than doubled with the binding of TSH. Using rat thyroid cells (FRTL-5) and a calcein fluorescence technique, we measured cell volumes after blockade of aquaporins 1 and 4, the major thyroid cell water transporters. TSH showed a dose-dependent ability to influence water transport, and similar effects were observed with stimulating TSHR autoantibodies. Small molecule TSHR agonists, which are allosteric activators of the TMD, also enhanced water transport, illustrating the role of the TMD in this phenomenon. Furthermore, the water channel pathway was also mapped across 2 activation motifs within the TSHR TMD, suggesting how water movement may influence activation of the receptor. In pathophysiological conditions such as hypothyroidism and hyperthyroidism where TSH concentrations are highly variable, this action of TSH may greatly influence water movement in thyroid cells and many other extrathyroidal sites where the TSHR is expressed, thus affecting normal cellular function.
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Seasonal changes in food intake and adiposity in many animal species are triggered by changes in the photoperiod. These latter changes are faithfully transduced into a biochemical signal by melatonin secreted by the pineal gland. Seasonal variations, encoded by melatonin, are integrated by third ventricular tanycytes of the mediobasal hypothalamus through the detection of the thyroid-stimulating hormone (TSH) released from the pars tuberalis. The mediobasal hypothalamus is a critical brain region that maintains energy homeostasis by acting as an interface between the neural networks of the central nervous system and the periphery to control metabolic functions, including ingestive behavior, energy homeostasis, and reproduction. Among the cells involved in the regulation of energy balance and the blood-hypothalamus barrier (BHB) plasticity are tanycytes. Increasing evidence suggests that anterior pituitary hormones, specifically TSH, traditionally considered to have unitary functions in targeting single endocrine sites, display actions on multiple somatic tissues and central neurons. Notably, modulation of tanycytic TSH receptors seems critical for BHB plasticity in relation to energy homeostasis, but this needs to be proven.
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Melatonina , Animais , Melatonina/fisiologia , Células Ependimogliais/metabolismo , Hipotálamo/fisiologia , Encéfalo/metabolismo , Tireotropina/metabolismo , Estações do Ano , HomeostaseRESUMO
Instructive signals that delineate the formation of thyroid follicles by thyrotropin (TSH) in stem cells are complex. Here, we have examined the role of protein kinase C (PKC) by using a unique Gαq/11 biased small molecule (MSq1) to develop thyroid progenitor cells. Mouse embryonic stem cells (mESCs) were differentiated into anterior endoderm cells and treated with either TSH or MSq1 in the presence or absence of PKC inhibitors. The transcriptional and translational response of key thyroid markers-sodium iodide symporter (NIS), thyroglobulin (TG), and thyrotropin receptor (TSHR) as well as potential signaling molecules-were then analyzed. The data confirmed that MSq1 is a potent Gαq/11 activator with a major increase in Gαq/11 signaling when compared to TSH. MSq1 activation resulted in an increase in thyroid-specific genes, demonstrating that enhanced PKC signaling was able to induce their expression. The specificity of the PKC signals over the protein kinase A (PKA) pathway in regulating thyroid gene expression was shown by using a specific PKC enzyme inhibitor. The data revealed that TG and NIS expression were suppressed in the presence of the PKC inhibition but, in contrast, were not influenced by PKA inhibition. This indicated that PKC activation was the dominant pathway in the inductive process for thyroid hormone production. Furthermore, by examining PKC isoforms we found that PKCξ was the predominant form in the ES cells that mediated the effects. Since PKCξ can lead to activation of transforming growth factor-ß-activated kinase (pTAK1), and its downstream effector nuclear factor κB (NFκB) complex, this demonstrated the involvement of the TAK1/NFκB pathway in thyroid speciation.
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Proteína Quinase C , Glândula Tireoide , Animais , Camundongos , Glândula Tireoide/metabolismo , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Tireotropina/farmacologia , Tireotropina/metabolismo , Receptores da Tireotropina/genética , Receptores da Tireotropina/metabolismo , Células-Tronco/metabolismoRESUMO
BACKGROUND: Graves' disease is associated with TSH receptor (TSHR) antibodies of variable bioactivity including "neutral" antibodies (N-TSHR-Ab) that bind to the hinge region of the TSHR ectodomain. We have previously found that such antibodies induced thyroid cell apoptosis via excessive mitochondrial and ER stress with elevated reactive oxygen species (ROS). However, the detailed mechanisms by which excess ROS was induced remained unclear. OBJECTIVES: To determine how ROS is induced by N-TSHR-monoclonal antibodies (mAb, MC1) mediated signaling and to measure stress in polyorganelles. METHODS: Total ROS and mitochondrial ROS was measured by fluorometry of live rat thyrocytes. Live-cell imaging of labelled organelles was carried out using red or green fluorescent dyes. Proteins were detected by Li-Cor Western immunoblots and immunocytochemistry. RESULTS: Endocytosis of N-TSHR-mAb induced ROS, disturbed vesicular trafficking, damaged organelles and failed to induce lysosomal degradation and autophagy. We found that the endocytosis triggered signaling cascades involving Gα13 and PKC-δ leading to intrinsic thyroid cell apoptosis. CONCLUSIONS: These studies define the mechanism of ROS induction in thyroid cells following the endocytosis of N-TSHR-Ab/TSHR complexes. We suggest that a viscous cycle of stress initiated by cellular ROS and induced by N-TSHR-mAbs may orchestrate overt intra-thyroidal, retro-orbital, and intra-dermal inflammatory autoimmune reactions in patients with Graves' disease.
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Doença de Graves , Ratos , Animais , Espécies Reativas de Oxigênio/metabolismo , Imunoglobulinas Estimuladoras da Glândula Tireoide , Receptores da Tireotropina , Anticorpos Monoclonais/farmacologiaRESUMO
Biophysical studies have established that the thyrotropin (TSH) receptor (TSHR) undergoes posttranslational modifications including dimerization. Following our earlier simulation of a TSHR-transmembrane domain (TMD) monomer (called TSHR-TMD-TRIO) we have now proceeded with a molecular dynamics simulation (MD) of TSHR-TMD dimerization using this improved membrane-embedded model. The starting structure was the TMD protein with all extracellular and intracellular loops and internal waters, which was placed in the relative orientation of the model originally generated with Brownian dynamics. Furthermore, this model was embedded in a DPPC lipid bilayer further solvated with water and added salt. Data from the MD simulation studies showed that the dimeric subunits stayed in the same relative orientation and distance during the 1000 ns of study. Comparison of representative conformations of the individual monomers when dimerized with the conformations from the monomer simulation showed subtle differences as represented by the backbone root mean square deviations. Differences in the conformations of the ligand-binding sites, suggesting variable affinities for these "hot spots," were also revealed by comparing the docking scores of 46 small-molecule ligands that included known TSHR agonists and antagonists as well as their derivatives. These data add further insight into the tendency of the TSHR-TMD to form dimeric and oligomeric structures and show that the differing conformations influence small-molecule binding sites within the TMD.
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Receptores da Tireotropina , Tireotropina , Receptores da Tireotropina/metabolismo , Dimerização , Domínios Proteicos , Tireotropina/metabolismo , LigantesRESUMO
(GPCR)The receptor for TSH receptor (TSHR), a G protein coupled receptor (GPCR), is of particular interest as the primary antigen in autoimmune hyperthyroidism (Graves' disease) caused by stimulating TSHR antibodies. To date, only one domain of the extracellular region of the TSHR has been crystallized. We have run a 1000 ns molecular dynamic simulation on a model of the entire TSHR generated by merging the extracellular region of the receptor, obtained using artificial intelligence, with our recent homology model of the transmembrane domain, embedded it in a lipid membrane and solvated it with water and counterions. The simulations showed that the structure of the transmembrane and leucine-rich domains were remarkably constant while the linker region (LR), known more commonly as the 'hinge region,' showed significant flexibility, forming several transient secondary structural elements. Furthermore, the relative orientation of the leucine-rich domain with the rest of the receptor was also seen to be variable. These data suggest that this LR is an intrinsically disordered protein. Furthermore, preliminary data simulating the full TSHR model complexed with its ligand (TSH) showed that (a) there is a strong affinity between the LR and TSH ligand and (b) the association of the LR and the TSH ligand reduces the structural fluctuations in the LR. This full-length model illustrates the importance of the LR in responding to ligand binding and lays the foundation for studies of pathologic TSHR autoantibodies complexed with the TSHR to give further insight into their interaction with the flexible LR.
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Inteligência Artificial , Receptores da Tireotropina , Receptores da Tireotropina/química , Receptores da Tireotropina/metabolismo , Ligantes , Leucina/metabolismo , Autoanticorpos , Receptores Acoplados a Proteínas G , Tireotropina/química , Tireotropina/metabolismoRESUMO
TSH receptor (TSHR) antibodies are the cause of Graves' disease and may also be found in patients with Hashimoto's thyroiditis. They come in at least three varieties: thyroid stimulating, thyroid blocking and neutral. The measurement of TSH receptor antibodies in Graves' disease and Hashimoto's thyroiditis is a common clinical activity and can be useful in diagnosis and prognosis. We show that it is not possible to detect the blocking variety of TSHR antibody in patients with Graves' disease because the stimulating antibody may overwhelm the measurement of blocking in the bioassays available for their measurement and may blind the valid interpretation of the results. To help explain this in more detail we show a series of studies with monoclonal TSHR antibodies which support this conclusion.
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Anticorpos Monoclonais , Doença de Graves , Doença de Hashimoto , Receptores da Tireotropina , Anticorpos Monoclonais/análise , Autoanticorpos/análise , Doença de Graves/diagnóstico , Doença de Hashimoto/diagnóstico , Humanos , Receptores da Tireotropina/análiseRESUMO
Background: Graves' eye disease, also called Graves' orbitopathy (GO), is a potentially debilitating autoimmune disease associated with retro-orbital inflammation and tissue expansion, involving both fibroblasts and adipocytes, resulting in periorbital edema, worsening proptosis, and muscle dysfunction with diplopia and may ultimately threaten sight. Accumulating evidence has indicated that autoantibodies to the thyrotropin receptor (TSHR), which induce the hyperthyroidism of Graves' disease, also help mediate the pathogenesis of the eye disease in susceptible individuals through TSHR expression on retro-orbital cells. Since it has long been known that the effects of insulin-like growth factor 1 (IGF-1) and thyrotropin are additive, recent clinical trials with a human monoclonal IGF-1 receptor blocking antibody (teprotumumab; IGF-1R-B-monoclonal antibody [mAb]) have demonstrated its ability to induce significant reductions in proptosis, diplopia, and clinical activity scores in patients with GO. However, the molecular mechanisms by which such an antibody achieves this result is unclear. Methods: We have used Li-Cor In-Cell Western, Western blot, and immunohistochemistry to define levels of different proteins in mouse and human fibroblast cells. Proteomic array was also used to define pathway signaling molecules. Using CCK-8 and BrdU cell proliferation ELISA, we have analyzed proliferative response of these cells to different antibodies. Results: We now show that a stimulating TSHR antibody was able to induce phosphorylation of the IGF-1R and initiate both TSHR and IGF-1R signaling in mouse and human fibroblasts. IGF-1R-B-mAb (1H7) inhibited all major IGF-1R signaling cascades and also reduced TSHR signaling. This resulted in the antibody-induced suppression of autophagy as shown by inhibition of multiple autophagy-related proteins (Beclin1, LC3a, LC3b, p62, and ULK1) and the induction of cell death by apoptosis as evidenced by activation of cleaved caspase 3, FADD, and caspase 8. Furthermore, this IGF-1R-blocking mAb suppressed serum-induced perkin and pink mitophagic proteins. Conclusions: Our observations clearly indicated that stimulating TSHR antibodies were able to enhance IGF-1R activity and contribute to retro-orbital cellular proliferation and inflammation. In contrast, an IGF-1R-B-mAb was capable of suppressing IGF-1R signaling leading to retro-orbital fibroblast/adipocyte death through the cell-extrinsic pathway of apoptosis. This is likely the major mechanism involved in proptosis reduction in patients with Graves' eye disease treated by IGF-1R inhibition.
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Doença de Graves , Oftalmopatia de Graves , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Apoptose , Diplopia , Fibroblastos , Oftalmopatia de Graves/metabolismo , Humanos , Imunoglobulinas Estimuladoras da Glândula Tireoide/metabolismo , Inflamação/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Proteômica , Receptor IGF Tipo 1/metabolismo , Receptores da Tireotropina , Tireotropina/metabolismoRESUMO
BACKGROUND: Graves' disease (GD) is associated with thyroid stimulating hormone (TSH) receptor (TSHR) antibodies of variable bioactivity. We have previously characterized "neutral" TSHR antibodies (N-TSHR-Abs) that bind to the hinge region of the TSHR ectodomain. We showed that an N-TSHR monoclonal antibody (mAb) failed to induce any G proteins to sustain survival signaling and lead to excessive stress and apoptosis. Furthermore, the addition of TSH, or the antioxidant N-acetyl-l-cysteine (NAC), rescued N-TSHR-mAb-induced apoptotic death. However, the detailed mechanisms of this rescue remained unclear. METHODS: Autophagy is activated in response to diverse stress related stimuli so we have, therefore, studied the autophagy response in rat thyroid cells (FRTL-5) during N-TSHR-mAb induced thyrocyte stress and apoptosis using the In Cell Western technique for quantitation along with immunocytochemistry. RESULTS: Under starvation conditions with N-TSHR-mAb the addition of TSH or NAC prevented thyroid cell death by enhancing autophagy. This was evidenced by elevated levels of autophagy related proteins including beclin 1, LC3A, LC3B, ULK1, p62, and also activated pink and perkin mitophagy related proteins. The phenomenon was further confirmed by image analyses using Cyto-ID and Mito-ID autophagy detection systems. We also found that either TSH or NAC enhanced PKA, Akt, mTORC, AMPK, Sirtuins, PGC1α, NRF-2, mitofusin-2, TFAM and catalase in the N-TSHR-mAb stressed cells. Thus TSH or NAC restored cell survival signaling which reduced cell stress and enhanced mitochondrial biogenesis. The N-TSHR-mAb also activated cytochrome-C, Bax, caspase-9, caspase-3A, and had less effect on FADD or caspase-8 indicating activation of the intrinsic pathway for apoptosis. CONCLUSIONS: These findings indicated that TSH or antioxidant can rescue thyroid cells from N-TSHR-mAb induced apoptosis via enhanced autophagy. These observations signify that N-TSHR-mAb in GD under low TSH conditions caused by the hyperthyroidism could be detrimental for thyrocyte survival which would be another factor able to precipitate ongoing autoinflammation.
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Receptores da Tireotropina , Glândula Tireoide , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacologia , Apoptose , Autofagia , RatosRESUMO
The synergistic activation of transcription factors can lead to thyroid progenitor cell speciation. We have previously shown in vitro that mouse or human stem cells, expressing the transcription factors NKx2-1 and Pax8, can differentiate into thyroid neo-follicular structures (TFS). We now show that syngeneic mouse TFS when implanted into hypothyroid TSH receptor knockout (TSHR-KO) mice can ameliorate the hypothyroid state for an extended period. ES cells derived from heterozygous TSHR-KO blastocysts were stably transfected with Nkx2-1-GFP and Pax8-mcherry constructs and purified into 91.8% double positive cells by flow cytometry. After 5 days of activin A treatment these double positive cells were then induced to differentiate into neo-follicles in Matrigel for 21 days in the presence of 500µU/mL of TSH. Differentiated TFS expressing thyroglobulin mRNA were implanted under the kidney capsule of 4-6 weeks old TSHR-KO mice (n=5) as well as hind limb muscle (n=2) and anterior chamber of one eye (n=2). Five of the mice tested after 4 weeks were all rendered euthyroid and all mice remained euthyroid at 20 weeks post implantation. The serum T4 fully recovered (pre-bleed 0.62 ± 0.03 to 8.40 ± 0.57 µg/dL) and the previously elevated TSH became normal or suppressed (pre-bleed 391 ± 7.6 to 4.34 ± 1.25 ng/dL) at the end of the 20 week observation period. The final histology obtained from the implanted kidney tissues showed only rudimentary thyroid follicular structures but which stained positive for thyroglobulin expression. The presence of only rudimentary structures at the site of implant on these extended animals suggested possible migration of cells from the site of implant or an inability of TFCs to maintain proper follicular morphology in these external sites for extended periods. However, there were no signs of tumor formation and no immune infiltration. These preliminary studies show that TSHR-KO mice are a useful model for orthotropic implantation of functional thyroid cells without the need for thyroidectomy, radioiodine ablation or anti thyroid drug control of thyroid function. This approach is also proof of principle that thyroid cells derived from mouse ES cells are capable of surviving as functional neo-follicles in vivo for an extended period of 20 weeks.
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Diferenciação Celular , Regulação da Expressão Gênica , Hipotireoidismo/terapia , Receptores da Tireotropina/fisiologia , Transplante de Células-Tronco/métodos , Células-Tronco/citologia , Glândula Tireoide/citologia , Animais , Feminino , Hipotireoidismo/etiologia , Hipotireoidismo/metabolismo , Hipotireoidismo/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos SCID , Testes de Função TireóideaRESUMO
Thyrotropin (TSH), traditionally seen as a pituitary hormone that regulates thyroid glands, has additional roles in physiology including skeletal remodeling. Population-based observations in people with euthyroidism or subclinical hyperthyroidism indicated a negative association between bone mass and low-normal TSH. The findings of correlative studies were supported by small intervention trials using recombinant human TSH (rhTSH) injection, and genetic and case-based evidence. Genetically modified mouse models, which disrupt the reciprocal relationship between TSH and thyroid hormone, have allowed us to examine an independent role of TSH. Since the first description of osteoporotic phenotype in haploinsufficient Tshr +/- mice with normal thyroid hormone levels, the antiosteoclastic effect of TSH has been documented in both in vitro and in vivo studies. Further studies showed that increased osteoclastogenesis in Tshr-deficient mice was mediated by tumor necrosis factor α. Low TSH not only increased osteoclastogenesis, but also decreased osteoblastogenesis in bone marrow-derived primary osteoblast cultures. However, later in vivo studies using small and intermittent doses of rhTSH showed a proanabolic effect, which suggests that its action might be dose and frequency dependent. TSHR was shown to interact with insulin-like growth factor 1 receptor, and vascular endothelial growth factor and Wnt pathway might play a role in TSH's effect on osteoblasts. The expression and direct skeletal effect of a biologically active splice variant of the TSHß subunit (TSHßv) in bone marrow-derived macrophage and other immune cells suggest a local skeletal effect of TSHR. Further studies of how locally secreted TSHßv and systemic TSHß interact in skeletal remodeling through the endocrine, immune, and skeletal systems will help us better understand the hyperthyroidism-induced bone disease.
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Doenças Ósseas/patologia , Osso e Ossos/patologia , Hipertireoidismo/complicações , Tireotropina/metabolismo , Animais , Doenças Ósseas/etiologia , Doenças Ósseas/metabolismo , HumanosRESUMO
The thyroid-stimulating hormone receptor (TSHR) is a G-protein-coupled receptor group A family member with 7 transmembrane helices. We generated 3 new models of its entire transmembrane region using a 600 ns molecular simulation. The simulation started from our previously published model, which we have now revised by also modeling the intracellular loops and the C-terminal tail, adding internal waters and embedding it into a lipid bilayer with a water layer and with ions added to complete the system. We have named this model TSHR-TMD-TRIO since 3 representative dominant structures were then extracted from the simulation trajectory and compared with the original model. These structures each showed small but significant changes in the relative positions of the helices. The 3 models were also used as targets to dock a set of small molecules that are known active compounds including a new TSHR antagonist (BT362), which confirmed the appropriateness of the model with some small molecules showing significant preference for one or other of the structures.
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Modelos Moleculares , Receptores da Tireotropina/química , Membrana Celular/química , Humanos , Ligação de Hidrogênio , Simulação de Acoplamento Molecular , Método de Monte Carlo , Domínios Proteicos , Estrutura Secundária de Proteína , Receptores da Tireotropina/antagonistas & inibidores , Tireotropina/farmacologiaRESUMO
Background: The development of the thyroid follicular cell has been well characterized as it progresses from the original stem cell, either embryonic or adult, through a series of transitions to form a differentiated and functional thyroid cell. Summary: In this review, we briefly outline what is known about this transitional process with emphasis on characterizing the thyroid progenitor stem cell by using data obtained from both in vitro and in vivo studies and both mouse and human cells. It is of particular importance to note the influence of independent factors that guide the transcriptional control of the developing thyroid cell as it is subjected to extracellular signals, often working via epigenetic changes, and initiating intrinsic transcriptional changes leading to a functional cell. Conclusion: Thyroid stem cells fall into the category of dispositional stem cells and are greatly influenced by their environment.
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Células-Tronco/fisiologia , Células Epiteliais da Tireoide/fisiologia , Glândula Tireoide/citologia , Glândula Tireoide/diagnóstico por imagem , Animais , Humanos , CamundongosRESUMO
Background: The success in rescuing thyroid deficiency in mice using thyroid cells derived from embryonic stem (ES) cells, together with the discovery of human induced pluripotent stem cells (iPSCs) from somatic cells, has raised the possibility of patient-specific thyroid cell replacement. In this study we demonstrate that human thyroid follicular cells can be derived from human iPSCs and show the ability of highly purified and differentiated cells to secrete thyroid hormone. Research Design and Methods: Human iPSCs were derived from adult skin fibroblasts using RNA reprogramming and differentiated in vitro into thyroid follicular cells by exposure to activin A, ethacridine and TSH as we have previously described for human ES cells. The resulting thyroid cells were then highly purified using double antibody cell sorting. Results: The iPSCs derived from human dermal fibroblasts showed stem cell-like morphologic changes and expressed pluripotent stem cell markers as assessed using qPCR, immunofluorescence staining, and FACS analysis. These cells retained their pluripotential characteristics as shown by teratoma formation after murine transplantation. Definitive endoderm cells were induced with activin A and the transcription factor TAZ was significantly induced on ethacridine treatment and translocated to the nucleus. Thyroid transcription factors NKX2-1 and PAX8 were also highly expressed in activin A derived endoderm cells and further induced by ethacridine. Following terminal differentiation with TSH, there was enhanced thyroid follicle formation, high expression of the thyroid specific genes-TG, TPO, TSHR and NIS, and secretion of thyroid hormone (T4) in vitro. Furthermore, we were able to achieve a 97% purification of TSHR+/NIS+ expressing cells after differentiation using a single purification procedure. Conclusions: These findings demonstrate that mature adult dermal fibroblasts can be matured into human iPSCs which have the potential to form functional thyroid follicular cells. This lays the groundwork for future person-specific thyroid regenerative therapy.
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Diferenciação Celular , Reprogramação Celular , Fibroblastos/citologia , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Teratoma/patologia , Células Epiteliais da Tireoide/citologia , Animais , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Teratoma/metabolismo , Células Epiteliais da Tireoide/metabolismoRESUMO
To gain further insight into the binding of the normal and variant human TSHß subunits (TSHß and TSHßv), we modeled the 2 monomeric proteins and studied their interaction with the TSH receptor ectodomain (TSHR-ECD) using molecular dynamics simulation Furthermore, analyzed their bioactivity in vitro using recombinant proteins to confirm that such binding was physiologically relevant. Examining the interaction of TSHß and TSHßv with the TSHR-ECD model using molecular dynamic simulation revealed strong binding of these proteins to the receptor ECD. The specificity of TSHß and TSHßv binding to the TSHR-ECD was examined by analyzing the hydrogen-bonding residues of these subunits to the FSH receptor ECD, indicating the inability of these molecules to bind to the FSH receptors. Furthermore, the modelling suggests that TSHß and TSHßv proteins clasped the concave surface of the leucine rich region of the TSHR ECD in a similar way to the native TSH using dynamic hydrogen bonding. These mutually exclusive stable interactions between the subunits and ECD residues included some high-affinity contact sites corresponding to binding models of native TSH. Furthermore, we cloned TSHß and TSHßv proteins using the entire coding ORF and purified the flag-tagged proteins. The expressed TSHß subunit proteins retained bioactivity both in a coculture system as well as with immune-purified proteins. In summary, we showed that such interactions can result in a functional outcome and may exert physiological or pathophysiological effects in immune cells.
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Domínios e Motivos de Interação entre Proteínas , Receptores da Tireotropina/química , Receptores da Tireotropina/metabolismo , Tireotropina Subunidade beta/química , Tireotropina Subunidade beta/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Subunidades ProteicasRESUMO
G protein coupled receptors (GPCRs) can lead to G protein and non-G protein initiated signals. By virtue of its structural property, the TSH receptor (TSHR) has a unique ability to engage different G proteins making it highly amenable to selective signaling. In this study, we describe the identification and characterization of a novel small molecule agonist to the TSHR which induces primary engagement with Gαq/11. To identify allosteric modulators inducing selective signaling of the TSHR we used a transcriptional-based luciferase assay system with CHO-TSHR cells stably expressing response elements (CRE, NFAT, SRF, or SRE) that were capable of measuring signals emanating from the coupling of Gαs , Gαq/11, Gßγ, and Gα12/13, respectively. Using this system, TSH activated Gαs , Gαq/11, and Gα12/13 but not Gßγ. On screening a library of 50K molecules at 0.1,1.0 and 10 µM, we identified a novel Gq/11 agonist (named MSq1) which activated Gq/11 mediated NFAT-luciferase >4 fold above baseline and had an EC50= 8.3 × 10-9 M with only minor induction of Gαs and cAMP. Furthermore, MSq1 is chemically and structurally distinct from any of the previously reported TSHR agonist molecules. Docking studies using a TSHR transmembrane domain (TMD) model indicated that MSq1 had contact points on helices H1, H2, H3, and H7 in the hydrophobic pocket of the TMD and also with the extracellular loops. On co-treatment with TSH, MSq1 suppressed TSH-induced proliferation of thyrocytes in a dose-dependent manner but lacked the intrinsic ability to influence basal thyrocyte proliferation. This unexpected inhibitory property of MSq1 could be blocked in the presence of a PKC inhibitor resulting in derepressing TSH induced protein kinase A (PKA) signals and resulting in the induction of proliferation. Thus, the inhibitory effect of MSq1 on proliferation resided in its capacity to overtly activate protein kinase C (PKC) which in turn suppressed the proliferative signal induced by activation of the predomiant cAMP-PKA pathway of the TSHR. Treatment of rat thyroid cells (FRTL5) with MSq1 did not show any upregulation of gene expression of the key thyroid specific markers such as thyroglobulin(Tg), thyroid peroxidase (Tpo), sodium iodide symporter (Nis), and the TSH receptor (Tshr) further suggesting lack of involvement of MSq1 and Gαq/11 activation with cellular differentation. In summary, we identified and characterized a novel Gαq/11 agonist molecule acting at the TSHR and which showed a marked anti-proliferative ability. Hence, Gq biased activation of the TSHR is capable of ameliorating the proliferative signals from its orthosteric ligand and may offer a therapeutic option for thyroid growth modulation.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores da Tireotropina/metabolismo , Transdução de Sinais , Animais , Células CHO , Proliferação de Células , Cricetulus , Proteínas de Ligação ao GTP/agonistas , Simulação de Acoplamento Molecular , Ligação Proteica , Células Epiteliais da Tireoide/metabolismoRESUMO
Graves' disease (GD) is an autoimmune disease that primarily affects the thyroid gland. It is the most common cause of hyperthyroidism and occurs at all ages but especially in women of reproductive age. Graves' hyperthyroidism is caused by autoantibodies to the thyroid-stimulating hormone receptor (TSHR) that act as agonists and induce excessive thyroid hormone secretion, releasing the thyroid gland from pituitary control. TSHR autoantibodies also underlie Graves' orbitopathy (GO) and pretibial myxoedema. Additionally, the pathophysiology of GO (and likely pretibial myxoedema) involves the synergism of insulin-like growth factor 1 receptor (IGF1R) with TSHR autoantibodies, causing retro-orbital tissue expansion and inflammation. Although the aetiology of GD remains unknown, evidence indicates a strong genetic component combined with random potential environmental insults in an immunologically susceptible individual. The treatment of GD has not changed substantially for many years and remains a choice between antithyroid drugs, radioiodine or surgery. However, antithyroid drug use can cause drug-induced embryopathy in pregnancy, radioiodine therapy can exacerbate GO and surgery can result in hypoparathyroidism or laryngeal nerve damage. Therefore, future studies should focus on improved drug management, and a number of important advances are on the horizon.
Assuntos
Antirretrovirais/efeitos adversos , Infecções por HIV/complicações , Antirretrovirais/farmacologia , Gerenciamento Clínico , Infecções por HIV/tratamento farmacológico , Infecções por HIV/fisiopatologia , Humanos , Qualidade de Vida/psicologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência Humana , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
Objective: It has been demonstrated that the transcription factors TAZ (transcriptional coactivator with PDZ-binding motif), paired box gene 8 (PAX8), and NK2 homeobox 1 (NKX2-1) are coexpressed in the nucleus of thyroid cells. Furthermore, TAZ is known to enhance the transcriptional activity of PAX8 and NKX2-1 as well as the key thyroid-specific gene, thyroglobulin (TG), suggesting a critical role for TAZ in the control of thyroid cell speciation. We previously reported that the small molecule ethacridine, identified as a TAZ activator, was able to induce thyroid-specific transcription in endodermal cells differentiated from human embryonic stem (hES) cells using activin A. Since transcription factors are epigenetically regulated in cell differentiation, we investigated the epigenetic changes in the promoter regions of these key transcription factors during in vitro differentiation of hES cells into thyrocytes. Methods: We initially profiled chromatin accessibility using the technique of Assay for Transposase Accessible Chromatin sequencing (ATAC-seq), and then examined DNA methylation and histone acetylation in the promoter regions of the three selected thyroid transcription factors and the thyroid-specific genes during hES cell differentiation. Results: ATAC-seq analysis showed enriched chromatin accessibility of TAZ, NKX2-1, and PAX8 after exposure to activin A and ethacridine. There were no methylation changes found in the NKX2-1, PAX8, and TAZ promoters by bisulfite sequencing. In contrast, acetylation of histone H4, specifically acetylation of lysine 16, was observed in each of the promoters when measured by chromatin immunoprecipitation polymerase chain reaction assays, which correlated with the activity and expression of NKX2-1 and PAX8 as well as sodium/iodide symporter, thyroid stimulating hormone receptor, and TG genes. Conclusions: These results indicate that ethacridine treatment of activin A-derived endodermal hES cells leads to enhanced chromatin accessibility, which, in turn, allows histone H4 acetylation in the regulation of active genes for speciation of thyroid follicular cells from hES cells.