Therese Carlsson

Down-regulation of the Sodium/Iodide Symporter Explains 131I-Induced Thyroid Stunning

(131)I radiation therapy of differentiated thyroid cancer may be compromised by thyroid stunning (i.e., a paradoxical inhibition of radioiodine uptake caused by radiation from a pretherapeutic diagnostic examination). The stunning mechanism is yet uncharacterized at the molecular level. We therefore investigated whether the expression of the sodium/iodide symporter (NIS) gene is changed by irradiation using (131)I. Confluent porcine thyroid cells on filter were stimulated with thyroid-stimulating hormone (TSH; 1 milliunit/mL) or insulin-like growth factor-I (IGF-I; 10 ng/mL) and simultaneously exposed to (131)I in the culture medium for 48 h, porcine NIS mRNA was quantified by real-time reverse transcription-PCR using 18S as reference, and transepithelial iodide transport was monitored using (125)I(-) as tracer. TSH increased the NIS expression >100-fold after 48 h and 5- to 20-fold after prolonged stimulation. IGF-I enhanced the NIS transcription at most 15-fold but not until 5 to 7 days. (131)I irradiation (7.5 Gy) decreased both TSH-stimulated and IGF-I-stimulated NIS transcription by 60% to 90% at all investigated time points. TSH and IGF-I stimulated NIS synergistically 15- to 60-fold after 5 days. NIS expression was reduced by (131)I also in costimulated cells, but the transcription level remained higher than in nonirradiated cells stimulated with TSH alone. Changes in NIS mRNA always correlated with altered (125)I(-) transport in cultures with corresponding treatments. It is concluded that down-regulation of NIS is the likely explanation of (131)I-induced thyroid stunning. Enhanced NIS expression by synergistically acting agents (TSH and IGF-I) partly prevents the loss of iodide transport expected from a given absorbed dose, suggesting that thyroid stunning might be pharmacologically treatable.

Radiation-Induced Thyroid Stunning: Differential Effects of 123I, 131I, 99mTc, and 211At on Iodide Transport and NIS mRNA Expression in Cultured Thyroid Cells

Recent clinical and experimental data demonstrate that thyroid stunning is caused by previous irradiation and may influence the efficacy of 131I radiation therapy of thyroid cancer and possibly hyperthyroidism. To avoid stunning, many clinics have exchanged 131I for 123I for pretherapeutic diagnostic imaging and dose planning. Furthermore, recent in vitro studies indicate that 131I irradiation reduces iodide uptake by downregulating the expression of the sodium iodide symporter (NIS). The rationale for this study was therefore to study effects on iodide transport and NIS messenger RNA (mRNA) expression in thyrocytes exposed to both 123I and 131I in addition to some other potentially interesting radionuclides. Methods: Thyrotropin-stimulated thyroid cell monolayers were exposed to 0.5 Gy of 123I, 131I, 99mTc, or 211At, all being radionuclides transported via NIS, in the culture medium for 6 h, or to various absorbed doses of 123I or 131I for 48 h. NIS mRNA expression was analyzed using quantitative reverse-transcriptase polymerase chain reaction. Results: Iodide transport and NIS mRNA expression were reduced by all radionuclides. At the same absorbed dose, iodide transport was reduced the most by 211At, followed by 123I and 99mTc (equally potent), whereas 131I was least effective. The onset of NIS downregulation was rapid (<1 d after irradiation) in cells exposed to 123I or 211At and was delayed in cells irradiated with 131I or 99mTc. Iodide transport and NIS expression were recovered only for 211At. 123I reduced the iodine transport and the NIS mRNA expression more efficiently than did 131I at an equivalent absorbed dose, with a relative biological effectiveness of about 5. Conclusion: The stunning effect per unit absorbed dose is more severe for 123I than for 131I. Despite the lower absorbed dose per unit activity for 123I than for 131I, stunning by 123I cannot be excluded in patients. The degree to which iodide transport capacity and NIS mRNA expression are reduced seems to be related to the biological effectiveness of the type of radiation delivering the absorbed dose to the target, with 211At (which has the highest relative biological effectiveness) causing the highest degree of stunning per unit absorbed dose in the present study.

Revising the embryonic origin of thyroid C cells in mice and humans

Current understanding infers a neural crest origin of thyroid C cells, the major source of calcitonin in mammals and ancestors to neuroendocrine thyroid tumors. The concept is primarily based on investigations in quail–chick chimeras involving fate mapping of neural crest cells to the ultimobranchial glands that regulate Ca2+ homeostasis in birds, reptiles, amphibians and fishes, but whether mammalian C cell development involves a homologous ontogenetic trajectory has not been experimentally verified. With lineage tracing, we now provide direct evidence that Sox17+ anterior endoderm is the only source of differentiated C cells and their progenitors in mice. Like many gut endoderm derivatives, embryonic C cells were found to coexpress pioneer factors forkhead box (Fox) a1 and Foxa2 before neuroendocrine differentiation takes place. In the ultimobranchial body epithelium emerging from pharyngeal pouch endoderm in early organogenesis, differential Foxa1/Foxa2 expression distinguished two spatially separated pools of C cell precursors with different growth properties. A similar expression pattern was recapitulated in medullary thyroid carcinoma cells in vivo, consistent with a growth-promoting role of Foxa1. In contrast to embryonic precursor cells, C cell-derived tumor cells invading the stromal compartment downregulated Foxa2, foregoing epithelial-to-mesenchymal transition designated by loss of E-cadherin; both Foxa2 and E-cadherin were re-expressed at metastatic sites. These findings revise mammalian C cell ontogeny, expand the neuroendocrine repertoire of endoderm and redefine the boundaries of neural crest diversification. The data further underpin distinct functions of Foxa1 and Foxa2 in both embryonic and tumor development. Highlighted article: Mouse thyroid C cell precursors arise in foregut endoderm, and not the neural crest, disproving the current concept of a neural crest origin of thyroid neuroendocrine cells.

Expression of Islet1 in thyroid development related to budding, migration, and fusion of primordia

The LIM homeodomain transcription factor Isl1 was investigated in mouse thyroid organogenesis. All progenitor cells of the midline thyroid diverticulum and lateral primordia (ultimobranchial bodies) expressed Isl1. This pattern persisted until the growing anlagen fused at embryonic day (E) 13.5. In Isl1 null mutants thyroid progenitors expressing Nkx2.1 and Pax8 were readily specified in the anterior endoderm but the size of the thyroid rudiment was reduced. In late development, only immature C‐cells expressed Isl1. In the adult gland the number of Isl1+ cells was small compared with cells expressing calcitonin. Analysis of microarray profiles indicated a higher level of Isl1 expression in medullary thyroid carcinomas than in tumors derived from follicular cells. Together, these findings suggest that Isl1 may be a novel regulator of thyroid development before terminal differentiation of the endocrine cell types. Isl1 is an embryonic C‐cell precursor marker that may be relevant also in cancer developed from the mature C‐cell. Developmental Dynamics 237:3820–3829, 2008.

Role of EphA4 Receptor Signaling in Thyroid Development: Regulation of Folliculogenesis and Propagation of the C-Cell Lineage

Transcriptome analysis revealed that the tyrosine kinase receptor EphA4 is enriched in the thyroid bud in mouse embryos. We used heterozygous EphA4-EGFP knock-in mice in which enhanced green fluorescent protein (EGFP) replaced the intracellular receptor domain (EphA4(+/EGFP)) to localize EphA4 protein in thyroid primordial tissues. This showed that thyroid progenitors originating in the pharyngeal floor express EphA4 at all embryonic stages and when follicles are formed in late development. Also, the ultimobranchial bodies developed from the pharyngeal pouch endoderm express EphA4, but the ultimobranchial epithelium loses the EGFP signal before it merges with the median thyroid primordium. Embryonic C cells invading the thyroid are exclusively EphA4-negative. EphA4 expression continues in the adult thyroid. EphA4 knock-out mice and EphA4-EGFP homozygous mutants are euthyroid and have a normal thyroid anatomy but display subtle histological alterations regarding number, size, and shape of follicles. Of particular interest, the pattern of follicular abnormality differs between EphA4(-/-) and EphA4(EGFP/EGFP) thyroids. In addition, the number of C cells is reduced by >50% exclusively in animals lacking EphA4 forward signaling (EphA4(EGFP/EGFP)). Heterozygous EphA4 mutants have no apparent thyroid phenotype. We conclude that EphA4 is a novel regulator of thyroid morphogenesis that impacts on postnatal development of the two endocrine cell lineages of the differentiating gland. In this process both EphA4 forward signaling (in the follicular epithelium) and reverse signaling mediated by its cognate ligand(s) (A- and/or B-ephrins expressed in follicular cells and C cells, respectively) are probably functionally important.

The Proteasome and Intracellular Redox Status: Implications for Apoptotic Regulation in Lens Epithelial Cells

Purpose: This study aimed to investigate redox regulation of the proteasome as well as the effect of proteasome inhibition on intracellular oxidative status and apoptosis. Methods: Oxidative stress was induced in cultured human lens epithelial cells (HLECs) and intact mouse lenses by 100 μ M H2O2. HLECs were also exposed to the reduced and the oxidized forms of glutathione (GSH/GSSG) and the reducing agent dithiotreitol (DTT). The chymotrypsin-like, the trypsin-like, and the peptidylglutamyl peptidase activities of the proteasome were measured using synthetic fluorogenic substrates. Superoxide as well as peroxide production, mitochondrial membrane potential, and the level of GSH was measured in HLECs after proteasome inhibition by MG-132 or lactacystin. Apoptosis was determined by measuring caspase-3 activation and by studying apoptotic nuclei after staining with Hoechst 33342. Results: All three peptidase activities of the proteasome were inhibited by 100 μ M H2O2 and by the oxidized form of glutathione (GSSG), whereas the reduced form (GSH) stimulated chymotrypsin-like and peptidylglutamyl peptidase activities in HLECs lysates. Intact mouse lenses exposed to 100 μ M H2O2 exhibited loss of transparency and trends of decreased chymotrypsin-like proteasome activity as well as decreased GSH levels. Inhibition of the proteasome in cultured HLECs caused significant increase in apoptosis and disturbed intracellular redox balance. Simultaneous addition of exogenous GSH completely abolished the increased apoptosis seen after MG-132 treatment. Conclusions: This study supports the hypothesis that intracellular proteolytic and oxidative regulatory systems are tightly coupled. The current data also indicate that apoptosis by proteasome inhibition is mediated through oxidative mechanisms.

Intracellular Effects of NSAIDs/ASA in Oxidatively Stressed Human Lens Epithelial Cells in Culture

The aim of the study was to examine the effects of nonsteroidal anti-inflammatory drugs (NSAIDs)/acetylsalicylic acid (ASA) on human lens epithelial cells (HLECs) during oxidative stress. HLECs were exposed to H2O2 in the absence or presence of indomethacin, diclofenac, celecoxib (NSAIDs) or ASA for 24 h. HLECs were assayed for changes in superoxide and peroxide production and for variations in glutathione. Mitochondrial depolarization was measured using the membrane potential-sensitive dye JC-1. The results of the study include reduction in superoxide and peroxide production as well as reduction in glutathione depletion in oxidatively stressed HLECs incubated with low concentrations of NSAIDs/ASA. However, no protection against H2O2-induced mitochondrial depolarization by NSAIDs/ASA could be seen. In conclusion, NSAIDs/ASA display reactive oxygen species-scavenging properties in H2O2-exposed HLECs in culture.

Misguided Migration of C Cell Precursors to Extra-Thyroidal LocationsRelated to Defective Pharyngeal Pouch Development in Shh DeficientMice

A possible role of sonic hedgehog (Shh) in recruitment of C cell precursors to the Ultimobranchial Body (UB) and embryonic thyroid was investigated in Shh-/- mice. Nkx2.1 and Foxa2 co-expression distinguished UB originating in the fourth pharyngeal pouch from other derivatives of pharyngeal endoderm. In mutants UB formed a single structure that failed to bud and instead of fusing with the midline thyroid primordium adhered to the thymic rudiments. Mature C cells appeared in the UB remnant and ectopically in the thymic parenchyma, foregut endoderm and trachea. Thyroid did not contain C cells except minute numbers close to the tracheal interface. Tracing progeny in Shh-CRE/Rosa26R mice showed the vast majority of both UB and thyroid progenitors derived from Shh negative endoderm, but Shh expressing cells appeared in both thyroid primordia before fusion of the two. The findings indicate that Shh determines the endoderm territory for C cell differentiation and guides the migration of C cell precursors into the thyroid, presumably by regulating the separation of glandular domains in the pharyngeal pouch endoderm. A cell-autonomous role of Shh in thyroid morphogenesis is suggested.

Linking loss of sodium-iodide symporter expression to DNA damage.

Radiotherapy of thyroid cancer with I-131 is abrogated by inherent loss of radioiodine uptake due to loss of sodium iodide symporter (NIS) expression in poorly differentiated tumor cells. It is also known that ionizing radiation per se down-regulates NIS (the stunning effect), but the mechanism is unknown. Here we investigated whether loss of NIS-mediated iodide transport may be elicited by DNA damage. Calicheamicin, a fungal toxin that specifically cleaves double-stranded DNA, induced a full scale DNA damage response mediated by the ataxia-telangiectasia mutated (ATM) kinase in quiescent normal thyrocytes. At sublethal concentrations (<1nM) calicheamicin blocked NIS mRNA expression and transepithelial iodide transport as stimulated by thyrotropin; loss of function occurred at a much faster rate than after I-131 irradiation. KU-55933, a selective ATM kinase inhibitor, partly rescued NIS expression and iodide transport in DNA-damaged cells. Prolonged ATM inhibition in healthy cells also repressed NIS-mediated iodide transport. ATM-dependent loss of iodide transport was counteracted by IGF-1. Together, these findings indicate that NIS, the major iodide transporter of the thyroid gland, is susceptible to DNA damage involving ATM-mediated mechanisms. This uncovers novel means of poor radioiodine uptake in thyroid cells subjected to extrinsic or intrinsic genotoxic stress.