MiR-200b categorizes patients into pancreas cystic lesion subgroups with different malignant potential.

Extracellular vesicles (EV) carry their cargo in a membrane protected form, however, their value in early diagnostics is not well known. Although pancreatic cysts are heterogeneous, they can be clustered into the larger groups of pseudocysts (PC), and serous and mucinous pancreatic cystic neoplasms (S-PCN and M-PCN, respectively). In contrast to PCs and S-PCNs, M-PCNs may progress to malignant pancreatic cancers. Since current diagnostic tools do not meet the criteria of high sensitivity and specificity, novel methods are urgently needed to differentiate M-PCNs from other cysts. We show that cyst fluid is a rich source of EVs that are positive and negative for the EV markers CD63 and CD81, respectively. Whereas we found no difference in the EV number when comparing M-PCN with other pancreatic cysts, our EV-based biomarker identification showed that EVs from M-PCNs had a higher level of miR-200b. We also prove that not only EV-derived, but also total cyst fluid miR-200b discriminates patients with M-PCN from other pancreatic cysts with a higher sensitivity and specificity compared to other diagnostic methods, providing the possibility for clinical applications. Our results show that measuring miR-200b in cyst fluid-derived EVs or from cyst fluid may be clinically important in categorizing patients.

Shared extracellular vesicle miRNA profiles of matched ductal pancreatic adenocarcinoma organoids and blood plasma samples show the power of organoid technology.

Extracellular vesicles (EV) are considered as a promising diagnostic tool for pancreatic ductal adenocarcinoma (PDAC), a disease with a poor 5-year survival that has not improved in the past years. PDAC patient-derived 3D organoids maintain the intratumoral cellular heterogeneity, characteristic for the tumor in vivo.Thus, they represent an ideal in vitro model system to study human cancers. Here we show that the miRNA cargo of EVs from PDAC organoids largely differs among patients. However, we detected a common set of EV miRNAs that were present in matched organoids and blood plasma samples of individual patients. Importantly, the levels of EV miR-21 and miR-195 were higher in PDAC blood EV preparations than in healthy controls, albeit we found no difference compared to chronic pancreatitis (CP) samples. In addition, here we report that the accumulation of collagen I, a characteristic change in the extracellular matrix (ECM) in both CP and PDAC, largely increases EV release from pancreatic ductal organoids. This provides a possible explanation why both CP and PDAC patient-derived plasma samples have an elevated amount of CD63 + EVs. Collectively, we show that PDAC patient-derived organoids represent a highly relevant model to analyze the cargo of tumor cell-derived EVs. Furthermore, we provide evidence that not only driver mutations, but also changes in the ECM may critically modify EV release from pancreatic ductal cells.

Extracellular vesicles transmit epithelial growth factor activity in the intestinal stem cell niche.

Extracellular vesicles (EV) are membrane-surrounded vesicles that represent a novel way of intercellular communication by carrying biologically important molecules in a concentrated and protected form. The intestinal epithelium is continuously renewed by a small proliferating intestinal stem cell (ISC) population, residing at the bottom of the intestinal crypts in a specific microenvironment, the stem cell niche. By using 3D mouse and human intestinal organoids, we show that intestinal fibroblast-derived EVs are involved in forming the ISC niche by transmitting Wnt and epidermal growth factor (EGF) activity. With a mouse model that expresses EGFP in the Lgr5+ ISCs, we prove that loss in ISC number in the absence of EGF is prevented by fibroblast-derived EVs. Furthermore, we demonstrate that intestinal fibroblast-derived EVs carry EGF family members, such as amphiregulin. Mechanistically, blocking EV-bound amphiregulin inhibited the EV-induced survival of organoids. In contrast, EVs have no role in transporting R-Spondin, a critical niche factor amplifying Wnt signaling. Collectively, we prove the important role of fibroblast-derived EVs as a novel transmission mechanism of factors in the normal ISC niche.

Extracellular vesicle release from intestinal organoids is modulated by Apc mutation and other colorectal cancer progression factors.

Extracellular vesicles (EVs) are membrane-surrounded structures that transmit biologically important molecules from the releasing to target cells, thus providing a novel intercellular communication mechanism. Since EVs carry their cargo in a protected form and their secretion is generally increased in tumorigenesis, EVs hold a great potential for early cancer diagnosis. By 3D culturing, we provide evidence that colorectal cancer (CRC) patient-derived organoids, representing a state-of-the-art established and essential approach for studying human CRC, is a suitable model for EV analysis. When testing the effects of major factors promoting CRC progression on EV release in the organoid model, we observed that Apc mutation, leading to uncontrolled Wnt activation and thus to tumorigenesis in the vast majority in CRC patients, critically induces EV release by activating the Wnt pathway. Furthermore, the extracellular matrix component collagen, known to accumulate in tumorigenesis, enhances EV secretion as well. Importantly, we show that fibroblast-derived EVs induce colony formation of CRC organoid cells under hypoxia. In contrast, there was no major effect of tumor cell-derived EVs on the activation of fibroblasts. Collectively, our results with CRC and Apc-mutant adenoma organoids identify Apc mutation and collagen deposition as critical factors for increasing EV release from tumors. Furthermore, we provide evidence that stromal fibroblast-derived EVs contribute to tumorigenesis under unfavorable conditions in CRC.

Plectin isoform P1b and P1d deficiencies differentially affect mitochondrial morphology and function in skeletal muscle.

Plectin, a versatile 500-kDa cytolinker protein, is essential for muscle fiber integrity and function. The most common disease caused by mutations in the human plectin gene, epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), is characterized by severe skin blistering and progressive muscular dystrophy. Besides displaying pathological desmin-positive protein aggregates and degenerative changes in the myofibrillar apparatus, skeletal muscle specimens of EBS-MD patients and plectin-deficient mice are characterized by massive mitochondrial alterations. In this study, we demonstrate that structural and functional alterations of mitochondria are a primary aftermath of plectin deficiency in muscle, contributing to myofiber degeneration. We found that in skeletal muscle of conditional plectin knockout mice (MCK-Cre/cKO), mitochondrial content was reduced, and mitochondria were aggregated in sarcoplasmic and subsarcolemmal regions and were no longer associated with Z-disks. Additionally, decreased mitochondrial citrate synthase activity, respiratory function and altered adenosine diphosphate kinetics were characteristic of plectin-deficient muscles. To analyze a mechanistic link between plectin deficiency and mitochondrial alterations, we comparatively assessed mitochondrial morphology and function in whole muscle and teased muscle fibers of wild-type, MCK-Cre/cKO and plectin isoform-specific knockout mice that were lacking just one isoform (either P1b or P1d) while expressing all others. Monitoring morphological alterations of mitochondria, an isoform P1b-specific phenotype affecting the mitochondrial fusion-fission machinery and manifesting with upregulated mitochondrial fusion-associated protein mitofusin-2 could be identified. Our results show that the depletion of distinct plectin isoforms affects mitochondrial network organization and function in different ways.

A novel pathway regulates thyroid hormone availability in rat and human hypothalamic neurosecretory neurons.

Hypothalamic neurosecretory systems are fundamental regulatory circuits influenced by thyroid hormone. Monocarboxylate-transporter-8 (MCT8)-mediated uptake of thyroid hormone followed by type 3 deiodinase (D3)-catalyzed inactivation represent limiting regulatory factors of neuronal T3 availability. In the present study we addressed the localization and subcellular distribution of D3 and MCT8 in neurosecretory neurons and addressed D3 function in their axons. Intense D3-immunoreactivity was observed in axon varicosities in the external zone of the rat median eminence and the neurohaemal zone of the human infundibulum containing axon terminals of hypophysiotropic parvocellular neurons. Immuno-electronmicroscopy localized D3 to dense-core vesicles in hypophysiotropic axon varicosities. N-STORM-superresolution-microscopy detected the active center containing C-terminus of D3 at the outer surface of these organelles. Double-labeling immunofluorescent confocal microscopy revealed that D3 is present in the majority of GnRH, CRH and GHRH axons but only in a minority of TRH axons, while absent from somatostatin-containing neurons. Bimolecular-Fluorescence-Complementation identified D3 homodimers, a prerequisite for D3 activity, in processes of GT1-7 cells. Furthermore, T3-inducible D3 catalytic activity was detected in the rat median eminence. Triple-labeling immunofluorescence and immuno-electronmicroscopy revealed the presence of MCT8 on the surface of the vast majority of all types of hypophysiotropic terminals. The presence of MCT8 was also demonstrated on the axon terminals in the neurohaemal zone of the human infundibulum. The unexpected role of hypophysiotropic axons in fine-tuned regulation of T3 availability in these cells via MCT8-mediated transport and D3-catalyzed inactivation may represent a novel regulatory core mechanism for metabolism, growth, stress and reproduction in rodents and humans.

Neuronal hypoxia induces Hsp40-mediated nuclear import of type 3 deiodinase as an adaptive mechanism to reduce cellular metabolism.

In neurons, the type 3 deiodinase (D3) inactivates thyroid hormone and reduces oxygen consumption, thus creating a state of cell-specific hypothyroidism. Here we show that hypoxia leads to nuclear import of D3 in neurons, without which thyroid hormone signaling and metabolism cannot be reduced. After unilateral hypoxia in the rat brain, D3 protein level is increased predominantly in the nucleus of the neurons in the pyramidal and granular ipsilateral layers, as well as in the hilus of the dentate gyrus of the hippocampal formation. In hippocampal neurons in culture as well as in a human neuroblastoma cell line (SK-N-AS), a 24 h hypoxia period redirects active D3 from the endoplasmic reticulum to the nucleus via the cochaperone Hsp40 pathway. Preventing nuclear D3 import by Hsp40 knockdown resulted an almost doubling in the thyroid hormone-dependent glycolytic rate and quadrupling the transcription of thyroid hormone target gene ENPP2. In contrast, Hsp40 overexpression increased nuclear import of D3 and minimized thyroid hormone effects in cell metabolism. In conclusion, ischemia/hypoxia induces an Hsp40-mediated translocation of D3 to the nucleus, facilitating thyroid hormone inactivation proximal to the thyroid hormone receptors. This adaptation decreases thyroid hormone signaling and may function to reduce ischemia-induced hypoxic brain damage.

Thyroid hormone and the neuroglia: both source and target.

Thyroid hormone plays a crucial role in the development and function of the nervous system. In order to bind to its nuclear receptor and regulate gene transcription thyroxine needs to be activated in the brain. This activation occurs via conversion of thyroxine to T3, which is catalyzed by the type 2 iodothyronine deiodinase (D2) in glial cells, in astrocytes, and tanycytes in the mediobasal hypothalamus. We discuss how thyroid hormone affects glial cell function followed by an overview on the fine-tuned regulation of T3 generation by D2 in different glial subtypes. Recent evidence on the direct paracrine impact of glial D2 on neuronal gene expression underlines the importance of glial-neuronal interaction in thyroid hormone regulation as a major regulatory pathway in the brain in health and disease.

Paracrine signaling by glial cell-derived triiodothyronine activates neuronal gene expression in the rodent brain and human cells.

Hypothyroidism in humans is characterized by severe neurological consequences that are often irreversible, highlighting the critical role of thyroid hormone (TH) in the brain. Despite this, not much is known about the signaling pathways that control TH action in the brain. What is known is that the prohormone thyroxine (T4) is converted to the active hormone triiodothyronine (T3) by type 2 deiodinase (D2) and that this occurs in astrocytes, while TH receptors and type 3 deiodinase (D3), which inactivates T3, are found in adjacent neurons. Here, we modeled TH action in the brain using an in vitro coculture system of D2-expressing H4 human glioma cells and D3-expressing SK-N-AS human neuroblastoma cells. We found that glial cell D2 activity resulted in increased T3 production, which acted in a paracrine fashion to induce T3-responsive genes, including ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), in the cocultured neurons. D3 activity in the neurons modulated these effects. Furthermore, this paracrine pathway was regulated by signals such as hypoxia, hedgehog signaling, and LPS-induced inflammation, as evidenced both in the in vitro coculture system and in in vivo rat models of brain ischemia and mouse models of inflammation. This study therefore presents what we believe to be the first direct evidence for a paracrine loop linking glial D2 activity to TH receptors in neurons, thereby identifying deiodinases as potential control points for the regulation of TH signaling in the brain during health and disease.

Knockdown of the type 3 iodothyronine deiodinase (D3) interacting protein peroxiredoxin 3 decreases D3-mediated deiodination in intact cells.

The type 3 iodothyronine deiodinase (D3) is the primary deiodinase that inactivates thyroid hormone. Immunoprecipitation of D3, followed by fluorescent two-dimensional difference gel electrophoresis and mass spectrometry, identified peroxiredoxin 3 (Prx3) as a D3-associated protein. This interaction was confirmed using reverse coimmunoprecipitation, in which pull-down of Prx3 resulted in D3 isolation, and by fluorescence resonance energy transfer between cyan fluorescent protein-D3 and yellow fluorescent protein-Prx3. Prx3 overexpression did not change D3 activity in transfected HEK 293 cells; however, Prx3 knockdown resulted in a 50% decrease in D3-mediated whole-cell deiodination. Notably, D3 activity of cell lysates with dithiothreitol as an exogenous reducing factor and D3 protein levels were not decreased with Prx3 knockdown, indicating that the observed reduction in whole-cell deiodination was not simply due to a decrease in D3 enzyme levels. Prx3 knockdown did not change D3's affinity for T3 because saturation of D3-mediated whole-cell deiodination occurred between 20 and 200 nm T3 both with and without Prx3. Furthermore, the decrease in D3 activity in whole cells was not attributable to nonspecific oxidative stress because pretreatment with the antioxidant N-acetyl cysteine did not reverse the effects of Prx3 knockdown. Thioredoxin, the cofactor needed for Prx3 regeneration, supported D3 microsomal activity; however, Prx3 knockdown did not change D3 activity in this system. In conclusion, knockdown of Prx3 decreases D3 activity in whole cells, whereas absolute levels of D3 are unchanged, consistent with Prx3 playing a rate-limiting role in the regeneration of the D3 enzyme.

Cellular and molecular basis of deiodinase-regulated thyroid hormone signaling.

The iodothyronine deiodinases initiate or terminate thyroid hormone action and therefore are critical for the biological effects mediated by thyroid hormone. Over the years, research has focused on their role in preserving serum levels of the biologically active molecule T(3) during iodine deficiency. More recently, a fascinating new role of these enzymes has been unveiled. The activating deiodinase (D2) and the inactivating deiodinase (D3) can locally increase or decrease thyroid hormone signaling in a tissue- and temporal-specific fashion, independent of changes in thyroid hormone serum concentrations. This mechanism is particularly relevant because deiodinase expression can be modulated by a wide variety of endogenous signaling molecules such as sonic hedgehog, nuclear factor-kappaB, growth factors, bile acids, hypoxia-inducible factor-1alpha, as well as a growing number of xenobiotic substances. In light of these findings, it seems clear that deiodinases play a much broader role than once thought, with great ramifications for the control of thyroid hormone signaling during vertebrate development and metamorphosis, as well as injury response, tissue repair, hypothalamic function, and energy homeostasis in adults.

Myofiber integrity depends on desmin network targeting to Z-disks and costameres via distinct plectin isoforms.

Dysfunction of plectin, a 500-kD cytolinker protein, leads to skin blistering and muscular dystrophy. Using conditional gene targeting in mice, we show that plectin deficiency results in progressive degenerative alterations in striated muscle, including aggregation and partial loss of intermediate filament (IF) networks, detachment of the contractile apparatus from the sarcolemma, profound changes in myofiber costameric cytoarchitecture, and decreased mitochondrial number and function. Analysis of newly generated plectin isoform-specific knockout mouse models revealed that IF aggregates accumulate in distinct cytoplasmic compartments, depending on which isoform is missing. Our data show that two major plectin isoforms expressed in muscle, plectin 1d and 1f, integrate fibers by specifically targeting and linking desmin IFs to Z-disks and costameres, whereas plectin 1b establishes a linkage to mitochondria. Furthermore, disruption of Z-disk and costamere linkages leads to the pathological condition of epidermolysis bullosa with muscular dystrophy. Our findings establish plectin as the major organizer of desmin IFs in myofibers and provide new insights into plectin- and desmin-related muscular dystrophies.

The thyroid hormone-inactivating deiodinase functions as a homodimer.

The type 3 deiodinase (D3) inactivates thyroid hormone action by catalyzing tissue-specific inner ring deiodination, predominantly during embryonic development. D3 has gained much attention as a player in the euthyroid sick syndrome, given its robust reactivation during injury and/or illness. Whereas much of the structure biology of the deiodinases is derived from studies with D2, a dimeric endoplasmic reticulum obligatory activating deiodinase, little is known about the holostructure of the plasma membrane resident D3, the deiodinase capable of thyroid hormone inactivation. Here we used fluorescence resonance energy transfer in live cells to demonstrate that D3 exists as homodimer. While D3 homodimerized in its native state, minor heterodimerization was also observed between D3:D1 and D3:D2 in intact cells, the significance of which remains elusive. Incubation with 0.5-1.2 m urea resulted in loss of D3 homodimerization as assessed by bioluminescence resonance energy transfer and a proportional loss of enzyme activity, to a maximum of approximately 50%. Protein modeling using a D2-based scaffold identified potential dimerization surfaces in the transmembrane and globular domains. Truncation of the transmembrane domain (DeltaD3) abrogated dimerization and deiodinase activity except when coexpressed with full-length catalytically inactive deiodinase, thus assembled as DeltaD3:D3 dimer; thus the D3 globular domain also exhibits dimerization surfaces. In conclusion, the inactivating deiodinase D3 exists as homo- or heterodimer in living intact cells, a feature that is critical for their catalytic activities.

Expression patterns of WSB-1 and USP-33 underlie cell-specific posttranslational control of type 2 deiodinase in the rat brain.

The type 2 deiodinase (D2) activates thyroid hormone and constitutes an important source of 3,5,3',-triiodothyronine in the brain. D2 is inactivated via WSB-1 mediated ubiquitination but can be rescued from proteasomal degradation by USP-33 mediated deubiquitination. Using an in silico analysis of published array data, we found a significant positive correlation between the relative mRNA expression levels of WSB-1 and USP-33 in a set of 56 mouse tissues (r = 0.08; P < 0.04). Subsequently, we used in situ hybridization combined with immunocytochemistry in rat brain to show that in addition to neurons, WSB-1 and USP-33 are differently expressed in astrocytes and tanycytes, the two main D2 expressing cell types in this tissue. Tanycytes, which are thought to participate in the feedback regulation of TRH neurons express both WSB-1 and USP-33, indicating the potential for D2 ubiquitination and deubiquitination in these cells. Notably, only WSB-1 is expressed in glial fibrillary acidic protein-positive astrocytes throughout the brain. Although developmental and environmental signals are known to regulate the expression of WSB-1 and USP-33 in other tissues, our real-time PCR studies indicate that changes in thyroid status do not affect the expression of these genes in several rat brain regions, whereas in the mediobasal hypothalamus, changes in gene expression were minimal. In conclusion, the correlation between the relative mRNA levels of WSB-1 and USP-33 in numerous tissues that do not express D2 suggests that these ubiquitin-related enzymes share additional substrates besides D2. Furthermore, the data indicate that changes in WSB-1 and USP-33 expression are not part of the brain homeostatic response to hypothyroidism or hyperthyroidism.

Ubiquitination-induced conformational change within the deiodinase dimer is a switch regulating enzyme activity.

Ubiquitination is a critical posttranslational regulator of protein stability and/or subcellular localization. Here we show that ubiquitination can also regulate proteins by transiently inactivating enzymatic function through conformational change in a dimeric enzyme, which can be reversed upon deubiquitination. Our model system is the thyroid hormone-activating type 2 deiodinase (D2), an endoplasmic reticulum-resident type 1 integral membrane enzyme. D2 exists as a homodimer maintained by interacting surfaces at its transmembrane and globular cytosolic domains. The D2 dimer associates with the Hedgehog-inducible ubiquitin ligase WSB-1, the ubiquitin conjugase UBC-7, and VDU-1, a D2-specific deubiquitinase. Upon binding of T4, its natural substrate, D2 is ubiquitinated, which inactivates the enzyme by interfering with D2's globular interacting surfaces that are critical for dimerization and catalytic activity. This state of transient inactivity and change in dimer conformation persists until deubiquitination. The continuous association of D2 with this regulatory protein complex supports rapid cycles of deiodination, conjugation to ubiquitin, and enzyme reactivation by deubiquitination, allowing tight control of thyroid hormone action.

Metabolic instability of type 2 deiodinase is transferable to stable proteins independently of subcellular localization.

Thyroid hormone activation is catalyzed by two deiodinases, D1 and D2. Whereas D1 is a stable plasma membrane protein, D2 is resident in the endoplasmic reticulum (ER) and has a 20-min half-life due to selective ubiquitination and proteasomal degradation. Here we have shown that stable retention explains D2 residency in the ER, a mechanism that is nevertheless over-ridden by fusion to the long-lived plasma membrane protein, sodium-iodine symporter. Fusion to D2, but not D1, dramatically shortened sodium-iodine symporter half-life through a mechanism dependent on an 18-amino acid D2-specific instability loop. Similarly, the D2-specific loop-mediated protein destabilization was also observed after D2, but not D1, was fused to the stable ER resident protein SEC62. This indicates that the instability loop in D2, but not its subcellular localization, is the key determinant of D2 susceptibility to ubiquitination and rapid turnover rate. Our data also show that the 6 N-terminal amino acids, but not the 12 C-terminal ones, are the ones required for D2 recognition by WSB-1.

Characterization of the nuclear factor-kappa B responsiveness of the human dio2 gene.

Type 2 iodothyronine deiodinase (D2) activates T4 by deiodination to T3, a process being the source of most T3 present in the brain. In the mediobasal hypothalamus, expression of the dio2 gene is potently activated by administration of bacterial lipopolysaccharide (LPS), which in turn mediates the modifications in thyroid homeostasis typically observed in patients with nonthyroidal illness syndrome. Here we show that LPS-induced D2 expression is also observed in human MSTO-211H cells that endogenously express D2. Exposure to LPS rapidly doubled D2 activity by a mechanism that was partially blocked by the nuclear factor-B (NF-B) inhibitor sulfasalazine. Next, the human dio2 5'-flanking region promoter assay was used in HC11 cells and the p65/NF-kappa B responsiveness mapped to the 3' approximately 600-bp region of hdio2 5'-flanking region, with an approximately 15-fold induction. Semiquantitative EMSA identified the strongest NF-B binding sites at the positions -683 bp (called no. 2) and -198 bp (no. 5) 5' to the transcriptional starting site. Despite the very similar NF-kappa B binding affinity of these two sites, site-directed mutagenesis and promoter assay indicated that only site no. 5 possessed transactivation potency in the presence of the p65 subunit of NF-kappa B. Other cytokine mediators such as signal transducer and activator of transcription-3 (STAT3) or signal transducer and activator of transcription-5 (STAT5) did not induce transcription of the dio2 gene. Our results indicate that inflammatory signals regulate D2 expression predominantly via the NF-kappa B pathway in a direct transcriptional manner and could contribute to the changes in thyroid economy observed in nonthyroidal illness syndrome during infection.

Plectin scaffolds recruit energy-controlling AMP-activated protein kinase (AMPK) in differentiated myofibres.

Plectin, a cytolinker protein greater than 500 kDa in size, has an important role as a mechanical stabiliser of cells. It interlinks the various cytoskeletal filament systems and anchors intermediate filaments to peripheral junctional complexes. In addition, there is increasing evidence that plectin acts as a scaffolding platform that controls the spatial and temporal localisation and interaction of signaling proteins. In this study we show that, in differentiated mouse myotubes, plectin binds to the regulatory gamma1 subunit of AMP-activated protein kinase (AMPK), the key regulatory enzyme of energy homeostasis. No interaction was observed in undifferentiated myoblasts, and plectin-deficient myotubes showed altered positioning of gamma1-AMPK. In addition we found that plectin affects the subunit composition of AMPK, because isoform alpha1 of the catalytic subunit decreased in proportion to isoform alpha2 during in vitro differentiation of plectin(-/-) myotubes. In plectin-deficient myocytes we could also detect a higher level of activated (Thr172-phosphorylated) AMPK, compared with wild-type cells. Our data suggest a differentiation-dependent association of plectin with AMPK, where plectin selectively stabilises alpha1-gamma1 AMPK complexes by binding to the gamma1 regulatory subunit. The distinct plectin expression patterns in different fibre types combined with its involvement in the regulation of isoform compositions of AMPK complexes could provide a mechanism whereby cytoarchitecture influences energy homeostasis.

The Hedgehog-inducible ubiquitin ligase subunit WSB-1 modulates thyroid hormone activation and PTHrP secretion in the developing growth plate.

WSB-1 is a SOCS-box-containing WD-40 protein of unknown function that is induced by Hedgehog signalling in embryonic structures during chicken development. Here we show that WSB-1 is part of an E3 ubiquitin ligase for the thyroid-hormone-activating type 2 iodothyronine deiodinase (D2). The WD-40 propeller of WSB-1 recognizes an 18-amino-acid loop in D2 that confers metabolic instability, whereas the SOCS-box domain mediates its interaction with a ubiquitinating catalytic core complex, modelled as Elongin BC-Cul5-Rbx1 (ECS(WSB-1)). In the developing tibial growth plate, Hedgehog-stimulated D2 ubiquitination via ECS(WSB-1) induces parathyroid hormone-related peptide (PTHrP), thereby regulating chondrocyte differentiation. Thus, ECS(WSB-1) mediates a mechanism by which 'systemic' thyroid hormone can effect local control of the Hedgehog-PTHrP negative feedback loop and thus skeletogenesis.