Defective thyroglobulin storage in LDL receptor-associated protein-deficient mice.

The molecular chaperone receptor-associated protein (RAP) is required for biosynthesis of megalin, an endocytic receptor for follicular thyroglobulin (Tg), the thyroid hormone precursor. RAP also binds to Tg itself, suggesting that it may affect Tg trafficking in various manners. To elucidate RAP function, we have studied the thyroid phenotype in RAP-knockout (RAP-KO) mice and found a reduction of Tg aggregates into thyroid follicles. Serum Tg levels were significantly increased compared with those of wild-type (WT) mice, suggesting a directional alteration of Tg secretion. In spite of these abnormalities, hormone secretion was maintained as indicated by normal serum thyroxine levels. Because Tg in thyroid extracts from RAP-KO mice contained thyroxine residues as in WT mice, we concluded that in RAP-KO mice, follicular Tg, although reduced, was nevertheless sufficient to provide normal hormone secretion. Serum TSH was increased in RAP-KO mice, and although no thyroid enlargement was observed, some histological features resembling early goiter were present. Megalin was decreased in RAP-KO mice, but this did not affect thyroid function, probably because of the concomitant reduction of follicular Tg. In conclusion, RAP is required for the establishment of Tg reservoirs, but its absence does not affect hormone secretion.

Thyroid dysfunction in megalin deficient mice.

Megalin mediates transcytosis of thyroglobulin (Tg), the thyroid hormone precursor, resulting in its passage into the bloodstream. The process involves especially hormone-poor Tg, which may favour hormone secretion by preventing competition with hormone-rich Tg for proteolytic degradation. To gain more insight into the role of megalin, here we studied thyroid function and histology in megalin deficient mice compared with WT mice. As expected from the knowledge that megalin mediates Tg transcytosis, serum Tg levels were significantly reduced in homozygous (megalin-/-) mice, which, more importantly, were found to be hypothyroid, as demonstrated by significantly reduced serum free thyroxine and significantly increased serum thyroid stimulating hormone (TSH) levels. In heterozygous (megalin+/-) mice, in which megalin expression was normal, thyroid function was unaffected. Although the serological phenotype in megalin-/- mice was not associated with histological alterations or goiter, our results support a major role of megalin in thyroid hormone secretion.

Preferential megalin-mediated transcytosis of low-hormonogenic thyroglobulin: a control mechanism for thyroid hormone release.

Hormone secretion by thyrocytes occurs by fluid phase uptake and lysosomal degradation of the prohormone thyroglobulin (Tg). However, some Tg internalized by megalin bypasses lysosomes and is transcytosed across cells and released into the bloodstream. Because the hormone content of Tg is variable, we investigated whether this affects transcytosis. We found that rat Tg with a low hormone content [low-hormonogenic rat Tg (low-horm-rTg)] is transcytosed by megalin across thyroid FRTL-5 cells to a greater extent than rat Tg with a high hormone content [hormonogenic rat Tg (horm-rTg)]. In immunoprecipitation experiments, the Tg sequence Arg-2489-Lys-2503 (required for binding to megalin and heparan sulfate proteoglycans) was found to be more exposed in low-horm-rTg, which accounted for its preferential transcytosis. Thus, removal of surface heparan sulfate proteoglycans from FRTL-5 cells or blocking of 2489-2503 reduced transcytosis of low-horm-rTg to a greater extent than that of horm-rTg. Preferential transcytosis of low-horm-rTg affected hormone release. Thus, the increase in hormone release from horm-rTg in FRTL-5 cells determined by megalin blocking (due to reduced transcytosis and enhanced Tg degradation) was rescued by low-horm-rTg, suggesting that megalin is required for effective hormone release. This finding was confirmed in a small number of megalin-deficient mice, which had serological features resembling mild hypothyroidism. Reduced hormone formation within Tg in vivo, due to treatment of rats with aminotriazole or of patients with Graves' disease with methimazole, resulted in increased Tg transcytosis via megalin, in confirmation of results with FRTL-5 cells. Our study points to a major role of megalin in thyroid homeostasis with possible implications in thyroid diseases.

Glycosaminoglycans provide a binding site for thyroglobulin in orbital tissues of patients with thyroid-associated ophthalmopathy.

The presence of thyroglobulin (Tg) in orbital tissues of patients with thyroid-associated ophthalmopathy (TAO) supports a role of Tg in TAO pathogenesis. To search for Tg-binding sites in orbital tissues, because Tg is a heparin-binding protein, we investigated its binding to glycosaminoglycans (GAGs) that are abundant in orbital tissues: chondroitin sulfate B (CSB) and C (CSC) and hyaluronic acid (HA). Both in solid phase and solution phase assays purified human Tg bound to GAGs. In solid-phase assays, binding was increased by coincubation with heparin or GAGs in solution, or with an antibody against a Tg heparin-binding sequence (Arg2489-Glu2503), possibly suggesting crosslinking of Tg molecules induced by GAGs or by the presumably bivalent antibody. Orbital tissue extracts from TAO patients that contained Tg were subjected to high-salt treatment, which resulted in separation of Tg from GAGs, as observed by column chromatography. After separation from GAGs, the Tg in orbital tissue extracts acquired the ability to bind to immobilized CSB, and heparin enhanced binding, resembling the findings with purified human Tg. Therefore, we conclude that GAGs provide binding sites for Tg in orbital tissues, which may explain the presence of Tg in orbital tissues of patients with TAO.

Serial ANCA titers: useful tool for prevention of relapses in ANCA-associated vasculitis.

BACKGROUND: The value of measuring serial antineutrophil cytoplasmic autoantibody (ANCA) titers in guiding therapy among patients with ANCA-associated vasculitis is controversial. METHODS: We measured serial titers of proteinase 3 (PR3)- and myeloperoxidase (MPO)-ANCA by antigen-specific enzyme-linked immunosorbent assays (ELISAs) in 48 patients with ANCA-associated vasculitis who were followed up during remission at the Massachusetts General Hospital from 1990 through 2000 (mean follow-up, 46.2 months). We retrospectively assessed disease activity by Birmingham Vasculitis Activity Score (BVAS). RESULTS: We found 21 episodes of fourfold or greater ANCA titer rises in 17 patients who were in complete remission (BVAS=0). Among eight patients who had 10 such titer rises and were not given increased immunosuppression, (group I), all suffered relapses after each episode (mean interval, 5.8 months), whereas among 11 patients, each with one titer rise, who received preemptive increased immunosuppression, (group II), only two relapses occurred, at 3 and 6 months. The difference in the cumulative incidence of relapses in a 1-year period between the two groups was 82% (P=0.0002). Changes in ANCA titers were also used to help guide therapy in the other 31 patients in the study; patients with slight titer rises often received incremental increases in immunosuppression, whereas those with falling titers received incremental decreases. The overall outcome in the entire group was favorable; 46 patients were alive at the end of the study; two died of unrelated diseases. CONCLUSION: Serial measurements of PR3- and MPO-ANCA titers in patients with ANCA-associated vasculitis during remission can help predict relapses, and preemptive increases in immunosuppression following fourfold titer rises reduces the risk of relapses. Moreover, adjustment of immunosuppression based on lesser titer changes appears to result in a favorable outcome.

Binding of heparin to human thyroglobulin (Tg) involves multiple binding sites including a region corresponding to a binding site of rat Tg.

OBJECTIVE: Binding of thyroglobulin (Tg) to heparin allows efficient Tg interaction with its endocytic receptor, megalin. Rat Tg (rTg) binds to heparin using an exposed carboxyl terminal region (RELPSRRLKRPLPVK, Arg2489-Lys2503) rich in positively charged residues which is, however, not entirely conserved in human Tg (hTg) (Arg2489-Glu2503, REPPARALKRSLWVE). Here, we investigated whether and how this difference affects binding of heparin. DESIGN: To compare binding of heparin to rTg and hTg. To investigate the role of the sequence 2489-2503 using a peptide-based approach. METHODS: Binding of biotin-labeled heparin to rTg, hTg and to Tg peptides was measured in solid phase assays. RESULTS: Heparin bound to rTg with moderately high affinity (K(d): 34.2 nmol/l, K(i): 37.6 nmol/l) and to hTg with lower affinity (K(d): 118 nmol/l, K(i): 480 nmol/l) and to a lower extent. Binding was dose-dependent and saturable, and was reduced by several specific competitors (Tg itself, unlabeled heparin, lactoferrin). Heparin bound to synthetic peptides corresponding to the rat (rTgP) and to the human (hTgP) Tg sequence 2489-2503. Heparin bound to rTgP to a greater extent and with greater affinity than to hTgP. An antibody against hTgP reduced binding of heparin to intact hTg by 30%, suggesting that in hTg this region is, in part, involved in heparin binding, but also that other regions account for most of the binding. Starting from the sequence of rTgP, we designed 6 synthetic 'mutant' peptides by replacing one amino acid residue of rTgP with the corresponding residue of the sequence of hTgP. Heparin bound to 5 of 6 mutant peptides to a lower extent and with lower affinity than to rTgP. CONCLUSIONS: In spite of a reduced binding ability of the sequence 2489-2503, hTg binds to heparin, in part, using alternative, as yet unidentified, binding sites. Substitution of both positive and neutral residues within the sequence 2489-2503 reduced heparin-binding, suggesting that not only charge, but also sequence and/or conformation, may account for the heparin-binding ability of this region of Tg.

Transcytosis of retinol-binding protein across renal proximal tubule cells after megalin (gp 330)-mediated endocytosis.

Plasma retinol-binding protein (RBP) combined with vitamin A (retinol) is partially filtered through the glomerulus and then absorbed by proximal tubule cells, leading to recycling of retinol to the circulation. Recently, it was shown that reabsorption of RBP-retinol complexes by proximal tubule cells is mediated by megalin (gp 330), an apical endocytic receptor. It was proposed that RBP is transported by megalin to lysosomes, where it is degraded, thus liberating retinol, which then combines with newly synthesized RBP to be secreted into the bloodstream. This study shows that passage of RBP through immortalized rat renal proximal tubule (IRPT) cells occurs by transcytosis after megalin-mediated endocytosis, which provides an alternative pathway for recycling of retinol. IRPT cells cultured as polarized monolayers with tight junctions were used on permeable filters in the upper chamber of dual-chambered devices, with megalin expression exclusively on the upper surface. After addition of RBP to the upper chamber and incubation at 37 degrees C, intact RBP was found in fluids that were collected from the lower chamber. In contrast, control substances (mannitol, lysozyme, albumin, and glutathione-S-transferase) were not appreciably transported across IRPT cells, indicating that passage of RBP was by transcytosis and not by paracellular leakage. Confocal microscopy analysis of IRPT cells after addition of RBP to the upper chamber revealed RBP-containing granules at the apical membrane, subapically, and also at basolateral membranes. When RBP was added to IRPT cells together with megalin competitors, the amount of transcytosed RBP was markedly reduced. We also found that some RBP was internalized and degraded by IRPT cells, but this process was not appreciably affected by megalin competitors, indicating that RBP endocytosed by megalin was not transported to lysosomes and degraded but rather transcytosed across IRPT cells.