Preferential megalin-mediated transcytosis of low- hormonogenic thyroglobulin: A control mechanism for thyroid hormone release Simonetta Lisi*, Aldo Pinchera*, Robert T. McCluskey†, Thomas E. Willnow‡, Samuel Refetoff§, Claudio Marcocci*, Paolo Vitti*, Francesca Menconi*, Lucia Grasso*, Fabiana Luchetti*, A. Bernard Collins†, and Michele Marino` *¶ *Department of Endocrinology, University of Pisa, Via Paradisa 2, 56124 Pisa, Italy; †Department of Pathology, Massachusetts General Hospital, Harvard Medical School, 55 Fruit Street, Boston, MA 02114; ‡Max Delbrueck Center for Molecular Medicine, Robert Roessle Strasse 10, D-13125 Berlin, Germany; and §Departments of Medicine and Pediatrics, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637 Edited by Marilyn Gist Farquhar, University of California at San Diego School of Medicine, La Jolla, CA, and approved September 25, 2003 (received for review April 17, 2003) Hormone secretion by thyrocytes occurs by fluid phase uptake and colloidal Tg that, however, is not iodinated to the same extent lysosomal degradation of the prohormone thyroglobulin (Tg). and has a variable hormone content (2, 3). However, some Tg internalized by megalin bypasses lysosomes Secretion of hormones by thyrocytes occurs by fluid-phase and is transcytosed across cells and released into the bloodstream. pinocytosis of Tg from the colloid followed by degradation in Because the hormone content of Tg is variable, we investigated lysosomes (2, 4). Newly synthesized Tg is soluble and readily whether this affects transcytosis. We found that rat Tg with a low available for endocytosis and hormone release (5), whereas a hormone content [low-hormonogenic rat Tg (low-horm-rTg)] is proportion of Tg in storage is insoluble and undergoes solubi- transcytosed by megalin across thyroid FRTL-5 cells to a greater lization by extracellular proteases before endocytosis (6). In addition to fluid-phase uptake, Tg is internalized by endocytic extent than rat Tg with a high hormone content [hormonogenic rat receptors, after which Tg is not degraded in lysosomes but is Tg (horm-rTg)]. In immunoprecipitation experiments, the Tg se- either recycled into the colloid or transported across cells by quence Arg-2489-Lys-2503 (required for binding to megalin and transcytosis (4). The multiligand endocytic receptor megalin is heparan sulfate proteoglycans) was found to be more exposed in responsible for transcytosis (4, 7, 8). low-horm-rTg, which accounted for its preferential transcytosis. Megalin was first identified by Kerjaschki and Farquhar (9) Thus, removal of surface heparan sulfate proteoglycans from and later found to be a member of the low-density lipoprotein FRTL-5 cells or blocking of 2489–2503 reduced transcytosis of receptor family, composed of a single-transmembrane domain, a low-horm-rTg to a greater extent than that of horm-rTg. Prefer- large ectodomain, and a short cytoplasmic tail (10–12). Megalin ential transcytosis of low-horm-rTg affected hormone release. mediates tubular uptake of low-molecular-weight proteins in the Thus, the increase in hormone release from horm-rTg in FRTL-5 cells kidney and is also involved in the development of the central Ϸ determined by megalin blocking (due to reduced transcytosis and nervous system, which is severely impaired in 98% of megalin- Ϫ/Ϫ enhanced Tg degradation) was rescued by low-horm-rTg, suggest- deficient (Meg ) mice (13). In the thyroid, megalin is ex- ing that megalin is required for effective hormone release. This pressed in a TSH-dependent manner on the apical surface of finding was confirmed in a small number of megalin-deficient mice, thyrocytes, where it binds and internalizes Tg, after which Tg is transported across cells by transcytosis (7). Tg is then released which had serological features resembling mild hypothyroidism. from basolateral membranes into the bloodstream, being in part Reduced hormone formation within Tg in vivo, due to treatment of complexed with secretory components of megalin ectodomain rats with aminotriazole or of patients with Graves’ disease with (8). Transcytosis also occurs in the kidney for retinol-binding methimazole, resulted in increased Tg transcytosis via megalin, in protein (14), another megalin ligand, but this is not the usual confirmation of results with FRTL-5 cells. Our study points to a function of the receptor; instead, for unknown reasons, the major role of megalin in thyroid homeostasis with possible impli- majority of megalin ligands undergo lysosomal degradation (13). cations in thyroid diseases. If the structure of thyroid follicles is intact, transcytosis is the major route by which Tg reaches the circulation (4). However, he thyroid plays a central role in the maintenance of body the physiological role of this pathway is unclear. An early homeostasis through the action of thyroid hormones [thy- interpretation was that transcytosis reduces hormone release by T diverting hormonogenic Tg from lysosomes (4). We have, how- roxine (T ) and triiodothyronine (T )]. Excessive (hyperthyroid- 4 3 ever, considered that transcytosis may rather serve to dispose Tg ism) or defective (hypothyroidism) function is avoided by a molecules with a low hormone content. In support of this negative-feedback control pathway involving the thyroid- hypothesis, here we show that transcytosis is preferential for stimulating hormone (TSH), which up-regulates hormone low-hormonogenic Tg, which plays an important role in the synthesis and is down-regulated by thyroid hormones them- control of thyroid function. selves (1). The follicle, the thyroid functional unit, is composed of a Methods single layer of epithelial cells (thyrocytes) surrounding a lumen Tg Preparations. Hormonogenic rat Tg (horm-rTg) was purified containing a material named colloid (2). The prohormone from frozen rat thyroid glands as described (7). Low- thyroglobulin (Tg) is the major protein component of colloid, into which it is secreted after synthesis by thyrocytes, which is up-regulated by TSH. At the cell–colloid interface, Tg under- This paper was submitted directly (Track II) to the PNAS office. goes iodination of tyrosyl residues, a process catalyzed by Abbreviations: T4, thyroxine; T3, triiodothyronine; TSH, thyroid-stimulating hormone; Tg, thyroglobulin; MegϪ/Ϫ, megalin-deficient mice; horm-rTg, hormonogenic rat Tg; low- thyroperoxidase, resulting in the formation of thyroid hormones horm-rTg, low-hormonogenic rat Tg; WB, Western blotting; anti-rTgP, antibody to rat Tg (3). If thyroid iodide uptake is effective, Tg hormone content 2489–2503; 1H2, mouse antimegalin antibody; HSPGs, heparan sulfate proteoglycans; depends on dietary iodine intake, which varies based on iodine mIgG, mouse IgG; RIgG, rabbit IgG; GD, Graves’ disease; FT4, free T4; TSH-R, TSH receptor. availability in the environment (3). To face conditions of low ¶To whom correspondence should be addressed. E-mail: [email protected]. iodine availability, large amounts of iodine are stored within © 2003 by The National Academy of Sciences of the USA 14858–14863 ͉ PNAS ͉ December 9, 2003 ͉ vol. 100 ͉ no. 25 www.pnas.org͞cgi͞doi͞10.1073͞pnas.2432267100 Downloaded by guest on September 25, 2021 hormonogenic rat Tg (low-horm-rTg) was obtained from media 500 ␮l of binding buffer. The lower chamber was rinsed with ␮ of FRTL-5 cells (American Type Culture Collection, Rockville, 1,000 l of Tg-free buffer. After6hat37°C, free-T3 was MD), as described (15). Thus, FRTL-5 cells, a differentiated rat measured in media from the upper and lower chamber with a kit thyroid cell line (16), are unable to form hormone residues from Lysophase (Sesto S. Giovanni, Milan). T3 was undetectable within endogenously synthesized Tg (17). By SDS͞PAGE and in media from cells incubated with Tg-free buffer. Western blotting (WB), Tgs resolved as 660- and 330-kDa bands. Ϫ/Ϫ The T4 content of Tg was determined as described (15). Radio- In Vivo Studies. (i) Mice. Sera from three homozygous Meg mice labeled Tgs were prepared with 125I-Na (NEN Life Science) by generated by targeted gene disruption as reported (22) and three using IODO beads (Pierce) (specific activity: 1,500–7,000 cpm͞ control littermates (Megϩ/Ϫ) were collected at the Max Del- ng). Tgs were labeled with biotin by using EZ-Link Sulfo-NHS- brueck Center according to institutional guidelines. Free T4 LC-Biotin (Pierce). (FT4) was measured by equilibrium dialysis immunoassay (Ni- chols Institute, San Juan Capistrano, CA). TSH was measured as Antibodies. Rabbit anti-Tg was from Axell (Westbury, NY). described (23). Tg was measured by ELISA. Mouse anti-Tg (unlabeled or horseradish peroxidase conju- (ii) Rats. Six female Lewis rats weighing 100–120 g (Charles River gated) was from DAKO. A rabbit antibody against the rat Tg Breeding Laboratories) received 0.04% aminotriazole (3-amino- sequence 2489–2503 (anti-rTgP) was described previously (18). 1,2,4-triazole, Sigma) in drinking water and L-thyroxine4 (L-I4) ␮ ͞ Rabbit anti-T4 was from Cappel. Two rabbit (A55 and antimega- (Sigma, 20 g day) by i.p. injection, for 6 days. Six untreated rats lin-GST) and one mouse (1H2) antimegalin antibodies were were used as controls. Animal care and death were in accordance described previously (8). None of these reacted with Tg (not with institutional guidelines. At death, serum samples were shown). A fluorescein isothiocyanate-conjugated mouse anti- collected and thyroid glands harvested and frozen for megalin body against heparan sulfate proteoglycans (HSPGs) was from immunofluorescence studies, which were performed as de- Seikagaku Kogyo (Tokyo). scribed (7). TSH (Amersham Pharmacia), total T3 (Diagnostic Products, Los Angeles), and Tg (by ELISA) were measured in Transcytosis and Uptake Experiments. For transcytosis assays, we all sera. To detect megalin secretory components complexed used confluent FRTL-5 cells cultured on 3-␮m filters in culture with Tg by immunoprecipitation, sera were incubated overnight inserts (Becton Dickinson) placed in 24-well plates, as described at 4°C with protein A beads coupled with rabbit anti-Tg, (7). Paracellular transport of 3H-mannitol was measured as antimegalin-GST, or RIgG and then subjected to WB for Tg.