Follicular Thyroglobulin (TG) Suppression of Thyroid-restricted Genes Involves the Apical Membrane Asialoglycoprotein Receptor and TG Phosphorylation

Abstract

Follicular thyroglobulin (TG) decreases expression of the thyroid-restricted transcription factors, thyroid transcription factor (TTF)-1, TTF-2, and Pax-8, thereby suppressing expression of the sodium iodide symporter, thyroid peroxidase, TG, and thyrotropin receptor genes (Suzuki, K., Lavaroni, S., Mori, A., Ohta, M., Saito, J., Pietrarelli, M., Singer, D. S., Kimura, S., Katoh, R., Kawaoi, A., and Kohn, L. D. (1997) Proc. Natl. Acad. Sci. U. S. A. 95, 8251-8256). The ability of highly purified 27, 19, or 12 S follicular TG to suppress thyroid-restricted gene expression correlates with their ability to bind to FRTL-5 thyrocytes and is inhibited by a specific antibody to the thyroid apical membrane asialoglycoprotein receptor (ASGPR), which is related to the ASGPR of liver cells. Phosphorylating serine/threonine residues of TG, by autophosphorylation or protein kinase A, eliminates TG suppression and enhances transcript levels of the thyroid-restricted genes 2-fold in the absence of a change in TG binding to the ASGPR. Follicular TG suppression of thyroid-restricted genes is thus mediated by the ASPGR on the thyrocyte apical membrane and regulated by a signal system wherein phosphorylation of serine/threonine residues on the bound ligand is an important component. These data provide a hitherto unsuspected role for the ASGPR in transcriptional signaling, aside from its role in endocytosis. They establish a functional role for phosphorylated serine/threonine residues on the TG molecule.Thyrotropin (TSH), 1 in concert with insulin and insulin-like growth factor-1 (IGF-1), regulates thyroid function (1-3). TSH increases expression of the sodium iodide symporter (NIS), thyroglobulin (TG), and thyroid peroxidase (TPO) genes; this increases concentrative iodide uptake, TG synthesis, and thyroid hormone formation (1-4). NIS, TG, and TPO expression are controlled by thyroid-restricted transcription factors: thyroid transcription factor (TTF)-1, TTF-2, and Pax-8 (5-12). TTF-2 is regulated by insulin/IGF-1 (9, 10), TTF-1 and Pax-8 by TSH/cAMP (13-16).We have recently shown that TG accumulated in the follicular lumen acts as a feedback suppressor of hormonally-increased thyroid function (17-19). Thus, follicular TG selectively suppresses expression of TTF-1, TTF-2, and Pax-8 (17, 18), thereby altering expression of the TG, TPO, NIS, and TSHR genes, and counter regulating TSH-and insulin/IGF-1-induced changes in these genes (17-19). The follicular TG acts transcriptionally; its suppressive effect is not duplicated by thyroid hormones or iodide (17-19). The mechanism by which follicular TG can act as a transcriptional suppressor is unknown.TG is synthesized as a 12 S molecule (330 kDa), but forms a 19 S dimer and 27 S tetramer; all three exist in the follicular lumen (20, 21). It has been suggested that newly synthesized TG attaches to a specific binding protein related to the lectinlike asialoglycoprotein receptor (ASPGR) of the liver (22-26) 2 and that the thyroid ASPGR vectorially transports newly synthesized TG to the fol...

Identification of thyroglobulin domain(s) involved in cell-surface binding and endocytosis

“…Also these binding properties were not restricted to thyroid tissue. The main role of Tg binding and endocytosis is lysosomal degradation and hormone release, although other functions, involving 'receptors', have been described (Ulianich et al 1999, Mezghrani et al 2000. Tg is the main component of the follicular lumen and is present at very high concentrations.…”

“…Thyroglobulin interactions with thyroid membranes have been observed for several years (Consiglio et al 1981). Several 'receptors' liable to mediate Tg binding to cell surfaces have been recently described: a receptor able to bind asialoagalacto Tg at acidic pH levels (Miquelis et al 1987); the protein disulfide isomerase (PDI) (Mezghrani et al 2000); histone H1 (Brix et al 1998); heparin (Marino et al 1999a); megalin (Zheng et al 1998, Marino et al 1999b; and rat hepatic lectin subunit of a rat asialoglycoprotein receptor (ASGPR) , Ulianich et al 1999. Some functions have been associated with various 'receptors': megalin was shown to be involved in transcytosis ; PDI was associated with Tg recycling (Miquelis et al 1987, Mezghrani et al 2000; histone H1 was involved in circulating Tg uptake by macrophages (Brix et al 1998); and a role was proposed for ASGPR in feedback regulation, ASGPR preventing thyroid-restricted gene expression under follicular Tg binding , Ulianich et al 1999.…”

Regulation of Dual Oxidase Expression and H2O2Production by Thyroglobulin

“…Thus, the possible presence of bioactive sites within the Tg molecule (660 kDa), cellular recognition systems for Tg (or its degradation fragments), and the signaling cascades that exert such unique and potent effects remain to be defined. However, the effects were shown to be specific to the Tg molecule and are not mimicked by other factors (30,(34)(35)(36). Although Tg prepared from iodide-deficient goiter and congenital goiter suppressed thyroid-specific gene expression more effectively than did Tg prepared from normal tissue (27,30), the effects of immature Tg under physiological conditions are not clear, especially because Tg moieties bind to several proteins in order to undergo vectorial transport to the follicular lumen (37)(38)(39)(40).…”

“…The cells were grown either in Coon's modified Ham's F-12 medium supplemented with 5% bovine serum (Invitrogen) and a six-hormone mixture (1 mU TSH/mL, 10 lg insulin/mL, 10 ng somatostatin/mL, 0.36 ng hydrocortisone/mL, 5 lg transferrin/mL, and 2 ng glycyl-L-histidyl-L-lysine acetate/mL) or with 0.2% bovine serum without TSH and insulin mixture as previously described (44,45). Bovine Tg (Sigma) was used at a final concentration of 1-30 mg/mL, which was previously confirmed as having essentially the same effect as highly purified 19S Tg (30,34,35).…”

Differential regulation of apical and basal iodide transporters in the thyroid by thyroglobulin

“…Bovine Tg, 8-bromo cAMP and forskolin were obtained from the Sigma Chemical Co. Bovine follicular 19S Tg prepared by salt extraction and agarose chromatography were used in some experiments and showed essentially the same results (Ulianich et al 1999). All experiments were repeated at least three times using different batches of cells.…”