Identification of thyroid hormone residues on serum thyroglobulin: a clue to the source of circulating thyroglobulin in thyroid diseases

Proc. Natl. Acad. Sci. U.S.A.

McCluskey

et al. 2003

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.

Section: Resultssupporting

confidence: 84%

“…with elevated serum Tg (35), and that serum Tg originated from transcytosis has a low hormone content (25,26).…”

Section: Discussionmentioning

confidence: 99%

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

Proc. Natl. Acad. Sci. U.S.A.

McCluskey

et al. 2003

“…Other mechanisms through which Tg can reach the bloodstream, namely, transcytosis or leakage from follicles (10), are unlikely to account for the elevated serum Tg levels. Thus megalin-mediated transcytosis, the main route of Tg transcytosis (14,15,17), could not be increased, because megalin on the apical membrane of thyrocytes was reduced.…”

Section: Discussionmentioning

confidence: 98%

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

Botta

American Journal of Physiology-Cell Physiology

et al. 2006

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.

“…Results presented here indicate that the Tg found in orbital tissues from patients with TAO originated in the thyroid. Thus, Tg was captured by an anti-T 4 antibody, showing that it contains thyroid hormone residues, which can be formed only in the thyroid (24)(25)(26)(27).…”

Section: Discussionmentioning

confidence: 99%

“…This issue is pertinent, because Tg mRNA has allegedly been found in the orbit (9), which raises the possibility that Tg can be locally synthesized and secreted. Because the formation of thyroid hormone residues within Tg molecules occurs exclusively in thyroid follicles (24), the finding of Tg containing thyroid hormone demonstrates a thyroid origin (25). As a positive control for hormone containing Tg, we used a purified preparation of rat Tg obtained from thyroid glands, and as a negative control a Tg preparation obtained from FRTL-5 cells supernatants.…”

Section: Tg In Orbital Tissues Originates In the Thyroidmentioning

confidence: 99%

Identification of Thyroglobulin in Orbital Tissues of Patients with Thyroid-Associated Ophthalmopathy

Marinò

et al. 2001

Thyroid

Thyroid-associated ophthalmopathy (TAO) is thought to have an autoimmune pathogenesis because of its association with autoimmune thyroid disease, in particular with Graves' disease. Nevertheless, the nature of the autoimmune reaction is unclear, and a target orbital autoantigen has not been conclusively identified. A widely discussed hypothesis is that antigens constitutively shared by the thyroid and orbital tissues are targets of an autoimmune reaction. It has been also postulated that a thyroid-soluble antigen, namely thyroglobulin (Tg), is transported to orbital tissues through the lymphatics, where it accumulates and elicits autoimmune damages in susceptible individuals. Here we have investigated whether Tg is present in orbital tissues from patients with TAO. Retrobulbar tissue specimens were obtained from three patients with Graves' disease and TAO who underwent decompressive orbitotomy, and at autopsy from two patients with no thyroid or eye disease. All patients with TAO had been previously treated with radioiodine to control Graves' hyperthyroidism. Western blot analysis with a monoclonal anti-Tg antibody showed the presence of intact Tg, both in soluble and membrane-associated fractions of orbital tissue extracts from the patients with TAO, in amounts estimated to range from approximately 320 to approximately 900 pg/microg of tissue protein. In contrast, Tg was not detected in orbital tissue extracts from patients with no thyroid or eye disease. Tg was also demonstrated in orbital tissue extracts from two of three patients with TAO by enzyme-linked immunosorbent assay (ELISA), in amounts estimated to range from approximately 450 to approximately 1000 pg/microg of protein. In addition, Tg in orbital tissue extracts from patients with TAO was immunoprecipitated by a rabbit anti-Tg antibody, suggesting that it retained its native conformation. An anti-thyroxine (T4) antibody captured in solid-phase Tg from orbital tissue extracts, showing that it contained thyroid hormone residues and had therefore originated in the thyroid. Tg-anti-Tg immune complexes were not found in orbital tissues, suggesting that if an autoimmune reaction to Tg occurs in TAO, it is likely to be cell mediated.