In vitro studies of the thyroglobulin degradation pathway: endocytosis and delivery of thyroglobulin to lysosomes, release of thyroglobulin cleavage products--iodotyrosines and iodothyronines.
Iodinated thyroglobulin stored in the thyroid follicular lumen is subjected to an internalization process and thought to be transferred into the lysosomal compartment for proteolytic cleavage and thyroid hormone release. In the present study, we have designed in vitro models to study: 1) the transfer of endocytosed thyroglobulin into lysosomes, and 2) the intracellular fate of free thyroid hormones and iodinated precursors generated by intralysosomal proteolysis of thyroglobulin. Open follicles prepared from pig thyroid tissue by collagenase treatment were used to probe the delivery of exogenous thyroglobulin to lysosomes via the differentiated apical cell membrane. Open follicles were incubated with pure [125I]thyroglobulin with or without unlabeled thyroglobulin in the presence or in the absence of chloroquine. Subcellular fractionation on a Percoll gradient showed that [125I]thyroglobulin was internalized and present in low (for the major part) and high density thyroid vesicles. In chloroquine-treated open follicles, we observed the appearance of a definite fraction of [125I]thyroglobulin in a lysosome subpopulation having the expected properties of phagolysosomes or secondary lysosomes. In contrast, in control open follicles, the amount of [125I]thyroglobulin or degradation products found in high density vesicles was lower and associated with the bulk of lysosomes, i.e., primary lysosomes. The content in thyroglobulin and degradation products of lysosomes at steady-state was analyzed by Western blot using polyclonal anti-pig thyroglobulin antibodies. Under reducing conditions, immunoreactive thyroglobulin species correspond to polypeptides with molecular weights ranging from 130,000 to less than 20,000. The presence of free thyroid hormones and iodotyrosines inside lysosomes and their intracellular fate was studied in dispersed thyroid cells labeled with [125I]iodide. Neo-iodinated [125I]thyroglobulin gave rise to free [125I]T4 which was secreted into the medium. In addition to released [125I]T4, a fraction of free [125I]T4 was identified inside the cells. Lysosomes isolated from dispersed thyroid cells did not contain significant amounts of free [125I]T4. The free intracellular [125I]T4 fraction seems to represent an intermediate 'hormonal pool' between thyroglobulin-bound T4 and secreted T4. Evidence for such a precursor-product relationship was obtained from pulse-chase experiments.


CONCLUSIONS
1) open thyroid follicles have the ability to internalize thyroglobulin by a mechanism of limited capacity and to address the endocytosed ligand to lysosomes.(ABSTRACT TRUNCATED AT 400 WORDS)
Rousset
B
B
Institut National de la Santé et de la Recherche Médicale, U197, Faculté de Médecine Alexis-Carrel, Lyon, France.
Selmi
S
S
Alquier
C
C
Bourgeat
P
P
Orelle
B
B
Audebet
C
C
Rabilloud
R
R
Bernier-Valentin
F
F
Munari-Silem
Y
Y
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D008247:Lysosomes / Q000276:immunology / Q000378:metabolism*; D008857:Microscopy, Interference; D007470:Monoiodotyrosine / Q000378:metabolism*; D011863:Radioimmunoassay; D013552:Swine; D013954:Thyroglobulin / Q000276:immunology / Q000378:metabolism*; D013961:Thyroid Gland / Q000166:cytology; D013963:Thyroid Hormones / Q000096:biosynthesis; D014284:Triiodothyronine / Q000378:metabolism*
D013963:Thyroid Hormones; D014284:Triiodothyronine; D013954:Thyroglobulin; D007470:Monoiodotyrosine