515 J. Endocrinol. Invest. 34: 515-520, 2011 DOI: 10.3275/7297 ABSTRACT. Thyroid hormone release requires degradation of thyroglobulin (Tg) by thyroid epithelial cells, which occurs mainly in the lysosomal pathway following Tg endocytosis. Non-specific fluid-phase endocytosis is thought to be the main route of Tg uptake leading to degradation, whereas re- ceptor-mediated endocytosis is believed to lead to post-en- docytic pathways other than degradation. To gain more in- sights into these issues, we investigated handling of Tg by various cell types. Tg bound similarly to thyroid (FRTL-5, FRT) and non-thyroid (COS-7, IRPT) cells, indicating the presence of membrane-binding sites, presumably receptors, in both cell types. Tg was internalized and degraded by all cells and degradation paralleled uptake, with the exception of FRTL- 5 cells, in which a lower proportion of Tg was degraded, sug- gesting that in FRTL-5 cells mechanisms that target Tg to the various post-endocytic pathways (either receptors or post- receptorial factors) are differently represented. Immuno- electronmicroscopy showed a common path of endocytosis in FRTL-5, COS-7, and IRPT cells, namely the formation of pseudopods engulfing Tg, followed by internalization and accumulation of Tg in cytoplasmic vesicles and lysosomes. The fastest rate was observed in COS-7 cells, probably re- flecting a lower impact of endocytic receptors. Our findings suggest that Tg uptake and degradation are not thyroid-spe- cific, that Tg binding sites exist in different cell types, and that uptake and/or degradation are differently regulated in differentiated thyroid cells, presumably because of a differ- ent impact of endocytic receptors or post-endocytic mecha- nisms, which are probably responsible for the regulation of hormone release. (J. Endocrinol. Invest. 34: 515-520, 2011) © 2011, Editrice Kurtis INTRODUCTION Synthesis and intrathyroidal metabolism of thyroglobu- lin(Tg)playacrucialroleinthesecretionofthyroidhor- monesbythyroidepithelialcells(TEC).Tgissynthesized by TEC and then secreted into the lumen of thyroid fol- licles, where it undergoes hormone formation by cou- plingofitstyrosylresidueswithiodide(1).Then,Tgisei- ther stored into follicles to form the colloid or it is pro- cessedfurthertoundergodegradationandhormonere- lease(1).ThemainsiteofTgdegradationisintracellular, namely the lysosomal pathway, where Tg is partly di- gestedfollowingitsuptakefromthecolloidbyTEC(2-4). Inaddition,Tgcanbedegradedwithinthefolliclelumen by extracellular proteases (5). In rodents, TEC can internalize Tg by macropinocytosis, butinmostspecies,includinghumans,uptakeofTgoc- cursmainlybymicropinocytosis(1,6).Themajorityofin- vestigatorsagreethatfluid-phasemicropinocytosisisthe main route of uptake leading to hormone release, be- cause of the very high concentrations of Tg within the colloid (1, 6). Thus, fluid-phase uptake is a constitutive processthatoccursbypassivegradientdiffusionofsub- stancesthatarehighlyconcentratedinextracellularfluid. Nevertheless, several studies have shown that also en- docytic receptors can mediate Tg endocytosis (6). Most authors believe that receptors contribute hormone re- leaseonlytoalimitedextentandthattheyaremorelike- ly to sort Tg molecules to post-endocytic pathways that do not lead to lysosomal degradation, namely recycling ortranscytosis,asdemonstratedforsomeofthem(6-14). Consideringthatfluid-phaseuptakecannotberegulated, the regulation of hormone release in TEC would be de- termined by the number and the types of endocytic re- ceptorsonthecellmembraneand/orbythepresenceof post-endocyticmechanismsthatdirectTgtothevarious intracellular pathways. In order to gain more insights in- totheseissues,weinvestigatedTgbinding,uptake,and degradationinvariouscellstypes,includingdifferentiat- edandnon-differentiatedthyroidcells,aswellascellsof non-thyroid origin. Our findings suggest that Tg uptake and degradation are not thyroid-specific, that Tg bind- ing sites exist in different cell types, and that uptake and/ordegradationaredifferentlyregulatedindifferen- tiated thyroid cells, presumably because of a different impactofendocyticreceptorsorpost-endocyticmecha- nisms,whichareprobablyresponsiblefortheregulation of hormone release. MATERIALS AND METHODS Materials HumanTg(hTg)waspurifiedfromGraves’diseasepatientsthy- roids, obtained at surgery upon informed consent. hTg was la- beled with 125 I-Na (Perkin Elmer, Waltham, MA) using IODO beads (Pierce, Rockford, IL) (specific activity: 1,500-7,000 cpm/ng). hTg was labeled with 20-nm gold particles as de- scribed (15). A rabbit anti-Tg antibody was from Axell (West- bury, NY). Key-words: Endocytosis, receptor, thyroglobulin, thyroid, thyroid hormone. Correspondence: M. Marinò, MD, Department of Endocrinology, University of Pisa, Via Paradisa 2, 56124, Pisa, Italy. E-mail: michele.marino@med.unipi.it Accepted May 5, 2010. First published online October 15, 2010. Binding, uptake, and degradation of internalized thyroglobulin in cultured thyroid and non-thyroid cells R. Botta 1 , S. Lisi 1 , A. Pinchera 1 , A.R. Taddei 2 , A.M. Fausto 3 , F. Giorgi 4 , and M. Marinò 1 1 Department of Endocrinology and Metabolism, University of Pisa, Pisa; 2 Interdepartimental Centre for Electron Microscopy and 3 Department of Environmental Science, University of the Tuscia, Viterbo; 4 Department of Neuroscience, University of Pisa, Pisa, Italy © 2011, Editrice Kurtis FOR PERSONAL USE ONLY