Connexin 32 fused to the green fluorescent protein retains its ability to control the proliferation of thyroid cells.

Cell-to-cell exchanges of signaling molecules are thought to be involved in the control of cell proliferation. Connexins, which are encoded by a family of genes expressed in a cell type-specific manner, are considered as tumor suppressors. Thyroid epithelial cells co-express connexin 32 (Cx32) and connexin 43 (Cx43) that form distinct and delocalized gap junctions in vivo. The communication-deficient rat thyroid-derived cell lines, FRTL-5 and FRT, stably transfected with the Cx32 cDNA, have a reduced proliferation rate related to a prolonged G1 cell cycle phase. To determine whether Cx32-gap junctions exert the same regulatory role in vivo, we have undertaken a program of production of transgenic mice over-expressing Cx32 specifically in thyrocytes. To this purpose, we designed a vector in which the Cx32 cDNA was fused to the gene encoding the enhanced green fluorescent protein (EGFP) and placed under the control of a strong and thyroid-specific promoter, the thyroglobulin gene promoter (pTg). In stably transfected FRTL-5 cells, the Cx32/EGFP chimeric protein forms functional gap junction channels and induces the same proliferation slowdown as native Cx32. The pTg-Cx32/EGFP construct should thus allow us to obtain the thyroid-targeted over-expression of Cx32 in the mouse to investigate the involvement of Cx32-gap junctions in thyroid growth, functional activity and propensity to form tumors.

Formation of three-dimensional thyroid follicle-like structures by polarized FRT cells made communication competent by transfection and stable expression of the connexin-32 gene.

Pig thyrocytes, either in the intact gland or cultured under conditions leading to thyroid follicle reconstitution, coexpress two gap junction proteins, connexin-32 (Cx32) and connexin-43 (Cx43). As thyrocytes cultured in the form of a monolayer only express Cx43, we hypothesized that Cx32 could play a role in thyroid folliculogenesis. In the present work, we analyzed the ability of polarized FRT cells (that are gap junction deficient) to form follicle-like structures after stable transfection with either Cx32 or Cx43 genes. Wild-type and transfected FRT cells, while growing, showed the capacity to form three-dimensional structures corresponding to domes that result from the accumulation of fluid underneath limited areas of the cell layer. The number of domes formed by FRT cells expressing Cx32 (FRT-Cx32) was 2- to 3-fold higher than that obtained with either wild-type or Cx43-transfected FRT cells (FRT-Cx43). Domes generated by FRT-Cx32 cells were stable (beyond 3 weeks of culture), whereas those formed from wild-type or FRT-Cx43 cells were transient, disappearing when cells reached confluence. Inspection of the cell organization within domes formed from FRT-Cx32 cells by phase contrast and confocal microscopy revealed a progressive transition from domes toward closed structures with a lumen. The tightness of the lumen was demonstrated by the retention of a fluorescent probe, lucifer yellow, introduced by microinjection. Electron microscope examinations showed that the neoformed follicle-like structures had an inside-out polarity. Analyses of cell motion and division with time, by fluorescence video microscopy, indicated that the transformation of domes into inside-out follicles brings into play the migration of cells and, to a lesser extent, cell multiplication underneath the domes. In conclusion, FRT cells forced to express Cx32 give rise to domes that transform into closed inside-out follicles. This gain of function appears Cx specific, as FRT-Cx43 cells did not form similar structures. Our data suggest that the formation and/or functioning of Cx32 gap junctions might represent a key event in thyroid epithelium morphogenesis, i.e. formation of a lumen from a tight epithelial cell layer.

Rho and Rac exert antagonistic functions on spreading of macrophage-derived multinucleated cells and are not required for actin fiber formation.

Multinucleated giant cells (MNGC) derived from avian blood monocytes present, like osteoclasts, an unusual cytoskeletal organization characterized by (1) cortical rings of actin filaments, (2) unique adhesion structures called podosomes and (3) vinculin containing focal complexes which are not visibly connected to F-actin structures. The Rho family of small GTPases plays an essential role in the regulation and organization of cellular cytoskeletal structures including F-actin and vinculin associated structures. Using bacterial toxins such as modified exoenzyme C3 (C3B) and toxin B or overexpression of constitutively active Rac and Rho proteins fused to the green fluorescent protein (GFP), we show that Rac and Rho play antagonistic roles in regulating the morphology of osteoclast-like cells. Inhibition of Rho by C3B triggered MNGC spreading whereas activated Rho promoted cell retraction. However, inhibition or activation of Rho led to complete disorganization of fibrillar actin structures, including podosomes. Toxin B inhibition of Rho, Rac and Cdc42 induced a time dependent F-actin and vinculin reorganization. Initially, actin fibers with associated adhesion plaques formed and disappeared subsequently. Finally, only small focal complexes remained at the MNGC periphery before retracting. At the time when actin fibers formed, we observed that Rac was already inhibited by toxin B. By combining C3B treatment and overexpression of a dominant negative form of Rac (N17Rac), we show that the formation of these focal adhesion and actin fiber structures required neither Rho nor Rac activity. Moreover, our results show that podosomes are extremely unstable structures since any modifications of Rho or Rac activity resulted in their dissociation.

Restoration of cell-to-cell communication in thyroid cell lines by transfection with and stable expression of the connexin-32 gene. Impact on cell proliferation and tissue-specific gene expression.

Normal thyroid epithelial cells coexpress connexin-32 and connexin-43, which form distinct gap junctions. In primary culture, connexin-43 is expressed by thyrocytes in monolayers or reorganized into follicles, whereas the expression of connexin-32 is dependent upon the reconstitution of follicles. To study the functional impact of connexin-32 gap junctions in thyroid cells, we transfected connexin-32 cDNA in two thyroid-derived communication-deficient cell lines, FRT and FRTL-5. The selected clones, which stably expressed connexin-32 at high levels and exhibited high gap junction-mediated dye-coupling, presented a reduced proliferation rate as compared with that of the corresponding wild-type FRT and FRTL-5 cells; the mean population doubling time was increased by approximately 35%. The proliferation of connexin-32-transfected FRTL-5 cells remained thyrotropin-dependent; the range of thyrotropin concentrations that stimulated growth was the same in transfected and control cells. The expression of connexin-32 led to an increase of thyroglobulin gene expression in FRTL-5 cells. The expression of two other tissue-specific proteins, thyroid transcription factor-1 and Pax-8, was unchanged. These findings provide evidence that connexin-32 gap junction-mediated cell-to-cell communication participates in the control of growth and differentiation of thyroid cells.

Cell-to-cell communication in the anterior pituitary: evidence for gap junction-mediated exchanges between endocrine cells and folliculostellate cells.

The ability of rat anterior pituitary cells to communicate through gap junctions (GJ) was studied using a fluorescent molecule, Lucifer Yellow (LY), which freely passes through GJ channels. The probe was introduced into the cell cytoplasm by using either the cut-end loading method on intact tissue, or cell microinjection on cultured cells. The identification of communicating cells was performed by immunofluorescence labeling of specific hormones in endocrine cells and of S100 protein in folliculostellate (FS) cells. Rat anterior pituitary cells in their physiological organization, i.e. in the intact tissue, exhibited a high level of coupling through GJ. LY-labeled cells were found up to 300-microns apart from its site of introduction. The communicating cells were primarily PRL cells, GH cells, and FS cells. Only a few LH, TSH, and ACTH cells were labeled with LY. Anterior pituitary cells, isolated from the rat tissue by mild protease treatment and cultured for 3 days, reestablished functional GJ as demonstrated by microinjection of LY into individual cells. By immunolabeling of specific hormones and/or S100 protein, we found a GJ coupling between FS cells, and between FS cells and endocrine cells, including PRL cells. The communication between FS cells was by far the most frequent. In conclusion, we demonstrate the presence of functional GJ between anterior pituitary cells of the same type and between anterior pituitary cells having distinct differentiated functions.

Endocytosis of albumin and thyroglobulin at the basolateral membrane of thyrocytes organized in follicles.

Serum proteins such as albumin are present inside thyroid follicles in both normal and pathological situations. To analyze the mechanism of entry of these proteins, we investigated the ability of polarized thyrocytes to internalize soluble molecules at their basolateral pole. Experiments were conducted on in vitro reconstituted thyroid follicles using BSA and pig thyroglobulin (Tg) coupled to gold particles for electron microscopy, conjugated to fluorescein for conventional and confocal fluorescence microscopy, or radioiodinated for biochemical measurements. Incubations were carried out at 37 C. BSA and Tg coupled to gold particles were rapidly internalized from the culture medium and sequentially found in small vesicles and early endosomes and in late endosomes and lysosomes. Fluorescence microscope analyses revealed that the majority of cells forming reconstituted thyroid follicles are capable of internalizing BSA and Tg, but that Tg was more efficiently endocytosed than BSA. Using radioiodinated ligands, it was observed that the endocytosis of Tg was 10 times higher than that of BSA. The internalization of [125I]Tg was inhibited by increasing concentrations of unlabeled Tg. In contrast, endocytosis of 125I-labeled BSA was independent of the unlabeled BSA concentration. Experiments performed at 4 C indicated the presence of a basolateral membrane binding activity for [125I]Tg; the Tg concentration that reduced the binding of labeled Tg by 50% ranged from 4-6 microM. These data are evidence of a process of internalization of soluble molecules at the basolateral pole of thyrocytes, with BSA being internalized by fluid phase endocytosis and Tg by selective endocytosis. Our findings explain how serum albumin can enter thyroid follicles and disclose a new cellular handling and transport pathway of Tg. We propose that selective uptake of Tg operating on molecules secreted at the basolateral surface of thyrocytes could control the amount of Tg released in the circulation.

[Gap junctions in the thyroid gland: distribution, regulation, function]. / Les jonctions gap dans la glande thyroïde: distribution, régulation, fonction.

As in most organized tissues, cells of the thyroid gland, thyrocytes, are connected by various types of intercellular junctions, among which gap junctions. Gap junctions are composed of channels that allow the direct cell-to-cell exchange of small cytoplasmic molecules (Mr < 1000). Proteins forming gap junction channels are the connexins (Cx). Thyrocytes coexpress two Cx: Cx32 and Cx43 that form distinct channels localized in different regions of the lateral plasma membrane domain; gap junctions formed of Cx43 are localized in tight junctions. The tissue-specific hormone, TSH, increases the synthesis of these two Cx and induces the opening of gap junction channels. Gap junction-mediated cell-to-cell communication is likely involved in the control of thyroid cell proliferation since the reinduction of cell-to-cell coupling in communication-deficient thyroid cell lines, by stable transfection of the Cx32 cDNA, induces a decrease of the proliferation rate of these cells.

Cell adhesion receptors and gap junctions in normal and neoplastic transformed thyrocytes.

In the thyroid cell adhesion receptors and gap junctions establish a complex modular network defining the structural properties of cells, their interactions with neighboring cells and the extracellular matrix. Neoplastic alterations of this network can lead to an imbalance of cell to cell communication and allows transformed cells to escape from the tissue to generate metastases. The present manuscript summarizes general and thyroid-specific aspects of the molecular basis of cell-cell contacts, an expanding field of tumor biology.

Gap junctions and cell polarity: connexin32 and connexin43 expressed in polarized thyroid epithelial cells assemble into separate gap junctions, which are located in distinct regions of the lateral plasma membrane domain.

Epithelial cells of the thyroid gland present an uncommon connexin expression pattern, they coexpress connexin32 and connexin43. In the present work, we have analyzed the membrane distribution of these two connexins to determine: (i) whether they co-assemble in the same gap junctions or form separate gap junctions; and (ii) whether their location is somehow related to the thyroid cell polarity. Immunofluorescence analyses of the localization of the two connexins in thyroid tissue sections revealed that connexin32 and connexin43 are located in different regions of the plasma membrane. We further analyzed the location of each of the two connexins with regard to that of the tight junction-associated protein, ZO1. Laser scanning confocal microscope observations of connexin32 or connexin43 and ZO1 double-immunolabelled thyroid cells, gave evidence for a separate localization of gap junctions made of each of these two connexins. Connexin32 gap junctions appeared as fluorescent spots scattered over the lateral membrane domain, while connexin43 gap junctions formed a meshed network superimposable with that of tight junctions in the subapical region of the cells. Western blot analyses of the distribution of connexins in thyroid plasma membrane subfractions obtained by ultracentrifugation on a sucrose gradient led to the identification of membrane sub-populations enriched in either connexin32 gap junctions or connexin43 gap junctions. Connexin32 gap junctions and connexin43 gap junctions were found to differ in their resistance to solubilization by N-lauroylsarcosine. Increasing concentrations of this detergent from 0.12% to 0.42% caused a progressive solubilization of connexin43 while connexin32 remained membrane-bound.(ABSTRACT TRUNCATED AT 250 WORDS)

Gap junction-mediated cell-to-cell communication in bovine and human adrenal cells. A process whereby cells increase their responsiveness to physiological corticotropin concentrations.

We have studied the role of gap junction-mediated intercellular communication on the steroidogenic response of bovine (BAC) and human (HAC) adrenal fasciculo-reticularis cells in culture to corticotropin (ACTH). Indirect immunofluorescence analyses showed that intact human and bovine adreno-cortical tissue as well as HAC and BAC in culture expressed the gap junction protein connexin43 (also termed alpha 1 connexin). Both HAC and BAC were functionally coupled through gap junctions as demonstrated by microinjection of a low molecular mass fluorescent probe, Lucifer yellow. The cell-to-cell transfer of the probe was blocked by 18 alpha-glycyrrhetinic acid (GA), an inhibitor of gap junction-mediated intercellular communication. GA markedly decreased the steroidogenic response (cortisol production) of both HAC and BAC to low (10 pM) but not to high (5 nM) concentrations of ACTH. GA had no inhibitory effect on the steroidogenic response to 8 Br-cAMP (at either low or high concentrations) and did neither modify the binding of 125I-ACTH to its receptor nor the ACTH-induced cAMP production. BAC cultured at high or low cell densities (2.4 x 10(5) vs. 0.24 x 10(5) cells/cm2) exhibited distinct levels of intercellular communication and were differently responsive to sub-maximal ACTH concentrations. The ACTH ED50 values for cortisol production were 8.5 +/- 1.3 and 45 +/- 14 pM (P < 0.02) for BAC cultured at high and low density, respectively. In the presence of GA, there was a shift of the ACTH concentration-response curves in the two culture conditions. The ACTH ED50 of high density and low density cultured BAC increased 25- and 5-fold, respectively, and became similar (220 +/- 90 and 250 +/- 120 pM). These results demonstrate that gap junction-mediated communication between hormone-responsive and nonresponsive cells is one mechanism by which adrenal cells increase their responsiveness to low ACTH concentrations.

Differential control of connexin-32 and connexin-43 expression in thyroid epithelial cells: evidence for a direct relationship between connexin-32 expression and histiotypic morphogenesis.

Thyroid epithelial cells cultured either as a monolayer or in the form of follicles, rapidly reconstitute functional gap junctions (Gj). We previously reported that the thyroid Gj gating is regulated by TSH. We have now performed molecular analyses of Gj proteins 1) to detect the connexin(s) (Cx) that is expressed in thyroid epithelial cells, 2) to determine whether the expression of Cx is hormonally regulated, and 3) to analyze the relationship between Cx expression and histiotypic morphogenesis, i.e. folliculogenesis. Studies were carried out on thyrocytes freshly isolated from the gland and on corresponding thyrocytes after 1-7 days in culture as monolayers or in the form of reconstituted follicles. The Cx gene transcription products were analyzed by Northern blot using specific complementary DNA probes for Cx26, Cx32, and Cx43. Cx proteins were identified and estimated by Western blot and indirect immunofluorescence using polyclonal antipeptide antibodies. Cx32 and Cx43 proteins and their corresponding messenger RNA (mRNA) were detected in thyrocytes freshly isolated from the gland. Thyrocytes contained a high amount of the 1.6-kilobase Cx32 mRNA and only traces of the 3-kilobase Cx43 transcript. No Cx26 transcripts could be detected. Thyrocytes cultured at a density of 0.2-0.5 x 10(6) cells/cm2 in the absence of TSH formed monolayers. Surprisingly, monolayer cells lost Cx32 protein within 24 h, and their Cx32 mRNA content decreased from high to barely detectable levels; Cx32 protein was no longer detected throughout the 1-week culture period. On the contrary, Cx43 mRNA and Cx43 protein rapidly increased in monolayer cells to reach very high levels within 2-4 days. Thyrocytes cultured at the same density, but in the presence of TSH also rapidly lost Cx32, but as soon as they reorganized into follicular structures, reexpressed Cx32 at a level (in terms of protein and mRNA) comparable to that found in cells freshly extracted from the gland. As observed for cell monolayers, reconstituted follicles overexpressed Cx43. The Cx43 protein and Cx43 mRNA contents of cultured thyrocytes were 20- to 50-fold higher than those found in isolated thyrocytes at the outset of culture. When thyrocytes were cultured with TSH, but at a low density (< 0.2 x 10(6) cells/cm2) to prevent follicle formation, a TSH-dependent increase in Cx43 was observed in monolayer cells. However, TSH did not cause any reexpression of Cx32.(ABSTRACT TRUNCATED AT 400 WORDS)

Thyroglobulin molecules internalized by thyrocytes are sorted in early endosomes and partially recycled back to the follicular lumen.

Thyroglobulin (Tg) molecules stored in thyroid follicle lumens are heterogeneous in terms of iodine and hormone contents. It has been suggested that thyroid hormone is preferentially produced from the most highly iodinated Tg molecules and that thyrocytes are capable of selecting these molecules. The cellular localization as well as the molecular basis of such a selection process are not known. The present work was undertaken to determine whether there is selectivity at the step of endocytosis and, if not, to discover other possible mechanisms. Studies were conducted on reconstituted thyroid follicles (RTF) in culture. We compared the ability of thyrocytes to internalize Tg and an exogenous protein, BSA, which is neither iodinated nor glycosylated. To identify the protein, Tg and BSA were coupled to gold particles of different size and microinjected in a fixed ratio into the lumen of RTF. Neither of the two protein gold probes detected by transmission electron microscope bound at the cell surface, and both entered the cells at a similar rate and were concentrated in early endosomes. After 20 min, both Tg-G and BSA-G were segregated into distinct vacuolar structures. At 60 min, the intracellular content of BSA-G (mainly in prelysosomes and lysosomes) was 2- to 3-fold higher than that of Tg-G. At the same time, there was a marked reduction in the BSA-G/Tg-G ratio in the lumen. The differences between the Tg-G and BSA-G distribution patterns that were amplified in TSH-treated RTF are in keeping with a back-transfer of internalized Tg toward the lumen. The existence of a cell to lumen transport of previously endocytosed Tg was further documented using intralumenal [125I]Tg as a marker. RTF pulse labeled with tracer amounts of [125I]iodide were shortly incubated with TSH to induce [125I]Tg endocytosis, and the fate of internalized [125I] Tg was studied in a chase incubation period of up to 4 h. At 20 C, where the degradation of internalized Tg is blocked, we observed a time-dependent decrease in intracellular [125I]Tg and a corresponding increase in the lumenal [125I]Tg content. This cell to lumen [125I]Tg transfer was inhibited by primaquine. In conclusion, our data show that 1) the thyroid apical endocytic process does not exhibit selectivity for Tg; 2) the thyrocyte possesses a sorting machinery for endocytosed ligands; and 3) internalized Tg molecules can be recycled back to the follicular lumen.(ABSTRACT TRUNCATED AT 400 WORDS)

Dynamic analysis of drug action on in vitro reconstituted thyroid follicle by microinjection of tracer molecules and videomicroscopy.

Thyroid cells isolated from the gland by trypsinization are capable in culture of reconstituting histiotypic structures, the thyroid follicles. This morphological differentiation requires the presence of the main thyroid regulator; thyrotropin. We have analyzed some structural and functional aspects of in vitro reconstituted thyroid follicles (RTF) using microinjection of fluorescent probes and videomicroscopy. This experimental approach allowed to visualize biological processes and actions of drugs, signalling factors, etc. in living cells. We describe here some examples of what can be studied with this powerful still-undervalued method. Microinjection of a cell-impermeant fluorescent probe of either high or low molecular mass into the lumen of RTF allowed to check the tightness of this compartment and therefore to analyze the control of tight junctions assembly. A small cell-impermeant probe like Lucifer Yellow microinjected into a cell was used to demonstrate and then to study the regulation of cell to cell communication via gap junctions. The presence of calcium in the lumen of RTF was detected by microinjection of a properly designed probe: Calcium Green which becomes fluorescent in the presence of the ligand. The lumen to cell transport or endocytosis of thyroglobulin, the thyroid prohormone, which is stored into the lumen of the follicles, is currently studied by microinjection of TRITC-labeled thyroglobulin. Coupled to image processing and videorecorder systems, kinetic analysis and quantitative measurements can be performed.

Thyroglobulin internalized by thyrocytes passes through early and late endosomes.

We have tried to characterize the intracellular compartments involved in the traffic of the thyroid prohormone thyroglobulin (Tg) from the site of storage, the follicular lumen, to the expected site(s) of proteolytic degradation, lysosomes. Electron microscope immunogold labeling with antibodies against Tg, cation-independent mannose-6-phosphate receptor (MPR), or arylsulfatase-A (ArS-A) was used to identify endocytic structures. The implication of these structures in the transport of Tg was analyzed by following the internalization and intracellular fate of Tg-colloidal gold complexes microinjected into the thyroid follicular lumen. Immunogold labeling was performed on ultrathin cryosections of intact pig tissue, in vitro reconstituted thyroid follicles (RTF), and isolated vesicles prepared by differential and isopycnic centrifugation. Microinjection experiments were carried out on RTF. Using double labeling for MPR and ArS-A, we characterized three types of structures: those slightly positive for MPR and ArS-A, those strongly positive for both markers, and those only positive for ArS-A. These compartments exhibited the properties of early endosomes (EE), late endosomes (LE), and lysosomes (L), respectively. Tg immunoreactivity was high in EE, low in LE, and undetectable in L. Similar morphological and immunochemical characteristics of EE, LE, and L were found in intact tissue, RTF, and isolated vesicles. Tg-gold complexes microinjected into the lumen of RTF were efficiently internalized within 5 min into structures with the appearance of EE. Sixty minutes after the injection, Tg-gold complexes were detected into LE and L. We present here the first direct experimental evidence for an involvement of endosomal compartments in the Tg internalization/degradation pathway. The data indicate that internalized Tg molecules are transported to EE and then transferred from EE to LE.

Hormonal control of cell to cell communication: regulation by thyrotropin of the gap junction-mediated dye transfer between thyroid cells.

By microinjection of Lucifer yellow (LY) and analysis of the cell to cell transfer of the fluorescent probe, we have examined 1) the ability of thyroid cells in primary culture to reconstitute gap junctions and 2) the effects of extracellular signals on the functional activity of these junctions. Isolated thyrocytes cultured in tissue culture-treated petri dishes either formed monolayers or reorganized in follicular structures in the presence of the glycoprotein hormone TSH. In both culture conditions, LY-coupled cells were evident after 24-36 h. The communication between cells forming a reconstituted thyroid follicle was maintained for up to 9 days. In contrast, the dye coupling between cells in monolayer progressively decreased with time. The cell to cell communication, i.e., the number of dye-coupled cells in thyroid cell monolayer, was increased by TSH in a time- and concentration-dependent manner. The TSH action was not related to de novo protein synthesis. (Bu)2cAMP exhibited stimulatory effects similar, in terms of time course and amplitude of action, to those of TSH. The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate rapidly inhibited both basal and TSH- or (Bu)2 cAMP-activated cell to cell communication. The dye coupling of cells in reconstituted follicles was also blocked by a short 12-O-tetradecanoyl phorbol 13-acetate treatment in both the presence and absence of TSH. Our data show that thyroid cells in culture, regardless of the full expression of the differentiated phenotype, rapidly reestablish intercellular gap junctions. The functional activity of gap junctions appears to be regulated 1) positively by a hormone, TSH, probably acting via the cAMP and protein kinase-A pathway, and 2) negatively by phorbol esters through the activation of protein kinase-C, the two regulatory pathways being interdependent.

Cell-cell interactions in the process of differentiation of thyroid epithelial cells into follicles: a study by microinjection and fluorescence microscopy on in vitro reconstituted thyroid follicles.

Thyroid cells, cultured in the presence of thyroid stimulating hormone, reorganized within 36-48 hr into follicular structures, the in vitro reconstituted thyroid follicles or RTF. By microinjection of fluorescent probes either into the neoformed intrafollicular lumen (IL) or into cells forming the follicles, we have studied the development and some functional properties of cell-cell contacts involved in a) the formation of the thyroid follicular lumen and b) the communication between thyrocytes within the follicle. The probes were compounds of either low (Lucifer Yellow: LY) or high molecular weight (Dextran labeled with fluorescein: FITC-Dextran and Cascade Blue conjugated to bovine serum albumin: CB-BSA). LY microinjected into IL of 2-9-day-old RTF was seen to label circular spaces with a diameter ranging from 10 to 100 microns. The cells delimiting the IL remained unlabeled. The fluorescent dye remained concentrated in IL for up to 24 hr. FITC-Dextran or CB-BSA microinjected into IL behaved as LY; the probes were restrained into the lumen. A 2 hr incubation of RTF with iodide induced alterations of the structure of IL; an effect mediated by an organic form of actively trapped iodide. A 15-30 min incubation of RTF in a low CA2+ medium caused the opening of IL visualized by the progressive decrease of the fluorescence of probes preinjected into the lumenal space. The same but more rapid effect was obtained by microinjection of EGTA into the IL. The low Ca2(+)-dependent opening of IL was also demonstrated by the release into the medium of thyroglobulin present in IL. Microinjection of LY in a cell involved in the follicle structure led to the rapid labeling of the other cells forming the follicle but LY did not penetrate the IL. Unlike LY, the distribution of FITC-Dextran or CB-BSA injected into cells delimiting the lumen was restricted to the microinjected cells. Alterations of medium or intralumenal Ca2+ concentration which caused the opening of IL did not affect the cell-to-cell transfer of LY. By using fluorescent probe microinjection, we show that the in vitro thyrocyte histiotypic differentiation leads to the reconstitution of functional intercellular junctions: tight junctions insuring the tightness of the neoformed lumen and gap junctions mediating the cell-to-cell exchange of small molecules. The structure of the thyroid follicles appears to be under the control of both extracellular and intralumenal Ca2+ concentrations.

Coated vesicles from thyroid cells carry iodinated thyroglobulin molecules. First indication for an internalization of the thyroid prohormone via a mechanism of receptor-mediated endocytosis.

We have tried to identify iodinated thyroglobulin molecules in purified thyroid-coated vesicles to determined whether the internalization of the thyroid prohormone could proceed via a mechanism of receptor-mediated endocytosis. Coated vesicles isolated from pig thyroids by differential centrifugation and centrifugation on 2H2O-sucrose cushion were characterized by transmission electron microscopy and analyses of the polypeptide composition by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and Western blot using anti-clathrin heavy chain and anti-thyroglobulin antibodies. Clathrin and thyroglobulin (Tg) appeared as the two major components of the purified thyroid coated vesicles (TCV). Purified TCV fraction was homogeneous when analyzed by isopycnic centrifugation on 30% Percoll gradient. TCV had an apparent buoyant density of 1.035 g/ml. The presence of Tg molecules inside TCV was ascertained by (a) immunogold labeling on cryosections of TCV pellet and (b) identification by gel electrophoresis and radio-immunoassay of a definite fraction of Tg (3-5% of total protein) in TCV treated by Triton X-100. The detergent-treated TCV also contained protein-bound iodine: 0.5-0.7 micrograms of iodine/mg protein. Pulse-chase experiments on in vitro reconstituted thyroid follicles have been used to further document the presence of iodinated Tg molecules in coated vesicles. TCV were isolated from reconstituted thyroid follicles previously labeled with [125I]iodide to radioiodinate Tg of the follicular lumen (the pre-endocytotic compartment) and incubated with or without thyrotropin or dibutyryl cyclic AMP to activate intraluminal 125I-Tg endocytosis. Autoradiographic analyses revealed the presence of 125I-Tg in purified TCV and Triton X-100-treated TCV. 125I-Tg present in TCV represented 1-2% of the total intracellular protein-bound radioactivity. Thyrotropin and dibutyryl cyclic AMP increased 2-3-fold the 125I-Tg content of TCV. Our results clearly show that iodinated Tg, the molecular form of the thyroid prohormone known to be internalized, is present into TCV. The data suggest that coated vesicles are involved in the uptake and transport of Tg from the follicular lumen to the lysosomal compartment and therefore, that the internalization of Tg could proceed, at least for a part, via a mechanism of receptor-mediated endocytosis.

Thyroid hormone residues are released from thyroglobulin with only limited alteration of the thyroglobulin structure.

We have tried to characterize thyroglobulin (Tg) degradation products in purified pig thyroid lysosomes to determine whether the release of thyroid hormone residues from Tg involves a random proteolytic attack or discrete and selective cleavage reactions. The intralysosomal soluble protein fraction was prepared by osmotic pressure-dependent lysis of lysosomes purified by isopycnic centrifugation on Percoll gradients. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed the presence of a fraction of Tg (5-10% of total lysosomal protein) with the same molecular weight as that of the intact Tg subunit. This high molecular weight Tg was the only intralysosomal species detected by Western blot using antipig Tg antibodies. In nondenaturing conditions, lysosomal Tg (LTg) identified by radioimmunoassay was in the form of a dimer with a sedimentation coefficient lower than that of either iodinated Tg (colloid Tg) or noniodinated Tg (microsomal Tg). LTg had a lower iodine content than colloid Tg:9-12 versus 39-42 iodine atoms/molecule. Pronase hydrolysates of LTg did not contain any 3,5,3',5'-tetraiodo-L-thyronine or 3,3',5-triiodo-L-thyronine residues detectable by reverse-phase high pressure liquid chromatography; iodine present in LTg was in the form of iodotyrosines. Under reducing conditions, LTg almost completely disappeared and gave rise to various polypeptides of smaller size. These results suggest that Tg transferred to lysosomes is subjected to selective proteolytic cleavage reaction(s) that release thyroid hormone residues. This early step would lead to the formation of hormone-depleted Tg molecules that are cleaved at discrete sites, the resulting polypeptides remaining bound through disulfide bonds to yield Tg molecules with an apparently normal size and a slightly altered structure.

In vitro studies of the thyroglobulin degradation pathway: endocytosis and delivery of thyroglobulin to lysosomes, release of thyroglobulin cleavage products--iodotyrosines and iodothyronines.

UNLABELLED: 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. IN CONCLUSION: 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)