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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-2351
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The Journal of Clinical Endocrinology & Metabolism Vol. 92, No. 7 2803-2810
Copyright © 2007 by The Endocrine Society

Thyrotropin Activates Guanosine 5'-Diphosphate/Guanosine 5'-Triphosphate Exchange on the Rate-Limiting Endocytic Catalyst, Rab5a, in Human Thyrocytes in Vivo and in Vitro

Marie-France van den Hove, Karine Croizet-Berger, Donatienne Tyteca, Charlotte Selvais, Philippe de Diesbach and Pierre J. Courtoy

Université Catholique de Louvain, Christian de Duve Institute of Cellular Pathology, Cell Biology Unit, B-1200 Brussels, Belgium

Address all correspondence and requests for reprints to: Marie-France van den Hove, Université Catholique de Louvain, Christian de Duve Institute of Cellular Pathology, Cell Biology Unit, 75 avenue Hippocrate, B-1200 Brussels, Belgium. E-mail: vandenhove{at}cell.ucl.ac.be.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: We have previously reported that the TSH receptor/cAMP cascade enhances the coordinate expression of the rate-limiting endocytic catalysts, Rab5a and Rab7, which respectively promote thyroglobulin (Tg) internalization and transfer to lysosomes, thereby accelerating thyroid hormone secretion.

Objective: We address whether TSH further controls Rab5a activity by promoting its GTP-bound state.

Design: We compared Rab5a activation in seven pairs of hyperactive and corresponding quiescent thyroid tissues; TSH effect was reproduced on polarized cultures of normal human thyrocytes.

Patients: We studied seven euthyroid patients bearing hyperactive autonomous adenomas; normal thyroid tissue for culture.

Main Outcome Measurements: Rab5a GDP/GTP exchange factor activity [Rab5a-guanine nucleotide exchange factor (GEF)], expression of Rabex-5 (a Rab5a-GEF), and function of thyrocytes in vitro were the main outcome measures.

Results: In autonomous adenomas, constitutive activation increased both total activity and sedimentability (membrane recruitment) of Rab5a-GEF, compared with perinodular tissues. Increased Rab5a-GEF activity correlated with increased expression of Rabex-5 and Rab5a, as well as with Tg store depletion. In polarized human thyrocyte monolayers, TSH did not affect total Rab5a-GEF activity after 2 h but promoted its membrane recruitment; after 4 d, TSH increased both Rab5a-GEF activity and Rabex-5 expression and recruitment onto membranes where Rabex-5 coimmunoprecipitated with Rabaptin-5 and Rab5a. Sedimentable Rab5a-GEF perfectly correlated with apical endocytosis and lysosomal transfer of 125I-Tg, and with basolateral secretion of 125I-derived hormones.

Conclusion: This study provides the first clinical and experimental evidence that regulation of the activity of a rate-limiting endocytic catalyst finely tunes a tightly controlled cellular function that ultimately governs whole body metabolism.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
THYROID HORMONE SECRETION is regulated by the TSH receptor (TSHR)/cAMP cascade, but molecular mechanisms that adjust this control at the level of thyrocytes remain poorly understood. The production of thyroid hormones depends on apical endocytosis of its large precursor, thyroglobulin (Tg), stored in the colloid at very high concentration, and its transfer to the late endocytic apparatus for lysosomal proteolysis, to release free T4 and T3 into the blood (1, 2, 3). Tg endocytosis, the limiting step of thyroid hormone secretion, is supported by macropinocytosis upon acute TSH stimulation, and by micropinocytosis in chronically stimulated thyroids (4) under physiological conditions (1, 2, 3). In man, both endocytic mechanisms are triggered by the activation of the cAMP cascade (3, 5). In view of the large excess of both Tg substrate and lysosomal proteases, we hypothesized that thyroid hormone generation must be finely tuned by their encounter, i.e. is controlled by rate-limiting endocytic catalysts regulating Tg apical uptake (Rab5) and transfer to lysosomes (Rab7). In a first report (5), we assessed the effect of TSH on Rab5 and Rab7 expression in thyroid samples from hyperactive autonomous adenomas (AAs), due to sporadic activating mutations of the TSHR or Gs{alpha} (6), with perinodular (PN) tissues put at rest by TSH suppression (observational study) and in normal human thyrocytes in polarized cultures upon TSH addition (experimental study). In pathological tissues, Rab5 and Rab7 expression was coordinately increased in AAs and correlated with the level of Tg store depletion. In polarized thyrocytes, sustained treatment by TSH or forskolin to increase cAMP production promoted Rab5 and Rab7 expression, apical uptake of 125I-Tg, and basolateral release of T4 and T3.

Here, we focused on further control of the activity of Rab5, a key rate-limiting endocytic catalyst for micropinocytosis and macropinocytosis (7, 8). Rab5 acts by recruiting effector proteins onto early endosomes, and by controlling, in time and space, their organization into distinct membrane subdomains (9, 10, 11, 12). Rab5 cycles between GDP-bound (inactive) and GTP-bound (active) states. In the current model, cytosolic GDP-Rabs become activated upon GTP exchange after membrane recruitment. The lifetime of the GTP-bound state, the ultimate determinant of final activity, depends on the balance between specific factors that respectively promote GTP loading [guanine nucleotide exchange factors (GEFs)] and GTP hydrolysis [GTPase-activating proteins (GAPs)]. Rab5-GEFs and -GAPs are thus potential regulators of Rab5 function.

Because spontaneous GDP release by Rab5 is very slow (13), GDP/GTP exchange by appropriate GEFs must be tightly regulated. Mammalian Rab5 and its yeast ortholog, Vps21p, can be activated by the paralogous GEFs, Rabex-5 and Vps9p (14, 15, 16). However, these GEFs have considerably lower affinity for GDP than Rab5 and are, therefore, weak exchangers (17), predicting that their function must be regulated by their abundance. Association of Rabex-5 via Rabaptin-5 increases Rab5 activity, allowing interaction of GTP-Rab5 with its various effectors of the endosome docking/fusion machinery long enough to support vesicular transfer (14, 18, 19). Another Rab5-GEF candidate is Rin1 (for "Ras interference"). When coexpressed with Rab5a, Rin1 accelerates endocytosis of the EGF receptor and fluid-phase endocytosis (20, 21). The exchange protein directly activated by cAMP (Epac) is another GEF active on the small GTPases Rap (22). Whether Epac can also activate Rab5 remains unknown.

The role of Rab5-GAPs is less clear. GTP hydrolysis on Rab5 is not conditional for membrane fusion but, rather, acts as a timer controlling the frequency of membrane docking/fusion events (23). The recently identified RabGAP-5 specifically accelerates GTP hydrolysis by Rab5 isoforms, thereby regulating endosomal trafficking (24). In vitro, the catalytic domain of p120 Ras-GAP also stimulates Rab5-GTPase activity (25).

Because TSH increases Rab5 membrane recruitment (5), here, we examined whether it promotes Rab5 activity via its GEFs and/or its GAPs. We found that constitutive thyrocyte activation in hyperactive AAs increases Rab5a-GEF activity (correlating with Rabex-5 expression) and membrane recruitment. A study of polarized human thyrocytes further discriminated two sequential levels, whereby TSH controls Rab5a-GEF function: membrane recruitment, followed by increased Rabex-5 expression.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patients and tissues

Seven new euthyroid patients bearing hyperactive AAs were included for this study. Clinical diagnosis was based on: 1) identification of a hot nodule by scintigraphy, 2) suppressed activity in PN tissue, and 3) very low or undetectable serum TSH. Unless specified, details on experimental procedures can be found in the study by Croizet-Berger et al. (5). Paired samples of the AAs and adjacent PN tissues were rapidly homogenized in 250 mM sucrose, 10 mM Tris (pH 7.4) (sucrose-Tris buffer), and sequentially centrifuged to isolate a low-speed nuclear fraction, high-speed sedimentable particles (MLP fraction), and the final supernatant. Because Tg represents a large and variable proportion of soluble proteins in homogenates, depending on endocytic activity, Tg concentration was measured in supernatants (5), and the amount of cellular protein in each assay was determined after correction for Tg contamination. To generate polarized human thyrocytes cultures, normal thyroid tissue was obtained from nine patients undergoing surgery for cold nodules. The study was approved by the Ethical Committee of Louvain University Medical School.

Culture and subcellular fractionation of human polarized thyrocytes

Human thyrocytes were cultured in 5 H Coon’s medium on 4.7 cm2 permeable filters until transepithelial resistance exceeded 1000 {Omega} x cm2. Then, 0 or 1 mU/ml bovine TSH (Sigma, St. Louis, MO) was added to the basolateral medium, and monolayers were further incubated for 4 d, insulin being removed for the last day. For subcellular fractionation, after 2 h of apical 125I-Tg uptake (see below) and extensive washing, cells from six to 12 filters were repeatedly extracted by a Dounce homogenizer in sucrose-Tris buffer (26). The postnuclear supernatant (total extract) was either fractionated into postnuclear particles and cytosol, or resolved in self-forming 20% (vol/vol) Percoll gradients (Amersham Bioscience, Roosendaal, Netherlands) (5, 27). To assign 125I counts to lysosomes, fraction nos. 10–12 (see Fig. 6Go) were selected. Their contamination by nonsedimentable counts in cytosols that equally distributed over the gradients was subtracted; lysosome-associated 125I was expressed as percentage of MLP fraction.


Figure 6
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  		FIG. 6. Two-stage activation of the endocytic machinery by the TSHR/cAMP cascade: rapid recruitment of Rab5a-GEF followed by increased expression of Rab5a/Rab5a-GEF. A, Assessment of 125I-Tg transfer to lysosomes. Polarized human thyrocytes were treated with TSH for 4 d, after which 125I-Tg was internalized from the apical medium at 37 C for 15 and 120 min, or at 17 C for 120 min. Postnuclear supernatants were resolved in self-generating Percoll gradients. Fractions were analyzed for radioactivity (upper panel, representative distribution for two, eight, and three experiments, respectively) and marker enzymes (bottom panel: 5'-nucleotidase, apical membranes; cathepsin D, lysosomes). B, Comparison between brief and sustained in vitro TSH effects on apical 125I-Tg endocytic uptake and lysosomal transfer. Polarized human thyrocytes were cultured without TSH or with TSH for 2 h or 4 d, as indicated. 125I-Tg was internalized from the apical medium for 2 h at either 37 C or 17 C. Percentage in lysosomes is defined as sedimentable radioactivity in fraction nos. 10–12 of Fig. 5AGo. Values are means ± SEM of three to eight experiments. C, Comparison of brief and sustained TSH effects on expression and membrane recruitment of Rab5a-GEF. Total and sedimentable Rab5a-GEF activities were measured in thyrocytes cultured with TSH for 2 h or 4 d. Results are expressed as percentage of untreated controls and are means ± SEM of four (2 h) or six paired experiments (4 d). Significance of differences between groups was tested by unilateral paired t test. D, Comparison between sedimentable Rab5a-GEF and 125I-Tg apical endocytic uptake (upper panel), transfer to lysosomes (central panel), and basolateral secretion of ethanol-soluble Tg-derived 125I-hormonal products (lower panel) for thyrocytes cultured without TSH (open circles), treated with TSH for 2 h (triangles), or 4 d (filled circles). prot, protein. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
125I-Tg endocytosis by, and 125I-hormone secretion from, cultured thyrocytes

Iodine-poor human Tg was radioiodinated to 20 -mol 125I/mol Tg (5). Endocytosis of 125I-Tg (50 nM) was measured at 2 h after addition to the apical medium. Basolateral medium was loaded on reverse-phase Sep-Pak Plus tC18 cartridges (Waters Corp., Milford, MA) conditioned with ethanol-ammoniac (99:1, vol/vol) and flushed with 15-mM ammonium acetate (pH 4.0). Hydrophilic products were washed with ammonium acetate. Free Tg-derived 125I-iodothyronines were then eluted with ethanol-ammoniac. Intact Tg and large peptides remaining on the cartridge never exceeded 0.1% of apical 125I-Tg, indicating tightness of thyrocyte monolayers. Because our previous analyses have shown that free 125I-tyrosines are essentially absent in the basolateral medium, the amount of ethanol-soluble radioactivity released therein is a fair index of hormone secretion.

GDP/GTP exchange assay

GST-Rab5a was expressed by a pGEX-Rab5a plasmid (gift of Dr. M. Zerial, Max-Planck Inst., Dresden, Germany) in competent Escherichia coli XL-1 blue bacteria (Stratagene, Amsterdam, Netherlands) and purified on glutathione-Sepharose beads. GDP/GTP exchange was measured at room temperature by a filter assay adapted from Hardt et al. (28). GST-Rab5a (62 pmol) was first loaded with GDP by incubation for 30 min in low Mg2+ buffer (3 mM MgCl2). Exchange reactions were performed in triplicates, on 1–2 µg protein samples in 20 µl (tissues) or 100 µl (cells) of high Mg2+ buffer (10-mM MgCl2) containing 0.175 pmol GTP[{gamma}-35S] (0.2 µCi; Amersham Bioscience). This exchange activity was destroyed upon boiling and inhibited by approximately 40% in 1 mM N-ethylmaleimide. Total GEF was assayed on homogenates or cell extracts. Membrane-bound GEF was assayed on postnuclear particles. The reaction was started by addition of approximately 1.2 pmol freshly prepared GDP-loaded Rab5a and usually stopped after 10 min by addition of 10 volumes of ice-cold stop buffer [100 mM NaCl, 20 mM Tris-HCl (pH 7.5), and 25 mM MgCl2]. Proteins were recovered on filters (HAWP025; Millipore Corp., Bedford, MA), and filter-bound radioactivity was measured. Each test was run with a blank at 4 C in which GDP-Rab5a was added after the stop solution. After blank subtraction (<10% of total), results were normalized to DNA (surgical specimens) or protein (cultured thyrocytes).

Western blotting

Samples (30 µg cell protein, nonreduced; + dithiothreitol for Epac and RabGAP-5) were resolved by 8% or 12% SDS-PAGE. After blocking with either 2% BSA (Rab5a) or 5% milk (GEFs and GAPs), and 0.5% Tween 20 in 150 mM NaCl, 5 mM EDTA, and 20 mM Tris (pH 7.4), membranes were probed overnight at 4 C with rabbit antiserum raised against Rab5a (1:200; Santa Cruz Biotechnology, Santa Cruz, CA), Rabex-5 (1:2000; from Dr. M. Zerial), Rin1 (1:500; Transduction Laboratories, Lexington, KY), Epac (1:1000; from Dr. S. Dremier, Institute of Interdisciplinary Research, Brussels, Belgium), or RabGAP-5 (1:500; from Dr. F. A. Barr, Max-Planck Institute, Martinsried, Germany), or mouse monoclonal antibodies to Rabaptin-5 (1:500; Transduction Laboratories), or p120 Ras-GAP (1:500; Santa Cruz Biotechnology). Blots were incubated with peroxidase-conjugated secondary antibodies (Biosource, Camarillo, CA), followed by enhanced chemiluminescence detection (PerkinElmer, Shelton, CT). Signals were quantified using Scion IMAGE 4.0.2 (Frederick, MD) and related to DNA content (5). Quantitative comparisons of paired samples were means of two to three gels; correlations were from single gels.

Immunoprecipitation

There were 20 µg postnuclear proteins from cultured thyrocytes lysed in 0.1% Triton X-100 in PBS (PBS-Triton) and precleared with 10 µl Protein A-Sepharose beads (Amersham Bioscience) for 60 min at 4 C. The cleared supernatant was immunoprecipitated using beads coated with 2 µl anti-Rabex-5 or nonrelevant antiserum at 4 C for 90 min. Immunoprecipitates were washed thrice with PBS-Triton, once with 500 mM NaCl in PBS-Triton, suspended in loading buffer without dithiothreitol, and analyzed by 9% SDS-PAGE and Western blotting.

Immunolocalization

For immunohistochemistry, 5-µm paraffin thyroid sections were processed as reported (29). After endogenous peroxidase inactivation with 1% H2O2 for 30 min and blocking, sections were incubated at 4 C overnight with rabbit antiserum to Rab5a (1:100), Rabex-5 (1:50), or human Tg (1:3000; DakoCytomation, Glosstrup, Denmark), followed by 1 h with secondary antibodies (Envision; DakoCytomation), and peroxidase activity was revealed for 5 min (29).

For double immunofluorescence, sections were briefly treated by microwaves in 10 mM citrate (pH 6.0), permeabilized with 0.3% PBS-Triton for 5 min, preincubated for 45 min with 0.3% Triton X-100/10% BSA/3% milk in PBS, incubated at 37 C for 2 h with rabbit anti-Rabex-5 antiserum (1:75) together with mouse monoclonal antibodies against human Tg (1:75; DakoCytomation), or nonrelevant antibodies of both species, washed, and further incubated for 1 h with Alexa488-antirabbit and Alexa568-antimouse antibodies (1:400; Molecular Probes, Eugene, OR). Sections were mounted in ProLong Gold (Invitrogen, Carlsbad, CA) and analyzed with an LSM 510 META confocal microscope (Zeiss, Jena, Germany) in sequential mode. Detection level was set over signal in sections incubated with nonrelevant antibodies.

Statistical analysis

Values are means ± SEM (n). Significance of differences between groups was tested by Student’s t tests (NS, not significant; P < 0.05; P < 0.01; and P < 0.001).


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Rab5a guanine nucleotide exchange assay in human thyrocytes

The assay of Rab5a-GEF was first validated using human thyroid supernatants from a pair of hyperactive AA and the corresponding quiescent PN tissue. Recombinant GST-Rab5a was loaded with GDP at low Mg2+, then GEF activity was measured upon addition of GTP[{gamma}-35S] at high Mg2+. This assay was tested as a function of time, amount of cell protein, and GTP-concentration (Fig. 1Go). Adequate sensitivity was obtained after 10 min. The assay was linear over a large range of protein concentration and showed an approximate 2-fold higher activity in this AA compared with its PN tissue. The Michaelis-Menten constant for GTP was identical at approximately 10–7 M, in good agreement with previous reports (17). In our assay conditions, total GEF activity in PN homogenates was remarkably constant, at 35 ± 3 pmol Rab5a-bound GTP/mg DNA after 10 min (n = 7; Fig. 2BGo). Postnuclear extracts of human thyrocytes in primary polarized cultures without TSH, to mimic quiescent PN tissues, showed a significantly higher basal activity (86 ± 3 pmol/mg DNA; n = 8). We concluded that Rab5a-GEF of thyroid tissue primarily reflected the contribution by thyrocytes. This was confirmed by immunohistochemistry (see Fig. 4AGo).


Figure 1
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  		FIG. 1. Validation of the Rab5a-GEF assay in supernatant from a pair of thyroid homogenates (patient 3 of Figs. 2–4GoGoGo). A, Time course. Two micrograms of protein of high-speed supernatant from the hyperactive AA (filled symbols) or corresponding PN quiescent tissue (open symbols) were incubated with GDP-loaded Rab5a-GST and GTP[{gamma}-35S] for the indicated times. GTP[{gamma}-35S]-Rab5a complex was measured by a filter assay. Results are means ± SEM (n = 3). B, Linearity. Exchange reaction for 10 min with the indicated amounts of cytosolic proteins (corrected for Tg content). C, GTP-concentration dependence. Exchange reaction for 45 min with 5-µg cytosolic proteins and the indicated concentrations of cold GTP, as competitor.

 

Figure 2
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  		FIG. 2. Constitutive activation of the TSHR/cAMP cascade increases total Rab5a-GEF activity and membrane recruitment and correlates with Rab5a expression and thyroid activation state. Paired homogenates from nodular (AA) and PN tissues of the seven patients, identified by numbers, were analyzed for total and sedimentable GEF activity. All individual values were normalized per milligram of DNA. A, Total Rab5a-GEF activity in AA samples relative to corresponding PN tissues, set to one. B–D, Comparison between total Rab5a-GEF activity and its sedimentable fraction (B), Rab5a expression (C), or Tg content (D) for individual AA samples (filled symbols) by reference to the average value of PN tissues (open symbols) (means ± SEM).

 

Figure 4
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  		FIG. 4. Rabex-5 colocalizes with Rab5a in human thyrocytes and is recruited on vesicles containing Tg. A, Immunohistochemical detection of Rabex-5 and Rab5a in serial paraffin sections from patient 3. The upper panel at low magnification shows high expression in the encapsulated AA and low expression in part of adjacent PN tissue. In a third section (data not shown), Tg was equally stained in the lumen of both AA and PN tissue. The lower immunoperoxidase panel at higher magnification of AA shows the almost exclusive expression of Rabex-5 and Rab5a in thyrocytes. B, Fixed-permeabilized paraffin section of patient 1 with double-immunofluorescence labeling for Rabex-5 (green) and Tg (red). Arrowheads point to structures labeled for the two antigens. Notice strong Tg labeling at the apical thyrocyte membranes. Lum, Follicle lumen (colloid was lost during tissue processing).

 
TSH increases total Rab5a-GEF activity and its membrane recruitment

We thus compared Rab5a-GEF activity in homogenates of seven AA/PN pairs (Fig. 2Go). Constitutive activation of the TSHR/cAMP cascade in AAs caused a moderate-to-strong increased total Rab5a-GEF activity (up to 3-fold) (Fig. 2AGo). The increase of total activity correlated with increased sedimentable fraction (up to 2-fold), reflecting membrane recruitment (Fig. 2BGo). Combining these two effects, GEF activity on target membranes was increased by up to 6-fold in patient 1. Individual total Rab5a-GEF activity also directly correlated with Rab5a level (Fig. 2CGo), but inversely with soluble Tg concentration, as expected for substrate depletion upon stimulation of endocytosis (Fig. 2DGo).

Rab5a-GEF activity level best correlates with Rabex-5 expression

Three GEF candidates were further analyzed: Rabex-5, Rin1, and Epac. To test if these could account for increased Rab5a-GEF in the AAs, we compared their expression by quantitative Western blotting in 5 AA/PN pairs (Fig. 3Go). Blot of Rabex-5 is illustrated at Fig. 3AGo. The expression of both Rabex-5 and Rin1 was increased in all cases of AA studied, as compared with corresponding PN tissue (2.4 ± 0.2 and 3.5 ± 0.5-fold, respectively) (Fig. 3BGo). In contrast, Epac showed no consistent variation. Comparing all individual levels of Rabex-5 expression and total GEF activity revealed a significant correlation (Fig. 3CGo). There was no such correlation for individual Rin-1 values, suggesting that Rin1 increase by TSH reflects another, still unknown function. We concluded that increased Rabex-5 expression best reflected Rab5a-GEF activity measured in AAs. These results also predicted that Epac did not play a crucial role for Rab5 activation.


Figure 3
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  		FIG. 3. Constitutive activation of the Rab5a-GEF activity by the TSHR/cAMP cascade best correlates with Rabex-5 expression. A, Western blot for Rabex-5 in homogenates from 5 AA/PN pairs run on the same gel (equal protein loads). B, After densitometry, values were normalized per milligram of DNA and presented as AA/PN ratios for the three candidate GEFs. C, Comparison between total Rab5a-GEF activity and normalized expression of Rabex-5 (left) or Rin1 (right) for individual AA (filled symbols) or their average value in PN tissues (open symbols) (means ± SEM, n = 5). Western blot values were normalized per milligram of DNA.

 
By immunohistochemistry, Rabex-5 displayed at low magnification a similar pattern as Rab5a, with a much stronger staining in AA than in PN tissues (Fig. 4AGo, upper panel). At higher magnification of AA tissues, both signals were almost restricted to thyrocytes (lower panel). By double immunofluorescence confocal microscopy, Rabex-5 was detected at apical punctate structures, several of which contained Tg (Fig. 4BGo). This result demonstrates that Rabex-5 can be recruited onto endosomes involved in apical Tg endocytosis.

Alternatively, we looked for the expression of two Rab5 GAPs, RabGAP-5 and p120 Ras-GAP. Both increased marginally [AA/PN ratios: 1.4 ± 0.2 (n = 6) and 1.2 ± 0.1 (n = 5), respectively] as compared with Rabex-5. This indicates that enhanced Rab5a-GTP-bound state in AAs is primarily caused by increased GTP exchange promoted by its Rab5-GEF, Rabex-5, not to decreased Rab5-GTPase activators.

In polarized human thyrocytes, sustained TSH exposure is sufficient to increase Rab5a-GEF activity and Rabex-5 expression

To complement observational studies in clinical samples by functional experimentation in vitro, human thyrocytes isolated from normal tissue were cultured as polarized monolayers, then treated or not with 1 mU/ml TSH for 4 d. TSH not only promoted apical endocytosis of 125I-Tg (Fig. 5AGo) and Rab5a expression (Fig. 5BGo), confirming our previous report (5), but also Rab5a-GEF activity (Fig. 5CGo), as well as Rabex-5 expression in total cell extracts (Fig. 5DGo).


Figure 5
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  		FIG. 5. In polarized human thyrocytes, TSH increases apical Tg endocytosis, Rab5a expression, Rab5a-GEF activity, and Rabex-5 expression, as well as Rabex-5 interaction with Rabaptin-5 and Rab5a. Human thyrocytes were isolated from normal thyroid tissue, polarized on a porous support, treated (filled symbols) or not (open symbols) with 1 mU/ml TSH for 4 d, then allowed to internalize 125I-Tg (50 nM) from the apical medium at 37 C for 2 h. Tg uptake was normalized to cell protein content (A). Cell extracts were further analyzed for Rab5a expression (B), total Rab5a-GEF activity (C), and Rabex-5 expression (D). Results are presented as percentages of untreated cultures (A–D). Gray symbols at (A) and (B) are means ± SEM of nine available values compiled from this paper and our previous work (5 ). Triangles refer to the samples analyzed by immunoprecipitation (E): lysed postnuclear particles (MLP fraction) of thyrocytes treated (+) or not (-) with TSH were immunoprecipitated (IP) with anti-Rabex-5 or irrelevant (CTL) antisera and immunoblotted (IB) for Rabex-5, Rabaptin-5, or Rab5a.

 
We verified the functional interaction of Rabex-5 with its known partners by coimmunoprecipitation of postnuclear particle lysates (Fig. 5EGo). Rabex-5/Protein A-Sepharose beads specifically retained Rabaptin-5 and Rab5a, and this association was increased by TSH. Beads with nonrelevant antibodies gave no signal. This result demonstrates that Rabex-5/Rabaptin-5 complexes may associate with Rab5a on membranes and promote nucleotide exchange on endosomes. In thyroid cultures, Rin1 expression was never detected without TSH but appeared under TSH; RabGAP-5 expression was readily detected but was not affected by TSH (data not shown).

A two-stage control by TSH of Tg endocytosis and intracellular trafficking: rapid membrane recruitment of Rab5a-GEF activity, then increased expression of Rab5a and its GEF

To measure transfer of apically internalized 125I-Tg to lysosomes, postnuclear extracts of thyrocytes treated with TSH for 4 d were analyzed by subcellular fractionation in Percoll density gradients that resolve plasma membrane/endosomes fractions (5'-nucleotidase/Rab5a) at low density from lysosomes (cathepsin D) at high density (Fig. 6AGo). Transport of 125I-Tg to lysosomal fractions at 37 C was obvious after 2 h, but negligible after 15 min or at 17 C. Because transfer to lysosomes is usually delayed by approximately 10–15 min at 37 C and is strongly inhibited at low temperatures (30), this experiment validated Percoll gradients for a functional assay of transport to lysosomes.

We then compared the effect of short (2 h) or sustained (4 d) TSH on Tg endocytosis and trafficking (Fig. 5BGo), in relation with Rab5a activation. After 2 h of TSH, both apical uptake of 125I-Tg and transport to dense lysosomes were increased (Fig. 6BGo); interestingly, whereas total Rab5a-GEF activity was unaffected, its membrane recruitment was significantly increased (Fig. 6CGo). Likewise, total Rabex-5 content did not change, but its sedimentable fraction was also increased (1.5 ± 0.2-fold; n = 3). These results indicate that TSH rapidly induces recruitment on endocytic structures of preexisting Rab5a-GEF/Rabex-5. In contrast, sustained TSH exposure increased both total and sedimentable Rab5a-GEF (Fig. 6CGo) and Rabex-5 (2.1 ± 0.1-fold; n = 3). Altogether, when comparing thyrocytes without TSH or with TSH for 2 h or 4 d, a remarkable correlation emerged between membrane-bound GEF activity and apical 125I-Tg uptake (Fig. 6DGo, upper panel), transport to lysosomes (central panel), or basolateral secretion of 125I-derived hormonal products (lower panel).


   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
Thyroid hormone secretion is regulated by Rab5 activity (5), which must be ultimately governed by the lifetime of its GTP-bound state. Here, we report that sustained stimulation of thyrocytes by the TSHR/cAMP cascade in vivo (AA patients) or in vitro (4 d with TSH) induces not only Rab5a expression, but also a Rab5a-GEF activity, best correlating with expression of the classical Rab5a-GEF, Rabex-5. Moreover, TSH rapidly (within 2 h) increases recruitment of available Rab5a-GEF/Rabex-5 onto membranes. Thus, TSH promotes Rab5a-GEF activity in two steps: rapid membrane recruitment, followed by increased expression. The coordinate expression of Rab5a, Rab7 (5), and Rabex-5 after sustained TSH exposure predicts cAMP-response elements in their respective promoters.

Membrane recruitment of Rab5a-GEF is a prerequisite and presumably rate-limiting step for Rab5 activation (12, 14, 23). Indeed, Rabex-5 concentration on isolated endosomes is much lower than Rab5 (19). We found that both brief and sustained TSH stimulation, which respectively induce macropinocytosis and micropinocytosis, favors Rab5a-GEF/Rabex-5 membrane recruitment, and that the absolute membrane-bound GEF activity perfectly correlates with apical Tg endocytosis, transfer to lysosomes, and basolateral hormonal production. This indicates that GTP exchange on Rab5a, likely mediated by Rabex-5, is a key rate-limiting factor for thyroid hormone production after both macropinocytosis and micropinocytosis.

Conceivably, the lifetime of GTP-bound Rab5 could also be extended by GAP depletion, but neither RabGAP-5 nor p120-GAP expression was lowered by TSH. However, this is not surprising because the half-time of intrinsic Rab5-GTPase at 37 C is compatible with completion of the process encompassing primary endocytic vesicles formation and their transfer to early endosomes (31). The subsequent early to late-endosomes transport likely involves a Rab5-negative feedback via a Rab5-GAP, such as RabGAP-5 we have identified in thyroid tissues. We previously reported that TSH also increases Rab7 expression (5). Whether TSH further stimulates the expression of a Rab7-GEF required for cargo progression from early to late endosomes (31) remains to be studied.

Rab5 is present in multiple intracellular compartments, suggesting its spatial compartmentalization, and selectively promotes apical endocytosis in polarized epithelial cells, although the mechanism underlying this polarity is not yet understood (8, 32). In polarized thyrocytes, the effect of TSH on Rab5 correlates with a strong stimulation of apical Tg endocytosis, while the constitutively higher basolateral endocytotic rate was not changed (33). Phosphoinositide 3-kinase has been reported to bind directly to Rab5 (34), an interaction required for protein kinase B/Akt activation by insulin (35). However, TSH-induced activation of Rab5 through this signaling cascade seems unlikely because: 1) TSH does not activate the phosphoinositide 3-kinase/protein kinase B pathway of thyrocytes; and 2) insulin, which is required for the mitogenic action of TSH-cAMP, does not interfere with cell differentiation (36).

At physiological TSH serum levels, apical endocytosis by human thyrocytes must be finely tuned at a very low rate, to avoid hyperthyroidism. In view of the vast excess of substrate, the challenge for individual thyrocytes is to harness apical Tg uptake to match the need of thyroid hormone production, at as low as approximately 1% of the Tg store per day in humans. Indeed, we measured a very low rate of apical endocytosis in nonstimulated human thyrocytes monolayers (1.4 nl/min·mg cell protein), close to the predicted level required for a basal hormone secretion. The challenge of thyrocytes is the exact opposite of kidney proximal tubule cells because the limited amount of ultrafiltrated provitamin D/binding protein complex must be avidly internalized by apical megalin-mediated uptake to achieve provitamin D capture and intracellular activation (37). Tg is also a ligand for megalin located at the apical membrane of thyrocytes (2, 38). Intriguingly, megalin-mediated Tg endocytosis was reported to avoid lysosomes (38). However, in our view, a prominent role of megalin for thyroid hormone secretion remains an open question. First, megalin-mediated endocytosis of Tg reported in polarized rat FRTL-5 cells (38) exceeds by at least 1000 times the requirement for normal T4 secretion. Second, megalin knockout mice do not develop thyroid abnormalities when compared with heterozygous mice with the same genetic background (39).

In conclusion, our results show that TSH rapidly recruits cytosolic Rab5a-GEF/Rabex-5 onto thyrocyte endosomes, then promotes the coordinate expression of Rab5 and Rabex-5. By controlling the level of GTP-bound (active) Rab5a, TSH stimulates apical Tg endocytosis, fueling its transport to lysosomes, so as to meet the demand of thyroid hormones. This study provides the first in vivo demonstration of fine tuning of Rab5 cycling activity by TSH, allowing thyrocytes to adapt thyroid hormone secretion to peripheral metabolic needs, and probably contributing to hypersecretion by AAs.


   Acknowledgments
 
We thank M. Eppe, M. Leruth, E. Marbaix, Y. Marchand, F. N'Kuli, L. Than, and P. Van Der Smissen for their assistance. We also thank Drs. M. Zerial (pGEX-Rab5a plasmid and antibodies to Rabex-5), F. A. Barr (antibodies to RabGAP-5), and S. Dremier (antibodies to Epac) for generously providing key reagents.


   Footnotes
 
This work was supported by grants from the Fonds de la Recherche Scientifique Médicale, Région Bruxelloise, Région Wallonne, Concerted Research Actions and Interuniversity Attraction Poles (Belgium), and a Delori postdoctoral fellowship (to K.C.-B.). M.-F.v.d.H. is a Fonds de la Recherche Scientifique research associate.

Disclosure Statement: M.-F.v.d.H., K.C.-B., D.T., C.S., P.d.D., and P.J.C. have nothing to declare.

First Published Online May 1, 2007

Abbreviations: AA, Autonomous adenoma; Epac, exchange protein directly activated by cAMP; GAP, GTPase-activating protein; GEF, guanine nucleotide exchange factor; MLP fraction, high-speed sedimentable particles; PBS-Triton, Triton X-100 in PBS; PN, perinodular; Rin1, Ras interference; Tg, thyroglobulin; TSHR, TSH receptor.

Received October 26, 2006.

Accepted April 24, 2007.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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