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. 2002 Jun 11;99(12):8277-82.
doi: 10.1073/pnas.122187699. Epub 2002 May 28.

The endocytic catalysts, Rab5a and Rab7, are tandem regulators of thyroid hormone production

Karine Croizet-Berger  1 , Chantal Daumerie, Marianne Couvreur, Pierre J Courtoy, Marie-France van den Hove
Affiliations

The endocytic catalysts, Rab5a and Rab7, are tandem regulators of thyroid hormone production

Karine Croizet-Berger et al. Proc Natl Acad Sci U S A. .

Abstract

Rab proteins are small GTPases that control distinct vesicular transport steps. Along the endocytic pathway, Rab5a is a rate-limiting catalyst of internalization, and Rab7 controls trafficking through late endosomes to lysosomes. The dependence of thyroid hormone production by thyrocytes on thyroglobulin endocytosis and intracellular processing in late endosomes/lysosomes suggests that its rate can be regulated by the expression or function of these endocytic catalysts. We compared the expression level and membrane recruitment of Rab5a and Rab7 in autonomous thyroid adenomas (where the cAMP cascade is constitutively activated) and surrounding quiescent tissues. The concentrations of Rab5a and Rab7, but not of Rab8, were coordinately increased up to 6-fold in adenomas, and correlated with a proportionate decrease in soluble thyroglobulin content (reflecting colloid depletion by accelerated endocytic uptake in hyperactive tissue). In adenomas, a higher proportion of Rab5a and Rab7 was membrane associated, and the equilibrium density of particulate Rab7 and iodine shifted toward lysosomal fractions, indicating that progression along the degradation pathway also was promoted. In cultures of polarized human thyrocytes from normal patients, thyroid-stimulating hormone or forskolin increased, to a similar extent, Rab5a and Rab7 but not Rab8 expression, apical endocytosis of thyroglobulin and lucifer yellow, and basolateral secretion of T(3) and T(4). Taken together, these in vivo and in vitro observations demonstrate that thyroid-stimulating hormone, via cAMP, coordinately enhances the expression of Rab5a and Rab7, which promote Tg endocytosis and transfer to lysosomes, respectively, resulting in accelerated thyroid hormone production.

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Figures

Figure 1
Figure 1
The coordinate increase in Rab5a and Rab7 levels inversely correlates with residual Tg content, according to the thyroid gland activation state. Homogenates from nodular AA and PN tissues of nine patients (each identified by a different number) were analyzed for Rabs (Western blotting) and Tg content (see Materials and Methods). Cathepsin D (lysosomal marker) and 5′-nucleotidase (apical membrane marker) activities were assayed in parallel. All values were normalized per mg DNA. (A) Specific content or activity in AA were expressed as a percentage of corresponding PN tissues. (B and C) Correlation in AA (filled circles) and corresponding PN tissues (white circles) between individual levels of Rab5a and Rab7, measured (B) on the same blot (r = 0.77, P < 0.01) or (C) between Tg content and Rab5a (r = −0.74, P < 0 01) or Rab7 (r = −0.82, P < 0.001). Samples from patients 4 and 5 were not available for this analysis.
Figure 2
Figure 2
Increased particulate recruitment of Rab5a and Rab7 in AAs. Four fractions obtained by differential centrifugation of thyroid homogenates (N, nuclear; ML, large particles; P, small particles; and S, soluble) from PN (white bars) and AA tissues (black bars) were analyzed for Rab5a and Rab7 (Western blotting) and for cathepsin B and D (enzyme activities). Results were expressed as percentages of the homogenate. Figure shows a representative pair of the three (Rabs) or nine (enzymes) analyzed.
Figure 3
Figure 3
(A) Density distributions in Percoll gradients. Particulate (ML) fractions from PN (white bars) and AA tissues (black bars) were mixed with Percoll and centrifuged. Pooled fractions are shown for the sake of simplicity (I, 1.032–1.052 in density; II, 1.052–1.056; III, 1.057–1.060; IV, 1.063–1.097). For each pool, content is expressed with respect to the initial content before centrifugation (C/Ci). Results are from one representative pair of the three analyzed. The shape of the gradient is superimposed (thin line). (B) Correlation between the increased content for Rab5a and Rab7 in total homogenates and for iodine in dense lysosomes (ML fraction, pool IV), in AA tissues with respect to corresponding PN tissues. Rab5a: r = 0.73, P < 0.05; Rab7: r = 0.84, P < 0.01).
Figure 4
Figure 4
Expression of Rab5a and Rab7, apical endocytosis, and thyroid hormone secretion are up-regulated in vitro by cAMP. In this representative experiment of three, polarized monolayers of thyrocytes from a normal thyroid gland (see Materials and Methods) were treated or not for 4 days with TSH or forskolin, as indicated. Cells then were allowed to internalize 50 nM radiolabeled Tg or 2 mg/ml lucifer yellow from the apical medium for 2 h at 37°C; cells then were washed, lysed, and tracer uptake was expressed as nl cleared per well (B). Alternatively, cells were incubated with 10 μM nonradioactive Tg in the apical medium for 18 h at 37°C, after which basolateral medium was collected for thyroid hormones assay (C). Aliquots of cell lysates then were analyzed for Rab5a and Rab7 content by Western blotting (A). Data are means ± SEM of four wells. Statistical analysis by unpaired Student's t test: *, P < 0.05; **, P < 0.01; NS, not significant.
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