Thyroid functions of mouse cathepsins B, K, and L (original) (raw)
In the thyroid, cysteine proteinases like cathepsins B, K, and L are not restricted to lysosomes of epithelial cells; rather, they are also detected at extracellular locations, i.e., they are associated with the apical plasma membrane, and secreted into the lumen of thyroid follicles (7–10). In vitro, cathepsins were shown to cleave Tg at neutral pH conditions, leading to the direct liberation of T4. We concluded that the cathepsins might be involved in thyroid hormone liberation by proteolytic processing of the prohormone Tg (2). To test this hypothesis in vivo, we have analyzed mice with deficiencies in cathepsin B, K, or L, and mice with double deficiencies in cathepsins B and K, or K and L.
Rearrangement of the endocytic system in cathepsin-deficient mice. Cryosections of thyroids were immunolabeled with antibodies against cathepsin B, K, D, or L as markers of endocytic vesicles in order to analyze their distribution and morphology within thyroid epithelial cells of mice with cathepsin deficiencies.
By immunolabeling, cathepsin B was detected within vesicles of thyroid epithelial cells of WT mice (Figure 1a, arrow). In addition, the apical plasma membrane of thyrocytes (Figure 1a and inset, arrowheads) was immunolabeled, as was the luminal content of follicles of WT mice (asterisks), indicating that cathepsin B is mainly located within lysosomes but also occurred at extracellular locations of WT thyroids. Cryosections of thyroids from mice with deficiencies in cathepsin B or cathepsins B and K were not immunolabeled (Figure 1, b and e, respectively), demonstrating the specificity of cathepsin B immunolabeling. In cathepsin K–/–, L–/–, or K–/–/L–/– mice, cathepsin B was localized within vesicles of thyroid epithelial cells (Figure 1, c, d, and f, arrows) and within the lumen of follicles (asterisks), whereas a staining of the apical plasma membrane was no longer observed. The results indicated that cathepsin B is associated with the apical plasma membrane in WT thyroids only, suggesting that deficiencies in cathepsins K and/or L led to the redistribution of binding partners of cathepsin B from the apical plasma membrane to other cellular locations.
Redistribution of cathepsin B in thyroids of cathepsin K–/–, L–/–, or K–/–/L–/– mice. Confocal fluorescence micrographs of cryosections of thyroid glands from WT mice (a) or cathepsin-deficient mice of the indicated genotypes (b–f) were immunolabeled with antibodies against cathepsin B. Thyroids from cathepsin B–deficient mice were not stained (b and e), indicating specificity of antibody labeling. Cathepsin B was detected within endocytic vesicles of thyroid epithelial cells (arrows). In WT mice, cathepsin B was also located at the apical plasma membrane of epithelial cells (arrowheads, inset) and within the lumina of thyroid follicles (asterisks). Deficiencies in cathepsin K and/or cathepsin L resulted in a redistribution of cell surface–associated cathepsin B, since immunolabelings of the apical plasma membranes were no longer observed. Rather, immunofluorescence became detectable over the follicle lumina (asterisks), and it was enhanced when compared with the WTs. Note the presence of non-immunolabeled inclusions within the thyroid follicle lumina of cathepsin L–/– or K–/–/L–/– mice (open circles). Bars: 50 μm.
In thyroids of WT mice, cathepsin K was detected within vesicles of the epithelial cells (Figure 2a, arrows), and within the follicle lumen (asterisks). Specificity of cathepsin K antibodies was proven by the absence of fluorescence signals when cryosections of thyroids from mice with cathepsin K deficiency were immunolabeled (not shown). In thyroids of cathepsin B–/– mice, cathepsin K was no longer detected within the follicle lumen, and cathepsin K–positive vesicles appeared smaller as compared with those of WT thyrocytes (Figure 2b, arrows; compare upper inset in b with inset in a). Furthermore, immunolabeling of cathepsin K at the apical surface of cathepsin B–deficient thyrocytes was observed (Figure 2b, lower inset in b, arrowheads). In contrast to its absence from the luminal content of cathepsin B–/– mice, cathepsin K was detected within the follicle lumen of thyroids from cathepsin L–/– mice (Figure 2c, asterisks), which often contained immunolabeled inclusions (open circles). In comparison with the WT, cathepsin K–labeled vesicles were enlarged in thyroid epithelial cells of cathepsin L–/– mice (Figure 2c, arrows in inset; compare inset in c with inset in a). The results indicated that deficiencies in cathepsin B or L had opposite effects on the sizes of cathepsin K–containing vesicles of thyrocytes.
Localization of cathepsins K and L. Confocal fluorescence micrographs of cryosections of thyroid glands from WT mice (+/+) or cathepsin-deficient mice of the indicated genotypes were immunolabeled with antibodies against cathepsin K (a–c) or cathepsin L (d–f). Compared with the WT (a, inset, arrows), cathepsin K–positive vesicles were smaller in cathepsin B–/– (b, top inset, arrows) and larger in cathepsin L–/– thyroid epithelial cells (c, inset, arrows). Cathepsin K was absent from thyroid follicle lumina of cathepsin B–/– mice but was detectable within lumina of WT or cathepsin L–/– thyroids (asterisks). Immunolabeling of cathepsin K was also observed at the apical plasma membrane of cathepsin B–/– thyroid epithelial cells (b, bottom inset, arrowheads). In addition, cathepsin K–positive inclusions were frequently detected within the luminal content of cathepsin L–/– mice (c, open circles). In WT mice, cathepsin L was detected mainly within endocytic vesicles (d, arrows) and, in a few follicles, in association with the apical plasma membrane of thyroid epithelial cells (d and inset in d, arrowheads). Deficiencies in cathepsin B and/or cathepsin K demonstrated a lack of cathepsin L at the apical plasma membrane and resulted in an enhancement of immunolabeling of thyroid follicle lumina (e and f, asterisks). The redistribution of cathepsin L was less obvious than that of cathepsin B (compare with Figure 1). Bars: 50 μm; in insets: 20 μm.
Cathepsin L was detected by immunolabeling within vesicles of WT thyrocytes (Figure 2d, arrows). In addition, a very faint immunolabeling was observed in association with the apical plasma membrane of WT cells (Figure 2, inset in d, arrowheads). Cell surface association of cathepsin L was not observed in thyroids from cathepsin B–/– (not shown), K–/–, or B–/–/K–/– mice, but staining of the follicle lumina occurred (Figure 2, e and f, asterisks). In thyroids from cathepsin L–/– or K–/–/L–/– mice, no immunolabeling with antibodies against cathepsin L was observed (not shown), proving specificity of antibody labeling.
In thyroid epithelial cells from WT mice, cathepsin D–positive vesicles, i.e., lysosomes, were distributed throughout the cells (Figure 3a). Lysosomes of cathepsin K–/– or B–/–/K–/– thyrocytes were comparable in size and distribution to the vesicles of WT cells (Figure 3b). However, immunolabeled vesicles of thyroid epithelial cells from mice with deficiencies in cathepsin B or L alone, or in both cathepsins K and L, demonstrated a significant enlargement when compared with WT lysosomes (Figure 3b; and compare c–e with a). Furthermore, lysosomes of thyroid epithelial cells of cathepsin L–/– mice often lacked cathepsin D immunolabeling within their lumina, i.e., they appeared as ringlike structures (Figure 3d, arrows), suggesting an association of immunolabeled cathepsin D with vesicular membranes. Similarly, cathepsin B was associated with lysosomal membranes in cathepsin L–/– thyrocytes (Figure 3d, inset, arrows). Accordingly, alterations in the ultrastructure of lysosomes were observed in keratinocytes and cardiomyocytes of cathepsin L–deficient mice (19, 20).
Swelling of cathepsin D–containing lysosomes in thyroids of cathepsin B–/–, L–/–, or K–/–/L–/– mice. Confocal fluorescence micrographs of cryosections of thyroid glands from WT mice (+/+) or cathepsin-deficient mice of the indicated genotypes were immunolabeled with antibodies against cathepsin D (a and c–e). Diameters of cathepsin D–containing lysosomes were determined morphometrically and are given as means ± SE (b). In WT thyroid epithelial cells, cathepsin D–positive vesicles were distributed throughout the cells (a). An immunolabeling indicative of cathepsin D at the apical cell surface or over the follicle lumina was not observed in either genotype. The sizes of lysosomes of cathepsin K–/– or B–/–/K–/– thyrocytes were similar to those of WT controls (b), whereas those from cathepsin B–/–, L–/–, or K–/–/L–/– thyroid epithelial cells were significantly enlarged (c–e). Cathepsin D was absent from the inner portions of enlarged lysosomes of thyroid epithelial cells with a deficiency in cathepsin L (d, arrows). Similarly, cathepsin B (Cath B) immunostainings revealed ringlike lysosomes in L–/– thyrocytes (d, inset, arrows), indicating a tight association of cathepsins B and D with vesicular membranes in cathepsin L–deficient thyrocytes. N, nuclei. *P < 0.05, **P < 0.01. In b, n = 16, 12, 16, 19, 18, and 14, respectively, for sections of the different genotypes indicated. Bars: 20 μm.
The results of immunolocalization of lysosomal enzymes indicated that deficiencies in cysteine proteinases in thyroid epithelial cells led to an extensive rearrangement of the endocytic system, and to a redistribution of extracellularly located enzymes.
Alterations of cathepsin levels in the thyroid. Because of the altered morphology of the endocytic system of thyroid epithelial cells from cathepsin-deficient mice, we thought to investigate potential compensatory effects on the translational level. For the analysis of the levels of cathepsin expression, lysates of thyroids from mice of the indicated genotypes were normalized to equal amounts of protein, separated on SDS gels, and transferred to nitrocellulose.
Probing of the blots with antibodies against cathepsin B showed, as expected, the lack of its expression in thyroids from cathepsin B–/– or B–/–/K–/– mice (Figure 4a). Levels of cathepsin B expression in thyroids from cathepsin K–/–, L–/–, or K–/–/L–/– mice were comparable to those in WT thyroids (Figure 4a). The mature forms of cathepsin B were expressed, i.e., single chain and smaller amounts of the heavy chain of two-chain cathepsin B were detected in immunoblots (Figure 4a).
Compensatory effects on the levels of cathepsin expression. Lysates of thyroids from mice of the indicated genotypes were normalized to equal amounts of protein, separated on 15% SDS gels, and transferred to nitrocellulose for subsequent incubation of the blots with antibodies against cathepsin (Cath) B (a), D (b), or L (c). Three to four blots each were used for densitometric evaluation as a measure of cathepsin expression levels. Levels are indicated by numbers below representative immunoblots and are expressed as the mean percentages ± SE of 100% expression in WT controls. Molecular mass markers are indicated in the left margins. Note that cathepsin B expression was absent from cathepsin B–/– and B–/–/K–/– thyroids, as expected, and that it was not altered by cathepsin K, L, or K/L deficiencies (a). In contrast, cathepsin D was downregulated in cathepsin B–/– thyroids, but upregulated under conditions of cathepsin L or K/L deficiency, whereas it was not significantly altered in cathepsin K–/– or B–/–/K–/– thyroids (b). Cathepsin L was absent from thyroid lysates of cathepsin L–/– or K–/–/L–/– mice, unaltered in cathepsin B–/– thyroids, and significantly upregulated in cathepsin K–/– or B–/–/K–/– mice (c). SC, single chain; HC, heavy chain; pro, proform. *P < 0.05, **P < 0.01.
Because it is known that cathepsin D might also be involved in Tg processing (11, 28), and because cathepsin D–positive lysosomes were enlarged in some genotypes (see Figure 3), the expression pattern of this aspartic protease was included in the analysis. In contrast to the principal expression of mature cathepsin B, immunoblotting of cathepsin D demonstrated that the expression of its proform dominated (Figure 4b), indicating that processing of cathepsin D to its fully mature form is negligible in thyroid epithelial cells. Expression of procathepsin D was significantly downregulated in thyroids from cathepsin B–/– mice (Figure 4b). The amounts of procathepsin D were slightly, but not significantly, enhanced in cathepsin K–/– thyroids (Figure 4b). Double deficiency in cathepsins B and K resulted in some reduction of the procathepsin D level, which was, however, not significant when compared with procathepsin D expression of WT thyroids (Figure 4b). In clear contrast, procathepsin D was significantly overexpressed in thyroids of cathepsin L–/– and K–/–/L–/– mice (Figure 4b). Because expression of procathepsin D was only slightly enhanced in thyroids of cathepsin K–/– mice when compared with WT (Figure 4b), the results indicated that it was primarily cathepsin L deficiency that induced overexpression of procathepsin D in the thyroid whereas cathepsin B deficiency resulted in a significant downregulation of procathepsin D levels. The result of cathepsin D overexpression in cathepsin L–deficient mouse thyroids might be indicative of a compensatory function of the aspartic lysosomal enzyme when cathepsin L is lacking. It remains unclear, however, whether cathepsin D compensates for cathepsin L action, because cathepsin D was present mainly in its proform within the thyroid (Figure 4b), hence, in its proteolytically inactive form.
When immunoblots were probed with various antibodies against cathepsin K purified from mouse or human tissues, or against the recombinant enzyme, or against distinct peptide regions of cathepsin K, it became obvious that the antibodies hardly recognized cathepsin K in lysates of mouse thyroids (not shown). However, several of the antibodies tested for immunoblotting indeed cross-reacted with cathepsin K of formaldehyde-fixed mouse thyroid tissue after immunolabeling of cryosections (compare Figure 2, a–c) — and did so in a specific manner, since immunolabeling was not observed in thyroids from cathepsin K–/– or cathepsin K–/–/L–/– mice. From the intensity of immunolabeling it might be concluded that the expression of cathepsin K was unaltered in cathepsin B–/– thyroids (Figure 2, compare b with a), whereas immunolabeling of cryosections from cathepsin L–/– thyroids might suggest a downregulation of cathepsin K (Figure 2, compare c with a). However, further studies are needed to analyze the levels of cathepsin K expression in cathepsin B– or L–deficient mouse thyroids by biochemical means.
Immunoblotting of cathepsin L proved, as expected, its deficiency in thyroids of cathepsin L–/– or K–/–/L–/– mice (Figure 4c). The major portion of cathepsin L expressed in WT thyroids was processed to its mature form (Figure 4c). Cathepsin B deficiency resulted in a nonsignificant upregulation of cathepsin L expression, whereas a significant cathepsin L overexpression was the result of a deficiency in cathepsin K alone or in cathepsins B and K (Figure 4c). These results indicated that cathepsin L might have a compensatory function when cathepsin K is lacking.
Survival of thyroid epithelial cells depends on expression of cathepsin L. A striking observation was the occurrence of inclusions within the lumina of thyroid follicles from cathepsin L–/– or K–/–/L–/– mice (Figures 1, 2, and 8, open circles; and Figure 5). Luminal inclusions were immunolabeled with antibodies against cathepsin K (Figure 2c, open circles) but appeared black after immunolabeling with antibodies against cathepsin B (Figure 1, d and f, open circles), T3 (Figure 5, d, e, g, and h), T4 (not shown), or Tg (Figure 8, d and f, open circles). In phase-contrast micrographs, the inclusions demonstrated a granular structure and were comparable in size to thyroid epithelial cells lining the follicle lumen (Figure 5, f and i), suggesting that luminal inclusions represented remnants of dead thyrocytes. Because such inclusions of remnants of dead cells were not present in the lumina of thyroid follicles of WT or cathepsin B–/–, K–/–, or B–/–/K–/– mice, the results indicated that cathepsin L is necessary for survival of thyroid epithelial cells. The molecular mechanism by which the survival of thyrocytes is maintained remains, however, unclear.
Multilayers of luminal Tg are lacking in thyroid follicles of cathepsin B–/–, L–/–, B–/–/K–/–, and K–/–/L–/– mice. Confocal fluorescence micrographs of cryosections of thyroid glands from WT mice (a) or cathepsin-deficient mice of the indicated genotypes (b–f) were immunolabeled with antibodies against Tg. As expected, Tg was detected within reticular structures and numerous intracellular vesicles (arrows), i.e., within compartments of the biosynthetic and of the endocytic route. Arrowheads indicate immunolabeled Tg in association with the ECM surrounding thyroid follicles. Open circles indicate non-immunolabeled inclusions of dead cells within the follicle lumina of cathepsin L–/– or K–/–/L–/– mice. In WT and cathepsin K–/– mice, immunolabeling revealed the characteristic multilayered appearance of Tg within the lumina of thyroid follicles (broken arrows), representing different states of Tg compaction. In contrast, Tg was homogeneously distributed within the follicle lumina of thyroids of cathepsin B–/–, L–/–, B–/–/K–/–, or K–/–/L–/– mice, indicating that deficiencies in either cathepsin B or cathepsin L resulted in the absence of differentially compacted luminal Tg. Bars: 50 μm; in insets: 20 μm.
Cathepsin L is essential for survival of thyroid epithelial cells. Confocal fluorescence (a, b, d, e, g, and h) and corresponding phase-contrast micrographs (insets in a, d, and g; and c, f, and i) of cryosections of thyroid glands from WT mice (+/+) or from mice with deficiencies in cathepsin L (L–/–, K–/–/L–/–) after immunolabeling with anti-T3 antibodies. Thyroid glands of mice with deficiencies in cathepsin L alone or in cathepsins K and L were characterized by numerous inclusions in the follicle lumina, which were not stained by the antibodies (d, e, g, and h). The inclusions were of irregular shapes with a granular appearance in phase-contrast micrographs (f and i), and their sizes were comparable to those of thyroid epithelial cells lining the follicle lumina, suggesting that luminal inclusions represented remnants of dead cells. Dead cells were not present under conditions of cathepsin B deficiency, i.e., in cathepsin B–/– or B–/–/K–/– mice. Bars: 50 μm; boxes in a, d, and g indicate regions that are shown in higher magnification in b and c, e and f, or h and i, respectively.
T4 liberation is mediated by cathepsins K and L. Because the endocytic system and the levels of protease expression of thyroid epithelial cells showed alterations in cathepsin-deficient mice, we postulated that Tg processing and Tg utilization, i.e., thyroid hormone liberation, might be affected.
Epithelia of thyroids from mice of all cathepsin-deficient genotypes were less extended than those of WTs (Figure 6a), indicating that flattening of thyroid epithelia was a result of cysteine proteinase deficiency. In euthyroid vertebrates, a demand of thyroid hormones is signaled by a rise in thyroid-stimulating hormone (TSH) levels in the blood as a result of negative feedback regulation (for reviews, see refs. 29, 30). Under such conditions of acute TSH stimulation, thyroid epithelial cells change from a cubic to a prismatic appearance, which reflects enhanced activity of thyrocytes, i.e., enhanced Tg turnover. Hence, flattening of epithelia might indicate a reduction in thyroid functional activity.
Alterations in thyroid physiological parameters. (a and b) Morphometric analysis of epithelial extensions, i.e., cell heights (a), and follicle dimensions (b) of thyroids from WT and cathepsin-deficient mice. Extensions of thyroid epithelia were significantly reduced (a) whereas follicle areas were significantly increased (b) in mice of all cathepsin-deficient genotypes. (c and d) To determine whether flattening of epithelia reflects a reduction in the functional activities of thyroid glands, serum T4 levels were determined by a radiometric immunoassay. (c) The scatter graph indicates distribution of serum T4 levels plotted against mouse age. (d) Data from mice of all ages were analyzed. Serum T4 levels were significantly but slightly reduced in cathepsin L–/– mice (c and d, blue). A systemic defect in thyroid function was apparent from the very significantly reduced serum T4 levels in cathepsin K–/–/L–/– mice (c and d, cyan). Mean values ± SD are given in a and d, mean values ± SE in b. Lines represent linear regressions of the single data plotted against mouse age in c. *P < 0.05, **P < 0.01. In a and b, arbitrarily chosen follicles were analyzed; n = 162, 70, 71, 114, 150, and 58, respectively. In c and d, T4 levels of different animals were determined; n = 25, 12, 10, 14, 28, and 13, respectively.
Because T4 is the major hormone released from the thyroid (29) and becomes converted into its biologically active form T3 by deiodinases upon entry of target cells, we considered serum T4 levels as a relevant systemic marker of thyroid function. In general, T4 levels were high in young animals and dropped with increasing age of WT or cathepsin B–/– or K–/– mice (Figure 6c, black, red, and green, respectively). An exception from this downregulation of T4 levels with aging were mice with double deficiencies in cathepsins B and K, in which T4 levels increased from significantly reduced levels in young mice to slightly elevated levels in mice older than 8 months (Figure 6c, orange). In cathepsin L–/– or K–/–/L–/– mice, the decline of serum T4 with age was less well pronounced as compared with WTs (Figure 6c, blue and cyan, respectively), indicating that cathepsin L–/– or K–/–/L–/– mice developed reduced T4 levels at an early age. The serum levels of FT4 were significantly reduced (P < 0.05) in the sera of mice with cathepsin L deficiency. In mice with double deficiency in cathepsins K and L (Figure 6d), the reduction was even greater (P< 0.01). reduced in mice with double deficiency in cathepsins K and L (Figure 6d). The systemic defect in thyroid function, i.e., reduction in serum T4, was established in both sexes of cathepsin K–/–/L–/– mice.
Reduced levels of thyroid hormones normally result in an increase in TSH levels, which may, after long intervals of several weeks, induce an enlargement of thyroid follicles due to hyperproliferation of thyroid epithelial cells. Therefore, the dimensions of thyroid follicles were analyzed morphometrically in mice with cathepsin deficiencies. In all cathepsin-deficient genotypes, an enlargement of thyroid follicles was observed when compared with the WTs (Figure 6b). The highest values of follicle areas were observed in cathepsin B–/– thyroids (Figure 6b), which were also characterized by extremely flat epithelia (Figure 6a) and by the highest amounts of Tg (see below). In mice with double deficiencies in cathepsins K and L, thyroid follicle areas were enhanced by about 80% as compared with the WTs (Figure 6b). Hence, the systemic defect in serum T4 in cathepsin K–/–/L–/– mice (Figure 6d, K–/–/L–/–) correlated with an enlargement of thyroid follicles (Figure 6b, K–/–/L–/–), a phenotype reminiscent of hypothyroidism. Deficiencies in cathepsin B, K, or L alone or in cathepsins B and K were not sufficient to induce this phenotype, indicating that a combinatory action of both cathepsins K and L is necessary for proper hormone liberation from the thyroid. Phenotypic alterations with the development of signs of hypothyroidism were observed in the mice with double deficiencies for cathepsins K and L only at the level of thyroid follicles, whereas the volumes or wet weights of thyroids from cathepsin K–/–/L–/– mice were unaltered compared with the WTs (not shown). This is most probably explained by enhanced cell death induced by cathepsin L deficiency (see above, Figure 5).
The results demonstrate that thyroid function is impaired by a deficiency in cysteine proteinases. An obvious phenotype with significantly reduced serum T4 levels was observed in cathepsin K–/–/L–/– double-deficient mice, indicating that a combinatory action of cathepsins K and L is necessary for T4 liberation, i.e., utilization of Tg.
Alterations in luminal Tg depositions are caused by deficiency in cathepsin B or L. Because reduced levels of serum T4 in cathepsin K–/–/L–/– mice are most probably caused by an impaired Tg degradation, the molecular status of Tg (Figure 7) and its localization (Figure 8) were analyzed next.
Tg persistence in thyroids of mice with deficiencies in cathepsin B or L. Lysates of thyroids from mice of the indicated genotypes were normalized to equal amounts of protein, separated on 10% SDS gels, and transferred to nitrocellulose for subsequent incubation of the blots with antibodies against Tg. (a) A representative immunoblot. (b) The uppermost portion of another immunoblot demonstrates the expression of dimeric (thick line) and monomeric Tg (thin line) in WT and all cathepsin-deficient thyroids, as well as the appearance of a high–molecular weight Tg fragment (broken line), which was observed in the lysates of cathepsin K–/–/L–/– thyroids only. Molecular mass markers are indicated in the left margin. (c) Densitometric profiles of the indicated bands representing intact Tg and several of its degradation fragments. (d) Three different immunoblots with two lanes per genotype were evaluated densitometrically. Mean values ± SD of the densities of the lanes are given. Note that the amounts of immunolabeled Tg were upregulated about five- to sixfold in thyroids of cathepsin B– or L–deficient mice, whereas cathepsin K–/– thyroids contained only about twofold the amounts of Tg present in WTs (d).
Equal amounts of protein of thyroid lysates from mice of the indicated genotypes were loaded onto SDS gels, and blots were probed with antibodies against Tg (Figure 7, a and b). Protein bands representing monomeric and dimeric Tg were present in all cathepsin-deficient genotypes (Figure 7b, thin and thick lines, respectively). Thyroid lysates from cathepsin K–/–/L–/– mice contained an additional band with higher electrophoretic mobility than monomeric Tg (Figure 7b, K–/–/L–/–, broken line). This band represented a high–molecular weight degradation fragment of Tg and indicated an alteration of Tg degradation in cathepsin K–/–/L–/– thyroids as compared with those of the other cathepsin-deficient genotypes. Densitometry of the gels demonstrated that the amounts of total Tg were higher in thyroids from mice of all cathepsin-deficient genotypes when compared with the WTs (Figure 7d). Comparable results were obtained when single bands representing intact Tg or its degradation fragments were analyzed by densitometry (Figure 7c). Five- to sixfold higher amounts of Tg were observed in thyroids from cathepsin B–/–, L–/–, B–/–/K–/–, or K–/–/L–/– mice, whereas cathepsin K deficiency alone resulted in Tg levels that were increased only about twofold over those of the WTs (Figure 7d), indicating that cathepsins B and L are more important than cathepsin K for the gross degradation of Tg.
Because Tg degradation is not restricted to intracellular locations like lysosomes but begins with its extracellular solubilization from the covalently cross-linked storage form, an alteration of Tg degradation might also affect its luminal and/or lysosomal depositions. Therefore, cryosections from thyroids were immunolabeled with antibodies against Tg (Figure 8). Within thyroid epithelial cells of all cathepsin-deficient genotypes, numerous reticular and vesicular structures were immunolabeled with Tg antibodies (Figure 8, insets, arrows). Such structures resemble cisternae of the endoplasmic reticulum as well as exo- and endocytic vesicles, indicating that Tg synthesis, export, and uptake generally occur in thyroid epithelial cells of all cathepsin-deficient genotypes. Additionally, immunofluorescence signals were often observed in association with the basal lamina surrounding individual thyroid follicles (Figure 8, insets, arrowheads), which are most probably representative of Tg reaching the circulation by transcytosis.
Luminal Tg showed the typical multilayered appearance within thyroid follicles of WT controls (Figure 8a). Storage of Tg within follicle lumina occurs in a highly compacted form in which Tg is multimerized and covalently cross-linked. Hence, antibodies are less able to interact with highly compacted Tg in the center of thyroid follicle lumina, resulting in dim fluorescence intensities. In contrast, the luminal portions apposed to the apical plasma membrane are characterized by intense immunofluorescence, i.e., soluble Tg is easily accessible for the antibodies (Figure 8a, broken arrows). Tg depositions within the lumina of thyroid follicles of cathepsin K–deficient mice (Figure 8c, broken arrows) were indistinguishable from those of WTs, whereas thyroids of all other cathepsin-deficient genotypes lacked the multilayered appearance of luminal Tg (Figure 8, b and d–f; compare with a), indicating that a deficiency in either cathepsin B or cathepsin L resulted in the absence of differentially compacted luminal Tg. These results are most probably explained by the notion that cathepsins B and L are involved in the solubilization of Tg from its covalently cross-linked storage form, which is achieved by limited extracellular proteolysis. Further experiments must show whether the extent of covalent cross-linkages in luminal Tg is altered in thyroid follicles of cathepsin B– or L–deficient mice.
The absence of multilayers of luminal Tg might also explain the observation of increased thyroid follicle dimensions in cathepsin B–/– mice, which could not be explained as a result of reduced serum T4 levels. If the rate of Tg export into the follicle lumen is unaltered, but the rate of removal of luminal Tg is reduced, then, as a consequence, the size of the luminal content must increase, resulting in an expansion of thyroid follicles. This is most clearly seen in cathepsin B–/– thyroid follicles (see B–/– in Figures 6, a and b; 7d; and 8b). A similar phenomenon is observed in cathepsin B–/–/K–/– thyroids but might be less pronounced when compared with the cathepsin B–/– genotype, because cathepsin L expression was upregulated in cathepsin B–/–/K–/– mice (Figure 4c) but not in cathepsin B–/– mice. These observations suggest a partial compensation of the double deficiency in cathepsins B and K by cathepsin L overexpression. Because systemic defects, i.e., significantly reduced serum levels of T4, were observed in cathepsin K–/–/L–/– mice only, the utilization of luminal Tg for T4 liberation is most probably mediated by a combinatory action of cathepsins K and L. The proposed sequential steps of proteolytic degradation of Tg for its solubilization and utilization are outlined in Figure 9.
Schematic drawing of the proposed sequence of proteolytic events leading to Tg degradation in mouse thyroid. Solubilization of extracellularly stored Tg from covalently cross-linked globules is mediated by cathepsins B and L. Soluble Tg is then subjected to limited proteolysis mediated by cathepsins K and L for Tg utilization, i.e., extracellular T4 liberation. Thereafter, partially degraded Tg re-enters thyroid epithelial cells by endocytosis and reaches endosomes and lysosomes for its complete degradation by the action of several lysosomal enzymes.