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Expression, Localization, and Thyrotropin Regulation of Cathepsin D in Human Thyroid Tissues

Biophysic Laboratory (T.M., F.B.), Department of Surgery (J-L.K., J.B.), Electron Microscopy (J-M.G., B.F., N.Q.), and Biostatistics (P.I.), Groupe de Recherche en Endocrinologie Expérimentale et Clinique, Jean Bernard Hospital, BP 577, 86021 Poitiers Cedex, France

Address all correspondence and requests for reprints to: Thierry Métayé, Laboratoire de Biophysique, Hôpital Jean Bernard, BP 577, 86021 Poitiers Cedex, France.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Enzymatic activity and isoform expression of cathepsin D (cath D) were studied in 107 cytosols from various human thyroid tissues including 21 normal tissues, 12 cold benign nodules, 17 toxic adenomas, 22 samples from Graves’ disease patients, and 35 thyroid carcinomas. Cath D assay was optimized for human thyroid tissues. We found that mean cath D specific activities, expressed as units per milligrams protein minus thyroglobulin, were higher in carcinomas (P = 0.0001), toxic adenomas (P = 0.0001), and specimens from Graves’ disease patients (P = 0.0001) than in normal thyroid tissues. Mean cath D activity in carcinomas was also significantly different from that in cold benign nodules (P < 0.001) and Graves’ disease tissues (P < 0.05) but not from that of toxic adenomas. To determine possible mechanisms by which the observed increase in cath D activity might be regulated, we used Western blotting to measure relative amounts of cath D isoforms in the various thyroid tissues. We found that the 31-kDa major processing form of cath D was significantly increased in carcinomas and toxic adenomas compared with normal tissues (P < 0.01), cold benign nodules (P < 0.05), and Graves’ disease tissues (P < 0.05). A positive correlation of cath D activity with relative expression of the 31-kDa form (r = 0.67, P = 0.0001) was observed in 104 thyroid cytosols. These data demonstrate a deregulation at the protein level, with resulting increases in cath D activity. Immunogold labeling of cath D showed particle concentration in lysosomes or phagosomes in both normal follicles and papillary carcinoma cells, indicating that cath D localization was not altered by malignant transformation in human thyroid cells. TSH induced cath D synthesis and secretion in extracellular fluid of normal human thyroid cells in primary culture; TSH had little effect on intracellular cath D level. In conclusion, TSH-induced cath D synthesis may explain high cath D levels in Graves’ disease tissues and toxic adenomas, because these tissues possess a permanently stimulated cAMP transduction pathway. Furthermore, the overexpression of cath D in thyroid carcinomas in comparison with normal controls adds further arguments for the potential role of cath D in tumor growth and metastasis.

	Introduction

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CATHEPSIN D (cath D) (EC 3.4.23.5) is a lysosomal proteolytic enzyme involved in protein catabolism, having an optimum activity at an acidic pH (3.0–5.0). It is a member of the aspartic protease gene family, which includes the secreted proteins pepsin, chymosin, and renin (1). Like other aspartic proteases, cath D is a pseudodimer in which the two parts of the molecule are mostly comprised of ß sheets flanking a deep active cleft (2). Each domain contributes an active site aspartate at the center of the cleft and contains a single carbohydrate group. Glycosylation at asparagine residues is responsible for the enzyme targeting to the lysosome via mannose 6-phosphate receptors (3). In certain tissues and cell types, a mannose 6-phosphate-independent pathway may also exist to direct proteins toward lysosomes (4). Cath D is synthesized in rough endoplasmic reticulum as a larger preproenzyme, which is subsequently converted to its mature form during its transport to lysosomes (5). The cleavage of prepeptide results in a 52- to 53-kDa catalytically inactive proenzyme (6). Next, the proenzyme is converted to a single-chain cath D (47- to 48-kDa) by removal of a 44-amino acid propeptide. The single-chain cath D undergoes further cleavage into a two-chain enzyme formed (7) by a light chain (14 kDa) and a heavy chain (31–34 kDa). The single-chain and two-chain enzyme appear to be equally active for cath D (8). During normal processing, various amounts of procathepsin D are secreted into the extracellular medium (9) and are available for receptor-mediated endocytosis (10).

Cath D is an ubiquitous protease involved in a variety of pathological situations such as Alzheimer’s disease (11), inflammatory processes (12), muscular dystrophy (13), and tumor metastasis (14). In human thyroid, cath D has been described as the main enzyme involved in thyroglobulin proteolysis (15, 16). Furthermore, this lysosomal aspartic endopeptidase appears to be quantitatively more important than thiol proteases in the initial phase of the digestion (17). Recently, we showed that cath D levels were significantly higher in thyroid tissues from patients with carcinomas, Graves’ disease, and toxic adenomas than in normal thyroid tissues (18). To gain further insight into the role and the regulation of cath D in pathological processes of the human thyroid gland, we present the results of enzymatic quantifications, isoform analysis, localization and TSH regulation of cath D in human thyroid tissues.

	Materials and Methods

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Patient thyroid samples

All samples from human thyroid tissues, including 21 histologically normal tissues, 12 benign nodules (8 multinodular goiters and 4 solitary nodules), 17 toxic adenomas, 22 tissues from patients with Graves’ disease, and 35 primary carcinomas, were obtained at operation and stored in liquid nitrogen for no more than 6 months. This study was approved by the Poitiers Hospital Ethics Committee. The complete nature of all thyroid tissues was characterized by histological examination. Normal thyroid tissues were adjacent to 9 benign nodules, 4 toxic adenomas, and 8 thyroid carcinomas. In patients with benign nodules, thyroid hormone levels were normal and ^99mTc scintigraphies showed cold nodules. Patients with toxic adenomas were hyperthyroid, and ^99mTc scintigraphies showed hot nodules. The diagnosis of Graves’ disease was based on a clinical hyperthyroid state with suppressed TSH, increased free T₃ and free T₄ in the presence of anti-TSH receptor antibodies, and a scintigraphically diffused hot thyroid. Patients with Graves’ disease were treated with carbimazole for at least 18 months before thyroidectomy and were in an euthyroid state. Patients with thyroid carcinomas were biologically euthyroid, and scintigraphies showed cold lesions. Calcitonin levels were assayed in all patients with thyroid nodules for diagnosis of medullary carcinomas.

The clinical characteristics of patients including age, sex, mean size of lesions, lymph node invasion, and presence or not of metastasis are summarized in Table 1.

Frozen tissues were cut into small pieces (2- to 3-mm cubes) with a razor blade and mechanically pulverized in an impact grinder at -180 C (Freezer mill, SPEX Industries, Edison, NJ). The powders were then homogenized at 4 C in three volumes of Tris buffer (10 mmol/L Tris-HCl, 1.5 mmol/L EDTA, 1.2 mmol/L dithiotreitol, and 5 mmol/L sodium molybdate, pH 7.4) using an Ultra-Turrax homogenizer (Janke and Kunkel, Staufen, Germany). The homogenates were centrifuged at 105,000 x g at 4 C for 1 h (L8–55, Beckman Instruments, Fullerton, CA) to isolate cytosols. Aliquots of cytosol samples were stored at -70 C until assays and immunoblottings of cath D.

Cath D enzymatic activity assay

Cath D enzymatic activity assay was derived from hemoglobin hydrolysis assay initially described by Anson (19). The reaction mixture containing 0.1 mol/L sodium formate buffer, pH 3.3, and 10–50 µL thyroid cytosol was preincubated 3 min at 37 C in a final volume of 700 µL. Then, 100 µL 4% acid-denatured hemoglobin was added, and incubation was done for 15 min at 37 C. The enzymatic reaction was stopped by 10% trichloroacetic acid. After centrifugation, absorbance of the supernatant fluid (containing the hemoglobin peptides) was determined at 280 nm. Cath D specificity was obtained using, in a parallel assay, 2 µmol/L pepstatin A (final concentration), a specific inhibitor of aspartic proteases (20). All incubations were done in duplicate. Assay results were linear between incubations for 5 min and 20 min, which resulted in the absorbance for 15 min being the difference between optical densities at these two periods of incubation. A unit of cath D activity is defined as that amount of enzyme necessary to cause an absorbance change of 1.0 at 280 nm for 60 min under the above-described experimental conditions. Specific activity is expressed as the units of cath D activity per milligram of total protein minus thyroglobulin (U/mg Pt-Tg). This expression was preferred to U/mg Pt because the major portion of thyroglobulin is not intracellular, and the different thyroid tissues did not contain the same amount of thyroglobulin. Protein concentrations were determined using the method of Lowry et al. (21) with BSA as standard, and cytosolic thyroglobulin levels using an immunoradiometric assay kit (Henning Berlin GmbH, Berlin, Germany).

Human thyroid cell culture

Normal human thyroid tissues were obtained aseptically from patients who underwent thyroid surgery, usually for uninodular or multinodular goiter. Subsequent steps were performed as previously described by Roger et al. (22). Thyroid tissues were subjected to enzymatic digestion in Ca²⁺- and Mg²⁺-free HBSS containing 1 g/L dispase (0.5 U/mg, Boehringer Mannheim, Germany) and 0.1 g/L collagenase (217 U/mg, Worthington Biochemical Corporation, Freehold, NJ). The isolated thyroid cells were cultured for 6 days in DMEM/Ham’s F-12 (1:1) (GIBCO BRL, Paisley, UK) containing 1.25 µg/mL human transferrin, 40 µg/mL vitamin C, and 5 µg/mL bovine insulin.

After cell culture, conditioned mediums were centrifugated to remove cell debris, then supernatants were pooled and concentrated to 50 µL using Microcon-10 concentrators (Amicon, Beverly, MA). Thyroid cells were detached from the culture dishes by treatment with 0.25% trypsin and 0.02% EDTA, washed, sonicated for 3 sec, and homogenized with a Dounce homogenizer in Tris buffer. Concentrated conditioned mediums and cell homogenates were stored at -70 C until use.

Electrophoresis and immunoblotting

SDS/PAGE was performed by the method of Laemmli (23) with a 12% separating gel. After electrophoresis, proteins were electrotransfered to a polyvinilidene difluoride membrane (PVDF) (Bio-Rad Labs., Hercules, CA) with a Bio-Rad Mini Trans-Blot apparatus (24). Unreactive sites on the PVDF membranes were blocked with nonfat dry milk. The PVDF membranes were then incubated for 1 h at room temperature in a 10^-3 dilution of polyclonal rabbit antihuman liver cath D (Athens Research and Technology, Athens, GA). After several washings, membranes were incubated for another hour in a 2 x 10^-3 dilution of horseradish peroxidase-labeled donkey antirabbit IgGs. Immunoreactive bands were visualized with commercial chemiluminescence system (ECL, Amersham International, Buckinghamshire, England) using Hyperfilm-ECL as described by the manufacturer. Films were optically scanned with a Hoefer Scientific model GS 300 densitometer (San Francisco, CA). The peak areas were analyzed with a GS 365W program, version 2.22 from Hoefer.

Transmission electron microscopy

The thyroid tissues were fixed in 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer (PB), pH 7.2, for 2 h at 4 C. After rinsing, samples were postfixed in 1% osmium tetroxide in PB for 1 h at 4 C, processed through a graded acetone series, embedded in Araldite (Fluka, Buchs, Switzerland), and polymerized overnight at 60 C. The sections (50 nm), stained with uranyl acetate and lead citrate, were examined with a 100CX Jeol electron microscope (Jeol, Tokyo, Japan)

Immunogold labeling procedure

Small samples of fresh thyroid tissues were fixed with 2% paraformaldehyde and 0.1% glutaraldehyde in PB. The samples were soaked for 1 h with 10 mmol/L glycine to block free aldehyde groups. After dehydratation in serial graded ethanol solution, samples were embedded in LR White resin (TAAB Labs., Aldermaston, UK). Thin sections (80 nm) were collected on nickel grids and etched successively in saturated aqueous sodium metaperiodate and 0.1 N HCl for 5 min. The sections were rinsed, blocked with 3% BSA, and then incubated overnight at 4 C with a 1/50 dilution of polyclonal rabbit anti-cath D (DAKO Corp., Carpinteria, CA). After several washings, sections were incubated for 1 h at 37 C with a 1/20 dilution of 10 nm gold-conjugated goat antirabbit IgGs (Sigma-Aldrich Chimie, St Quentin Fallavier, France). The procedure was specificity controlled using normal rabbit serum instead of anti-cath D antibody. Sections were then stained with uranyl acetate and examined in a Jeol 100CX electron microscope.

Statistical methods

Differences in cath D values and relative isoform quantities in the various thyroid tissues were analyzed using the Kruskall-Wallis and Mann-Whitney nonparametric tests. An association between relative percentages of each isoform and cath D activity was sought using the Spearman rank correlation coefficient.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cath D enzymatic activity

Enzymatic activity assay of cath D was realized with respect to reaction linearity between incubations of 5–20 min. We also documented that reaction velocity was proportional to cytosol quantity for an absorbance at 280 nm between 0.06–0.12 optical density. Cath D assay values were obtained using pepstatin A, an inhibitor of aspartic proteases. Because cath D is the main intracellular enzyme in this protease group (1), pepstatin-inhibited acid protease gives a close estimation of cath D activity. These assay conditions were used to compare cath D activity levels in thyroid tissues from patients with benign nodules, Graves’ disease, toxic adenomas, carcinomas, and normal controls (Table 2). Cath D-specific activities of carcinomas were significantly different from those in normal tissues (P = 0.0001), cold benign nodules (P < 0.001), and Graves’ disease specimens (P < 0.05) but not from those in toxic adenomas. Because of small sample numbers, results from follicular/trabecular, anaplastic, and medullary carcinomas could not be compared with the other groups. However, it is worth noting that mean values from anaplastic carcinomas were greater than those from well-differentiated thyroid carcinomas. Toxic adenomas and Graves’ disease samples had mean cath D activities statistically different from those of normal tissues and cold benign nodules.

Cath D isoforms in human thyroid tissues were analyzed using SDS/PAGE and immunoblotting. Results from a trabecular carcinoma, a toxic adenoma, a cold benign nodule with their adjacent normal tissues, together with a tissue cytosol from a patient with Graves’ disease are shown in Fig. 1. In each case, the major cath D form was a 52-kDa protein as determined by molecular mass standards. Another 31-kDa form that comigrated with a purified human liver cath D (lane 8) was prominent in the carcinoma (lane 1), the toxic adenoma (lane 3), and the Graves’ disease (lane 5) tissues. The 31-kDa protein level was visually decreased in the carcinoma, the toxic adenoma, and the cold benign nodule (lane 6) with respect to their adjacent normal tissues (lanes 2, 4, and 7). Other minor bands of 27 kDa, 28 kDa, 34 kDa, and 40 kDa were seen with varying detectable amounts in the different thyroid tissues. In the trabecular carcinoma, each of these minor bands had a greater intensity than in the normal tissue. To test antibody specificity, polyclonal anti-cath D was preincubated with purified cath D before Western blotting, which resulted in either the disappearance or a significant reduction in intensity of each bands with the exception of the 28-kDa band (data not shown).

View larger version (51K): [in this window] [in a new window]   Figure 1. Immunoblot analysis of cath D isoforms in different normal and pathological thyroid cytosols. Experiment was as described in Materials and Methods with same amounts of protein (10 µg) run in each lane. Lanes 1 and 2 represent a trabecular carcinoma and its adjacent normal tissue, respectively. Lanes 3 and 4 represent a toxic adenoma and its adjacent normal tissue, respectively. Lane 5 is a tissue cytosol from a patient with Graves’ disease. Lanes 6 and 7 represent a cold benign nodule and its adjacent normal tissue, respectively. Lane 8 is a purified human liver cath D (35.6 mU). Arrowheads indicate 52-, 34-, 31-, and 27-kDa proteins as calculated by mobility of molecular weight markers.

Cath D immunogold labeling

We present the results of immunocytochemical cath D localization in normal human thyroid tissue and primary papillary adenocarcinoma. Ultrastructural studies of normal thyroid (Fig. 2A) showed that follicular cells were arranged in a single layer around the central colloid. Microvilli were easily identified; the nuclei contained homogeneous chromatin. Within the cytoplasm, lysosomes were prominent; endoplasmic reticulum and mitochondria were also observed. Papillary adenocarcinoma revealed a papillae growth pattern; the nuclei were irregular with invaginations (Fig. 2C). The cancerous cell cytoplasm contained many inclusions and phagosomes.

View larger version (195K): [in this window] [in a new window]   Figure 2. Ultrastructural localization of cath D in normal human thyroid (A and B) and papillary adenocarcinoma (C and D). Samples were treated for transmission electron microscopy (A and C) and for immunogold labeling (B and D) as described in Materials and Methods. Immunogold particules were present predominantly in lysosomes and phagosomes of normal human thyroid and papillary adenocarcinoma, respectively. Ly, lysosome; Ph, phagosome; V, vessel; N, nucleus; C, colloid.

TSH regulation of cath D in human thyroid cell culture

Cellular and secreted cath D isoforms from human thyroid cell cultures were analyzed by SDS/PAGE and immunoblotting (Fig. 3). In cell homogenate, a single major band corresponding to the mature cath D form (31 kDa) was specifically detected by anti-cath D antibody. Treatment of thyroid cells for 6 days with TSH produced a weak increase (1.10-fold) in cellular 31-kDa isoform when compared with the control. After concentration of conditioned mediums, two bands were detected in the extracellular compartment. They correspond to secreted cath D isoforms and were identified by their molecular masses as the precursor (52 kDa) and the mature (31 kDa) cath D forms. TSH cell treatment increased the 52- and 31-kDa protein secretion by 3.1- and 5.3-fold, respectively, when compared with the control (Fig. 3). Increase of cath D level cannot be attributed to the mitogenic action of TSH, because identical cell numbers were counted in the presence and absence of TSH at the end of thyroid cell culture. Summation of the densitometry analysis of cath D bands in the cell homogenate and medium provided a measure of cath D contents. Using the difference between TSH and controls in human primary thyroid cell culture, we calculated TSH-stimulated newly synthesized cath D to be 40% secreted.

View larger version (50K): [in this window] [in a new window]   Figure 3. Immunoblot analysis of cellular and secreted cath D isoforms from normal human thyroid cell culture. Thyroid cells were cultured for 6 days without (C) or with 5 mU/mL TSH. Conditioned mediums then were concentrated and cells treated as described in Materials and Methods. Aliquots (6 µL) of concentrated mediums and similar amounts of protein (10 µg) from cell homogenates were analyzed by Western blotting. Arrowheads indicate 52- and 31-kDa proteins as calculated by mobility of molecular weight markers.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We previously reported the absence of a significant difference between neoplastic and normal human thyroid tissues in terms of estrogen receptor content (18). These results, associated with the very low estrogen receptor level, indicated that cath D was probably not regulated by estrogen in human thyroid contrary to human breast cancer cells (30). In this study, we show that cath D is induced by TSH in normal human thyroid cell culture. Because most of TSH effects in thyroid cells are mediated by cAMP (31), activation of cAMP transduction system in Graves’ disease (32), toxic adenomas (33), and differentiated thyroid carcinomas (34) may increase cath D activity. However, in anaplastic and medullary thyroid carcinomas, cAMP transduction system is not stimulated by TSH. This suggests that in carcinomas, cath D levels may be controlled by both a cAMP-dependent mechanism as well as a cAMP-independent mechanism.

Interest in cath D initially came from breast cancer studies, in which it appeared to be involved in tumor growth and metastasis (35). Our data indicate a positive correlation between the overexpression of cath D and the degree of thyroid malignancy in patients with benign nodule formation, well-differentiated malignant tumors, and anaplastic cancer. Although these histological changes are not necessarily sequential, there is evidence (36) that proliferative gradation and differentiative potential exist among cells in each thyroid follicle. Because toxic adenomas and Graves’ disease tissues are able to produce increased cath D activities and yet remain nontumorigenic, cath D may not be directly involved in the process of thyroid tumor development, but it probably gives a selective growth advantage to a precancerous lesion. The cooperative role of cath D in actions of thyroid growth factors such as insulin-like growth factor-I (37) and basic fibroblast growth factor (38) but also the participation of cath D in TSH-induced thyroid cell proliferation may explain cath D mitogenic effects in thyroid tissue. Furthermore, the positive correlation existing between tissular cath D level and thyroid cancer size (39) corroborates cath D effects in cellular growth processes.

In summary, our study shows that cath D activity is significantly increased in thyroid carcinomas, toxic adenomas, and tissues from Grave’ disease when compared with cold benign nodules and normal controls. TSH induces cath D synthesis and secretion in normal human thyroid cell culture. We cannot make conclusions about the involvement of cath D in the process of thyroid tumor development, however, the gradual overexpression of this protease in normal thyroid tissues, benign goiters, and thyroid carcinomas adds further arguments for the potential mitogenic effect of cath D.

	Acknowledgments

 
We are grateful to N. Met and J.-P. Reynaud for their excellent technical assistance. We warmly thank M. Garcia from Pr. H. Rochefort Laboratory, INSERM U148, Montpellier, for helpful discussion and careful reading of the manuscript.

Received March 5, 1997.

Revised June 4, 1997.

Accepted June 20, 1997.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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