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Gender-Specific Pattern of Insulin-Like Growth Factor-Binding Protein-3 Protease Activity in Mouse Thyroid

Department of Molecular Medicine, Karolinska Institute, Stockholm S-171 76, Sweden

Address all correspondence and requests for reprints to: Moira S. Lewitt, Karolinska Institutet, Department of Molecular Medicine, Karolinska Hospital, M1:02, Stockholm S-171 76, Sweden. E-mail: Moira.Lewitt{at}molmed.ki.se.

	Abstract

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There is evidence that the IGF system plays an important role in the growth and function of the thyroid gland. Proteolysis is an important posttranslational process that modulates the affinity of IGF binding proteins (IGFBPs) to IGFs, thus regulating their activity. IGFBP-3 has been shown to be cleaved by members of the kallikrein family, some of which are expressed in human thyroid and are characterized by regulation by steroid hormones. The aim of this study was to determine whether IGFBP-3 protease activity is present in mouse thyroid tissue and to characterize its activity by gender and nutrition. Male and female BALB/c mice, aged 16 wk, were studied in the fasted state, or after 1-h or 4-h refeeding. IGFBP protease activity was present in thyroid tissue and resulted in a decrease in IGFBP-3 affinity for IGF-I. The activity was inhibited by 10 mM ZnCl₂, activated by CaCl₂, and was substantially greater in tissue from male mice compared with that from female animals. These properties and the pattern of effect of a panel of protease inhibitors were consistent with this protease being a member of the tissue kallikrein family. Serum inhibited the proteolytic effect of thyroid extracts. There was no effect of nutrition. In conclusion, the degree of activity of IGFBP-3 protease in mouse thyroid tissue is gender specific and is likely to lead to an increased IGF availability in male mice.

	Introduction

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THE IGF SYSTEM, comprising IGF-I and -II, IGF-binding proteins (IGFBPs) 1–6, and their receptors and proteases, plays a key role in normal cellular physiology by regulating proliferation, apoptosis, and differentiation in a variety of tissues. In thyroid cells, IGF-I has been shown to potentiate TSH-induced proliferation in vitro (1). Elevated circulating IGF-I, such as occurs in acromegaly, is associated with goiter (2). As well as deriving from endocrine sources, the components of the IGF system are present within thyroid tissue (3) and may be involved in thyrocyte proliferation via autocrine or paracrine mechanisms. Transgenic mice overexpressing IGF-I and the type 1 IGF receptor in thyroid have goiter, increased circulating thyroid hormone levels, and reduced TSH concentrations (4). Thyroid hormones in turn modify the IGF system in a variety of ways including a central effect on GH secretion (5) and a stimulating action on the secretion of IGFBP-1 by liver cells (6). TSH inhibits IGFBP expression and secretion by thyroid (3, 7, 8), including a 46-kDa IGFBP consistent with IGFBP-3 (8).

The IGF system is also believed to play an important role in the growth of thyroid cancer cells, with an autocrine loop of increased IGF-II secretion and the insulin receptor-A isoform having been implicated (9). IGF-I expression is also increased in thyroid tumors (10).

The IGFBPs are cleaved by proteases that may have tissue-specific roles (11). Whereas thyroid tissue is known to express a number of proteases, the role and regulation of IGFBP-specific proteases is not known. Some of the proteases identified in human thyroid tissue (12, 13) have also been shown to degrade IGFBP-3, e.g. human kallikrein 2 (hK2) (14), prostate-specific antigen (PSA or hK3) (14, 15), and cathepsin D (16). The rat thyroid follicular cell line FRTL-5 has been shown to have a cell-associated IGFBP-3 protease (17). Proteases in the thyroid have the potential to cleave locally secreted IGFBP-3 as well as IGFBP-3 derived from the circulation. Proteolytic cleavage is likely to result in decreased affinity for IGFs; however, additional direct, IGF-independent effects have been documented for IGFBP-3 fragments, for example to induce apoptosis (18).

The aim of this study was to determine whether IGFBP-3 protease activity is present in mouse thyroid tissue. We have previously characterized the IGF system in normal mice and peroxisome proliferator-activated receptor (PPAR)- knockout animals and identified a gender-specific IGFBP-3 protease in plasma that was induced by refeeding after fasting in PPAR- knockout mice (19). In the present study, therefore, we also determined the effect of gender and nutrition on any IGFBP-3 activity detected in mouse thyroid.

	Materials and Methods

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Reagents
Recombinant nonglycosylated human IGFBP-3 (hIGFBP-3) was purchased from Diagnostic Systems Laboratories (Webster, TX), and 2.5-µg aliquots were iodinated by the chloramine T method. Glycosylated hIGFBP-3 was obtained from GroPep Limited (Thebarton, Australia). Recombinant human IGF-I was kindly provided by Kabi Pharmacia (Stockholm, Sweden), and 5-µg aliquots were iodinated using lactoperoxidase and purified by HPLC. Disuccinimidyl suberate (DSS) was purchased from Pierce (Rockford, IL). All other reagents, including human PSA (minimum purity 95%), the serine protease inhibitors phenylmethylsulfonyl fluoride (PMSF) and aprotinin; a kallikrein inhibitor cyclohexylacetyl-Phe-Arg-Ser-Val-Gln amide (C₃₆H₅₈N₁₀O₈, Sigma C-6922); the cysteine protease inhibitors leupeptin and E64; and the aspartic protease inhibitor, pepstatin A, were obtained from Sigma (Stockholm, Sweden). The protease inhibitors were dissolved in Dulbecco’s PBS (Invitrogen, Stockholm, Sweden) before use.

Animals
All animal studies were approved by the local animal ethics committee (North Stockholm). BALB/c mice were brought into the animal facility 2 wk before the experiment to adapt to the environment. They were housed under a constant light-dark cycle (light from 0700–1700 h) and received a standard diet (R70, Lactamin) and free access to water. Male and female mice, aged 16 wk, were fasted overnight for 16–18 h, with free access to water during this time. At the end of the fasting period, they were each weighed and then randomly divided into three groups: one group was studied in the fasting state (seven males, seven females), another was allowed free access to food for 1 h (1-h refed; seven males, five females), and the third was given access to food for 4 h before sampling (4-h refed; five males, seven females). At the end of the experiment, body weight was measured again in the refed animals. Blood was taken by cardiac puncture under CO₂ inhalation anesthesia, serum separated, and stored at -20 C. Thyroid tissues were dissected, weighed, frozen in liquid nitrogen, and stored at -80 C.

PPAR- knockout mice were kindly provided by Professor Gustav Dallner (Stockholm University, Stockholm, Sweden). We have previously characterized the IGF system and the effect of feeding in these mice (19). In each mouse, serum was collected, after CO₂ anesthesia, both by cardiac puncture and after decapitation. Serum was separated and stored at -20 C.

Preparation of tissue extracts
The entire thyroid gland was homogenized in 500 µl PBS, 1 mM CaCl₂, and centrifuged to remove tissue and cell debris, and the supernatant was stored at -20 C. The protein contents of tissue extracts were then measured by Bio-Rad protein microassay according to the manufacturer’s instruction (Bio-Rad, Sundbyberg, Sweden), using transferrin for the standard curve. The protein content of the extract represented 20.6 ± 0.9% and 14.1 ± 1.0% of the original thyroid weight, for males and females, respectively.

Protease assay for nonglycosylated IGFBP-3
Proteolysis of nonglycosylated IGFBP-3 was assayed after the method of Lamson et al. (20). Radioiodinated nonglycosylated IGFBP-3 (20,000 cpm) was incubated with mouse thyroid extract, PSA, or 0.1 µl mouse serum in PBS containing 1 mM CaCl₂, unless otherwise indicated, in a final volume of 10 µl at 37 C for 2 h. The reaction was terminated by the addition of Laemmli buffer. [¹²⁵I]IGFBP forms were separated on 14% nonreducing SDS-PAGE. The gels were then fixed, dried, and analyzed after phosphoimaging using the Fuji PhosphoImage program (Fuji Co., Stockholm, Sweden). The gels shown in the figures are representative of at least three experiments with similar results.

Protease assays for glycosylated IGFBP-3
Proteolysis of glycosylated IGFBP-3 was determined by ligand-binding assays using radioiodinated IGF-I. Glycosylated IGFBP-3 was incubated in the absence or presence of mouse thyroid extract in PBS containing 1 mM CaCl₂ in a final volume of 5 µl at 37 C for 2 h. Radioiodinated IGF-I (100,000 cpm) was added to each sample in a final volume of 9 µl (0.035% BSA) in the presence and absence of increasing concentrations of cold IGF-I for an additional 2 h at 22 C. Cross-linking was carried out by adding DSS in dimethylsulfoxide to a final concentration of 1 mM, and after 30 min at 22 C, the reaction was terminated by adding a 0.1 vol of 0.5 M Tris-HCl (pH 8.0). Samples were subjected to SDS-PAGE as described above. On each gel, samples of intact IGFBP-3 were run alongside those of IGFBP-3 that had been proteolysed by thyroid extract from a single mouse. Gels demonstrating the competitive displacement of radioiodinated IGF-I by cold IGF-I were repeated on the thyroid extract from four individual mice.

Ligand binding was also carried out in a solution assay. Glycosylated IGFBP-3 was incubated in the absence and presence of mouse thyroid extract in PBS containing 1 mM CaCl₂ in a final volume of 5 µl at 37 C for 2 h. Radioiodinated IGF-I (20,000 cpm) was added to each sample in a final volume of 150 µl of 0.05 M sodium phosphate (pH 6.5) with 0.25% BSA. After a 2-h incubation at 22 C, unbound IGF-I tracer was precipitated by the addition of 1 ml ice-cold 0.05 M sodium phosphate (pH 6.5) containing 0.25% charcoal and 2% BSA. After 1 h at 4 C, samples were centrifuged at 3200 rpm for 15 min, and 575 µl of supernatant was counted in a -counter. Nonspecific binding was between 5 and 12% of total counts, and results are expressed as a percentage of IGF-I tracer binding to intact IGFBP-3 in the absence of protease.

Statistics
Unless otherwise indicated, the statistical analysis was by two-way ANOVA followed, if appropriate, by the Student-Newman-Keuls post hoc test (SigmaStat, Jandel Scientific Software, San Rafael, CA). The effect of protease inhibitors and activators was determined by one-way ANOVA followed by the Student-Newman-Keuls test. Statistical significance was set at a P value less than 0.05. Results are expressed as the mean ± SEM.

	Results

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The thyroid weight was larger in male mice, both in terms of absolute weight and after correction for body weight (see Table 1). We studied the presence of IGFBP-3 protease activity in thyroid tissue. The thyroid was isolated and homogenized in PBS, and identical concentrations of extract were incubated with radioiodinated nonglycosylated hIGFBP-3 and subjected to SDS-PAGE. As shown in Fig. 1, IGFBP-3 protease activity was present in 80-ng samples extracted from thyroids of male mice, whereas very little activity was observed under these experimental conditions using the same amount of thyroid tissue extract from female animals. The activity in the extract from each mouse was determined on at least two occasions alongside other samples from each gender and nutritional group. The degree of activity between individual mice was similar in the same gender group (Fig. 1), and there was no obvious change in IGFBP-3 protease activity with feeding in either male or female animals (data not shown). Because there was a similar degree of activity within each gender group, a pool of material containing equal volumes of thyroid extract from the 4-h refed male mice (n = 5) was used to further characterize the IGFBP-3 protease activity.

View larger version (87K): [in this window] [in a new window]   FIG. 1. Gender-specific pattern of IGFBP-3 protease activity in mouse thyroid. Thyroid tissue extracts representing 80 ng protein were incubated with iodinated nonglycosylated hIGFBP-3 for 2 h at 37 C and then subjected to gel electrophoresis under nonreducing conditions as described in Materials and Methods. IGFBP-3 fragments were detected by autoradiography after drying the gel. [125I]IGFBP-3 was incubated alone (lane 1) or in the presence of thyroid extracts from 4-h refed mice. In a representative gel, samples from three males (lanes 2–4) and three females (lanes 7–9) are shown alongside a pooled sample from five males (lane 5) and seven females (lane 6). Molecular mass markers are indicated by the arrows.

View larger version (96K): [in this window] [in a new window]   FIG. 2. Pattern of fragmentation of nonglycosylated IGFBP-3 by protease(s) in mouse thyroid, compared with that of human PSA. Decreasing concentrations of an extract of thyroid tissue (pooled from five male mice, 4 h after refeeding) were incubated with iodinated nonglycosylated human IGFBP-3 for 2 h at 37 C and then subjected to gel electrophoresis under nonreducing conditions as described in Materials and Methods. IGFBP-3 fragments were detected by autoradiography after drying the gel. The pattern of fragmentation generated by increasing concentrations of PSA is shown for comparison. [125I]IGFBP-3 alone, subjected to the same incubation conditions, is shown in the first lane (*). Molecular mass markers are indicated by the arrows.

View larger version (68K): [in this window] [in a new window]   FIG. 3. Effect of protease inhibitors and cations on IGFBP-3 protease activity. A, The effect of protease inhibitors on the IGFBP-3 protease activity generated by 40 ng of thyroid tissue extract (pooled from 4-h refed male mice; n = 5). Samples were incubated with thyroid extract, alone and in the presence of the protease inhibitors PMSF, aprotinin, cyclohexylacetyl-Phe-Arg-Ser-Val.Gln amide (kallikrein inhib), leupeptin, and pepstatin A. Also shown is the effect of zinc chloride (0.1–10 mM) or calcium chloride (10 mM) on the IGFBP-3 protease activity derived from mouse thyroid tissue (A) and on human PSA (2 µg) activity (B) in the presence and absence of EDTA, as indicated. Samples were incubated with iodinated nonglycosylated human IGFBP-3 for 2 h at 37 C and then subjected to gel electrophoresis under nonreducing conditions as described in Materials and Methods. IGFBP-3 fragments were detected by autoradiography after drying the gel. [125I]IGFBP-3 alone, subjected to the same incubation conditions, is shown on each gel (*). Molecular mass markers are indicated by the arrows. C, Densitometry results from three gels. Results are expressed as a percentage of intact [125I]IGFBP-3 run in the absence of extract (mean ± SEM). , P < 0.05, compared with thyroid extract alone.

View larger version (38K): [in this window] [in a new window]   FIG. 4. Effect of increasing concentrations of cations on IGFBP-3 proteolysis by mouse thyroid extract. Samples containing 40 ng of thyroid tissue extract (pooled from 4-h refed male mice; n = 5) were incubated with iodinated nonglycosylated human IGFBP-3 in the absence and presence of increasing concentrations of ZnCl2 (0.001–10 mM) and CaCl2 (0.1–10 mM) with and without EDTA (10 mM). A, After incubation for 2 h at 37 C, samples were then subjected to gel electrophoresis under nonreducing conditions as described in the Materials and Methods. IGFBP-3 fragments were detected by autoradiography after drying the gel. [125I]IGFBP-3 alone, subjected to the same incubation conditions, is shown in the first lane (*). Molecular mass markers are indicated by the arrows. B, Pooled densitometry results from six gels using 10 mM ZnCl2 or 10 mM CaCl2 in the absence and presence of EDTA (10 mM). Results are expressed as a percentage of intact [125I]IGFBP-3 run in the absence of extract (mean ± SEM). , P < 0.001, compared with thyroid extract alone; , P < 0.001, effect of EDTA + CaCl2 compared with CaCl2 alone.

View larger version (46K): [in this window] [in a new window]   FIG. 5. Potential inhibitory effect of serum on the IGFBP-3 protease activity derived from mouse thyroid. A, The protease activity from two PPAR- knockout male mice (KO1 and KO2) was obtained after 4-h refeeding after a 16-h fasting period, both by cardiac puncture (lanes 2 and 4) and from trunk blood (lanes 3 and 5). B, The effect of 0.1 µl serum on protease activity in increasing concentrations of thyroid tissue extract. The serum sample and tissue extract represented a pool from five normal male mice after 4-h refeeding. For each gel, samples were incubated with iodinated nonglycosylated human IGFBP-3 for 2 h at 37 C and then subjected to gel electrophoresis under nonreducing conditions as described in the Materials and Methods. IGFBP-3 fragments were detected by autoradiography after drying the gel. [125I]IGFBP-3 alone, subjected to the same incubation conditions, is shown on each gel (*). Molecular mass markers are indicated by the arrows.

View larger version (37K): [in this window] [in a new window]   FIG. 6. Glycosylated IGFBP-3 has a reduced affinity for IGF-I after proteolysis by mouse thyroid tissue extract. IGFBP-3 (8 ng) was incubated with 100 ng thyroid tissue extract from each of four male 4-h refed mice for 2 h at 37 C. Samples containing 8 ng intact IGFBP-3 or proteolysed IGFBP-3 were then incubated with radioiodinated IGF-I in the presence of increasing concentrations of cold IGF-I for an additional 2 h at 22 C. After cross-linking with DSS, samples were subjected to gel electrophoresis under nonreducing conditions and detected by autoradiography after drying the gel, as outlined in the Materials and Methods. A, A representative gel showing the results from one animal. B, Densitometry results from four animals (mean ± SEM). , P < 0.001, compared with intact IGFBP-3 at the same concentration of cold IGF-I.

View larger version (15K): [in this window] [in a new window]   FIG. 7. Effect of gender on the proteolysis of glycosylated IGFBP-3 by mouse thyroid tissue. Increasing concentrations of pooled extracts of mouse thyroid, from male and female mice, 4-h refed after a 16-h fasting period, were incubated with 8 ng of glycosylated human IGFBP-3 for 2 h at 37 C. Samples were then incubated with radioiodinated IGF-I and subjected to charcoal precipitation as outlined in the Materials and Methods. The nonspecific counts (in the absence of IGFBP-3) in this experiment represented 9.1% of the total counts. Results are expressed as percent specific binding to intact IGFBP-3 in the absence of extract.

	Discussion

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The members of the tissue kallikrein gene family are serine proteases that localize to the same chromosomal locus and have considerable structural similarities (21, 22). Several proteins expressed by this family have been shown to cleave IGFBP-3. Two of these are human kallikreins, hK2 (23, 24) and hK3 (PSA) (15, 24). The kallikreins are characterized by hormonal regulation by sex steroids (22). Of interest, both of these are androgen up-regulated genes that are expressed in thyroid (12, 22). Three mouse kallikreins degrade IGFBP-3 and are encoded by the genes mGK3 [-subunit of 7S nerve growth factor (NGF)] (25), mGK21 (26), and mGK27 (27). In mice, mGK21 (26) and mGK27 (27) are expressed in male-specific tissues. The protease that we have identified in mouse thyroid appears to relate closely in function to hK2, generating a similar pattern of fragmentation (14); however, this gene is said to be lacking in the mouse (21). It is possible of course that another androgen-dependent serine protease such as kallikrein 4 (22), which does have a mouse homolog (21) but which is not yet characterized in terms of IGFBP-3 proteolysis, is responsible. It is also possible that more than one protease is present and responsible for IGFBP-3 degradation in this crude thyroid extract. Of note, we observed that the potency of IGFBP-3 protease activity in crude extracts of mouse thyroid is much higher than that of PSA, exceeding its potency more than 100-fold, assuming that these proteases have a similar molecular mass.

The protease activity in mouse thyroid was inhibited by high, supraphysiological concentrations of zinc chloride (10 mM). A similar phenomenon has been observed for hK2 (23) and PSA (15). Zinc also prevents NGF from dissociating and releasing active -subunit, and 1 µM concentrations should result in negligible -subunit protease activity (28). Cohen and colleagues (25), however, found that 1 mM zinc had no effect on the IGFBP-3 proteolysis induced by 7 S NGF. In our study 10 mM zinc was required to effectively inhibit the protease activity of mouse thyroid extract. However, the effect of zinc, taken together with the observation that concentrations of NGF in the submandibular gland are greater in male mice (28), suggest that mGK3 could still be a candidate for the IGFBP-3 protease activity that we have observed in thyroid. In our studies, the dramatic effect of calcium, to activate IGFBP-3 proteolysis, occurred at concentrations closer to those present in the cellular environment. Of interest, a calcium-dependent serine protease that cleaves IGFBP-3 has been observed in rat thyroid FRTL-5 cells (17). Because our tissue was homogenized, it is not clear in which cell compartment our protease was found, although one can speculate on a role for calcium in regulating intracellular IGFBP-3 availability, e.g. for apoptosis (29). We consistently observed an inhibitory effect on protease activity of the combination of calcium and EDTA. At present we cannot offer an explanation for this phenomenon.

Endogenous IGFBP-3 is glycosylated, a feature that is proposed to protect it from proteolytic degradation (30). In our study, we show that glycosylated IGFBP-3 was degraded using rather similar concentrations of mouse thyroid extract to those used for nonglycosylated IGFBP-3. Furthermore, cleavage of glycosylated IGFBP-3 generated a single IGF-binding form with reduced IGF-I affinity, as indicated by an approximately 10-fold increase in cold IGF-I required to displace IGF-I tracer binding. Proteolysis of any endocrine or locally derived IGFBP-3 is therefore likely to lead to increased IGF availability to the type 1 IGF-I receptor. In the thyrocyte milieu, such an increase in IGF action may lead to a potentiation of TSH signaling (1). Testosterone is an important mitogen to thyrocytes in male rats, enhancing the action of TSH and having an independent stimulatory effect on proliferation (31). We speculate on the role of IGFBP-3 proteolysis and therefore increased IGF availability in this phenomenon. It is interesting that T₃ stimulates the production of the hK2 in prostate carcinoma cells (32). Whether local thyroid hormone production has a local paracrine effect on protease activity in thyroid is unknown.

The IGF system, including IGFBP-3 proteolysis, potentially plays a role in determining gender-related differences in thyroid disease. We noted that the size of the thyroid gland was greater in male mice, independent of body weight. In aging rats, there are gender differences in thyroid function, with male animals having more pronounced decreases in thyroid hormone secretion with age (33). In humans, the incidence of differentiated thyroid cancer is higher in women; however, men with thyroid cancer are reported to have reduced survival (34).

The system in vivo, however, is likely to be complex. Many kallikreins are associated with activation cascades, and there may be more than one IGFBP-3 protease present in the tissue. Protease inhibitors may also be present. We found that mouse serum inhibited the proteolytic effect of thyroid extracts, but this was overcome at high concentrations of thyroid tissue. Circulating PSA is also associated with inhibitors of IGFBP-3 protease activity (24). We have previously identified a nutritionally regulated IGFBP-3 protease in trunk blood from male PPAR- knockout mice (19). Absence of this activity in samples obtained by cardiac puncture now suggests that it may have represented contamination by thyroid tissue. Our findings highlight the importance of avoiding contamination by tissue fluids of blood samples in studies of IGFBP proteolysis. In the current study, nutritional regulation of IGFBP-3 proteolysis was not observed in thyroid extracts from control mice. We can only speculate that variation in the concentration or pattern of circulating inhibitors is likely to be responsible for the effect of nutrition in the previous study. Whether IGFBP-3 protease in thyroid is able to reach the circulation, and whether it has an endocrine role, is entirely speculative. It is interesting that hK2 mRNA is detectable in the circulation of patients with thyroid cancer, where it is proposed to be a marker of disease (35).

	Acknowledgments

 
We thank Professors Kerstin Brismar and Kerstin Hall for helpful discussions and their support and Inga-Lena Wivall-Helleryd for expert technical assistance.

	Footnotes

 
This work was supported by the Swedish Research Council (4224), the Novo Nordisk Foundation, the Swedish Diabetes Association, and the Persson Family Foundation.

Abbreviations: DSS, Disuccinimidyl suberate; hK2, human kallikrein 2; IGFBP, IGF binding protein; NGF, nerve growth factor; PMSF, phenylmethylsulfonyl fluoride; PPAR, peroxisome proliferator-activated receptor; PSA, prostate-specific antigen.

Received October 14, 2003.

Accepted for publication December 4, 2003.

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