Type 2 Iodothyronine Selenodeiodinase Is Expressed throughout the Mouse Skeleton and in the MC3T3-E1 Mouse Osteoblastic Cell Line during Dif
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内分泌学杂志 2005年第1期
Department of Anatomy (C.H.A.G., C.R.Z.), Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo 05508, Brazil; Thyroid Section (M.A.C., J.W.H., A.C.B.), Division of Endocrinology, Diabetes, and Hypertension, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115; and Endocrine Division (J.M.D., A.L.M.), Hospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, Porto Alegre 90035, Brazil
Address all correspondence and requests for reprints to: Antonio C. Bianco, M.D., Ph.D., Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, Harvard Institutes of Medicine Building 643, Boston Massachusetts 02115. E-mail: abianco@partners.org.
Abstract
Thyroid hormone affects multiple aspects of bone metabolism, but little is known about thyroid hormone deiodination in bone cells except that cultures of skeletal cells and bone organ express types 1 and 2 iodothyronine deiodinases (D1 and D2) mRNAs. In the present study, outer ring deiodination (ORD) activity was detected in bone extracts of multiple sites of the mouse skeleton, bone marrow, and the MC3T3-E1 osteoblastic cell line. In all tissues, ORD was detected using 125I-rT3 or 125I-T4 as substrates and was found to be 6-n-propylthiouracil insensitive, display a Michaelis constant (T4) of approximately 1 nM, increase about 3-fold in hypo- and virtually disappear in thyrotoxicosis. Extracts of calvaria had the lowest ORD activity, whereas tibial and femoral extracts had roughly three times as much. The absence of ORD activity in bone extracts from mice with targeted disruption of the Dio2 gene confirms the principal role of D2 in this tissue. In the MC3T3-E1 osteoblasts, D2 activity increased in a time-dependent manner after plating, and with the content of selenium in the media, reaching a maximum 5–7 d later as cells attained more than 90% confluence. In these cells D2 half-life is about 30–40 min, which is further accelerated by exposure to substrate and stabilized by the proteasome inhibitor, MG132. Treatment with vitamin D [1,25(OH)2VD]-induced D2 activity by 2- to 3-fold as early as 24 h, regardless of the level of cell confluence, but estradiol, PTH, forskolin, leptin, TNF, TGF?, and dexamethasone did not affect D2. Given the role of D2 in other cell types and processes, it is likely that bone ORD not only plays a role in bone development and adult bone T3 homeostasis but also contributes to extrathyroidal T3 production and maintenance of serum T3.
Introduction
IN BRAIN, PITUITARY gland, and brown adipose tissue (BAT), the intracellular thyroid status is largely determined by the isolated or combined expression of types 2 (D2) and 3 (D3) iodothyronine deiodinase, independent of serum T3. These enzymes are involved in the activation of the prohormone T4 via outer ring deiodination (ORD) to T3 or inactivation of T4 and T3 via inner ring deiodination to reverse T3 and T2, respectively (for review see Ref. 1). Because D2 is an endoplasmic reticulum-resident protein distributed in the perinuclear region (2), T3 produced via D2 reaches the nucleus and thus contributes significantly to the thyroid hormone receptor saturation. D3, on the other hand, is a plasma membrane protein with its active center located in the extracellular space (3), thus constituting an efficient barrier against the entrance of T4 or T3 into the cells and decreasing the overall thyroid hormone receptor saturation.
T4 activation via ORD can also be catalyzed by type 1 iodothyronine deiodinase (D1). However, D1 has a 3 orders of magnitude higher Michaelis constant (Km) for T4 and is located in the plasma membrane, and thus its product (T3) equilibrates rapidly with the plasma (2, 4). Therefore, D2 is considered the critical T4-activating enzyme (1). Studies in mice with targeted disruption of the Dio2 gene, which have normal serum T3, demonstrate that intracellular D2-catalyzed T4 activation to T3 is critical for thyroid hormone-dependent cochlear development (5), TSH feedback mechanism (6), and adaptive lipogenesis and thermogenesis in BAT (7, 8). D2 expression is predominantly regulated at the posttranslational level by well-conserved endoplasmic reticulum-associated ubiquitination mechanisms that are accelerated during the conversion of T4 to T3 (9, 10). Therefore, the approximately 40-min half-life of D2 is prolonged to approximately 300 min in the absence of T4 or shortened to approximately 25 min in its presence, constituting an intracellular feedback mechanism to control T3 homeostasis. In addition, D2 responsiveness to cAMP constitutes the basis for its rapid neural stimulation in several tissues, linking D2 expression with the hypothalamus and widening the spectrum of environmental and endogenous stimuli that potentially influence adaptive T3 production (for review see Ref. 1).
D2 mRNA was reported to be induced by 1,25-dihydroxyvitamin D3 [1,25(OH)2VD] in primary osteoblastic cells derived from mouse calvaria and ST2 cells, a murine bone marrow-derived stromal cell line that has the phenotypes of osteoblast- and osteoclast-supporting cells (11). In addition, mRNAs for D1 and D2 were detected in organ cultures of fetal mouse tibias and ATDC5 cells (12), a mouse chondrogenic cell line. These findings are potentially relevant because thyroid hormone is an important regulator of bone development and metabolism. Hypothyroidism causes a generalized delay in endochondral and intramembranous ossification in addition to important alterations in the epiphyseal growth plates, such as reduced thickness, disorganized columns of chondrocytes, and impaired differentiation of hypertrophic chondrocytes (13, 14), resulting in reduced growth and skeletal abnormalities (15). In adults, hyperthyroidism increases the activity of both osteoblasts and osteoclasts; however, the latter predominate, favoring resorption, negative balance of calcium, and bone loss (16). Thyrotoxicosis has been related to reduced bone mineral density at multiple skeletal sites and increased risk of hip fracture (16, 17). It is interesting that, as opposed to the generalized osteopenic effects of the T3 in experimental animals (18), long-term administration of T4 leads to a preferential femoral over vertebral bone loss, regardless of the type of bone, i.e. cortical vs. trabecular bone (12, 19, 20). This finding raises the possibility that thyroid hormone activation plays a differential role in the regulation of the local thyroid status within the bone tissue, depending on the skeletal site.
In the present investigation, we demonstrate the presence of D2 activity in both bone and bone marrow of several skeletal sites of the adult mouse, thus suggesting that D2 helps determine the intracellular thyroid status of differentiating as well as mature bone cells. In addition, D2 activity was found in the MC3T3-E1 mouse osteoblastic cell line, in which it exhibits similar biochemical and cellular properties as described in the other tissues in which D2 is involved in the regulation of intracellular thyroid status. No D1 activity was found in adult bone or the mouse cell line.
Materials and Methods
MG132, a proteasome inhibitor, was obtained from Calbiochem (La Jolla, CA). T4; rT3; cycloheximide (CX); Na2SeO3; 1,25(OH)2VD, the active metabolite of vitamin D; forskolin; PTH; TGF?; dexamethasone (Dexa); leptin; 17?-estradiol; and 6-n-propylthiouracil (PTU), a specific inhibitor of D1, were obtained from Sigma-Aldrich (St. Louis, MO). 125I-T4 and 125I-rT3 (specific activity, 4400 Ci/mmol) were from PerkinElmer Life Sciences (Norwalk, CT). All other reagents were of analytical grade.
Cell culture and D2 activity
MC3T3-E1 cells were kindly provided by Dr. Henry Kronenberg (Endocrine Unit, Massachusetts General Hospital and Harvard Medical School). Cells were maintained in MEM (Life Technologies, Inc.-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (Life Technologies, Inc.-BRL), 100 U/ml penicillin, 100 μg/ml streptomycin (Life Technologies, Inc.-BRL), and 1–200 nM Na2SeO3. Seven days after seeding, at approximately 90% confluence, cells were treated with T4 (10–8 M); rT3 (10–8 M); MG132 (10–5 M); CX (10–6 M); 1,25(OH)2VD (10–8 M); forskolin (10–7 M); PTH (10–8 M);. TGF? (5 ng/ml); Dexa (10–7 M); leptin (10–7 M); 17?-estradiol (10–8 M); or vehicle for 24 h. Each experiment was performed with duplicate dishes for each condition. At the appropriate times, cells were harvested and processed for D2 activity as described previously (21).
Animal studies
All experiments were performed under a protocol approved by the Harvard Medical School Standing Committee on Animals. C57BL/6J (B6) mice were obtained from the Animal Resource colony of the Jackson Laboratory (Bar Harbor, ME). Mice with targeted disruption of the D2 gene were developed in a C57BL/6–129SV strain background (6). All mice were males and approximately 2 months old. Animals were housed in plastic cages (four per cage) at 21 C; light cycles were 12 h, and access to food and water was ad libitum. B6 animals were left untreated or treated with methimazole (0.1%) and sodium perchlorate (1%), added to the drinking water, to induce hypothyroidism or were treated with T4, receiving 10 μg T4 per 100 g body weight per day (ip). Treatments with methimazole + sodium perchlorate or T4 lasted 10 d.
All mice were killed by cervical dislocation, and the bones were dissected and cooled with liquid nitrogen. Bone marrow from femur, tibia, and humerus were collected by centrifugal isolation as previously described (23) with modifications. Briefly, bones were dissected and their proximal or distal ends were removed. Bones were placed in Microfuge tubes supported by plastic inserts (cut from 0.5-ml Microfuge tubes) and centrifuged at 4000 rpm for 2 min, 4 C. The bone tissue was transferred to another tube and stored at –80 C. The bone marrow pellet was washed with 1 ml ice-cold PBS and centrifuged at 2000 x g for 1 min at 4 C. The supernatant was removed and the pellet resuspended in 80–100 μl ice-cold 0.1 M phosphate buffer (pH 7.0), containing 1 mM EDTA, 0.25 M sucrose, and 10 mM dithiothreitol (PE-DTT buffer). Samples were sonicated, and the protein concentration was quantified by the Bradford method (24). The bones, freed from bone marrow, were crushed in a steel mortar and pestle set (Fisher Scientific International, Inc, Hampton, NH) precooled in dry ice. The crushed bones were transferred to Microfuge tubes precooled in ice and containing 100 μl ice-cold PE-DTT buffer with 5 mM taurodeoxycholate per bone (femur, tibia, etc.). The samples were vortexed for 30 sec, centrifuged at 4,000 rpm for 1 min at 4 C, sonicated, and centrifuged at 12,000 x g for 5 min, 4 C. Supernatants were transferred to clean tubes, and 100 μl ice-cold PE-DTT buffer containing 5 mM taurodeoxycholate per bone were added to the pellets. Samples were vortex for 30–60 sec, recentrifuged (12,000 x g for 5 min at 4 C); the supernatant was transferred to the respective clean tubes, and the protein concentration was quantified. The deiodination activity was assayed in the bone marrow and bone tissue extracts as previously described in the presence of 20 mM dithiothreitol (25).
Statistical analysis
Multiple comparisons were performed by ANOVA followed by the Student-Newman-Keuls test.
Results
D2 activity in bone
To verify the presence of ORD activity in the bone tissue, mouse bone extracts (200–300 μg protein from femur or tibia) were incubated with 0.5 nM 125I-rT3, a substrate suitable for measuring ORD via D1 or D2. Under these conditions the substrate consumption was about 6% and the rate of ORD about 0.3 fmol/min·mg protein (Fig. 1A). The addition of 1 mM PTU, a specific D1 inhibitor, to the reaction mixture did not affect ORD, indicating a predominant PTU-insensitive pathway, suggestive of D2 (data not shown). The presence of D2 activity was further supported by data obtained from bone extracts assayed in the presence of 100 nM 125I-rT3, an approach that has been used to saturate the low-Km D2 enzyme with unlabeled substrate (26). Substrate-saturation reduced 125I-rT3 deiodination in about 65% in the bone extracts, confirming the presence of substantial low-Km ORD activity (Fig. 1A). That this saturable component is D2 was demonstrated by assaying bone samples obtained from mice with targeted disruption of the Dio2 gene (Dio2–/–), which showed no saturable component when assayed at 0.5 and 100 nM rT3 (Fig. 1A). Using 1 mM PTU and 100 nM 125I-rT3 to establish the level of nonsaturable ORD, we obtained an apparent Km (T4) of approximately 1 nM, the D2 kinetic identity (Fig. 1B). Additional studies performed in mice displayed the expected thyroid hormone regulation of this enzyme, an approximately 3-fold increase in ORD activity observed in femur extracts of hypothyroid mice, and suppression to near background levels in the T4-treated mice (Fig. 1C).
FIG. 1. D2 activity in bone extracts. A, D2 activity in the femur and tibia of wild-type and Dio2–/– mice. B, Lineweaver-Burke plot of the substrate saturation curve of D2 activity in femoral extracts. C, D2 activity in the femur of hypothyroid (hypo), control, and T4-treated mice. D, D2 activity in bone extracts of several skeletal sites. D2 assay was performed using the indicate concentrations of 125I-rT3 in the presence of 1 mM PTU and 20 mM dithiothreitol. All results are the mean ± SD of four to six mice. *, P < 0.05 vs. control by ANOVA.
One interesting observation was the presence of a substantial (35%), nonsaturable, PTU-insensitive 125I-rT3 metabolizing pathway in both 100 nM rT3 and Dio2–/– samples, which cannot be attributed to D1 or D2 (Fig. 1A). This pathway was more than 80% inhibited by reducing the incubation temperature to 4 C, supporting an enzymatic nature to this reaction (data not shown). Although rT3 is not a good substrate for D3, inner ring deiodination was also excluded by the absence of deiodination with 0.5 nM 125I-T3 as substrate (data not shown). It is notable that the activity of this 125I-rT3 metabolizing pathway was present in femur and tibia of Dio2–/– mice and was not affected by thyroid status (Fig. 1C), which suggests that this pathway is not deiodination and could represent, for example, ether bond cleavage of the 125I-rT3 by monocyte/macrophage bone cells such as observed in peripheral leukocytes (27). The resulting catabolite would still be detected in the assay together with the free 125I–, but conveniently this can be discriminated by the strategy outlined above.
D2 activity was then measured in mouse bone tissue extracts obtained from several skeletal sites (Fig. 1D). Calvaria had the lowest D2 activity of all sites tested, about 0.1 fmol/min·mg protein, whereas tibia and femur had roughly 3 times as much. D2 activity in vertebrae, scapula, humerus, and radioulna was between 0.1 and 0.2 fmol/min·mg protein (Fig. 1D). We also evaluated D2 activity in bone marrow extracts of humerus, femur, and tibia. In these sites D2 activity varied between 0.15 and 0.2 fmol/min·mg protein.
D2 is expressed in the MC3T3-E1 mouse osteoblastic cell line
We extended our studies to evaluate the deiodination pathway in MC3T3-E1 osteoblasts. D2 activity was found in the MC3T3-E1 osteoblasts at about the same velocity observed in bone extracts (Fig. 2A). However, the presence of that nonsaturable PTU-insensitive 125I-rT3 metabolization pathway, which cannot be attributed to D1 or D2, was not observed in these cells, suggesting that such pathway in bone is unrelated to osteoblasts. Because D2 is a selenoprotein, endogenous expression of D2 depends on the concentration of Na2SeO3 in the medium (25). To establish optimal conditions for studying D2 activity, MC3T3-E1 osteoblasts were grown for 5 d in the presence of 1–200 nM Na2SeO3. It is notable that D2 activity increased with the Na2SeO3 concentration, reaching a plateau of about 6-fold at 100–200 nM (Fig. 2A). Therefore, all subsequent experiments were performed with medium containing 100 nM Na2SeO3.
FIG. 2. Effects of selenium, 1,25(OH)2VD, and proteasome inhibitor on D2 activity in MC3T3-E1 osteoblastic cells. A, D2 activity in cells grown for 5 d with 1–200 nM Na2SeO3. B, Time-dependent induction of D2 activity in vehicle- and 1,25(OH)2VD-treated (10–8 M) cells. C, D2 activity in cells exposed to 30 nM rT3 and treated with MG132 (10–5 M), a proteasome inhibitor, or vehicle. All results are the mean ± SD of three to six points.
Interestingly, D2 activity in MC3T3-E1 osteoblasts increased in a time-dependent manner after the cells were plated, reaching a maximum at about d 7 when cells attained more than 90% confluence (Fig. 2B). Longer culture times (up to 4 wk) in the presence of differentiating agents (25 μg/ml ascorbic acid and 2 mM ?-glycerol phosphate) did not induce further D2 activity (data not shown). Because of the reported induction of D2 mRNA by 1,25(OH)2VD in murine primary osteoblastic cells (11), we tested whether this was the case in MC3T3-E1 osteoblasts. In fact, treatment with 1,25(OH)2VD induced D2 activity by 2- to 2.5-fold after as little as 24 h of exposure. The same fold induction was observed if treatment was extended up to 96 h, regardless of the level of cell confluence (Fig. 2B).
One of the hallmarks of D2 expression is sensitivity with loss of activity as a result of exposure to its own substrate, T4 or rT3, which is due to selective ubiquitination and proteasomal degradation (28). Substrate-induced loss of D2 activity was also observed in MC3T3-E1 osteoblasts as early as 30 min after addition of rT3 to the medium (Fig. 2C). Treatment with MG132, a proteasome inhibitor, not only increased basal D2 activity but also prevented the loss of D2 activity induced by rT3 (Fig. 2C). In addition, D2 displays a relatively rapid turnover rate in MC3T3-E1 osteoblasts, as determined by the loss of D2 activity in cells exposed to CX, which is also prevented by MG132 added to the cells just 10 min before CX (Fig. 3A), indicating that D2 inactivation is mediated by the ubiquitin-proteasomal system (1). These experiments were repeated in MC3T3-E1 osteoblasts that had been treated with 1,25(OH)2VD for 48 h to verify whether such treatment affects D2 expression at posttranslational level; similar results were obtained.
FIG. 3. Effects of multiple treatments on D2 activity in MC3T3-E1 osteoblastic cells. A, Loss of D2 activity 0–120 min after vehicle- or MG132-treated cells were exposed to CX. B, Same as in A except that cells were previously treated for 48 h with 1,25(OH)2VD (10–8 M). C, D2 activity in cells previously treated with vehicle or 1,25(OH)2VD for 24 h and then exposed to the indicated substances, as described in Materials and Methods. All results are the mean ± SD of three to six points.
The presence of D2 in bone and MC3T3-E1 osteoblasts as well as its inducibility by 1,25(OH)2VD prompted us to study what other bone relevant substances could influence D2 activity in these cells. Thus, MC3T3-E1 osteoblasts were treated for 24 h with TGF?, Dexa, TNF, leptin, forskolin, PTH, or 17?-estradiol, but no significant change in D2 activity was observed (Fig. 3C). These various treatments were then applied to cells that had been pretreated with 10–8 M 1,25(OH)2VD for 24 h, and still no effect was observed (Fig. 3C).
Discussion
To our knowledge this is the first demonstration that D2 activity is present in extracts of bone tissue as well as bone marrow throughout the skeleton of the adult mouse. The presence of D2 activity in bone was suggested in a previous study of organ-cultured mouse tibias (12). However, given its incomplete characterization, the PTU-insensitive apparent ORD pathway described in that study is more likely to represent the nonsaturable, PTU-insensitive 125I-rT3 metabolizing pathway described here in the femur and tibia of wild-type and Dio2–/– mice (Fig. 1, A and C). In the present study, bone D2 activity has been identified by its PTU insensitivity (Fig. 1), Km (T4) in the nano molar range (Fig. 1B), short half-life that is stabilized in the presence of a proteasome inhibitor (Fig. 3, A and B), and typical induction during hypothyroidism and suppression by hyperthyroidism (Fig. 1C) or exposure to substrate (Fig. 2C). The existence of D2 in bone supports a tightly controlled local T3 homeostasis, similar to what has been extensively demonstrated in other D2-expressing tissues such as brain and BAT (1). Given the relatively large volume of bone tissue, skeletal D2 is also likely to play a previously unanticipated role in systemic T3 homeostasis, contributing to extrathyroidal T3 production and maintenance of serum T3.
In addition to bone, we found D2 activity in the bone marrow of different skeletal sites, which contains multipotent stromal and hematopoietic cells that differentiate, respectively, into osteoblasts and osteoclasts (29, 30). D2 activity was also found in MC3T3-E1 cells, which are known to differentiate in culture from a preosteoblastic to a mature osteoblastic phenotype, a process that is accelerated by 1,25(OH)2VD (31). Remarkably, D2 activity in MC3T3-E1 cells was not only stimulated by 1,25(OH)2VD but also was increased with time after cells were seeded, compatible with D2-generated T3 playing a role in osteoblastic differentiation. In fact, it is known that thyroid hormone induces osteoblast differentiation (32, 33) and increases osteoclastogenesis (34).
D2-specific activity and the biochemical and biological properties of D2 in MC3T3-E1 cells are similar to those found in other cell lines endogenously expressing this enzyme. In addition, MSTO-211 cells are the only other cell line in which basal D2 expression is enhanced by exposure to Na2SeO3. However, MC3T3-E1 cells fail to respond to other agents known to affect D2 activity in other cell types, such as adrenergic stimulators, leptin, and TNF. It is also notable that other substances known to affect bone metabolism such as PTH, estradiol, and Dexa also did not affect D2 activity in these cells, suggesting a high degree of specificity of the 1,25(OH)2VD effect. Exposure of MC3T3-E1 cells to 1,25(OH)2VD did not affect D2 half-life, the main pathway by which D2 activity is controlled. This finding, along with the previous report that D2 mRNA is increased by treatment with 1,25(OH)2VD (11), confirms that the induction of the Dio2 gene is the main mechanism by which vitamin D receptor or vitamin D receptor-dependent signals increase D2 activity.
In accord with the reported induction of D2 mRNA by 1,25(OH)2VD in murine primary osteoblastic cells and ST2 cells (11), we found that, in MC3T3-E1 cells, 1,25(OH)2VD treatment increased D2 activity by 2.5-fold as early as 24 h and independently of cell confluence. This result prompted us to investigate the effect of other bone-resorbing as well as bone-formation agents on D2 activity in these cells. These various treatments, alone or in combination with 1,25(OH)2VD, did not affect basal or 1,25(OH)2VD-induced D2 activity. Nevertheless, the positive effect of 1,25(OH)2VD on D2 activity suggests a possible synergism between thyroid hormone and 1,25(OH)2VD on the regulation of osteoblastic function. In fact, these two hormones have been shown to influence MC3T3-E1 cells at different levels, such as in the regulation of osteocalcin and matrix metalloproteinase-13 mRNA expression (35) and the molecular organization of adherens junctions among these osteblastic cells (36). Interaction of thyroid hormone with 1,25(OH)2VD was also shown in mouse bone marrow cell cultures, in which T3 in combination with 1,25(OH)2VD, but not alone, induced osteoclastogenesis in a synergic manner (34).
Interestingly, in preosteoblastic cells, which were shown to express D2 after 1,25(OH)2VD treatment, T3 and T4 were shown to induce mRNA expression of receptor activator of nuclear factor-B (RANK) ligand (11). RANK ligand is a key factor that binds to its cognate receptor, RANK, in osteoclast precursors to activate osteoclastogenesis (22). In addition, it was shown that either T4 or T3 was able to enhance the 1,25(OH)2VD-induced osteoclastogenesis in coculture of bone marrow cells with primary osteoblasts, which suggested that T4 was converted to T3 in this system (11).
The presence of D2 activity throughout the skeleton of the mouse suggests that local deiodinase activity is important to bone physiology, ensuring that intracellular T3 homeostasis is attained in the skeleton to promote differentiation and/or regulate the activity of bone cells. The identification of D2 activity in MC3T3-E1 cells and its induction by 1,25(OH)2VD support the view that D2 is important to bone metabolism and that there is a cross-talk between thyroid hormone and 1,25(OH)2VD, involving D2 to control the differentiation or function of bone cells.
Acknowledgments
The authors are grateful to Dr. Stephen A. Huang and Michelle Mulcahey for performing D3 assays and Dr. Henry Kronenberg for providing the MC3T3-E1 cells.
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Address all correspondence and requests for reprints to: Antonio C. Bianco, M.D., Ph.D., Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, Harvard Institutes of Medicine Building 643, Boston Massachusetts 02115. E-mail: abianco@partners.org.
Abstract
Thyroid hormone affects multiple aspects of bone metabolism, but little is known about thyroid hormone deiodination in bone cells except that cultures of skeletal cells and bone organ express types 1 and 2 iodothyronine deiodinases (D1 and D2) mRNAs. In the present study, outer ring deiodination (ORD) activity was detected in bone extracts of multiple sites of the mouse skeleton, bone marrow, and the MC3T3-E1 osteoblastic cell line. In all tissues, ORD was detected using 125I-rT3 or 125I-T4 as substrates and was found to be 6-n-propylthiouracil insensitive, display a Michaelis constant (T4) of approximately 1 nM, increase about 3-fold in hypo- and virtually disappear in thyrotoxicosis. Extracts of calvaria had the lowest ORD activity, whereas tibial and femoral extracts had roughly three times as much. The absence of ORD activity in bone extracts from mice with targeted disruption of the Dio2 gene confirms the principal role of D2 in this tissue. In the MC3T3-E1 osteoblasts, D2 activity increased in a time-dependent manner after plating, and with the content of selenium in the media, reaching a maximum 5–7 d later as cells attained more than 90% confluence. In these cells D2 half-life is about 30–40 min, which is further accelerated by exposure to substrate and stabilized by the proteasome inhibitor, MG132. Treatment with vitamin D [1,25(OH)2VD]-induced D2 activity by 2- to 3-fold as early as 24 h, regardless of the level of cell confluence, but estradiol, PTH, forskolin, leptin, TNF, TGF?, and dexamethasone did not affect D2. Given the role of D2 in other cell types and processes, it is likely that bone ORD not only plays a role in bone development and adult bone T3 homeostasis but also contributes to extrathyroidal T3 production and maintenance of serum T3.
Introduction
IN BRAIN, PITUITARY gland, and brown adipose tissue (BAT), the intracellular thyroid status is largely determined by the isolated or combined expression of types 2 (D2) and 3 (D3) iodothyronine deiodinase, independent of serum T3. These enzymes are involved in the activation of the prohormone T4 via outer ring deiodination (ORD) to T3 or inactivation of T4 and T3 via inner ring deiodination to reverse T3 and T2, respectively (for review see Ref. 1). Because D2 is an endoplasmic reticulum-resident protein distributed in the perinuclear region (2), T3 produced via D2 reaches the nucleus and thus contributes significantly to the thyroid hormone receptor saturation. D3, on the other hand, is a plasma membrane protein with its active center located in the extracellular space (3), thus constituting an efficient barrier against the entrance of T4 or T3 into the cells and decreasing the overall thyroid hormone receptor saturation.
T4 activation via ORD can also be catalyzed by type 1 iodothyronine deiodinase (D1). However, D1 has a 3 orders of magnitude higher Michaelis constant (Km) for T4 and is located in the plasma membrane, and thus its product (T3) equilibrates rapidly with the plasma (2, 4). Therefore, D2 is considered the critical T4-activating enzyme (1). Studies in mice with targeted disruption of the Dio2 gene, which have normal serum T3, demonstrate that intracellular D2-catalyzed T4 activation to T3 is critical for thyroid hormone-dependent cochlear development (5), TSH feedback mechanism (6), and adaptive lipogenesis and thermogenesis in BAT (7, 8). D2 expression is predominantly regulated at the posttranslational level by well-conserved endoplasmic reticulum-associated ubiquitination mechanisms that are accelerated during the conversion of T4 to T3 (9, 10). Therefore, the approximately 40-min half-life of D2 is prolonged to approximately 300 min in the absence of T4 or shortened to approximately 25 min in its presence, constituting an intracellular feedback mechanism to control T3 homeostasis. In addition, D2 responsiveness to cAMP constitutes the basis for its rapid neural stimulation in several tissues, linking D2 expression with the hypothalamus and widening the spectrum of environmental and endogenous stimuli that potentially influence adaptive T3 production (for review see Ref. 1).
D2 mRNA was reported to be induced by 1,25-dihydroxyvitamin D3 [1,25(OH)2VD] in primary osteoblastic cells derived from mouse calvaria and ST2 cells, a murine bone marrow-derived stromal cell line that has the phenotypes of osteoblast- and osteoclast-supporting cells (11). In addition, mRNAs for D1 and D2 were detected in organ cultures of fetal mouse tibias and ATDC5 cells (12), a mouse chondrogenic cell line. These findings are potentially relevant because thyroid hormone is an important regulator of bone development and metabolism. Hypothyroidism causes a generalized delay in endochondral and intramembranous ossification in addition to important alterations in the epiphyseal growth plates, such as reduced thickness, disorganized columns of chondrocytes, and impaired differentiation of hypertrophic chondrocytes (13, 14), resulting in reduced growth and skeletal abnormalities (15). In adults, hyperthyroidism increases the activity of both osteoblasts and osteoclasts; however, the latter predominate, favoring resorption, negative balance of calcium, and bone loss (16). Thyrotoxicosis has been related to reduced bone mineral density at multiple skeletal sites and increased risk of hip fracture (16, 17). It is interesting that, as opposed to the generalized osteopenic effects of the T3 in experimental animals (18), long-term administration of T4 leads to a preferential femoral over vertebral bone loss, regardless of the type of bone, i.e. cortical vs. trabecular bone (12, 19, 20). This finding raises the possibility that thyroid hormone activation plays a differential role in the regulation of the local thyroid status within the bone tissue, depending on the skeletal site.
In the present investigation, we demonstrate the presence of D2 activity in both bone and bone marrow of several skeletal sites of the adult mouse, thus suggesting that D2 helps determine the intracellular thyroid status of differentiating as well as mature bone cells. In addition, D2 activity was found in the MC3T3-E1 mouse osteoblastic cell line, in which it exhibits similar biochemical and cellular properties as described in the other tissues in which D2 is involved in the regulation of intracellular thyroid status. No D1 activity was found in adult bone or the mouse cell line.
Materials and Methods
MG132, a proteasome inhibitor, was obtained from Calbiochem (La Jolla, CA). T4; rT3; cycloheximide (CX); Na2SeO3; 1,25(OH)2VD, the active metabolite of vitamin D; forskolin; PTH; TGF?; dexamethasone (Dexa); leptin; 17?-estradiol; and 6-n-propylthiouracil (PTU), a specific inhibitor of D1, were obtained from Sigma-Aldrich (St. Louis, MO). 125I-T4 and 125I-rT3 (specific activity, 4400 Ci/mmol) were from PerkinElmer Life Sciences (Norwalk, CT). All other reagents were of analytical grade.
Cell culture and D2 activity
MC3T3-E1 cells were kindly provided by Dr. Henry Kronenberg (Endocrine Unit, Massachusetts General Hospital and Harvard Medical School). Cells were maintained in MEM (Life Technologies, Inc.-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (Life Technologies, Inc.-BRL), 100 U/ml penicillin, 100 μg/ml streptomycin (Life Technologies, Inc.-BRL), and 1–200 nM Na2SeO3. Seven days after seeding, at approximately 90% confluence, cells were treated with T4 (10–8 M); rT3 (10–8 M); MG132 (10–5 M); CX (10–6 M); 1,25(OH)2VD (10–8 M); forskolin (10–7 M); PTH (10–8 M);. TGF? (5 ng/ml); Dexa (10–7 M); leptin (10–7 M); 17?-estradiol (10–8 M); or vehicle for 24 h. Each experiment was performed with duplicate dishes for each condition. At the appropriate times, cells were harvested and processed for D2 activity as described previously (21).
Animal studies
All experiments were performed under a protocol approved by the Harvard Medical School Standing Committee on Animals. C57BL/6J (B6) mice were obtained from the Animal Resource colony of the Jackson Laboratory (Bar Harbor, ME). Mice with targeted disruption of the D2 gene were developed in a C57BL/6–129SV strain background (6). All mice were males and approximately 2 months old. Animals were housed in plastic cages (four per cage) at 21 C; light cycles were 12 h, and access to food and water was ad libitum. B6 animals were left untreated or treated with methimazole (0.1%) and sodium perchlorate (1%), added to the drinking water, to induce hypothyroidism or were treated with T4, receiving 10 μg T4 per 100 g body weight per day (ip). Treatments with methimazole + sodium perchlorate or T4 lasted 10 d.
All mice were killed by cervical dislocation, and the bones were dissected and cooled with liquid nitrogen. Bone marrow from femur, tibia, and humerus were collected by centrifugal isolation as previously described (23) with modifications. Briefly, bones were dissected and their proximal or distal ends were removed. Bones were placed in Microfuge tubes supported by plastic inserts (cut from 0.5-ml Microfuge tubes) and centrifuged at 4000 rpm for 2 min, 4 C. The bone tissue was transferred to another tube and stored at –80 C. The bone marrow pellet was washed with 1 ml ice-cold PBS and centrifuged at 2000 x g for 1 min at 4 C. The supernatant was removed and the pellet resuspended in 80–100 μl ice-cold 0.1 M phosphate buffer (pH 7.0), containing 1 mM EDTA, 0.25 M sucrose, and 10 mM dithiothreitol (PE-DTT buffer). Samples were sonicated, and the protein concentration was quantified by the Bradford method (24). The bones, freed from bone marrow, were crushed in a steel mortar and pestle set (Fisher Scientific International, Inc, Hampton, NH) precooled in dry ice. The crushed bones were transferred to Microfuge tubes precooled in ice and containing 100 μl ice-cold PE-DTT buffer with 5 mM taurodeoxycholate per bone (femur, tibia, etc.). The samples were vortexed for 30 sec, centrifuged at 4,000 rpm for 1 min at 4 C, sonicated, and centrifuged at 12,000 x g for 5 min, 4 C. Supernatants were transferred to clean tubes, and 100 μl ice-cold PE-DTT buffer containing 5 mM taurodeoxycholate per bone were added to the pellets. Samples were vortex for 30–60 sec, recentrifuged (12,000 x g for 5 min at 4 C); the supernatant was transferred to the respective clean tubes, and the protein concentration was quantified. The deiodination activity was assayed in the bone marrow and bone tissue extracts as previously described in the presence of 20 mM dithiothreitol (25).
Statistical analysis
Multiple comparisons were performed by ANOVA followed by the Student-Newman-Keuls test.
Results
D2 activity in bone
To verify the presence of ORD activity in the bone tissue, mouse bone extracts (200–300 μg protein from femur or tibia) were incubated with 0.5 nM 125I-rT3, a substrate suitable for measuring ORD via D1 or D2. Under these conditions the substrate consumption was about 6% and the rate of ORD about 0.3 fmol/min·mg protein (Fig. 1A). The addition of 1 mM PTU, a specific D1 inhibitor, to the reaction mixture did not affect ORD, indicating a predominant PTU-insensitive pathway, suggestive of D2 (data not shown). The presence of D2 activity was further supported by data obtained from bone extracts assayed in the presence of 100 nM 125I-rT3, an approach that has been used to saturate the low-Km D2 enzyme with unlabeled substrate (26). Substrate-saturation reduced 125I-rT3 deiodination in about 65% in the bone extracts, confirming the presence of substantial low-Km ORD activity (Fig. 1A). That this saturable component is D2 was demonstrated by assaying bone samples obtained from mice with targeted disruption of the Dio2 gene (Dio2–/–), which showed no saturable component when assayed at 0.5 and 100 nM rT3 (Fig. 1A). Using 1 mM PTU and 100 nM 125I-rT3 to establish the level of nonsaturable ORD, we obtained an apparent Km (T4) of approximately 1 nM, the D2 kinetic identity (Fig. 1B). Additional studies performed in mice displayed the expected thyroid hormone regulation of this enzyme, an approximately 3-fold increase in ORD activity observed in femur extracts of hypothyroid mice, and suppression to near background levels in the T4-treated mice (Fig. 1C).
FIG. 1. D2 activity in bone extracts. A, D2 activity in the femur and tibia of wild-type and Dio2–/– mice. B, Lineweaver-Burke plot of the substrate saturation curve of D2 activity in femoral extracts. C, D2 activity in the femur of hypothyroid (hypo), control, and T4-treated mice. D, D2 activity in bone extracts of several skeletal sites. D2 assay was performed using the indicate concentrations of 125I-rT3 in the presence of 1 mM PTU and 20 mM dithiothreitol. All results are the mean ± SD of four to six mice. *, P < 0.05 vs. control by ANOVA.
One interesting observation was the presence of a substantial (35%), nonsaturable, PTU-insensitive 125I-rT3 metabolizing pathway in both 100 nM rT3 and Dio2–/– samples, which cannot be attributed to D1 or D2 (Fig. 1A). This pathway was more than 80% inhibited by reducing the incubation temperature to 4 C, supporting an enzymatic nature to this reaction (data not shown). Although rT3 is not a good substrate for D3, inner ring deiodination was also excluded by the absence of deiodination with 0.5 nM 125I-T3 as substrate (data not shown). It is notable that the activity of this 125I-rT3 metabolizing pathway was present in femur and tibia of Dio2–/– mice and was not affected by thyroid status (Fig. 1C), which suggests that this pathway is not deiodination and could represent, for example, ether bond cleavage of the 125I-rT3 by monocyte/macrophage bone cells such as observed in peripheral leukocytes (27). The resulting catabolite would still be detected in the assay together with the free 125I–, but conveniently this can be discriminated by the strategy outlined above.
D2 activity was then measured in mouse bone tissue extracts obtained from several skeletal sites (Fig. 1D). Calvaria had the lowest D2 activity of all sites tested, about 0.1 fmol/min·mg protein, whereas tibia and femur had roughly 3 times as much. D2 activity in vertebrae, scapula, humerus, and radioulna was between 0.1 and 0.2 fmol/min·mg protein (Fig. 1D). We also evaluated D2 activity in bone marrow extracts of humerus, femur, and tibia. In these sites D2 activity varied between 0.15 and 0.2 fmol/min·mg protein.
D2 is expressed in the MC3T3-E1 mouse osteoblastic cell line
We extended our studies to evaluate the deiodination pathway in MC3T3-E1 osteoblasts. D2 activity was found in the MC3T3-E1 osteoblasts at about the same velocity observed in bone extracts (Fig. 2A). However, the presence of that nonsaturable PTU-insensitive 125I-rT3 metabolization pathway, which cannot be attributed to D1 or D2, was not observed in these cells, suggesting that such pathway in bone is unrelated to osteoblasts. Because D2 is a selenoprotein, endogenous expression of D2 depends on the concentration of Na2SeO3 in the medium (25). To establish optimal conditions for studying D2 activity, MC3T3-E1 osteoblasts were grown for 5 d in the presence of 1–200 nM Na2SeO3. It is notable that D2 activity increased with the Na2SeO3 concentration, reaching a plateau of about 6-fold at 100–200 nM (Fig. 2A). Therefore, all subsequent experiments were performed with medium containing 100 nM Na2SeO3.
FIG. 2. Effects of selenium, 1,25(OH)2VD, and proteasome inhibitor on D2 activity in MC3T3-E1 osteoblastic cells. A, D2 activity in cells grown for 5 d with 1–200 nM Na2SeO3. B, Time-dependent induction of D2 activity in vehicle- and 1,25(OH)2VD-treated (10–8 M) cells. C, D2 activity in cells exposed to 30 nM rT3 and treated with MG132 (10–5 M), a proteasome inhibitor, or vehicle. All results are the mean ± SD of three to six points.
Interestingly, D2 activity in MC3T3-E1 osteoblasts increased in a time-dependent manner after the cells were plated, reaching a maximum at about d 7 when cells attained more than 90% confluence (Fig. 2B). Longer culture times (up to 4 wk) in the presence of differentiating agents (25 μg/ml ascorbic acid and 2 mM ?-glycerol phosphate) did not induce further D2 activity (data not shown). Because of the reported induction of D2 mRNA by 1,25(OH)2VD in murine primary osteoblastic cells (11), we tested whether this was the case in MC3T3-E1 osteoblasts. In fact, treatment with 1,25(OH)2VD induced D2 activity by 2- to 2.5-fold after as little as 24 h of exposure. The same fold induction was observed if treatment was extended up to 96 h, regardless of the level of cell confluence (Fig. 2B).
One of the hallmarks of D2 expression is sensitivity with loss of activity as a result of exposure to its own substrate, T4 or rT3, which is due to selective ubiquitination and proteasomal degradation (28). Substrate-induced loss of D2 activity was also observed in MC3T3-E1 osteoblasts as early as 30 min after addition of rT3 to the medium (Fig. 2C). Treatment with MG132, a proteasome inhibitor, not only increased basal D2 activity but also prevented the loss of D2 activity induced by rT3 (Fig. 2C). In addition, D2 displays a relatively rapid turnover rate in MC3T3-E1 osteoblasts, as determined by the loss of D2 activity in cells exposed to CX, which is also prevented by MG132 added to the cells just 10 min before CX (Fig. 3A), indicating that D2 inactivation is mediated by the ubiquitin-proteasomal system (1). These experiments were repeated in MC3T3-E1 osteoblasts that had been treated with 1,25(OH)2VD for 48 h to verify whether such treatment affects D2 expression at posttranslational level; similar results were obtained.
FIG. 3. Effects of multiple treatments on D2 activity in MC3T3-E1 osteoblastic cells. A, Loss of D2 activity 0–120 min after vehicle- or MG132-treated cells were exposed to CX. B, Same as in A except that cells were previously treated for 48 h with 1,25(OH)2VD (10–8 M). C, D2 activity in cells previously treated with vehicle or 1,25(OH)2VD for 24 h and then exposed to the indicated substances, as described in Materials and Methods. All results are the mean ± SD of three to six points.
The presence of D2 in bone and MC3T3-E1 osteoblasts as well as its inducibility by 1,25(OH)2VD prompted us to study what other bone relevant substances could influence D2 activity in these cells. Thus, MC3T3-E1 osteoblasts were treated for 24 h with TGF?, Dexa, TNF, leptin, forskolin, PTH, or 17?-estradiol, but no significant change in D2 activity was observed (Fig. 3C). These various treatments were then applied to cells that had been pretreated with 10–8 M 1,25(OH)2VD for 24 h, and still no effect was observed (Fig. 3C).
Discussion
To our knowledge this is the first demonstration that D2 activity is present in extracts of bone tissue as well as bone marrow throughout the skeleton of the adult mouse. The presence of D2 activity in bone was suggested in a previous study of organ-cultured mouse tibias (12). However, given its incomplete characterization, the PTU-insensitive apparent ORD pathway described in that study is more likely to represent the nonsaturable, PTU-insensitive 125I-rT3 metabolizing pathway described here in the femur and tibia of wild-type and Dio2–/– mice (Fig. 1, A and C). In the present study, bone D2 activity has been identified by its PTU insensitivity (Fig. 1), Km (T4) in the nano molar range (Fig. 1B), short half-life that is stabilized in the presence of a proteasome inhibitor (Fig. 3, A and B), and typical induction during hypothyroidism and suppression by hyperthyroidism (Fig. 1C) or exposure to substrate (Fig. 2C). The existence of D2 in bone supports a tightly controlled local T3 homeostasis, similar to what has been extensively demonstrated in other D2-expressing tissues such as brain and BAT (1). Given the relatively large volume of bone tissue, skeletal D2 is also likely to play a previously unanticipated role in systemic T3 homeostasis, contributing to extrathyroidal T3 production and maintenance of serum T3.
In addition to bone, we found D2 activity in the bone marrow of different skeletal sites, which contains multipotent stromal and hematopoietic cells that differentiate, respectively, into osteoblasts and osteoclasts (29, 30). D2 activity was also found in MC3T3-E1 cells, which are known to differentiate in culture from a preosteoblastic to a mature osteoblastic phenotype, a process that is accelerated by 1,25(OH)2VD (31). Remarkably, D2 activity in MC3T3-E1 cells was not only stimulated by 1,25(OH)2VD but also was increased with time after cells were seeded, compatible with D2-generated T3 playing a role in osteoblastic differentiation. In fact, it is known that thyroid hormone induces osteoblast differentiation (32, 33) and increases osteoclastogenesis (34).
D2-specific activity and the biochemical and biological properties of D2 in MC3T3-E1 cells are similar to those found in other cell lines endogenously expressing this enzyme. In addition, MSTO-211 cells are the only other cell line in which basal D2 expression is enhanced by exposure to Na2SeO3. However, MC3T3-E1 cells fail to respond to other agents known to affect D2 activity in other cell types, such as adrenergic stimulators, leptin, and TNF. It is also notable that other substances known to affect bone metabolism such as PTH, estradiol, and Dexa also did not affect D2 activity in these cells, suggesting a high degree of specificity of the 1,25(OH)2VD effect. Exposure of MC3T3-E1 cells to 1,25(OH)2VD did not affect D2 half-life, the main pathway by which D2 activity is controlled. This finding, along with the previous report that D2 mRNA is increased by treatment with 1,25(OH)2VD (11), confirms that the induction of the Dio2 gene is the main mechanism by which vitamin D receptor or vitamin D receptor-dependent signals increase D2 activity.
In accord with the reported induction of D2 mRNA by 1,25(OH)2VD in murine primary osteoblastic cells and ST2 cells (11), we found that, in MC3T3-E1 cells, 1,25(OH)2VD treatment increased D2 activity by 2.5-fold as early as 24 h and independently of cell confluence. This result prompted us to investigate the effect of other bone-resorbing as well as bone-formation agents on D2 activity in these cells. These various treatments, alone or in combination with 1,25(OH)2VD, did not affect basal or 1,25(OH)2VD-induced D2 activity. Nevertheless, the positive effect of 1,25(OH)2VD on D2 activity suggests a possible synergism between thyroid hormone and 1,25(OH)2VD on the regulation of osteoblastic function. In fact, these two hormones have been shown to influence MC3T3-E1 cells at different levels, such as in the regulation of osteocalcin and matrix metalloproteinase-13 mRNA expression (35) and the molecular organization of adherens junctions among these osteblastic cells (36). Interaction of thyroid hormone with 1,25(OH)2VD was also shown in mouse bone marrow cell cultures, in which T3 in combination with 1,25(OH)2VD, but not alone, induced osteoclastogenesis in a synergic manner (34).
Interestingly, in preosteoblastic cells, which were shown to express D2 after 1,25(OH)2VD treatment, T3 and T4 were shown to induce mRNA expression of receptor activator of nuclear factor-B (RANK) ligand (11). RANK ligand is a key factor that binds to its cognate receptor, RANK, in osteoclast precursors to activate osteoclastogenesis (22). In addition, it was shown that either T4 or T3 was able to enhance the 1,25(OH)2VD-induced osteoclastogenesis in coculture of bone marrow cells with primary osteoblasts, which suggested that T4 was converted to T3 in this system (11).
The presence of D2 activity throughout the skeleton of the mouse suggests that local deiodinase activity is important to bone physiology, ensuring that intracellular T3 homeostasis is attained in the skeleton to promote differentiation and/or regulate the activity of bone cells. The identification of D2 activity in MC3T3-E1 cells and its induction by 1,25(OH)2VD support the view that D2 is important to bone metabolism and that there is a cross-talk between thyroid hormone and 1,25(OH)2VD, involving D2 to control the differentiation or function of bone cells.
Acknowledgments
The authors are grateful to Dr. Stephen A. Huang and Michelle Mulcahey for performing D3 assays and Dr. Henry Kronenberg for providing the MC3T3-E1 cells.
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