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Endocrinology Vol. 140, No. 4 1649-1656
Copyright © 1999 by The Endocrine Society

ARTICLES

1{alpha},25-Dihydroxyvitamin D3 Up-Regulates Bcl-2 Expression and Protects Normal Human Thyrocytes from Programmed Cell Death1

Su He Wang, Ronald J. Koenig, Thomas J. Giordano, Andrzej Myc, Norman W. Thompson and James R. Baker, Jr.

Departments of Medicine (S.H.W., R.J.K., A.M., J.R.B.), Pathology (T.J.G., J.R.B.) and Surgery (N.W.T.), University of Michigan Medical School, Ann Arbor, Michigan 48109-0648

Address all correspondence and requests for reprints to: James R. Baker, Jr., M.D., Department of Medicine, University of Michigan Medical School, 9240 Medical Science Research Building III, Ann Arbor, Michigan 48109-0648. E-mail: jbakerjr{at}umich.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Apoptosis is thought to play an important role in the pathogenesis of autoimmune thyroid disease. 1{alpha},25-dihydroxyvitamin D3 (VD3) has been shown to suppress several autoimmune diseases. However, the mechanism by which VD3 has these effects is not known. We evaluated the alterations in apoptosis, induced by VD3. Thyrocytes were treated with VD3, and the expression of the Bcl-2 family molecules was studied at both the messenger RNA and protein levels. It was found that VD3 significantly induced the expression of Bcl-2 messenger RNA and protein in thyrocytes but had no effect on the expression of Bcl-xl and Bax. The increase in Bcl-2 expression, mediated by VD3, correlated with protection of thyrocytes against the induction of apoptosis by either staurosporine or UV irradiation. VD3-induced increases in the expression of Bcl-2 could be mimicked by VD3 analogs with high nuclear receptor affinity, but not by analogs only with nongenomic actions. These data indicate a role for Bcl-2 in the regulation of apoptosis in thyrocytes and raise the possibility that VD3 or its agonists may have therapeutic benefit in thyroid disorders.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CHRONIC (HASHIMOTO’S) thyroiditis is an extremely frequent autoimmune disease, affecting approximately 5% of women, and is the most common cause of hypothyroidism in the United States (1). Previous studies indicate that apoptosis plays an important role in the destruction of thyrocytes in thyroiditis (2, 3, 4). However, the regulation of apoptosis in thyroid cells has not been fully defined. Several studies have documented that the induction of apoptosis in thyroid cells through different pathways is highly regulated, but the studies differed on the exact manner and effect of this regulation (2, 3, 4, 5, 6). Bcl-2 is one member of a family of protooncogenes that regulates apoptosis (7). The strong inhibitory effect of Bcl-2 on programmed cell death is evidenced by the apoptosis of Bcl-2-negative cortical thymocytes during T cell development, whereas positively selected, surviving thymocytes in the medulla show strong staining for Bcl-2 (8). Several members of the Bcl-2 family are expressed in thyroid follicular cells in both normal and pathologic conditions (9). In light of the putative role of apoptotic regulation in the development of autoimmune hypothyroidism, it is of interest to determine whether Bcl-2 has a role in the regulation of apoptosis in thyrocytes.

1{alpha},25-dihydroxyvitamin D3 (VD3), an active form of vitamin D, is a potent secosteroid hormone that produces a wide array of biological effects in its target tissues. In addition to the long recognized role of VD3 in calcium homeostasis, VD3 also has immunomodulatory and antiinflammatory properties, as well as a regulatory role in the growth and differentiation of a variety of cell types and tissues (10, 11). VD3 acts through an intracellular vitamin D receptor (VDR), which belongs to the nuclear hormone receptor gene superfamily (12). The hormone-receptor complex binds to sequence-specific sites in promoters and mediates its effects by altering transcriptional activity of specific genes (13). As might be expected from VD3’s multiple activities, VDRs are present in both classical target tissues, regulating calcium homeostasis, and a wide variety of nonclassical target tissues that include breast cancer cells, lymphocytes, and thyroid follicular cells (11). VD3 has also been suggested to have a variety of nongenomic actions, mediated without the requirement of nuclear receptor binding (11, 13).

VD3 has been reported to alter the induction of apoptosis in breast cancer cells (14). Furthermore, VD3 (in the presence of cyclosporin) significantly reduced the incidence of thyroid pathology and the severity of thyroiditis in a mouse model of experimental autoimmune thyroiditis (15). In this report, we demonstrate that VD3 induces the expression of Bcl-2 messenger RNA (mRNA) and protein in human thyroid follicular cells, and this action is associated with protection against apoptosis. These studies indicate a role for Bcl-2 in regulating susceptibility to apoptosis in Hashimoto’s thyroiditis and other thyroid disorders, and they suggest a possible therapeutic role for VD3 in these diseases.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Thyroid tissues
Thyroid tissue was obtained from 10 patients undergoing thyroidectomy for thyroid cancer (follicular or papillary). Normal tissue was recovered, harvesting tissue either from areas adjacent to the carcinomas or from the contralateral lobe. All patients were females who had received no steroid hormone treatment, and their mean age was 39.5 ± 10.5 yr. Three of the patients were postmenopausal, and one with papillary carcinoma was pregnant.

Cell culture conditions
Thyroid tissue was digested with collagenase, hyaluronidase, and DNase I (Sigma Chemical Co., St. Louis, MO) in RPMI-1640. Red blood cells were lysed with ammonium chloride lysis buffer (0.15 M NH4Cl, 10 mM KPO4, and 1 mM EDTA, pH7.3); and cells were cultured in Cellgro media (Mediatech, Herndon, VA) with 20% NuSerum IV (Collaborative Biomedical Products, Bedford, MA) at 37 C in 5% CO2. After 24 h, nonadherent cells were removed by extensive washing with Cellgro media, and remaining adherent cells were maintained in the same media for 3 additional days. The cells were confirmed to be thyrocytes by staining for thyroglobulin. One day before initiation of experiments, cells were plated in Cellgro media supplemented with 5% charcoal-stripped FBS. After changing media the next day, VD3 or analogs or vehicle was added and cultured with the cells for an additional 48 h.

Vitamin D3 and its related compounds
The structures of VD3 and its analogs used in these experiments are shown in Fig. 1Go. VD3, 1{alpha},25-(OH)2–16-Enc-23-yne-D3 (analog V), 1{alpha},25-dihydroxytachysterol3 (analog JB), and 1{alpha},25(OH)2-lumisterol3 (analog JN) were received as gracious gifts from Dr. Anthony W. Norman (University of California, Riverside, CA). VD3 and its analogs were stored in the dark as stock solutions in absolute ethanol at -70 C. The maximal concentration of ethanol in the culture (0.01%) did not influence either cell growth or Bcl-2 expression. TSH (bovine), T3, all-transretinoic acid (RA, >98% pure), and 9-cis RA (cis-RA, 98% pure) were obtained from Sigma Chemical Co.



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  		Figure 1. Structures of VD3 and analogs V, JB, and JN.

 
RT-competitive PCR
RT-competitive PCR was used to quantitate Bcl-2 expression levels in thyrocytes. RNA from cells was prepared by TRI reagent (Molecular Research Center, Inc., Cincinnati, OH). One microgram of total RNA was used in a first-strand complementary DNA synthesis with 100 ng oligo (dT)18. The forward and reverse primer sequences for human Bcl-2 were 5'-ACC TGG ATG TTC TGT GCC-3' and 5'-CCC CTC TAC TGC TCT TTC-3', respectively. The amplified PCR product was 801 bp. A 620-bp competitive internal standard for human Bcl-2 was constructed from the 801-bp PCR fragment by deleting an internal 181-bp sequence. This 620-bp internal standard and the 801-bp target sequence were amplified with similar efficiencies by PCR. For competitive PCR, a known amount of the internal standard (620 bp) was coamplified with target RT products in the same PCR tube. PCR products were then size-fractionated through a 1.5% agarose gel, and the bands were visualized using ethidium bromide. The PCR produced two different sized products, a 801-bp fragment for the target and a 620-bp fragment for the internal standard. The densities of the two bands corresponding to the endogenous target and the synthetic competitor amplification products were determined with Scan Analysis, a densitometer for the Macintosh computer (Biosoft, Ferguson, MO).

Immunoblot analysis for Bcl-2 family members
The expression of Bcl-2, Bcl-xl, and Bax proteins was determined by Western blot analyses. A hamster monoclonal antibody to human Bcl-2, and rabbit antihuman Bcl-x and Bax polyclonal antibodies (PharMingen, San Diego, CA) were used, respectively, in the Western blot analyses. Lysates of thyroid follicular cells were prepared by scraping cells from plates and suspending them into PBS (pH 7.4). The cells were collected by centrifugation, and the resulting pellets were suspended in ice-cold lysis buffer [150 mM NaCl, 10 mM Tris (pH 7.4), 5 mM EDTA, 1% Triton X-100] containing protease inhibitor cocktail tablet (Boehringer Mannheim). After incubation on ice for 30 min, samples were centrifuged at 15,000 x g for 20 min. A Triton-soluble fraction was collected, and total protein concentration was determined using BCA protein assay reagent (Pierce Chemical Co., Rockford, IL). Appropriate protein amounts were subjected to SDS-PAGE. After electrophoresis, proteins were transferred to nitrocellulose membranes. The blots were blocked in TBST buffer (20 mM Tris (pH 7.4), 150 mM NaCl, 0.05% Tween 20) with 5% nonfat dry milk, overnight at 4 C. This was followed by incubation with the primary antibody for 3 h and then a horseradish peroxidase-conjugated second antibody for 1 h at room temperature. Immune complexes were detected with an enhanced chemiluminescence detection method (Amersham, Buckinghamshire, UK) and then exposed to Hyperfilm ECL. The bands on Hyperfilm were quantified by a Scan Analysis program.

Induction of apoptotic cell death
Staurosporine, a protein kinase inhibitor, was used to induce apoptosis in thyrocytes (2). The cells were treated with 0.5–1 µM of staurosporine for 24 h. After treatment, cells displayed the morphological features of apoptosis, including membrane blebbing, with the cells rounding up and becoming nonadherent. To further confirm that these changes were caused by apoptosis, the ApopTag plus in situ apoptosis detection kit (Oncor, Gaithersburg, MD) was used to detect fragmented DNA specific for apoptosis in thyrocytes treated with staurosporine. UV radiation, an effective apoptosis-inducer (16), was also used to trigger apoptosis in thyroid cells. Cells were preincubated for 48 h, with or without 100 nM VD3, in Cellgro media containing TSH (but in the absence of FBS). The cells then were irradiated with 300, 600, or 900 J/M2 UV using a UV Stratalinker 1800 (Stratagene, La Jolla, CA). After irradition, the culture media were replaced with fresh media, with or without VD3, and incubated for another 48 h. The cell viability was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay (2).

Assessment of cell death
The thyrocytes were treated with or without 100 nM VD3 or its analogs for 48 h. After this incubation, the cells were further cultured with or without designated concentrations of staurosporine for indicated times. The MTT assay was used to examine the viability of cells. The percentage of cell survival was defined as absorbance of the treated well/absorbance of the control well x 100%. To quantify apoptosis, Annexin-V-FLUOS Staining Kit (Boehringer Mannheim, Indianapolis, IN) was used according to the manufacturer’s instruction. Fluorescien isothiocyanate-conjugated annexin V (annexin V-FITC) and propidium iodide-stained cells were determined on a flow cytometer, and the data were analyzed by CellQuest software (Becton Dickinson and Co., San Jose, CA).

Statistics
Data are expressed as mean ± SD and analyzed by Student’s t test by using a computer program (InStat 2.01). A P value of 0.05 or less was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The induction of Bcl-2 mRNA by VD3
Thyrocytes incubated with 100 nM VD3 were found to have an increase in Bcl-2 mRNA levels at 12 h, with a maximum level achieved at 48 h (Fig. 2aGo). Fig. 2bGo shows the increased amounts of Bcl-2 mRNA in two representative samples. The ratio of target to internal standard increased from 59 ± 0.96 in control samples to 206 ± 34, a 3.5-fold elevation.



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  		Figure 2. Effects of VD3 on the expression of Bcl-2 mRNA. A RT-competitive PCR was performed to assess Bcl-2 mRNA levels. A 801-bp Bcl-2 target band and a 620-bp internal standard band were detected. The densities of the bands were measured, and the ratio of target to internal standard was then calculated. A, Thyrocytes were incubated with 100 nM VD3 for different time periods. Total RNA were isolated at the different lengths of incubation (0, 12, 24, 48, and 72 h) for Bcl-2 mRNA analysis. The values of the Bcl-2/internal standard ratio were calculated at different time points. B, Cells from thyrocyte samples were incubated with 100 nM VD3 for 48 h, and total RNA was isolated for analysis of Bcl-2 mRNA. Results from two representative samples were shown.

 
Induction of Bcl-2 protein by VD3 is time and concentration dependent
Thyrocytes were incubated with various concentrations of VD3 for 48 h, and protein lysates were analyzed by Western blot. This revealed dose-dependent expression of Bcl-2, with a maximal expression found at a concentration of 100 nM VD3 (Fig. 3aGo). The expression of Bcl-2 protein was increased after 12 h of incubation with VD3, reached its maximum at 48 h, and was maintained out to 72 h (Fig. 3bGo).



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  		Figure 3. Effects of VD3 on Bcl-2 protein expression. A 26-kDa band of Bcl-2 protein was detected by Western analysis using a monoclonal antibody to human Bcl-2. The densities of the bands were determined by a densitometer. A, Thyrocytes were incubated with various concentrations of VD3 (0–1000 nM) for 48 h, and protein (10 µg), in each lane, was used for analysis of Bcl-2 protein signals. The values of band densities at different concentrations of VD3 were plotted. B, Thyrocytes were incubated with 100 nM VD3 for different time periods. Protein (10 µg), in each lane, was obtained after different lengths of incubation (0, 12, 24, 48, and 72 h) for Bcl-2 protein analysis by Western blot. The values of band densities at different time points were plotted.

 
Effects of VD3 on the expression of Bcl-xl and Bax protein
Like Bcl-2, Bcl-xl has an antiapoptotic function, whereas Bax is proapoptotic. It is thought that Bax is the direct antagonist of Bcl-2, and therefore, the balance between Bcl-2 and Bax is believed to determine whether the process of apoptosis occurs in cells (7). Although VD3 induced the expression of Bcl-2 protein, it did not change either Bax or Bcl-xl protein levels (Fig. 4aGo). Therefore, the induction of Bcl-2 protein results in an increase in the Bcl-2/Bax ratio.



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  		Figure 4. Effects of VD3 on Bcl-xl, Bax, and Bcl-2 protein expression and influence of T3, TSH, RA, and cis-RA on Bcl-2 protein. A, Thyrocytes were incubated with 100 nM VD3 for 48 h, and Western analysis was carried out to detect Bcl-xl, Bax, and Bcl-2 proteins. Ten micrograms of total protein were loaded per lane. B, Thyrocytes were incubated with 100 nM T3, 10 mIU/ml TSH, 100 nM RA, or 100 nM cis-RA, in combination with either 100 nM of VD3 or vehicle for 48 h. Ten micrograms of total protein were loaded per lane for analysis by Western blot.

 
Effects of other nuclear hormone receptor superfamily ligands on Bcl-2 protein concentrations
Other nuclear hormone receptor superfamily ligands bind to receptors that are structurally related to the VD3 receptor (12). These agents include T3, RA, and cis-RA. None of these compounds exerted significant regulation of Bcl-2 expression in either a positive or negative direction (Fig. 4bGo). TSH, a pituitary hormone that is the major GH for thyroid cells, also had no effect on Bcl-2 levels. The use of VD3, in combination with any of these agents, showed no effect, compared with VD3 alone.

VD3 does not increase Bcl-2 levels in a breast cancer cell line (MCF-7)
Others have found that Bcl-2 levels in a breast cancer cell line are decreased by VD3 treatment (14), which is in contrast to our findings in thyrocytes. Therefore, we included MCF-7 cells, a human breast cancer cell line, as a control in this study to examine Bcl-2 expression. As found in the previous study (14), VD3-treated MCF-7 cells showed a reduction of Bcl-2 protein levels after treatment with 10–100 nM VD3 (data not shown).

Effects of VD3 analogs on Bcl-2 expression
Three VD3 analogs (JB, JN, and V), with different structural features and functional activities (Fig. 1Go), were carefully selected to analyze the action of VD3. The biological effects of VD3 are reported to occur via either genomic or nongenomic pathways, and VD3 possesses the ability to function via both pathways (11). Like VD3, the V analog, which possesses genomic function, increased the expression of both Bcl-2 mRNA (Fig. 5AGo) and protein (Fig. 5BGo). In contrast, neither JN (an analog without genomic function) nor JB (an inactive analog without genomic and nongenomic function) induced Bcl-2 RNA or protein levels, compared with the control (Fig. 5Go, A and B). These data suggest that the induction of Bcl-2 by VD3 is the result of a VDR-mediated event.



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  		Figure 5. Effects of VD3 and its analogs V, JB, and JN on Bcl-2 expression. Thyrocytes were incubated with 100 nM VD3, 100 nM V, 100 nM JB, or 100 nM JN, respectively, for 48 h; and total RNA and protein were isolated. A, Bcl-2 mRNA was assayed by RT-competitive PCR. The target band is 801 bp, and the internal control is 620 bp. M, DNA markers. Protein (10 µg), in each lane, was used to detect Bcl-2 by Western blot.

 
Effects of VD3 on thyroid cell susceptibility to the induction of apoptosis
Anti-Fas monoclonal antibody or tumor necrosis factor (TNF){alpha} alone did not trigger receptor-mediated apoptosis in thyrocytes; only in the presence of cycloheximide did anti-Fas monoclonal antibody induce apoptosis (2). In the cell death pathway mediated by Fas receptor, cycloheximide inhibits the expression of Bcl-2 up-regulated by VD3. Therefore, staurosporine or UV irradiation were used to determine whether VD3 blocks apoptosis in thyrocytes. As expected from our prior published studies (2), staurosporine induced apoptosis in primary human thyrocytes, analyzed by ApopTag (data not shown). An MTT assay was performed to provide quantitative evidence of VD3 protection of thyroid cells from staurosporine-induced apoptosis. In the presence of VD3, cell survival rate was increased from a mean of 27.2 ± 3.5% to 59.7 ± 4.0% (P < 0.01), at 0.5–1 µM staurosporine (Fig. 6aGo). Cells treated by UV irradiation of 300 J/M2 or more showed a significant lose of viability, as quantitated by MTT assay. The percentage of nonapoptotic cells post irradiation was significantly higher in cells supplemented with 100 nM VD3 (51 ± 3.8%) than in cells without VD3 (24.9 ± 1.4%, P < 0.01) (Fig. 6bGo). Both VD3 and the VD3 analog V significantly protected thyroid cells against apoptosis induced by staurosporine (0.5 µM), but VD3 analogs JB and JN had no significant protective effect (Fig. 7Go). This correlated exactly with ability of these compounds to induce Bcl-2 expression.



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  		Figure 6. VD3 protects against staurosporine- and radiation-induced apoptosis. Thyrocytes were pretreated with 100 nM VD3 or vehicle for 48 h. A, Staurosporine (0.025–1 µM) was then added into the cell culture for a further 24 h to induce apoptosis; B, thyrocytes were irradiated by 300–900 J/M2 and cultured for another 48 h with fresh media and VD3 or vehicle. An MTT assay was performed to assay cell viability. The results were expressed as percentage of normalized cell survival (absorbance of the treated well/absorbance of the control well x 100%).

 


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  		Figure 7. Protection against apoptosis by VD3 analogs V, JB, and JN. Thyrocytes were pretreated with 100 nM VD3, or analog V, JB, or JN for 48 h; 0.5 µM staurosporine was then added into the cell culture for a further 24 h to induce apoptosis. After this incubation, an MTT assay was performed to assay cell viability. The results were expressed as percentage of normalized cell survival (absorbance of the treated well/absorbance of the control well x 100%). CTRL, Untreated cells.

 
Quantitation of VD3 protection of thyroid cell from apoptosis
To further examine VD3 protection of primary human thyrocytes against apoptosis induced by staurosporine, thyroid cells were pretreated with VD3 and incubated with or without staurosporine for 4 h. The resulting cell samples were then subjected to staining with annexin V-FITC. As shown in Fig. 8Go, VD3 also protects cells from the early events involved in apoptosis. Using annexin V as an early marker of apoptosis, VD3 protection is evident as early as 4 h after staurosporine treatment. Quantitatively, cells treated with VD3 and staurosporine showed a 40% reduction in the number of cells stained positive by annexin V, compared with cells treated with staurosporine alone.



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  		Figure 8. VD3 protects primary human thyrocytes from early apoptosis induced by staurosporine. Cells were pretreated with either 100 nM VD3 or vehicle for 48 h and incubated with or without 0.5 µM staurosporine for an additional 4 h. The treated cells were then subjected to stain with annexin V-FITC, and fluorescence was analyzed using a flow cytometer. The results were expressed as percentage of positive stained cells normalized to negative control (untreated cells stained with annexin V-FITC). Annexin V-FITC fluorescence histograms of cells treated with VD3 and staurosporine showed a 40% reduction in annexin V stained cells, as compared those treated with staurosporine alone, and were significantly different (P < 0.001), as tested with Kolmogorov-Smirnov statistics.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There is increasing evidence that apoptosis has a crucial role in the pathogenesis of autoimmune thyroid diseases, particularly thyroiditis. In Hashimoto’s thyroiditis, a high occurrence of apoptosis in thyroid follicular cells has been observed, associated with lymphocytic infiltrates (4). This may result, over time, in the scarcity of intact thyrocytes present at the advanced stage of thyroiditis (17). It is believed that apoptosis in Hashimoto’s thyroiditis is triggered by autoimmune responses, and the Fas pathway has been proposed as playing an important role in this process (2, 3, 4, 5). However, there is disagreement as to how this process is regulated and the degree to which other pathways of apoptosis are involved (2, 3, 5). Bcl-2 can function as an antiapoptotic force in thyrocytes, as demonstrated in this study, and this concept is further strengthened by the fact that there are significant baseline levels of Bcl-2 protein expressed in thyrocytes (18). In contrast to Graves’ disease, thyroid tissues from Hashimoto’s disease are reported to express lower levels of Bcl-2 (19). This is associated with the high rate of apoptotic cell death in Hashimoto’s thyrocytes, indicating an inverse relationship between Bcl-2 levels and the rate of apoptosis in thyrocytes. Though the ability of Bcl-2 to block Fas-induced apoptosis is still open to debate (20, 21), Bcl-2 may have a role in the pathogenesis of thyroiditis by inhibiting other pathways of apoptosis in thyrocytes. Elevated Bcl-2 expression might be particularly beneficial at the early stages of thyroiditis (before Fas mediated, CD8 cytotoxicity is induced). At this early time point, apoptosis may be induced by oxidative stress or cytokines, and this might then result in a specific immune response. Therefore, increasing Bcl-2 concentrations may have a role in preventing thyroiditis-induced hypothyroidism by impeding the development of specific autoimmunity or blocking immune mediated cytotoxicity.

The present study is the first to examine the regulation of the Bcl-2 family of proteins in normal human thyroid cells. VD3 increased Bcl-2 mRNA and protein levels, elevated the Bcl-2/Bax ratio, and protected thyrocytes from apoptosis. The data suggest a role for VD3 in the regulation of apoptosis in the thyroid, as well as a potential role for pharmacotherapy, with VD3 or its analogs, in thyroiditis. We also sought to determine the mechanism by which the VD3 protective effect occurs. VD3 generally exerts its biological effects via two distinguishable modes of action; genomic and nongenomic. These two pathways seem to be mediated by different receptors located in different compartments of cells (11). The receptor for the genomic actions is a well-characterized nuclear protein, whereas the receptor for the nongenomic actions is poorly characterized and is presumably located on cell membranes (11, 13). To assess which pathway leads to the induction of Bcl-2 by VD3, three structurally different analogs (V, JB, and JN) were chosen to be included in this study (Fig. 1Go). Among the three analogs, only V has affinity for nuclear VDRs and genomic effects (11, 22). Our results are consistent with a nuclear receptor-mediated induction of Bcl-2, because VD3 and V demonstrated this activity, but JB and JN did not. Signaling via the nuclear receptor also is supported by prior findings of the expression of nuclear VDRs in thyrocytes and is compatible with the timeframe required for the induction of a regulatory effect by nuclear hormone (11).

Whereas VD3-mediated changes in Bcl-2 initiate through the nuclear receptor, subsequent events in this process are not entirely clear. VD3 has been known to regulate the production of several cytokines, such as IL-1ß, IL-2, IL-6, TNF{alpha}, and INF[{gamma} (23, 24, 25). Experiments from our laboratory have shown that IL-1ß, TNF{alpha}, and INF[{gamma} do not affect the expression of Bcl-2 (unpublished data); however, the regulation of Bcl-2 by VD3-induced changes in other cytokine concentrations has not been examined. The direct regulation of target genes by VDR is achieved via binding to specific vitamin D response elements (VDREs) located in promoter regions of the genes (26, 27, 28). VDREs are typically characterized by direct repeats of AG (G/T) TCA-like sequences separated by 3 bp (29). Inspection of the Bcl-2 gene 5' flanking region (30) reveals several such motifs that could potentially function as VDREs, although no canonical motifs are present. It will be of interest to determine whether these sequences indeed function as VDREs and thus indicate the direct induction of Bcl-2 transcription by VD3.

VDRs are members of the nuclear hormone receptor gene superfamily. This superfamily includes structurally related intracellular receptors for glucocorticoids, androgen, progestin, estrogen, thyroid hormone, RA, and 9-cis RA (31). RA, a derivative of vitamin A, is able to induce apoptosis by down-regulation of Bcl-2 in embryonal stem cells (32). However, none of the above agents altered Bcl-2 expression in the thyroid, either alone or in combination with VD3. These findings indicate that stimulation of Bcl-2 expression by VD3 in thyrocytes is via a distinctive pathway unrelated to other members of the nuclear hormone receptor gene superfamily. It is intriguing that VD3 up-regulates Bcl-2 expression in thyrocytes but decreases Bcl-2 expression in breast cancer cells (14). The basis for this cell type specificity is unclear but is worthy of future study. Also of interest is whether the up-regulation of Bcl-2 in thyroid cancer alters the sensitivity of these cells to either therapeutic radiation or chemotherapeutic agents. This has been reported in prostate cancer cells, where Bcl-2 overexpression protects against radiation-induced apoptosis (33). If this is true in thyroid cells, VD3 starvation or agents that block the VDR may have utility in thyroid cancer therapy.

The finding that VD3 analogs with genomic action, such as the V analog, have a significant regulatory effect on Bcl-2 expression is important. Clinical applications of VD3 in patients are limited by the side-effect of hypercalcemia. By adding a double bond between carbons 16 and 17 and a triple bond between carbons 23 and 24, the V analog is more powerful in the generation of genomic action than VD3, but significantly less potent than VD3, in the accumulation of serum calcium (34). This information, coupled with our data, suggest that VD3 analogs may have a therapeutic role in the treatment of Hashimoto’s thyroiditis.


   Acknowledgments
 
We gratefully acknowledge G. Chen and J. D. Bretz for helpful discussions, and P. Arscott and J. Bartron for their technical assistance.


   Footnotes
 
1 This work was supported by Grant R01-AI37141 and a supplemental grant from the office for Research in Women’s Health at NIH. Back

Received September 17, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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