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The Vitamin D Receptor in Thyroid Development and Function

The Vitamin D Receptor in Thyroid Development and Function Translational Thyroidology / Original Paper Received: April 10, 2012 Eur Thyroid J 2012;1:168–175 Accepted after revision: August 2, 2012 DOI: 10.1159/000342363 Published online: September 22, 2012 The Vitamin D Receptor in Thyroid Development and Function a b c Isabelle Clinckspoor Anne-Catherine Gérard Jacqueline Van Sande b a a a Marie-Christine Many Lieve Verlinden Roger Bouillon Geert Carmeliet a, d a a, d Chantal Mathieu Annemieke Verstuyf Brigitte Decallonne a b Clinical and Experimental Endocrinology, Katholieke Universiteit Leuven, Pôle de Morphologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, and IRIBHM, Université Libre de Bruxelles, Brussels , and Department of Endocrinology, University Hospitals Leuven, Leuven , Belgium Key Words and 6 40 ng/ml, respectively), but no difference was ob- Vitamin D receptor  T hyroid  Calcitonin  Vitamin D  served. Conclusions: In mice, the VDR is redundant for nor- Calcium mal thyrocyte function, but not for C cell function, where it mediates the negative control of calcitonin by 1,25-dihy- droxy vitamin D . In patients, vitamin D status does not affect Abstract basal serum calcitonin levels. A study in healthy individuals Background and Objective: Vitamin D is known to modu- is needed to confirm these findings. late thyroid neoplastic and autoimmune disease. We investi- Copyright © 2012 European Thyroid Association Published by S. Karger AG, Basel gated the role of the vitamin D receptor (VDR) in normal thy- roid development and function (thyrocytes and C cells). Methods: The thyroid phenotype of VDR knockout mice was studied in comparison to wild-type controls. The mice were Introduction fed a normal diet or a calcium-rich diet to circumvent effects induced by hypocalcemia. Results: Thyroid morphology 1,25-dihydroxyvitamin D [1,25(OH) D ], the active 3 2 3 was unaltered in VDR knockout mice. Also, expression of dif- form of vitamin D, is mainly known for its effects on cal- ferent parameters of thyrocyte function was comparable cium and phosphate homeostasis with bone, intestine (immunohistochemistry). C cell physiology was, however, af- and kidney as principal target tissues. However, fected in the absence of the VDR, resulting in increased 1,25(OH) D has pleiotropic effects which include anti- 2 3 thyroidal calcitonin expression (immunohistochemistry), proliferative, anti-inflammatory and prodifferentiating paralleled by increased serum calcitonin levels, but only in effects [1] . Several lines of evidence suggest a role for vi- normocalcemic mice. To study a possible effect of vitamin D tamin D in thyroid disease. The ligand for 1,25(OH) D , 2 3 status on basal calcitonin levels in humans, serum calcitonin the nuclear vitamin D receptor (VDR), is expressed in concentrations were compared between vitamin D-defi- many tissues including benign and malignant thyroid tis- cient and -sufficient patients (serum 25-OH vitamin D ^ 10 sue [2, 3] . © 2012 European Thyroid Association B. Decallonne Published by S. Karger AG, Basel University Hospitals Leuven, Departement of Endocrinology Fax +41 61 306 12 34 2235–0640/12/0013–0168$38.00/0 Herestraat 49 E-Mail [email protected] Accessible online at: BE–3000 Leuven (Belgium) www.karger.com www.karger.com/etj Tel. +32 16 34 6994, E-Mail brigitte.decallonne   @   uzleuven.be Downloaded from Bioscientifica.com at 01/31/2022 07:09:12PM via Deepdyve 1,25(OH) D concentration dependently inhibits tumor marker in the workup of thyroid nodular disease 2 3 TSH-stimulated iodide uptake in rat thyroid follicular and in the follow-up of patients with medullary thyroid (FRTL-5) cells [4] . We and others have shown that high cancer [27] , further insight is needed in the role of doses of 1,25(OH) D or structural analogues have anti- 1,25(OH) D -VDR signaling in thyroid physiology. 2 3 2 3 proliferative effects in human thyroid cancer cell lines In this study we used systemic VDR knockout (KO) [5 –7] . On the other hand, data on a possible association mice to investigate the effect of impaired VDR signaling between vitamin D deficiency and increased risk for thy- on thyroid development and function, studying both thy- roid cancer are scarce and debated [8–10] . Several studies rocyte and C cell function. VDR-KO mice are character- also suggest a link between vitamin D and autoimmune ized by hypocalcemia, secondary hyperparathyroidism thyroid disease. 1,25(OH) D protects human thyrocytes and bone deformities. These effects can be prevented by 2 3 from programmed cell death via increased Bcl-2 expres- a calcium-rich diet from weaning on [28] . sion and prevents autoimmune thyroiditis in mice [11, 12] . The immunomodulatory effect of 1,25(OH) D or the 2 3 analogue elocalcitol is exerted through decreased expres- Materials and Methods sion of HLA class II molecules on thyrocytes and im- Mice paired Th1-mediated inf lammatory responses in thyro- Systemic Leuven VDR-KO mice were generated as described cytes, while the T cell response is shifted towards a Th2 [2 8] and housed in our animal facility under conventional condi- phenoty pe [13, 14] . Furthermore, polymorphisms of VDR tions. Wild-type (WT) sex-matched littermates were used as con- and CYP27B1 – the enzyme responsible for the activation trols. The mice were kept on a normal diet (Ssniff R/M-H; Ssniff, of the vitamin D precursor 25-hydroxyvitamin D (25- Soest, Germany) or a calcium-rich diet (20% lactose/2% calci- um/1.25% phosphorous, diet TD96348; Harlan, Horst, The Neth- OHD ) into active 1,25(OH) D – have been associated 3 2 3 erlands) in order to exclude effects induced by hypocalcemia. with increased risk for autoimmune thyroid disease and Food and water were given ad libitum. The diets were started im- thyroid cancer in some studies [15, 16] , although other mediately after weaning. Animals were sacrificed at 10–12 weeks studies found no association [17, 18] . Finally, vitamin D of age and the entire thyroid as well as blood (heart puncture) deficiency is reported to correlate with the presence of were collected. The thyroid was embedded in OCT, snap-frozen in liquid nitrogen and stored at –80  °   C until use. Serum was stored antithyroid antibodies and abnormal thyroid function in at –20   °   C until use. All experimental procedures were approved by humans [19, 20] , and to modulate autoimmune hyperthy- the Ethical Committee of the KU Leuven. roidism in mice [21] . Apart from inhibiting osteoclast-mediated bone re- Histology and Histomorphometry sorption and increasing renal calcium and phosphate Mouse thyroid cr yostat sections (7  m) were mounted on glass slides followed by hematoxylin and eosin staining according to excretion, calcitonin (produced by the parafollicular standard protocols. Morphometric analysis of the thyroid glands C cells) is known to be involved in the homeostasis of VDR-WT and -KO mice on both diets (n = 3 mice per group) of 1,25(OH) D . More specifically, together with parat- 2 3 was performed using the point-counting method described by hormone (PTH), calcitonin is a positive regulator of Weibel et al. [29] . One thousand points were counted for each thy- CYP27B1. Whereas PTH is mainly active under hypocal- roid, and the relative volumes of epithelium and follicular lumen (colloid) were calculated and expressed as percentage relative vol- cemic conditions, calcitonin is the main regulator under ume. normocalcemic to hypercalcemic conditions [22, 23] . On the other hand, CYP27B1 is negatively regulated by fibro- Immunohistochemistry blast growth factor 23 (FGF23). PTH and FGF23 are Iodinated thyroglobulin (Tg-I), thyroxin (T4), dual oxidase known to be regulated by 1,25(OH) D through the VDR (Duox), calcitonin, caspase-6 and 4-hydroxynonenal (4-HNE) 2 3 immunostaining were performed on frozen sections. With the [24] . High 1,25(OH) D levels decrease calcitonin pro- 2 3 exception of 4-HNE, sections were rehydrated with PBS-BSA (1%) duction in C cells in rats, suggesting that 1,25(OH) D 2 3 and thereafter incubated in PBS-BSA (1%) containing 1: 50 goat negatively controls calcitonin. However, the involvement serum. 4-HNE sections were incubated with PBS-BSA (5%) con- of the VDR in this negative feedback has not yet been taining 1: 30 goat serum. The conditions for immunohistochem- demonstrated [25, 26] . istry are summarized in table 1 . All sections were incubated with the first antibody (Tg-I, T4, Duox, calcitonin, caspase-6 and In human physiology and pathophysiology, the role of 4-HNE) at room temperature. The binding of antibodies was de- 1,25(OH) D -VDR signaling in the regulation of thyro- 2 3 tected using a second antibody conjugated to a peroxidase-labeled cyte and C cell homeostasis is unclear, but as vitamin D polymer (EnVision detection; DakoCytomation, Heverlee, Bel- deficiency and thyroid dysfunction are both very preva- gium). Peroxidase activity was revealed by AEC substrate (3-ami- no-9-ethylcarbazole, Dako). Finally, sections were counterstained lent and as serum calcitonin is widely used as a specific Eur Thyroid J 2012;1:168–175 The Thyroid Phenotype in VDR-KO 169 Mice Downloaded from Bioscientifica.com at 01/31/2022 07:09:12PM via Deepdyve Table 1. Experimental conditions for immunohistochemistry Antibody 1° Antibody Tg-I (J.J.M. De Vijlder, Amsterdam, The Netherlands) mouse monoclonal, 1:2,000, 1 h T4 (MyBiosource, San Diego, Calif., USA) mouse monoclonal, 1:800, overnight Duox 1/2 (F. Miot, ULB, Brussels, Belgium) rabbit polyclonal, 1:75, 4 h Caspase-6 (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) rabbit polyclonal antibody, 1:75, 1 h 4-HNE (Calbiochem, Darmstadt, Germany) rabbit polyclonal, dilution 1:500, overnight Calcitonin (Dako, Heverlee, Belgium) rabbit polyclonal, 1:500, 3 h with Mayer’s hematoxylin, rinsed and mounted in Faramount Statistical Analysis Aqueous Mounting Medium (Dako). Each immunostaining was Data are expressed as means 8 SEM. For comparisons of nu- performed on thyroid sections of VDR-WT and -KO mice on both merical data between VDR-WT and -KO mice, and between pa- diets (n = 3–6 mice per group), and evaluated by two independent tients with low and high 25-OHD levels, an unpaired Student’s investigators (I.C. and A.G.). One representative experiment for t test was used, unless otherwise stated. A two-sided p value of each immunostaining is shown. ! 0.05 was considered statistically significant. Mouse Serum Biochemistry Total serum calcium and creatinine was analyzed by Synchron Clinical Systems (Beckman Coulter, Suarlée, Belgium). Serum Results calcitonin levels were measured with a two-site immunoradio- metric assay (Immutopics, San Clemente, Calif., USA). Briefly, Disrupted VDR Signaling Does Not Affect Thyroid two antibodies namely I-labeled calcitonin antibody and calci- Morphology in Mice tonin antibody immobilized on plastic beads were added to the samples. After an 18-hour incubation period, the beads were We f irst investigated whether thyroid histolog y of 10- washed three times and the counts were recorded in a gamma to 12-week-old VDR-KO mice was different from their counter. Serum TSH was evaluated by bioassay using a line of WT littermates. On hematoxylin and eosin staining, no Chinese hamster ovary cells (CHO-K1) stably transfected with difference was observed in the thyroid follicles or thy- human TSH receptor cDNA, as previously described [30] . Five to rocytes between both groups fed a normal diet. As ex- eleven animals per group were analyzed separately. pected, serum calcium levels were significantly lower in Patient Serum Biochemistry VDR-KO mice compared to WT mice (7.5 8 0.42 vs. Serum samples from patients were randomly selected in a 1: 1 10.0 8 0.2 mg/dl, respectively; p ! 0.0001). Renal func- male:female ratio in our hospital laboratory department based tion (creatinine) was not different (data not shown). on the presence of either low or high 25-OHD levels (^ 10 and Since many of the observed effects in VDR-KO mice are 6 40 ng/ml, respectively), using an equilibrium radioimmuno- secondary to their hypocalcemic status, we subsequent- assay (DiaSorin, Stillwater, Minn., USA). Intra- and interassay coeff icients of variation were 11 and 9%, respectively. The detec- ly evaluated thyroid morphology in mice fed a calcium- tion limit of the radioimmunoassay kit was 1.5 ng/ml. For each rich diet, resulting in a correction of the hypocalcemia patient the following data were retrieved from the medical chart: (9.6 8 1.6 and 10.7 8 0.9 mg/dl for VDR-WT and -KO age, sex, previous or current thyroid or parathyroid disease, cur- mice, respectively). Also under normocalcemic condi- rent use of proton pump inhibitor, serum calcium, renal func- tion (serum creatinine and eGFR), and serum TSH level in the tions, we did not observe morphological differences be- same serum sample as used for the 25-OHD determination. Pa- tween VDR-WT and -KO thyroid glands (f ig. 1 a). Fur- tients hospitalized at the intensive care unit, patients with thermore, thyroid sections also showed no signs of im- known thyroid or parathyroid disease, or with neuroendocrine mune infiltration or neoplasia in any of the groups. tumors were excluded for further analysis, as well as patients Further quantitative morphometric investigation using with severe hypocalcemia or hypercalcemia or with an abnor- mal serum TSH. the point-counting method confirmed the absence of A total of 48 patient sera (4 groups, n = 12 patients per group) difference in thyrocyte size or colloid/thyrocyte ratio were thus collected and stored for additional calcitonin determi- between VDR-WT and -KO mice ( fig. 1 b). nation, using a radioimmunoassay (Biosource, Nijvel, Belgium). Intra- and interassay coefficients of variation were 1.4 and 4.6%, respectively. The detection limit of the radioimmunoassay kit was 0.9 pg/ml. Eur Thyroid J 2012;1:168–175 170 Clinckspoor et al.   Downloaded from Bioscientifica.com at 01/31/2022 07:09:12PM via Deepdyve Normal diet Calcium-rich diet VDR-WT VDR-KO VDR-WT VDR-KO % % Fig. 1. The effect of VDR disruption on VDR-WT 50 70 mouse thyroid morphology. a Hematoxy- VDR-KO lin and eosin staining of thyroids isolated from VDR-WT and -KO mice on normal 40 and calcium-rich diets. Original magnifi- cation ! 40. b Relative volume of colloid 10 (left) and epithelium (right) in VDR-WT 0 0 Normal diet Calcium-rich Normal diet Calcium-rich and -KO mice on normal and calcium-rich b diet diet diets according to the point-counting method. Normal diet Calcium-rich diet VDR-WT VDR-KO VDR-WT VDR-KO Fig. 2. Immunohistochemical staining of thyroid sections (7  m) of VDR-WT and b -KO mice on normal and calcium-rich di- ets. Sections were stained for Tg-I (a), T4 (b) and Duox (c). Immunostaining for Tg-I and T4 was similar in all groups. Immu- nostaining for Duox tended to be more apical in the VDR-WT mice and more in the cytoplasm in the VDR-KO mice (indi- cated by arrows), irrespective of the diet. Original magnification ! 40. Thyrocyte and C Cell Function in Mice in the Absence was observed for Tg-I and T4 between VDR-WT and -KO of VDR Signaling mice on either diet. Duox tended to be located more at the Thyrocyte functionality was assessed by immuno- apical pole of the thyrocytes in WT mice and more in the staining of thyroid sections of VDR-WT and -KO mice cytoplasm in the VDR-KO mice, again irrespective of the for Tg-I, T4 and Duox. As shown in figure 2 , no difference diet. As altered expression of Duox could be caused by Eur Thyroid J 2012;1:168–175 The Thyroid Phenotype in VDR-KO 171 Mice Downloaded from Bioscientifica.com at 01/31/2022 07:09:12PM via Deepdyve Color version available online Normal diet Calcium-rich diet VDR-WT VDR-KO VDR-WT VDR-KO Fig. 3. Calcitonin expression in thyroid tis- sue and serum of VDR-WT and -KO mice on a normal and calcium-rich diet. a Im- VDR-WT munohistochemical staining for calcito- VDR-KO nin on thyroid sections (7  m) showing a clearly higher expression (indicated by ar- rows) in VDR-KO mice compared to VDR- WT mice, irrespective of the diet. Original magnification ! 10. b Serum calcitonin levels were elevated in VDR-KO mice fed a calcium-rich diet, but not in VDR-KO 0 b Normal diet Calcium-rich diet mice on a normal diet, suggesting that cal- cium constitutes a major trigger for calci- tonin secretion. oxidative stress, staining for 4-HNE (a marker of oxida- f luence on basal serum calcitonin levels. We studied se- tive stress) was additionally performed, as well as for cas- rum calcitonin levels in males and females with low and pase-6 (a marker of apoptosis). Both 4-HNE and cas- high serum 25-OHD levels ( ^ 10 and 6 40 ng/ml, re- pase-6 staining were not different between the groups spectively) separately. As can be observed in table 2 , 25- (data not shown). Under the normal diet, serum TSH was OHD serum levels were significantly different in both not different between VDR-WT and -KO mice (0.39 8 the male and the female patient groups. Furthermore, 0.005 and 0.53 8 0.08  U/ml, respectively), but under the both the vitamin D-deficient and vitamin D-sufficient calcium-rich diet TSH was slightly lower in the VDR-KO groups were normocalcemic and comparable for age, re- mice (0.33 8 0.02  U/ml) compared to the VDR-WT nal function and proton pump inhibitor use. mice (0.58 8 0.1 U /ml; p = 0.021). As shown in figure 4 , basal serum calcitonin levels C cell function was studied by calcitonin staining of were not different between the groups with low or high thyroid sections, revealing a strongly increased calcito- 25-OHD , neither when studying both sexes separately, nin expression in VDR-KO compared to the WT mice, nor when evaluating both sexes grouped. Also, after ex- irrespective of the diet ( fig. 3 a). Subsequent serum calci- clusion of patients with renal failure (eGFR ! 30 ml/min/ tonin measurements confirmed increased serum calcito- 1.73 m ) or users of proton pump inhibitors (both known nin levels in VDR-KO mice (125.1 8 36.4 ng/ml) com- confounders increasing serum calcitonin), no difference pared to VDR-WT mice (46.4 8 22.8 ng/ml) fed a calci- in basal serum calcitonin level was observed between the um-rich diet. However, this increase was not observed in vitamin D-deficient and vitamin D-sufficient group hypocalcemic VDR-KO mice fed a normal diet ( fig. 3 b). (data not shown). In Humans, Vitamin D Status Does Not Inf luence Basal Serum Calcitonin Levels Discussion Since C cell function was clearly altered in the absence of normal 1,25(OH) D -VDR signaling in mice, we inves- In the present study we investigated the role of the 2 3 tigated whether these findings could also be relevant for VDR in normal thyroid physiology by studying systemic humans. More specifically, we studied whether vitamin VDR-KO mice in comparison to WT age-matched and D deficiency – a widespread health problem – has an in- sex-matched littermates. Thyrocyte function was not al- Eur Thyroid J 2012;1:168–175 172 Clinckspoor et al.   Downloaded from Bioscientifica.com at 01/31/2022 07:09:12PM via Deepdyve Calcitonin (μU/ml) Color version available online ++ Low 25-OHD 2+ 1,25(OH) D Low Ca PTH CYP27B1 2 3 High 25-OHD VDR 1,25(OH) D FGF23 CYP27B1 2 3 VDR 2 + + 2+ Calcitonin CYP27B1 1,25(OH) D High Ca 2 3 VDR Males Females All Fig. 4. Serum calcitonin levels in vitamin D-deficient (low 25- Fi g. 5. Overview of 1,25(OH) D homeostasis maintained by 2 3 OHD ) and vitamin D-sufficient (high 25-OHD ) patients with- PTH, FGF23 and calcitonin. Low calcium levels trigger PTH re- 3 3 out history of known thyroid or parathyroid disease, medullary lease which stimulates CYP27B1. High calcium levels stimulate cancer or neuroendocrine cancer. Serum calcitonin levels were calcitonin release, also stimulating CYP27B1. FGF23 represents not significantly different, neither when studying both sexes sep- the negative regulator of CYP27B1. PTH, FGF23 and calcitonin arately, nor when both sexes were grouped. are regulated by 1,25(OH) D via the VDR. Stimulating effects are 2 3 indicated with full arrows, inhibitory effects are indicated with dashed lines. Table 2. Characteristics of patients with low and high serum 25- tered but C cell activity was clearly increased in the ab- OHD level sence of the VDR, confirming that 1,25(OH) D nega- 2 3 tively regulates calcitonin via the VDR. Low vit. D High vit. D p value Supraphysiological doses of active vitamin D have been described to inhibit proliferation and reduce the se- Serum 25-OHD , ng/ml verity of autoimmunity in case of underlying malignant Males 7.180.74 49.382.5 <0.0001 Females 6.680.59 53.684.8 <0.0001 or autoimmune thyroid disease, respectively [5, 6, 11] . In Age, years the present study, global thyroid morphology was un- Males 60.884.0 59.084.6 0.78 changed in VDR-KO mice, also after correction of the Females 58.786.8 54.583.0 0.58 hypocalcemic state, and no signs of autoimmune thy- creatinine, mg/dl roiditis or thyroid neoplasia were present. Further analy- Males 1.580.5 2.380.6 0.32 Females 1.180.4 1.080.1 0.78 sis of thyrocyte functionality by studying the expression eGFR, ml/min/1.73 m2 of T4 and Tg-I showed no differences. Duox was located Males 49.288.9 35.388.0 0.28 more in the cytoplasm (instead of apically in the VDR- Females 0.87 59.5812.0 50.387.3 WT mice), possibly as a result of oxidative stress. How- calcium, mg/dl ever, no signs of increased oxidative stress (4-HNE) or Males 8.880.2 9.080.2 0.56 Females 0.21 9.180.2 9.480.2 apoptosis (caspase-6) were observed. Serum TSH was not PPI use, % different under the normal diet, but was slightly lower a b Males 45 (5/11) 42 (5/12) 0.85 under the calcium-rich diet. A similar observation was Females 33 (4/12) 17 (2/12) 0.35 made by others in rats with severe vitamin D deficiency, P PI = Proton pump inhibitor. but serum T4 levels remained unchanged [31] . These re- PPI use was not known in 1 male with low vitamin D. sults suggest that the 1,25(OH) D -VDR signaling path- 2 3 b 2 test. way has a redundant role in normal thyrocyte develop- ment and function in rodents, although a mild effect on the TSH setpoint is possible. Eur Thyroid J 2012;1:168–175 The Thyroid Phenotype in VDR-KO 173 Mice Downloaded from Bioscientifica.com at 01/31/2022 07:09:12PM via Deepdyve Calcitonin (ng/ml) Second, we studied the role of the VDR in parafollic- On the other hand, it is known that C cell physiology, in ular C cell physiology. The enzyme CYP27B1, responsi- contrast to thyrocyte physiology, is largely different in ble for the final activation of 25-OHD into active rodents as compared to humans [33, 34] . We found no 1,25(OH) D , is known to be positively regulated by PTH increased basal serum calcitonin levels in a cohort of 2 3 and calcitonin, and negatively regulated by FGF23. The patients with severe vitamin D deficiency compared to transcription factor C/EBP and the SWI/SNF chroma- patients with vitamin D sufficiency (all patients were nor- tin remodeling complex mediate CYP27B1 transcription mocalcemic and matched for sex, age, renal function, use induced by calcitonin [23] . In the present study we ob- of proton pump inhibitors). The variable comorbidity of served clearly increased thyroidal calcitonin expression the patients, however, represents a limitation of this study. in VDR-KO mice, independent from the serum calcium In conclusion, VDR disruption did not inf luence thy- level. In the normocalcemic mice the increased calcitonin roid morphology or thyrocyte function in mice. How- expression at the C cell level was paralleled by increased ever, VDR constitutes an important determinant in C cell serum calcitonin levels. This was not observed in VDR- physiology in mice, negatively controlling calcitonin. In KO mice fed a normal diet, which is most probably ex- a pilot study in patients, there was no evidence for altera- plained by their hypocalcemic status inhibiting secretion tions of serum calcitonin levels in case of very low or high of calcitonin by the C cells. In a rat model, it has been es- levels of serum 25-OHD . These findings need to be con- tablished by others that calcium constitutes the main reg- firmed in a study in healthy individuals, preferably by a ulator for calcitonin secretion, but that 1,25(OH) D and prospective randomized placebo-controlled clinical trial, 2 3 not calcium constitutes the main regulator for calcitonin evaluating serum calcitonin before and after vitamin D transcription [25, 32] . We hereby demonstrate for the first supplementation along with serum levels of 25-OHD , time that the inhibitory action of 1,25(OH) D on calci- 1,25(OH) D , PTH, FGF23 and VDR polymorphism. At 2 3 2 3 tonin production acts via the VDR. Due to the absence of present, there is no indication for a systematic evaluation VDR signaling, the inhibitory effect of 1,25(OH) D on C of the patient’s vitamin D status along with a serum cal- 2 3 cells and thus the physiological negative feedback control citonin measurement. is lost. Thyroiditis and renal insufficiency were ruled out as potential other causes for increased calcitonin by in- creased bystander C cell activation and decreased renal Acknowledgements clearance, respectively. Furthermore, Egrise et al. [31 ] de- We are grateful to Christine de Ville de Goyet, Paul Drent, Jos scribed that rats fed a vitamin D-deficient diet showed Laureys, Suzanne Marcelis, Ingrid Stockmans and Erik Van strongly increased thyroidal calcitonin mRNA expres- Herck for their invaluable technical assistance. sion, which suggests that deficiency of the ligand for VDR This work was supported by the Flemish Research Foundation also results in uncontrolled calcitonin expression in (clinical FWO fellowship for C.M. and B.D. and grant G021207N). rodents. Whether chronically defective signaling could eventually lead to C cell proliferation is not known. Disclosure Statement Thus, as summarized in figure 5 , we demonstrate in the present study that besides the known positive regulation The authors declare that no competing financial interest ex- of CYP27B1 by PTH and its negative feedback via ists. 1,25(OH) D -VDR signaling, calcitonin (the other k nown 2 3 positive regulator of CYP27B1, but mainly active under normocalcemic to hypercalcemic conditions) is also neg- atively controlled by 1,25(OH) D via the VDR. FGF23 2 3 represents the negative regulator of CYP27B1, with a pos- 1 Bouillon R, Carmeliet G, Verlinden L, van References itive feedback by 1,25(OH) D via the VDR. 2 3 Etten E, Verstuyf A, Luderer HF, Lieben L, In the last part of the study, we investigated whether Mathieu C, Demay M: Vitamin D and hu- man health: lessons from vitamin D receptor increased calcitonin caused by defective 1,25(OH) D - 2 3 null mice. Endocr Rev 2008; 29: 726–776. VDR signaling as observed in rodents also applies to hu- 2 Khadzkou K, Buchwald P, Westin G, Dralle mans. If so, this would imply that the vitamin D status H, Akerstrom G, Hellman P: 25hydroxy vita- min D 1alpha-hydroxylase and vitamin D and also VDR polymorphism might inf luence the basal receptor expression in papillary thyroid car- calcitonin level. As vitamin D deficiency is very preva- cinoma. J Histochem Cytochem 2006; 54: 355–361. lent, this could have important clinical consequences. Eur Thyroid J 2012;1:168–175 174 Clinckspoor et al.   Downloaded from Bioscientifica.com at 01/31/2022 07:09:12PM via Deepdyve 3 Clinckspoor I, Hauben E, Verlinden L, Van Crescioli C: Elocalcitol inhibits inf lamma- al control of other bone anabolic and cata- den Bruel A, Vanwalleghem L, Vander tory responses in human thyroid cells and bolic genes that orchestrate the regulation of Poorten V, Delaere P, Mathieu C, Verstuy f A, T cells. Endocrinology 2008; 149: 3626– phosphate and calcium mineral metabolism. Decallonne B: Altered expression of key 3634. J Steroid Biochem Mol Biol 2007; 103: 381– players in vitamin D metabolism and signal- 14 Tokuda N, Mano T, Levy RB: 1,25-Dihy- 388. ing in malignant and benign thyroid tumors. droxyvitamin D antagonizes interferon- 25 Naveh-Many T, Silver J: Regulation of calci- J Histochem Cytochem 2012; 60: 502–511. gamma-induced expression of class II major tonin gene transcription by vitamin D me- 4 Lamberg-Allardt C, Valtonen E, Polojarvi histocompatibility antigens on thyroid fol- tabolites in vivo in the rat. J Clin Invest 1988; M, Stewen P: Characterization of a 1,25-di- licular and testicular Leydig cells. Endocri- 81: 270–273. hydroxy-vitamin D r eceptor in FRTL-5 nology 1990; 127: 1419–1427. 26 Martin-Lacave I, Ramos F, Utrilla JC, Conde cells. Evidence for an inhibitory effect of 15 Lin WY, Wan L, Tsai CH, Chen RH, Lee CC, E, Hevia A, Fernandez R, Moreno AM, 1,25-dihydroxy-vitamin D on thyrotropin- Tsai FJ: Vitamin D receptor gene polymor- Fernandez-Santos JM, Galera-Davidson H: induced iodide uptake. Mol Cell Endocrinol phisms are associated with risk of Hashimo- Chronic hypervitaminosis D determines a 1991; 81: 25–31. to’s thyroiditis in Chinese patients in Tai- decrease in C-cell numbers and calcitonin 5 Liu W, Asa SL, Fantus IG, Walfish PG, Ezzat wan. J Clin Lab Anal 2006; 20: 109–112. levels in rats. J Endocrinol Invest 1998; 21: S: Vitamin D arrests thyroid carcinoma cell 16 Stefanic M, Papic S, Suver M, Glavas-Obro- 102–108. growth and induces p27 dephosphorylation vac L, Karner I: Association of vitamin D re- 27 Costante G, Durante C, Francis Z, Schlum- and accumulation through PTEN/akt-de- ceptor gene 3  -variants with Hashimoto’s berger M, Filetti S: Determination of calcito- pendent and -independent pathways. Am J thyroiditis in the Croatian population. Int J nin levels in C-cell disease: clinical interest Pathol 2002; 160: 511–519. Immunogenet 2008; 35: 125–131. and potential pitfalls. Nat Clin Pract Endo- 6 Clinckspoor I, Verlinden L, Overbergh L, 17 Collins JE, Heward JM, Nithiyananthan R, crinol Metab 2009; 5: 35–44. Korch C, Bouillon R, Mathieu C, Verstuyf A, Nejentsev S, Todd JA, Franklyn JA, Gough 28 Van Cromphaut SJ, Dewerchin M, Hoen- Decallonne B: 1,25-Dihydroxyvitamin D SC: Lack of association of the vitamin D derop JG, Stockmans I, Van HE, Kato S, Bin- and a superagonistic analog in combination receptor gene with Graves’ disease in UK dels RJ, Collen D, Carmeliet P, Bouillon R, with paclitaxel or suberoylanilide hy- Caucasians. Clin Endocrinol 2004; 60: 618– Carmeliet G: Duodenal calcium absorption droxamic acid have potent antiproliferative 624. in vitamin D receptor-knockout mice: func- effects on anaplastic thyroid cancer. J Steroid 18 Maalej A, Petit-Teixeira E, Chabchoub G, tiona l and molecular aspects. Proc Natl Acad Biochem Mol Biol 2011; 124: 1–9. Hamad MB, Rebai A, Farid NR, Cornelis F, Sci USA 2001; 98: 13324–13329. 7 Okano K, Usa T, Ohtsuru A, Tsukazaki T, Ayadi H: Lack of association of VDR gene 29 Weibel ER, Kistler GS, Scherle WF: Practical Miyazaki Y, Yonekura A, Namba H, Shindoh polymorphisms with thyroid autoimmune stereological methods for morphometric cy- H, Yamashita S: Effect of 22-oxa-1,25-dihy- disorders: familial and case/control studies. tology. J Cell Biol 1966; 30: 23–38. droxyvitamin D on human thyroid cancer J Clin Immunol 2008; 28: 21–25. 30 Perret J, Ludgate M, Libert F, Gerard C, Du- cell growth. Endocr J 1999; 46: 243–252. 19 Goswami R, Marwaha RK, Gupta N, Tan- mont JE, Vassart G, Parmentier M: Stable ex- 8 Lee S, Morimoto S, Onishi T, Tsuji M, Okada don N, Sreenivas V, Tomar N, Ray D, Kan- pression of the human TSH receptor in CHO Y, Seino Y, Ishida M, Yamaoka K, Takai S, war R, Agarwal R: Prevalence of vitamin D cells and characterization of differentially Miyauchi A, Kumahara Y: Normal serum deficiency and its relationship with thyroid expressing clones. Biochem Biophys Res 1,25-dihydroxyvitamin D in patients with autoimmunity in Asian Indians: a commu- Commun 1990; 171: 1044–1050. medullary carcinoma of the thyroid. J Clin nity-based survey. Br J Nutr 2009; 102: 382– 31 Egrise D, Burniat A, Lefort A, Libert F, Miot Endocrinol Metab 1982; 55: 361–363. 386. F, Goldman S, Cor vilain B, Moreno-Reyes R: 9 Stepien T, Krupinski R, Sopinski J, Kuzdak 20 Kivity S, Agmon-Levin N, Zisappl M, Sha- Vitamin D deficiency modulates the regula- K, Komorowski J, Lawnicka H, Stepien H: pira Y, Nag y EV, Danko K, Szekanecz Z , Lan- tion of the pituitar y-thyroid a xis and thyroid Decreased 1-25 dihydroxyvitamin D con- gevitz P, Shoenfeld Y: Vitamin D and auto- gene expression (abstract No. 2614). 14th In- centration in peripheral blood serum of pa- immune thyroid diseases. Cell Mol Immu- ternational Thyroid Congress (ITC), Paris, tients with thyroid cancer. Arch Med Res nol 2011; 8: 243–247. September 2010. 2010; 41: 190–194. 21 Misharin A, Hewison M, Chen CR, Lagishet- 32 Naveh-Many T, Raue F, Grauer A, Silver J: 10 Laney N, Meza J, Lyden E, Erickson J, Treude ty V, Aliesky HA, Mizutori Y, Rapoport B, Regulation of calcitonin gene expression by K, Goldner W: The prevalence of vitamin D McLachlan SM: Vitamin D deficiency mod- hypocalcemia, hypercalcemia, and vitamin deficiency is similar between thyroid nodule ulates Graves’ hyperthyroidism induced in D in the rat. J Bone Miner Res 1992; 7: 1233– and thyroid cancer patients. Int J Endocrinol BALB/c mice by thyrotropin receptor immu- 1237. 2010; 2010: 805716. nization. Endocrinology 2009; 150: 1051– 33 Bjerre KL , Madsen LW, Andersen S, Almholt 11 Fournier C, Gepner P, Sadouk M, Charreire 1060. K, de Boer AS, Drucker DJ, Gotfredsen C, J: In vivo beneficial effects of cyclosporin A 22 Shinki T, Ueno Y, DeLuca HF, Suda T: Calci- Egerod FL , Hegelund AC, Jacobsen H, Jacob- and 1,25-dihydroxyvitamin D on the in- tonin is a major regulator for the expression sen SD, Moses AC, Molck AM, Nielsen HS, duction of experimental autoimmune thy- of renal 25-hydroxyvitamin D -1alpha-hy- Nowak J, Solberg H, Thi TD, Zdravkovic M: roiditis. Clin Immunol Immunopathol 1990; droxylase gene in normocalcemic rats. Proc Glucagon-like Peptide-1 receptor agonists 54: 53–63. Natl Acad Sci USA 1999; 96: 8253–8258. activate rodent thyroid C-cells causing calci- 12 Wang SH, Koenig RJ, Giordano TJ, Myc A, 23 Zhong Y, Armbrecht HJ, Christakos S: Cal- tonin release and C-cell proliferation. Endo- Thompson NW, Baker JR Jr: 1Alpha,25- di- citonin, a regulator of the 25-hydroxyvita- crinology 2010; 151: 1473–1486. hydroxyvitamin D up-regulates Bcl-2 ex- min D 1alpha-hydroxylase gene. J Biol 34 Hegedus L, Moses AC, Zdravkovic M, Le TT, 3 3 pression and protects normal human thyro- Chem 2009; 284: 11059–11069. Daniels GH: GLP-1 and calcitonin concen- cy tes from programmed cell death. Endocri- 24 Barthel TK, Mathern DR, Whitfield GK, tration in humans: lack of evidence of calci- nology 1999; 140: 1649–1656. Haussler CA, Hopper HA, Hsieh JC, Slater tonin release from sequential screening in 13 Borgogni E, Sarchielli E, Sottili M, Santar- SA, Hsieh G, Kaczmarska M, Jurutka PW, over 5,000 subjects with type 2 diabetes or lasci V, Cosmi L, Gelmini S, Lombardi A, Kolek OI, Ghishan FK, Haussler MR: nondiabetic obese subjects treated with the Cantini G, Perigli G, Luconi M, Vannelli 1,25-Dihydroxyvitamin D /VDR-mediated human GLP-1 analog, liraglutide. J Clin En- GB, Annunziato F, Adorini L, Serio M, induction of FGF23 as well as transcription- docrinol Metab 2011; 96: 853–860. Eur Thyroid J 2012;1:168–175 The Thyroid Phenotype in VDR-KO 175 Mice Downloaded from Bioscientifica.com at 01/31/2022 07:09:12PM via Deepdyve http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png European Thyroid Journal Bioscientifica

The Vitamin D Receptor in Thyroid Development and Function

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Bioscientifica
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© 2012 European Thyroid Association Published by S. Karger AG, Basel
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10.1159/000342363
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Abstract

Translational Thyroidology / Original Paper Received: April 10, 2012 Eur Thyroid J 2012;1:168–175 Accepted after revision: August 2, 2012 DOI: 10.1159/000342363 Published online: September 22, 2012 The Vitamin D Receptor in Thyroid Development and Function a b c Isabelle Clinckspoor Anne-Catherine Gérard Jacqueline Van Sande b a a a Marie-Christine Many Lieve Verlinden Roger Bouillon Geert Carmeliet a, d a a, d Chantal Mathieu Annemieke Verstuyf Brigitte Decallonne a b Clinical and Experimental Endocrinology, Katholieke Universiteit Leuven, Pôle de Morphologie, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, and IRIBHM, Université Libre de Bruxelles, Brussels , and Department of Endocrinology, University Hospitals Leuven, Leuven , Belgium Key Words and 6 40 ng/ml, respectively), but no difference was ob- Vitamin D receptor  T hyroid  Calcitonin  Vitamin D  served. Conclusions: In mice, the VDR is redundant for nor- Calcium mal thyrocyte function, but not for C cell function, where it mediates the negative control of calcitonin by 1,25-dihy- droxy vitamin D . In patients, vitamin D status does not affect Abstract basal serum calcitonin levels. A study in healthy individuals Background and Objective: Vitamin D is known to modu- is needed to confirm these findings. late thyroid neoplastic and autoimmune disease. We investi- Copyright © 2012 European Thyroid Association Published by S. Karger AG, Basel gated the role of the vitamin D receptor (VDR) in normal thy- roid development and function (thyrocytes and C cells). Methods: The thyroid phenotype of VDR knockout mice was studied in comparison to wild-type controls. The mice were Introduction fed a normal diet or a calcium-rich diet to circumvent effects induced by hypocalcemia. Results: Thyroid morphology 1,25-dihydroxyvitamin D [1,25(OH) D ], the active 3 2 3 was unaltered in VDR knockout mice. Also, expression of dif- form of vitamin D, is mainly known for its effects on cal- ferent parameters of thyrocyte function was comparable cium and phosphate homeostasis with bone, intestine (immunohistochemistry). C cell physiology was, however, af- and kidney as principal target tissues. However, fected in the absence of the VDR, resulting in increased 1,25(OH) D has pleiotropic effects which include anti- 2 3 thyroidal calcitonin expression (immunohistochemistry), proliferative, anti-inflammatory and prodifferentiating paralleled by increased serum calcitonin levels, but only in effects [1] . Several lines of evidence suggest a role for vi- normocalcemic mice. To study a possible effect of vitamin D tamin D in thyroid disease. The ligand for 1,25(OH) D , 2 3 status on basal calcitonin levels in humans, serum calcitonin the nuclear vitamin D receptor (VDR), is expressed in concentrations were compared between vitamin D-defi- many tissues including benign and malignant thyroid tis- cient and -sufficient patients (serum 25-OH vitamin D ^ 10 sue [2, 3] . © 2012 European Thyroid Association B. Decallonne Published by S. Karger AG, Basel University Hospitals Leuven, Departement of Endocrinology Fax +41 61 306 12 34 2235–0640/12/0013–0168$38.00/0 Herestraat 49 E-Mail [email protected] Accessible online at: BE–3000 Leuven (Belgium) www.karger.com www.karger.com/etj Tel. +32 16 34 6994, E-Mail brigitte.decallonne   @   uzleuven.be Downloaded from Bioscientifica.com at 01/31/2022 07:09:12PM via Deepdyve 1,25(OH) D concentration dependently inhibits tumor marker in the workup of thyroid nodular disease 2 3 TSH-stimulated iodide uptake in rat thyroid follicular and in the follow-up of patients with medullary thyroid (FRTL-5) cells [4] . We and others have shown that high cancer [27] , further insight is needed in the role of doses of 1,25(OH) D or structural analogues have anti- 1,25(OH) D -VDR signaling in thyroid physiology. 2 3 2 3 proliferative effects in human thyroid cancer cell lines In this study we used systemic VDR knockout (KO) [5 –7] . On the other hand, data on a possible association mice to investigate the effect of impaired VDR signaling between vitamin D deficiency and increased risk for thy- on thyroid development and function, studying both thy- roid cancer are scarce and debated [8–10] . Several studies rocyte and C cell function. VDR-KO mice are character- also suggest a link between vitamin D and autoimmune ized by hypocalcemia, secondary hyperparathyroidism thyroid disease. 1,25(OH) D protects human thyrocytes and bone deformities. These effects can be prevented by 2 3 from programmed cell death via increased Bcl-2 expres- a calcium-rich diet from weaning on [28] . sion and prevents autoimmune thyroiditis in mice [11, 12] . The immunomodulatory effect of 1,25(OH) D or the 2 3 analogue elocalcitol is exerted through decreased expres- Materials and Methods sion of HLA class II molecules on thyrocytes and im- Mice paired Th1-mediated inf lammatory responses in thyro- Systemic Leuven VDR-KO mice were generated as described cytes, while the T cell response is shifted towards a Th2 [2 8] and housed in our animal facility under conventional condi- phenoty pe [13, 14] . Furthermore, polymorphisms of VDR tions. Wild-type (WT) sex-matched littermates were used as con- and CYP27B1 – the enzyme responsible for the activation trols. The mice were kept on a normal diet (Ssniff R/M-H; Ssniff, of the vitamin D precursor 25-hydroxyvitamin D (25- Soest, Germany) or a calcium-rich diet (20% lactose/2% calci- um/1.25% phosphorous, diet TD96348; Harlan, Horst, The Neth- OHD ) into active 1,25(OH) D – have been associated 3 2 3 erlands) in order to exclude effects induced by hypocalcemia. with increased risk for autoimmune thyroid disease and Food and water were given ad libitum. The diets were started im- thyroid cancer in some studies [15, 16] , although other mediately after weaning. Animals were sacrificed at 10–12 weeks studies found no association [17, 18] . Finally, vitamin D of age and the entire thyroid as well as blood (heart puncture) deficiency is reported to correlate with the presence of were collected. The thyroid was embedded in OCT, snap-frozen in liquid nitrogen and stored at –80  °   C until use. Serum was stored antithyroid antibodies and abnormal thyroid function in at –20   °   C until use. All experimental procedures were approved by humans [19, 20] , and to modulate autoimmune hyperthy- the Ethical Committee of the KU Leuven. roidism in mice [21] . Apart from inhibiting osteoclast-mediated bone re- Histology and Histomorphometry sorption and increasing renal calcium and phosphate Mouse thyroid cr yostat sections (7  m) were mounted on glass slides followed by hematoxylin and eosin staining according to excretion, calcitonin (produced by the parafollicular standard protocols. Morphometric analysis of the thyroid glands C cells) is known to be involved in the homeostasis of VDR-WT and -KO mice on both diets (n = 3 mice per group) of 1,25(OH) D . More specifically, together with parat- 2 3 was performed using the point-counting method described by hormone (PTH), calcitonin is a positive regulator of Weibel et al. [29] . One thousand points were counted for each thy- CYP27B1. Whereas PTH is mainly active under hypocal- roid, and the relative volumes of epithelium and follicular lumen (colloid) were calculated and expressed as percentage relative vol- cemic conditions, calcitonin is the main regulator under ume. normocalcemic to hypercalcemic conditions [22, 23] . On the other hand, CYP27B1 is negatively regulated by fibro- Immunohistochemistry blast growth factor 23 (FGF23). PTH and FGF23 are Iodinated thyroglobulin (Tg-I), thyroxin (T4), dual oxidase known to be regulated by 1,25(OH) D through the VDR (Duox), calcitonin, caspase-6 and 4-hydroxynonenal (4-HNE) 2 3 immunostaining were performed on frozen sections. With the [24] . High 1,25(OH) D levels decrease calcitonin pro- 2 3 exception of 4-HNE, sections were rehydrated with PBS-BSA (1%) duction in C cells in rats, suggesting that 1,25(OH) D 2 3 and thereafter incubated in PBS-BSA (1%) containing 1: 50 goat negatively controls calcitonin. However, the involvement serum. 4-HNE sections were incubated with PBS-BSA (5%) con- of the VDR in this negative feedback has not yet been taining 1: 30 goat serum. The conditions for immunohistochem- demonstrated [25, 26] . istry are summarized in table 1 . All sections were incubated with the first antibody (Tg-I, T4, Duox, calcitonin, caspase-6 and In human physiology and pathophysiology, the role of 4-HNE) at room temperature. The binding of antibodies was de- 1,25(OH) D -VDR signaling in the regulation of thyro- 2 3 tected using a second antibody conjugated to a peroxidase-labeled cyte and C cell homeostasis is unclear, but as vitamin D polymer (EnVision detection; DakoCytomation, Heverlee, Bel- deficiency and thyroid dysfunction are both very preva- gium). Peroxidase activity was revealed by AEC substrate (3-ami- no-9-ethylcarbazole, Dako). Finally, sections were counterstained lent and as serum calcitonin is widely used as a specific Eur Thyroid J 2012;1:168–175 The Thyroid Phenotype in VDR-KO 169 Mice Downloaded from Bioscientifica.com at 01/31/2022 07:09:12PM via Deepdyve Table 1. Experimental conditions for immunohistochemistry Antibody 1° Antibody Tg-I (J.J.M. De Vijlder, Amsterdam, The Netherlands) mouse monoclonal, 1:2,000, 1 h T4 (MyBiosource, San Diego, Calif., USA) mouse monoclonal, 1:800, overnight Duox 1/2 (F. Miot, ULB, Brussels, Belgium) rabbit polyclonal, 1:75, 4 h Caspase-6 (Santa Cruz Biotechnology, Santa Cruz, Calif., USA) rabbit polyclonal antibody, 1:75, 1 h 4-HNE (Calbiochem, Darmstadt, Germany) rabbit polyclonal, dilution 1:500, overnight Calcitonin (Dako, Heverlee, Belgium) rabbit polyclonal, 1:500, 3 h with Mayer’s hematoxylin, rinsed and mounted in Faramount Statistical Analysis Aqueous Mounting Medium (Dako). Each immunostaining was Data are expressed as means 8 SEM. For comparisons of nu- performed on thyroid sections of VDR-WT and -KO mice on both merical data between VDR-WT and -KO mice, and between pa- diets (n = 3–6 mice per group), and evaluated by two independent tients with low and high 25-OHD levels, an unpaired Student’s investigators (I.C. and A.G.). One representative experiment for t test was used, unless otherwise stated. A two-sided p value of each immunostaining is shown. ! 0.05 was considered statistically significant. Mouse Serum Biochemistry Total serum calcium and creatinine was analyzed by Synchron Clinical Systems (Beckman Coulter, Suarlée, Belgium). Serum Results calcitonin levels were measured with a two-site immunoradio- metric assay (Immutopics, San Clemente, Calif., USA). Briefly, Disrupted VDR Signaling Does Not Affect Thyroid two antibodies namely I-labeled calcitonin antibody and calci- Morphology in Mice tonin antibody immobilized on plastic beads were added to the samples. After an 18-hour incubation period, the beads were We f irst investigated whether thyroid histolog y of 10- washed three times and the counts were recorded in a gamma to 12-week-old VDR-KO mice was different from their counter. Serum TSH was evaluated by bioassay using a line of WT littermates. On hematoxylin and eosin staining, no Chinese hamster ovary cells (CHO-K1) stably transfected with difference was observed in the thyroid follicles or thy- human TSH receptor cDNA, as previously described [30] . Five to rocytes between both groups fed a normal diet. As ex- eleven animals per group were analyzed separately. pected, serum calcium levels were significantly lower in Patient Serum Biochemistry VDR-KO mice compared to WT mice (7.5 8 0.42 vs. Serum samples from patients were randomly selected in a 1: 1 10.0 8 0.2 mg/dl, respectively; p ! 0.0001). Renal func- male:female ratio in our hospital laboratory department based tion (creatinine) was not different (data not shown). on the presence of either low or high 25-OHD levels (^ 10 and Since many of the observed effects in VDR-KO mice are 6 40 ng/ml, respectively), using an equilibrium radioimmuno- secondary to their hypocalcemic status, we subsequent- assay (DiaSorin, Stillwater, Minn., USA). Intra- and interassay coeff icients of variation were 11 and 9%, respectively. The detec- ly evaluated thyroid morphology in mice fed a calcium- tion limit of the radioimmunoassay kit was 1.5 ng/ml. For each rich diet, resulting in a correction of the hypocalcemia patient the following data were retrieved from the medical chart: (9.6 8 1.6 and 10.7 8 0.9 mg/dl for VDR-WT and -KO age, sex, previous or current thyroid or parathyroid disease, cur- mice, respectively). Also under normocalcemic condi- rent use of proton pump inhibitor, serum calcium, renal func- tion (serum creatinine and eGFR), and serum TSH level in the tions, we did not observe morphological differences be- same serum sample as used for the 25-OHD determination. Pa- tween VDR-WT and -KO thyroid glands (f ig. 1 a). Fur- tients hospitalized at the intensive care unit, patients with thermore, thyroid sections also showed no signs of im- known thyroid or parathyroid disease, or with neuroendocrine mune infiltration or neoplasia in any of the groups. tumors were excluded for further analysis, as well as patients Further quantitative morphometric investigation using with severe hypocalcemia or hypercalcemia or with an abnor- mal serum TSH. the point-counting method confirmed the absence of A total of 48 patient sera (4 groups, n = 12 patients per group) difference in thyrocyte size or colloid/thyrocyte ratio were thus collected and stored for additional calcitonin determi- between VDR-WT and -KO mice ( fig. 1 b). nation, using a radioimmunoassay (Biosource, Nijvel, Belgium). Intra- and interassay coefficients of variation were 1.4 and 4.6%, respectively. The detection limit of the radioimmunoassay kit was 0.9 pg/ml. Eur Thyroid J 2012;1:168–175 170 Clinckspoor et al.   Downloaded from Bioscientifica.com at 01/31/2022 07:09:12PM via Deepdyve Normal diet Calcium-rich diet VDR-WT VDR-KO VDR-WT VDR-KO % % Fig. 1. The effect of VDR disruption on VDR-WT 50 70 mouse thyroid morphology. a Hematoxy- VDR-KO lin and eosin staining of thyroids isolated from VDR-WT and -KO mice on normal 40 and calcium-rich diets. Original magnifi- cation ! 40. b Relative volume of colloid 10 (left) and epithelium (right) in VDR-WT 0 0 Normal diet Calcium-rich Normal diet Calcium-rich and -KO mice on normal and calcium-rich b diet diet diets according to the point-counting method. Normal diet Calcium-rich diet VDR-WT VDR-KO VDR-WT VDR-KO Fig. 2. Immunohistochemical staining of thyroid sections (7  m) of VDR-WT and b -KO mice on normal and calcium-rich di- ets. Sections were stained for Tg-I (a), T4 (b) and Duox (c). Immunostaining for Tg-I and T4 was similar in all groups. Immu- nostaining for Duox tended to be more apical in the VDR-WT mice and more in the cytoplasm in the VDR-KO mice (indi- cated by arrows), irrespective of the diet. Original magnification ! 40. Thyrocyte and C Cell Function in Mice in the Absence was observed for Tg-I and T4 between VDR-WT and -KO of VDR Signaling mice on either diet. Duox tended to be located more at the Thyrocyte functionality was assessed by immuno- apical pole of the thyrocytes in WT mice and more in the staining of thyroid sections of VDR-WT and -KO mice cytoplasm in the VDR-KO mice, again irrespective of the for Tg-I, T4 and Duox. As shown in figure 2 , no difference diet. As altered expression of Duox could be caused by Eur Thyroid J 2012;1:168–175 The Thyroid Phenotype in VDR-KO 171 Mice Downloaded from Bioscientifica.com at 01/31/2022 07:09:12PM via Deepdyve Color version available online Normal diet Calcium-rich diet VDR-WT VDR-KO VDR-WT VDR-KO Fig. 3. Calcitonin expression in thyroid tis- sue and serum of VDR-WT and -KO mice on a normal and calcium-rich diet. a Im- VDR-WT munohistochemical staining for calcito- VDR-KO nin on thyroid sections (7  m) showing a clearly higher expression (indicated by ar- rows) in VDR-KO mice compared to VDR- WT mice, irrespective of the diet. Original magnification ! 10. b Serum calcitonin levels were elevated in VDR-KO mice fed a calcium-rich diet, but not in VDR-KO 0 b Normal diet Calcium-rich diet mice on a normal diet, suggesting that cal- cium constitutes a major trigger for calci- tonin secretion. oxidative stress, staining for 4-HNE (a marker of oxida- f luence on basal serum calcitonin levels. We studied se- tive stress) was additionally performed, as well as for cas- rum calcitonin levels in males and females with low and pase-6 (a marker of apoptosis). Both 4-HNE and cas- high serum 25-OHD levels ( ^ 10 and 6 40 ng/ml, re- pase-6 staining were not different between the groups spectively) separately. As can be observed in table 2 , 25- (data not shown). Under the normal diet, serum TSH was OHD serum levels were significantly different in both not different between VDR-WT and -KO mice (0.39 8 the male and the female patient groups. Furthermore, 0.005 and 0.53 8 0.08  U/ml, respectively), but under the both the vitamin D-deficient and vitamin D-sufficient calcium-rich diet TSH was slightly lower in the VDR-KO groups were normocalcemic and comparable for age, re- mice (0.33 8 0.02  U/ml) compared to the VDR-WT nal function and proton pump inhibitor use. mice (0.58 8 0.1 U /ml; p = 0.021). As shown in figure 4 , basal serum calcitonin levels C cell function was studied by calcitonin staining of were not different between the groups with low or high thyroid sections, revealing a strongly increased calcito- 25-OHD , neither when studying both sexes separately, nin expression in VDR-KO compared to the WT mice, nor when evaluating both sexes grouped. Also, after ex- irrespective of the diet ( fig. 3 a). Subsequent serum calci- clusion of patients with renal failure (eGFR ! 30 ml/min/ tonin measurements confirmed increased serum calcito- 1.73 m ) or users of proton pump inhibitors (both known nin levels in VDR-KO mice (125.1 8 36.4 ng/ml) com- confounders increasing serum calcitonin), no difference pared to VDR-WT mice (46.4 8 22.8 ng/ml) fed a calci- in basal serum calcitonin level was observed between the um-rich diet. However, this increase was not observed in vitamin D-deficient and vitamin D-sufficient group hypocalcemic VDR-KO mice fed a normal diet ( fig. 3 b). (data not shown). In Humans, Vitamin D Status Does Not Inf luence Basal Serum Calcitonin Levels Discussion Since C cell function was clearly altered in the absence of normal 1,25(OH) D -VDR signaling in mice, we inves- In the present study we investigated the role of the 2 3 tigated whether these findings could also be relevant for VDR in normal thyroid physiology by studying systemic humans. More specifically, we studied whether vitamin VDR-KO mice in comparison to WT age-matched and D deficiency – a widespread health problem – has an in- sex-matched littermates. Thyrocyte function was not al- Eur Thyroid J 2012;1:168–175 172 Clinckspoor et al.   Downloaded from Bioscientifica.com at 01/31/2022 07:09:12PM via Deepdyve Calcitonin (μU/ml) Color version available online ++ Low 25-OHD 2+ 1,25(OH) D Low Ca PTH CYP27B1 2 3 High 25-OHD VDR 1,25(OH) D FGF23 CYP27B1 2 3 VDR 2 + + 2+ Calcitonin CYP27B1 1,25(OH) D High Ca 2 3 VDR Males Females All Fig. 4. Serum calcitonin levels in vitamin D-deficient (low 25- Fi g. 5. Overview of 1,25(OH) D homeostasis maintained by 2 3 OHD ) and vitamin D-sufficient (high 25-OHD ) patients with- PTH, FGF23 and calcitonin. Low calcium levels trigger PTH re- 3 3 out history of known thyroid or parathyroid disease, medullary lease which stimulates CYP27B1. High calcium levels stimulate cancer or neuroendocrine cancer. Serum calcitonin levels were calcitonin release, also stimulating CYP27B1. FGF23 represents not significantly different, neither when studying both sexes sep- the negative regulator of CYP27B1. PTH, FGF23 and calcitonin arately, nor when both sexes were grouped. are regulated by 1,25(OH) D via the VDR. Stimulating effects are 2 3 indicated with full arrows, inhibitory effects are indicated with dashed lines. Table 2. Characteristics of patients with low and high serum 25- tered but C cell activity was clearly increased in the ab- OHD level sence of the VDR, confirming that 1,25(OH) D nega- 2 3 tively regulates calcitonin via the VDR. Low vit. D High vit. D p value Supraphysiological doses of active vitamin D have been described to inhibit proliferation and reduce the se- Serum 25-OHD , ng/ml verity of autoimmunity in case of underlying malignant Males 7.180.74 49.382.5 <0.0001 Females 6.680.59 53.684.8 <0.0001 or autoimmune thyroid disease, respectively [5, 6, 11] . In Age, years the present study, global thyroid morphology was un- Males 60.884.0 59.084.6 0.78 changed in VDR-KO mice, also after correction of the Females 58.786.8 54.583.0 0.58 hypocalcemic state, and no signs of autoimmune thy- creatinine, mg/dl roiditis or thyroid neoplasia were present. Further analy- Males 1.580.5 2.380.6 0.32 Females 1.180.4 1.080.1 0.78 sis of thyrocyte functionality by studying the expression eGFR, ml/min/1.73 m2 of T4 and Tg-I showed no differences. Duox was located Males 49.288.9 35.388.0 0.28 more in the cytoplasm (instead of apically in the VDR- Females 0.87 59.5812.0 50.387.3 WT mice), possibly as a result of oxidative stress. How- calcium, mg/dl ever, no signs of increased oxidative stress (4-HNE) or Males 8.880.2 9.080.2 0.56 Females 0.21 9.180.2 9.480.2 apoptosis (caspase-6) were observed. Serum TSH was not PPI use, % different under the normal diet, but was slightly lower a b Males 45 (5/11) 42 (5/12) 0.85 under the calcium-rich diet. A similar observation was Females 33 (4/12) 17 (2/12) 0.35 made by others in rats with severe vitamin D deficiency, P PI = Proton pump inhibitor. but serum T4 levels remained unchanged [31] . These re- PPI use was not known in 1 male with low vitamin D. sults suggest that the 1,25(OH) D -VDR signaling path- 2 3 b 2 test. way has a redundant role in normal thyrocyte develop- ment and function in rodents, although a mild effect on the TSH setpoint is possible. Eur Thyroid J 2012;1:168–175 The Thyroid Phenotype in VDR-KO 173 Mice Downloaded from Bioscientifica.com at 01/31/2022 07:09:12PM via Deepdyve Calcitonin (ng/ml) Second, we studied the role of the VDR in parafollic- On the other hand, it is known that C cell physiology, in ular C cell physiology. The enzyme CYP27B1, responsi- contrast to thyrocyte physiology, is largely different in ble for the final activation of 25-OHD into active rodents as compared to humans [33, 34] . We found no 1,25(OH) D , is known to be positively regulated by PTH increased basal serum calcitonin levels in a cohort of 2 3 and calcitonin, and negatively regulated by FGF23. The patients with severe vitamin D deficiency compared to transcription factor C/EBP and the SWI/SNF chroma- patients with vitamin D sufficiency (all patients were nor- tin remodeling complex mediate CYP27B1 transcription mocalcemic and matched for sex, age, renal function, use induced by calcitonin [23] . In the present study we ob- of proton pump inhibitors). The variable comorbidity of served clearly increased thyroidal calcitonin expression the patients, however, represents a limitation of this study. in VDR-KO mice, independent from the serum calcium In conclusion, VDR disruption did not inf luence thy- level. In the normocalcemic mice the increased calcitonin roid morphology or thyrocyte function in mice. How- expression at the C cell level was paralleled by increased ever, VDR constitutes an important determinant in C cell serum calcitonin levels. This was not observed in VDR- physiology in mice, negatively controlling calcitonin. In KO mice fed a normal diet, which is most probably ex- a pilot study in patients, there was no evidence for altera- plained by their hypocalcemic status inhibiting secretion tions of serum calcitonin levels in case of very low or high of calcitonin by the C cells. In a rat model, it has been es- levels of serum 25-OHD . These findings need to be con- tablished by others that calcium constitutes the main reg- firmed in a study in healthy individuals, preferably by a ulator for calcitonin secretion, but that 1,25(OH) D and prospective randomized placebo-controlled clinical trial, 2 3 not calcium constitutes the main regulator for calcitonin evaluating serum calcitonin before and after vitamin D transcription [25, 32] . We hereby demonstrate for the first supplementation along with serum levels of 25-OHD , time that the inhibitory action of 1,25(OH) D on calci- 1,25(OH) D , PTH, FGF23 and VDR polymorphism. At 2 3 2 3 tonin production acts via the VDR. Due to the absence of present, there is no indication for a systematic evaluation VDR signaling, the inhibitory effect of 1,25(OH) D on C of the patient’s vitamin D status along with a serum cal- 2 3 cells and thus the physiological negative feedback control citonin measurement. is lost. Thyroiditis and renal insufficiency were ruled out as potential other causes for increased calcitonin by in- creased bystander C cell activation and decreased renal Acknowledgements clearance, respectively. Furthermore, Egrise et al. [31 ] de- We are grateful to Christine de Ville de Goyet, Paul Drent, Jos scribed that rats fed a vitamin D-deficient diet showed Laureys, Suzanne Marcelis, Ingrid Stockmans and Erik Van strongly increased thyroidal calcitonin mRNA expres- Herck for their invaluable technical assistance. sion, which suggests that deficiency of the ligand for VDR This work was supported by the Flemish Research Foundation also results in uncontrolled calcitonin expression in (clinical FWO fellowship for C.M. and B.D. and grant G021207N). rodents. Whether chronically defective signaling could eventually lead to C cell proliferation is not known. Disclosure Statement Thus, as summarized in figure 5 , we demonstrate in the present study that besides the known positive regulation The authors declare that no competing financial interest ex- of CYP27B1 by PTH and its negative feedback via ists. 1,25(OH) D -VDR signaling, calcitonin (the other k nown 2 3 positive regulator of CYP27B1, but mainly active under normocalcemic to hypercalcemic conditions) is also neg- atively controlled by 1,25(OH) D via the VDR. FGF23 2 3 represents the negative regulator of CYP27B1, with a pos- 1 Bouillon R, Carmeliet G, Verlinden L, van References itive feedback by 1,25(OH) D via the VDR. 2 3 Etten E, Verstuyf A, Luderer HF, Lieben L, In the last part of the study, we investigated whether Mathieu C, Demay M: Vitamin D and hu- man health: lessons from vitamin D receptor increased calcitonin caused by defective 1,25(OH) D - 2 3 null mice. Endocr Rev 2008; 29: 726–776. VDR signaling as observed in rodents also applies to hu- 2 Khadzkou K, Buchwald P, Westin G, Dralle mans. If so, this would imply that the vitamin D status H, Akerstrom G, Hellman P: 25hydroxy vita- min D 1alpha-hydroxylase and vitamin D and also VDR polymorphism might inf luence the basal receptor expression in papillary thyroid car- calcitonin level. As vitamin D deficiency is very preva- cinoma. J Histochem Cytochem 2006; 54: 355–361. lent, this could have important clinical consequences. Eur Thyroid J 2012;1:168–175 174 Clinckspoor et al.   Downloaded from Bioscientifica.com at 01/31/2022 07:09:12PM via Deepdyve 3 Clinckspoor I, Hauben E, Verlinden L, Van Crescioli C: Elocalcitol inhibits inf lamma- al control of other bone anabolic and cata- den Bruel A, Vanwalleghem L, Vander tory responses in human thyroid cells and bolic genes that orchestrate the regulation of Poorten V, Delaere P, Mathieu C, Verstuy f A, T cells. Endocrinology 2008; 149: 3626– phosphate and calcium mineral metabolism. Decallonne B: Altered expression of key 3634. J Steroid Biochem Mol Biol 2007; 103: 381– players in vitamin D metabolism and signal- 14 Tokuda N, Mano T, Levy RB: 1,25-Dihy- 388. ing in malignant and benign thyroid tumors. droxyvitamin D antagonizes interferon- 25 Naveh-Many T, Silver J: Regulation of calci- J Histochem Cytochem 2012; 60: 502–511. gamma-induced expression of class II major tonin gene transcription by vitamin D me- 4 Lamberg-Allardt C, Valtonen E, Polojarvi histocompatibility antigens on thyroid fol- tabolites in vivo in the rat. J Clin Invest 1988; M, Stewen P: Characterization of a 1,25-di- licular and testicular Leydig cells. Endocri- 81: 270–273. hydroxy-vitamin D r eceptor in FRTL-5 nology 1990; 127: 1419–1427. 26 Martin-Lacave I, Ramos F, Utrilla JC, Conde cells. Evidence for an inhibitory effect of 15 Lin WY, Wan L, Tsai CH, Chen RH, Lee CC, E, Hevia A, Fernandez R, Moreno AM, 1,25-dihydroxy-vitamin D on thyrotropin- Tsai FJ: Vitamin D receptor gene polymor- Fernandez-Santos JM, Galera-Davidson H: induced iodide uptake. Mol Cell Endocrinol phisms are associated with risk of Hashimo- Chronic hypervitaminosis D determines a 1991; 81: 25–31. to’s thyroiditis in Chinese patients in Tai- decrease in C-cell numbers and calcitonin 5 Liu W, Asa SL, Fantus IG, Walfish PG, Ezzat wan. J Clin Lab Anal 2006; 20: 109–112. levels in rats. J Endocrinol Invest 1998; 21: S: Vitamin D arrests thyroid carcinoma cell 16 Stefanic M, Papic S, Suver M, Glavas-Obro- 102–108. growth and induces p27 dephosphorylation vac L, Karner I: Association of vitamin D re- 27 Costante G, Durante C, Francis Z, Schlum- and accumulation through PTEN/akt-de- ceptor gene 3  -variants with Hashimoto’s berger M, Filetti S: Determination of calcito- pendent and -independent pathways. Am J thyroiditis in the Croatian population. Int J nin levels in C-cell disease: clinical interest Pathol 2002; 160: 511–519. Immunogenet 2008; 35: 125–131. and potential pitfalls. Nat Clin Pract Endo- 6 Clinckspoor I, Verlinden L, Overbergh L, 17 Collins JE, Heward JM, Nithiyananthan R, crinol Metab 2009; 5: 35–44. Korch C, Bouillon R, Mathieu C, Verstuyf A, Nejentsev S, Todd JA, Franklyn JA, Gough 28 Van Cromphaut SJ, Dewerchin M, Hoen- Decallonne B: 1,25-Dihydroxyvitamin D SC: Lack of association of the vitamin D derop JG, Stockmans I, Van HE, Kato S, Bin- and a superagonistic analog in combination receptor gene with Graves’ disease in UK dels RJ, Collen D, Carmeliet P, Bouillon R, with paclitaxel or suberoylanilide hy- Caucasians. Clin Endocrinol 2004; 60: 618– Carmeliet G: Duodenal calcium absorption droxamic acid have potent antiproliferative 624. in vitamin D receptor-knockout mice: func- effects on anaplastic thyroid cancer. J Steroid 18 Maalej A, Petit-Teixeira E, Chabchoub G, tiona l and molecular aspects. Proc Natl Acad Biochem Mol Biol 2011; 124: 1–9. Hamad MB, Rebai A, Farid NR, Cornelis F, Sci USA 2001; 98: 13324–13329. 7 Okano K, Usa T, Ohtsuru A, Tsukazaki T, Ayadi H: Lack of association of VDR gene 29 Weibel ER, Kistler GS, Scherle WF: Practical Miyazaki Y, Yonekura A, Namba H, Shindoh polymorphisms with thyroid autoimmune stereological methods for morphometric cy- H, Yamashita S: Effect of 22-oxa-1,25-dihy- disorders: familial and case/control studies. tology. J Cell Biol 1966; 30: 23–38. droxyvitamin D on human thyroid cancer J Clin Immunol 2008; 28: 21–25. 30 Perret J, Ludgate M, Libert F, Gerard C, Du- cell growth. Endocr J 1999; 46: 243–252. 19 Goswami R, Marwaha RK, Gupta N, Tan- mont JE, Vassart G, Parmentier M: Stable ex- 8 Lee S, Morimoto S, Onishi T, Tsuji M, Okada don N, Sreenivas V, Tomar N, Ray D, Kan- pression of the human TSH receptor in CHO Y, Seino Y, Ishida M, Yamaoka K, Takai S, war R, Agarwal R: Prevalence of vitamin D cells and characterization of differentially Miyauchi A, Kumahara Y: Normal serum deficiency and its relationship with thyroid expressing clones. Biochem Biophys Res 1,25-dihydroxyvitamin D in patients with autoimmunity in Asian Indians: a commu- Commun 1990; 171: 1044–1050. medullary carcinoma of the thyroid. J Clin nity-based survey. Br J Nutr 2009; 102: 382– 31 Egrise D, Burniat A, Lefort A, Libert F, Miot Endocrinol Metab 1982; 55: 361–363. 386. F, Goldman S, Cor vilain B, Moreno-Reyes R: 9 Stepien T, Krupinski R, Sopinski J, Kuzdak 20 Kivity S, Agmon-Levin N, Zisappl M, Sha- Vitamin D deficiency modulates the regula- K, Komorowski J, Lawnicka H, Stepien H: pira Y, Nag y EV, Danko K, Szekanecz Z , Lan- tion of the pituitar y-thyroid a xis and thyroid Decreased 1-25 dihydroxyvitamin D con- gevitz P, Shoenfeld Y: Vitamin D and auto- gene expression (abstract No. 2614). 14th In- centration in peripheral blood serum of pa- immune thyroid diseases. Cell Mol Immu- ternational Thyroid Congress (ITC), Paris, tients with thyroid cancer. Arch Med Res nol 2011; 8: 243–247. September 2010. 2010; 41: 190–194. 21 Misharin A, Hewison M, Chen CR, Lagishet- 32 Naveh-Many T, Raue F, Grauer A, Silver J: 10 Laney N, Meza J, Lyden E, Erickson J, Treude ty V, Aliesky HA, Mizutori Y, Rapoport B, Regulation of calcitonin gene expression by K, Goldner W: The prevalence of vitamin D McLachlan SM: Vitamin D deficiency mod- hypocalcemia, hypercalcemia, and vitamin deficiency is similar between thyroid nodule ulates Graves’ hyperthyroidism induced in D in the rat. J Bone Miner Res 1992; 7: 1233– and thyroid cancer patients. Int J Endocrinol BALB/c mice by thyrotropin receptor immu- 1237. 2010; 2010: 805716. nization. Endocrinology 2009; 150: 1051– 33 Bjerre KL , Madsen LW, Andersen S, Almholt 11 Fournier C, Gepner P, Sadouk M, Charreire 1060. K, de Boer AS, Drucker DJ, Gotfredsen C, J: In vivo beneficial effects of cyclosporin A 22 Shinki T, Ueno Y, DeLuca HF, Suda T: Calci- Egerod FL , Hegelund AC, Jacobsen H, Jacob- and 1,25-dihydroxyvitamin D on the in- tonin is a major regulator for the expression sen SD, Moses AC, Molck AM, Nielsen HS, duction of experimental autoimmune thy- of renal 25-hydroxyvitamin D -1alpha-hy- Nowak J, Solberg H, Thi TD, Zdravkovic M: roiditis. Clin Immunol Immunopathol 1990; droxylase gene in normocalcemic rats. Proc Glucagon-like Peptide-1 receptor agonists 54: 53–63. Natl Acad Sci USA 1999; 96: 8253–8258. activate rodent thyroid C-cells causing calci- 12 Wang SH, Koenig RJ, Giordano TJ, Myc A, 23 Zhong Y, Armbrecht HJ, Christakos S: Cal- tonin release and C-cell proliferation. Endo- Thompson NW, Baker JR Jr: 1Alpha,25- di- citonin, a regulator of the 25-hydroxyvita- crinology 2010; 151: 1473–1486. hydroxyvitamin D up-regulates Bcl-2 ex- min D 1alpha-hydroxylase gene. J Biol 34 Hegedus L, Moses AC, Zdravkovic M, Le TT, 3 3 pression and protects normal human thyro- Chem 2009; 284: 11059–11069. Daniels GH: GLP-1 and calcitonin concen- cy tes from programmed cell death. Endocri- 24 Barthel TK, Mathern DR, Whitfield GK, tration in humans: lack of evidence of calci- nology 1999; 140: 1649–1656. Haussler CA, Hopper HA, Hsieh JC, Slater tonin release from sequential screening in 13 Borgogni E, Sarchielli E, Sottili M, Santar- SA, Hsieh G, Kaczmarska M, Jurutka PW, over 5,000 subjects with type 2 diabetes or lasci V, Cosmi L, Gelmini S, Lombardi A, Kolek OI, Ghishan FK, Haussler MR: nondiabetic obese subjects treated with the Cantini G, Perigli G, Luconi M, Vannelli 1,25-Dihydroxyvitamin D /VDR-mediated human GLP-1 analog, liraglutide. J Clin En- GB, Annunziato F, Adorini L, Serio M, induction of FGF23 as well as transcription- docrinol Metab 2011; 96: 853–860. Eur Thyroid J 2012;1:168–175 The Thyroid Phenotype in VDR-KO 175 Mice Downloaded from Bioscientifica.com at 01/31/2022 07:09:12PM via Deepdyve

Journal

European Thyroid Journal – Bioscientifica

Published: Oct 22, 2012

Keywords: Vitamin D receptor

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