Disruption of Nuclear Vitamin D Receptor Gene Causes Enhanced Thrombogenicity in Mice

Abstract

Vitamin D metabolites influence the expression of various genes involved in calcium homeostasis, cell differentiation, and regulation of the immune system. Expression of these genes is mediated by the activation of the nuclear vitamin D receptor (VDR). Previous studies have shown that a hormonally active form of vitamin D, 1α,25-dihydroxyvitamin D3, exerts anticoagulant effects in cultured monocytic cells. To clarify whether activation of VDR plays any antithrombotic actions in vivo, hemostatic/thrombogenic systems were examined in normocalcemic VDR knock-out (KO) mice on a high calcium diet and compared with wild type and hypocalcemic VDRKO mice that were fed a regular diet. Platelet aggregation was enhanced significantly in normocalcemic VDRKO mice compared with wild type and hypocalcemic VDRKO mice. Aortic endothelial nitric-oxide (NO) synthase expression and urinary NOx excretions were reduced in hypocalcemic VDRKO mice, but not in normocalcemic VDRKO mice. Northern blot and RT-PCR analyses revealed that the gene expression of antithrombin in the liver as well as that of thrombomodulin in the aorta, liver and kidney was down-regulated in hypo- and normocalcemic VDRKO mice. Whereas tissue factor mRNA expression in the liver and kidney was up-regulated in VDRKO mice regardless of plasma calcium level. Furthermore, VDRKO mice manifested an exacerbated multi-organ thrombus formation after exogenous lipopolysaccharide injection regardless of the calcemic conditions. These results demonstrate that activation of nuclear VDR elicits antithrombotic effects in vivo, and suggest that the VDR system may play a physiological role in the maintenance of antithrombotic homeostasis. Vitamin D metabolites influence the expression of various genes involved in calcium homeostasis, cell differentiation, and regulation of the immune system. Expression of these genes is mediated by the activation of the nuclear vitamin D receptor (VDR). Previous studies have shown that a hormonally active form of vitamin D, 1α,25-dihydroxyvitamin D3, exerts anticoagulant effects in cultured monocytic cells. To clarify whether activation of VDR plays any antithrombotic actions in vivo, hemostatic/thrombogenic systems were examined in normocalcemic VDR knock-out (KO) mice on a high calcium diet and compared with wild type and hypocalcemic VDRKO mice that were fed a regular diet. Platelet aggregation was enhanced significantly in normocalcemic VDRKO mice compared with wild type and hypocalcemic VDRKO mice. Aortic endothelial nitric-oxide (NO) synthase expression and urinary NOx excretions were reduced in hypocalcemic VDRKO mice, but not in normocalcemic VDRKO mice. Northern blot and RT-PCR analyses revealed that the gene expression of antithrombin in the liver as well as that of thrombomodulin in the aorta, liver and kidney was down-regulated in hypo- and normocalcemic VDRKO mice. Whereas tissue factor mRNA expression in the liver and kidney was up-regulated in VDRKO mice regardless of plasma calcium level. Furthermore, VDRKO mice manifested an exacerbated multi-organ thrombus formation after exogenous lipopolysaccharide injection regardless of the calcemic conditions. These results demonstrate that activation of nuclear VDR elicits antithrombotic effects in vivo, and suggest that the VDR system may play a physiological role in the maintenance of antithrombotic homeostasis. In many target organs, vitamin D exerts a variety of biological functions such as calcium homeostasis, cell proliferation, and cell differentiation. Most of these actions are exerted through the transcriptional control of target genes by the activation of the nuclear vitamin D receptor (VDR). 1The abbreviations used are: VDR, vitamin D receptor; AT, antithrombin; eNOS, endothelial nitric-oxide synthase; GFD, glomeruli with fibrin deposition; KO, knock-out; LPS, lipopolysaccharide; NO, nitric oxide; NOx, NO metabolites; PTH, parathyroid hormone; RARE, retinoic acid-responsive element; RT, reverse transcription; TF, tissue factor; TM, thrombomodulin; VDRE, vitamin D-responsive element; VDRKO, vitamin D receptor knock-out; WT, wild type.1The abbreviations used are: VDR, vitamin D receptor; AT, antithrombin; eNOS, endothelial nitric-oxide synthase; GFD, glomeruli with fibrin deposition; KO, knock-out; LPS, lipopolysaccharide; NO, nitric oxide; NOx, NO metabolites; PTH, parathyroid hormone; RARE, retinoic acid-responsive element; RT, reverse transcription; TF, tissue factor; TM, thrombomodulin; VDRE, vitamin D-responsive element; VDRKO, vitamin D receptor knock-out; WT, wild type. VDR is a ligand-inducible transcription factor that belongs to the nuclear receptor superfamily (1Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6043) Google Scholar). VDR forms a heterodimer with the retinoid X receptor and binds to specific vitamin D-responsive elements on target genes (2Haussler M.R. Whitfield G.K. Haussler C.A. Hsieh J.C. Thompson P.D. Selznick S.H. Dominguez C.E. Jurutka P.W. J. Bone Miner. Res. 1998; 13: 325-349Crossref PubMed Scopus (1217) Google Scholar, 3Takeyama K. Masuhiro Y. Fuse H. Endoh H. Murayama A. Kitanaka S. Suzawa M. Yanagisawa J. Kato S. Mol. Cell. Biol. 1999; 19: 1049-1055Crossref PubMed Scopus (190) Google Scholar), which initiate sequences of events that lead to the activation or repression of target gene transcription by recruiting transcriptional cofactor complexes. Previous studies demonstrated that VDR knock-out (KO) mice manifest a variety of phenotypic abnormalities, including hypocalcemia, osteopenia, growth retardation, alopecia (4Yoshizawa T. Handa Y. Uematsu Y. Takeda S. Sekine K. Yoshihara Y. Kawakami T. Arioka K. Sato H. Uchiyama Y. Masushige S. Fukamizu A. Matsumoto T. Kato S. Nat. Genet. 1997; 16: 391-396Crossref PubMed Scopus (971) Google Scholar, 5Li Y.C. Pirro A.E. Amling M. Delling G. Baron R. Bronson R. Demay M.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9831-9835Crossref PubMed Scopus (805) Google Scholar), impaired immunity (6Mathieu C. Van Etten E. Gysemans C. Decallonne B. Kato S. Laureys J. Depovere J. Valckx D. Verstuyf A. Bouillon R. J. Bone Miner. Res. 2001; 16: 2057-2065Crossref PubMed Scopus (140) Google Scholar), hypertension with cardiac hypertrophy (7Li Y.C. Kong J. Wei M. Chen Z.F. Liu S.Q. Cao L.P. J. Clin. Invest. 2002; 110: 229-238Crossref PubMed Scopus (1737) Google Scholar), and abnormal skeletal muscle development (8Endo I. Inoue D. Mitsui T. Umaki Y. Akaike M. Yoshizawa T. Kato S. Matsumoto T. Endocrinology. 2003; 144: 5138-5144Crossref PubMed Scopus (345) Google Scholar) and that most, but not all, of these phenotypic abnormalities can be rescued by normalization of serum mineral levels (6Mathieu C. Van Etten E. Gysemans C. Decallonne B. Kato S. Laureys J. Depovere J. Valckx D. Verstuyf A. Bouillon R. J. Bone Miner. Res. 2001; 16: 2057-2065Crossref PubMed Scopus (140) Google Scholar, 9Li Y.C. Amling M. Pirro A.E. Priemel M. Meuse J. Baron R. Delling G. Demay M.B. Endocrinology. 1998; 139: 4391-4396Crossref PubMed Google Scholar). In addition to its classical target tissues, VDR is also expressed in monocytic cells (10Koyama T. Shibakura M. Ohsawa M. Kamiyama R. Hirosawa S. Blood. 1998; 92: 160-167Crossref PubMed Google Scholar) and vascular endothelial cells (11Merke J. Milde P. Lewicka S. Hugel U. Klaus G. Mangelsdorf D.J. Haussler M.R. Rauterberg E.W. Ritz E. J. Clin. Invest. 1989; 83: 1903-1915Crossref PubMed Scopus (297) Google Scholar), suggesting potential roles of vitamin D in antithrombotic functions. Koyama and colleagues (10Koyama T. Shibakura M. Ohsawa M. Kamiyama R. Hirosawa S. Blood. 1998; 92: 160-167Crossref PubMed Google Scholar, 12Ohsawa M. Koyama T. Yamamoto K. Hirosawa S. Kamei S. Kamiyama R. Circulation. 2000; 102: 2867-2872Crossref PubMed Scopus (118) Google Scholar) have found that a hormonally active form of vitamin D, 1α,25-dihydroxyvitamin D3, as well as retinoic acid, exert anticoagulant effects by up-regulating the expression of the anticoagulant glycoprotein, thrombomodulin (TM), and by down-regulating the expression of a critical coagulation factor, tissue factor (TF), in cultured monocytic cells and human peripheral monocytes. However, it remains unclear whether activation of nuclear VDR can elicit antithrombotic actions in vivo. The present study was undertaken to clarify the physiological role of VDR-mediated actions in the hemostatic/thrombogenic system in vivo. Animal Preparation—VDR homozygous mutants (4Yoshizawa T. Handa Y. Uematsu Y. Takeda S. Sekine K. Yoshihara Y. Kawakami T. Arioka K. Sato H. Uchiyama Y. Masushige S. Fukamizu A. Matsumoto T. Kato S. Nat. Genet. 1997; 16: 391-396Crossref PubMed Scopus (971) Google Scholar) and wild type (WT) control littermates were maintained as hybrids with C57BL/6J and CBA genetic backgrounds in a specific pathogen-free facility with a 12-h light and dark cycle. As reported previously (4Yoshizawa T. Handa Y. Uematsu Y. Takeda S. Sekine K. Yoshihara Y. Kawakami T. Arioka K. Sato H. Uchiyama Y. Masushige S. Fukamizu A. Matsumoto T. Kato S. Nat. Genet. 1997; 16: 391-396Crossref PubMed Scopus (971) Google Scholar, 9Li Y.C. Amling M. Pirro A.E. Priemel M. Meuse J. Baron R. Delling G. Demay M.B. Endocrinology. 1998; 139: 4391-4396Crossref PubMed Google Scholar), a lactose-rich high calcium diet can normalize hypocalcemia in VDRKO mice, whereas a high calcium diet causes considerable hypercalcemia in WT mice. Therefore, after weaning (at 3 weeks of age) we divided the mice into three groups as follows: group 1 are WT mice fed a regular diet (containing 1.2% calcium and 1.0% phosphorus, Japan Clea, Tokyo, Japan); group 2 are VDRKO mice fed a regular diet (hypocalcemic); and group 3 are VDRKO mice fed a lactose-rich high calcium diet (normocalcemic). Experiments were conducted in 12-week-old mice that had been weaned then fed a specified diet for 9 weeks. Blood Collection—Blood was collected from the inferior vena cava of mice under ether anesthesia. The blood was drawn with 21-gauge needles into plastic syringes containing 0.1 volume of 3.8% trisodium citrate solution. Some whole blood samples were used immediately for platelet aggregation analysis, and other whole blood samples were centrifuged at 2,000 × g for 10 min to obtain plasma. The plasma was stored at -80 °C until assayed. Measurement of Plasma Calcium and Parathyroid (PTH) Hormone— Plasma levels of calcium and PTH were determined using the OCPC method and a rat PTH IRMA Kit (Immunotopics, Inc., San Clemente, CA), respectively. Comparison of Platelet Aggregation with a Screen Filtration Pressure Method—Measurements of platelet aggregation with a screen filtration pressure aggregometer (WBA analyzer from SSR Engineering Co., Ltd., Yokohama, Japan) were performed according to a method described previously (13Ozeki Y. Sudo T. Toga K. Nagamura Y. Ito H. Ogawa T. Kimura Y. Thromb. Res. 2001; 101: 65-72Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 14Sudo T. Ito H. Kimura Y. Platelets. 2003; 14: 239-246Crossref PubMed Scopus (24) Google Scholar). Reaction tubes containing 200-μl aliquots of whole blood were placed in an incubation chamber at 37 °C for 30 s followed by the addition of 22.2 μl of a serial concentration (1, 2, 4, 8 or 16 μm) of ADP (Sigma). 2 min after the addition of ADP, the blood samples were filtered through microsieve screens connected to a pressure sensor. A negative pressure of -130 mm Hg was established as 100%. The 0% pressure base line was established as -6 mm Hg, rather than 0 mm Hg because of the viscosity of whole blood. The platelet aggregation pressure of each reaction tube was determined as the pressure rate (percent). For the screen filtration pressure aggregometer study, the pressure rate was standardized using a grading curve produced by plotting four or five concentrations of ADP on the x axis and pressure rate (percent) on the y axis. The concentration of ADP causing a 50% increase in pressure rate was calculated and applied as the platelet aggregatory threshold index. Measurement of Daily Urinary Excretion of Nitric Oxide Metabolites—To evaluate urinary excretion of nitric oxide (NO) metabolites (NOx), mice were housed individually in metabolic cages that provided free access to tap water and food for 24 h. NOx levels, from the murine urine, were determined by high performance liquid chromatography (15Yamada K. Nabeshima T. J. Neurochem. 1997; 68: 1234-1243Crossref PubMed Scopus (156) Google Scholar). Estimation of Plasma Prothrombin Time and Activated Partial Thromboplastin Time—Plasma prothrombin time and activated partial thromboplastin time were determined by a scattered light detection method (16Poller L. Thromb. Haemostasis. 1988; 60: 18-20Crossref PubMed Scopus (22) Google Scholar). Measurement of Plasma Antithrombin (AT) Activity—Plasma AT activity was measured using N-test ATIII-S (Nittobo, Tokyo, Japan), which determines anticoagulant activity using a chromogenic substrate, according to the manufacturer's instructions. Western Blot Analysis—Aortic tissue samples were homogenized in an ice-cold tissue protein extraction reagent (T-PER™, Pierce) containing protease inhibitors. Equal amounts of protein (50 μg) were loaded onto 10% acrylamide gels and then transferred to nitrocellulose membranes using a wet blot apparatus. Membranes were blocked overnight at 4 °C in blocking buffer (10 mm Tris-HCl, 10 mm NaCl, 0.1% polyoxyethylenesorbitan monolaurate) with 5% skim milk. The first antibody, mouse anti-human endothelial nitric-oxide synthase (eNOS) monoclonal antibody (1:1000 dilution, BD Transduction Laboratories, San Jose, CA), was applied for 1 h at room temperature. After a 30-min wash with the blocking buffer, membranes were probed with the second antibody (1:5000 dilution anti-mouse IgG whole antibody, for 1 h at room temperature. Membranes were for 30 The membranes were then with Western for min and then to were using a system. and were with physiological was by an extraction using concentrations were measured at and then samples were stored in water at -80 Northern Blot of of the liver was on gels and transferred to membranes by in a high × were in a for 1 h at °C followed by overnight with a specific Northern were in 2 × and 0.1% at room for min and then in × and 0.1% at °C for min to The and reverse sequences of the AT and respectively. AT mRNA was after for by the of RT-PCR of TF, TM, and mRNA in aorta, and kidney tissue were by a RT-PCR method (8Endo I. Inoue D. Mitsui T. Umaki Y. Akaike M. Yoshizawa T. Kato S. Matsumoto T. Endocrinology. 2003; 144: 5138-5144Crossref PubMed Scopus (345) Google Scholar). used were as follows: TF, and reverse TM, and reverse and reverse were on with by and Expression levels of and were expressed to the Experiments and were with lipopolysaccharide h mice were and aorta, and kidney tissue were The tissue samples were placed immediately in and stored After samples were in and were These tissue were and with a protease for 10 min at room temperature. After with was blocked with for and then was blocked with the After with water and blocking by incubation with 10% serum in for 10 min at room the were with anti-human antibody overnight at 4 The were then with and with of for 30 min at room temperature. After with the were with for 30 min at room temperature. incubation in of containing of μl of and of for min at room the were with for 1 the were after with water and The of the glomeruli with fibrin were calculated in of each of the glomeruli were as for each a group were expressed as the For was using a of was at Plasma Calcium and PTH in WT and VDRKO The plasma calcium levels of VDRKO mice fed a regular diet were significantly compared with WT mice VDRKO mice were fed a high calcium plasma calcium levels were to levels to in WT mice As in plasma PTH levels were in VDRKO mice compared with WT mice. with the normalization of hypocalcemia from a high calcium diet plasma PTH levels to levels in WT mice. Platelet Aggregation in VDRKO the of VDR activation on platelet we first whole blood aggregation using a screen filtration pressure method in 12-week-old mice Platelet aggregatory threshold in hypocalcemic VDRKO mice were compared with in WT mice In platelet aggregatory threshold in normocalcemic VDRKO mice were significantly than in WT mice These results demonstrate that hypocalcemia is for the of platelet aggregation in VDRKO mice fed a regular diet and that VDR a on platelet Urinary of Nitric Oxide in VDRKO by a Calcium platelet aggregation is by the of NO, we then urinary excretion of NOx Daily urinary excretion of NOx in VDRKO mice was significantly compared with WT mice. a high calcium diet the urinary of NOx in VDRKO mice to a to that in WT mice. it is that the enhanced platelet aggregation in normocalcemic VDRKO mice is mediated by a in NO level. Aortic Expression in VDRKO by Calcium blot demonstrated that protein levels in hypocalcemic VDRKO mice to of WT mice. may be a of levels of urinary NOx excretion in hypocalcemic VDRKO mice a high calcium protein levels in VDRKO mice to levels to in WT mice These results that reduced protein levels in VDRKO mice are mediated by VDRKO Prothrombin Time and Activated Partial Thromboplastin prothrombin time and activated partial thromboplastin time in WT, and normocalcemic VDRKO mice were were these mice in prothrombin time s in WT, and normocalcemic VDRKO mice, or activated partial thromboplastin time s in WT, and normocalcemic VDRKO mice, These results demonstrate that the of VDR not lead to impaired blood coagulation and is with the that VDRKO mice not a Plasma and Expression of Antithrombin in VDRKO examined the plasma activity of AT in these mice. of plasma AT in VDRKO mice regardless of plasma calcium levels compared with in WT mice In gene expression levels of AT in the liver were reduced by in hypo- and normocalcemic VDRKO mice compared with in WT mice These results demonstrate that activated VDR AT gene expression in the Expression of and in VDRKO shown in mRNA expression levels in the liver and kidney were enhanced in hypo- and normocalcemic VDRKO mice compared with in WT mice and to be in the of normocalcemic VDRKO mice. In mRNA levels in the aorta, and kidney were reduced in VDRKO mice compared with in WT mice of plasma calcium levels to the expression of and genes in VDRKO mice and These results that activation of VDR elicits of and of gene in mRNA levels in the aorta, and kidney were by RT-PCR as described under was from each of WT mice and VDRKO mice at weeks of aorta, and kidney tissue samples were examined in each are expressed as the in VDRKO the that the VDR system was to WT and VDRKO mice. mice the until revealed that hypo- and normocalcemic VDRKO mice fibrin in the glomeruli and of the kidney compared with WT mice fibrin was also in the of VDRKO mice regardless of plasma calcium levels fibrin was in the or from any group of mice not To the of fibrin in the we compared the three groups of mice. As shown in the was significantly in hypo- and normocalcemic VDRKO mice than that in WT mice. fibrin in the liver and kidney was a was in of VDRKO mice not of glomeruli with fibrin after injection in WT mice and VDRKO mice at weeks of was into mice as described under After were to The of glomeruli with fibrin was significantly in VDRKO mice than WT mice. tissue samples were examined in each are expressed as the The present study that platelet aggregation was enhanced significantly in normocalcemic VDRKO mice. are of that to thrombus formation C. Thromb. Haemostasis. PubMed Scopus Google Scholar). and abnormal on various Thromb. Google Scholar). plasma calcium concentration is the for platelet of calcium through to calcium the thrombus to thrombus growth S. S. J. Biol. 2003; PubMed Scopus Google Scholar). it is to that the impaired platelet aggregation in hypocalcemic VDRKO mice was by the hypocalcemia in these mice. In the platelet aggregation in normocalcemic VDRKO mice are and are to VDR in and have been in studies that a hormonally active form of vitamin D gene expression and gene expression in monocytic cells (10Koyama T. Shibakura M. Ohsawa M. Kamiyama R. Hirosawa S. Blood. 1998; 92: 160-167Crossref PubMed Google Scholar, 12Ohsawa M. Koyama T. Yamamoto K. Hirosawa S. Kamei S. Kamiyama R. Circulation. 2000; 102: 2867-2872Crossref PubMed Scopus (118) Google Scholar). However, in effects of the vitamin system on these have been In the present study, the gene expression of AT in the liver as well as that of in the aorta, and kidney in VDRKO mice was whereas mRNA expression in the liver and kidney was up-regulated in VDRKO mice regardless of plasma calcium the vitamin system is shown to the expression of antithrombotic the expression of a factor, AT is a plasma by and is of the of blood coagulation through mice were by gene and homozygous mice were shown to be because of in the and liver with K. T. K. Y. A. M. Takeda Ito M. Yamamoto K. T. K. T. H. J. Clin. Invest. 2000; PubMed Scopus Google Scholar). In mice a thrombus formation in the kidney after injection M. T. K. Y. Yamamoto K. T. K. M. T. H. Blood. 2002; PubMed Scopus Google Scholar). The present suggest that the vitamin system may AT activity through transcriptional control of AT gene M. A. J. PubMed Scopus Google Scholar) reported of human AT gene expression by retinoid X receptor and receptor study revealed of 1α,25-dihydroxyvitamin on AT The were to the of the vitamin system on AT gene transcription may be the of a AT gene in study M. A. J. PubMed Scopus Google Scholar). expression in monocytic and vascular endothelial cells the retinoic acid-responsive transcriptional activation of the gene (10Koyama T. Shibakura M. Ohsawa M. Kamiyama R. Hirosawa S. Blood. 1998; 92: 160-167Crossref PubMed Google Scholar), and because on the gene is to the vitamin D-responsive is a that the of the vitamin system on the of gene expression is mediated the of VDR to the of the gene (10Koyama T. Shibakura M. Ohsawa M. Kamiyama R. Hirosawa S. Blood. 1998; 92: 160-167Crossref PubMed Google Scholar, 12Ohsawa M. Koyama T. Yamamoto K. Hirosawa S. Kamei S. Kamiyama R. Circulation. 2000; 102: 2867-2872Crossref PubMed Scopus (118) Google Scholar). In is for transcriptional regulation of gene expression by the vitamin system through or The gene and a nuclear and these are for of gene transcription by in vascular endothelial cells and by in monocytic cells Thromb. Haemostasis. 1997; PubMed Scopus Google Scholar). from activated monocytic cells is by 1α,25-dihydroxyvitamin through the of nuclear activation D. M. D. P. R. P. J. Clin. Invest. 1998; 101: PubMed Scopus Google Scholar), the vitamin system may gene expression of nuclear The present study also that VDRKO mice manifest an exacerbated thrombus after exogenous injection regardless of the calcemic conditions. In with the present H. K. Y. M. E. M. K. S. Thromb. Haemostasis. 2001; PubMed Scopus Google Scholar) demonstrated a of the active form of vitamin using a coagulation rat platelet down-regulated expression of AT and with of expression in hypo- and normocalcemic VDRKO mice can to the enhanced of VDRKO mice. In of NO is with and thrombus formation in the of and with endothelial J. J. A. Clin. 2000; Google Scholar, B. J. 1998; Google Scholar). However, because reduced levels and urinary NOx excretion in hypocalcemic VDRKO mice were in normocalcemic VDRKO mice, the exacerbated thrombus formation by injection in normocalcemic VDRKO mice be by a in NO is to clarify the effects of vitamin D3, the present results are with the that the vitamin system plays an role in antithrombotic in vivo. for in the of the