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Vitamin K2 Regulation of Bone Homeostasis Is Mediated by the Steroid and Xenobiotic Receptor SXR

2003; Elsevier BV; Volume: 278; Issue: 45 Linguagem: Inglês

10.1074/jbc.m303136200

ISSN

1083-351X

Autores

Michelle M. Tabb, Aixu Sun, Changcheng Zhou, Felix Grün, Jody Errandi, Kimberly M. Romero, Hang Pham, Satoshi Inoue, Shyamali Mallick, Min Lin, Barry M. Forman, Bruce Blumberg,

Tópico(s)

Hormonal Regulation and Hypertension

Resumo

Vitamin K2 is a critical nutrient required for blood clotting that also plays an important role in bone formation. Vitamin K2 supplementation up-regulates the expression of bone markers, increases bone density in vivo, and is used clinically in the management of osteoporosis. The mechanism of vitamin K2 action in bone formation was thought to involve its normal role as an essential cofactor for γ-carboxylation of bone matrix proteins. However, there is evidence that suggests vitamin K2 also has a transcriptional regulatory function. Vitamin K2 bound to and activated the orphan nuclear receptor SXR and induced expression of the SXR target gene, CYP3A4, identifying it as a bona fide SXR ligand. Vitamin K2 treatment of osteosarcoma cells increased mRNA levels for the osteoblast markers bone alkaline phosphatase, osteoprotegerin, osteopontin, and matrix Gla protein. The known SXR activators rifampicin and hyperforin induced this panel of bone markers to an extent similar to vitamin K2. Vitamin K2 was able to induce bone markers in primary osteocytes isolated from wild-type murine calvaria but not in cells isolated from mice deficient in the SXR ortholog PXR. We infer that vitamin K2 is a transcriptional regulator of bone-specific genes that acts through SXR to favor the expression of osteoblastic markers. Thus, SXR has a novel role as a mediator of bone homeostasis in addition to its role as a xenobiotic sensor. An important implication of this work is that a subset of SXR activators may function as effective therapeutic agents for the management of osteoporosis. Vitamin K2 is a critical nutrient required for blood clotting that also plays an important role in bone formation. Vitamin K2 supplementation up-regulates the expression of bone markers, increases bone density in vivo, and is used clinically in the management of osteoporosis. The mechanism of vitamin K2 action in bone formation was thought to involve its normal role as an essential cofactor for γ-carboxylation of bone matrix proteins. However, there is evidence that suggests vitamin K2 also has a transcriptional regulatory function. Vitamin K2 bound to and activated the orphan nuclear receptor SXR and induced expression of the SXR target gene, CYP3A4, identifying it as a bona fide SXR ligand. Vitamin K2 treatment of osteosarcoma cells increased mRNA levels for the osteoblast markers bone alkaline phosphatase, osteoprotegerin, osteopontin, and matrix Gla protein. The known SXR activators rifampicin and hyperforin induced this panel of bone markers to an extent similar to vitamin K2. Vitamin K2 was able to induce bone markers in primary osteocytes isolated from wild-type murine calvaria but not in cells isolated from mice deficient in the SXR ortholog PXR. We infer that vitamin K2 is a transcriptional regulator of bone-specific genes that acts through SXR to favor the expression of osteoblastic markers. Thus, SXR has a novel role as a mediator of bone homeostasis in addition to its role as a xenobiotic sensor. An important implication of this work is that a subset of SXR activators may function as effective therapeutic agents for the management of osteoporosis. Osteoporosis is a common disease affecting the elderly, particularly postmenopausal women, although a significant minority of older men is also affected. It is defined as the gradual reduction in bone strength with advancing age that is manifested by such observations as bone fracture following minimal trauma (1Orwoll E.S. Bauer D.C. Vogt T.M. Fox K.M. Ann. Intern. Med. 1996; 124: 187-196Crossref PubMed Scopus (199) Google Scholar, 2Seeman E. Tsalamandris C. Formica C. Hopper J.L. McKay J. J. Bone Miner. 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Vitam. Nutr. Res. 1997; 67: 350-356PubMed Google Scholar). Post-translational conversion of 9–12 Glu to Gla residues is required for the function of proteins such as prothrombin and Factors VII, IX, and X in the blood clotting cascade (for reviews, see Refs. 21Shearer M.J. Lancet. 1995; 345: 229-234Abstract PubMed Scopus (370) Google Scholar and 22Suttie J.W. Annu. Rev. Biochem. 1985; 54: 459-477Crossref PubMed Scopus (374) Google Scholar). In addition, Gla-containing proteins such as osteocalcin and matrix Gla protein are abundant in bone tissues where they are thought to play important roles in regulating mineralization (for reviews, see Refs. 23Binkley N.C. Suttie J.W. J. Nutr. 1995; 125: 1812-1821Crossref PubMed Scopus (163) Google Scholar and 24Vermeer C. Jie K.S. Knapen M.H. Annu. Rev. Nutr. 1995; 15: 1-22Crossref PubMed Google Scholar). Recent studies have demonstrated that the orphan nuclear receptor SXR 1The abbreviations used are: SXR, steroid and xenobiotic receptor; PXR, pregnane X receptor; CYP, cytochrome P-450; RT-PCR, reverse transcriptase PCR; QRT-PCR, quantitative real time RT-PCR; RIF, rifampicin; ALP, alkaline phosphatase; OPN, osteopontin; MGP, matrix Gla protein; OPG, osteoprotegerin; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; SRC-1, steroid receptor coactivator-1; ACTR, activator for thyroid hormone and retinoid receptors; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; F, forward; R, reverse; PCN, pregnenolone 16α-carbonitrile; PBP, peroxisome proliferator-activated receptor (PPAR)-binding protein; GRIP, glucocorticoid receptor-interacting protein; SMRT, silencing mediator of retinoid and thyroid receptors; NCoR, nuclear receptor corepressor; CHX, cycloheximide; WT, wild-type. (25Blumberg B. Kang H. Bolado Jr., J. Chen H. Craig A.G. Moreno T.A. Umesono K. Perlmann T. De Robertis E.M. Evans R.M. Genes Dev. 1998; 12: 1269-1277Crossref PubMed Scopus (67) Google Scholar) (also known as PXR (26Kliewer S.A. Moore J.T. Wade L. Staudinger J.L. Jones M.A. McKee D.D. Oliver B.M. Willson T.M. Zetterstrom R.H. Perlmann T. Lehmann J. Cell. 1998; 92: 73-82Abstract Full Text Full Text PDF PubMed Scopus (1339) Google Scholar), PAR (27Bertilsson G. Heidrich J. Svensson K. Asman M. Jendeberg L. Sydow-Backman M. Ohlsson R. Postlind H. Blomquist P. Berkenstam A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12208-12213Crossref PubMed Scopus (795) Google Scholar), and NR1I2) plays a central role in the transcriptional regulation of xenobiotic detoxifying enzymes and transporters such as CYP3A4 and MDR1 (28Dussault I. Lin M. Hollister K. Wang E.H. Synold T.W. Forman B.M. J. Biol. Chem. 2001; 276: 33309-33312Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 29Staudinger J.L. Goodwin B. Jones S.A. Hawkins-Brown D. MacKenzie K.I. LaTour A. Liu Y. Klaassen C.D. Brown K.K. Reinhard J. Willson T.M. Koller B.H. Kliewer S.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3369-3374Crossref PubMed Scopus (1139) Google Scholar, 30Synold T.W. Dussault I. Forman B.M. Nat. Med. 2001; 7: 584-590Crossref PubMed Scopus (760) Google Scholar, 31Willson T.M. Kliewer S.A. Nat. Rev. Drug Discov. 2002; 1: 259-266Crossref PubMed Scopus (413) Google Scholar, 32Xie W. Barwick J.L. Downes M. Blumberg B. Simon C.M. Nelson M.C. Neuschwander-Tetri B.A. Brunt E.M. Guzelian P.S. Evans R.M. Nature. 2000; 406: 435-439Crossref PubMed Scopus (608) Google Scholar). SXR is activated by a diverse array of pharmaceutical agents including Taxol, rifampicin, SR12813, clotrimazole, phenobarbital, hyperforin (33Jones S.A. Moore L.B. Shenk J.L. Wisely G.B. Hamilton G.A. McKee D.D. Tomkinson N.C. LeCluyse E.L. Lambert M.H. Willson T.M. Kliewer S.A. Moore J.T. Mol. Endocrinol. 2000; 14: 27-39Crossref PubMed Scopus (543) Google Scholar, 34Moore L.B. Parks D.J. Jones S.A. Bledsoe R.K. Consler T.G. Stimmel J.B. Goodwin B. Liddle C. Blanchard S.G. Willson T.M. Collins J.L. Kliewer S.A. J. Biol. Chem. 2000; 275: 15122-15127Abstract Full Text Full Text PDF PubMed Scopus (746) Google Scholar, 35Wentworth J.M. Agostini M. Love J. Schwabe J.W. Chatterjee V.K. J. Endocrinol. 2000; 166: R11-R16Crossref PubMed Scopus (228) Google Scholar), the herbal antidepressant St. John's wort (36Moore L.B. Goodwin B. Jones S.A. Wisely G.B. Serabjit-Singh C.J. Willson T.M. Collins J.L. Kliewer S.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7500-7502Crossref PubMed Scopus (868) Google Scholar), and peptide mimetic human immunodeficiency virus protease inhibitors such as ritonavir (28Dussault I. Lin M. Hollister K. Wang E.H. Synold T.W. Forman B.M. J. Biol. Chem. 2001; 276: 33309-33312Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). These studies indicate that SXR functions as a xenobiotic sensor to coordinately regulate drug clearance in the liver and intestine. Indeed gene knockout studies have confirmed a role for SXR in regulating the metabolism of endogenous steroids and dietary and xenobiotic compounds (29Staudinger J.L. Goodwin B. Jones S.A. Hawkins-Brown D. MacKenzie K.I. LaTour A. Liu Y. Klaassen C.D. Brown K.K. Reinhard J. Willson T.M. Koller B.H. Kliewer S.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3369-3374Crossref PubMed Scopus (1139) Google Scholar, 32Xie W. Barwick J.L. Downes M. Blumberg B. Simon C.M. Nelson M.C. Neuschwander-Tetri B.A. Brunt E.M. Guzelian P.S. Evans R.M. Nature. 2000; 406: 435-439Crossref PubMed Scopus (608) Google Scholar). During our original screening experiments that led to the discovery of SXR as a steroid and xenobiotic sensor, we noted that vitamin K2 could also activate SXR. 2B. Blumberg, unpublished observations. This observation led us to consider the possibility that vitamin K2 might act as a bona fide SXR ligand to mediate biological processes other than xenobiotic metabolism and clearance. Since vitamin K2 was previously suspected to have a transcriptional regulatory function in addition to its role as an enzyme cofactor (37Koshihara Y. Hoshi K. J. Bone Miner. Res. 1997; 12: 431-438Crossref PubMed Scopus (121) Google Scholar), we hypothesized that SXR might be the mediator of this activity. In this report, we demonstrate that vitamin K2 transcriptionally activates SXR in a dose-dependent manner and binds directly to SXR in vitro and in vivo. SXR mRNA is expressed in osteosarcoma cell lines, and vitamin K2 induced the expression of the prototypical SXR target gene CYP3A4 in these cells. Vitamin K2 up-regulates the steady state mRNA levels for a panel of osteoblastic bone markers in the osteosarcoma cell lines HOS, MG-63, and Saos-2, demonstrating a mechanistic connection between vitamin K2 and bone development. The known SXR activators rifampicin and hyperforin induce the same panel of bone markers as does vitamin K2, further confirming a role for SXR in the regulation of these genes. Finally we found that vitamin K2 was able to induce bone markers in primary osteocytes isolated from wild-type murine calvaria but not in cells isolated from PXR knockout mice. From these data, we conclude that vitamin K2 modulates the expression of osteoblastic bone markers through SXR and infer that vitamin K2 activation of SXR could be an important factor favoring the deposition of bone over its resorption. Therefore, SXR is likely to be involved in the maintenance of bone homeostasis in addition to its known role in hormonal homeostasis. This reveals a novel biological function for SXR and suggests that a subset of SXR activators may function as effective therapeutic agents for the management of osteoporosis. SXR Detection by RT-PCR—HOS, MG-63, Saos-2, LS180, and HeLa cells were cultured in phenol red-free DMEM supplemented with 10% resin charcoal-stripped FBS. Total RNA was isolated using TriZol reagent (Invitrogen). For RT-PCR analysis, 1 μg of total RNA was reverse transcribed using Superscript II reverse transcriptase according to the manufacturer-supplied protocol (Invitrogen). SXR was detected with the following primer set: forward primer, 5′-CAAGCGGAAGAAAAGTGAACG-3′; reverse primer, 5′-CTGGTCCTCGATGGGCAAGT-3′. PCR was carried out at 37 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 45 s. Cell Culture and Transfection—COS-7 cells were cultured and transfected as described previously (38Grun F. Venkatesan R.N. Tabb M.M. Zhou C. Cao J. Hemmati D. Blumberg B. J. Biol. Chem. 2002; 277: 43691-43697Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Typically COS-7 cells were cultured in phenol red-free DMEM supplemented with 10% FBS. For transient transfection experiments, COS-7 cells were seeded into 96-well plates at a density of 5000 cells/well. The next day cells were transfected with either CMX-GAL-SXR or CMX-GAL4 (control) together with tk(MH100)4-luc reporter (39Forman B.M. Umesono K. Chen J. Evans R.M. Cell. 1995; 81: 541-550Abstract Full Text PDF PubMed Scopus (567) Google Scholar) and CMX-β-galactosidase transfection control plasmids using standard calcium phosphate precipitation methodology. 22–24 h after transfection, the cells were washed twice with phosphate-buffered saline supplemented with 1 mm MgCl2 or DMEM-ITLB (DMEM containing 5 μg/ml insulin, 5 μg/ml holotransferrin, 5 μg/ml selenium, 0.5% defined lipid mix (Invitrogen), 0.12% (w/v) delipidated bovine serum albumin (Sigma)) (40Buck J. Grun F. Derguini F. Chen Y. Kimura S. Noy N. Hammerling U. J. Exp. Med. 1993; 178: 675-680Crossref PubMed Scopus (89) Google Scholar). Ligands were typically purchased from Sigma and BIOMOL Research Laboratories Inc., made freshly from powder in Me2SO as 0.1 m stocks, diluted in Me2SO to appropriate concentrations, and added to media with vigorous vortex mixing. Ligands were added in DMEM-ITLB, and the cells were incubated for an additional 24–48 h. The cells were lysed in situ, and extracts were prepared and assayed for β-galactosidase and luciferase activity as described previously (41Blumberg B. Sabbagh W. Juguilon H. Bolado Jr., J. Ong E.S. Evans R.M. Genes Dev. 1998; 12: 3195-3205Crossref PubMed Scopus (819) Google Scholar). Reporter gene activity was normalized to the β-galactosidase transfection controls, and the results were expressed as normalized relative luciferase units per OD β-galactosidase per minute to facilitate comparisons between plates. Fold induction was calculated relative to solvent controls. Each data point represents the average of triplicates ± S.E. The experiments were repeated three times with similar results. For coactivator recruitment experiments, GAL4-coactivator plasmids were generated by cloning the receptor interaction domains of human TIF2 (GenBank™ accession number NM_006540, amino acids 563–790), human SRC-1 (GenBank™ accession number U59302, amino acids 600–800), or human ACTR (GenBank™ accession number AF036892, amino acids 600–788) into pCMX-GAL4. The GAL4-PBP construct was described previously (30Synold T.W. Dussault I. Forman B.M. Nat. Med. 2001; 7: 584-590Crossref PubMed Scopus (760) Google Scholar). To construct herpesvirus VP16 activation domain fusion proteins, full-length SXR was PCR-amplified and ligated in-frame into pCDG-VP16 vector (25Blumberg B. Kang H. Bolado Jr., J. Chen H. Craig A.G. Moreno T.A. Umesono K. Perlmann T. De Robertis E.M. Evans R.M. Genes Dev. 1998; 12: 1269-1277Crossref PubMed Scopus (67) Google Scholar). All constructs were sequenced to verify that no errors were introduced during the PCR. Ligand Binding Assays—N-terminal His6-tagged human SXR ligand binding domain was expressed in Escherichia coli together with the SRC-1 receptor interaction domain essentially as described previously (28Dussault I. Lin M. Hollister K. Wang E.H. Synold T.W. Forman B.M. J. Biol. Chem. 2001; 276: 33309-33312Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Active protein was refolded from inclusion bodies solubilized in denaturation buffer (6 m guanidinium-HCl, 50 mm HEPES, pH 7.4, 0.2 m NaCl, 25 mm dithiothreitol, 1% (w/v) Triton X-100) by rapid 10-fold dilution into binding buffer (50 mm HEPES, pH 7.4, 1 m sucrose, 0.2 m NaCl, 0.1 mm dithiothreitol, 0.1% (w/v) CHAPS) followed by dialysis overnight at 4 °C against binding buffer. Binding assays were performed by coating 96-well nickel chelate FlashPlates (PerkinElmer Life Sciences) with a 10-fold molar excess of protein for 1 h at 22 °C in binding buffer (50 mm HEPES, pH 7.4, 200 mm NaCl, 1 m sucrose, 0.1% CHAPS). Unbound protein was removed from the wells by washing four times with binding buffer. [3H]SR12813 (33Jones S.A. Moore L.B. Shenk J.L. Wisely G.B. Hamilton G.A. McKee D.D. Tomkinson N.C. LeCluyse E.L. Lambert M.H. Willson T.M. Kliewer S.A. Moore J.T. Mol. Endocrinol. 2000; 14: 27-39Crossref PubMed Scopus (543) Google Scholar) (Amersham Biosciences) was added to a final concentration of 50 nm in each well either alone or together with competitor ligands in binding buffer as indicated. Incubation was continued for 3 h at room temperature. Total counts were measured using a Topcount scintillation counter (Packard Instrument Co.). Counts remaining after the addition of 10 μm clotrimazole were taken as nonspecific background and subtracted from all wells (33Jones S.A. Moore L.B. Shenk J.L. Wisely G.B. Hamilton G.A. McKee D.D. Tomkinson N.C. LeCluyse E.L. Lambert M.H. Willson T.M. Kliewer S.A. Moore J.T. Mol. Endocrinol. 2000; 14: 27-39Crossref PubMed Scopus (543) Google Scholar). All assays were performed in triplicate and reproduced in independent experiments. Alkaline Phosphatase (ALP) Activity Assay—ALP activity was measured as described previously (42AnonymousScand. J. Clin. Lab. Investig. 1974; 33: 291-306Crossref PubMed Google Scholar). Briefly, cells were harvested by washing twice with phosphate-buffered saline, then collected with a cell scraper, and transferred to 1.5-ml microcentrifuge tubes. Cell pellets were obtained by centrifugation at 14,000 rpm at 4 °C, and lysates were prepared with a solution containing 0.2% (v/v) Nonidet P-40 and 1 mm MgCl2. Aliquots of lysate were combined with reaction buffer (1 m diethanolamine, pH 9.8, 1 mm MgCl2, and 10 mm p-nitrophenyl phosphate) and incubated at 37 °C for 30 min. Absorbance at 405 nm was measured using a Spectra MAX Plus spectrophotometer (Amersham Biosciences), and the enzyme activity was calculated as described previously (6Akedo Y. Hosoi T. Inoue S. Ikegami A. Mizuno Y. Kaneki M. Nakamura T. Ouchi Y. Orimo H. Biochem. Biophys. Res. Commun. 1992; 187: 814-820Crossref PubMed Scopus (54) Google Scholar). ALP activity was corrected for protein content, which was determined using the Bio-Rad protein assay kit. Quantitative Real Time RT-PCR Analysis of Bone Biomarker Genes in Osteosarcoma Cell Lines—Human osteosarcoma cell lines HOS, MG-63, and Saos-2 were obtained from American Type Culture Collection (Manassas, VA) and cultured in phenol red-free DMEM supplemented with 10% resin charcoal-stripped FBS. Cells were treated with vitamin K2, 1α,25-(OH)2 vitamin D3, rifampicin, or solvent controls for 48 h. Total RNA was isolated and reverse transcribed as described above. Quantitative real time RT-PCR (QRT-PCR) was performed using the following primer sets: ALP (F, 5′-CATGGCTTTGGGCAGAAGGA-3′; R, 5′-CTAGCCCCAAAAAGAGTTGCAA-3′), osteopontin (OPN) (F, 5′-CAGACGAGGACATCACCTCA-3′; R, 5′-TGGCTGTGGGTTTCAGCA-3′), matrix Gla protein (MGP) (F, 5′-ATCGCTACTTCAGGAAGCGCC-3′; R, 5′-TGACTCTCCTTTGACCCTGACCCTCAC-3′), osteoprotegerin (OPG) (F, 5′-CCTCTCATCAGCTGTTGTGTG-3′; R, 5′-TATCTCAAGGTAGCGCCCTTC-3′), glyceraldehyde-3-phosphate dehydrogenase (F, 5′-TGGACCTCATGGCCCACA-3′; R, 5′-TCAAGGGGTCTACATGGCAA-3′), CYP3A4 (F, 5′-GGCTTCATCCAATGGACTGCATAAAT-3′; R, 5′-TCCCAAGTATAACACTCTACACAGACAA-3′), and the SYBR green PCR kit (Applied Biosystems) in a DNA Engine Opticon-Continuous Fluorescence Detection system (MJ Research). All samples were quantitated by the comparative cycle threshold (Ct) method for relative quantitation of gene expression, normalized to glyceraldehyde-3-phosphate dehydrogenase (43Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (124899) Google Scholar). Isolation of Calvaria, Culture of Primary Bone Cells, and QRT-PCR—Calvaria were isolated from newborn wild-type and PXR knockout mice (postnatal day 1–5) and were digested sequentially with 0.1% collagenase, 0.05% trypsin, 4 mm EDTA in 1× phosphate-buffered saline essentially as described in Refs. 44Wong G.L. Cohn D.V. Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 3167-3171Crossref PubMed Scopus (308) Google Scholar and 45Partridge N.C. Alcorn D. Michelangeli V.P. Kemp B.E. Ryan G.B. Martin T.J. Endocrinology. 1981; 108: 213-219Crossref PubMed Scopus (253) Google Scholar. Bone cells released upon digestion were cultured in phenol-red free DMEM, 10% FBS and treated for 48 h with 10 μm vitamin K2 or pregnenolone 16α-carbonitrile (PCN). Total RNA was isolated, reverse transcribed, and analyzed using the following mouse primer sets: MGP (F, 5′-TCTCACGAAAGCATGGAGTC-3′; R, 5′-ATCTCGTAGGCAGGCTTGTT-3′), OPG (F, 5′-CTGCTGAAGCTGTGGAAACA-3′; R, 5′-AAGCTGCTCTGTGGTGAGGT-3′), and glyceraldehyde-3-phosphate dehydrogenase (F, 5′-AACTTTGGCATTGTGGAAGG-3′; R, 5′-GGATGCAGGGATGATGTTCT-3′). Statistical Analysis—Differences between two groups were analyzed using two-sample, two-tailed Student's t test. A p value less than 0.05 was considered to be significant. All data are presented in the text and figures as the mean ± S.E. SXR Is Expressed in Osteosarcoma Cell Lines—SXR functions as a xenobiotic sensor and is expressed at high levels in the liver and intestine where it modulates the levels of CYP enzymes and ATP-binding cassette family transporters (31Willson T.M. Kliewer S.A. Nat. Rev. Drug Discov. 2002; 1: 259-266Crossref PubMed Scopus (413) Google Scholar, 46Xie W. Evans R.M. J. Biol. Chem. 2001; 276: 37739-37742Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). SXR is expressed at lower levels in normal and neoplastic breast tissues (47Dotzlaw H. Leygue E. Watson P. Murphy L.C. Clin. Cancer Res. 1999; 5: 2103-2107PubMed Google Scholar) and breast cancer cell lines (MCF-7, T47D, MDA-MB-231, and MDA-MB-435) (47Dotzlaw H. Leygue E. Watson P. Murphy L.C. Clin. Cancer Res. 1999; 5: 2103-2107PubMed Google Scholar). 3M. M. Tabb and B. Blumberg, unpublished data. It is not clear at present what role SXR is playing in other tissues. We were intrigued by the ability of vitamin K2 to activate SXR in preliminary experiments. To ascertain whether SXR might be mediating the effects of vitamin K2, we first determined whether SXR was expressed in a panel of osteosarcoma cell lines using RT-PCR. SXR expression was observed in the LS180 human colon adenocarcinoma cells and in the osteosarcoma cell lines HOS, MG-63, and Saos-2. SXR mRNA was not detected in HeLa cells or in negative controls (Fig. 1). It has been previously reported that SXR is expressed in LS180 cells (28Dussault I. Lin M. Hollister K. Wang E.H. Synold T.W. Forman B.M. J. Biol. Chem. 2001; 276: 33309-33312Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 30Synold T.W. Dussault I. Forman B.M. Nat. Med. 2001; 7: 584-590Crossref PubMed Scopus (760) Google Scholar), whereas it is not expressed in HeLa cells (48Masuyama H. Inoshita H. Hiramatsu Y. Kudo T. Endocrinology. 2002; 143: 55-61Crossref PubMed Scopus (51) Google Scholar). SXR is expressed at highest levels in LS180 cells with lower levels in the osteosarcoma cell lines (Fig. 1). Vitamin K2 Activates SXR—SXR is activated by a diverse array of pharmaceutical agents including Taxol, rifampicin, SR12813, clotrimazole, phenobarbital, and hyperforin. As noted above, our early screening efforts aimed at identifying SXR ligands also demonstrated SXR activation by vitamin K2. Accordingly we tested the ability of vitamin K2 to activate SXR in dose-response experiments. As shown in Fig. 2A, vitamin K2 activates CMX-GAL-SXR robustly with the highest levels of activation approximately equivalent to 1 μm rifampicin (RIF). In contrast, no activation was observed using CMX-GAL4 alone, demonstrating that the activation results from a specific interaction with the SXR ligand binding domain. Next we tested the ability of vitamin K2 to induce the SXR target gene CYP3A4 in cultured osteosarcoma cells. It has been reported previously that CYP3A4 expression is induced by RIF and vitamin D3 in cultured HepG2 and LS180 cells (49Schmiedlin-Ren P. Thummel K.E. Fisher J.M. Paine M.F. Watkins P.B. Drug Metab. Dispos. 2001; 29: 1446-1453PubMed Google Scholar, 50Sumida A. Fukuen S. Yamamoto I. Matsuda H. Naohara M. Azuma J. Biochem. Biophys. Res. Commun. 2000; 267: 756-760Crossref PubMed Scopus (56) Google Scholar), although the 1α,25-(OH)2 vitamin D3 induction of CYP3A4 is mediated by the vitamin D3 receptor rather than by SXR (51Makishima M. Lu T.T. Xie W. Whitfield G.K. Domoto H. Evans R.M. Haussler M.R. Mangelsdorf D.J. Science. 2002; 296: 1313-1316Crossref PubMed Scopus (976) Google Scholar). RIF induced the expression of CYP3A4 in all three lines (Fig. 2B). Vitamin K2 was able to induce CYP3A4 expression at both 1 and 10 μm in all three lines. 1α,25-(OH)2 vitamin D3 could not induce CYP3A4 expression in Saos-2 or HOS cells (Fig. 2B). Vitamin K2 Specifically Binds to SXR in Vitro and in Vivo— Since vitamin K2 activates SXR in transient transfections (Fig. 2A) and induces the expression of a prototypical SXR target gene in osteosarcoma cells (Fig. 2B), we next sought to determine whether vitamin K2 binds to SXR. One important measure of ligand binding is the ability of a compound to induce a nuclear receptor to interact with coactivator proteins. Accordingly we conducted coactivator recruitment experiments that utilized VP16-SXR together with fusions between the GAL4 DNA binding domain and the receptor-interacting domains of the nuclear hormone receptor coactivators SRC-1, TIF2, ACTR, and PBP (30Synold T.W. Dussault I. Forman B.M. Nat. Med. 2001; 7: 584-590Crossref PubMed Scopus (760) Google Scholar). As shown in Fig. 3A, VP16-SXR was able to interact with PBP, SRC-1, and ACTR in the presence of vitamin K2 or the known SXR ligand RIF. The results from the coactivator recruitment experiments paralleled those of the activation assays. As is the case for other SXR ligands (30Synold

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