Biochemical Evidence for a 170-Kilodalton, AF-2-Dependent Vitamin D Receptor/Retinoid X Receptor Coactivator That Is Highly Expressed in Osteoblasts

Jurutka, Peter W.; Remus, Lenore S.; Whitfield, G. Kerr; Galligan, Michael A.; Haussler, Carol A.; Haussler, Mark R.

doi:10.1006/bbrc.1999.2044

Cited by 6 publications

(3 citation statements)

References 29 publications

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“…pGEX fusion constructs were transformed into Escherichia coli (strain BL21 for VDR and Hr; strain DH5␣ for RXR␣). The detailed procedure for overexpression of GST fusion proteins has been described previously (46). GST alone was expressed from pGEX-4T in E. coli strain DH5␣ and linked to glutathione-Sepharose beads to serve as a control for background protein association.…”

Section: Methodsmentioning

confidence: 99%

Physical and Functional Interaction between the Vitamin D Receptor and Hairless Corepressor, Two Proteins Required for Hair Cycling

Hsieh

Sisk

Jurutka

et al. 2003

Journal of Biological Chemistry

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Both the vitamin D receptor (VDR) and hairless (hr) genes play a role in the mammalian hair cycle, as inactivating mutations in either result in total alopecia. VDR is a nuclear receptor that functions as a ligand-activated transcription factor, whereas the hairless gene product (Hr) acts as a corepressor of both the thyroid hormone receptor (TR) and the orphan nuclear receptor, ROR␣. In the present study, we show that VDR-mediated transactivation is strikingly inhibited by coexpression of rat Hr. The repressive effect of Hr is observed on both synthetic and naturally occurring VDR-responsive promoters and also when VDR-mediated transactivation is augmented by overexpression of its heterodimeric partner, retinoid X receptor. Utilizing in vitro pull down methods, we find that Hr binds directly to VDR but insignificantly to nuclear receptors that are not functionally repressed by Hr. Coimmunoprecipitation data demonstrate that Hr and VDR associate in a cellular milieu, suggesting in vivo interaction. The Hr contact site in human VDR is localized to the central portion of the ligand binding domain, a known corepressor docking region in other nuclear receptors separate from the activation function-2 domain. Coimmunoprecipitation and functional studies of Hr deletants reveal that VDR contacts a C-terminal region of Hr that includes motifs required for TR and ROR␣ binding. Finally, in situ hybridization analysis of hr and VDR mRNAs in mouse skin demonstrates colocalization in cells of the hair follicle, consistent with a hypothesized intracellular interaction between these proteins to repress VDR target gene expression, in vivo.Nuclear receptors comprise a family of ligand-activated transcription factors that coordinate physiological and developmental processes by regulating specific changes in gene expression (1, 2). The vitamin D receptor (VDR) 1 mediates signaling by 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) and is a member of the thyroid hormone/retinoic acid receptor subfamily of nuclear receptors that heterodimerize with the retinoid X receptor (RXR) on direct repeat hormone-responsive elements in the promoters of regulated genes (3, 4). Binding of liganded VDR⅐RXR to a vitamin D-responsive element (VDRE) in target genes such as osteocalcin, osteopontin, and vitamin D 24-hydroxylase (CYP24) is accompanied by the recruitment of coactivator proteins (5). VDR coactivators such as steroid receptor coactivator-1 (6), NCoA-62 (7), and vitamin D receptor interacting protein 205 (8) stimulate transcriptional activation by facilitating chromatin remodeling and/or attraction of RNA polymerase II. Although some unliganded nuclear receptors (thyroid hormone and retinoic acid receptors) can mediate repression through association with corepressors such as nuclear receptor corepressor (N-CoR) (9) and silencing mediator for retinoic acid and thyroid hormone receptors (SMRT) (10), evidence suggests that VDR does not associate strongly with these corepressors (9 -12).Targeted gene deletion studies in mice (13, 14) and inactivat...

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Section: Methodsmentioning

confidence: 99%

Physical and Functional Interaction between the Vitamin D Receptor and Hairless Corepressor, Two Proteins Required for Hair Cycling

Hsieh

Sisk

Jurutka

et al. 2003

Journal of Biological Chemistry

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207

201

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“…For GST pulldown assays, expression plasmids for VDR interacting proteins (VIPs) (1.0 mg each) were used to generate [ 35 S] methionine-labeled VIPs by in vitro transcription/translation (TNT Coupled Reticulocyte lysate kit, Promega Corp., Madison, WI). As described in detail elsewhere [Jurutka et al, 2000], GST-control and GST-hVDR Sepharose beads (25 ml each) were incubated in KETZD-0.15 M buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.3 mM ZnCl 2 , 1 mM dithiothreitol, 10% glycerol, 200 ng/ml pefabloc SC (Roche Molecular Biochemicals, Indianapolis, IN), 15 mg/ml aprotinin, 1 mg/ml leupeptin, and 1 mg/ml pepstatin A, plus 0.15 M KCl, 0.1% Tween-20, and 1 mg/ml BSA) at 48C for 1.5 h on a rocking platform in the absence or presence of lipophilic ligands and/or VDREs: 1,25(OH) 2 D 3 (10…”

Section: Gst Pulldown Assaysmentioning

confidence: 99%

Molecular and functional comparison of 1,25‐dihydroxyvitamin D₃ and the novel vitamin D receptor ligand, lithocholic acid, in activating transcription of cytochrome P450 3A4

Jurutka

Thompson

Whitfield

et al. 2004

J of Cellular Biochemistry

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The vitamin D receptor (VDR) binds to and mediates the effects of the 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) hormone to alter gene transcription. A newly recognized VDR ligand is the carcinogenic bile acid, lithocholic acid (LCA). We demonstrate that, in HT-29 colon cancer cells, both LCA and 1,25(OH)(2)D(3) induce expression of cytochrome P450 3A4 (CYP3A4), an enzyme involved in cellular detoxification. We also show that LCA-VDR stimulates transcription of gene reporter constructs containing DR3 and ER6 vitamin D responsive elements (VDREs) from the human CYP3A4 gene. Utilizing gel mobility shift, pulldown, and mammalian two-hybrid assays, we observe that: (i) 1,25(OH)(2)D(3) enhances retinoid X receptor (RXR) heterodimerization with VDR more effectively than LCA, (ii) the 1,25(OH)(2)D(3)-liganded VDR-RXR heterodimer recruits full-length SRC-1 coactivator, whereas this interaction is minimal with LCA unless LXXLL-containing fragments of SRC-1 are employed, and (iii) both 1,25(OH)(2)D(3) and LCA enhance the binding of VDR to DRIP205/mediator, but unlike 1,25(OH)(2)D(3)-VDR, LCA-VDR does not interact detectably with NCoA-62 or TRIP1/SUG1, suggesting a different pattern of LCA-VDR comodulator association. Finally, residues in the human VDR (hVDR) ligand binding domain (LBD) were altered to create mutants unresponsive to 1,25(OH)(2)D(3)- and/or LCA-stimulated transactivation, identifying S237 and S225/S278 as critical for 1,25(OH)(2)D(3) and LCA action, respectively. Therefore, these two VDR ligands contact distinct residues in the binding pocket, perhaps generating unique receptor conformations that determine the degree of RXR and comodulator binding. We propose that VDR is a bifunctional regulator, with the 1,25(OH)(2)D(3)-liganded conformation facilitating high affinity endocrine actions, and the LCA-liganded configuration mediating local, lower affinity cellular detoxification by upregulation of CYP3A4 in the colon.

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“…28 The FokI translational start codon polymorphism has structural consequences for the VDR which result in a less transcriptionally potent protein. 29,30 In addition, 2 intronic polymorphisms, BsmI and ApaI, and the TaqI polymorphism in exon 9 have been reported to alter VDR expression, probably through linkage disequilibrium with variants in the 3 0 UTR altering VDR-mRNA stability. 31 Epidemiological studies have reported associations between VDR polymorphism genotype and risk of both CRA [32][33][34][35][36] and CRC, [37][38][39] and in some studies, the associations were modified by dietary vitamin D and calcium intakes.…”

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confidence: 99%

Dairy products, polymorphisms in the vitamin D receptor gene and colorectal adenoma recurrence

Muir

Liu

Logan

et al. 2008

Intl Journal of Cancer

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Vitamin D receptor (VDR) activation inhibits proliferation and angiogenesis in the colorectal epithelium, and inhibits metastasis of colorectal tumors. Polymorphisms in the VDR gene alter receptor cellular levels and functioning, and may confer altered susceptibility to colorectal neoplasia. We aimed to investigate the influence of VDR polymorphisms and dietary factors impacting on vitamin D metabolism on colorectal adenoma (CRA) recurrence. Data on dietary intakes of calcium, vitamin D and dairy products were collected from 853 participants in the United Kingdom Colorectal Adenoma Prevention trial, a randomized trial of aspirin and folate for CRA recurrence prevention. The VDR Cdx2, FokI, BsmI, ApaI and TaqI polymorphisms were genotyped in 546 participants with available DNA, and gene-diet interaction analyses performed in 480. Dairy product intake was inversely related to CRA recurrence risk independent of calcium and vitamin D [relative risk (RR) 5 0.64; 95% confidence intervals (CIs): 0.47-0.88, for subjects in the highest compared to lowest intake tertile, p trend 5 0.005]. Milk accounted for 60% of dairy product intake, and on analysis of milk and nonmilk dairy products separately recurrence risk in individuals in the highest tertile of milk intake was half that of lowest tertile individuals (RR 5 0.52; 95% CI: 0.38-0.72, p trend 5 3.2 3 10 25 ), whereas nonmilk dairy products did not influence recurrence. VDR polymorphism genotypes and haplotypes did not directly alter recurrence risk, but the reduction in risk associated with high dairy product intake was confined to individuals with ApaI aA/AA genotype (p interaction 5 0.02). These findings indicate dairy products, and in particular milk, have chemopreventive activity against CRA recurrence. ' 2008 Wiley-Liss, Inc.Key words: colorectal adenoma recurrence; dairy products; VDR; polymorphism Colorectal cancer (CRC) is the third commonest cancer worldwide after lung and breast cancer, and two-thirds of CRC occurs in developed countries. 1 Up to 90% of CRCs develop from colorectal adenomas (CRAs) and colonoscopic removal of CRA reduces the incidence of CRC. 2,3 In some individuals, the recurrence rate for CRA is as high as 40-50% after 3 years, 4 CRA recurrence is a surrogate marker for CRC risk and prevention of CRA recurrence is a useful endpoint for evaluating chemopreventive agents. 5 Activation of vitamin D receptors (VDRs) present in many cell types, including colorectal epithelial cells, can inhibit proliferation, angiogenesis and invasiveness, and studies in animal models indicate that vitamin D supplementation can lower the incidence and metastatic potential of intestinal tumors. 6-9 Ecological studies correlating sunlight exposure with cancer incidence provided the first evidence that vitamin D levels are inversely related to CRC development in humans. 10 Subsequent epidemiological studies have reported lower risks of CRA and CRC in individuals with high intakes and circulating levels of vitamin D. 11-14 Vitamin D plays a crucial role in calcium me...

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Biochemical Evidence for a 170-Kilodalton, AF-2-Dependent Vitamin D Receptor/Retinoid X Receptor Coactivator That Is Highly Expressed in Osteoblasts

Cited by 6 publications

References 29 publications

Physical and Functional Interaction between the Vitamin D Receptor and Hairless Corepressor, Two Proteins Required for Hair Cycling

Physical and Functional Interaction between the Vitamin D Receptor and Hairless Corepressor, Two Proteins Required for Hair Cycling

Molecular and functional comparison of 1,25‐dihydroxyvitamin D₃ and the novel vitamin D receptor ligand, lithocholic acid, in activating transcription of cytochrome P450 3A4

Dairy products, polymorphisms in the vitamin D receptor gene and colorectal adenoma recurrence

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Biochemical Evidence for a 170-Kilodalton, AF-2-Dependent Vitamin D Receptor/Retinoid X Receptor Coactivator That Is Highly Expressed in Osteoblasts

Cited by 6 publications

References 29 publications

Physical and Functional Interaction between the Vitamin D Receptor and Hairless Corepressor, Two Proteins Required for Hair Cycling

Physical and Functional Interaction between the Vitamin D Receptor and Hairless Corepressor, Two Proteins Required for Hair Cycling

Molecular and functional comparison of 1,25‐dihydroxyvitamin D3 and the novel vitamin D receptor ligand, lithocholic acid, in activating transcription of cytochrome P450 3A4

Dairy products, polymorphisms in the vitamin D receptor gene and colorectal adenoma recurrence

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Molecular and functional comparison of 1,25‐dihydroxyvitamin D₃ and the novel vitamin D receptor ligand, lithocholic acid, in activating transcription of cytochrome P450 3A4