Ligands, 1α,25-Dihydroxyvitamin D3 and Vitamin D Anal

53 Pages·2009·4.97 MB·English

JPET Fast Forward. Published on May 4, 2009 as DOI: 10.1124/jpet.108.149815
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JPET #149815
Upregulation of Transporters and Enzymes by the Vitamin D Receptor (VDR)
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Ligands, 1 ,25-Dihydroxyvitamin D and Vitamin D Analogues, in the Caco-2 Cell Monolayer
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Jianghong Fan, Shanjun Liu, Yimin Du, Jodi Morrison, Robert Shipman, n
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and K. Sandy Pang d fro
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Department of Pharmaceutical Sciences (JF, SL, YD, KSP), Leslie Dan Faculty of Pharmacy, E
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University of Toronto, Toronto, ON, Canada M5S 3M2, and NoAb BioDiscoveries Inc. (JM, RS), rn
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Mississauga, ON, Canada L5N 8G4 F
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Copyright 2009 by the American Society for Pharmacology and Experimental Therapeutics.
JPET Fast Forward. Published on May 4, 2009 as DOI: 10.1124/jpet.108.149815
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Running Title: VDR Regulation of transporters and enzymes
Correspondence: Dr. K. Sandy Pang
Faculty of Pharmacy, University of Toronto
144 College Street, Toronto, Ontario
Canada M5S 3M2
TEL: [phone]
FAX: [phone]
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E-mail: [email protected] n
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Abstract: 250 words d fro
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Introduction: 743 words jp
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Discussion: 1490 words tjo
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Tables: 3 ls
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Figures: 11 t A
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References: 40 J
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ABBREVIATIONS: VDR, vitamin D receptor; PXR, pregnane X receptor; CAR, constitutive
androstane receptor; FXR, farnesoid X receptor; GR, glucocorticoid receptor; RXR, retinoid X
receptor; VDRE, vitamin D response element; DBP, vitamin D binding protein; 1,25(OH) D ,
2 3
α α α
1 ,25-dihydroxyvitamin D ; 25(OH)D , 25-hydroxyvitamin D ; l (OH)D , 1 -hydroxyvitamin D ;
3 3 3 3 3
α α
l (OH)D , 1 -hydroxyvitamin D or Hectorol; CDF-DA, 5-(and-6)-carboxy-2',7'-
2 2
dichlorofluorescein diacetate; RTqPCR, real-time quantitative polymerase chain reactions;
CYP3A4, cytochrome P-450 3A4; SULT, sulfotransferase; ASBT, apical sodium dependent bile
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acid transporter; PEPT1, oligopeptide transporter 1; P-gp, P-glycoprotein; MDR1, multidrug n
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resistance 1 gene; MRPs, multidrug resistance-associated proteins; DMEM, Dulbecco's modified d fro
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Eagle's medium; FBS, fetal bovine serum; ITS, insulin-transferrin-selenium; TBS, tris-buffered- jp
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saline; TEER, transepithelial electrical resistance; A, apical; B, basolateral; Papp, apparent tjo
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permeability; EfR, efflux ratio. ls
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JPET Fast Forward. Published on May 4, 2009 as DOI: 10.1124/jpet.108.149815
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ABSTRACT
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The effects of 1 ,25-dihydroxyvitamin D (1,25(OH) D ) on gene expression and function
3 2 3
were studied in Caco-2 cells. Microarray analyses, RT-qPCR, and Western blotting were used to
determine the mRNA and protein expression of transporters and enzymes after 1,25(OH) D or
2 3
vehicle (0.1% ethanol) treatment for 1, 3, 6 and 10 days. The mRNA and protein expressions of
ASBT, PEPT1, MRP3 and SULT1E1 remained unchanged with 1,25(OH) D treatment, whereas
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those for CYP3A4, MDR1, and MRP2 were significantly increased (P<.05). 1,25(OH) D
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treatment significantly enhanced MRP4 protein expression by increasing protein stability without n
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affecting mRNA expression, as confirmed in cycloheximide experiments. Marked increase in 6 - d fro
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hydroxylation of testosterone by CYP3A4 was also observed in the 6-day, 1,25(OH) D -treated jp
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(100 nM) cell lysate. The transport of [3H]digoxin, the P-gp substrate, after treatment with 100 nM etjo
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1,25(OH) D for 3 days revealed a higher apparent permeability (P ) in the B to A direction over ls
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that of vehicle-treatment (15.1±0.53 x 10-6 vs.11.8±0.58 x10-6 cm/sec, P <.05), while the Papp in t A
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the A to B direction was unchanged; the efflux ratio was increased (5.8 to 8.0). Reduced cellular J
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retention of 5-(and-6)-carboxy-2',7'-dichlorofluorescein, suggestive of higher MRP2 activity, was ls
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observed in the 3-day, 100 nM 1,25(OH) D -treated cells over controls. Higher protein expression eb
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of CYP3A4, MRP2, P-gp, and MRP4 was also observed after a 6-day treatment with other vitamin 5
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α α 2
D analogues (100 nM of 1 -hydroxyvitamin D , 1 -hydroxyvitamin D or Hectorol®, and 25- 3
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hydroxyvitamin D ) in Caco-2 cells, suggesting a role of 1,25(OH) D and analogues in the
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activation of enzymes and transporters via the vitamin D receptor.
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INTRODUCTION
The intestine plays an important role in the absorption of orally administered drugs. The
expression and proximity of metabolic enzymes and efflux transporters in the enterocyte
contribute to intestinal first-pass removal and delimit the tissue accumulation of endo- and xeno-
biotics. In the small intestine, cytochrome P450 3A4 (CYP3A4) accounts for approximately 70%
of total cytochrome P450 content and is responsible for the metabolism of approximately 50% of
drugs currently in use (Pelkönen et al., 2008). The ATP-dependent drug efflux protein, P- D
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glycoprotein (P-gp), encoded by the multidrug resistance 1 gene (MDR1) located in the apical a
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membrane of the enterocyte, is involved in the active excretion of a wide variety of lipophilic from
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cationic drugs from intestine (Artursson et al., 2001). The multidrug resistance-associated proteins t.a
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(MRPs), another important subfamily of ATP-binding cassette transporters, are involved in the urn
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transport of unconjugated amphiphilic anions and glutathione, glucuronide and sulfate conjugates. rg
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MRP2 is an efflux transporter localized in the apical membrane, whereas MRP1, MRP3 and P
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MRP5 are basolateral efflux transporters. The localization of MRP4 in enterocytes has been u
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inferred to exist at the basolateral membrane for efflux (Prime-Chapman et al., 2004). n
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The expression and function of drug metabolic enzymes and transporters are under ry
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regulation of nuclear receptors. In addition to the pregnane X receptor (PXR), the constitutive 2
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androstane receptor (CAR), farnesoid X receptor (FXR) and glucocorticoid receptor (GR) (for
review, see Urquhart et al., 2007), the vitamin D receptor (VDR) is known to induce enzyme and
transporter genes in the intestine. These include the human CYP3A4 (Thummel et al., 2001),
SULT2A1 (Song et al., 2006), murine Mrp3 (McCarthy et al., 2005), and the rat apical sodium-
dependent bile acid transporter (Asbt) (Chen et al., 2006).
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1α,25-Dihydroxyvitamin D (1,25(OH) D ), the biologically active form of vitamin D, is the
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natural ligand of the VDR. Vitamin D is synthesized in the skin from its precursor, 7-
dehydrocholesterol, in response to ultraviolet light, and is converted to 25-hydroxyvitamin D
3
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(25(OH)D ) in the liver, then 1,25(OH) D by the 1 -hydroxylase in the kidney (Darwish and
3 2 3
DeLuca, 1996). Upon activation, the ligand-receptor complex recruits coactivators and
heterodimerizes with the retinoid X receptor (RXR), then binds to the vitamin D response element
(VDRE) and regulates target gene expression. During the last three decades, evidence has
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accumulated that 1,25(OH) D mediates its biological activities through specific binding to the n
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nuclear VDR. The therapeutic potential of 1,25(OH)2D3 is limited by its tendency to induce fro
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hypercalcemia (Prudencio et al., 2001). Vitamin D analogues that have lower hypercalcemic jpe
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effects are potential, therapeutic agents that can be used for treatment of human diseases and tjo
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disorders (Stein and Wark, 2003). 1 -hydroxyvitamin D3 (1 (OH)D3) is a prodrug of the active ls.o
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form of vitamin D , and undergoes metabolic conversion by 25-hydroxylase in the liver or t A
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intestine to 1,25(OH)2D3 before exerting its effect (Brickman et al., 1976). 1 -hydroxyvitamin D2 Jo
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(1 (OH)D ) or Hectorol is activated by 25-hydroxylase to produce 1,25(OH) D , a biologically ls
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active form of vitamin D , and is reported to display significantly reduced hypercalcemia at b
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therapeutic dosages for the treatment of secondary hyperparathyroidism (Brown, 2001). 5, 2
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The Caco-2 cell monolayer is a well-established human carcinoma cell line that
differentiates spontaneously in culture upon reaching confluence to become enterocyte-like cells
that contain transporters and enzymes (Artursson et al., 2001). Effects of 1,25(OH) D and the
2 3
vitamin D analogues on the expression and activity of enzymes and transporters in the human
intestine may thus be conveniently ascertained in Caco-2 cells due to high expression level of the
VDR. Most transporters, except BCRP, are present at reasonably high levels (Taipalensuu et al.,
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JPET Fast Forward. Published on May 4, 2009 as DOI: 10.1124/jpet.108.149815
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2001). It had been demonstrated that CYP3A4 expression was upregulated by 1,25(OH) D via
2 3
binding to the VDRE (Schmiedlin-Ren et al., 1997). More recently, a functional VDRE has been
identified in the human MDR1 gene (Saeki et al., 2008). However, the role of the VDR on other
target genes is unknown. In this study, we investigated the effects of 1,25(OH) D and the vitamin
2 3
D analogues on the expression and function of major metabolic enzymes and transporters in Caco-
2 cells. To confirm that CYP3A4 function was catalytically induced by 1,25(OH) D in Caco-2
2 3
cells, the activity of testosterone 6β-hydroxylase, a marker of induction of CYP3A4 (Chan et al.,
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2004), was measured. Digoxin, a prototypic substrate of P-gp, was used to evaluate P-gp function n
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in Caco-2 cells (Cavet et al., 1996), while the fluorescent MRP2 substrate, 5-(and-6)-carboxy-2',7'- fro
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dichlorofluorescein (CDF), whose diacetate prodrug, CDF-DA, readily diffuses across the cell jpe
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membrane for hydrolysis by intracellular esterases to form CDF, was used to evaluate MRP2 tjo
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function (Tian et al., 2004). ls.o
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JPET Fast Forward. Published on May 4, 2009 as DOI: 10.1124/jpet.108.149815
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METHODS
Materials
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1 ,25(OH) D , l (OH)D , 25(OH)D , cycloheximide, and glucose were purchased from
2 3 3 3
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Sigma-Aldrich Canada (Mississauga, ON, Canada). Hectorol® (l (OH)D ) was kindly provided
2
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by Dr. Peter Bonate from Genzyme. Testosterone and 6 -hydroxytestosterone were obtained from
Sigma (St. Louis, MO). 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate (CDF-DA),
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, 0.05% trypsin– D
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ethylenediaminetetraacetic acid, penicillin–streptomycin and non-essential amino acids were all a
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obtained from Invitrogen (Carlsbad, CA). The 12-well Transwell plate (Cat. No. 3401) was from
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purchased from Corning Incorporated Life Sciences (Lowell, MA). [3H]Digoxin (specific activity, t.a
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μ tjo
40 mCi/ mole), whose purity exceeded 98% as verified by HPLC, and [14C]mannitol (specific urn
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activity, 51 mCi/mmol) were procured from Perkin Elmer Life and Analytical Sciences (Waltham, rg
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MA). The materials and reagents for RT-qPCR were purchased from Applied Biosystems (Foster P
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City, CA, USA). Reagents for mRNA sample preparation were from Sigma, Ambion (Austin, TX). u
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Anti-CYP3A4, MRP2 and P-gp antibodies were purchased from Abcam (Cambridge, MA). Anti- on
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villin antibody was obtained from Santa Cruz (Santa Cruz, CA). Anti-VDR and anti-Cyp3A4 rua
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antibody were from Thermo Scientific (Waltham, MA) and BD Biosciences (Mississauga, ON), 0
2
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respectively. Donkey anti-rabbit IgG, sheep anti-mouse IgG and the enhanced chemiluminescence
(ECL) were purchased from Amersham-Pharmacia Biotech Inc. (Baie d'Urfe, Quebec). Other
antibodies were kindly provided by different investigators: anti-PEPT1 (Dr. Wolfgang Sadee, Ohio
State University, Columbus, Ohio); anti-ASBT (Dr. Paul A. Dawson, Wake Forest University
School of Medicine, NC); anti-MRP3 (Dr. Yuichi Sugiyama, University of Tokyo, Japan); anti-
MRP4 (Dr. John D. Schuetz, St. Jude Children’s Research Hospital, TN).
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Cell Culture
Caco-2 cells were obtained from the American Type Culture Collection (Manassas, VA).
Cells were allowed to undergo two passages prior to use. Then cells were cultured in DMEM
supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 100 U/ml penicillin and
100 μg/ml streptomycin under an atmosphere of 5% CO and 95% relative humidity at 37 °C.
2
Cells used in the studies were between passage numbers 30 to 50. Caco-2 cells were seeded in a
density of 2.5× 104 cells per cm2 in 60 mm dishes; the medium was changed every other day
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except for treatment days. For the treatment with 1,25(OH) D and vitamin D analogues (10 or n
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100 nM), cells were treated with control medium containing 0.1% ethanol or 1,25(OH)2D3 and fro
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vitamin D analogues daily on day 15 for 6 consecutive days, on day 18 for 3 consecutive days, or jpe
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day 20 for one day; cells were then harvested on day 21. For studies investigating the effect of tjo
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1,25(OH)2D3 on MRP4 protein stability, Caco-2 cells were first treated with 100 nM 1,25(OH)2D3 ls.o
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or ethanol (0.1%) for 6 days and then with cycloheximide (10 μg/ml) to arrest protein synthesis, t A
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followed by protein isolation for MRP4 determination at 0, 8, 24 and 48 h after the cycloheximide J
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treatment. For studies determining the effect of 25(OH)D on gene expression in the presence or ls
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absence of FBS, Caco-2 cells were treated with 100 nM 1,25(OH)2D3, 25(OH)D3 or ethanol (0.1%) bru
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for 6 days in the presence of 10% FBS or in the serum free condition, wherein a 1% mixture of , 2
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insulin-transferrin-selenium (ITS, Invitrogen, Carlsbad, CA) was used instead.
DTEx Microarray Analysis
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Total RNA (~2 g) was converted to aRNA using the MessageAmp II aRNA Amplification kit
(ABI/Ambion, Chicago IL/Austin TX) according to the manufacturer’s instructions. Labeled
cDNA was prepared from purified aRNA using CY5-labelled random nonamers and SuperScript
II reverse transcriptase (Invitrogen, Burlington ON) according to the manufacturer’s instructions.
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CY5-labeled cDNA was purified on QIAquick PCR purification columns (Qiagen, Mississauga
ON). Purified CY5-labeled cDNA was quantified using a NanoDrop spectrophotometer
(NanoDrop Inc., Wilmington DE). CY5-labeled cDNA (~3 μg) was denatured at 95oC for 10 min
and hybridized to a DTEx microarray slide [145 ADME-associated genes printed on Nexterion E
slides (Schott, Louisville KY)] in Nexterion 1X Hyb solution for 18 h at 45 oC. Prior to
hybridization, each DTEx microarray slide was denatured in boiled water for 60 sec and blocked
in Nexterion Block E for 15 min at 45o C. Following hybridization, the DTEx microarray slides
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were washed once for 15 min at 45oC in each of the following solutions; 2xSSC:0.2% SDS, n
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2xSSC and 0.2xSSC. The DTEx microarray slides were “spin-dried” at 1200 rpm for 2 min at fro
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room temperature, and then scanned in a ProScanArray HT (Perkin Elmer, Woodbridge ON). jpe
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Following image acquisition, fluorescent signals from the DTEx microarray were assessed using tjo
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QuantArray. Results were exported to GeneLinker Gold (Improved Outcomes Software, Kingston, ls.o
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ON) for normalization, hierarchical cluster analysis and matrix plot images. Four independent t A
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DTEx microarray experiments were performed for each RNA sample in this study. Each gene on J
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the DTEx microarray was printed in quadruplicate resulting in n = 16 for each gene interrogated ls
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in the DTEx microarray analysis. b
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Real-time Quantitative Polymerase Chain Reactions: RT-qPCR , 2
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RNA levels of the drug transporters and enzymes were analyzed by RT-qPCR. Total RNA
was isolated from cells using TRI Reagent (Sigma Chemical Company, Oakville, ON). The first-
strand cDNA was synthesized by using the First Strand cDNA Synthesis Kit (Fermentas,
Burlington, ON). Then cDNA samples were subjected to qPCR assays with the fluorescent dye
SYBR Green methodology and an ABI 7500 detector (Applied Biosystems, Foster City, CA).
Gene-specific primers, designed with the Primer3 software (Whitehead Institute for Biomedical
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