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DRUG METABOLISM AND DISPOSITION
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/dmd.113.052704
Drug Metab Dispos 41:1934–1948, November 2013
Expression of Efflux Transporters in Human Ocular Tissues
Peng Chen, Hao Chen,1 Xinjie Zang, Min Chen, Haoran Jiang, Shasha Han, and Xianggen Wu
State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong
Academy of Medical Sciences, Qingdao, People’s Republic of China
Received May 24, 2013; accepted August 26, 2013
ABSTRACT
transporters in mRNA and protein levels in ocular tissues. At the
protein level, MRP1-7, MDR1, and LRP were expressed in the corneal epithelium; MRP1-7, MDR1, LRP, and BCRP were expressed
in the conjunctival epithelium; MRP1-2, MRP6-7, MDR1, and LRP
were expressed in the ICB; MRP1-3, MRP6-7, MDR1, and LRP were
expressed in the retina; MRP1-3, MRP6-7, MDR1, and LRP were expressed in the HCEC; and MRP7, MDR1, LRP, and BCRP were
expressed in the ARPE-19. This quantitative and systematic study
of efflux transporters in normal ocular tissues and cell lines provides
evidence of cross-ocular tissue transporter expression differences,
implying that efflux transporter expression variability should be
taken into consideration for better understanding of ocular pharmacokinetic and pharmacodynamic data.
Introduction
Toit et al., 2011; Pahuja et al., 2012; Tartara et al., 2012). Additional
factors preventing drugs from reaching the target site of the anterior
chamber involve drug elimination through aqueous humor outflow
and entering the uveal blood circulation via the BAB (Gaudana et al.,
2010). Systemic administration is one possible route for delivery of
drugs to the back of the eye; however, retinal transfer of drugs from
the circulating blood is strictly regulated by two blood-ocular barrier
systems: the BAB and the BRB (Gaudana et al., 2010).
The multidrug resistance (MDR) phenotype, a major type of cell
resistance toward xenobiotics, remains the main cause of failure in
cancer chemotherapy (Wu et al., 2011). The MDR phenomenon is
associated with a decrease in intracellular xenobiotics/drug accumulation by efflux transporters, such as multidrug resistance 1 (MDR1)–
P-glycoprotein, multidrug resistance–associated proteins (MRPs),
breast cancer-resistance protein (BCRP, also known as ABCG2),
and lung-resistance protein (LRP). These proteins have been subsequently detected in normal tissues of kidney, intestine, adrenal
gland, liver, blood-brain barrier, and cells of the immune system. The
presence of these proteins in the apical membranes of normal tissues,
such as renal tubular cells and intestinal absorptive cells, can interfere
with drug availability, metabolism, and toxicity, because they reduce
intestinal absorption and increase renal secretion of a wide range of
drugs and metabolites, which are substrates for these membrane
transporters (Bodo et al., 2003; Delou et al., 2009).
Many transporters have been discovered in tissues of the liver,
kidney, and intestine (Obligacion et al., 2006; Klaassen and Lu, 2008;
Delou et al., 2009; Klaassen and Aleksunes, 2010). Among them,
efflux transporters and influx transporters are related to drug delivery
The eye is a complex organ with sophisticated anatomic and
physiologic structures (Sasaki et al., 1999; Edelhauser et al., 2010;
Ulbrich and Lamprecht, 2010). These structures include various ocular
barriers, such as corneal epithelium, the blood-aqueous barrier (BAB),
and the blood-retina barrier (BRB), which govern the entry and exit of
nutrients and xenobiotics into and out of ocular tissues in the anterior
and posterior parts of the eye, making it a challenge to deliver drugs
effectively into ocular tissues (Mannermaa et al., 2006; Zhang et al.,
2008; Mitra, 2009). Topically applied drugs may be rapidly eliminated
from the precorneal area (Wu et al., 2010). The cornea, a major
pathway for eye drops or eye ointments to enter the eye, is also an
important barrier to drug penetration (Ghate and Edelhauser, 2006; du
This work was supported by the National Natural Science Foundation of China
[Project Nos. 81000369 and 81271036]; the Young and Middle-Aged Scientists
Research Awards Fund of Shangdong Province, China [Project No. BS2009SV053];
and the Science and Technology Foundation of Qingdao, Shandong Province,
China [Project No. 13-1-4-177-jch].
This manuscript was previously presented: Chen P, Chen H, Zang X, Chen M,
Jiang H, Han S, Wu X (2013) Expression of Efflux Transporters in Human Ocular
Tissues. International Symposium on Ocular Pharmacology and Pharmaceutics;
2013 Mar 7–10; Paris, France.
None of the authors has a proprietary or commercial interest in any of the
mentioned drugs or companies.
1
Current affiliation: Department of Microbiology, Key Laboratory of Medicine
and Biotechnology of Qingdao, Qingdao University Medical College.
dx.doi.org/10.1124/dmd.113.052704.
ABBREVIATIONS: ARPE-19, human retinal pigment epithelial cell line; BAB, blood-aqueous barrier; BCRP, breast cancer–resistance protein; BRB,
blood-retina barrier; CE, cornea epithelium; HCE, human corneal epithelial; HCEC, human corneal epithelial cell line; ICB, iris-ciliary body; LRP, lung
resistance protein; MDR, multidrug resistance; MDR1, multidrug resistance 1; MRP1-7, multidrug resistance-associated protein 1-7; PCR,
polymerase chain reaction; RC, retina-choroid; RPE, retinal pigment epithelial; TBST, Tris-buffered saline Tween-20.
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To investigate the expression profiles of efflux transporters in human ocular tissues, quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry were used to
obtain the relative mRNA and protein expressions of various efflux
transporters in human ocular tissues. The cornea, conjunctiva, irisciliary body (ICB), retina and choroid, human corneal epithelial cell
line (HCEC), and human retinal pigment epithelial cell line (ARPE19) were examined for the expressions of multidrug resistance–
associated proteins 1–7 (MRP1–7), multidrug resistance 1 (MDR1)
P-glycoprotein, lung resistance protein (LRP), and breast cancer–
resistance protein (BCRP). The expression sites and patterns of
efflux transporters were significantly different in ocular tissues,
HCEC, and ARPE-19, as well as the expression profiles of efflux
Efflux Transporters in Human Ocular Tissues
(Gaudana et al., 2010; Su et al., 2011). The role of efflux transporters
has been investigated more intensively than that of influx transporters, due to the defense mechanism of efflux transporters against
penetration of xenobiotics (Eechoute et al., 2011). Prominent efflux
transporters identified in tissues belong to the ATP binding cassette
superfamily, including MDR1, MRPs, and BCRP (Giacomini et al.,
2010). LRP is not an ATP binding cassette transporter but a major
vault protein (Bouhamyia et al., 2007). As it is thought to drive drugs
away from the nucleus, LRP has also been reported as an efflux
transporter (Leonard et al., 2003; Bouhamyia et al., 2007), although
there are discrepancies about its MDR functions (Mossink et al.,
2002).
Efflux transporters lower bioavailability by effluxing the molecules out of the cell membrane and cytoplasm, and they are important determinants of drug accumulation in target organs or cells and
of overall drug uptake, distribution, and excretion. Moreover, some
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efflux transporters have been discovered in ocular tissues, such as the
cornea and retina (Kajikawa et al., 1999; Kennedy and Mangini, 2002;
Becker et al., 2007; Vellonen et al., 2010). Similar to the liver, kidney,
and intestine tissues, the expression and function of efflux transporters at various layers of ocular epithelial and endothelial cells may
significantly influence ocular drug efficacy by means of absorption,
distribution, and elimination (Dey et al., 2004; Hariharan et al., 2009).
However, knowledge about these efflux transporters in ocular tissues
is very limited, despite the potentially important role they may play in
ocular drug disposition. The data of expression and function available
only involve lower species and cell lines, and some even remain
controversial.
Expression and location profiles are important prefactors to functional protein research (Dallas et al., 2006). With some differences in
the results, expression profiles of efflux transporters in human corneas have been evaluated (Becker et al., 2007; Zhang et al., 2008;
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Fig. 1. mRNA expression levels of efflux transporters in human ocular tissues. The data are expressed as the mean 6 S.D. a: PCE-conjunctiva,0.05, b: PCE-ICB,0.05,
c: PCE-RC,0.05, d: Pconjunctiva-ICB,0.05, e: Pconjunctiva-RC,0.05, f: PICB-RC,0.05. (n = 6 for CE, ICB, and RC; n = 4 for conjunctiva). As MRP3 in ICB and RC, and MRP4
in CE had no expression, the statistical analysis was not performed here.
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Chen et al.
Vellonen et al., 2010). However, there have been few reports on the
expression pattern of efflux transporters in the other human ocular
drug-absorption barrier tissues, such as conjunctiva, iris-ciliary body
(ICB), and retina-choroid (RC). In the present study, we characterized
the expression and location profiles of 10 major efflux transporters in
ocular tissues. In addition, the expression profiles of human corneal
epithelial cells (HCEC) and human retinal pigment epithelial cells
(ARPE-19), two cell lines commonly used as in vitro models for
corneal and retinal drug absorption studies, respectively, were investigated and compared.
Materials and Methods
TABLE 1
Patient demographic information
Tissues
Donor No.
Age (yrs)
Gender
Eye
Endothelial Cell
Density (/mm2)
Cornea, Conjunctiva, Iris-Ciliary Body,
and Retina-Choroid
1
18
Male
2
38
Male
3
15
Female
4
29
Female
5
29
Female
6
45
Female
1
15
Male
2
19
Male
3
27
Male
4
16
Female
5
18
Female
6
25
Female
OD
OS
OD
OS
OD
OS
OD
OS
OD
OS
OD
OS
OD
OS
OD
OS
OD
OS
OD
OS
OD
OS
OD
OS
2197
2341
2550
2470
3250
3180
2790
2830
2790
2830
2570
2390
2812
2963
2759
2688
2518
2637
3022
3087
2975
2891
2367
2470
Cornea Epithelium
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Human Ocular Tissues. The human ocular tissues from donors with no
history of eye disease were provided by the Eye Bank of Shandong,
International Federation of Eye and Tissue Banks (Qingdao, China). The eye
globes, enucleated within 10 hours of death, were immediately dissected, and
the conjunctiva, ICB, and RC were collected. Patient data are summarized in
Table 1. Human corneal epithelium (CE) tissues were obtained from patients
who underwent photorefractive keratectomy surgery for correction of refractive
errors at Qingdao Eye Hospital, Shandong Eye Institute (Qingdao, China).
Written informed consent for photorefractive keratectomy and CE to be used
in this research was obtained from each patient. Patients with manifested ocular pathology or topical ocular drug therapy were excluded. The CE was
scraped off before photoablation. Each tissue was snap-frozen in liquid nitrogen and stored at 280°C for protein and RNA analysis. Normal human
corneas that were left after corneal transplantation surgery and were not suitable for further clinical application were provided by Qingdao Eye Hospital,
as well as the conjunctiva, ICB, and retina used for immunohistochemical
analysis. All tissues were from donors free of ocular disease and other systemic
complications. This study was approved by the Ethical Review Committee of
Shandong Eye Institute, and the handling of donor tissues was consistent with
the tenets of the Declaration of Helsinki regarding the protection of donor
confidentiality.
Cell Culture. Simian virus 40–immortalized HCECs were kindly provided
by Prof. Choun-Ki Joo (School of Medicine, the Catholic University of Korea,
Seoul, Korea). The cells were cultured in Dulbecco’s modified Eagle’s
medium/F-12 (1:1) media, 5% fetal bovine serum (Gibco-BRL, Grand Island,
NY), 5 mg/ml insulin (Sigma, St. Louis, MO), 0.1 ng/ml cholera toxin (EMD
Biosciences, San Diego, CA), 10 ng/ml human epidermal growth factor (R&D
Systems, Minneapolis, MN), and 0.5% dimethyl sulfoxide (Sigma) in a humidified
5% CO2 incubator at 37°C.
ARPE-19 (ATCC, catalog no. CRL-2302) cells were cultured in 1:1 of
Dulbecco’s modified Eagle’s medium/nutrient mixture F12 (Invitrogen, Carlsbad,
CA), containing 10% fetal bovine serum (Invitrogen). The cells were incubated
at 37°C in a humidified atmosphere of 5% CO2 and 95% air. The culture medium
was changed every 3 days.
Construction of Plasmid and Standard Curves. The target DNA
fragments of corresponding genes obtained by chemical synthesis were ligated
into the pMD19-T vector (Takara, Dalian, China) at 16°C overnight. The
ligation mixtures were transformed into competent Escherichia coli DH5a cells.
After being confirmed by polymerase chain reaction (PCR) and sequencing, the
positive clones were designated as recombinant plasmid pMD19-MRP1, pMD19MRP2, pMD19-MRP3, pMD19-MRP4, pMD19-MRP5, pMD19-MRP6, pMD19MRP7, pMD19-MDR1, pMD19-LRP, and pMD19-BCRP.
The correct recombinant plasmids were extracted as standard plasmids for
fluorescence quantitative polymerase chain reaction (PCR) using a plasmid
extraction kit (Tiangen Corp, Beijing, China). A Smartspec3000 spectrophotometer (Bio-Rad Corp, Hercules, CA) was used to determine the concentration
of recombinant plasmids by measuring the absorbance at 260 nm, and purity
was confirmed using a 260/280 nm ratio. The plasmid copy number was
calculated using the following formula: molecules ml21 = (A 6.022 1023)
(660 B)-1, where A is the plasmid concentration (g ml21), B is the plasmid
length containing the cloned sequence, 6.022 1023 is the Avogadro’s number,
and 660 is the average molecular weight of one base pair.
The recombinant plasmids were serially diluted 10-fold in ddH2O, from 1021
to 1027. Each dilution was tested in triplicate and used as an amplification
template to construct standard curves by plotting the plasmid copy number
logarithm against the Ct values under optimum conditions. Sequence detection
system software (7500 System; Applied Biosystems, Singapore) was used to
create the standard curves and calculate the correlation coefficients. Positive
control (standard plasmid without dilution) and negative control experiments
were conducted in parallel for quality control.
Quantitative Detection of Samples. Quantitative real-time PCR was
performed according to previous descriptions with minor modification (Perini
et al., 2011). Total RNA was isolated from each tissue according to the
manufacturer’s protocol (NucleoSpin RNA II System; Macherey-Nagel, Düren,
Germany) and subjected to reverse transcription at 42°C for 60 minutes in a
40-ml reaction mixture using a first-strand cDNA synthesis kit (Takara). The
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Efflux Transporters in Human Ocular Tissues
TABLE 2
Sequences of oligonucleotide primers and probes used for fluorescence quantitative PCR
Gene Name
Primer Sequences (F)
Primer Sequences (R)
Probe Sequences
MRP1
MRP2
MRP3
MRP4
MRP5
MRP6
MRP7
MDR1
BCRP
LRP
GACCATGAATGTGCAGAAGG
CCTCACAAACTGCCTCTTCA
ACAACCTCATCCAGGCTACC
GGAACTGTTTGATGCACACC
CAGGTGGTGGAGTTTGACAC
TTCTCCTCTACGCTGGGTTT
GCTTATCTCTTGGGCAGAGG
TCCTTCATCTATGGTTGGCA
GTTGTGATGGGCACTCTGAC
GACGTCATCACCATCGAAAC
GGATCTTCGTCTTCCTCAGC
AGCCCAATGGAAGCAATATC
GGTTGGCTGGAGAATCAAAT
TGATGACAAACATGGCACAG
GGCATAGAATCGGGAACTGT
ACAGACGAGTAGCTGGCTGA
GTGGCCTCATCGATACACAG
TAGCGATCTTCCCAGAACCT
CCCTGTTAATCCGTTCGTTT
TCAAAGTGCCAGTTGTAGGC
CTTGTGTGCCTAGCCCGGGC
AATCTGACCACCGGCAGCCTC
CTGACCATCGCACACCGGCT
TTTGCCGTCCGTCTGGATGC
CCATCGGTCCTTCTGTCCAACG
CCAAGCGTCTCAGCTGGCATG
TGGCCAGGGCTCTCCTCACA
CCCATCATTGCAATAGCAGGAGTTGT
TCAGCAGCTCTTCGGCTTGCA
CATGCCAGGCTGCAACTGCA
were conducted at 25°C, and three washes with 10 ml TBST were performed
between each step. The membranes were then developed with SuperSignal West
Femto Maximum Sensitivity substrate (Pierce Biotechnology, Rockford, IL) and
exposed to X-ray film (Kodak, Rochester, NY). The immunoreactive bands were
quantified using National Institutes of Health Image 1.62 Software (National
Institutes of Health, Bethesda, MD). All the experiments in this study were
performed three times, and the results were reproducible. For each sample, the
levels of proteins of interest were normalized to that of GAPDH. Primary
antibodies included antibodies against MRP1,MRP2, MRP3, MRP4, MRP6,
MRP7, BCRP, MDR1, and LRP (Santa Cruz Biotechnology, Santa Cruz, CA);
anti-MRP5 antibodies (Abcam, Cambridge, MA); and anti-GAPDH antibody
(Kangchen, Shanghai, China).
Immunohistochemistry and Immunocytochemical Staining. Each tissue
was fixed in 10% buffered formalin and embedded in paraffin for immunohistochemical analysis. Paraffin sections, 4 mm in thickness, were deparaffinized, rehydrated, and stained with antibodies, using routine protocols. The
antibody information is shown in Table 3. 3,39-Diaminobenzidine and 3amino-9-ethylcarbazole, originally introduced for the localization of horseradish peroxidase, have been widely used for staining in immunohistochemistry.
The cornea and conjunctiva were stained with 3,39-diaminobenzidine, which
can form a brown precipitate upon oxidation, while the ICB and RC were
stained with 3-amino-9-ethylcarbazole, which can form a red precipitate upon
oxidation.
Cells were plated in a glass culture dish (Biousing Biotech Co., Wuxi,
China) at a density of approximately 4.0 104 cells/35-mm dish in complete
medium and grown to subconfluence. The medium was removed, and the cells
were fixed in 4% paraformaldehyde in phosphate-buffered saline for 15
TABLE 3
Antibody information
Product Name
Catalog Number
Subtype
Immunogen
MRP1 (MRPm6)
MRP2 (M2III-5)
sc-59606
sc-59611
Mouse IgG1
Mouse IgG2b
MRP3 (H-150)
sc-20767
Rabbit IgG
MRP4 (H-280)
sc-20768
Rabbit IgG
MRP5
ab77369
Goat IgG
MRP6 (H-70)
sc-25505
Rabbit IgG
MRP7 (H-300):
sc-67241
Rabbit IgG
MDR1 (JSB-1)
sc-59593
Mouse IgG1
C-terminus of human origin
A fusion protein of
MRP2 of rat origin
Amino acids 241–390 of
MRP3 of human origin
N-terminus of MRP4 of
human origin
N-terminus of MRP5 of
human origin
Amino acids 1–70 of MRP6 of
human origin
Amino acids 331–630 mapping
within an internal region of
MRP7 of human origin
MDR of hamster origin
LRP (1014)
BCRP (B-25)
sc-23916
sc-130933
Mouse IgG1
Rabbit IgG
LRP purified from MCF7 cells
Synthetic BCRP peptide of
human origin
Dilution
(immunohistochemistry/
immunofluorescence)
Dilution
(Western blotting)
Human
Human and rat
1:50
1:50
1:200
1:200
Human, mouse,
and rat
Human
1:200
1:500
1:200
1:500
Human, mouse,
and rat
Mouse, rat,
and human
Mouse, rat,
and human
1:200
1:500
1:200
1:500
1:200
1:500
Mouse, rat, human,
and hamster
Human
Mouse, rat,
and human
1:50
1:200
1:200
1:200
1:500
1:500
Reacts with
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reagents (Tiangen) and sequence detection system were used in fluorescence
quantitative PCR as recommended by the manufacturer. The primers and
oligonucleotide probes used are listed in Table 2. Cycling conditions were as
follows: 10 minutes at 95°C, followed by 40 cycles of amplification for 15
seconds at 95°C and 1 minute at 60°C. The expected fragment length was 150–
300 bp. Quantification data were analyzed by the Sequence Detection System
software. The log-linear portion of the fluorescence versus cycle plot was
extended to determine a fractional cycle number at which a threshold fluorescence was obtained (threshold cycle), and this number was used as a
reference for each analyzed gene. The cDNA samples were amplified in parallel
with plasmid standards in each run, and their Ct values were plotted together
with the standard curves, from which the target gene mRNA copy numbers
were determined.
Western Blotting and Antibodies. Western blotting was performed as
previously described (Chen et al., 2012), and the antibody information is listed
in Table 3. The total protein was prepared from each tissue using radio immunoprecipitation assay buffer (50 mmol/l Tris, 150mmol/l NaCl, 1% Triton
X-100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate, sodium orthovanadate, and sodium fluoride, pH 7.4; Galen, Beijing, China) and quantified.
The protein (50 mg in 15 ml loading buffer) was resolved in 10% sodium
dodecyl sulfate polyacrylamide gel electrophoresis gel before being transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA).
The blots were blocked in 5% nonfat dry milk dissolved in Tris-buffered saline
Tween-20 (TBST; 20 mmol/l Tris, pH 7.5, 0.5 mmol/l NaCl, 0.05% Tween-20)
for 1 hour and then incubated with the primary antibody in TBST for 1 hour,
followed by incubation with horseradish peroxidase-conjugated secondary antibody (Amersham Biosciences, Uppsala, Sweden) for 1 hour. All incubations
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Chen et al.
minutes, followed by three phosphate-buffered saline washes. The fixed cells
were incubated with 0.2% Triton-100 in phosphate-buffered saline for 5
minutes, blocked in 5% bovine serum albumin, and incubated with primary
antibodies for 30 minutes, followed by incubation with corresponding
fluorescence-conjugated secondary antibodies for 1 hour. Images were obtained
using an Eclipse TE2000-U confocal laser-scanning microscope (Nikon, Tokyo,
Japan).
Data Analysis. Data are presented as mean 6 S.D. Difference comparison
of measurement data among groups was determined with single factor variance
analysis, and comparisons between two groups were performed with the least
significant difference t test (Fig. 1). The differences among groups were
analyzed using Student’s t test (Fig. 2). SPSS 11.5 software (SPSS Inc.,
Chicago, IL) was used, and P , 0.05 was considered significant.
Results
mRNA Expression in Human Ocular Tissues, HCEC, and
ARPE-19. The rank order of MRP1 expression was RC.ICB.CE.
conjunctiva; MRP2 was RC.ICB.conjunctiva.CE; MRP3 was
conjunctiva.CE, and it was absent in RC and ICB; MRP4 was ICB.
conjunctiva.RC, and it was absent in CE; MRP5 was RC.ICB.
CE.conjunctiva; MRP7 was RC.CE.conjunctiva.ICB; MDR1 and
BCRP were ICB.RC . conjunctiva.CE; and LRP was CE.ICB.
conjunctiva.RC. MRP6 was absent in all the ocular tissues (Fig. 1).
The mRNA expression of MDR1 in HCEC was absent, while that
of other efflux transporters in the HCEC was significantly higher
than the CE; even MRP4 had a high expression, but it was absent in
the CE. However, in ARPE-19, the mRNA expression profile was
somewhat sophisticated—MDR1was absent in ARPE-19; significantly higher expression than the RC in MRP1, MRP4, and LRP;
and significantly lower in MRP2, MRP5, MRP7, and BCRP. As in
the ocular tissues, MRP6 was absent from the HCEC and ARPE-19
(Fig. 2).
Western Blotting Studies of Protein Expression in Human
Ocular Tissues. Western blotting analysis of protein expression in
human ocular tissues is presented in Fig. 3. The quantitative comparison
of the fold difference in the expression of efflux transporters is shown in
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Fig. 2. mRNA expression levels of efflux transporters in HCEC, APRE-19, CE, and RC. Data are expressed as mean 6 S.D. (n = 6 for CE and RC; n = 3 for HCEC and
ARPE-19). *P , 0.05. As MRP3 in RC, MRP4 in CE, and MDR1 in HCEC and ARPE-19 had no expression, the statistical analysis was not performed here.
Efflux Transporters in Human Ocular Tissues
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Fig. 4. MRP1, MRP3, MRP6, MRP7, and LRP were found in the CE,
while only faint MDR1 expression was observed in the CE. MRP1,
MRP3, MRP4, MRP6, MRP7, MDR1, BCRP, and LRP were detected
in the conjunctiva. MRP1-7, MDR1, and LRP were found in ICB.
MRP1, MRP2, MRP3, MRP5, MRP6, MRP7, MDR1, BCRP, and LRP
were detected in the RC. MRP1, MRP2, MRP3, MRP7, MDR, BCRP,
and LRP were observed in the HCEC. Expression of MRP7, MDR1,
BCRP, and LRP was evident in APRE-19.
Localization of Efflux Transporters in Human Ocular Tissues.
The expression sites of efflux transporters differed among the ocular
tissues. In the human cornea, MRP1, MRP2, and MRP6 were found to
be predominant in the basal cell layer of the corneal epithelium, as
well as faint MRP3 and MRP4; MRP7 and MDR1 were detected in
the whole corneal epithelium, with a mild expression of LRP (Fig. 5).
In the human conjunctiva, the MRP1, MRP7, and LRP expressions
were found in the whole conjunctival epithelium; the MRP2, MRP3,
Fig. 4. The quantitative comparison of the fold difference in the expression of efflux transporters. Data are expressed as mean 6 S.D., and n = 4.
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Fig. 3. Efflux transporter expressions in the human ocular tissues, HCEC, and ARPE-19 by Western blotting. Each sample contained 50 mg protein in 15 ml loading buffer.
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Chen et al.
MRP4, MRP6, MDR1, and BCRP expressions were detected in the
basal cell layer. The MRP5 expression was detected in the upper layer
of the conjunctival epithelium (Fig. 6).
In the human ICB, expressions of MRP1, MRP2, and MDR1 were
observed in the stromal cells (Fig. 7). High MRP1, MRP2, MRP6, and
MDR1 expressions, as well as faint MRP3, MRP7, and LRP expressions
were detected in the whole retina (Fig. 8). The immunohistochemical
localization of drug transporters in human ocular tissues is summarized
in Table 4.
Localization of Efflux Transporters in the HCEC and ARPE19. The results of immunofluorescence microscopy are summarized in
Figs. 9 and 10. There were also great differences in the expression
sites of efflux transporters between the HCEC and ARPE-19. In the
HCEC, MRP1, MRP2, MRP3, MRP6, MRP7, MDR1, and LRP were
observed in the whole cytoplasm, while MRP4, MRP5, and BCRP
were absent. In ARPE-19, MRP7, MDR1, LRP, and BCRP were
detected in the whole cytoplasm, while MRP1-6 was absent (Table 4).
Discussion
Transporter functions may reflect factors of location (within a tissue,
as well as within cells), the level of expression, substrate and inhibitor
specificity, and functional kinetics (Dallas et al., 2006). It is important
to formulate a clear description of the expression profiles of the efflux
transporters in ocular tissues. In this study, several barriers in ocular
drug absorption, through topical or systemic administration, were chosen
to elucidate their efflux transporter expression profiles in mRNA and
protein levels. A summary of this study’s results, as well as some expression reports of efflux transporters in human ocular tissues, is shown in
Table 5.
The Cornea and HCEC. The corneal epithelium is lipoidal in
nature and contains 90% of the total cells in the cornea; it displays
significant resistance toward permeation of topically administered
hydrophilic drugs. Kawazu et al., (1999) first reported the presence of
MDR1 in cultured rabbit corneal epithelial cells, where it could help
protect cells from being damaged by uptake of substances (Kawazu
et al., 1999). In 2003, Dey et al. (2003) found, for the first time, that
the human cornea can express MDR1. Since then, individual efflux
transporters in human and animal corneas and corneal epithelial cells
have been widely investigated (Karla et al., 2007a,b, 2009; Pelis et al.,
2009; Vellonen et al., 2010; Barot et al., 2011; Li et al., 2012).
Functional activity of some efflux transporters has also been evaluated,
but the results of efflux transporter expressions and functions were very
different. Dey et al. (2003) and Becker et al. (2007) investigated the
mRNA expression of MDR1 in human corneas, as well as the MDR1
expression in a human corneal cell line by Western blotting, but no
detectable or very low MDR1 mRNA and/or protein expressions were
observed in human corneas according to other reports (Dey et al., 2003;
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 12, 2017
Fig. 5. Representative figures of immunolocalization of efflux transporters in human cornea. Arrow indicates the localization of efflux transporters. The brown color was
seen in positive cells. Nuclei were counterstained in blue with hematoxylin.
Efflux Transporters in Human Ocular Tissues
1941
Becker et al., 2007), and these discrepancies may be due to variation in
sensitivity of different experimental methods and different tissues used
(Vellonen et al., 2010). Regarding the function of efflux transporters,
Dey et al. (2003) evaluated the functional activity of MDR1 efflux
pump with cultured rabbit primary corneal epithelial cells and a corneal
cell line (Statens Seruminstitut rabbit cornea [SIRC] cells) as the model,
with the results that MDR1 affected corneal uptake. Becker et al. (2007)
evaluated the potential value of different epithelial cell culture systems
as in vitro models for investigation of corneal permeability. Transformed human corneal epithelial cells, SIRC cells, SkinEthic human
corneal epithelium, Clonetics human corneal epithelium, excised rabbit
corneas, and human corneas were involved; however, MDR1 and
similar efflux systems were observed to have no significant effects on
corneal permeability. Discrepancies have been found not only in the
expression of efflux transporters at the mRNA and protein levels, but
also in different corneal models; expressions may even fluctuate in the
same cell models, depending on the maturation status of cells (Vellonen
et al., 2006; Verstraelen, 2011; Juuti-Uusitalo et al., 2012).
There have been many reports on the expression and function of
efflux transporters in animal corneal cell models; however, to our
knowledge, only a few of them are related to the efflux transporter expression in human corneas, and the results are conflicting. In the current
study, MDR1 displayed 9.11 6 2.33 Qty/ng RNA expression in the CE,
similar to the results of other reports (Becker et al., 2007; Vellonen
et al., 2010). MRP1, MRP2, MRP3, MRP7, and BCRP also had low
mRNA expressions, while MRP5 and LRP had relatively high mRNA
expressions. However, discrepancies in the expression of efflux transporters at the mRNA and protein levels were also found in our results,
such as MRP5 and MRP6. BCRP, as a stem cell marker, which was
reported to express in human corneas (Saghizadeh et al., 2011), showed
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 12, 2017
Fig. 6. Representative figures of immunolocalization of efflux transporters in human conjunctiva. Arrow indicates the localization of efflux transporters. The brown color
was seen in positive cells. Nuclei were counterstained in blue with hematoxylin.
1942
Chen et al.
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 12, 2017
Fig. 7. Representative figures of immunolocalization of efflux transporters in human iris-ciliary body. Arrow indicates the localization of efflux transporters. The red color
was seen in positive cells. Nuclei were counterstained in blue with hematoxylin.
Efflux Transporters in Human Ocular Tissues
1943
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Fig. 8. Representative figures of immunolocalization of efflux transporters in human retina. Arrow indicates the localization of efflux transporters. The red color was seen in
positive cells. Nuclei were counterstained in blue with hematoxylin.
1944
Chen et al.
very faint mRNA and failed to be detected by Western blotting in this
study. The reason may be that stem cells in the cornea amounted to ,1%
of total cells, and BCRP existed in the limbal epithelium but not in the
stem cell–free corneal epithelium (Budak et al., 2005).
Polarization and stratification seem to have only slight effects on the
efflux protein expression in HCE cells, since only small differences
were detected between the HCE model and nonconfluent HCE cells
(Vellonen et al., 2010), so only nondifferentiated state of the HCEC
TABLE 4
Summary of immunohistochemical localization of drug transporters in human ocular tissues and immunofluorescence
microscopy in the HCEC and ARPE-19
Ocular Tissue
MRP1
MRP2
MRP3
MRP4
MRP5
MRP6
MRP7
MDR1
LRP
BCRP
Corneal Epithelium
Conjunctiva Epithelium
Iris-Ciliary Body
Retina
HCEC
ARPE-19
+
+++
++
+++
+
—
+
+
++
+++
+
—
+
+
—
+
+
—
+
+
—
—
—
—
+
+
—
—
—
—
+
+
+
++
+
—
+
++
+
+
+
++
+
++
++
++
++
++
+
++
+
+
+
+
—
+
—
—
—
+
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 12, 2017
Fig. 9. Representative figures of immunolabeling of efflux transporters in the HCEC. Arrow
indicates the expression of efflux transporters.
Efflux Transporters in Human Ocular Tissues
1945
was used in the current study (The ARPE-19 was performed like this).
Our results showed that cell lines or primary cells differed substantially
from freshly isolated human corneas or corneal epithelium in terms of
efflux transporter expressions, as previously reported (Becker et al.,
2007; Xiang et al., 2009; Vellonen et al., 2010).
Conjunctiva. The conjunctival layer plays a role in the drug
absorption of timolol maleate and carbonic anhydrase inhibitors to the
posterior segment of the eye, and the conjunctival epithelium is the
main barrier (Urtti, 2006; Gaudana et al., 2010). It was reported that
MDR1 existed in rabbit conjunctival epithelial cells and impaired the
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 12, 2017
Fig. 10. Representative figures of immunolabeling of
efflux transporters in ARPE-19. Arrow indicates the
expression of efflux transporters.
1946
Chen et al.
TABLE 5
Summary of some expression reports of efflux transporters in human ocular tissues
Ocular Tissue
Corneal
Epithelium
Conjunctiva
Epithelium
mRNA
MRP2
MRP3
+a
+b
2c
+d
2a
2b
2c
+d
2a
+b
+d
+e
Protein
IH or IF
RFTM
RFR
RFTM
RFR
RFTM
2a
+b
2c
+e
+d
+
+a
2e
—
2c
+d
+g
—
NR
+
NR
+
NR
+
+b
+d
+
NR
—
NR
—
+b
+d
+
NR
+
+d
—
—
+d
—
+a
2d
+
NR
+
NR
+
NR
+
NR
—
NR
+
2b
+
NR
+
NR
+
NR
+
+b
+
NR
+
NR
+
NR
+
+d
+
NR
+
NR
—
NR
+
+d
+
NR
—
NR
+
NR
+
NR
+
NR
+
NR
+
NR
+
NR
+
NR
+
NR
+
+b
—
NR
+
NR
—
+b
+
NR
+
NR
—
NR
+
NR
—
NR
—
+d
—
NR
+
NR
+
NR
+
NR
+
NR
+
NR
+
NR
+
NR
+
—
NR
+
NR
+
+
NR
—
NR
—
+
NR
+
2d
—
—
NR
+
NR
+
+
NR
+
NR
+
+
NR
+
NR
+
NR
+
+b
+d
+
NR
+
+h
+
+b
+d
+
NR
+
+i
+
+
NR
+
+c
+
NR
+
NR
+
NR
+
NR
+
NR
+
NR
+
NR
+
NR
+
NR
+
RFR, results from reports; RFTM, results from this manuscript; IH, immunohistochemistry; IF, immunofluorescence; NR, no report.
a
Vellonen et al., 2010.
b
Zhang et al., 2008.
c
Becker et al., 2007.
d
Dahlin et al., 2013.
e
Verstraelen J and Reichl S (2013) Expression analysis of MDR1, BCRP and MRP3 transporter proteins in different in vitro and ex vivo cornea models for drug
absorption studies. Int J Pharm 441:765–775.
f
Dey et al., 2003.
g
Deprez MP, Nicolas MJ, Moulin AP, Leonidas Z, and Francois M (2011) Expression of the stem cell markers ABCG2/BCRP, Bmi-1, P63alpha And C/EBPdelta in
the normal human cornea: an immunohistochemical study. Invest Ophthalmol Vis Sci 52:5115.
h
Schlingemann RO, Hofman P, Klooster J, Blaauwgeers HG, Van der Gaag R, and Vrensen GF (1998) Ciliary muscle capillaries have blood-tissue barrier
characteristics. Exp Eye Res 66:747–754.
i
Esser P, Tervooren D, Heimann K, Kociok N, Bartz-Schmidt KU, Walter P, and Weller M (1998) Intravitreal daunomycin induces multidrug resistance in
proliferative vitreoretinopathy. Invest Ophthalmol Vis Sci 39:164–170.
absorption of the immunosuppression drugs such as cyclosporine A
and b-blocker propranolol (Saha et al., 1998; Yang et al., 2000).
Therefore, it is important to understand the expression and regulation of
efflux transporters, as well as their effects on drug transport in the conjunctiva. Our study seems to be the first report on efflux transporter
expression profiles in the human conjunctiva. In mRNA expressions, all
of the efflux transporters except MRP6 were observed; at the protein
level, MRP1, MRP3, MRP4, MRP6, MRP7, MDR1, BCRP, and LRP
could be detected in the conjunctiva.
Iris-Ciliary Body. The blood-aqueous barrier resides in the iris and
ciliary body epithelium. In the iris, the capillary endothelium is not fenestrated, but there are tight junctions. In the ciliary body, there are tight
connections between the apical ends of the nonpigmented epithelial cells
(Goel et al., 2010). The nonpigmented epithelium of the ciliary body
represents an important component of the BAB of the eye. Many therapeutic drugs penetrate poorly across the nonpigmented epithelium into
the aqueous humor of the eye interior (Pelis et al., 2009). Previously, only
individual efflux transporters in the ICB were investigated. Zhang et al.
(2008) observed MDR1, MRP1-3, and BCRP mRNA expressions in the
ICB, finding that MRP2 had an extremely low expression; MDR1,
MRP3, and BCRP had a low expression; MRP1 had a medium expression in this tissue. Dey et al. (2003) confirmed the BCRP expression
in the ciliary epithelium and iris pigment epithelium in adult human eyes
by RT-PCR and immunofluorescence studies. In this study, we first
reported efflux transporter expression profiles in the human ICB. Only
MRP3 and MRP6 could not be detected in mRNA expressions; MRP5
and BCRP could not be detected at the protein level. The results of
immunohistochemistry also showed that MRP1, MRP2, and MDR1 had
high expressions in the stromal cells of the ciliary body.
Retina-Choroid and ARPE-19. The retina is composed of neural
retina and retinal pigment epithelium cells, which are adjacent to the
choroid (Zhang et al., 2008). The BRB resides in the retina and consists of
two major components: the endothelium of retinal blood vessels (inner
BRB) and the retinal pigment epithelium (outer BRB). Although the inner
and outer BRBs together create a “privileged” environment in the retina
and vitreous, the outer BRB constitutes the major absorption barrier
for transsclerally or topically administrated drugs. The retina-choroid is
commonly used as a tissue-level model to study the permeability of the
outer BRB. As an ex vivo model, the retina-choroid can also be a useful
tool for identification and characterization of carrier-mediated systems on
Downloaded from dmd.aspetjournals.org at ASPET Journals on May 12, 2017
mRNA
BCRP
+c
+
NR
—
2c
2d
IH or IF
LRP
2a
+b
2c
+e
+f
+
2a
2e
+
2c
—
2a
+
+a
2c
2d
+
NR
+
NR
+
NR
+
+b
+d
+
NR
+
NR
+
+b
+d
+
NR
+
2d
+
RFTM
RFR
RFTM
RFR
RFTM
RFR
MDR1
NR
+
+a
RFTM
RFR
Protein
MRP7
2a
—
2a
IH or IF
mRNA
MRP6
+
2a
2e
+
NR
+
2a
RFTM
RFR
RFTM
RFR
RFTM
RFR
RFTM
RFR
MRP5
+a
+d
+
+a
mRNA
MRP4
2a
+d
RFTM
RFR
IH or IF
Retina
MRP1
Protein
Protein
ICB
RFR
Efflux Transporters in Human Ocular Tissues
Conclusion
This article showed the mRNA and protein expression profiles of
efflux transporters in human ocular tissues. The wide differences in
expression among tissues and cell lines indicate that investigations on
efflux transporter functional activity should be based on their natural
expressions in ocular tissues, and cell lines should be used with
caution when in vitro models are involved.
Acknowledgments
The authors thank Dr. Ting Liu for his assistance in immunofluorescence
microscopy, Dr. Ye Wang for her assistance in Western blotting, Yao Wang for
her assistance in cell culture, Weiyun Shi for his critical review of the manuscript,
and Ping Lin for her editorial assistance.
Authorship Contributions
Participated in research design: Wu, P. Chen.
Conducted experiments: P. Chen, H. Chen, Jiang, Han.
Contributed new reagents or analytic tools: Zang, M. Chen.
Performed data analysis: Wu, P. Chen.
Wrote or contributed to the writing of the manuscript: Wu, P. Chen.
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E-mail: [email protected]
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