IF in JCR CiteScore
Rank About IJO Current
Issue Featured Articles Articles In Press Recent Accepted
International Journal
of Ophthalmology
2017; 10(9): 1370-1373
·Basic Research·
Effect of dopamine on bone morphogenesis protein-2
expression in human retinal pigment epithelium
Hong-Hui Li1, Yan-Li Sun2,
Dong-Mei Cui3, Juan Wu3, Jun-Wen Zeng3
1Chengdu
University of TCM, Chengdu 610075, Sichuan Province, China
2Ophthalmology
Center, Yinzhou Hospital Affiliated to Medical School of Ningbo University,
Ningbo 315040, Zhejiang Province, China
3State Key
Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen
University, Guangzhou 510060, Guangdong Province, China
Correspondence to: Jun-Wen
Zeng. State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun
Yat-sen University, Guangzhou 510060, Guangdong Province, China. cier72@163.com
Received: 2016-03-15
Accepted: 2017-06-05
Abstract
AIM: To
investigate the effect of dopamine on bone morphogenesis protein-2 (BMP-2)
expression in retinal pigment epithelium (RPE) cells in vitro.
METHODS: ARPE-19
cells as a human RPE cell line were cultured with dopamine for different times
(2, 4, 6, 8, 12, 16 and 24h) or with different concentrations (0.1, 1, 2, 5,
10, 20, and 100 μg/mL) in
vitro. BMP-2 mRNA expression level in ARPE-19 cells was analyzed with
real-time polymerase chain reaction (PCR) analysis and BMP-2 protein level was
measured with Western blot analysis. The active form of BMP-2 in the culture
medium was measured with enzyme-linked immunosorbent assay (ELISA).
RESULTS: The
expression level of BMP-2 increased significantly cultured with 20 μg/mL
dopamine, at different time points (P<0.05). BMP-2 mRNA level peaked
2h and the protein level peaked at 6 and 8h after treatment. The concentrations
of secreted BMP-2 elevated at 12h and peaked at 24h (P<0.05) in a
time-dependent manner. Treated with 100 μg/mL
dopamine for 6h, the expression levels of BMP-2 mRNA and protein in ARPE-19
cells were enhanced significantly compared to that in the untreated cells (P<0.05).
And secreted BMP-2 protein in the cell culture supernatant was also increased (P<0.05).
CONCLUSION: Dopamine
up-regulate BMP-2 expression in RPE cells, and this may be associated with its
inhibitive effect on myopia development.
KEYWORDS: dopamine; retinal
pigmental epithelieum; bone morphogenetic protein-2
Citation: Li HH, Sun YL, Cui DM, Wu J, Zeng JW. Effect of dopamine on bone
morphogenesis protein-2 expression in human retinal pigment epithelium. Int
J Ophthalmol 2017;10(9):1370-1373
INTRODUCTION
Dopamine is a chemical messenger for light
adaptation, and it involves trophic functions of the retina, including growth,
development, cell death and experimental myopia[1].
In animal models of myopia, dopamine receptor agonist and dopamine have
inhibitory effect on form-deprived myopia (FDM)[2]
and lens-induced myopia (LIM)[3]. So dopamine
serves as a signaling molecular in regulating ocular growth[4],
but its mechanism is unclear. Dopamine is released from the dopaminergic
amacrine cells in the inner plexiform layer and the vast majority of retinal
cells respond to dopamine[5]. All of the retinal
cells bear dopamine receptors, including retinal pigment epithelium (RPE) cells[1]. Study has shown that dopamine pathways might be
involved in the control of interphotoreceptor matrix properties and retina/RPE interaction[4,6].
In LIM and FDM, the retinal dopamine level
decreases[4-5], but the
choroidal and scleral dopamine levels are not changed[4].
So we proposed that dopaminergic mechanisms of regulating ocular growth may lie
in modulating the secretion of growth factors by the RPE cells to relay the
information from the retina to the sclera[7].
Bone morphogenesis protein-2 (BMP-2) is an
important factor for early eye morphogenesis, ocular development and growth[8-9]. BMP-2 expression in chick
retina/RPE is down-regulated in FDM[8]. BMP-2 and
its receptors are found in human sclera fibroblasts and it influences extra
cellular matrix synthesis in vitro[10-11]. The gene-analysis of humans confirmed that BMP-2
related to refractive error and myopia[12-13].
In the present study, we investigate the
effect of dopamine on the level of BMP-2 expression in RPE cells in vitro.
MATERIALS AND METHODS
Cell Culture and Handling ARPE-19 (ATCC Number: CRL-2502) cells as a human RPE cell line were
routinely cultured in a DMEM/F12 (1:1) medium containing 10% fetal bovine serum
(FBS, Invitrogen Life Technologies, Carlsbad, CA, USA) and 100 IU/mL penicillin/streptomycin
(Gibco, CA, USA) at 37℃ in 5% (vol/vol) CO2 humidified atmosphere. The medium was changed
every 2-3d. Cells were routinely passaged at a confluence between 80% and 90%
and at a split ratio ranging from 1:3 to 1:5 by digestion in 0.05% trypsi-0.02%
ethylene diamine tetraacetic acid (Invitrogen Life Technologies, Carlsbad, CA,
USA). Stock solution was prepared by dissolving dopamine (Sigma-Aldrich, St.
Louis, MO, USA) in ddH2O at a concentration of 100 mg/mL. ARPE-19
cells were cultured with 20 μg/mL dopamine for 2, 4, 6, 8, 12, 16 and
24h respectively, or with different concentration of dopamine (0.1, 1, 2, 5,
10, 20, 100 μg/mL) for 6h. The treament was repeated for 3 times respectively.
Real-time Polymerase Chain Reaction
Total RNA was isolated from cells with
Trizol reagent (Invitrogen Corp. Carlsbad, USA) according to the manufacturer’s
instructions. The cDNA was synthesized from 500 ng of total RNA (Takara
Biotechnology, Japan). Real-time polymerase chain reaction (PCR) was then
performed with Roche 4800 real-time-PCR system with SYBR Green I Master mix
(Roche Diagnostics GmbH, Mannheim, Germany). The relative quantification of the
target sequence and the reference sequence requires that the PCR efficiencies
of the BMP-2 gene and the β-actin gene PCR reactions be close to the
identical. During the log-linear phase, amplification can be described by
N=No(1+E)n, where N is the number of amplified molecules, No is the initial
number of molecules, E is the amplification efficiency, and n is the number of
cycles. If the amplification efficiency is similar for the two reactions, the
initial concentration of the sample is calculated on the basis of the above
formula by using the comparative delta Ct method and the gene copy number was
given by the formula 2−(∆∆Ct). The sequences of the
primer pairs were: BMP-2 [GenBank: NM_001200.2]: forward primer
5’-ACTCGAAATTCCCCGTGACC-3’ and reverse primer 5’-CCACTTCCACCACGAATCCA-3’; β-actin [GenBank: NM_001101.3]: forward primer 5’-CCAGAGGCGTACAGGGATAG-3’
and reverse primer 5’-CCAACCGCGAGAAGATGA-3’. The expected sizes for the BMP-2
and β-actin PCR products were 144 and 97 bp, respectively. After
amplification, the products were melted and the melting curve of the two genes
has only one peak respectively, so the primer has great specificity.
Western Blot Analysis The total protein of cultured cells was collected in lysis buffer with
1% phenylmethanesulfonyl fluoride (PMSF, all from Beyotime, Shanghai, China)
and its concentration was assayed by the BCA protein assay kit (Beyotime,
Shanghai, China). Totally 20 μg protein samples were separated by 12%
SDS-PAGE and electrotransferred to PVDF membranes (Millipore, Billerica, MA)
with 200 mA for 1h, which were blocked with 5% milk (Sigma Chemical Co. St.
Louis, USA) for 2h at room temperature. Subsequently, the membranes were
incubated with anti-β-actin antibody (ab8226, 1:3000 dilution;
Abcam, San Francisco, USA), anti-BMP-2 antibody (ab6285, 1:500 dilution; Abcam,
San Francisco, USA) overnight at 4℃, followed by the HRT-conjugated goat anti-mouse IgG antibodies (BA1051, 1:1000 dilution, Boster Biological
Technology) antibody for 1h at room temperature. Proteins were detected with
the enhanced chemiluminescence (ECL) detection system and exposed onto a
negative film, developed, and fixed. The film was scanned and then analyzed
with Bio-Rad Quautity Imaging software (version 1, Bio-Rad Laboratories). β-actin served as an internal reference, and the experiments were
repeated at least three times.
Enzyme Linked Immunosorbent Assay Determination APRE-19 cells were plated at a concentration of 2×104 per
well in 24 well plates containing DMEM plus 10% FBS and allowed to attach for
18h. Cells were washed with phosphate buffered solution (PBS) and re-fed with
0.5 mL of DMEM without FBS for 7h. Dopamine was added to cells at the different
concentrations or for different times, and then the culture supernatants were
collected to analyze the BMP-2 protein concentration using BMP-2 Quantikine
enzyme linked immunosorbent assay (ELISA) Kit (DBP200, R&D System, MN,
USA). Luciferase units obtained were normalized to the protein content of each
well. All experiments were performed at least three times with four independent
wells per condition.
Statistical Analysis Each experiment was repeated at least
three times. Data were expressed as mean±standard error of the mean (SEM). The
statistical significance of differences between cell groups in vitro was
determined by one-way ANOVA followed by Tukey’s post hoc multiple comparison
tests using the Mann-Whitney U test when significance was detected. All
the data analysis was performed using SPSS (version19.0, SPSS Inc., Chicago,
USA). P<0.05 was regarded as statistically significant.
RESULTS
To investigate the effect of dopamine on
BMP-2 expression in ARPE-19 cells in vitro, the levels of BMP-2 mRNA and
protein expression in the ARPE-19 cells and the concentrations of BMP-2
production in the supernatants of ARPE-19 cells were measured. When ARPE-19
cells were cultured with 20 μg/mL dopamine, the BMP-2 mRNA and protein levels
increased significantly at 2, 4, 6, 8, 12, 16 and 24h (P<0.05; Figure
1). BMP-2 mRNA expression level in treated cells peaked after 2h and it
increased 4.0-fold (Figure 1A), and the protein level peaked after 6 and 8h and
it elevated 2.1-fold (Figure 1B, 1C) when compared wtih the control. The
concentrations of secreted BMP-2 had no obvious changes after 6 and 8h
treatment but it elevated significantly at 16h and peaked at 24h increasing
2.0-fold (P<0.05; Figure 2). Our results indicated that the
production of BMP-2 by ARPE-19 could be induced by dopamine in a time-dependent
manner.
Figure 1 The
effect of dopamine on BMP-2 expression in ARPE-19 cells for different times Dopamine (20 µg/mL) enhanced the expression
of BMP-2 mRNA (A) and protein (B, C) in ARPE-19 cells. This effect existed
after 2h treatment with dopamine and lasted after 24h. The data were means±SEM
(n=3, aP<0.05 vs the control).
Figure 2 Dopamine stimulated secretion of
BMP-2 in the supernatants of ARPE-19 cells BMP-2 protein in the media was measured
by ELISA and normalized to cell counts (106). With 20 µg/mL dopamine
in culture, the BMP-2 concentration increased significantly after 16 and 48h
(A). There were no statistically significant differences when comparing the
concentrations of secreted BMP-2 after 2, 4, 6, 8 and 12h. When ARPE-19 cells
were treated with 20 and 100 µg/mL dopamine for 6h, the concentration of
secreted BMP-2 increased significantly (B). The data were means±SEM (n=3,
aP<0.05 vs the control).
With 20 and 100 µg/mL dopamine in the culture
for 6h, BMP-2 mRNA level increased 10.9-fold and 9.4-fold seperately (Figure
3A), and BMP-2 protein level increased nearly 4.0-fold compared to the
untreated cells (Figure 3B, 3C). The active form of BMP-2 in the supernatant
was also enhanced significantly with 20 and 100 µg/mL dopamine (Figure 2B).
Between the two concentration of dopamine, their effect on BMP-2 expression and
secration has no differences (P>0.05). There were no statistical
differences in BMP-2 mRNA and protein expression levels , and the
concentrations of secreted BMP-2 also had no differences when comparing the
cells cultured with 0.5, 1, 2, 5 and 10 µg/mL dopamine with the control cells (P>0.05).
Figure 3 BMP-2 expression in ARPE-19 cells
cultured with dopamine for 6h With 20 or
100 µg/mL dopamine in the culture, levels of BMP-2 mRNA (A) and protein (B, C)
increased significantly. The data were means±SEM (n=3, aP<0.05
vs the control).
DISCUSSION
This study demonstrated that dopamine
stimulated BMP-2 expression in RPE cells in vitro and this may be associated
with the inhibitive effect of dopamine on ocular growth and myopia development.
Many studies have shown that dopamine
pathways are involved in myopia development[14]
and the inhibitory effects of dopamine on FDM and LIM are mediated through
stimulation of the dopamine D1-receptor and D2-receptor, especially the
D2-receptor[15]. Apomorphine blocking myopia may
be by a dopamine D2-receptor mechanism acting in retina or pigmented epithelium[5]. RPE plays a role in eye growth and refractive
development by relaying signals between the retina and adjacent choroidal and
scleral layers[16-17]. RPE can
modulate fluid exchange between the retina and choroid inducing the choroidal
thickness changes and scleral growth [17-19].
In vitro RPE cells directly influenced the scleral glycosi aminoglyca
good (GAG) synthesis[19]. Therefore, we proposed
that dopamine and its agonist influced the production and release of some
growth factors from RPE cells, which normally affected the growth of scleral
chondrocytes. In our study, dopamine increased the secretion of BMP-2 and the
expression levels of BMP-2 mRNA and protein in RPE cells.
BMP-2 belongs to the BMPs family and its
expression has been detected in both adult and embryonic tissues of cornea,
trabecular meshwork, optic nerve head, retina and conjunctiva[20]. BMP-2 is involved in the pathophysiology of several
ocular diseases and regulating myopia development[21].
During myopia development, choroidal BMP-2 gene expression involves in vascular
regulation and/or angiogenesis and its expression alternation may be part of a
common choroidal response[22]. In vitro,
BMP-2 influences scleral extracellular matrix synthesis[11].
In relative terms, BMP-2 appears more highly expressed in RPE layer compared to
retina and choroid[21], suggesting BMP-2 may be
produced by RPE cells. In this study, we demonstrated that the secreted BMP-2
increased with dopamine, so it may reach retina, choroid and sclera by
diffusion to affect their function.
In our study, we presented that dopamine
stimulated secretion of BMP-2 by RPE cells in vitro. This suggested that
dopamine blocking myopia development was possibly through modulation RPE
secretory activity of growth factors such as BMP-2 into retina, choroid and
sclera.
ACKNOWLEDGEMENTS
Conflicts of Interest: Li HH, None; Sun
YL, None; Cui DM, None; Wu J, None; Zeng JW, None.
REFERENCES
1 Popova E.
Role of dopamine in distal retina. J Comp
Physiol A Neuroethol Sens Neural Behav Physiol 2014;200(5):333-358. [CrossRef] [PubMed]
2 Mao J, Liu
S, Qin W, Li F, Wu X, Tan Q. Levodopa inhibits the development of
form-deprivation myopia in guinea pigs. Optom
Vis Sci 2010;87(1):53-60. [CrossRef] [PubMed]
3 Nickla DL,
Totonelly K. Dopamine antagonists and brief vision distinguish lens-induced-
and form-deprivation-induced myopia. Exp
Eye Res 2011;93(5):782-785. [CrossRef] [PMC free article]
[PubMed]
4
Feldkaemper M, Schaeffel F. An updated view on the role of dopamine in myopia. Exp Eye Res 2013;114:106-119. [CrossRef] [PubMed]
5 Yan T,
Xiong W, Huang F, Zheng F, Ying H, Chen JF, Qu J, Zhou X. Daily injection but
not continuous infusion of apomorphine inhibits form-deprivation myopia in
mice. Invest Ophthalmol Vis Sci
2015;56(4):2475-2485. [CrossRef]
[PubMed]
6 Lan W,
Yang Z, Feldkaemper M, Schaeffel F. Changes in dopamine and ZENK during
suppression of myopia in chicks by intense illuminance. Exp Eye Res 2016;145:118-124. [CrossRef] [PubMed]
7 Hung GK,
Mahadas K, Mohammad F. Eye growth and myopia development: unifying theory and
Matlab model. Comput Biol Med
2016;70:106-118. [CrossRef]
[PubMed]
8 McGlinn
AM, Baldwin DA, Tobias JW, Budak MT, Khurana TS, Stone RA. Form-deprivation
myopia in chick induces limited changes in retinal gene expression. Invest Ophthalmol Vis Sci
2007;48(8):3430-3436. [CrossRef]
[PMC free
article] [PubMed]
9 Sakuta H,
Takahashi H, Shintani T, Etani K, Aoshima A, Noda M. Role of bone morphogenic
protein 2 in retinal patterning and retinotectal projection. J Neurosci 2006;26(42):10868-10878. [CrossRef] [PubMed]
10 Wang Q,
Zhao G, Xing S, Zhang L, Yang X. Role of bone morphogenetic proteins in form-deprivation
myopia sclera. Mol Vis
2011;17:647-657. [PMC
free article] [PubMed]
11 Hu J, Cui
D, Yang X, Wang S, Hu S, Li C, Zeng J. Bone morphogenetic protein-2: a
potential regulator in scleral remodeling. Mol
Vis 2008;14: 2373-2380. [PMC free article]
[PubMed]
12 Yoshikawa
M, Yamashiro K, Miyake M, Oishi M, Akagi-Kurashige Y, Kumagai K, Nakata I,
Nakanishi H, Oishi A, Gotoh N, Yamada R, Matsuda F, Yoshimura N; Nagahama Study
Group. Comprehensive replication of the relationship between myopia-related
genes and refractive errors in a large Japanese cohort. Invest Ophthalmol Vis Sci 2014;55(11):7343-7354. [CrossRef] [PubMed]
13 Verhoeven
VJ, Hysi PG, Wojciechowski R, et al.
Genome-wide meta-analyses of multiancestry cohorts identify multiple new
susceptibility loci for refractive error and myopia. Nat Genet 2013;45(3):314-318. [CrossRef] [PMC free article]
[PubMed]
14 Iribarne
M, Canto-Soler MV, Torbidoni V, Suburo AM. Controlling retinal pigment epithelium
injury after experimental detachment of the retina. Invest Ophthalmol Vis Sci 2007;48(3):1348-1354. [CrossRef] [PubMed]
15 Nickla
DL, Totonelly K, Dhillon B. Dopaminergic agonists that result in ocular growth
inhibition also elicit transient increases in choroidal thickness in chicks. Exp Eye Res 2010;91(5):715-720. [CrossRef] [PMC free article]
[PubMed]
16 Zhang Y,
Wildsoet CF. RPE and choroid mechanisms underlying ocular growth and myopia. Prog Mol Biol Transl Sci
2015;134:221-240. [CrossRef]
[PMC free
article] [PubMed]
17 Wang S,
Liu S, Mao J, Wen D. Effect of retinoic acid on the tight junctions of the
retinal pigment epithelium-choroid complex of guinea pigs with lens-induced
myopia in vivo. Int J Mol Med
2014;33(4):825-832. [CrossRef]
[PMC free
article] [PubMed]
18 Zhang H,
Wong CL, Shan SW, Li KK, Cheng AK, Lee KL, Ge J, To CH, Do CW. Characterisation
of Cl(-) transporter and channels in experimentally induced myopic chick eyes. Clin Exp Optom 2011;94(6): 528-535. [CrossRef] [PubMed]
19 Sheng C,
Zhu X, Wallman J. In vitro effects of insulin and RPE on choroidal and scleral
components of eye growth in chicks. Exp
Eye Res 2013;116:439-448. [CrossRef] [PubMed]
20 Rymer J,
Wildsoet CF. The role of the retinal pigment epithelium in eye growth
regulation and myopia: a review. Vis
Neurosci 2005;22(3): 251-261. [CrossRef] [PubMed]
21 Zhang Y,
Liu Y, Wildsoet CF. Bidirectional, optical sign-dependent regulation of BMP2
gene expression in chick retinal pigment epithelium. Invest Ophthalmol Vis Sci 2012;53(10):6072-6080. [CrossRef] [PMC free article]
[PubMed]
22 He L,
Frost MR, Siegwart JT Jr, Norton TT. Gene expression signatures in tree shrew
choroid during lens-induced myopia and recovery. Exp Eye Res 2014;123:56-71. [CrossRef] [PMC free article]
[PubMed]
--------------------------------------------------------------------------------------------------------------------------------
All rights reserved by Press of International Journal of Ophthalmology (IJO
PRESS)