p38 MAPK Mediates the Expression of Type I Collagen
Induced by TGF-␤2 in Human Retinal Pigment
Epithelial Cells ARPE-19
Kenichi Kimoto,1,2 Kazuo Nakatsuka,1 Noritaka Matsuo,2 and Hidekatsu Yoshioka2
PURPOSE. Transforming growth factor (TGF)-␤ has been implicated as the key mediator of proliferative vitreoretinopathy,
but the cellular mechanisms by which TGF-␤ induces extracellular matrix protein (ECM) synthesis are not fully understood.
The current study was conducted to examine whether the
mitogen-activated protein kinase (MAPK) pathway is involvedin TGF-␤2–induced collagen expression in retinal pigment epithelial cells.
METHODS. Human retinal pigment epithelial cells ARPE-19 were
cultured and stimulated with various concentrations of TGF␤2. The type I collagen gene (COL1A1, COL1A2) expression
induced by TGF-␤2 was evaluated by real-time RT-PCR. Synthesis of type I collagen was evaluated by the concentration of the
C-terminal propeptide of type I (PICP) in the medium. The
activation of MAPK pathways by TGF-␤2 was assessed by immunoblot with anti-phospho-p38 and anti-phospho-extracellular signal-regulated kinase (ERK)1/2 antibodies. The role of
MAPK was assessed using biochemical inhibitors. To examine
the transcriptional activities of COL1A1 and COL1A2, luciferase reporter assays were also performed.
RESULTS. mRNA expression of COL1A1 and COL1A2 and type I
collagen synthesis were activated by TGF-␤2. Both ERK and
p38 MAPK pathways were also activated by TGF-␤2. The biochemical blockade of p38 MAPK activation, but not ERK activation, inhibited TGF-␤2–induced type I collagen mRNA expression and type I collagen synthesis. Moreover, blockade of
the p38 MAPK pathway inhibited the increase in both COL1A1
and COL1A2 promoter activities when induced by TGF-␤2.
CONCLUSIONS. TGF-␤2 activates p38 MAPK and p38 MAPK plays
a role in relaying the TGF-␤2 signal to type I collagen production in the retinal pigment epithelium. (Invest Ophthalmol Vis
Sci. 2004;45:2431–2437) DOI:10.1167/iovs.03-1276

Proliferative vitreoretinopathy (PVR) is a major cause for
failure of rhegmatogenous retinal detachment surgery.1,2 It
is characterized by cellular proliferation and membrane formation within the vitreous cavity and on the surface of theretina.3,4 When the blood–retinal barrier is breached, serum
components and inflammatory cells enter the vitreous cavity
and subretinal space, exposing the retinal pigment epithelial

From the Departments of 1Ophthalmology and 2Anatomy, Biology, and Medicine, Faculty of Medicine, Oita University, Oita, Japan.
Supported in part by Grants-in-Aid for Scientific Research
11470312 and 14370468 (HY) from the Ministry of Education, Culture,
Sports, Science and Technology of Japan.
Submitted for publication November 22, 2003; revised March 1,
2004; accepted March 9, 2004.
Disclosure: K. Kimoto, None; K. Nakatsuka, None; N. Matsuo,
None; H. Yoshioka, None
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Corresponding author: Kenichi Kimoto, Department of Ophthalmology, Faculty of Medicine, Oita University, Hasama-machi, Oita 8795593, Japan; kimotok@med.oita-u.ac.jp.
Investigative Ophthalmology & Visual Science, July 2004, Vol. 45, No. 7
Copyright © Association for Research in Vision and Ophthalmology(RPE) cells to a variety of cytokines. This exposure leads toactivation of the RPE cells and subsequent separation from theestablished monolayer. Activated RPE cells proliferate and migrate through retinal tears to form pathologic membranes onboth surfaces of the neural retina.3 These PVR membranes arecomposed of RPE cells, retinal glial cells, fibroblasts, and macrophages, as well as a host of accumulated extracellular matrix
proteins (ECM).5–7
Type I collagen, the major component of the ECM in PVR
membranes,8 is a heterotrimer composed of coordinately expressed two ␣1 chains and one ␣2 chain. They are encoded by
distinct genes, COL1A1 and COL1A2, respectively, and theirexpression is modulated by various cytokines.9
Transforming growth factor (TGF)-␤ is a member of a superfamily of multifunctional cytokines which function during
development, wound repair, and other pathologic processes. It
is a potent inducer of ECM protein synthesis and accumulationand has been implicated as the key mediator of various fibrogenous diseases, including PVR.10 Evidence for high concentrations of TGF-␤ in vitreous aspirates from patients diagnosed
with PVR has highlighted its importance in PVR.11 Of note,
immunologic assays showed that most TGF-␤ in the vitreous
aspirates was from the TGF-␤2 isoform, not TGF-␤1.11,12 However, to our knowledge, there are no reports describing theeffect of TGF-␤2 on the expression of type I collagen mRNA in
the RPE. Furthermore, despite many data demonstrating theeffects of TGF-␤ on ECM induction in glomerulonephritis, livercirrhosis, and lung fibrosis,13 the cellular mechanisms bywhich TGF-␤ exerts its effects have not been fully elucidated.
Recent evidence has indicated that TGF-␤ transduces signals
through two different pathways, Smad, and mitogen activatedprotein kinase (MAPK), through extracellular signal regulated
kinase (ERK) and p38 MAPK.14 –16
This investigation was undertaken to determine whether
TGF-␤2 activates specific MAPK signaling pathways in a cultured human retinal pigment epithelial cell line, ARPE-19, andwhether this MAPK activation mediates TGF-␤2–induced promoter activity, mRNA expression, and protein synthesis of type
I collagen.

MATERIALS AND METHODS
Reagents
Recombinant human TGF-␤2 was purchased from R&D Systems (Minneapolis, MN). SB203580 and PD98059 were purchased from Calbiochem (San Diego, CA). Antibodies were purchased from the following
vendors: anti-phospho-p38 MAPK (Thy180/Tyr182) and anti-phosphoSmad2/3 (Ser465/Ser467) rabbit polyclonal antibodies were from Cell
Signaling Technology (Beverly, MA). Anti-p38 MAPK, anti-phosphoERK1/2 (Tyr204), and anti-ERK1/2 rabbit polyclonal antibodies werefrom Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Smad2/3 rabbit
polyclonal antibody was from Upstate Biotechnology (Lake Placid,
NY).

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Kimoto et al.

Cell Culture and Stimulation by TGF-␤2
Cells of the human retinal pigment epithelial line ARPE-19 were obtained from the American Type Culture Collection (ATCC; Manassas,
VA). ARPE-19 is an immortalized, nontransfected cell line that spontaneously arose from cultures of human RPE.17 Cells were maintained in
1:1 (vol/vol) mixture of Dulbecco’s modified Eagle’s and Ham’s F12
medium (DMEM/F12; ATCC) supplemented with 10% heat-inactivatedfetal bovine serum (FBS; Sanko Junyaku, Tokyo, Japan) and antibiotics(100 U/mL penicillin G and 100 mg/mL streptomycin sulfate) in ahumidified incubator at 37°C in 5% CO2. Cells at passage numbers 13
to 18 were used in the present studies.
When cultures achieved confluence, the medium was removed andreplaced with serum-free DMEM/F12. After 24 hours of serum starvation, various concentrations of TGF-␤2 were added to the medium, andthe cultures were incubated for another 24 hours for RNA isolation or
48 hours for protein analysis. In the experiments using the MEK-1
inhibitor, PD98059, and the p38 MAPK inhibitor, SB203580, we preincubated cells for 30 minutes before treatment with or without
exogenous TGF-␤2.

RNA Isolation and cDNA Synthesis
Total RNA was prepared using a minikit (RNeasy; Qiagen, Valencia,
CA). Cells in lysis buffer containing 1% ␤-mercaptoethanol were passed
through a separation column (QIAShredder; Qiagen), and total RNA
was obtained according to the supplier’s protocol. cDNA was produced with a kit (Omniscript RT; Qiagen), using random hexamers. To
confirm TGF-␤2–treated ARPE-19 cells continued to express RPE65
mRNA, cDNA was amplified (HotStarTaq DNA Polymerase; Qiagen).
The specific primers used for testing the presence of RPE65 mRNA
(NM_000329) are as follows: 5⬘-TTCTGAGTGTGGTGGTGAGC-3⬘(sense) and 5⬘-GGCCTGTCTCACAGAGGAAG-3⬘ (antisense).
The thermal cycling conditions included 1 cycle at 95°C for 15
minutes, 30 cycles at 94°C for 30 seconds, 57°C for 30 seconds, and72°C for 30 seconds. The PCR product was confirmed by sequence
analysis using a sequencer (model 310; Applied Biosystems, Foster
City, CA).

Real-Time PCR
Real-time PCR was performed using a sequence-detection system(Prism TM 7700; Applied Biosystems). This system is based on theability of the 5⬘ nuclease activity of Taq polymerase to cleave adual-labeled fluorogenic hybridization probe during DNA chain extension. The probe is labeled with a reporter fluorescent dye, FAM, at the5⬘ end and a quencher fluorescent dye, TAMRA, at the 3⬘ end. During
the extension phase of PCR, the nucleolytic activity of the DNApolymerase cleaves the hybridization probe and releases the reporterdye from the probe with a concomitant increase in reporter fluorescence. The sequence-specific primers and probe mixtures for human
COL1A1, human COL1A2, and human GAPDH were from predeveloped assays (Assays-on-Demand; Applied Biosystems). The thermal
cycling conditions included 1 cycle at 50°C for 2 minutes, 1 cycle at
95°C for 10 minutes, 40 cycles at 95°C for 15 seconds, 60°C for 1
minute. The relative mRNA expression levels of COL1A1 and COL1A2
were normalized against that of the GAPDH gene from the same RNA
preparations, using a comparative threshold cycle method.18

Measurement of the C-Terminal Propeptide ofType I Collagen
Cells were placed in six-well dishes, incubated in medium containing
10% FBS and grown to confluence. The medium was changed toserum-free DMEM/F12 for 24 hours, followed by incubation in thepresence or absence of various concentrations of TGF-␤2, with orwithout PD98059 or SB203580 in the medium containing 200 ␮M of
L-ascorbic acid 2-phosphate (Wako Pure Chemical Industries, Osaka,
Japan).19 After 48 hours, 20 ␮L of the conditioned medium was used insubsequent assays.

IOVS, July 2004, Vol. 45, No. 7
The concentration of PICP in the medium was measured with a
PICP enzyme-linked immunosorbent assay (ELISA) kit, according to the
manufacturer’s method (TaKaRa Biochemicals Co., Osaka, Japan).

Preparation of Cell Lysates and Western
Blot Analysis
Cells grown to confluence were cultured for 24 hours with serum-free
medium and then treated with 10 ␮g/mL of TGF-␤2 for different
periods. The cells were washed twice with ice-cold phosphate-buffered saline (PBS), lysed on ice in RIPA buffer (50 mM Tris-HCl [pH 7.4],
150 mM NaCl, 1 mM EDTA, 1% NP-40, 0.5% Na-deoxycholate) containing protease and phosphatase inhibitors (1 mM phenylmethylsulfonyl
fluoride, 1 mM Na3VO4, 1 mM NaF, 1 ␮g/mL aprotinin, 1 ␮g/mL
leupeptin, 1 ␮g/mL pepstatin). Cell extracts were then centrifuged at
15,000g for 10 minutes at 4°C, supernatants were collected, andprotein content was determined using a bicinchoninic acid (BCA)
protein assay reagent kit (Pierce, Rockford, IL). Fresh cell extracts
were prepared in sample buffer (0.125 M Tris-HCl [pH 6.8]) 4% SDS,
20% glycerol, 0.002% bromophenol blue, and 10% 2-mercaptoethanol).
Samples were resolved by SDS-PAGE and electroblotted onto a polyvinylidene difluoride (PVDF) membrane. Equal loading and appropriate
transfer of each lane was confirmed by staining the PVDF membranewith the ponceau S solution (Sigma-Aldrich, St. Louis, MO). After they
were blocked with 5% skim milk or 5% BSA in Tris-buffered saline
containing 0.1% Tween-20 for 1 hours at room temperature, membranes were incubated with anti-ERK2 antibody (C-14), anti-phosphoERK antibody (E-4), anti-p38 MAPK antibody, anti-phosph-p38 MAPK
antibody, anti-Smad2/3 antibody, or anti-phospho-Smad2/3 antibody,for 1 hour at room temperature or overnight at 4°C, followed byincubation with the horseradish peroxidase (HRP)– conjugated antirabbit IgG for 1 hour at room temperature. The signals were enhanced
using a chemiluminescence system (ECL plus; Amersham Pharmacia
Biotech, Buckinghamshire, UK) and exposed to x-ray film.

Plasmid Constructs
For generating the COL1A2-luciferase (COL1A2/Luc) construct in the
pGL3 vector (Promega, Madison, WI), a 436-bp fragment containing
the sequences from ⫺378 to ⫹58 of the human COL1A2 promoter was
amplified by PCR from a COL1A2/Luc plasmid,20 –23 which was kindly
provided by Yutaka Inagaki (Tokai University, Kanagawa, Japan). The
primers, which are SacI site-linked sense and XhoI site-linked antisense
primers specific for the promoter, are as follows: 5⬘-GGAGCTCAGATCTGCAAATTCTGCC-3⬘ (sense); 5⬘-TCTCGAGGCATGCAGTCGTGGCCAG-3⬘ (antisense). The SacI and XhoI sites are italic. The PCR
product was cloned into a vector (pGEM-T Easy; Promega), followedby digestion with SacI and XhoI and subcloned into the SacI/XhoI sitesof the pGL3-Basic vector.
Similarly, for generating the COL1A1/Luc, a 560 bp-fragment containing the TGF-␤ response elements of the human COL1A1 gene24
from ⫺341 to ⫹119 was amplified by PCR from genomic DNA using
KpnI site-linked sense and XhoI site-linked antisense. The primers are
as follows: 5⬘-CGGTACCCAGTTCCACTTCTTCTAG-3⬘ (sense), 5⬘-TCTCGAGGTCTAGACCCTAGACATG-3⬘ (antisense). The KpnI and XhoI
sites are italic. The PCR product was finally cloned into the pGL3 basic
vector. The creation of the mutated Smad binding site (Smad-mut/Luc)
was generated by site-directed mutagenesis using the COL1A2/Luc
plasmid as a template. The primers used in the PCR amplification wereas follows: 5⬘-GGAATTCACGAGTCAGAGTTTCCCC (sense), 5⬘-GGAATTCCATACCTCCGCCCTCCGC (antisense).
The EcoRI site for the mutated Smad binding site construct is italic.
The PCR product was digested with EcoRI, followed by self-ligation.
This plasmid was digested with Sac I and HindIII and recloned into the
Sac I/HindIII sites of the pGL3-basic vector. All constructs were confirmed by sequence analysis using the sequencer (model 310; Applied
Biosystems).

IOVS, July 2004, Vol. 45, No. 7

Involvement of MAPK in Collagen Expression by TGF-␤2

2433

collagen genes, but, when treated with exogenous TGF-␤2,
type I collagen mRNA expression increased dramatically in adose-dependent fashion (Fig. 2A). To examine whether theincrease of collagen mRNA expression was followed by anincrease in collagen protein synthesis, we measured the concentration of PICP in the medium. Similarly, exogenous
TGF-␤2 increased type I collagen protein levels in the culture
media of ARPE-19 in a dose-dependent fashion (Fig. 2B).

Activation of p38 MAPK and ERK 1/2 by TGF-␤2
in ARPE-19 Cells
Some of the biological effects of TGF-␤1 are mediated by
activation of the MAPK signaling pathway in certain cell
types.25–27 We first examined the levels of p38 MAPK and
ERK1/2 proteins expression in ARPE-19 cells treated with exogenous TGF␤2 (10 ng/mL) by Western analyses, using antip38 MAPK, anti-phospho-p38 MAPK, anti-ERK1/2, and antiphospho-ERK1/2 antibodies. The anti-phospho-p38 MAPK and

FIGURE 1. (A) Phase-contrast photomicrographs of confluent culturesof ARPE-19 cells before and after treatment of TGF-␤2 (10 ng/mL) for
24 hours. (B) RT-PCR analysis of RPE65 mRNA expression in ARPE-19
cells before and after treatment of TGF-␤2 (10 ng/mL) for 24 hours.
Lane M: 100-bp ladder; lane 1: before treatment of TGF-␤2; and lane
2: after treatment of TGF-␤2. Magnification, ⫻100. Bar, 50 ␮m.

Transient Transfection and Luciferase Assays
Transcriptional activities of the COL1A1 and COL1A2 gene were determined by transient transfections into ARPE-19 cells. A kit (EndoFree
Plasmid Maxi Kit; Qiagen) was used to purify the plasmids. For transient expression reporter assays, ARPE-19 cells were then transfected(Effectene Transfection Reagent; Qiagen). Briefly, cells were seeded
the day before transfection in six-well plates. Cells at 60% to 80%
confluence were transfected according to the manufacturer’s recommendations. Six hours later, cells were stimulated with or without
TGF-␤2 (10 ng/mL) in the presence or absence of PD98059 or
SB203580 for an additional 24 hours. Cells were lysed and assayedusing the dual-luciferase reporter assay system (Promega). Luciferase
activities were analyzed using a luminometer (Lumat LB.9507; EG and
G, Berthold, Germany). A construct containing the renilla luciferase(pRL-TK; Promega) was cotransfected together with COL1A1/Luc or
COL1A2/Luc or Smad-mut/Luc and used as a control for transfection
efficiency. The values were expressed relative to the activities in
nontreated cells.

Statistical Analysis
The values represented the mean ⫾ SD of multiple independent tests
and Student’s t-test was used to evaluate the statistical differences
between groups, and a P ⬍ 0.01 was considered to be significant.

RESULTS
Stimulation of Type I Collagen mRNA Expression
and Collagen Synthesis in ARPE-19 Cells
by TGF-␤2
ARPE-19 cells retained the characteristic features of native RPE.
The cells exhibited defined cell borders, a cobblestone appearance, and expressed RPE 65 mRNA before and after TGF-␤2
treatment (Fig. 1). The cells expressed a basal level of type I

FIGURE 2. (A) Analysis of type I collagen mRNA expression using
real-time RT-PCR. ARPE-19 cells were treated with various amounts of
TGF-␤2 for 24 hours. The relative levels of mRNA were normalizedagainst that of GAPDH from the same cDNA preparation. Data represent the mean ⫾ SD of results in six independent experiments. (B) The
concentration of PICP in the culture media was measured by ELISA.
Quiescent cells were treated with various amounts of TGF-␤2 for 48
hours. Data represent the mean ⫾ SD of results in four independent
experiments. *P ⬍ 0.01, compared with no stimulation with TGF-␤2.

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IOVS, July 2004, Vol. 45, No. 7
The effect of SB203580 or PD98059 on TGF-␤2 induction of
the COL1A2/Luc promoter was investigated in ARPE-19 (Fig.
6). Treatment with SB203580 significantly reduced TGF-␤2
induction. Similar to the COL1A2/Luc promoter, SB203580
also reduced TGF-␤2 induction of the COL1A1/Luc promoter
activity. PD98059, however, failed to reduce TGF-␤2 inductionof both promoters. The inhibitor treatment had very little
effect on the basal promoter activities of COL1A1/Luc and
COL1A2/Luc.

Effects of SB203580 or PD98059 on the Activity of
the Smad-mut/Luc Promoter and on the
Phosphorylation of Smad2/3 Induced by TGF-␤2

FIGURE 3. Activation of MAPK by TGF-␤2 in ARPE-19 cells. Lysatesfrom cells incubated in the presence of exogenous TGF-␤2 (10 ng/mL)
for the indicated times were subjected to Western blot analysis using
various antibodies. (A) Bands corresponding to the phosphorylated
form of p38 MAPK (top) and total p38 MAPK (bottom) were detected.
(B) Bands corresponding to the phosphorylated form of ERK1/2 (top)and total ERK1/2 (bottom) were detected. In each lane, two bands
corresponded to ERK1 (top) and ERK2 (bottom).

anti-phospho-ERK1/2 antibodies detected specifically the phosphorylated forms of p38 MAPK and ERK1/2, respectively,
whereas, the anti-p38 MAPK and anti-ERK1/2 antibodies detected total p38 MAPK or ERK1/2 proteins, respectively (Fig.
3). Exogenous TGF-␤2 increased the phosphorylation of p38
MAPK within 30 minutes after stimulation, and its activation
persisted for 24 hours (Fig. 3A). In contrast, ERK1/2 phosphorylation increased as early as 5 minutes after stimulation with
TGF-␤2 and persisted for as long as 60 minutes (Fig. 3B).
Preincubation of ARPE-19 cells with the p38 inhibitor
SB203580 or ERK1/2 route inhibitor PD98059 blocked TGF␤2–induced p38 or ERK1/2 activation at 10 ␮M concentration,
respectively (Fig. 4).

TGF-␤ signals from the cell surface to the nucleus are transduced by Smad2, -3, and -4, and phosphorylation of Smad-2/3 is
crucial for this downstream signaling cascade.28,29 Studies
have shown that Smad proteins mediate TGF-␤ signaling andstimulate COL1A2 mRNA expression.30 –33 Therefore, we evaluated the role of Smads on regulating the COL1A2/Luc promoter when induced by TGF-␤2 using the mutated Smad binding site construct (Smad-mut/Luc) in ARPE-19 cells. As
predicted, TGF-␤2 induction of Smad-mut/Luc promoter activity was significantly diminished, compared with the activity of
COL1A2/Luc. Moreover, the treatment with SB203580 reduced
TGF-␤2 induction of Smad-mut/Luc promoter activity, but
treatment with PD98059 had no effect (Fig. 7A). Furthermore,
we examined the involvement of two inhibitors in regulating
the Smad cascade. Cell extracts from ARPE-19 cells, pretreatedwith either SB203580 or PD98059 for 30 minutes before administration of TGF-␤2 (10 ng/mL), were analyzed by Western
blot analysis for phosphorylated Smad2/3 (top) and total
Smad2/3 (bottom) using an anti-phospho-Smad2/3 antibody
and an anti-Smad2/3 antibody, respectively (Fig. 7B). The ⬃58kDa Smad2 and ⬃50-kDa Smad3 proteins were present in both

Effects of SB203580 or PD98059 on the Type I
Collagen mRNA Expression and Collagen
Synthesis by TGF-␤2
Given that TGF-␤2 induced increases in both type I collagengenes and MAPK phosphorylation, we posed the question ofwhether the MAPK pathway is involved in mediating TGF-␤2–
induced COL1A1 and COL1A2 gene expression. Preincubationof ARPE-19 with SB203580, a specific inhibitor of p38 MAPK,
efficiently prevented approximately two thirds of the TGF-␤2–
induced COL1A1 and COL1A2 mRNA expression (Fig. 5A). In
addition, SB203580 inhibited approximately one half of the
TGF-␤2–induced collagen synthesis (Fig. 5B). In contrast,
PD98059, a specific inhibitor of MEK1, which prevents theactivation of ERK1/2 pathway, failed to inhibit TGF-␤2–induced type I collagen mRNA expression and protein synthesis(Fig. 5). Both inhibitor treatments had very little effect on thebasal levels of type I collagen mRNA expression and protein
synthesis.

Effects of SB203580 or PD98059 on the Activitiesof the COL1A1/Luc and COL1A2/Luc Promoter
Induced by TGF-␤2
The COL1A2 promoter is a widely used experimental model
system to study transcriptional regulation of the type I collagengene at the basal level and in response to exogenous stimuli.

FIGURE 4. Effects of MAPK inhibitors on TGF-␤2–induced phosphorylation. (A) SB203580 inhibited TGF-␤2–induced p38 activation. Cell
lysates were harvested after incubation with or without TGF-␤2 for 60
minutes in the presence or absence of SB203580. (B) PD98059 inhibited TGF-␤2–induced ERK1/2 activation. Cell lysates were harvested
after incubation with or without TGF␤2 for 30 minutes in the presenceor absence of PD98059.

IOVS, July 2004, Vol. 45, No. 7

Involvement of MAPK in Collagen Expression by TGF-␤2

FIGURE 5. Effects of MAPK inhibitors on TGF-␤2–induced COL1A1
and COL1A2 mRNA expression (A) and type I collagen synthesis (B) in
ARPE19 cells. (A) Analysis of COL1A1 and COL1A2 mRNA expression
using real-time RT-PCR. Quiescent cells were pretreated with or without SB203580 (10 ␮M) or PD98059 (10 ␮M) and incubated in thepresence or absence of TGF-␤2 (10 ng/mL) for 24 hours. The relative
levels of mRNA were normalized against that of GAPDH from the same
cDNA preparation. Data are the mean ⫾ SD of results in six independent experiments. (B) Concentration of PICP in the culture media wasmeasured by ELISA. Cells were treated the same as in (A) for 48 hours.
Data represent the mean ⫾ SD of results in four independent experiments. *P ⬍ 0.01, compared with stimulation with TGF-␤2 and withoutboth inhibitors.

2435

within 5 to 30 minutes. The activation of p38 MAPK continued
for at least 24 hours. To examine the relationship of MAPK
phosphorylation and collagen synthesis, we used chemical
inhibitors of the MAPK pathway on type I collagen expression.
Blockade of the ERK 1/2 pathway by PD98059, a specificinhibitor of MEK1 that prevents downstream activation of ERK
1/2, did not prevent induction of COL1A1 and COL1A2 mRNAor type I collagen synthesis by TGF-␤2 in the ARPE-19 cells. In
contrast, the presence of SB203580, a specific inhibitor of the
p38 MAPK pathway, significantly reduced promoter activity,
mRNA, and protein expression after induction by exogenous
TGF-␤2. These data show that p38 MAPK could be involved in
TGF-␤2–induced collagen synthesis in ARPE-19 cells.
Smads are a family of proteins that operate downstream ofvarious members of the TGF-␤ superfamily.34 Smad2 and -3 aredownstream effectors of the TGF-␤ signaling pathway. On
ligand binding, they are phosphorylated by the TGF-␤ type I
receptor kinase and translocated to the nucleus in a complex
with Smad4.35 This complex may either bind directly to thepromoters of its target genes, or associate with other transcription factors to induce gene transcription.36 A conserved
Smad3/4 DNA binding site, GTCTAGAC,37 is present within
the COL1A2 promoter.22 Indeed, Smad-mut/Luc promoter activity was significantly diminished when induced by TGF-␤2.
Therefore, Smads such as p38 MAPK contribute to collagengene expression in the presence of TGF-␤2 in ARPE-19 cells.
Over the last few years, cross-talk between the MAPK and
Smad signaling pathways in response to TGF-␤ stimulation
have been suggested.15,38 – 42 Therefore, we chose to examine
the effect of SB203580 and PD98059 on the activity of theSmad-mut/Luc promoter and on the phosphorylation of
Smad2/3 induced by TGF-␤2. Treatment with SB203580 reduced TGF-␤2 induction of the Smad-mut/Luc promoter activity; however, SB203580 and PD98059 had no significant effecton the TGF-␤2–induced phosphorylation of Smad2/3 in
ARPE-19 cells. These data suggest that p38 MAPK mediates theexpression of type I collagen induced by TGF-␤2 in ARPE-19,
which is independent of the Smad pathway. Similar data havebeen observed in dermal fibroblasts,16,43 hepatic stellate cells44
and mesangial cells,45 all of which play important roles infibrogenic diseases.
In ARPE-19 cells, blockage of the ERK1/2 pathway did not
significantly prevent induction of collagen gene expression bycontrol and TGF-␤2–treated cells, and an increase in phosphorylated Smad2/3 was noticed after TGF-␤2 treatment. Thus,
Smad2/3 phosphorylation in ARPE-19 cells is increased after
TGF-␤2 treatment, indicating that these cells can also respondto the ligand through a Smad-dependent pathway. However,SB203580 and PD98059 had no effect on TGF-␤2–induced
phosphorylation levels of Smad2/3 in ARPE-19 cells.

DISCUSSION
In this study, we examined the role of MAPK signaling cascades
in mediating the effects of TGF-␤2 on collagen synthesis in thehuman retinal pigment epithelial cell line, ARPE-19. Exogenous
TGF-␤2 increased the expression of collagen type I at thetranscriptional level, leading to an increase in the synthesis of
the protein. Treatment of ARPE-19 cells with exogenous
TGF-␤2 resulted in p38 MAPK and ERK 1/2 phosphorylation

FIGURE 6. Effects of MAPK inhibitors on TGF-␤2–induced COL1A1
and COL1A2 promoter activity in ARPE19 cells. Transfected cells werepretreated with or without SB203580 (10 ␮M) or PD98059 (10 ␮M)
and incubated in the presence or absence of TGF-␤2 (10 ng/mL) for 24hours. Results are expressed as multiples of increase compared withuntreated control cells. Data represent the mean ⫾ SD of results fiveindependent experiments. *P ⬍ 0.01, compared with the condition of
stimulation with TGF-␤2 and without both inhibitors.

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IOVS, July 2004, Vol. 45, No. 7
MAPK signaling pathways play important roles in various processes of ocular disease. In our study, we have demonstrated
that p38 MAPK signaling pathway mediates TGF-␤2 induced
type I collagen synthesis in RPE cells. The MAPK pathway may
be a new therapeutic target for treating many ocular diseases,
especially PVR.

Acknowledgments
The authors thank Stephen Gee (University of North Carolina at ChapelHill), an IOVS volunteer editor, for editing the manuscript.

References

FIGURE 7. Effects of MAPK inhibitors on the TGF-␤2–induced Smadmut/Luc promoter activity (A) and phosphorylation of Smad2/3 (B) in
ARPE-19 cells. (A) Transfected cells were pretreated with or without
SB203580 (10 ␮M) or PD98059 (10 ␮M) and incubated in the presenceor absence of TGF-␤2 (10 ng/mL) for 24 hours. Results are expressed
as multiples of increase compared with the untreated control. Values
represent the mean ⫾ SD of five independent experiments. *P ⬍ 0.01,
compared with stimulation with TGF-␤2 and without both inhibitors in
the Smad-mut/Luc. (B) Quiescent cells were pretreated with or without SB203580 (10 ␮M) or PD98059 (10 ␮M) and incubated in thepresence or absence of TGF-␤2 (10 ng/mL) for 30 minutes. Bands
corresponding to the phosphorylated form of Smad2/3 (top) and total
Smad2/3 (bottom) were detected.

TGF-␤2. In mesangial cells,46 ERK1/2 activity is essential forcollagen synthesis on induction by TGF-␤1, but in dermal
fibroblasts,47 activation of ERK1/2 inhibits type I collagen expression. It seems that MAPK activity could vary, depending on
different cell types, culture conditions, and ambient levels ofother cytokines.23
With respect to the role of MAPK in retinal diseases, such as
PVR, ERK1/2 are phosphorylated in RPE cells within 15 minutes and remain phosphorylated for several days after retinal
detachment (RD).48 Thus, ERK1 and -2 play key roles in thecontrol of RPE cell proliferation and migration.49,50 In pathologic conditions, RPE cells are exposed to a variety of stimuli,
including TGF-␤, that may induce cell death, in which JNK1
and p38 MAPK induce RPE cell death.51 In addition, the inflammatory process, due to breakdown of the blood–retinal
barrier, causes activation of leukocytes and their subsequent
infiltration into the choroid, retina, and vitreous. Bian et al.52
demonstrated that RPE cells secrete IL-8 and MCP-1 when incoculture with monocytes through activation of p38 MAPK
and ERK1/2 signaling pathways. These studies suggest that

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