Biosci. Rep. (2008) / 28 / 307–317 (Printed in Great Britain)/ doi 10.1042/BSR20080083
PKC isoenzymes differentially modulate the effect
of thrombin on MAPK-dependent RPE proliferation
Jose P. PALMA-NICOLAS, Edith LÓPEZ and Ana Marı́a LÓPEZ-COLOMÉ1
Departamento de Neurociencias, Instituto de Fisiologı́a Celular, Universidad Nacional Autónoma de México (UNAM),
Apartado Postal 70-253, Ciudad Universitaria, C.P. 04510, México, D.F, Mexico
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Synopsis
Thrombin signalling through PAR (protease-activated receptor)-1 is involved in cellular processes, such as proliferation,
differentiation and cell survival. Following traumatic injury to the eye, thrombin signalling may participate in disorders,
such as PVR (proliferative vitreoretinopathy), a human eye disease characterized by the uncontrolled proliferation,
transdifferentiation and migration of otherwise quiescent RPE (retinal pigment epithelium) cells. PARs activate the
Ras/Raf/MEK/ERK MAPK pathway (where ERK is extracellular-signal-regulated kinase, MAPK is mitogen-activated
protein kinase and MEK is MAPK/ERK kinase) through the activation of Gα and Gβγ heterotrimeric G-proteins, and
the downstream stimulation of the PLC (phospholipase C)-β/PKC (protein kinase C) and PI3K (phosphoinositide 3kinase) signalling axis. In the present study, we examined the molecular signalling involved in thrombin-induced RPE
cell proliferation, using rat RPE cells in culture as a model system for PVR pathogenesis.
Our results showed that thrombin activation of PAR-1 induces RPE cell proliferation through Ras-independent
activation of the Raf/MEK/ERK1/2 MAPK signalling cascade. Pharmacological analysis revealed that the activation
of ‘conventional’ PKC isoforms is essential for proliferation, although thrombin-induced phosphorylation of ERK1/2
requires the activation of atypical PKCζ by PI3K. Consistently, thrombin-induced ERK1/2 activation and RPE cell
proliferation were prevented completely by PI3K or PKCζ inhibition. These results suggest that thrombin induces RPE
cell proliferation by joint activation of PLC-dependent and atypical PKC isoforms and the Ras-independent downstream
stimulation of the Raf/MEK/ERK1/2 MAPK cascade. The present study is the first report demonstrating directly
thrombin-induced ERK phosphorylation in the RPE, and the involvement of atypical PKCζ in this process.
Key words: extracellular-signal-regulated kinase (ERK), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC),
proliferative vitreoretinopathy (PVR), retinal pigment epithelium-65 (RPE-65), thrombin
&
INTRODUCTION
The serine protease thrombin has been shown to affect a wide
variety of processes within diverse cell types, including RPE (retinal pigment epithelium) cells [1,2]. Intracellular thrombin signalling is triggered by activation of the PARs (protease-activated
receptors), a family of GPCRs (G-protein-coupled receptors) activated by proteolytic cleavage of the extracellular domain, which
unmasks a new sequence that functions as an intramolecular
ligand [3]. Four members of this family have been identified:
PAR-1, PAR-3 and PAR-4, which are activated by thrombin,
and the closely related PAR-2, which is sensitive to cleavage
by trypsin [4]. PAR-1 is the prototype of this receptor family,
and its cleavage at the Arg41 –Ser42 bond by thrombin exposes
a new N-terminus (S42 FLLRN47 ) that acts as a tethered ligand
%
[4]. Synthetic ligands corresponding to the cleaved N-terminus
can displace the tethered ligand from the binding site and fully
activate PAR-1 in an intermolecular mode [5].
PAR-1 expression at the cell surface is highly regulated by
ligand cleavage. In resting cells, an intracellular pool of functional receptors maintains surface expression through a largely
undefined recycling mechanism. Upon thrombin stimulation, the
cleaved receptors are targeted to the lysosome for degradation,
and new functional receptors from the intracellular pool restore
PAR membrane expression [6] and sensitivity to thrombin. Although in endothelial cells and fibroblasts receptor replenishment
does not require new protein synthesis [7,8], in megakaryoblasts
this process depends on de novo protein synthesis [9].
PARs have been linked to the activation of an extraordinarily
diverse array of physiologic responses by interacting with several
GPCR Gα subunits, in particular, Gq11α , G12/13α and Gαi , which
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Abbreviations used: DAG, diacylglycerol; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK, extracellular-signal-regulated kinase; FBS, fetal bovine serum;
GEF, guanine-nucleotide-exchange factor; GPCR, G-protein-coupled receptor; IP2 , inositol 1,4-bisphosphate; IP3 , inositol 1,4,5-trisphosphate; KRB, Krebs–Ringer bicarbonate; MAPK,
mitogen-activated protein kinase; MEK, MAPK/ERK kinase; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; PAR, protease-activated
receptor; PI, phosphoinositide; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; cPKC, classic PKC; nPKC, novel PKC; PLC, phospholipase C; PTX, pertussis toxin; PVR,
proliferative vitreoretinopathy; RPE, retinal pigment epithelium; SOS, Son of sevenless.
1 To whom correspondence should be addressed (email acolome@ifc.unam.mx).
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J. P. Palma-Nicolas, E. López and A. M. López-Colomé
accounts for the pleiotropic action of its ligands [3,10]. Activation of PARs by thrombin in platelets and CCL-39 cells showed
receptor coupling to PI (phosphoinositide) hydrolysis and to the
inhibition of adenylate cyclase via at least two distinct effectors, most likely Gq -like and Gi -like G-proteins [11]. Consistent
with previous studies, thrombin-induced PI hydrolysis and cell
growth via PTX (pertussis toxin)-insensitive and -sensitive Gproteins has been observed in human airway smooth muscle [12].
PAR-1 coupling to G12/13 activation has also been demonstrated
[13], although the downstream modulation of G12/13 effectors,
such as Rho, GEFs (guanine-nucleotide-exchange factors) and
PLC (phospholipase C)-β, by PAR-1 stimulation remains to be
established [14,15].
PAR-1 signalling activates the MAPK (mitogen-activated protein kinase) cascade in several cell types [16,17]. MAPKs play
a pivotal role in a variety of cellular functions [18,19], including the induction of cell proliferation, chemokine expression and
epithelial–mesenchymal transformation [20–22].
Three major mammalian MAPK subfamilies have been described: ERK (extracellular-signal-regulated kinase) 1 and ERK2,
JNKs (c-Jun N-terminal kinases) and the p38 kinases. Among
these, p42/p44 MAPK (ERK1/2) activation is typically associated with cell survival, proliferation and differentiation, given
their activation by mitogens and cell-survival factors [18,23].
The activation of ERK1/2 triggers their translocation from the
cytoplasm to the nucleus, which appears to be an important regulatory step for mitogen-induced gene expression and cell cycle
re-entry to promote cell proliferation [24].
The best-characterized MAPK linear pathway includes the
small GTPase Ras and the kinases Raf, MEK (MAPK/ERK
kinase) and ERK. Receptor and non-receptor tyrosine kinases,
as well as GPCRs, lacking intrinsic tyrosine kinase activity, have
been shown to activate Ras, the first step in this signalling cascade.
Upon Ras-mediated activation of Raf, which, in turn, activates the
dual-function kinase MEK, ERK1/2 phosphorylation on Tyr204
and Thr202 by MEK leads to the activation of nuclear and cytoplasmic ERK substrates [25]. This signalling cascade has been
shown to play an important role in the control of cell proliferation
by ERK-induced activation of the transcription factors NF-κB
(nuclear factor κB), c-Myc, CREB (cAMP-response-elementbinding protein) and AP-1 (activator protein-1) [26], as well as
by promoting the expression of regulatory proteins involved in
the cell cycle [27,28]. Thus MAPKs are important integrators
of GPCR- and tyrosine-kinase-receptor-mediated signals for cell
proliferation [24].
In the RPE, thrombin stimulates PLC activity [1] and IL-8
(interleukin-8) gene expression [29], and also induces cell proliferation in vitro [30], suggesting a role for thrombin in the
pathogenesis of proliferative disorders, such as PVR (proliferative vitreoretinopathy), a human retinal disease that involves the
proliferation, de-differentiation and migration of RPE cells into
the vitreous [31]. The direct activation of the MAPK signalling
pathway by thrombin in the RPE has not been demonstrated,
although the induction of VEGF (vascular endothelial growth
factor) expression in these cells by thrombin has been shown
to depend on ERK1/2 activation [32], indirectly suggesting a
role for MAPK in PAR-1 downstream signalling. In vitro experiments have shown that serum stimulates RPE cell proliferation
by activating the Ras/Raf/MEK/ERK signalling pathway [33].
Furthermore, chick [34], rat [35] and human [34] RPE cells in
culture are also induced to proliferate by glutamate, through the
activation of the MEK/ERK1/2 MAPK pathway.
GPCRs, such as PARs, activate the MAPK pathway through
multiple signalling pathways [36]. PKC (protein kinase C) and
Ca2+ /calmodulin-dependent pathways have been shown to regulate the MAPK cascade by the activation of the small GTPase
Ras, the first step in this signalling mechanism [37]. Additionally, it has been suggested that the sustained activation of ERK
required for the induction of gene expression by MAPK pathway
activity is MEK-independent [38,39] and might depend on PKC
activation [40].
In the present study, we analysed the kinetics of thrombininduced activation of the Raf/MEK/ERK1/2 MAPK cascade in rat
RPE cells in culture, its contribution to RPE cell proliferation and
its transactivation by PKC isoenzymes. Our results show, for the
first time, the requirement for the joint activation of two distinct
intracellular signalling pathways for thrombin stimulation of RPE
cell proliferation.
MATERIALS AND METHODS
Reagents
All reagents used were of cell-culture grade. The PAR-1 peptide (Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-ProPhe), thrombin, hirudine, Ro-32-0432 and the PKCζ pseudosubstrate (myristic acid–Ser-Ile-Tyr-Arg-Arg-Gly-Ala-Arg-Arg-TrpArg-Lys-Leu) were obtained from Calbiochem. Staurosporine,
wortmannin and PD98058 were from Sigma, and AG-1478,
U-73122 and U0126 were purchased from Tocris Bioscience.
PAR-3 (Ser-Phe-Asn-Gly-Gly-Pro) and PAR-4 (Gly-Tyr-ProGly-Lys-Phe) peptide agonists were obtained from Bachem.
Serum-free Opti-MEM® (Invitrogen) was used as the standard
medium for all assays.
Long-Evans rat RPE cell culture
RPE cells were isolated as described previously [35]. Briefly, 8–
10-day old Long-Evans rats were anaesthetized by inhaled chloroform and killed following the animal care and use guidelines
established by our institution. The eyes were enucleated, rinsed
in Dulbecco’s modified Eagle’s medium (Gibco BRL) containing
100 i.u./ml penicillin and 100 μg/ml streptomycin, and incubated
for 30 min at 37◦ C in the presence of 2% (v/v) dispase. After
removal of the sclera and the choroid, the RPE was detached
from the neural retina in calcium- and magnesium-free HBSS
(Hanks balanced salt solution), and incubated in the presence of
0.1% trypsin for 5 min at 37◦ C. Trypsin digestion was stopped
by a 1:1 dilution with Opti-MEM® . The dissociated cells were
suspended in Opti-MEM® containing 4% (v/v) FBS (fetal bovine
serum), and seeded at a density of 50 000 cells/cm2 in 96-well or
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6-well culture plates for cell proliferation assays and Western blot
analyses respectively. The purity of the culture was established by
expression of the RPE marker RPE-65, and cell viability (>90%)
was assessed by the Trypan Blue exclusion method.
RPE cell proliferation assay
RPE cells cultured for 24 h at 37◦ C in 4% (v/v) FBS-supplemented Opti-MEM® were serum-deprived for 24 h prior to stimulation with thrombin (0–4 units/ml) in serum-free Opti-MEM®
for a further 24 h. Cell proliferation was quantified in nonconfluent cultures using the colorimetric MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium] reduction method (Cell Titer 96® Aqueous One
Solution Reagent; Promega) according to the manufacturer’s instructions. Cultures maintained in serum-free Opti-MEM® were
used as negative controls and the specificity of thrombin effects
was assessed, in all cases, using the thrombin inhibitor hirudine
(4 units/ml). When tested, enzyme inhibitors [AG-1478, U0126,
U-73122, staurosporine, Ro-32-0432 (various concentrations, see
the Figure legends for details), 20 μM manumycine (Calbiochem) and 50 nM Raf-1 inhibitor (Calbiochem)] were added 1 h
prior to thrombin stimulation. The absorbance measured at 490
and 630 nm was corrected for background, and the absorbance
from unstimulated cultures was arbitrarily set as 100% (basal)
proliferation. When the effect of inhibitors was tested, proliferation in thrombin-stimulated cultures was set as 100% (control).
Western blot analysis
RPE cells from confluent 6-well plates were serum-deprived for
24 h and washed three times with RPE KRB (Krebs–Ringer bicarbonate) buffer (118 mM NaCl, 5 mM KCl, 1.2 mM MgSO4 ,
2.5 mM CaCl2 , 1 mM NaHPO4 , 5.6 mM glucose and 25 mM
NaHCO3 ). Cultures were then incubated at 37◦ C in thrombinsupplemented (2 units/ml) serum-free Opti-MEM® for 0–72 h to
determine the time course of ERK1/2 phosphorylation. When
tested, inhibitors were added to the culture 1 h prior to thrombin
stimulation (2 units/ml). PAR-1, PAR-3 and PAR-4 agonists were
used at a final concentration of 25 μM in serum-free Opti-MEM® .
At the indicated time points, cells were washed twice with KRB
buffer, and disrupted in lysis buffer containing 50 mM Tris/HCl
(pH 7.4), 150 mM NaCl, 10 mM EDTA, 0.1% SDS, 1% Triton
X-100, 1% CHAPS, 0.5% NP-40 (Nonidet P40), 0.1% BSA,
40 mM β-glycerophosphate, 10 mM sodium pyrophosphate and
a protease inhibitor cocktail (10%; Sigma). Proteins in total-cell
lysates (25 μg) were resolved by SDS/PAGE (12% gels) and
transferred on to PVDF membranes.
After blocking for 30 min at room temperature (25◦ C) with 5%
(w/v) non-fat dried skimmed milk powder in 20 mM Tris/HCl
(pH 7.5) containing 500 mM NaCl and 0.1% Tween 20, the PVDF
membranes were probed at 4◦ C with the following primary antibodies: mouse anti-[MAPK pERK1/2 (pThr202 /Tyr204 )] antibody (where pERK is phospho-ERK and pThr202 /Tyr204 are
phospho-Thr202 and phospho-Tyr204 ) (1:1000 dilution; Calbiochem), rabbit anti-ERK1/2 antibody (1:10000 dilution; Calbiochem) and mouse anti-β-actin antibody (1:5000 dilution;
Chemicon). HRP (horseradish peroxidase)-conjugated second-
ary antibodies (Invitrogen), raised in the corresponding species, were used at the same dilution as the corresponding
primary antibodies and developed using the Immobilon Western
AP Chemiluminescent Substrate (Millipore). Kodak® film images were digitized using an Alpha Digi-Doc system (Alpha
Innotech), and densitometric analysis was performed using the
Quantity One Software V4.6 from Bio-Rad. Basal ERK1/2 activation from unstimulated cells was arbitrarily set as 100% for the
time course assays. When inhibitors were tested, ERK1/2 activation in thrombin-stimulated cultures was set as 100% (control).
Measurement of [3 H]inositol phosphate
accumulation
PLC-β activation by thrombin was quantified by [3 H]inositol
phosphate production as described previously [41]. RPE cell cultures were incubated at 37◦ C in the presence of 2 μCi of myo-[23
H(N)]inositol (PerkinElmer) per well for 16–24 h. Cells were
then washed three times with 1 ml of pre-warmed (37◦ C) KRB
buffer containing 10 mM LiCl. RPE cells were stimulated with
2 units/ml thrombin for 10 min, and total-cell lysates were obtained by adding 1 ml of chloroform/methanol [1:2 (v/v)] to each
well. The aqueous phase was extracted and 3 H-labelled inositol phosphates were eluted individually, or in batches, using a
Dowex AG1 (X8, 100–200 mesh) column. Radioactivity in the
samples was determined usng a liquid scintillation counter (LS
6000 SC; Beckman Coulter). Total PI formation [inositol monophosphate + IP2 (inositol 1,4-bisphosphate) + IP3 (inositol 1,4,5trisphosphate)] from non-stimulated cells was used as control
wells for basal activation of PLC-β. The inhibition of inositol
phosphate formation by hirudine (4 units/ml) was used as a control for specificity.
Statistical analysis
Raw data for analysis were obtained from pooled RPE cells
from 10–15 Long-Evans rats in three independent experiments.
An unpaired Student’s t test was applied for statistical analysis, using the GraphPad Prism V4.0 program *P < 0.05,
**P < 0.01 and ***P < 0.001.
RESULTS
Thrombin induces RPE cell proliferation
by activating the MAPK cascade
Thrombin has been shown to induce the proliferation of human
RPE cells in culture [30]. As a first approach towards understanding the role of thrombin in PVR pathogenesis, we studied
the effect of thrombin on rat RPE cell proliferation. RPE cells
were serum-deprived for 24 h prior to stimulation with thrombin
at 1, 2 and 4 units/ml in serum-free Opti-MEM® , and cell proliferation was quantified using the colorimetric MTS assay after 24 h
incubation in the presence of thrombin. As shown in Figure 1(a),
the proliferative response of RPE cells was dose-dependent and
was totally inhibited by the thrombin-specific inhibitor hirudine
(4 units/ml).
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Figure 1 Thrombin induces RPE cell proliferation through the
MAPK pathway
Non-confluent cultures of rat RPE cells were serum-deprived for 24 h
prior to thrombin stimulation. (a) Thrombin (black bars) stimulates proliferation in a dose-dependent manner. Inhibition by hirudine (4 units/ml)
(white bars) demonstrates that the effect of thrombin is mediated
by activation of PARs. Values from unstimulated cultures were set
as 100%. U/mL, units/ml. (b) Dose-dependent inhibition of thrombin
(2 units/ml)-induced proliferation by the MEK inhibitor U0126 demonstrates the involvement of MAPK signalling in thrombin-induced proliferation. Cell proliferation was measured by the colorimetric MTS reduction method following 24 h stimulation by thrombin. Values from
thrombin-stimulated cultures were set as 100%. Basal, proliferation in
unstimulated cells (control). Results are means +
− S.E.M. (n = 3), with
each experiment performed in triplicate.
In order to define the molecular mechanisms underlying
thrombin stimulation of RPE proliferation, we assessed the involvement of the MAPK signalling pathway by testing the effect
of MEK inhibition on thrombin-induced proliferation. As shown
in Figure 1(b), U0126, known to interfere with ERK phosphorylation by MEK, induced a dose-dependent inhibition of thrombininduced proliferation. Correspondingly, the inhibition of MEK
activation by 30 mM PD98059 produced a similar effect (results
not shown). U0126 was used in subsequent experiments, since it
has been shown to inhibit ERK phosphorylation by active or inactive MEK, which eliminates any effect caused by residentactivated MEK.
Thrombin induces biphasic activation
of ERK1/2 through PAR-1
The ERK1/2 MAPK signalling pathway has been linked to cell
proliferation in a variety of cells, as well as to thrombin-mediated
effects on platelets, in which distinct effects have been related
to variations in the duration of ERK activation [16,42]. In order to
Figure 2 PAR-1 activation by thrombin stimulates ERK1/2
phosphorylation
Confluent RPE cell cultures were serum-deprived for 24 h prior to
thrombin stimulation. Total-cell lysates were obtained at the indicated
time points, and 25 μg of total protein was resolved by SDS/PAGE.
PVDF membranes were probed with mouse anti-[MAPK pERK1/2
(pThr202 /Tyr204 )] (p-ERK) and rabbit anti-ERK1/2 antibodies. (a) Time
course of ERK1/2 activation by thrombin (2 units/ml). Values from unstimulated cultures were set as 100% ERK1/2 phosphorylation. Densitometric analysis results are means +
− S.E.M. (n = 3), with each experiment performed in triplicate. (b) RPE cell cultures were stimulated for
10 min with the agonist peptides for PAR-1, PAR-3 and PAR-4 (25 μM)
and PAR-3 and PAR-4 (PAR-3 + 4), in serum-free medium. ERK1/2 phosphorylation was assessed by Western blotting. The gel shows a representative experiment which has been performed in triplicate. pERK, phospho-ERK; pThr202 /Tyr204 , phospho-Thr202 and phospho-Tyr204 ; U/ml,
units/ml.
determine the kinetics of ERK1/2 activation by thrombin, serumdeprived RPE cells were stimulated with 2 units/ml thrombin,
and ERK phosphorylation was measured at early (5, 10, 15 and
30 min), intermediate (1, 2, 4, 8 and 12 h) and late time points (1,
2 and 3 days). Western blot analysis of phosphorylated ERK1/2
showed that thrombin induced transient and biphasic activation
of ERK, which peaked at 10 and at 120 min post-stimulation
(Figure 2a). The ERK phosphorylation level returned to baseline
after 4 h, and remained unchanged up to 8–12 h. Replenishment
of the medium with thrombin every 24 h did not modify the ERK
activation profile at later time periods (results not shown).
In order to identify the PAR subtype involved in the thrombinmediated effects on ERK1/2 activation, we stimulated RPE cells
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with specific TRAPs (thrombin receptor agonist peptides). The
PAR-1 agonist (25 μM) was able to induce the first (10 min) and
second (120 min) peak of ERK1/2 phosphorylation at the same
level as thrombin. Neither PAR-3 nor PAR-4 agonist peptides,
alone or in combination, modified ERK activation, as shown in
Figure 2(b) (only the first 10 min peak is shown).
Thrombin-induced ERK1/2 activation is specific
and unrelated to EGFR [EGF (epidermal growth
factor) receptor] transactivation
The specificity of the effect of thrombin on ERK1/2 activation at
10 and 120 min was demonstrated by the abolition of both phosphorylation peaks by the inclusion of the thrombin-specific inhibitor hirudine 30 min prior to stimulation (Figure 3a). Moreover,
replacement of the medium with thrombin-free Opti-MEM® at
20 min post-stimulation, or the addition of hirudine following
the first ERK1/2 activation peak, prevented ERK1/2 activation at
120 min (results not shown). These results rule out the possibility
that the second (120 min) peak of ERK activation resulted from
early thrombin-induced release of growth factors into the culture
medium.
Specifically, ERK1/2 activation by thrombin has been ascribed
to the transactivation of EGFR in some cells [43–45]. To address
this possibility in the RPE, cells were treated with the specific
EGFR inhibitor AG-1478 prior to thrombin stimulation. Our results showed that ERK1/2 activation by thrombin does not result
from EGFR transactivation (Figure 3b) and, moreover, that RPE
cell proliferation was not prevented by AG-1478 (Figure 3c).
Thrombin-induced ERK1/2 activation bypasses Ras
activation
Although the linear activation of Ras/Raf/MEK/ERK is the common sequence followed by receptor-induced MAPK pathway
activation in most cell types, it has not been defined for thrombin-activated PAR-1 in the RPE. In order to analyse the sequential activation of the MAPK pathway components by thrombin
in RPE cells, we used the Ras farnesylation inhibitor manumycine, the Raf-1 inhibitor and the MEK1/2 inhibitor U0126. The
activation status of the terminal kinase ERK1/2 was analysed by
Western blotting following 10 min stimulation with thrombin
in cultures pre-treated with the above-mentioned inhibitors. As
shown in Figure 4, although the Raf-1 inhibitor (50 nM) and
the MEK inhibitor U0126 (10 μM) prevented thrombin-induced
ERK1/2 activation, the Ras-activation inhibitor manumycine (20
and 40 μM) did not. The same result was observed for the late
peak (120 min) of ERK1/2 activation (results not shown). These
results suggest that thrombin activates ERK in a Ras-independent
manner.
Thrombin-induced RPE cell proliferation depends on
PLC-β activation
PLC-β has been involved in thrombin-induced signalling in
human RPE cells [1]. We analysed the effect of thrombin stimulation on PLC-β activity by measuring [3 H]inositol phosphate
formation in cells incubated overnight in the presence of myo[2-3 H (N)]inositol. Figure 5(a) shows that thrombin (2 units/ml)
Figure 3 ERK1/2 activation by thrombin is unrelated to EGFR
(a) Confluent RPE cell cultures were treated with or without hirudine (4 units/ml) in serum-free medium prior to thrombin stimulation
(2 units/ml) for 10 min or 2 h, and ERK1/2 activation was quantified
by Western blotting with mouse anti-[MAPK pERK1/2 (pThr202 /Tyr204 )]
(p-ERK) and rabbit anti-ERK1/2 antibodies. ERK phosphorylation in
thrombin-stimulated cultures was set as 100%. Thrombin-induced
ERK1/2 phosphorylation (b) and cell proliferation (c) were measured in
non-confluent cultures treated with the EGFR inhibitor AG-1478 prior to
thrombin stimulation. The histogram results are means +
− S.E.M. (n = 3)
and the gels show a representative experiment. Values from thrombinstimulated cultures were set as 100%. Basal, phosphorylated ERK1/2
in unstimulated cells (control). pERK, phospho-ERK; pThr202 /Tyr204 ,
phospho-Thr202 and phospho-Tyr204 ; U/ml, units/ml.
increases 3 H-labelled PI formation by approx. 200%. This effect
was prevented by hirudine, as well as by the PLC-β inhibitor
U-73122. Moreover, U-73122 also abolished thrombin-induced
RPE cell proliferation (Figure 5b). Since the MEK inhibitors
PD98058 and U0126 also prevented the effect of thrombin on
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Figure 4 ERK1/2 activation by thrombin is Ras independent
Confluent RPE cell cultures were serum-deprived for 24 h prior to stimulation with thrombin (2 units/ml; 10 min). Cells were disrupted in lysis
buffer, and 25 μg of total protein was resolved by SDS/PAGE (12%
gels). PVDF membranes were probed with mouse anti-[MAPK pERK1/2
(pThr202 /Tyr204 )] (p-ERK) and rabbit anti-ERK1/2 antibodies. The MEK
inhibitor U0126 (10 μM), Raf-1 inhibitor (50 nM) and the Ras farnesylation inhibitor manumycine (20 μM) were added 1 h prior to thrombin stimulation. The histogram results are means +
− S.E.M. (n = 3) and
the gel shows a representative experiment. Values from thrombinstimulated cultures were set as 100%. Basal, phosphorylated ERK1/2
in unstimulated cells (control). pERK, phospho-ERK; pThr202 /Tyr204 ,
phospho-Thr202 and phospho-Tyr204 .
proliferation, these results suggest that the upstream activation
of PLC-β is required for thrombin induction of MEK activation
and proliferation.
Thrombin-induced RPE cell proliferation requires
the activation of cPKC (classic PKC) and nPKC
(novel PKC) isoenzymes
The activation of both cPKC (α, β and γ) and nPKC (δ, ε, η and
θ) isoenzymes has been shown to require membrane-associated
phosphatidylinositol bisphosphate breakdown of DAG (diacylglycerol) and IP3 . The IP3 -mediated release of Ca2+ from intracellular pools recruits classic isoenzymes to lipid rafts, where
membrane-associated DAG fully activates the cPKC isoenzymes
[46]. nPKCs, however, do not require DAG for full activation.
In order to establish the downstream effectors of PLC-β activation leading to proliferation, we examined the participation of
PKC isoenzymes in thrombin-stimulated RPE cells by measuring
the effect of the broad-range protein kinase inhibitor staurosporine and that of Ro-32-0432, a specific inhibitor of the cPKCs
(α, β and γ) and the nPKC PKCε on cell proliferation. As
shown in Figure 6, both staurosporine (Figure 6a) and Ro-320432 (Figure 6b) inhibited thrombin-induced cell proliferation
Figure 5 Thrombin activation of PLC-β stimulates RPE cell
proliferation
(a) Thrombin-induced PI synthesis (inositol monophosphate + IP2 + IP3 )
was measured in cultures incubated with 2 μCi of myo-[2-3 H(N)]inositol
per well for 16–24 h in the presence of LiCl (10 mM). Following
10 min stimulation with 2 units/ml thrombin, cells were disrupted and
[3 H]inositol phosphates were extracted using chloroform/methanol [1:2
(v/v)] and eluted in batches in a Dowex AG1 (X8, 100–200 mesh)
column. Radioactivity in the samples was quantified using a liquid scintillation counter. PI synthesis (inositol monophosphate + IP2 + IP3 ) is expressed as a percentage of the values in unstimulated cultures (Basal),
which was set as 100%. Hirudine (4 units/ml) and U-73122 (2.5 μM)
were used as specificity controls for the thrombin effect and PLC-β activation respectively. (b) Effect of PLC-β inhibition on thrombin-induced
proliferation. Non-confluent cultures were stimulated with 2 units/ml
thrombin for 24 h in the absence or presence of U-73122, and proliferation was measured using the colorimetric MTS reduction method.
Values for thrombin-stimulated cultures were set as 100%. Basal, PLC-β
activity in unstimulated cells (control). For both (a) and (b), results are
means +
− S.E.M. (n = 3), with each experiment performed in triplicate.
U/ml, units/ml.
in a dose-dependent manner. However, although staurosporine
partially inhibited proliferation, the effect of thrombin was completely prevented by Ro-32-0432. This result suggests that both
cPKC (α, β and γ) and nPKC (ε) isoenzymes are involved in this
process.
Thrombin-induced ERK1/2 phosphorylation is
unrelated to PLC-β, cPKC and nPKC activity
The cPKC isoenzyme PKCα has been shown to activate Raf-1
by direct phosphorylation [47], and PKCε has also been shown
to activate Raf-1 in a Ras-independent manner [48]. Hence the
Ras-independent activation of MEK/ERK by thrombin could
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PKCζ and thrombin-induced RPE proliferation
Figure 6 cPKC and nPKC isoenzymes are involved in thrombininduced RPE cell proliferation
Non-confluent cultures of RPE cells from Long-Evans rats were serum-deprived for 24 h, and stimulated with thrombin (2 units/ml) for 24 h.
Cell proliferation was quantified using the colorimetric MTS reduction
method. Absorbance from unstimulated cultures (Basal) was set as
100%. Participation of PKC signalling in thrombin-induced proliferation
was assessed by treating the cultures prior to thrombin stimulation
with (a) staurosporine or (b) Ro-32-0432. Results are means +
− S.E.M.
(n = 3) with each experiment performed in triplicate.
be elicited by the activation of cPKC and nPKC isoenzymes
derived from PLC-β activity shown in the present study (Figures 5 and 6). In order to explore this possibility, we measured
the effect of thrombin on ERK1/2 phosphorylation in the presence of the PLC-β inhibitor U-73122 and the PKC inhibitors
staurosporine and Ro-32-0432. The results showed that none of
the inhibitors affected thrombin-induced activation of ERK at
concentrations shown to inhibit cell proliferation (Figure 7).
These results reveal that thrombin stimulates RPE cell proliferation through the activation of a PLC-β/PKC signalling pathway,
independent from ERK activation. Moreover, since cell proliferation was abolished by the independent inhibition of MEK or
PKC, our results suggest a co-operative interaction of the MAPK
pathway and PLC-β/PKC signalling in the induction of proliferation by thrombin.
Figure 7 ERK1/2 activation by thrombin is unrelated to PLC-β,
cPKC and nPKC
The involvement of PLC-β and PKC in thrombin-induced ERK activation was analysed in serum-deprived RPE cell cultures (see the Materials and methods section for details). Thrombin (2 units/ml) was
added for 10 min, and cells were disrupted in lysis buffer. The protein
mixture (25 μg) was resolved by SDS/PAGE (12% gels). PVDF membranes were probed with mouse anti-[MAPK pERK1/2 (pThr202 /Tyr204 )]
(p-ERK) and rabbit anti-ERK1/2 antibodies. The inhibitors U-73122
(5 μM), staurosporine (25 nM) and Ro-32-0432 (20 μM) were included
in the medium 30 min prior to thrombin stimulation. Values for thrombinstimulated cultures were set as 100%. Basal, phosphorylated ERK1/2
in unstimulated cells (control). Results are means +
− S.E.M. (n = 3) with
each experiment performed in triplicate. The gel shows a representative experiment. pERK, phospho-ERK; pThr202 /Tyr204 , phospho-Thr202
and phospho-Tyr204 .
In order to analyse further the mechanism involved in Rasindependent activation of ERK by thrombin, we examined the
possible involvement of atypical PKC isoforms in this process, using a myristoylated peptide pseudosubstrate for PKCζ
(myristic acid–Ser-Ile-Tyr-Arg-Arg-Gly-Ala-Arg-Arg-Trp-ArgLys-Leu). As shown in Figure 8(a), this pseudosubstrate prevented Ras-independent ERK1/2 activation by thrombin. Moreover,
inhibition of PI3K, the upstream activator of PKCζ, by wortmannin also prevented thrombin stimulation of ERK phosphorylation
(Figure 8a). As expected, PKCζ inhibition also abolished RPE
cell proliferation (Figure 8b).
DISCUSSION
Atypical PKCζ mediates Ras-independent ERK1/2
activation by thrombin
Within the PKC family of serine/threonine kinases, PKC isoenzymes ζ and λ/ι are considered atypical, since their activation
does not require Ca2+ or DAG, but depends on the activity of
PI3K (phosphoinositide 3-kinase) [49].
Proliferative eye diseases, which eventually lead to blindness,
represent an important cause of failure in surgery aimed at correcting retinal detachment [50]. In response to ocular stress, such
as trauma, photocoagulation, retinal detachment or ischaemia,
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Figure 8 PI3K and atypical PKCζ activation is required for thrombin-induced ERK1/2 phosphorylation and RPE cell proliferation
(a) ERK activation. Serum-deprived RPE cell cultures were stimulated
with 2 units/ml thrombin for 10 min. Cells were disrupted in lysis buffer, and the proteins in the lysate were examined as described in the
Materials and methods section and as described for Figure 7. The PKCζ
pseudosubstrate (PKCζ PS) peptide inhibitor (myristic acid–Ser-Ile-TyrArg-Arg-Gly-Ala-Arg-Arg-Trp-Arg-Lys-Leu) (25 μM) and wortmannin (1 μM)
were included in the culture medium 30 min prior to thrombin stimulation. Western blotting of total-cell lysates resolved by SDS/PAGE was
performed with mouse anti-[MAPK pERK1/2 (pThr202 /Tyr204 )] (p-ERK)
and rabbit anti-ERK1/2 antibodies. (b) Cell proliferation. Non-confluent
RPE cell cultures were stimulated with thrombin in the absence (control) or presence of increasing concentrations of the PKCζ pseudosubstrate. Following stimulation, the cultures were incubated for 24 h
in the same medium containing thrombin (control) or thrombin with
the PKCζ pseudosubstrate (thrombin + PKCζ PS). Proliferation was
quantified by the colorimetric MTS assay. Values for thrombin-stimulated cultures were set as 100%. Basal, phosphorylated ERK1/2
(a) and cell proliferation (b) in unstimulated cells (control). Res202
204
ults are means +
− S.E.M. (n = 3). pERK, phospho-ERK; pThr /Tyr ,
phospho-Thr202 and phospho-Tyr204 ; U/ml, units/ml.
the blood–retina barrier is compromised, allowing ocular tissues,
such as the RPE, to come in contact with blood constituents
[51]. Thrombin has been shown to stimulate cell proliferation via
the activation of GPCRs in several cell types, including the RPE
[52], although the molecular mechanisms leading to this outcome
remain largely undefined.
The results in the present study demonstrate that thrombin,
which is contained in blood serum, induces rat RPE cell proliferation through the GPCR PAR-1, via Ras-independent activation
of the Raf/MEK/ERK cascade. We provide evidence for the involvement of PLC-β, PI3K and PKC isoenzymes in the regulation
of this signalling pathway. We demonstrate, for the first time, the
requirement for PKCζ in thrombin-induced effects on RPE cells.
MAPK signalling plays a central role in several cellular processes, including proliferation [16,53–55]. Our results demonstrate that thrombin induces RPE cell proliferation by PAR-1
activation of the MEK/ERK1/2 module of the MAPK pathway.
The specificity of the effect of thrombin through PAR-1 was established by the inhibition of proliferation and MAPK activation
by hirudine, and by the lack of effect of the agonist peptides for
PAR-3 and PAR-4 on ERK1/2 phosphorylation (Figure 2).
ERK1/2 activation was found to be both transient and biphasic,
peaking at 10 and 120 min following stimulation, which is in
agreement with previous observations in astrocytes [36], fibroblasts [56] and endothelial cells [54]. In astrocytes, the late response has been ascribed to receptor recycling, whereas thrombin
promotes the cis-transactivation of PAR-1 gene expression in endothelial cells [54]. Although the membrane recycling dynamics
for PAR-1 in the RPE remain to be established, unresponsiveness
to thrombin up to 72 h following the late ERK phosphorylation
peak (Figure 2) rules out the appearance of newly synthesized uncleaved receptors at the membrane. Additionally, suppression of
the increase in ERK phosphorylation at both time points by hirudine eliminates a possible non-specific effect mediated by
thrombin-induced release of neurotrophins or neuroactive compounds known to activate the MAPK pathway. Among these
compounds, EGF activation of the MAPK pathway in the RPE
has been reported [57], and, in order to determine if an early
release of EGF by thrombin could be responsible for the late
ERK activation peak, we tested the effect of the specific EGFR
inhibitor AG-1478 and showed that it did not affect the effect of
thrombin on ERK or proliferation (Figure 3).
Canonical activation of the MEK/ERK module by ligandgated receptors with intrinsic receptor tyrosine kinase activity
has been studied extensively and shown to depend on the activation of the monomeric GTPase Ras by GEFs, such as SOS
(Son of sevenless)-1/SOS-2 [58]. A major downstream target
of activated Ras-GTP is the serine/threonine kinase Raf-1, which
subsequently activates MEK/ERK1/2. Thrombin has been shown
to activate the classic Ras-dependent MAPK pathway in human
[16] and canine [20] tracheal smooth muscle cells. Our results,
however, showed that thrombin-induced ERK phosphorylation
in RPE cells bypasses Ras, since it was not prevented by the Ras
farnesylation inhibitor manumycine, which has been shown to
suppress the membrane anchoring of Ras [59] and Ras activation
by serum in RPE cells [60].
Ras-independent activation of MEK/ERK has been observed
in human platelets and ascribed to PLC-β activity, leading to
the downstream activation of PKC [61]. Upon activation, PAR-1
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PKCζ and thrombin-induced RPE proliferation
signalling to Gq/11α , Gαi and G12/13α could be involved in ERK
activation and/or cell proliferation. We showed that, in contrast
with fibroblasts in which ERK activation has been shown to depend on the activation of Gi by PAR-1 [56], in RPE cells, neither
PTX nor the inhibitor of Rho kinase (Y-27632), the downstream
effector of G12/13α , inhibited the effect of thrombin on ERK or cell
proliferation (results not shown), suggesting that Gq/11α could be
responsible for thrombin actions through PAR-1.
We explored this possibility and demonstrated that the activation of PLC-β by thrombin increases IP3 formation in RPE cells
by approx. 200%. This effect was prevented by hirudine as well
as by the PLC-β inhibitor U-73122. Furthermore, the inhibition
of PLC-β also prevented thrombin-induced proliferation, suggesting a causal relationship from PAR-1 to PLC-β and proliferation.
MEK activation [62], as well as direct phosphorylation of Raf-1
by cPKCα [47], has been demonstrated, and the activation of
Raf-1 by PKCε in a Ras-independent manner has also been reported [48]. Consistent with these results, we demonstrated that
the inhibition of DAG-dependent PKC isoforms by staurosporine and Ro-32-0432 prevented thrombin-induced proliferation
of RPE cells. However, in contrast with results showing that
Ras-independent ERK1/2 phosphorylation depends on the activation of cPKC or nPKC isoenzymes [48,61,63], neither of these
compounds prevented Ras-independent activation of ERK1/2 by
thrombin. Since Ro-32-0432 has been shown to inhibit the cPKC
(α, βI, βII and γ) and nPKC (ε) isoforms, but not PKCδ or
PKCθ, the possible involvement of these isoforms in ERK1/2
activation by thrombin in our system cannot be disregarded.
Together, these results suggested that thrombin action on
PAR-1 promotes proliferation by activating two distinct intracellular signalling pathways: PLC-β/PKC and the Ras-independent
Raf/MEK/ERK MAPK cascade. Since the pharmacologic inhibition of either individual pathway completely suppressed proliferation, we conclude that the joint activation of these pathways
is required for the effect of thrombin on proliferation. In support
of this assumption, although the inhibition of cPKC isoenzymes
prevented proliferation, the direct activation of PKC by PMA
alone, or in combination with a Ca2+ ionophore (ionomycin),
had no effect (results not shown).
Previous studies on this subject have shown that, although
insulin-induced ERK phosphorylation does not relate to DAGdependent PKC isoforms, PI3K and PKCζ (or PKCλ, which is
72% homologous with PKCζ and shares an identical pseudosubstrate sequence), as well as MEK1, are required for insulininduced activation of ERK in rat adipocytes [64]. Because
atypical PKCζ is known to serve as an effector of PI3K in different cell types [49], we tested the involvement of PKCζ in
Ras-independent activation of ERK by thrombin.
Our results demonstrate that the pseudosubstrate peptide for
the inhibitory region of PKCζ concomitantly inhibited ERK
phosphorylation and cell proliferation induced by thrombin.
Although direct phosphorylation of ERK by PKCζ was not
observed, since MEK is considered to be the exclusive upstream kinase for ERK, the phosphorylation of 14-3-3 scaffold protein by PKCζ has been associated with Raf-1 activa-
tion [65], which could explain the requirement for PKCζ activity
in Ras-independent ERK phosphorylation by thrombin in the
RPE.
Findings regarding the participation of PKCζ in ERK activation are controversial, possibly as a result of the particular cell
types or receptors analysed. Inhibition of PI3K, the upstream
activator of PKCζ, has been shown to inhibit ERK activity induced by interleukins [66,67] and growth factors in different cell
lines [68,69]. In contrast, PI3K inhibition by wortmannin and LY294002 had no effect on ERK phosphorylation in response to the
ligand-stimulated chemokine receptor CXCR3 (CXC chemokine
receptor 3) in hepatic stellate cells [70] or on activation of ERK
in response to EGF in glioblastoma cells [71].
Gβγ subunits couple PAR-1 to distinct signalling pathways,
notably activation of PI3K. In astrocytes, the effect of PAR-1
agonists on activation of ERK1/2 and proliferation are strongly
inhibited by the PI3K blocker wortmannin. PAR-1 activation of
ERK and proliferation in these cells depends on a PTX-sensitive
pathway mediated by Gβγ , PI3K and Ras, and a PTX-insensitive
pathway involving PKC and Raf [36]. These mixed observations
led us to examine whether PI3K in RPE cells may be activated in
response to stimulation of PAR-1 by thrombin.
In agreement with the requirement of PKCζ activity for the
activation of ERK and proliferation by thrombin (Figure 8), inhibition of PI3K by wortmannin also inhibited ERK1/2 activation in
RPE cells. These findings demonstrate that, although PI3K activity is essential for the activation of ERK, its activity may also
be required to stimulate additional proliferative pathways that do
not involve ERK and which remain to be defined.
In conclusion, in the present study we show that the activation
of the GPCR PAR-1 by thrombin triggers RPE cell proliferation
by the joint activation of the ERK1/2 MAPK signalling cascade, in a Ras-independent manner, and that of PI/PLC-β-PKC,
upstream of MAPK activation. The present study shows, for the
first time, the involvement of PKCζ-mediated phosphorylation of
ERK1/2 in the proliferative response of RPE cells to thrombin,
and further supports an important role for thrombin in the pathogenesis of PVR induced by injury or retinal surgery.
FUNDING
This work was supported by the Programa de Apoyo a Proyectos
de Investigación e Innovación Tecnológica/Universidad Nacional
Autónoma de México [grant number IN203507]; and the Consejo
Nacional de Ciencia y Tecnologı́a [grant number 80398] to
A.M.L.-C.
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Received 9 July 2008; accepted 17 July 2008
Published as Immediate Publication 17 July 2008, doi 10.1042/BSR20080083
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