FAK phosphorylation plays a central role in thrombin-induced RPE cell migration

Accepted Manuscript FAK phosphorylation plays a central role in thrombin-induced RPE cell migration

E.D. Aguilar-Solis, A.M. López-Colomé, I. Lee-Rivera, A. Alvarez-Arce, E. López PII: DOI: Reference:

S0898-6568(17)30116-X doi: 10.1016/j.cellsig.2017.04.016 CLS 8905

To appear in:

Cellular Signalling

Received date: Revised date: Accepted date:

16 February 2017 21 April 2017 22 April 2017

Please cite this article as: E.D. Aguilar-Solis, A.M. López-Colomé, I. Lee-Rivera, A. Alvarez-Arce, E. López , FAK phosphorylation plays a central role in thrombin-induced RPE cell migration. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Cls(2017), doi: 10.1016/j.cellsig.2017.04.016

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

FAK phosphorylation plays a central role in thrombin-induced RPE cell migration. Aguilar-Solis E.D.1, López-Colomé, A.M.1*, Lee-Rivera, I.1, Alvarez-Arce A.1, López,

CR

IP

T

E.1

Instituto de Fisiología Celular, Universidad Nacional Autónoma de México. Mexico

ED

M

AN

US

city, México.

*Corresponding Author. Instituto de Fisiología Celular, UNAM. Apartado Postal 70-

PT

253, Ciudad Universitaria, Mexico city, CdMx, 04510 Mexico. Tel 52(55) 56225617 Fax: 52(55) 56225607. E-mail: [email protected]

CE

E.D. A-S e-mail: [email protected] A. A-A e-mail: [email protected]

AC

I.L-R e-mail: [email protected] E.L e-mail: [email protected]

Running Title: FAK in RPE migration Keywords: Retina; proliferative vitreoretinopathy; signal transduction; Focal adhesion

1

ACCEPTED MANUSCRIPT Abstract. The migration of retinal pigment epithelial (RPE) cells is an important step in various pathologic conditions including subretinal neovascularization (SRN), proliferative vitreoretinopathy (PVR) and, importantly, as a consequence of retinal surgery. Therefore, the elucidation of the mechanisms underlying RPE trans-

T

differentiation and migration is essential for devising effective treatments aimed to

IP

the prevention of these disorders. A common event in these pathologies is the alteration of the blood-retina barrier (BRB), which allows the interaction of RPE

CR

cells with thrombin, a pro-inflammatory protease contained in serum. Our previous work has demonstrated that thrombin induces RPE cell cytoskeletal remodeling

US

and migration, hallmark processes in the development of PVR; however, the molecular mechanisms involved are still unclear. Cell migration requires the

AN

disassembly of focal adhesions induced by focal adhesion kinase (FAK) phosphorylation, together with the formation of actin stress fibers. The aim of the

M

present work was to identify thrombin-activated signaling pathways leading to FAK phosphorylation and to determine FAK participation in thrombin-induced RPE cell

ED

migration. Results demonstrate that the activation of PAR1 by thrombin induces FAK autophosphorylation at Y397 and the subsequent phosphorylation of

PT

Y576/577 within the activation loop. FAK phosphorylation was shown to be under the control of c/nPKC and PI3K/PKC-δ, as well as by Rho/ROCK, since the

CE

inhibition of these pathways prevented thrombin-induced FAK phosphorylation and the consequent disassembly of focal adhesions, in parallel to FAK-dependent actin

AC

stress fiber formation and RPE cell migration. These findings demonstrate, for the first time, that thrombin stimulation of RPE cell transformation and migration are regulated by FAK tyrosine phosphorylation. Thus, targeting FAK phosphorylation may provide a strategical basis for PVR treatment.

2

ACCEPTED MANUSCRIPT 1. Introduction The retinal pigment epithelium (RPE) is the predominant component of the outer blood-retina barrier (BRB), and plays an essential role in the survival of retinal neurons and the maintenance of visual function [1]. The breakdown of the BRB has particularly grievous consequences to vision, since increased permeability of the

T

BRB is an early event in the establishment of retinal diseases such as subretinal

IP

neovascularization, diabetic retinopathy, and proliferative vitreoretinopathy (PVR),

CR

a major cause of retinal surgery failure and the loss of vision [2].

The barrier integrity of cellular monolayers is directly related to the actin

US

cytoskeleton, as has been shown by studies demonstrating an increase in the paracellular permeability by actin-disrupting agents [3, 4]. Under pathological

AN

conditions involving the alteration of the BRB due to ocular trauma, retinal detachment or metabolic imbalance as occurs in diabetes, quiescent RPE cells

M

undergo epithelial–mesenchymal transition (EMT) and uncontrolled proliferation, migrate to the vitreous, and develop into contractile membranes on retinal

ED

surfaces, characteristic of PVR and other fibro-proliferative eye diseases leading to blindness [5, 6]. RPE cell exposure to serum-contained thrombin upon the

PT

breakdown of the BRB has been associated with the development of PVR [7]. In addition to its well known role in hemostasis, thrombin, a pro-inflammatory

CE

multifunctional serine protease activated upon tissue injury, has been shown to regulate cell proliferation, invasiveness and tumor growth in several cell types [8,

AC

9]. Our previous work has demonstrated that thrombin promotes RPE cell proliferation, cytoskeletal remodeling and migration [10-12]. Although the molecular mechanisms involved in these processes are still unclear, these data indicate that thrombin-induced activation of signaling cascades could be involved in the development of PVR. In fact, more than 95% PVR cases occur as a result of retinal surgical procedures, a condition in which the RPE comes in direct contact with blood-contained thrombin [7]. Thrombin intracellular signaling is mediated by G protein coupled receptors (GPCRs) termed proteinase-activated receptors (PARs), activated by the 3

ACCEPTED MANUSCRIPT proteolytic unmasking of a new N-terminal sequence that functions as a tethered ligand which binds intramolecularly to the receptor. Four types of PARs are currently known: PARs -1, -3 and -4, activated by thrombin, and PAR2, activated by trypsin, tryptase, and other serine proteases [13]. PARs can also be activated by short synthetic peptides mimicking the amino terminus created by cleavage [14]. Besides the established paradigm of PAR activation, additional modes of PAR

T

activation occur and involve cofactoring between different PARs, which can modify

IP

each other’s signaling activity through different mechanisms, including the

CR

modulation of the efficiency of G protein signaling [15, 16].

PAR 1, the prototype of PAR family, couples to members of the Gq/11, Gi, and G12/13

US

families of GPCRs. Gαq subunits activate phospholipase Cβ (PLC-β), with the formation of inositol 1,4,5- trisphosphate (IP3) and diacylglycerol (DAG), the

AN

endogenous activator of conventional/novel PKC isoforms (c/n PKC). Gαi inhibits adenylyl cyclase. The α-subunits of G12 and G13 activate Rho GTPases, known to

M

be involved in the regulation of the assembly and organization of the actin cytoskeleton, through the activation of Rho kinase (ROCK). Additionally, the Gβγ

ED

subunits can activate phosphoinositide 3-kinase (PI3K) and other lipid modifying

PT

enzymes, protein kinases and ion channels [14]. Among these signaling pathways, the proteolytic activation of PAR1 by thrombin

CE

has been shown to activate PLC, PKC and PI3K signaling pathways in RPE cells [10, 12, 17], all involved in the regulation of cell adhesion properties and stress

AC

fiber formation [18-20], which further supports thrombin participation in the development of PVR. Additionally, evidence from studies in platelets, fibroblasts and endothelial cells among others, have demonstrated that PAR1 signaling promotes the activation of Src family tyrosine kinases, Rho kinase and Focal Adhesion Kinase (pp125FAK; FAK) [21], suggesting that PAR1-activated signals could be responsible for cell transformation and migration through the activation of FAK [22].

4

ACCEPTED MANUSCRIPT The non-receptor tyrosine kinase Focal Adhesion Kinase (FAK) is a central component of focal adhesions (FAs), which regulates cytoskeleton dynamics essential for cell motility including integrin activation, actin fiber anchorage, actomyosin contraction and adhesion turnover [23, 24]. Integrin receptor activation upon cell adhesion to extracellular matrix promotes FAK autophosphorylation at tyrosine 397 (Y397), which generates a binding site for the cytoplasmic tyrosine

T

kinase Src [25, 26], PI3K [27] and PLC [28], and the subsequent phosphorylation

IP

of FAK by Src at tyrosine residues Y407 and Y576/577 in the putative activation

CR

loop of the kinase domain [29]. The phosphorylation of Y397 and Y576/577 is critical for adhesion-induced FAK activation and enhancement of cell spreading

US

and migration [30, 31]. Src also phosphorylates Y871 and Y925 at FAK C-terminal region [32], which generates interaction domains for the recruitment of enzymes

AN

and structural proteins including p130Cas, Shc, Grb2, PI3K and paxillin, thus allowing FAK participation in a wide signaling network activated by integrins [23,

M

33]. Additionally, FAK/Src phosphorylation of α-actinin at Y12 promotes FA interaction with actin stress fibers leading to cell contraction and movement [34, 35].

ED

Due to its central role in the control of cell movement, the participation of FAK in pathological conditions such as cancer, which involve cell spreading, migration and

PT

invasion, has been postulated [23, 31, 36-38]. FA turnover is required for cell movement and migration and is strictly regulated by

CE

the concerted action of several kinases and phosphatases [23, 35, 39]. Although thrombin as well as G protein-coupled receptor (GPCR) agonists have been shown

AC

to induce FAK phosphorylation, thus promoting the disassembly of adhesion complexes [30], to date, the mechanisms through which FAK regulates the complex interactions among the cell components which define cell mechanics are largely undefined. Although the involvement of some growth factors in the development of PVR has been documented, the specific receptors and intracellular signaling pathways leading to PVR remain largely unexplored. Our previous work has demonstrated that thrombin promotes the assembly of actin stress fibers and the migration of RPE cells, prominent features of PVR [12, 40]. Since the design of clinical or pharmacological procedures aimed to the prevention 5

ACCEPTED MANUSCRIPT of PVR clearly requires the elucidation of the molecular mechanisms involved in this process, the aim of the present work was to investigate the participation of FAK phosphorylation/activation in thrombin effect, the intracellular signals mediating this effect, and the repercussion of these processes on RPE cell motility. We here demonstrate that thrombin stimulates FAK autophosphorylation at Y397 as well as phosphorylation at Y576/577 within the activation loop, critical for

T

adhesion-induced FAK activation and for FAK-induced cell spreading and

CR

IP

migration responses.

US

2. Materials and Methods

All reagents used were cell culture grade. Dulbecco’s modified Eagle’s medium

AN

(DMEM), Opti-MEM, Trypsin–EDTA, fetal bovine serum (FBS), F12 and PenicillinStreptomycin were purchased from Gibco | Thermo Fisher Scientific (Waltham, MA

M

USA). Dispase, Wortmannin, Protease inhibitor cocktail (P8340), FAK Inhibitor 14, Trypsin, Bicinchoninic acid kit and Mitomycin C were from Sigma-Aldrich (St. Louis,

ED

MO). Thrombin, Hirudin, PAR1 receptor Agonist peptide (Ser- Phe-Leu-Leu-ArgAsn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe), PKC inhibitor pseudosubstrate (Myr-

PT

SIYRRGARRWRKL), Ro-32-0432 and Y-27632, and mouse monoclonal antibody anti-Actin, were obtained from Calbiochem/ EMD Millipore (Billerica,

MA,

USA).

CE

PAR 3 (H-SFNGGP-NH2) and PAR 4 (H-GYPGKF-NH2) agonist peptides were from Bachem (Bubendorf, Switzerland). Trizol, Murine Moloney Leukemia Virus (MMLV-RT),

the

RNAse

inhibitor

(RNAseOut),

and

AC

Retrotranscriptase

Lipofectamine RNAi-MAX reagent were from Invitrogen| Thermo Fisher Scientific (Waltham, MA USA). Taq recombinant polymerase was from Altaenzymes (Alberta, Canada). Rabbit monoclonal antibodies anti-phospho-FAK (Y576/577), antiVinculin, anti-Paxillin and anti-GAPDH as well as HRP conjugated secondary antibodies rabbit anti-IgG and mouse anti-IgG were from Cell Signaling Technology (Danvers, MA, USA). Mouse monoclonal antibody anti-FAKH1 and Type IV collagen were from Santa Cruz Biotechnology (Dallas, TX, USA). G proteinsepharose protein was from Amersham Biosciences (GE Healthcare Life Sciences; 6

ACCEPTED MANUSCRIPT Chicago, IL, USA). Cell Titer 96® Aqueous One Solution Reagent for MTS reduction viability assessment was from Promega (Madison, WI, USA). SCH79797 was purchased from Tocris Bioscience. 2.1 Cell culture Retinal pigment epithelial cells were isolated as previously described [41]. Briefly,

T

8- to 10-day old Long Evans rats were anesthetized by inhaled chloroform and

IP

sacrificed following the guidelines for animal care of the ARVO Statement for the

CR

Use of Animals in Ophthalmic and Vision Research. The eyes were enucleated, rinsed in DMEM containing penicillin (100 U/ml) and streptomycin (100 mg/ml), and incubated for 30 min at 37°C in medium containing 2% dispase. After removal of

US

the sclera and the choroid, the RPE was detached from the neural retina in calcium- and magnesium-free Hank’s balanced salt solution, and incubated in the

AN

presence of 0.1% Trypsin–EDTA for 5 min. at 37°C. The dissociated cells were suspended in Opti-MEM supplemented with 4% fetal bovine serum (FBS), and

M

seeded at a density of 2x104 cells/cm2 in 8-well chamber slides (Nalgene Nunc International, Rochester, NY) for actin staining, or at a density of 1x105 cells/cm2 in

ED

12-well culture plates coated with 10 μg/cm2 Type IV collagen, following manufacturer instructions. The purity of the culture (99.1%) was established by

PT

immunofluorescence against the specific RPE biochemical marker RPE65, as previously described [11], and cell viability (>90%) was assessed by MTS [(3 - (4,5-

CE

dimetiltiazol-2-il) -5 - (3 - carboxymethoxyphenyl) -2 - (4-sulfofenil)-2H-tetrazolio] reduction method (Cell Titer 96® Aqueous One Solution Reagent, Promega),

AC

following manufacturer instructions. Unless stated, experiments were performed in confluent RPE cell monolayers. Cells were serum-deprived for 24 h, and incubated in the presence of 2 U/ml thrombin or specific PAR agonists (50 μM) for the indicated period of time. When tested, pharmacological inhibitors were included 20 min prior to thrombin stimulation, and cell viability was examined in order to discard a toxic effect from these drugs (Supplementary Figure 1).

2.2 Quantitation of FAK mRNA.

7

ACCEPTED MANUSCRIPT Semiquantitative RT-PCR was used for measuring FAK mRNA expression. Total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. Messenger RNA retrotranscription was performed using 1 μg total RNA, 20 U Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT; Invitrogen), 1 U RNase inhibitor (RNAseOut, Invitrogen), and 0.25 μg oligo dT primer (Invitrogen), in the manufacturer’s recommended buffer (50 mM Tris–HCl,

T

pH 8.3, 75 mM KCl, 3 mM MgCl2, 10mM dithiotreitol, DTT). Complementary DNA

IP

(5 μl) was used as substrate for PCR with oligonucleotides for FAK

CR

(5'CTGTACTTCGGACAGCGTGA-3' y 5'-ATGTCGTGAGCGCATAGACC-3') and β-actin

(5’-GCTCGTCGTCGACAACGGCT-3’

y

5’-

US

CAAACATGATCTGGGTCATCTTCTC-3’). PCR reaction was performed using 2.5 U of recombinant Taq Polymerase (Invitrogen), 0.4 mM of each oligonucleotide

AN

and dNTPs, 1.5 mM MgCl2, in manufacturer’s recommended buffer (Tris–HCl 20 mM, pH 8.4, KCl 50 mM). Retro-transcription was carried at 80 °C for 10 min, 42 °C for 60 min and 75 °C for 15 min. For PCR, samples were incubated at 94 °C

M

for 5 min and FAK expression was amplified for 26 cycles and β-actin for 22 cycles:

Densitometric

analysis

ED

94 °C for 30 seconds; 59 °C for 30 seconds and 72 °C for 45 seconds. was

performed

using

ImageJ

software

PT

(http://imagej.nih.gov/ij/ [in the publicdomain]). β-actin amplification was used as loading control; PAR expression was normalized to β-actin expression in the same

CE

sample.

AC

2.3 Western Blot Analysis. Following stimulation, samples were processed as described previously [41]. Protein in the lysates was quantified using Bradford reagent, and 10 μg total protein samples were resolved by 7.5% SDS/PAGE and electrotransferred onto polyvinyldiene difluoride (PVDF) membranes (GE Healthcare, Piscataway, NJ, USA). After blocking for 1 hour at room temperature with 5% non-fat milk, 3% bovine serum albumin in Tween-Tris-buffered saline (TBS), the PVDF membranes were incubated at 4 °C overnight with the primary antibodies vs. α-phospho-FAK (Y576/Y577) (1:1000), α-FAK (1:1500), α-GAPDH (1:5000), α-Vinculin (1:1000) or 8

ACCEPTED MANUSCRIPT α-Paxillin (1:1000) overnight at 4°C. Secondary HRP-conjugated rabbit anti-IgG (1:1000) or mouse anti-IgG (1:1000) antibodies (Invitrogen) were incubated for 2 hours at 4°C and membranes were developed using the Immobilon Western AP Chemiluminescent Substrate (EMD Millipore, Billerica, MA, USA). Kodak film images were digitized using an Alpha Digi-Doc system (Alpha-Innotech, San Leandro, CA, USA), and densitometric analysis was performed using ImageJ

T

software (http://imagej.nih.gov/ij/). Anti-β-actin was used as loading control, and

percentage of control (basal levels). 2.4 FAK expression silencing. small

interfering

(SASI_Rn02_00260847).

RNA

(siRNA)

Transfection

was

US

FAK

CR

IP

expression was normalized in relation to these values. Data were graphed as a

of

siRNA

purchased was

from

performed

Sigma using

AN

Lipofectamine RNAi-MAX reagent (Invitrogen) in an antibiotic-free 4% FBS supplemented Opti-MEM medium for 24 h. Following transfection, thrombin-

M

induced FAK activation or actin stress fiber formation was assessed, respectively, by Western blot and immunocytochemistry. Cultures treated with Lipofectamine,

ED

were used as control (100%). The effect of FAK knockdown was tested in 30–50%

PT

confluent cultures.

2.5 Immunofluorescent F-actin detection.

CE

In order to assess the reorganization of the actin cytoskeleton induced by thrombin in RPE cells in primary culture, cells from confluent 8-well chamber slides were

AC

serum deprived for 24 h, and then stimulated with thrombin (2 U/ml) for the indicated period of time. When tested, the pharmacological inhibitors were added to the medium 20 min. prior to thrombin stimulation. After treatment, cells were fixed in 2% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 5 min then blocked for 30 min with 1% BSA. For actin staining, fixed cells were incubated with rhodamine-conjugated phalloidin for 1 h at room temperature. Samples were mounted with Dako Fluorescence Mounting Medium (Dako North America, Inc., Carpinteria, CA) and visualized using an Olympus FluoView FV1000 confocal microscope (Olympus Tokyo, Tokyo, Japan). The exposure time and 9

ACCEPTED MANUSCRIPT intensity gain were adjusted for providing optimal visualization, and were kept constant for all experiments. Fixed cells were viewed a single time and maintained in the dark until laser exposure in order to avoid photo-bleaching. F-actin fluorescence was quantitated in three representative fields (15 cells per field) at 40X magnification using Image-Pro Plus software. Data were normalized by calculating fluorescence intensity/cell area. All experiments were performed in

IP

T

triplicate.

CR

2.6 Wound Healing Assays.

Rat RPE cells were grown to 60% confluence in six-well plates coated with collagen IV (Costar; Corning, Inc., Washington, DC, USA) in DMEM containing

US

10% FBS. Cells transfected with FAK siRNA were serum-deprived for 24 hours, followed by 30 min. incubation with 1 μg/ml Mitomycin C. To assess cell migration,

AN

the cell monolayers were scratched mechanically with a pipette tip drawing a 1.2mm line. Scratched monolayers were rinsed with serum-free DMEM and further

M

incubated for 24 hours at 37°C in the presence of 2U/mL thrombin. DMEM containing 4% FBS and serum-free DMEM were used as positive and negative

ED

controls. At the end of the stimulation period, cultures were fixed with 4% paraformaldehyde, stained with 0.1 cresyl violet, and visually examined by phase-

PT

contrast microscopy (Nikon Eclipse TS100 inverted microscope).

CE

2.7 Immunoprecipitation.

Following lysis in 50 mM Tris–HCl; pH 7.4, 150 mM NaCl, 1 mM EDTA and 0.5%

AC

Triton X-100 buffer (TNTE), 700 μg of protein were incubated overnight at 4°C in the presence of 1 μg anti-FAK-loaded sepharose beads. Following solubilization in 60 μl of Laemmli buffer (0.75 mM Tris– HCl pH 8.8, 5%; SDS, 20% glycerol and 0.01% bromophenol blue), 20 μl of the extract were resolved by SDS-PAGE and Western blot. 2.8 Statistical Analysis Results are expressed as the mean ± SEM. Raw data for analysis were obtained from at least three independent experiments as specified in the figure legends. 10

ACCEPTED MANUSCRIPT Student’s t-test was applied to results in which only two groups (negative control versus thrombin stimulation, or thrombin stimulation versus inhibitor) were compared. Unpaired one-way ANOVA was used to compare different conditions. Prism V5.0 for MacOSX program from GraphPad (La Jolla, CA, USA) was used.

IP

3.1 Thrombin stimulates the tyrosine phosphorylation of FAK.

T

3. Results

CR

In order to demonstrate a role for FAK in the stimulation of RPE cell migration by thrombin, confluent cultures of rat RPE cells were treated with 2U/ml thrombin, and the phosphorylation of FAK was determined. The auto-phosphorylation of Y397

US

and of Y576/577 within the catalytic domain was measured using specific antibodies and Western blot. Our results show that whereas Y397 is transiently

AN

phosphorylated following 2 minutes stimulation (Figure 1A), a highly significant increase in the phosphorylation of Y576/577 was attained at 5 minutes and

M

sustained up to 60 minutes in the presence of thrombin (Figure 1B). The phosphorylation of Y576/577, required for attaining the full catalytic activity of FAK

ED

[30, 42], showed to be dose dependent with maximal value at 2 U/ml thrombin

PT

(Figure 1C).

3.2 Thrombin induction of FAK Y576/577 phosphorylation is induced by PAR 1

CE

activation.

The specificity of thrombin (2 U/ml) stimulation of FAK Y576/577 phosphorylation

AC

was determined in confluent cultures of RPE cells. As shown in Figure 2A, thrombin-induced Y576/577 FAK phosphorylation was completely prevented by coincubation of thrombin with the specific thrombin inhibitors Hirudin, which prevents thrombin interaction with the receptor, or the thrombin catalytic inhibitor PPACK. In order to demonstrate that thrombin effect is receptor-mediated, and to identify the receptor responsible for thrombin induction of FAK phosphorylation, confluent RPE cell cultures were stimulated with specific agonist peptides (AP) for PAR1, PAR3 and PAR4. Results in Figure 2B show that whereas PAR1 AP stimulated FAK phosphorylation to the same extent as thrombin, stimulation by PAR3 or PAR4 11

ACCEPTED MANUSCRIPT agonist peptides had no effect. To further confirm thrombin effect through PAR1, RPE cells were incubated for 30 min in the presence of the specific PAR1 inhibitor SCH79797 (30μM), prior to stimulation with 2U/ml thrombin. Results in Figure 2C demonstrate that thrombin-induced FAK phosphorylation is abolished by SCH79797 inhibition of PAR1, as well as by siRNA suppression of FAK expression. These

results

demonstrate

that

thrombin

induction

of

FAK

IP

T

phosphorylation/activation is mediated mainly by PAR1 activation.

CR

3.3 Thrombin induction of RPE cell migration requires FAK expression. Considerable evidence implicates FAK as an essential component of transduction

US

signals leading to the turnover of focal adhesions in migrating cells [43]. In spite of this evidence, FAK participation in thrombin induction of RPE cell migration

AN

remains unexplored. In order to address this question, we analyzed RPE cell migration in a wound-healing assay in control versus FAK siRNA-transfected RPE

M

cells stimulated with 2 U/ml thrombin or 4% FBS (positive control). Figure 3B shows that thrombin and FBS promote RPE cell migration into the wounded area;

ED

this response was abolished by the siRNA-induced suppression of FAK expression (Figure 3A, and 3B). Since wound healing involves cell proliferation in addition to

PT

migration, experiments were performed in the presence of the DNA synthesis inhibitor Mitomycin C (1 μg/ml). The inhibition of proliferation in this condition was

CE

assessed by the MTS reduction method (Supplementary Figure 2). The graphical analysis of results in Figure 3B is depicted in Figure 3C. This result demonstrates

AC

that FAK is essential for thrombin signaling to RPE cell migration.

3.4 Thrombin-induced FAK phoshorylation promotes the disassembly of focal adhesions and actin stress fiber formation in RPE cells. FAK phosphorylation induced by distinct stimuli leads to the disassembly of FAs required for cell movement and migration [43]. FAK association with the structural proteins paxillin and vinculin is a central requirement for the maintenance of FA complexes linking the actin cytoskeleton with the extracellular matrix. In order to test if thrombin-induced FAK phosphorylation results in FA disassembly, we 12

ACCEPTED MANUSCRIPT measured the association of vinculin and paxillin with FAK in thrombin-stimulated RPE cells using co-immunoprecipitation assays. Results showed that thrombin stimulation abolishes the association of FAK with vinculin (Figure 4A) and paxillin (Figure 4B), indicating the dissociation of FA complexes. In addition to the disassembly of FAs, cell migration requires the formation of contractile actin stress fibers, which allow cell movement. As depicted in Figure 5,

T

thrombin stimulation induced a significant increase in actin stress fiber assembly,

IP

which was prevented by the FAK inhibitor F14. These results suggest that thrombin

CR

promotes actin stress fiber assembly through FAK phosphorylation.

US

3.5 PI3K/PKCζ signaling mediates thrombin-induced FAK phosphorylation at Y576/577.

AN

Within the PKC family of serine/threonine kinases, the stimulation of PAR1 is coupled to Gβγ signaling to PI3K, a major activator of atypical PKCδ [44]. Our

M

previous work demonstrated the involvement of PI3K/PKCδ in thrombin-induced RPE cell migration [40]. In order to define the molecular mechanism involved in this

ED

outcome, we investigated if thrombin-induced FAK activation is mediated by PI3K/PKCδ signaling pathway. To this end, we examined the effect of PI3K/PKCδ

PT

inhibition on thrombin-induced FAK Y576/577 phosphorylation. As shown in Figure 6A, inhibition of PI3K, an upstream activator of PKCδ by wortmannin prevented

CE

thrombin stimulation of FAK Y576/577 phosphorylation. Moreover, PKCδ inhibition by the myristoylated peptide pseudosubstrate (myristic acid–Ser-Ile-Tyr-Arg-Arg-

AC

Gly-Ala-Arg-Arg-Trp-Arg-Lys-Leu) also prevented thrombin-induced FAK Y576/577 phosphorylation. (Figure 6B). Of notice, PKCδ inhibitory pseudosubstrate (PS) not only prevented thrombin-induced FAK phosphorylation, but apparently decreased total FAK expression. Hence, in order to discard a non-specific effect of PKCδ PS on RPE cell survival or FAK gene expression, the viability of RPE cells and of FAK gene (PTK2) transcription following wortmannin or PKCδ inhibitory PS treatment was assessed. As shown in Supplementary Figure 1B and 1F, neither wortmannin nor PKCδ-PS affected cell survival or FAK gene expression.

13

ACCEPTED MANUSCRIPT Gq-coupled receptors have been shown to regulate the activity of Rho/ROCK, known to play critical regulatory roles in cytoskeletal rearrangements underlying changes in cell shape, motility, and polarization [20]. Furthermore, PKCδ has been proposed as the downstream effector of Rho/ROCK leading to actin polymerization [45] and cell migration [46]. Since the inhibition of PKCδ prevented thrombininduced FAK phosphorylation (Figure 6B), we tested the effect of Rho/ROCK

T

inhibition on thrombin-induced FAK Y576/577 phosphorylation. Results in Figure

IP

6C show that the inhibition of ROCK by the specific inhibitor Y27632 completely

CR

prevented thrombin effect.

US

3.6 The inhibition of c/n PKC isoforms induces hyperphosphorylation of FAK at Y576/577.

AN

PAR 1 coupling to members of the Gq family of G proteins results in the activation of PLC-β and the downstream activation of c/n PKC isoforms [13]. Our previous

M

work has demonstrated that PAR1 activation by thrombin induces PLCβ/ c/nPKC signaling in RPE cells [10]. In order to analyze the possible contribution of c/nPKC

ED

isoforms to thrombin-induced FAK Y576/577 phosphorylation, we tested the effect of pharmacologic inhibition of c/nPKC isoforms on thrombin-induced FAK

PT

phosphorylation. Unexpectedly, FAK Y576/577 phosphorylation was significantly increased by the specific c/nPKC inhibitor Ro 32-0432 alone (Figure 7A).

CE

Furthermore, stimulation by the PKC inhibitor showed to be additive with stimulation of FAK phosphorylation induced by thrombin. To further confirm this

AC

result, we tested the effect of the broad action PKC inhibitor staurosporine. Figure 7B shows that staurosporine alone, promotes FAK phoshorylation to the same level as thrombin; however, the joint inclusion of thrombin and staurosporine stimulated FAK phosphorylation to the same level as the individual compounds, indicating a synergistic mechanism. These results suggest that stimulation of FAK Y576/577 phosphorylation by Ro 32-0432 and staurosporine is achieved through distinct molecular mechanisms, which adds novel information regarding the specificity of c/nPKC inhibitors, possibly related to the cell type examined (RPE). Cell viability was not compromised by the inhibitory agents (Supplementary Figure 14

ACCEPTED MANUSCRIPT 1C-E). On this line, DMSO used as a solvent for inhibitor stock solutions, had no effect on FAK phosphorylation (Supplementary Figure 1A) or cell viability (Data not shown).

3.7 Thrombin-induced RPE cell migration is prevented by the inhibition of c/nPKC isoforms.

T

Since inhibition of c/nPKC by Ro 32-0432 or staurosporine significantly increased

IP

FAK phosphorylation (Figure 8), we next analyzed if FAK hyperphosphorylation

CR

translated into RPE cell migration in a wound-healing assay. Results in Figure 8 clearly show that although inhibition of c/nPKC inhibitors induced FAK

US

phosphorylation to a higher level than thrombin, c/n PKC inhibition did not promote cell migration, suggesting the involvement of c/nPKC in the control of FA turnover.

AN

4. Discussion.

M

Cell migration is a highly complex and regulated process in which intracellular and extracellular signals conjoin to produce a coordinated response [47-49]. As a

ED

consequence of ocular damage involving BRB breakdown, exposure of RPE cells to blood-contained thrombin is a common feature in the development of PVR

PT

following trauma, retinal detachment, metabolic alterations in diabetes and retinal surgery aimed to reattach the retina [2, 50]. Although thrombin-induced alteration

CE

of endothelial cell functions has been extensively studied [51], RPE responses to this protease are still not clear.

AC

PVR is characterized by the epithelial–mesenchymal transformation (EMT) and uncontrolled proliferation of otherwise quiescent RPE cells which, upon the assembly of actin stress fibers, migrate to the vitreous and generate contractile cellular membranes on both surfaces of the retina. The contraction of transformed RPE cells causes retinal detachment [2]. In spite of abundant evidence supporting FAK participation in cancer cell transformation and metastasis [36], the possible role of FAK in diseases involving RPE cell transformation and migration has not been established. Based on our previous work demonstrating the induction of RPE cell migration by thrombin [40], the aim of the present work was to investigate the 15

ACCEPTED MANUSCRIPT participation of FAK in this effect in order to gain insight into the molecular mechanisms involved in thrombin induction of RPE cell migration. We showed that the specific activation of PAR1 by thrombin concentrations found in blood serum (2U/ml) induces FAK phosphorylation at Y397 and the further phosphorylation at Y576/577 within the catalytic site carried by Src, both required for the full activation of FAK, the disassembly of FAs, and the promotion of cell

T

movement. In fact, we showed that FAK expression is essential for thrombin

IP

induction of RPE cell transformation (Figure 5) and migration (Figure 3).

CR

The coordinated interaction of PKC family isoforms has been implicated in a wide range of cellular responses [52], including cancer cell transformation and the

US

achievement of migratory properties [53-56]. Among PAR1-activated pathways, PKC is known to play an important role in cell migration [40, 42].

AN

The PKC family of serine/ threonine kinases includes 12 isoforms classified in 3 subfamilies, based on their activation mechanism: conventional isoforms α, β1, β2,

M

and γ (cPKC) activated by diacylglycerol (DAG) and calcium, novel ε, δ, ζ, ε isoforms (nPKC) activated by DAG in a calcium-independent manner, and atypical

ED

isoforms δ and η/λ (aPKC), which are not responsive to either DAG or Ca2+ [35] and instead, possess a Phox–Bem (PB)1 domain that facilitates interactions with

PT

scaffolding proteins leading to constitutive activation. Ten of these isoforms: PKCα, PKCβI, PKCβII, PKCδ, PKCɛ, PKCζ, PKCμ, PKCξ, PKC1, PKCδ and PKCη are

CE

expressed in cultured human RPE cells [57]. Due to the known role of FAK in cell movement and migration [42], we first

AC

demonstrated that thrombin stimulation of actin stress fiber assembly and migration of RPE cells are mediated by the promotion of FAK phosphorylation/activation, since they were prevented by FAK inhibition (Figure 5) or FAK suppression (Figure 3). The analysis of PKC isoform participation in thrombin-induced FAK Y576/577 phosphorylation demonstrated that thrombin effect is mediated by the activation of PI3K/PKCδ. Moreover, activation of FAK by PI3K/PKCδ signaling showed to be responsible for thrombin-induced RPE cell migration since it was prevented by the inhibition of PKC-δ, or that of its known upstream activator PI3K. Also on this line, FAK has been shown to regulate cell migration by modulating the turnover of focal 16

ACCEPTED MANUSCRIPT adhesions and the assembly of actin cytoskeleton through the activation of RhoA and its downstream effector ROCK [20, 45, 58]. FAK activates small GTPases by directly binding to- and phosphorylating their exchange factors. FAK binds to p190RhoGEF directly via a sequence within the FAT domain, and co-expression of the two molecules results in enhancement of their phosphorylation as well as GTP loading of Rho [59]. Thrombin activation of PAR1-coupled G12/13 has been shown

T

to promote the activation of Rho/ROCK, leading to the assembly of actin stress

IP

fibers, the increase in cell contractility and migration [60, 61]. Furthermore, PKC-δ

polymerization

[45,

62].

Our

results

show

CR

has been proposed as the downstream effector of Rho, leading to actin that

thrombin-induced

FAK

US

phosphorylation is prevented by the inhibition of PKCδ (Figure 6B) or by that of its putative activators PI3K (Figure 6A) and ROCK (Figure 6C). This is consistent with

AN

our previous work demonstrating that thrombin promotes actin stress fiber formation in RPE cells through PI3K/PKCδ and also by Rho/ROCK-mediated MLC

M

phosphorylation [12]. Moreover, studies in cancer cells have shown that activation of PAR1 promotes RhoA activation and the reorganization of FAs leading to cell

ED

contraction, decreased cell adhesion, and cell migration [46]. A novel finding of this work was that the inhibition of c/nPKC isoforms significantly FAK

Y576/577

phosphorylation

(Figure

7).

Since

tyrosine

PT

increased

dephosphorylation is as important as tyrosine phosphorylation in regulating the

CE

signaling events at focal adhesions, a possible explanation for this result could be that c/nPKC activity is required for maintaining the basal (non-stimulated) level of

AC

FAK phosphorylation through a dephosphorylation process. Two tyrosine phosphatases implicated in fibroblast migration: SHP-2 and PTEN are known to regulate the phosphorylation level of FAK [63]. Specifically, SHP-2 regulates FAK activation by dephosphorylating Y397, the auto-phosphorylation site that primes FAK function, thus preventing the subsequent phosphorylation of Y576/577 carried by Src [64-67]. Moreover, phosphorylation by PKC isoforms α, β1, β2, and η has been shown to activate SHP-2 by a process completely blocked by the PKC inhibitor bisindolylmaleimide [68, 69], which would explain the increase in

17

ACCEPTED MANUSCRIPT phosphorylated FAK observed in our system upon the inhibition of c/nPKC by the bisindolylmaleimide Ro-32-0432 (Figure 7A). Unexpectedly, in spite of the requirement of FAK phosphorylation for thrombin induction of focal adhesion disassembly (Figure 4), the highly significant phosphorylation of FAK upon c/nPKC inhibition did not promote RPE cell migration (Figure 8). On this regard, in parallel with the disassembly of FAs at the rear end,

T

cell migration requires the assembly of new FAs at the leading edge of the cell;

IP

hence, the hyperphosphorylation of FAK induced by c/n PKC inhibition, which

CR

results in the cytoplasmic retention of FAK [70], could hamper FAK recruitment into new adhesions at the leading edge, thus preventing cell migration. Collectively,

US

these results unveil a novel mechanism by which PKCδ and c/nPKC isoforms may regulate thrombin-induced focal adhesion turnover and migration of RPE cells

AN

through the regulation of FAK phosphorylation.

In summary, we demonstrate for the first time, that thrombin promotes RPE cell

M

morphologic transformation and migration through the phosphorylation of FAK, leading to the downstream assembly of actin stress fibers, the disassembly of focal

ED

adhesions, and the induction of cell migration. These effects showed to be differentially modulated by PKCδ and c/nPKC isoforms, suggesting that the control

PT

of FAK phosphorylation is critical for the induction of RPE cell migration upon exposure to thrombin due to BRB alteration, thus providing a putative target for the

AC

Declarations

CE

design of pharmacological treatments aimed to the prevention of PVR.

Competing interests The authors declare that they have no competing interests

Funding This work was partially supported by Consejo Nacional de Ciencia y Tecnología (CONACyT; grant 254333) and Programa de Apoyo a Proyectos de Investigación

18

ACCEPTED MANUSCRIPT e

Innovación

Tecnológica/

Universidad

Nacional

Autónoma

de

México

(PAPIIT/UNAM; grant IN20015) to A.M.L-C.

Authors' contributions A.M.L-C. contributed the conception, design, writing and critical review of the manuscript, as well as the final approval of the version to be published. E.D.A-S,

T

performed the experiments. I.L-R contributed to drafting and critical review of the

IP

manuscript. A.A-A performed some experiments and contributed to the preparation

CR

of the figures, and E.L. performed experiments and contributed to the drafting of

US

the manuscript.

References

AN

[1] O. Strauss, The retinal pigment epithelium in visual function, Physiological reviews, 85 (2005) 845-881.

M

[2] J.C. Pastor, E.R. de la Rua, F. Martin, Proliferative vitreoretinopathy: risk factors and pathobiology, Progress in retinal and eye research, 21 (2002) 127-144.

ED

[3] N.S. Harhaj, D.A. Antonetti, Regulation of tight junctions and loss of barrier function in pathophysiology, The international journal of biochemistry & cell biology,

PT

36 (2004) 1206-1237.

[4] S. Hayashi, K. Takeuchi, S. Suzuki, T. Tsunoda, C. Tanaka, Y. Majima, Effect

(2006) 46-52.

CE

of thrombin on permeability of human epithelial cell monolayers, Pharmacology, 76

AC

[5] R.P. Casaroli-Marano, R. Pagan, S. Vilaro, Epithelial-mesenchymal transition in proliferative vitreoretinopathy: intermediate filament protein expression in retinal pigment epithelial cells, Investigative ophthalmology & visual science, 40 (1999) 2062-2072.

[6] J.P. Thiery, J.P. Sleeman, Complex networks orchestrate epithelialmesenchymal transitions, Nature reviews. Molecular cell biology, 7 (2006) 131-142. [7] J. Bastiaans, J.C. van Meurs, V.C. Mulder, N.M. Nagtzaam, M. Smits-te Nijenhuis, D.C. Dufour-van den Goorbergh, P.M. van Hagen, H. Hooijkaas, W.A.

19

ACCEPTED MANUSCRIPT Dik, The role of thrombin in proliferative vitreoretinopathy, Investigative ophthalmology & visual science, 55 (2014) 4659-4666. [8] E.W. Davie, J.D. Kulman, An overview of the structure and function of thrombin, Seminars in thrombosis and hemostasis, 32 Suppl 1 (2006) 3-15. [9] S.F. Hackett, J.H. Singer, K.H. Leschey, P.A. Campochiaro, Thrombin Is a Stimulator of Retinal-Pigment Epithelial-Cell Proliferation, Experimental eye

T

research, 53 (1991) 95-100.

IP

[10] J.P. Palma-Nicolas, E. Lopez, A.M. Lopez-Colome, PKC isoenzymes

CR

differentially modulate the effect of thrombin on MAPK-dependent RPE proliferation, Bioscience Rep, 28 (2008) 307-317.

US

[11] A. Parrales, J.P. Palma-Nicolas, E. Lopez, A.M. Lopez-Colome, Thrombin stimulates RPE cell proliferation by promoting c-Fos-mediated cyclin D1

AN

expression, Journal of cellular physiology, 222 (2010) 302-312. [12] A.Y. Ruiz-Loredo, E. Lopez, A.M. Lopez-Colome, Thrombin promotes actin

M

stress fiber formation in RPE through Rho/ROCK-mediated MLC phosphorylation, Journal of cellular physiology, 226 (2011) 414-423.

ED

[13] S.R. Coughlin, Thrombin signalling and protease-activated receptors, Nature, 407 (2000) 258-264.

PT

[14] V.S. Ossovskaya, N.W. Bunnett, Protease-activated receptors: Contribution to physiology and disease, Physiological reviews, 84 (2004) 579-621.

CE

[15] H. Lin, A.P. Liu, T.H. Smith, J. Trejo, Cofactoring and dimerization of proteinase-activated receptors, Pharmacological reviews, 65 (2013) 1198-1213.

AC

[16] M. Nakanishi-Matsui, Y.W. Zheng, D.J. Sulciner, E.J. Weiss, M.J. Ludeman, S.R. Coughlin, PAR3 is a cofactor for PAR4 activation by thrombin, Nature, 404 (2000) 609-+.

[17] H. Wang, J.J. Ubl, R. Stricker, G. Reiser, Thrombin (PAR-1)-induced proliferation in astrocytes via MAPK involves multiple signaling pathways, American journal of physiology. Cell physiology, 283 (2002) C1351-1364. [18] A. Schmidt, M.N. Hall, Signaling to the actin cytoskeleton, Annual review of cell and developmental biology, 14 (1998) 305-338.

20

ACCEPTED MANUSCRIPT [19] B. Klages, U. Brandt, M.I. Simon, G. Schultz, S. Offermanns, Activation of G(12)/G(13) results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets, J Cell Biol, 144 (1999) 745-754. [20] H. Chikumi, J. Vazquez-Prado, J.M. Servitja, H. Miyazaki, J.S. Gutkind, Potent activation of RhoA by Galpha q and Gq-coupled receptors, The Journal of biological chemistry, 277 (2002) 27130-27134.

T

[21] S.R. Macfarlane, M.J. Seatter, T. Kanke, G.D. Hunter, R. Plevin, Proteinase-

IP

activated receptors, Pharmacological reviews, 53 (2001) 245-282.

CR

[22] T. Thennes, D. Mehta, Heterotrimeric G proteins, focal adhesion kinase, and endothelial barrier function, Microvasc Res, 83 (2012) 31-44.

US

[23] D.J. Webb, K. Donais, L.A. Whitmore, S.M. Thomas, C.E. Turner, J.T. Parsons, A.F. Horwitz, FAK-Src signalling through paxillin, ERK and MLCK regulates

AN

adhesion disassembly, Nat Cell Biol, 6 (2004) 154-+.

[24] M. Schober, S. Raghavan, M. Nikolova, L. Polak, H.A. Pasolli, H.E. Beggs, L.F.

M

Reichardt, E. Fuchs, Focal adhesion kinase modulates tension signaling to control actin and focal adhesion dynamics, J Cell Biol, 176 (2007) 667-680.

ED

[25] M.D. Schaller, J.D. Hildebrand, J.D. Shannon, J.W. Fox, R.R. Vines, J.T. Parsons, Autophosphorylation of the focal adhesion kinase, pp125FAK, directs

PT

SH2-dependent binding of pp60src, Molecular and cellular biology, 14 (1994) 1680-1688.

CE

[26] B.S. Cobb, M.D. Schaller, T.H. Leu, J.T. Parsons, Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase,

AC

pp125FAK, Molecular and cellular biology, 14 (1994) 147-155. [27] H.C. Chen, P.A. Appeddu, H. Isoda, J.L. Guan, Phosphorylation of tyrosine 397 in focal adhesion kinase is required for binding phosphatidylinositol 3-kinase, Journal of Biological Chemistry, 271 (1996) 26329-26334. [28] X. Zhang, A. Chattopadhyay, Q.S. Ji, J.D. Owen, P.J. Ruest, G. Carpenter, S.K. Hanks, Focal adhesion kinase promotes phospholipase C-gamma1 activity, Proceedings of the National Academy of Sciences of the United States of America, 96 (1999) 9021-9026.

21

ACCEPTED MANUSCRIPT [29] M.B. Calalb, T.R. Polte, S.K. Hanks, Tyrosine Phosphorylation of Focal Adhesion Kinase at Sites in the Catalytic Domain Regulates Kinase-Activity - a Role for Src Family Kinases, Molecular and cellular biology, 15 (1995) 954-963. [30] J.T. Parsons, Focal adhesion kinase: the first ten years, J Cell Sci, 116 (2003) 1409-1416. [31] J.D. Owen, P.J. Ruest, D.W. Fry, S.K. Hanks, Induced focal adhesion kinase

T

(FAK) expression in FAK-null cells enhances cell spreading and migration requiring

IP

both auto- and activation loop phosphorylation sites and inhibits adhesion-

CR

dependent tyrosine phosphorylation of Pyk2, Molecular and cellular biology, 19 (1999) 4806-4818.

US

[32] D.D. Schlaepfer, C.R. Hauck, D.J. Sieg, Signaling through focal adhesion kinase, Prog Biophys Mol Bio, 71 (1999) 435-478.

AN

[33] D.D. Schlaepfer, S.K. Hanks, T. Hunter, P. van der Geer, Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion

M

kinase, Nature, 372 (1994) 786-791.

[34] J.C. Wu, Y.C. Chen, C.T. Kuo, H.W. Yu, Y.Q. Chen, A. Chiou, J.C. Kuo, Focal

ED

adhesion kinase-dependent focal adhesion recruitment of SH2 domains directs SRC into focal adhesions to regulate cell adhesion and migration, Sci Rep-Uk, 5

PT

(2015).

[35] Y.Q. Wu, K.W. Zhang, J. Seong, J. Fan, S. Chien, Y.X. Wang, S.Y. Lu, In-situ

CE

coupling between kinase activities and protein dynamics within single focal adhesions, Sci Rep-Uk, 6 (2016).

AC

[36] G.W. McLean, N.O. Carragher, E. Avizienyte, J. Evans, V.G. Brunton, M.C. Frame, The role of focal-adhesion kinase in cancer - a new therapeutic opportunity, Nature reviews. Cancer, 5 (2005) 505-515. [37] M. Luo, J.L. Guan, Focal adhesion kinase: A prominent determinant in breast cancer initiation, progression and metastasis, Cancer Lett, 289 (2010) 127-139. [38] J. Zhao, J.L. Guan, Signal transduction by focal adhesion kinase in cancer, Cancer metastasis reviews, 28 (2009) 35-49.

22

ACCEPTED MANUSCRIPT [39] Y.L. Hu, S.Y. Lu, K.W. Szeto, J. Sun, Y.X. Wang, J.C. Lasheras, S. Chien, FAK and paxillin dynamics at focal adhesions in the protrusions of migrating cells, Sci Rep-Uk, 4 (2014). [40] J.P. Palma-Nicolas, E. Lopez, A.M. Lopez-Colome, Thrombin stimulates RPE cell motility by PKC-zeta- and NF-kappaB-dependent gene expression of MCP-1 and CINC-1/GRO chemokines, Journal of cellular biochemistry, 110 (2010) 948-

T

967.

IP

[41] R.L. Pacheco-Dominguez, J.P. Palma-Nicolas, E. Lopez, A.M. Lopez-Colome,

CR

The activation of MEK-ERK1/2 by glutamate receptor-stimulation is involved in the regulation of RPE proliferation and morphologic transformation, Experimental eye

US

research, 86 (2008) 207-219.

[42] S.K. Mitra, D.A. Hanson, D.D. Schlaepfer, Focal adhesion kinase: In command

AN

and control of cell motility, Nat Rev Mol Cell Bio, 6 (2005) 56-68. [43] D.J. Webb, J.T. Parsons, A.F. Horwitz, Adhesion assembly, disassembly and

M

turnover in migrating cells - over and over and over again, Nat Cell Biol, 4 (2002) E97-E100.

ED

[44] M.M. Chou, W. Hou, J. Johnson, L.K. Graham, M.H. Lee, C.S. Chen, A.C. Newton, B.S. Schaffhausen, A. Toker, Regulation of protein kinase C zeta by PI 3-

PT

kinase and PDK-1, Current biology : CB, 8 (1998) 1069-1077. [45] C. Laudanna, D. Mochly-Rosen, T. Liron, G. Constantin, E.C. Butcher,

CE

Evidence of zeta protein kinase C involvement in polymorphonuclear neutrophil integrin-dependent adhesion and chemotaxis, The Journal of biological chemistry,

AC

273 (1998) 30306-30315. [46] R.D. Loberg, K. Tantivejkul, M. Craig, C.K. Neeley, K.J. Pienta, PAR1mediated RhoA activation facilitates CCL2-induced chernotaxis in PC-3 cells, Journal of cellular biochemistry, 101 (2007) 1292-1300. [47] D.A. Lauffenburger, A.F. Horwitz, Cell migration: A physically integrated molecular process, Cell, 84 (1996) 359-369. [48] A.J. Ridley, M.A. Schwartz, K. Burridge, R.A. Firtel, M.H. Ginsberg, G. Borisy, J.T. Parsons, A.R. Horwitz, Cell migration: Integrating signals from front to back, Science, 302 (2003) 1704-1709. 23

ACCEPTED MANUSCRIPT [49] M. Vicente-Manzanares, D.J. Webb, A.R. Horwitz, Cell migration at a glance, J Cell Sci, 118 (2005). [50] T. Sakamoto, H. Sakamoto, S.J. Sheu, K. Gabrielian, S.J. Ryan, D.R. Hinton, Intercellular Gap Formation Induced by Thrombin in Confluent Cultured Bovine Retinal-Pigment Epithelial-Cells, Investigative ophthalmology & visual science, 35 (1994) 720-729.

T

[51] A.A. Birukova, K. Smurova, K.G. Birukov, K. Kaibuchi, J.G.N. Garcia, A.D.

CR

barrier dysfunction, Microvasc Res, 67 (2004) 64-77.

IP

Verin, Role of Rho GTPases in thrombin-induced lung vascular endothelial cells

[52] S.F. Steinberg, Structural basis of protein kinase C isoform function,

US

Physiological reviews, 88 (2008) 1341-1378.

[53] E.M. Griner, M.G. Kazanietz, Protein kinase C and other diacylglycerol

AN

effectors in cancer, Nature reviews. Cancer, 7 (2007) 281-294. [54] H. Guo, F. Gu, W. Li, B. Zhang, R. Niu, L. Fu, N. Zhang, Y. Ma, Reduction of

M

protein kinase C zeta inhibits migration and invasion of human glioblastoma cells, Journal of neurochemistry, 109 (2009) 203-213.

ED

[55] R.H. Sun, P. Gao, L. Chen, D.L. Ma, J.M. Wang, J.J. Oppenheim, N. Zhang, Protein kinase C zeta is required for epidermal growth factor-induced chemotaxis

PT

of human breast cancer cells, Cancer Res, 65 (2005) 1433-1441. [56] Y. Liu, B. Wang, J.N. Wang, W.Z. Wan, R.H. Sun, Y.L. Zhao, N. Zhang, Down-

CE

regulation of PKC zeta expression inhibits chemotaxis signal transduction in human lung cancer cells, Lung Cancer, 63 (2009) 210-218.

AC

[57] K. Yu, P. Ma, J. Ge, C.D. Willey, P. Yang, Z. Wang, Q. Gao, Expression of protein kinase C isoforms in cultured human retinal pigment epithelial cells, Graefe's archive for clinical and experimental ophthalmology = Albrecht von Graefes Archiv fur klinische und experimentelle Ophthalmologie, 245 (2007) 993999. [58] S. Etienne-Manneville, A. Hall, Rho GTPases in cell biology, Nature, 420 (2002) 629-635. [59] M.A. Wozniak, K. Modzelewska, L. Kwong, P.J. Keely, Focal adhesion regulation of cell behavior, Biochimica et biophysica acta, 1692 (2004) 103-119. 24

ACCEPTED MANUSCRIPT [60] D.L. Greenberg, G.J. Mize, T.K. Takayama, Protease-activated receptor mediated RhoA signaling and cytoskeletal reorganization in LNCaP cells, Biochemistry-Us, 42 (2003) 702-709. [61] M. Raftopoulou, A. Hall, Cell migration: Rho GTPases lead the way, Dev Biol, 265 (2004) 23-32. [62] J. Gomez, A. Garcia, L.R. Borlado, P. Bonay, C. MartinezA, A. Silva, M.

T

Fresno, A.C. Carrera, C. EicherStreiber, A. Rebollo, IL-2 signaling controls actin

CR

protein kinase C-zeta, J Immunol, 158 (1997) 1516-1522.

IP

organization through Rho-like protein family, phosphatidylinositol 3-kinase, and

[63] A. Angers-Loustau, J.F. Cote, M.L. Tremblay, Roles of protein tyrosine

US

phosphatases in cell migration and adhesion, Biochemistry and cell biology = Biochimie et biologie cellulaire, 77 (1999) 493-505.

AN

[64] Z.R. Hartman, M.D. Schaller, Y.M. Agazie, The tyrosine phosphatase SHP2 regulates focal adhesion kinase to promote EGF-induced lamellipodia persistence

M

and cell migration, Molecular cancer research : MCR, 11 (2013) 651-664. [65] S. Manes, E. Mira, C. Gomez-Mouton, Z.Z.J. Zhao, R.A. LaCalle, C. Martinez-

ED

A, Concerted activity of tyrosine phosphatase SHP-2 and focal adhesion kinase in regulation of cell motility, Molecular and cellular biology, 19 (1999) 3125-3135.

PT

[66] K. Inagaki, T. Noguchi, T. Matozaki, T. Horikawa, K. Fukunaga, M. Tsuda, M. Ichihashi, M. Kasuga, Roles for the protein tyrosine phosphatase SHP-2 in organization,

cell

adhesion

and

cell

migration

revealed

by

CE

cytoskeletal

overexpression of a dominant negative mutant, Oncogene, 19 (2000) 75-84.

AC

[67] D.H. Yu, C.K. Qu, O. Henegariu, X.L. Lu, G.S. Feng, Protein-tyrosine phosphatase Shp-2 regulates cell spreading, migration, and focal adhesion, Journal of Biological Chemistry, 273 (1998) 21125-21131. [68] V. Strack, J. Krutzfeldt, M. Kellerer, A. Ullrich, R. Lammers, H.U. Haring, The protein-tyrosine-pbosphatase SHP2 is phosphorylated on serine residues 576 and 591 by protein kinase C Isoforms alpha, beta 1, beta 2, and eta, Biochemistry-Us, 41 (2002) 603-608. [69] K. Mussig, H. Staiger, H. Fiedler, K. Moeschel, A. Beck, M. Kellerer, H.U. Haring, Shp2 is required for protein kinase C-dependent phosphorylation of serine 25

ACCEPTED MANUSCRIPT 307 in insulin receptor substrate-1, Journal of Biological Chemistry, 280 (2005) 32693-32699. [70] A. Hamadi, T.B. Deramaudt, K. Takeda, P. Ronde, Hyperphosphorylated FAK Delocalizes from Focal Adhesions to Membrane Ruffles, Journal of oncology, 2010 (2010).

IP

T

Figure Legends

CR

Figure 1. Thrombin induces FAK phosphorylation at tyrosine residues 397 and 576/577 in RPE cells. Rat RPE cells in primary culture were serum-deprived for 24 hours prior to stimulation with 2 U/ml thrombin. FAK phosphorylation was

US

analyzed by Western Blot, as described in Methods. Thrombin promotes Y397 phosphorylation at 2 min stimulation (A) and of Y576/577 at 5 min stimulation,

AN

which was sustained up to 60 min (B). (C) Y576/577 phosphorylation is dosedependent: maximal phosphorylation was attained by addition of 2 U/ml thrombin.

M

Values for non-stimulated cultures maintained in serum-free OPTIMEM were set as 100%. Data were normalized relative to total FAK expression. Data are expressed **

p<0.01, p< 0.05,

***

p< 0.001 Student’s t-test, compared to control.

PT

*

ED

as the mean ± SEM of three independent experiments performed in triplicate.

CE

Figure 2. Thombin specific activation of PAR 1 induces FAK phosphorylation in RPE. Confluent cultures of RPE cells were serum-deprived for 24 hours prior to

AC

stimulation with thrombin (2 U/ml) or 50 μM PAR-1, -3 or -4 agonist peptides (AP) for 5 min. FAK phosphorylation was assessed by Western blot. Values for control, non-stimulated cultures were set as 100%. (A) Thrombin stimulation of FAK phosphorylation at Y576/577 was prevented by the inclusion of the specific thrombin inhibitors Hirudin (H) and PPACK (PP) 30 min. prior to stimulation. FBS was included as positive control. (B) PAR 1 Agonist Peptide (PAR 1 AP) mimicked thrombin effect; PAR 3 or PAR 4 APs had no effect. Results are expressed as the *

mean ± SEM of three independent experiments performed in triplicate. p<0.01, **

p< 0.05,

26

***

p< 0.001, Student’s t-test, referred to control. (C) Thrombin stimulation

ACCEPTED MANUSCRIPT of FAK phosphorylation was prevented by the PAR-1 inhibitor SCH79797 (30μM), and suppressed by FAK siRNA transfection prior to thrombin stimulation. Results are expressed as the mean ± SEM of three independent experiments performed in triplicate.* p<0.01,

**

p< 0.05,

***

p< 0.001, Student’s t-test, referred to thrombin-

stimulation.

T

Figure 3. FAK expression is essential for thrombin induction of RPE cell

IP

migration. (A) FAK siRNA suppressed FAK expression by ~50% at 24 hours. (B)

CR

Confluent RPE cell cultures were serum-deprived for 24 hours prior to stimulation with 2 U/ml thrombin or 4% FBS (positive control). Cell migration was measured in

US

a “scratch-wound” assay. Mitomycin C (1 μg/ml) was included to discard the contribution of proliferation to the closure of the wound. Treatment with thrombin or

AN

FBS promoted cell migration into the wounded area in control cultures. Thrombininduced cell migration was abolished by FAK suppression. Cultures were fixed,

M

stained and photographed. (C) Graphical representation of the data obtained in (B). Results are the mean ± SEM of three independent experiments performed in *

**

***

p< 0.001, one-way ANOVA, Dunnett´s post- hoc.

ED

triplicate. p<0.01, p< 0.05,

PT

Figure 4. Thrombin promotes the disassembly of adhesion complexes through the phosphorylation of FAK. Primary confluent cultures of RPE cells

CE

were serum-deprived for 24 hrs prior to stimulation by 2 U/ml thrombin for 5 min. FAK expression was suppressed by FAK siRNA transfection using Lipofectamine

AC

RNAi-MAX for 24 hrs. The effect of thrombin on the disassembly of focal adhesion complexes was assessed by the loss of FAK/paxillin and FAK/vinculin interaction using co-immunoprecipitation. Exposure of RPE cells to thrombin abolished FAK interaction with vinculin (A) and paxillin (B). FAK suppression by siRNA induced FAK dissociation from vinculin and paxillin, indicating a thrombin-induced, FAKmediated effect. Association in non-stimulated cultures was set as 100% (control). Results are expressed as the mean ± SEM of three independent experiments *

**

performed in triplicate. p<0.01, p< 0.05, post-hoc. 27

***

p< 0.001, One-way ANOVA, Dunnett´s

ACCEPTED MANUSCRIPT

Figure 5. Thrombin-induced assembly of actin stress fibers requires FAK activation. (A) RPE cells were serum-starved for 24 h and treated for 20 min. with the FAK inhibitor F14 prior to the addition of 2 U/ml thrombin for 5 min. Cells were fixed and stained for F-actin using rhodamine-conjugated phalloidin. (B) Relative quantification of the F-actin fluorescence modification induced by thrombin. Results

T

are the mean ±SEM of three independent experiments. *P< 0.05, **P< 0.01

CR

IP

Student’s t-test relative to thrombin stimulation.

Figure 6. Thrombin stimulation of FAK Y576/577 phosphorylation requires

US

PI3K/PKCζ activity. RPE cells were serum-starved for 24 hours prior to the inclusion of the specific inhibitors for 20 min. Following pre-incubation with the

AN

inhibitors, cells were stimulated with 2 U/ml thrombin for 5 min. Relative FAK phosphorylation was determined by Western blot. FAK phosphorylation at Y576/577 was assessed in the absence (control) or in the presence of (A): 100 nM

M

of the PI3K inhibitor wortmannin (W); (B): 10 μM of the Inhibitor Myristoylated

ED

Pseudosubstrate peptide of PKCδ (PS), or (C): Rho/ROCK inhibitor Y27632 (10μM). Results show that the inhibition of PI3K or of its downstream target PKCδ

PT

completely prevented thrombin effect. Rho/ROCK activation is required for thrombin-induced Y576/577 FAK phosphorylation. Values for thrombin stimulation

CE

were set as 100%. Data are expressed as the mean ± SEM of three independent experiments performed in triplicate.

***

p< 0.001, Student’s t-test, referred to

AC

thrombin stimulation.

Figure 7. Inhibition of c/nPKC induces FAK Y576/577 hyperphosphorylation. Confluent cultures of RPE cells were serum-deprived for 24 hrs. (A): Cells were pre-incubated with the specific c/nPKC inhibitor Ro 32-0432 (10μM) for 20 min. prior to stimulation with 2 U/ml thrombin for 5 min. (B): RPE cells were preincubated for 20 min. with increasing doses of the broad-spectrum c/nPKC inhibitor staurosporine (2.5-10 nM) prior to thrombin addition. Whole cell extracts were immunoblotted using specific anti-phospho Y576/577 FAK antibody. Data 28

ACCEPTED MANUSCRIPT are expressed as the mean ± SEM of three independent experiments performed in **

triplicate. p< 0.05,

***

p< 0.001, unpaired Student’s t-test, referred to control.

Figure 8. c/n PKC inhibition prevents thrombin-induced RPE cell migration Cultures were serum-deprived for 24 hrs prior to stimulation by 2 U/ml thrombin for

T

24 hrs. (A): Cell migration was assessed in a “wound-healing” assay in the

IP

absence (control) or presence of 10 μM of the specific c/nPKC inhibitor Ro32-0432 for 20 min prior to stimulation or 24 hrs alongside with thrombin. Cultures were

CR

fixed, stained and photographed in order to visually count migrated cells. Graphical representation of results is shown in (B). Values in unstimulated cultures were set

US

as 100%. Results are the mean ± SEM of three independent experiments. ****P <

AC

CE

PT

ED

M

AN

0.0001 two-way ANOVA, Dunnett’s post- hoc.

29

CE

AC

Figure 1

PT

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

30

AC

Figure 2

CE

PT

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

31

AC

CE

PT

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

Figure 3

32

AC

CE

PT

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

Figure 4

33

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

AC

CE

PT

ED

Figure 5

34

AC

CE

PT

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

Figure 6

35

AC

Figure 7

CE

PT

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

36

AC

Figure 8

CE

PT

ED

M

AN

US

CR

IP

T

ACCEPTED MANUSCRIPT

37

ACCEPTED MANUSCRIPT

Highlights 

Thrombin induces FAK tyrosine phosphorylation through the specific activation of PAR 1.



Thrombin-induced FAK phosphorylation is differentially controlled by c/n PKC, PI3K/PKC-δ, and Rho/ROCK. Thrombin promotes FAK-dependent actin stress fiber assembly.



FAK expression is required for thrombin-induced RPE cell migration.

AC

CE

PT

ED

M

AN

US

CR

IP

T



38