Molecular Vision 2007; 13:2320-2327 <http://www.molvis.org/molvis/v13/a262/> Received 12 February 2007 | Accepted 2 November 2007 | Published 21 December 2007

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Phorbol 12-myristate 13-acetate-activated protein kinase C increased migratory activity of subconjunctival fibroblasts via stress-activated protein kinase pathways

Naoko Nomura,¹ Motohiro Nomura,² Masayuki Takahira,¹ Kazuhisa Sugiyama¹
 
 

¹Department of Ophthalmology and Visual Science, Kanazawa University Graduate School of Medical Science, Kanazawa, ²Department of Neurosurgery, Yokohama Sakae Kyosai Hospital, Yokohama, Japan

Correspondence to: Naoko Nomura, Department of Ophthalmology and Visual Science, Kanazawa University Graduate School of Medical Science, 13-1 Takara-machi, Kanazawa 920-8641, Japan; Phone: +81-76-265-2403; FAX: +81-76-222-9660; email: nomura413jp@yahoo.co.jp

Abstract

Purpose: The aim of this study was to clarify the mechanism of subconjunctival fibroblast migration, focusing on the effect of protein kinase C (PKC).

Methods: Subconjunctival fibroblasts were isolated from rats and cultured in 10% fetal bovine serum-containing culture medium. The fibroblasts were treated with phorbol 12-myristate 13-acetate (PMA) to activate PKC. Migration was assessed using a wound-healing assay. Western blot analysis was employed to examine activation of stress-activated protein kinase (SAPK) pathways. Immunofluorescent analysis was performed to examine the actin cytoskeleton dynamics.

Results: Seven PKC isoforms (α, β, γ, δ, ε, ι, and λ) were present in rat subconjunctival fibroblasts. PMA induced lamellipodia formation and subsequent migration of the subconjunctival fibroblasts. PMA treatment in the subconjunctival fibroblasts elicited phosphorylation of SAPKs, including c-jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAPK). Treatment of the subconjunctival fibroblasts with an inhibitor of PKC abrogated the phosphorylation of these proteins. Furthermore, specific inhibitors of JNK and p38MAPK blocked PMA-induced migration of the subconjunctival fibroblasts. The phosphorylation of c-jun and heat shock protein 27, downstream effectors of JNK and p38MAPK, respectively, was upregulated by PMA treatment.

Conclusions: PMA-activated PKC increased migratory activity, and SAPK pathways were critical for migration of subconjunctival fibroblasts.

Introduction

Glaucoma filtering surgery (GFS) is commonly performed to treat glaucoma when medication fails to control intraocular pressure adequately [1]. The main cause of failure of GFS is wound contraction and scarring at the filtering bleb site. Fibroblasts from the subconjunctival space play a fundamental role in this process through proliferation, migration, production of extracellular matrix components, and subsequent contraction of the extracellular matrix [2]. Repair and remodeling of conjunctival and subconjunctival tissue after GFS require the integration of multiple external signals from growth factors, cytokines, and chemokines [1,2].

Protein kinase C (PKC) pathway has been shown to have a profound influence on cellular activity. PKC is also to be involved in the wound healing process in several types of tissue [3,4]. PKC constitutes a multifunctional family of serine/threonine protein kinases with at least 10 different isoforms. The PKC gene family is divided into three subgroups based on sequence homology and cofactor requirements: classic-conventional PKC isoforms (PKC-α, β, and γ), novel PKC isoforms (PKC-δ, ε, ι, and η), and atypical PKC isoforms (PKC-ι, λ, and ζ). Cell signaling pathways involving the PKC family are initiated by binding of a ligand, such as a growth factor, to its respective cell surface receptor, which triggers the breakdown of phospholipids by phospholipases C and D and production of diacylglycerol (DAG). DAG binds to and activates most PKC isoforms, which then translocate to specific subcellular compartments that vary depending on the PKC isoform and cell type. Phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), are potent tumor promoters and can substitute for DAG in stimulating PKC [5]. PKCs have been described as important regulators of cytoskeletal function [6]. In endothelial cells, PKC-α have been reported to display a positive effect on endothelial cell migration [7]. In glioblastoma cells, PKC-ε was required for PMA-induced adhesion and migration [8]. The role of PKC in migration in some kinds of cells has been revealed, however, the effect of PKC on subconjunctival fibroblasts remains mostly unclear.

Recent studies have demonstrated that mitogen-activated protein kinase (MAPK) including the extracellular-signal-regulated protein kinase (ERK), c-jun N-terminal kinase (JNK), and p38MAPK play a crucial role in cell migration [9]. ERKs are predominantly activated by mitogenic factors, while JNK and p38MAPK, members of the stress-activated protein kinase (SAPK) family, are preferentially activated by stress-inducing stimuli such as ultraviolet light, heat shock, and pro-inflammatory cytokines [9].

In this study, we investigated the effect of PMA-activated PKC on subconjunctival fibroblast migration. We also analyzed the mechanism of PMA-induced migration of the subconjunctival fibroblasts.

Methods

Reagents and antibodies

PMA and bisindolylmaleimide I (BIS) were purchased from Sigma-Aldrich Corporation (St. Louis, MO). SP600125 and SB203580, inhibitors of JNK and p38MAPK, were purchased from Calbiochem (San Diego, CA). PMA, BIS, SP600125, and SB203580 were prepared as 10 mM stock solution in dimethyl sulfoxide (DMSO).

The primary antibodies used were mouse anti-p38MAPK monoclonal antibody, rabbit anti-phospho-p38MAPK (Thr180/Tyr182) polyclonal antibody, rabbit anti-JNK polyclonal antibody, rabbit anti-phospho-JNK (Thr183/Tyr185) polyclonal antibody, rabbit anti-phospho-c-jun (Ser63) polyclonal antibody, rabbit anti-phospho-c-jun (Ser73) polyclonal antibody, mouse anti-heat shock protein (Hsp) 27 monoclonal antibody, and rabbit anti-phospho-Hsp27 (Ser82) polyclonal antibody. The aforementioned antibodies were purchased from Cell Signaling Technology (Beverly, MA). A PKC sampler kit (BD Biosciences, Franklin Lakes, NJ) was used to detect PKC isoforms.

Cell cultures

All procedures were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the University of Kanazawa's Institutional Animal Care and Use Committee.

Adult male Wister rats (200-300 g) were purchased from (Ninox, Kanazawa, Japan). Rats were allowed unlimited access to water and rat chow and were exposed to a 12 h light/dark cycle and a room temperature of 24 °C. Subconjunctival fibroblasts were obtained as previously described with minor modification [10]. Briefly, subconjunctival tissues were taken from the inferior bulbar conjunctiva of 12 Wister rats. The tissue was cut into a small sections and anchored onto the bottom of 6 cm culture dishes (BD Biosciences) with a sterile coverslip and overlaid with Dulbecco's modified Eagle medium (DMEM; Sigma-Aldrich Corporation) supplemented with 10% fetal bovine serum. Subconjunctival fibroblasts were obtained from outgrowth of subconjunctival tissue. In case a colony of epithelial cells with cobble stone appearance was observed, the colony was scraped with a sterile micro-pipette tip and washed with PBS. Once the monolayers had reached subconfluence, the cells were passaged and cultured in 10-cm culture dishes. The fibroblasts were characterized morphologically and stained positively with vimentin and negatively with cytokeratines. The third- to fifth-passage cells that maintained proliferative potential and fibroblast-like elongated morphology were used for the study.

Stimulation of protein kinase C by phorbol 12-myristate 13-acetate and inhibition of kinase activity by specific inhibitors

The fibroblasts were incubated in serum-free medium for 24 h. Then, PMA was added to the serum-free culture medium, and the cells were cultured for various times.

BIS, SP600125, and SB203580 were used for inhibition of PKC, JNK, and p38MAPK, respectively. Thirty min prior to addition of PMA, 5 μM BIS was added to the culture medium. Alternatively, either SP600125 or SB203580 were similarly added to the culture medium to the concentration of 10 or 20 μM, respectively. The cells treated with inhibitors were incubated with PMA for various times and subjected to further analysis.

Evaluation of cell migration using wound healing assay

Wound-healing assay was performed as previously described with minor modification [11]. Briefly the cells were seeded in 12-well culture dishes (Asahi Techno Glass, Funabashi, Japan) and cultured until they reached confluence. The cells were then incubated with serum-free culture medium for 24 h, and scraped with a 200 μl micro-pipette tip, denuding a strip of the monolayer approximately 500 μm in diameter. Variation in the wound diameter within experiments was approximately 5%. Cultures were washed twice with PBS to remove cell debris and incubated with serum-free culture medium for an additional 16 h with PMA or vehicle. When specific inhibitors were used, an appropriate concentration of each inhibitor was added to the culture medium 30 min before PMA treatment. After incubation, the cells were photographed with a digital camera, and the migrated area was measured using NIH Image (v1.63) software. To ensure that the same wounds were compared, we used a permanent marker to make positioning marks at the bottom of the culture dishes. The migration area in the wound was calculated according to the following formula: cell free area at 0 h-cell free area at 16 h. At least 10 fields were analyzed and the migrated area was expressed as a percentage of that in untreated control cells.

Protein extraction and Western blot analysis

The cells were harvested from each culture condition at the appropriate times, and washed with ice-cold PBS. Total protein was extracted using a lysis buffer containing 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 5 μg/ml phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 5 μg/ml leupeptin, and 2 μg/ml pepstatin. The samples were centrifuged at 18000 xg for 30 min at 4 °C.

To extract of cytosolic and membrane fractions of cells, we washed the cells with PBS and harvested them in ice-cold homogenization buffer (20 mM HEPES, 2 mM EDTA, 2 mM EGTA, 50 mM NaF, 0.25 M sucrose and protease inhibitors) [12]. The cells were dounce homogenized for 15 strokes on ice and centrifuged at 15000 rpm for 45 min. The supernatant was recovered as the cytosolic fraction. The pellet was resuspended in homogenization buffer containing 1% Triton X-100 and incubated on ice for 30 min. Then the suspension was centrifuged at 15000 rpm for 45 min, and the supernatant was recovered as the membrane fraction. Extract was stored at -80 °C until use. Protein concentration was determined using the BCA assay (Pierce, Rockford, IL).

Western blot analysis was done by electrophoresing equal amounts of protein (15-20 μg) on SDS-PAGE gels, transferring them to nitrocellulose membranes (0.45 μm, Trans-Blot Transfer Medium, Bio-Rad, Hercules, CA) and staining with Ponceau S (Sigma). After the confirmation of protein transfer, proteins were detected with the specific antibodies. All primary antibodies were used at 1:1000 dilution. Actin protein was detected as a control with mouse anti-β-actin monoclonal antibody used at 1:10000 dilution (Chemicon). Sheep anti-mouse IgG and donkey anti-rabbit IgG horseradish peroxidase-linked secondary antibodies (Amersham, Piscataway, NJ) at 1:4000 dilution were used as secondary antibodies. Protein detection was performed using a SuperSignal West Femto Maximum Sensitivity Substrate (Pierce) and visualized using Hyperfilm ECL (Amersham).

Immunofluorescent analysis

Cells were plated in eight-well chamber slides and incubated overnight. After the cells attached to the slides, they were incubated with serum-free culture medium for 24 h. After a 30 min initial treatment with specific inhibitors or vehicle, 10 nM PMA was added to the culture medium and the cells were incubated for 120 min. Then the cells were fixed with 3.7% paraformaldehyde for 20 min and washed with PBS three times. The cells were permeabilized with 0.2% Triton X-100 for 5 min and washed with PBS three times. Next, cells were blocked with 2% BSA, then incubated at room temperature with Alexa fluor 488 phalloidin (Invitrogen). Samples were washed with PBS, then mounted. Alexa fluor 488 phalloidin was used for detection of F-actin, which appeared green under a fluorescence microscope.

Statistical analysis

All experiments were performed at least three times. The data were expressed as the mean±standard error of the mean (S.E.M.). Multiple comparisons were performed by ANOVA to determine the over all impact of treatment within an experiment. Additional post-hoc testing was performed by Tukey-Kramer procedure to determine the statistical significance when necessary. P-values lower than 0.05 were considered significant.

Results

Expression of protein kinase C isoforms in subconjunctival fibroblasts

As a first step to study the involvement of PKC signaling in rat subconjunctival fibroblasts, we investigated the expression of PKC isoforms. Subconjunctival fibroblasts grown in primary culture were subcultured at low density, and total proteins were extracted and evaluated for the expression of PKC isoforms by Western blot analysis. The cells were found to express three classic PKCs (α, β, and γ), two novel PKCs (δ and ε), and two atypical PKCs (ι and λ). PKC-η and θ were not detected by Western blot analysis (Figure 1). The expression of PKC-ζ was not examined in this study.

Phorbol 12-myristate 13-acetate increased migratory activity of subconjunctival fibroblasts

The migration of subconjunctival fibroblasts has important roles in wound healing [2,12,13]. PMA interacts with and activates both classical (α, β, and γ) and novel (δ, ε, η, and θ) PKC isoforms [5]. We next investigated the effect of PMA on subconjunctival fibroblast migration. As shown in Figure 2B, the fibroblasts treated with PMA migrated to the center of the wound. The fibroblasts showed the highest ability of migration at the concentration of 10 nM PMA. The migration of the PMA (10 nM)-treated cells was 4.7 times higher than that of the control cells (Figure 2A). And the PMA-stimulated migration was time-dependent (data not shown).

To confirm that PMA-induced migration was dependent on PKC activity, we initially treated cells with BIS, a selective inhibitor of both classical (α, β, and γ) and novel (δ, ε, and η) PKC isoforms [14,15], and then treated them with PMA. Initial treatment with BIS inhibited PMA-induced migration (Figure 2A). These results indicated that PKC activity was required for PMA-induced subconjunctival fibroblast migration.

Phorbol 12-myristate 13-acetate induced lamellipodia formation in subconjunctival fibroblasts

When cells migrate, dynamic reorganization of the actin cytoskeleton occurs. Actin is polymerized at the leading edge of moving cells. On cell migration, lamellipodia, which are actin filament-rich organelles, form at the leading edge of moving cells [6,9].

The effects of PMA on the actin cytoskeleton were determined by staining cells with fluorescently labeled phalloidin to detect F-actin. As shown in the middle panel of Figure 3, lamellipodia formation was observed in the subconjunctival fibroblasts 2 h after PMA treatment. This phenomenon was abrogated by initial treatment of the cells with BIS (Figure 3, right panel). These results indicated that the PMA-induced formation of lamellipodia in the subconjunctival fibroblasts was dependent on PKC activation.

Phorbol 12-myristate 13-acetate induced activation of stress-activated protein kinase in subconjunctival fibroblasts

Recent studies have demonstrated that SAPK activation plays crucial roles in cell migration [9]. Thus, we hypothesized that PMA-induced subconjunctival fibroblast migration was also dependent on SAPK activation.

To investigate the activation of JNK and p38MAPK by PMA treatment, we performed Western blot analysis. Phosphorylation-specific antibodies were used to detect the activation of JNK or p38MAPK. As shown in Figure 4, phosphorylation of both kinases was observed 5 min after PMA treatment.

BIS was added to the cells before PMA treatment to confirm whether the activation of JNK or p38MAPK was induced by PKC activation. Furthermore, inhibitors of JNK and p38MAPK were also used, and phosphorylation of the kinases was analyzed.

As shown in Figure 4A, phosphorylation of JNK was inhibited by initial treatment with BIS or SP600125. Phosphorylation of p38MAPK was also inhibited by BIS or SB203580 (Figure 4B). No significant change was observed in the expression of total JNK or p38MAPK by the treatment with PMA or the inhibitors. These results indicated that the activation of JNK and p38MAPK by PMA might be due to PKC activation.

Activation of stress-activated protein kinase was critical for phorbol 12-myristate 13-acetate-induced subconjunctival fibroblast migration

Next we investigated whether JNK as well as p38MAPK was involved in PMA-induced migration of subconjunctival fibroblasts. We analyzed PMA-induced migration of the fibroblasts treated with specific inhibitors for JNK or p38MAPK.

As shown in Figure 5, the inhibition of JNK activation by SP600125 caused a decrease of migration to 9% of that in the cells treated with PMA alone. Similarly, cell migration was also reduced to 12% of that of the PMA-treated cells by inhibition of p38MAPK by SB203580. These findings showed that PMA-induced migration of subconjunctival fibroblasts was dependent on the activation of JNK or p38MAPK.

Activation of downstream targets of c-jun N-terminal kinase

To elucidate the mechanism by which PMA induced the migration of subconjunctival fibroblasts via the SAPK pathways, we analyzed the roles of downstream effectors. Javelaud, et al. showed that c-jun phosphorylation by JNK was critical for TGF-β-induced fibroblast migration [16]. We analyzed the phosphorylation of c-jun, a key substrate of JNK, in PMA stimulation.

We examined the phosphorylation of JNK and c-jun by performing Western blot analysis at various times during the culture period after PMA treatment. As shown in Figure 6A, JNK activation peaked at 5 min after PMA treatment, and returned to the basal level at 60 min. In contrast, the phosphorylation of c-jun (Ser63 and Ser73) was detected after 5 min of PMA treatment and persisted for at least 120 min.

Then, to investigate whether the phosphorylation of c-jun was dependent on JNK activation, we added SP600125 to the cells prior to PMA treatment. In addition, to confirm that the phosphorylation of c-jun by PMA was a consequence of PKC activation, we used BIS. Figure 6B shows that pretreatment with BIS or SP600125 decreased the phosphorylation of c-jun.

Activation of downstream targets of p38MAPK

One of the indirect downstream effectors of p38MAPK is Hsp27, which is known to be an F-actin polymerization modulator [9,17].

We examined the effect of PMA on the phosphorylation of p38MAPK and Hsp27 by Western blot analysis. As shown in Figure 7A, p38MAPK phosphorylation was induced 5 min after PMA treatment and persisted for at least 120 min. Hsp27 was phosphorylated at 60 min of PMA treatment, and the phosphorylation persisted until at least 120 min. On the other hand, there was no change in total p38MAPK or Hsp27 expression.

Then, to investigate whether the phosphorylation of Hsp27 was dependent on p38MAPK or PKC activation, we added SB203580 or BIS to the cells prior to PMA treatment. Figure 7B shows that pretreatment with BIS or SB203580 decreased the phosphorylation of Hsp27.

Previous reports have shown that phosphorylation of Hsp27 is necessary for the formation of F-actin, and phosphorylated Hsp27 increased the rate and extent of actin polymerization in lamellipodia [18]. Based on those results, together with our finding that PMA induced lamellipodia formation (Figure 3), we examined the subcellular distribution of phosphorylated Hsp27 after PMA treatment.

Cell fractionation experiments indicated that unphosphorylated Hsp27 was present in both the cytosolic and membrane fractions in the control, and its subcellular distribution was not significantly changed after PMA treatment (Figure 8). Phosphorylated Hsp27 was significantly increased in not only the cytosolic but also the membrane fraction after PMA treatment (Figure 8). These results suggested that Hsp27 might be involved in PMA-induced subconjunctival fibroblast migration.

Effect of SP600125 and SB203580 on stress-activated protein kinase pathways

We further examined the effect of SP600125 on p38MAPK/Hsp27 phosphorylation induced by PMA. SP600125 failed to block the PMA-induced phosphorylation of p38MAPK and Hsp27 (Figure 9A).

Next, we examined the effect of SB203580 on JNK/c-jun phosphorylation induced by PMA. SB203580 failed to block the PMA-induced phosphorylation of JNK and c-jun (Figure 9B). These observations are consistent with previous reports that showed the specificity of SB203580 and SP600125 toward p38MAPK and JNK, respectively [19-21].

Discussion

PKC is one of the intracellular mediators of signal transduction pathways, and it participates in various cell functions throughout the body, such as membrane dynamics and migration. However, the role of PKC in subconjunctival fibroblast migration has not been extensively studied. In this study, we analyzed the role of PMA-activated PKC in the migration of subconjunctival fibroblasts, a key step in tissue scarring after GFS [2,22].

First, we determined that at least seven isoforms of PKC were expressed in the subconjunctival fibroblasts by Western blot analysis (Figure 1).

Next, we showed that PMA-activated PKC was one of the essential signals that stimulated subconjunctival fibroblast migration (Figure 2). Furthermore, an immunofluorescent study indicated that PMA induced actin reorganization, which was followed by lamellipodia formation in the subconjunctival fibroblasts (Figure 3). We also showed that PMA-activated PKC induced migration via JNK as well as p38MAPK (Figure 4 and Figure 5).

While SAPK activation in the PKC signaling pathway has been implicated in regulating cell migration, the exact roles of each protein may differ depending on cell type and context [9,23]. Therefore, we further investigated the roles of SAPK pathways in the PMA-induced migration of subconjunctival fibroblasts.

In the JNK pathway, c-jun is a substrate that is activated directly by JNK. The sites of phosphorylation of c-jun are at Ser63 and Ser73, and phosphorylation of c-jun by JNK is generally thought to increase transcriptional activity [9]. In our study, inhibition of JNK by SP600125 blocked c-jun phosphorylation, suggesting that PMA-induced c-jun phosphorylation was mediated via JNK activation in subconjunctival fibroblasts. Recently, it was reported that expression of a c-jun mutant whose Ser63 and Ser73 were mutated to alanine residues significantly inhibited the TGF-β-induced migration of dermal fibroblasts [16]. Similarly, c-jun was also shown to be involved in the platelet derived growth factor-BB-induced migration of rat aortic vascular smooth muscle [24] and periodontal cells [25]. It is possible that JNK contributes to PMA-induced migration through regulation of gene expression. We found that blocking protein synthesis or transcription using cycloheximide or actinomycin also inhibited PMA-induced migration of subconjunctival fibroblasts (unpublished data). Hence it appears that JNK has functions required for PMA-induced migration and may also be involved in the transcription and production of protein.

Regarding the p38MAPK pathway, it is well known that p38MAPK participates in inflammation, apoptosis, and cell differentiation [9,26]. In addition, p38MAPK has been reported to be involved in growth factor- and cytokine-induced cell migration in several cell types [9,18]. Activation of p38MAPK results in activation of MAP kinase-activated protein kinase-2/3 and phosphorylation of Hsp27. Phosphorylation of Hsp27 was crucial for the migration of smooth muscle and endothelial cells [17,27]. In our study, we showed that the induction of Hsp27 phosphorylation by PMA treatment was mediated via p38MAPK pathway (Figure 7B). Furthermore, phosphorylated Hsp27 appeared in the membrane fraction after PMA treatment (Figure 8). The accumulation of phosphorylated Hsp27 in the membrane might participate in F-actin polymerization and the subsequent induction of lamellipodia formation in subconjunctival fibroblasts. These results indicated that p38MAPK also had functions required for cell migration that might be dependent on Hsp27.

In summary, PMA-activated PKC induced lamellipodia formation and subsequent increased migratory activity of subconjunctival fibroblasts. Furthermore, PMA-activated PKC-induced migration of subconjunctival fibroblasts was mediated by the activation of SAPKs such as JNK and p38MAPK. These findings indicated that inhibition of the activity of PKC might lead us to develop novel therapeutic modalities for treatment of scarring after GFS. Further analysis should be necessary to elucidate which PKC isoforms are involved in subconjunctival fibroblast migration.

Acknowledgements

This study was supported in part by grants-in aid for Encouragement of Scientists (Grant No.17924036 to N.N. and 18924037 to M.N.) from Japan Society for the Promotion of Science.

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