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Originally published In Press as doi:10.1074/jbc.M610740200 on February 9, 2007

J. Biol. Chem., Vol. 282, Issue 18, 13610-13616, May 4, 2007
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Protein C Is an Autocrine Growth Factor for Human Skin Keratinocytes*

Meilang Xue1, David Campbell, and Christopher J. Jackson

From the Sutton Research Laboratories, The University of Sydney at Royal North Shore Hospital, St Leonards, New South Wales 2065, Australia

Received for publication, November 20, 2006 , and in revised form, January 30, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The protein C (PC) pathway plays an important role in coagulation and inflammation. Many components of the PC pathway have been identified in epidermal keratinocytes, including endothelial protein C receptor (EPCR), which is the specific receptor for PC/activated PC (APC), but the core member of this pathway, PC, and its function in keratinocytes has not been defined. In this study, we reveal that PC is strongly expressed by human keratinocytes at both gene and protein levels. When endogenous PC was blocked by siRNA the proliferation of keratinocytes was significantly decreased. This inhibitory effect was restored by the addition of recombinant APC. PC siRNA treatment also increased cell apoptosis by 3-fold and inhibited cell migration by more than 20%. When keratinocytes were pretreated with RCR252, an EPCR-blocking antibody, or PD153035, an epidermal growth factor receptor (EGFR) inhibitor, cell proliferation was hindered by more than 30%. These inhibitors also completely abolished recombinant APC (10 µg/ml)-stimulated proliferation. Blocking PC expression or inhibiting its binding to EPCR/EGFR decreased the phosphorylation of ERK1/2 but increased p38 activation. Furthermore, inhibition of ERK decreased cell proliferation by ~30% and completely abolished the stimulatory effect of APC on proliferation. Taken together, these results indicate that keratinocyte-derived PC promotes cell survival, growth, and migration in an autocrine manner via EPCR, EGFR, and activation of ERK1/2. Our results highlight a novel role for the PC pathway in normal skin physiology and wound healing.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein C (PC),2 a vitamin K-dependent zymogen, is converted to activated protein C (APC) on the endothelial surface when thrombin binds to thrombomodulin (1, 2). The activation of PC is augmented by its specific receptor, endothelial protein C receptor (EPCR) (1–3), a 46-kDa, type I transmembrane glycoprotein homologous to major histocompatibility complex class I/CD1 family proteins (4, 5). APC plays a key role in the regulation of blood coagulation and also has significant anti-inflammatory properties associated with inhibition of proinflammatory cytokines and a reduction of leukocyte recruitment (6, 7). APC prevents lipopolysaccharide-induced pulmonary vascular injury and protects against ischemia/reperfusion-induced renal injury by inhibiting the accumulation and activation of leukocytes (6, 8). In vitro, APC suppresses the nuclear factor-{kappa}B (NF-{kappa}B) pathway in both human monocytes (7, 9) and endothelial cells (10). APC also inhibits lipopolysaccharide-induced tumor necrosis factor-{alpha} expression in a monocytic cell line (9) and inhibits endothelial cell apoptosis (11). The effectiveness of APC as an anticoagulant and anti-inflammatory agent is demonstrated by its efficacy as a treatment for patients with severe sepsis (12).

APC can activate endothelial matrix metalloproteinase (MMP)-2 (13), a member of the MMP family of zinc-dependent endopeptidases that plays a vital role in the tissue repair process by remodeling the extracellular matrix (14). In cultured human keratinocytes, APC enhances cell proliferation, migration, and MMP-2 activity (15). Recently, a novel function of APC as a promoter of cutaneous wound healing was identified. In a rat healing model APC accelerated full thickness wound closure by stimulating re-epithelialization, promoting angiogenesis, and preventing inflammation (16).

The epidermis is the outermost skin layer and provides the first line of defense against the external environment. Keratinocytes, the predominant cell type in human skin, exist at various stages of differentiation corresponding to different epidermal layers (17, 18). Dividing cells in the basal layer progressively differentiate and withdraw from the cell cycle as they are displaced toward the skin surface. Keratinocytes play a fundamental role in skin metabolism and in wound closure by proliferating and migrating, to compensate for superficial cell loss or to cover the exposed connective tissue, and by producing various mediators including cytokines/chemokines, the T cell receptor, and antimicrobial peptides (19). Keratinocytes in the basal and suprabasal layers of the epidermis express many components of the PC pathway, including thrombomodulin (20), PC inhibitor (21), and EPCR (22). Prior to this study, PC was thought to be synthesized almost exclusively by the liver and vascular endothelial cells with a circulatory half-life of {approx}20 min (23). The current study shows that PC is strongly expressed by skin keratinocytes. Furthermore, this keratinocyte-derived PC promotes cell survival, growth, and migration in an autocrine manner via EPCR, epidermal growth factor receptor (EGFR), and activation of ERK1/2.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Keratinocyte Culture and Reagents—Normal keratinocytes were isolated from neonatal foreskins (n = 30) as described previously (15) in accordance with the local ethics regulations. Extracted cells were cultured in keratinocyte-serum free medium (K-SFM, Invitrogen). When more than 70% confluent, primary cultured cells were trypsinized and used in experiments. Cells were seeded into either 24-well culture plates at 5 x 105 cells/well or 8-well PermanoxTM slides (Nalge Nunc International Corp., Rochester, NY) and incubated for 12 h to allow for adhesion. The confluent cells were then treated with recombinant APC (Xigris, Eli Lilly, Indianapolis, IN), PC (Sigma), EPCR-blocking antibody RCR252, EPCR-nonblocking antibody RCR92 (gift from Prof. Fukudome, Dept. of Immunology, Saga Medical School, Nabeshima, Saga, Japan), PD153035, an inhibitor of EGFR, and inhibitors of c-Jun (2 µM), p38 (70 nM), and ERK (10 µM) (EMD Biosciences, Inc., San Diego). Medium used for cells treated with PD153035 and the related control contained 0.01% dimethyl sulfoxide (Me2SO), as PD153035 was originally dissolved in Me2SO. MAP kinase inhibitors and PD153035 did not exert any cytotoxic effect on keratinocytes when used at indicated concentrations (data not shown). Cells and culture supernatants were collected for detection of mRNA and protein expression.

Small Interfering RNA (siRNA) Preparation and Nucleofection—Small interfering RNA duplex oligonucleotides were purchased from Proligo (Sigma-Proligo). The siRNAs designed for PC were: sense 5'-GAGGUGAGCUUCCUCAAUUGC-3' and antisense 5'-AAUUGAGGAAGCUCACCUCGC-3'. A scrambled form of PC siRNA was used as a negative control. Keratinocytes were adjusted to 1 x 106 cells/ml in growth medium and subjected to nucleofection using the human keratinocyte NucleofectorTM kit and Amaxa NucleofectorTM II machine according to the manufacturer's instructions (Amaxa Biosystems, Cologne, Germany). Cells were allowed to attach overnight, then trypsinized and seeded into either 24-well plates (4 x 105 cells/ml) or 96-well plates (1 x 104 cells/ml), and incubated for 48 and 72 h. The specificity of siRNAs was confirmed by a validated short hairpin RNA (Superarray, Frederick, MD).

RNA Extraction and Reverse Transcription Real-time PCR— Total RNA was extracted from keratinocytes using Tri Reagent (Sigma) according to the manufacturer's instructions. Single-stranded cDNA was synthesized from total RNA using avian myeloblastosis virus-reverse transcriptase and oligo(dT)15 as a primer (Promega Corp., Madison, WI). The levels of mRNA were semiquantified using real-time PCR on a Rotor-gene 3000A (Corbett Research, Sydney, Australia). Samples were normalized to the housekeeping gene RPL13A, and results were reported for each sample relative to the control. PC PCR product was also separated on a 2% agarose gel and imaged using the Infinity-Capt gel documentation system (Vilber, Lourmat, France). The primers used were as follows: PC (213 bp), sense 5'-TCTTCGTCCACCCCAACTAC-3' and antisense 5'-GGTTTCTCTTGGCCTCCTTC-3'; RPL13A (152 bp), sense 5'-AAGCCTACAAGAAAGTTTGCCTATC-3' and antisense 5'-TGTTTCCGTAGCCTCATGAGC-3'.

APC Activity Assay—The activity of APC in culture supernatants and cell lysates was quantitated using the chromogenic substrate Spectrozyme PCa (American Diagnostica Inc., Stamford, CT) according to the manufacturer's instructions. A standard curve was generated using human recombinant APC.

MTT Assay—The colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay was performed to quantitate the effect of different test agents on cell growth and viability. Briefly, 1 x 104 cells/well were seeded into a 96-well microplate to a final volume of 200 µl and then incubated for 4 h to allow cells to attach. Cells were then treated with different test agents. Three hours prior to the completion of the treatment, 10 µl of 5 mg/ml MTT (Sigma) was added to cells. After a further incubation for 3 h, the MTT solution was removed and replaced by 100 µl Me2SO. The optical density of each well was determined at a wavelength of 570 nm with a reference wavelength of 630 nm.

In Vitro Migration Assay—Cells were seeded into 24-well plates and cultured to confluence. Cell monolayers were then scratched with a 1000-µl blue plastic pipette tip (Greiner Bioone, Greiner Int., Longwood, FL), creating a cell-free area ~2 mm in width. "Wounded" monolayers were washed twice with PBS to remove loose cell debris, and a defined area of the wound was photographed under phase-contrast microscopy. To standardize the position of the wound when photographing, small indents were made in the plastic well using a sterile 31-gauge needle. To prevent cell proliferation, cells were pretreated with mitomycin C (10 µg/ml, Sigma), which was applied to the cells 2 h before wounding and removed with three PBS washes. Cell migration was determined after 24 h by counting the cells that had moved into the wounded area; the percentage of cell migration was calculated as [number of migrated PC siRNA-treated cells/number of migrated scrambled siRNA-treated cells] x 100.

Western Blot—Keratinocytes were washed three times with PBS and lysis buffer (0.15 M NaCl, 0.01 mM phenylmethylsulfonyl fluoride, 1% Nonidet P-40, 0.02 M Tris, 6 M urea/H2O) was added. Cell lysates were centrifuged at 10,000 x g for 15 min, and supernatants were separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The primary antibodies used were: rabbit anti-human EPCR antibody (1:500 dilution, Invitrogen); mouse anti-human PC (HC-2, 1:500 dilution, Sigma); rabbit anti-phosphorylated forms of p38, c-Jun, and ERK2 (1:1000 dilutions, Santa Cruz Biotechnology Inc., Santa Cruz, CA); and rabbit anti-phospho-EGFR (Y1173) and rabbit anti-human active caspase-3 (R&D Systems, Minneapolis, MN). Immunoreactivity was detected using the ECL detection system (Amersham Biosciences). Anti-human beta-actin antibody was included to normalize against unequal loading.

Immunohistochemical Staining—Cultured keratinocytes in Permanox slides were fixed with 4% paraformaldehyde. Human foreskin was fixed with 10% PBS-buffered formalin. Paraffin-embedded tissue was deparaffinized and subjected to immunohistochemistry. After quenching with 2% H2O2 in methanol and equilibrating in PBS, samples were incubated with mouse anti-human PC antibody or mouse IgG (Dako Corp., Carpinteria, CA) overnight at 4 °C. Samples were then processed for staining using Dako LSAB+ systems stain kit and counter-stained with hematoxylin and Scott's bluing solution. After mounting, tissue sections were observed under a light microscope (ECLIPSE 80i, Nikon Corp.).


Figure 1
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  		FIGURE 1.
Expression of human PC/APC in keratinocytes and neonatal foreskin. A, PC mRNA expression by primary human keratinocytes was detected by reverse transcription-PCR. Left lane, DNA markers. B, PC protein was detected in human keratinocytes by immunohistochemistry using mouse anti-human PC antibody. Scale bar:40 µm. Mouse IgG was used as a negative control. C, PC protein was detected in human neonatal foreskin. D, negative control. Arrows indicate vascular endothelial cells. Arrowheads indicate epidermal basal layer. Scale bar: 100 µm. Images represent one of three independent experiments.

 
For dual staining, deparaffinized foreskin tissues were blocked by 5% horse serum in PBS and incubated with goat anti-human EPCR and rabbit anti-human EGFR antibodies (R&D Systems) for 2 days at 4 °C. After washing with PBS, tissue sections were incubated with anti-rabbit IgG conjugated with Cy3 (red) and anti-goat IgG conjugated with FITC (green) (1:400, Sigma-Aldrich). Tissue sections were washed with PBS and observed under a fluorescence microscope (Nikon ECLIPSE 80i). Images were acquired and processed using a Nikon digital camera and software (Diagnostic Instruments) and Image J (rsb.info.nih.gov/ij).

Apoptosis Detection—Apoptotic keratinocytes were detected using an in situ cell death detection kit according to manufacturer's instructions (Roche Diagnostics).

Statistical Analysis—Significance was determined using one-way analysis of variance and the Student-Newman-Keuls test. p values less than 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
PC Is Expressed by Human Keratinocytes—To investigate whether epidermal keratinocytes produce PC, mRNA from unstimulated primary cultured cells was subjected to reverse transcription-PCR. PC mRNA was detected in cultured keratinocytes as shown by the prominent band at 213 bp (Fig. 1A). In concordance with gene expression, immunohistochemistry results showed that cultured keratinocyte monolayers stained strongly positive with an antibody against APC/PC (Fig. 1B). The expression of PC was also assessed in neonatal foreskin tissues by immunohistochemistry. The basal and suprabasal layers of the epidermis were strongly positive for PC, whereas the outer stratum corneum where the keratinocytes lose their viability showed weak staining (Fig. 1C). PC was also immunolocalized to dermal vascular endothelial cells (Fig. 1C).

Inhibition of Endogenous PC with siRNA Suppresses Keratinocyte Proliferation and Migration and Promotes Apoptosis—Because recombinant APC stimulates keratinocyte proliferation (15), we investigated whether endogenous PC/APC could stimulate the growth of keratinocytes in an autocrine manner. PC siRNA was used to suppress the endogenous PC expression by keratinocytes. The efficacy of PC siRNA was examined at 48 h by real-time PCR and Western blot (Fig. 2, A and B). PC siRNA dose-dependently reduced PC mRNA levels by up to 80% when used at 0.5 µM. This concentration of siRNA was used in subsequent experiments unless otherwise specified.


Figure 2
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  		FIGURE 2.
PC siRNA treatment inhibits the growth of keratinocytes. A, efficacy of PC siRNA at blocking PC mRNA expression by keratinocytes at 48 h, detected by reverse transcription real-time PCR. Data are expressed as mean ± S.E. (n = 3). B, PC/APC levels in the supernatant and cell lysate of keratinocytes following PC siRNA treatment for 48 h detected by Western blot. C, APC activity in cell lysates after treatment for 48 h with either scrambled control siRNA or PC siRNA (0.5 µM). D, growth rate of keratinocytes in response to PC siRNA (0.5 µM) and APC (0.1, 1, 10, and 20 µg) treatment after 72 h as detected by MTT assay. Cell proliferation is expressed as a percentage of control (mean ± S.D.). Graphs represent one of three independent experiments. *, p < 0.05; **, p < 0.01.

 


Figure 3
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  		FIGURE 3.
Endogenous PC/APC prevents keratinocyte apoptosis. Keratinocytes were treated with PC siRNA (0.5 µM). After 48 h, cells were harvested, and cell lysates were used to detect the activation of caspase-3 by Western blot (A) and semiquantified using image analysis software (B). Cells were used for a TUNEL (terminal dUTP nick-end labeling) assay to detect apoptotic cells (black arrows indicate apoptotic cells) (C) and quantitated by counting apoptotic cells under high power microscopy (x20) (D). Data were expressed as the average number of apoptotic cells per field of 15 fields (mean ± S.E., n = 3). Images represent one of three independent experiments. **, p < 0.01. Scale bar:40 µm.

 
After a 24-h incubation, APC activity (1.53 µg of APC activity/106 cells) was detected in the whole cell lysates but not the culture medium (data not shown) of unstimulated keratinocytes, as measured using the Spectrozyme PCa activity assay. Because the basal culture medium contained no exogenous APC, the activity detected in cell lysates was most likely derived from the activation of endogenous PC. This was confirmed by the finding that the level of APC was reduced by ~50% in PC siRNA-treated cells (Fig. 2C).

PC siRNA reduced keratinocyte proliferation by ~35% after 72 h compared with control (Fig. 2D). This inhibition was dose-dependently reversed by adding recombinant APC, with the growth rate being restored at a concentration of 20 µg/ml APC (Fig. 2D).

The effect of PC siRNA on keratinocyte apoptosis was detected by measuring the amount of active caspase-3, a marker for apoptosis, using Western blotting. An 8-fold increase in active caspase-3 was observed in PC siRNA-treated cells when compared with that in cells treated with scrambled control (Fig. 3, A and B). To confirm this result, apoptotic cells were evaluated by an in situ cell death detection kit. PC siRNA treatment resulted in ~3 times more apoptotic cells than with scrambled siRNA treatment at 48 h (Fig. 3, C and D).

The migration of PC siRNA-treated cells was also measured using a scratch wounding assay. Confluent keratinocytes were scratch-wounded, and cell migration was evaluated 24 h after wounding. PC siRNA-treated cells exhibited ~20% less migration into the wounded areas than control cells (Fig. 4).

EPCR and EGFR Are Required for Endogenous PC-stimulated Keratinocyte Proliferation—To test whether the effect of endogenous PC is mediated through EPCR, unstimulated keratinocytes were treated with RCR252, an antibody that binds to EPCR and prevents PC/APC from binding. After 72 h, this treatment resulted in a dose-dependent decrease in the proliferation of keratinocytes, showing a greater than 30% reduction when cells were treated with 10 µg/ml EPCR (Fig. 5A). These results indicate that keratinocyte-derived PC acts via EPCR to stimulate cell proliferation under normal unstimulated conditions. In addition, the stimulatory effect of recombinant APC on cell proliferation was abrogated by blocking EPCR (Fig. 5A). The nonblocking control antibody, RCR92, had no effect (Fig. 5A).


Figure 4
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  		FIGURE 4.
PC siRNA treatment decreases migration of keratinocytes. Cell monolayers were pretreated with mitomycin C and then scratched with a 1000-µl blue plastic pipette tip. A, the defined area of the wound was photographed under a phase-contrast microscopy at time 0 h and 24 h. To standardize the position of the wound for photography, small indents were made in the plastic well (marked by outlined arrows). B, cell migration was determined after 24 h by counting the cells that had moved out of the initial area, and the percentage of cell migration was calculated as [number of migrated PC siRNA-treated cells/number of migrated scrambled siRNA-treated cells] x 100. Data are expressed as the number of migrated cells as a percentage of control (mean ± S.E., n = 3). Images represent one of three independent experiments. *, p < 0.05. Scale bar:10 µm.

 


Figure 5
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  		FIGURE 5.
Blocking EPCR and EGFR decreases the growth of unstimulated and APC-stimulated keratinocytes. Cell proliferation was detected using the MTT assay. A, the growth rate of keratinocytes in response to the EPCR-blocking antibody RCR252 or control nonblocking antibody RCR92 in the presence or absence of APC (1 µg/ml) after 72 h. B, cells were incubated in keratinocyte-serum free medium without EGF and treated with PD153035, an inhibitor of EGFR, or with PD153035 plus RCR252 1 h prior to the addition of recombinant APC (0, 1 or 10 µg). After incubation for 72 h, cell proliferation was measured by MTT assay. Data are expressed as cell proliferation as a percentage of control (mean ± S.D.), and graphs represent one of three independent experiments. *, indicates comparison with normal control; +, indicates comparison with APC treatment. {clubsuit}, indicates blocking both EPCR and EGFR in comparison with individual treatment; +, {clubsuit}, or *, p < 0.05; ++ or **, p < 0.01.

 


Figure 6
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  		FIGURE 6.
EPCR and EGFR are co-localized in skin and up-regulated by PC/APC in keratinocytes. Keratinocytes were treated with recombinant APC or PC siRNA, and the expression of EPCR (A) and the phosphorylated form of EGFR (P-EGFR) (B) were detected in whole cell lysates by Western blot. C, co-localization of activated EGFR and EPCR on foreskin epidermis: red, EGFR; green, EPCR. In the merged image, yellow indicates co-localization of EPCR and EGFR. White arrows indicate basal epidermis, and white arrowheads indicate vascular endothelium. Images represent one of three independent experiments. Scale bar:50 µm.

 


Figure 7
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  		FIGURE 7.
PC/APC regulates the activation of MAP kinases. A, keratinocytes were treated with PC siRNA (0.5 µM) for 72 h or with RCR252 (10 µg) or PD153035 (2.5 µM) for 24 h, and cells were collected for Western analysis. Images represent one of three independent experiments. B, Western blotting results were analyzed by image analysis software. Data are expressed as a percentage of control protein expression (mean ± S.E., n = 3). C, keratinocyte proliferation was measured at 72 h in response to inhibitors of c-Jun (2 µM), p38 (70 nM), and ERK (10 µM). Data are expressed as a percentage of control cell proliferation (mean ± S.D.). Images represent one of three independent experiments. In B and C, *, p < 0.05; **, p < 0.01.

 
EGFR is highly expressed in human keratinocytes in vivo and in vitro (24, 25) and plays a central role in numerous aspects of keratinocyte biology. Functional activation of EGFR results from phosphorylation of specific tyrosine residues in the C-terminal cytoplasmic domain. This activation can be blocked specifically by tyrosine kinase inhibitors such as PD153035. In this study, PD153035 (2.5 µm) inhibited the proliferation of unstimulated keratinocytes by more than 30% (Fig. 5B), suggesting that EGFR is required for normal cell growth. The stimulatory effect of 1 or 10 µg/ml recombinant APC on proliferation was nearly abolished by 2.5 µm PD153035 (Fig. 5B), indicating that APC requires EGFR to stimulate keratinocyte growth. Inhibition of both EPCR and EGFR together caused a further modest but significant (p < 0.05, under basal conditions and in response to 10 µg/ml APC) reduction in proliferation compared with individually blocking either EPCR or EGFR (Fig. 5).

APC Up-regulates the Expression/Activation of EPCR and EGFR— When cells were stimulated with 1 or 10 µg/ml recombinant APC, the expression of EPCR and the phosphorylation of EGFR were markedly increased (Fig. 6, A and B). In contrast, when cells were treated with PC siRNA, EPCR protein in cell lysates was reduced by more than 50% (Fig. 6A), and the phosphorylated form of EGFR was also dramatically inhibited (Fig. 6B). Thus, APC appears to enhance its effect by increasing the expression/activation of EPCR and EGFR. Using dual immunofluorescent staining, we found that both EPCR and activated EGFR were co-localized in the same areas of basal and suprabasal keratinocytes in the epidermis (Fig. 6C), which is similar to PC localization in skin epidermis (Fig. 1C), Endothelial cells in the dermis stained positively for EPCR but not for activated EGFR.

APC Regulates the Activation of MAP Kinases—APC-induced cell proliferation is controlled through the activation of MAP kinases in various cell types (15, 26). Here, PC siRNA-treated keratinocytes were evaluated for their levels of MAP kinase activity. Seventy-two hours after PC siRNA treatment, phosphorylation of ERK2 and c-Jun was attenuated (Fig. 7, A and B). In contrast, the phosphorylated form of p38 was increased by ~50% (Fig. 7, A and B).

Blocking of either EPCR with RCR252 (10 µg/ml) or EGFR with PD153035 (2.5 µM) for 24 h resulted in a similar MAP kinase response as PC siRNA, with a decrease in phosphorylated forms of ERK2, minimal change in c-Jun and an increase in the phosphorylation of p38 (Fig. 7, A and B).

Specific inhibitors of ERK, p38, and c-Jun (10 µM, 2 µM, and 70 nM, respectively) were added to normal cultured keratinocytes for 72 h in the presence or absence of recombinant APC (1 µg/ml), and then cell growth was assessed. Inhibition of ERK decreased cell proliferation by ~30% and completely abolished the stimulatory effect of APC on proliferation. Inhibition of p38 or c-Jun had no effect on cell proliferation (Fig. 7C).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Although PC is thought to be synthesized almost exclusively by the liver, the current study shows that it is strongly expressed by skin keratinocytes both in vitro and in vivo. The immunohistochemical localization for PC in the basal and suprabasal layers of the epidermis is similar to the pattern of expression of other members of the PC pathway, including EPCR (22) and thrombomodulin (20). Both of these receptors augment the conversion of PC to APC (27, 28) in the presence of thrombin, which is also produced by epidermal keratinocytes (29). The ability of keratinocytes to secrete and activate PC is demonstrated by our finding that in the absence of exogenous APC, the Spectrozyme activity assay detected substantial levels of APC in cultured cells, which was reduced by treating the cells with PC siRNA (Fig. 2C). Thus, the epidermis possesses its own independent PC system that can synthesize PC, activate PC to APC, and mediate the function of PC/APC by its receptors/inhibitors.

Keratinocyte growth is thought to be controlled by the autocrine induction of heparin binding factors of the EGF family, by up-regulation and dimerization of receptors, and by cross-induction between receptors and ligands (30). Here we show that PC also accounts for an essential part of autocrine growth capacity of cultured human keratinocytes by promoting cell proliferation. In addition to its effect on proliferation, cells treated with PC siRNA had a more than 3-fold higher apoptotic rate than control cells, indicating that PC/APC inhibits apoptosis. PC regulates the activation of caspase-3, a major effector caspase, of which the inactive form is expressed in a wide range of tissues including the epidermis (31). In normal oral epithelium, cleaved caspase-3 clearly distinguishes apoptotic keratinocytes from cells that are terminally differentiated (32). Recent findings indicate that caspase-14, but not caspase-3, is activated during normal keratinocyte differentiation (33). Thus caspase-3 activation appears to be restricted to keratinocytes undergoing apoptosis. Blocking PC by siRNA greatly increased the activation of caspase-3, which is consistent with the increased apoptotic cells.

The PC pathway is a critical regulator of the blood coagulation system and plays an important role in inflammatory and immunomodulatory processes (6, 7). Clinical trial results have shown that recombinant APC reduces mortality in patients with sepsis-induced multi-organ failure when administered by infusion (12). Similar to these systemic effects, local production of APC may regulate cutaneous inflammation and immune defense in the skin. Keratinocytes of the epidermis provide the major cellular component of the outermost barrier to the environment (19). When the skin is broken, a critical response is triggered to restore its protective function. Within 24 h of wounding, keratinocytes from the wound margins begin to migrate and invade the wound bed, where they proliferate to form the new epithelium. Strong evidence is now emerging that the PC pathway contributes to cutaneous wound repair. In a rat healing model, APC induced angiogenesis and re-epithelialization while inhibiting inflammation to promote cutaneous wound healing (16). In human skin keratinocytes, recombinant APC stimulates proliferation, MMP-2 activity, and migration and prevents apoptosis, all vital processes of re-epithelialization (22). The current study extends on these findings to show that endogenously derived PC can stimulate a wound healing phenotype in keratinocytes by stimulating proliferation, migration and preventing apoptosis. These results highlight a new important autocrine action of PC/APC during wound re-epithelization.

Recent reports show that recombinant APC acts via EPCR to stimulate keratinocyte proliferation (22) and that APC acts via EPCR and EGFR in human peripheral blood lymphocytes to inhibit cell migration (34). In the current study, the decrease in cell growth brought about by inhibitors of EPCR and EGFR could not be recovered by exogenous APC, indicating that endogenous PC/APC promotes cell growth via these two receptors. Simultaneously using inhibitors to both EPCR and EGFR resulted in a modest additional inhibition of proliferation compared with individually blocking either receptor (Fig. 5), suggesting that the two receptors can partially act independently of each other to mediate the proliferative effect of APC. Both EPCR and EGFR are strongly expressed in human skin epidermis (22, 35). Dual immunofluorescent staining clearly showed that EPCR and activated EGFR are exclusively co-localized in basal and suprabasal keratinocytes (Fig. 5), similar to the location of PC. Together, these three components comprise a potentially powerful autocrine regulator of the biological functions of keratinocytes.

EGFR, a member of the ErbB family of receptor tyrosine kinases, plays an important role in regulating the development of the epidermis and its appendages (36). In normal epidermis, EGFR is essential for numerous aspects of keratinocyte biology including growth, suppression of terminal differentiation and regulation of cell migration (37). Functional activation of EGFR results from increased phosphorylation of specific tyrosine residues in its C-terminal cytoplasmic domain. In wounded skin, EGFR is transiently up-regulated and is the major effector of the proliferative and migratory responses during wound re-epithelialization. Accumulating evidence shows that EGFR not only mediates responses to EGF-like ligands but is also a major transducer of diverse signaling systems and a switch point for cellular communication networks (38, 39). PC/APC is not known to act as a ligand for EGFR, but it may activate this receptor via other mechanisms, for example by utilizing G protein-coupled receptors (GPCRs). In human keratinocytes, recombinant APC can cleave the GPCR, PAR-1, to stimulate cell proliferation and MMP-2 activity (22). Signaling pathways linking GPCR and EGFR have recently been revealed in some cell types. Sabri et al. (40) reported that thrombin activation of PAR-1 in cardiac fibroblasts leads to intracellular transactivation of EGFR through rapid phosphorylation of phospholipase C, formation of inositol polyphosphate-3, and mobilization of intracellular calcium resulting in activation of Src and Fyn kinases, which associate with and activate EGRF tyrosine kinase. An alternative pathway termed "triple membrane-passing signal" has been proposed in which a GPCR activates an ADAM (a disintegrin and metalloprotease), which in turn releases an EGFR ligand, such as EGF, to activate EGFR (41). Whether endogenous PC/APC acts via these pathways to promote keratinocyte proliferation is yet to be determined.

The MAPK pathway is a prerequisite for growth factor-stimulated mitogenesis in many cell types (42). Three major downstream MAPK cascades are mitogen-activated ERK1/2 and stress/cytokine-activated p38 and c-Jun N-terminal kinases. Mice lacking Jun in epidermal keratinocytes are born with open eyes and eyelid cells and show reduced expression of the EGFR (43). Although c-Jun is primarily a positive regulator of cell proliferation (44), our data show that PC/APC caused minimal change in c-Jun activation in keratinocytes. Instead, three lines of evidence indicate that ERK1/2 is the major MAP kinase associated with PC/APC enhancement of cell proliferation. Firstly, using siRNA to block keratinocyte-derived PC reduced the activation of ERK. Secondly, preventing PC/APC from binding EPCR or activating EGFR inhibited ERK activation; and thirdly, a specific ERK inhibitor prevented PC/APC from inducing proliferation. In contrast, p38 exhibited opposite effects to ERK. These results are in keeping with previous studies presenting evidence that p38 MAP kinase functions to promote differentiation and apoptosis, whereas signaling through ERK promotes keratinocyte proliferation and survival (45) and endothelial cell proliferation (26).

In summary, the key findings of this study are: (i) PC is strongly expressed by keratinocytes, the major cell type of the skin epidermis; (ii) endogenous PC/APC stimulates proliferation and migration and inhibits apoptosis in keratinocytes in an autocrine manner; and (iii) stimulation of proliferation by endogenous PC/APC is mediated via two receptors, EPCR and EGFR, and the MAP kinase ERK1/2. These novel findings highlight the importance of the PC pathway in skin physiology and help elucidate keratinocyte function in normal wound healing.


   FOOTNOTES
 
* This work was supported by a University of Sydney postdoctoral fellowship, The Lincoln Centre, Rebecca Cooper Foundation, a grant from Northern Sydney Area Health (to M. X.) and the Sutton Arthritis Research Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Level One, Block Four, Sutton Research Labs, The University of Sydney at Royal North Shore Hospital, St Leonards, New South Wales 2065, Australia. Tel.: 612-99266045; Fax: 612-99266269; E-mail: mlxue{at}med.usyd.edu.au.

2 The abbreviations used are: PC, protein C; APC, activated protein C; EPCR, endothelial protein C receptor; ERK, extracellular signal-regulated kinase; GPCR, G protein-coupled receptor; NF-{kappa}B, nuclear factor-{kappa}B; MAP, mitogen-activated protein; MAPK, MAP kinase; MMP, matrix metalloproteinase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; MTT, colorimetric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide; PBS, phosphate-buffered saline; siRNA, small interfering RNA. Back


   ACKNOWLEDGMENTS
 
Foreskins were provided by Dr. Gordon Campbell, Sydney Adventist Hospital. We thank Susan Smith for preparation of RA tissue sections, Aiqun Xue and Dr. Qun Li for assistance in apoptosis detection and dual staining, and Dr. Nghia Le for advice.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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