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• W2023688094 abstract "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. 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. Protein C (PC), 2The 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-κB, nuclear factor-κB; 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. 2The 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-κB, nuclear factor-κB; 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. a vitamin K-dependent zymogen, is converted to activated protein C (APC) on the endothelial surface when thrombin binds to thrombomodulin (1Fukudome K. Ye X. Tsuneyoshi N. Tokunaga O. Sugawara K. Mizokami H. Kimoto M. J. Exp. Med. 1998; 187: 1029-1035Crossref PubMed Scopus (99) Google Scholar, 2Stearns-Kurosawa D.J. Kurosawa S. Mollica J.S. Ferrell G.L. Esmon C.T. Proc. Natl. Acad. Sci. U. S. A. 1996; 19: 10212-10216Crossref Scopus (449) Google Scholar). The activation of PC is augmented by its specific receptor, endothelial protein C receptor (EPCR) (1Fukudome K. Ye X. Tsuneyoshi N. Tokunaga O. Sugawara K. Mizokami H. Kimoto M. J. Exp. Med. 1998; 187: 1029-1035Crossref PubMed Scopus (99) Google Scholar, 2Stearns-Kurosawa D.J. Kurosawa S. Mollica J.S. Ferrell G.L. Esmon C.T. Proc. Natl. Acad. Sci. U. S. A. 1996; 19: 10212-10216Crossref Scopus (449) Google Scholar, 3Xu J. Esmon N.L. Esmon C.T. J. Biol. Chem. 1999; 274: 6704-6710Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), a 46-kDa, type I transmembrane glycoprotein homologous to major histocompatibility complex class I/CD1 family proteins (4Fukudome K. Esmon C.T. J. Biol. Chem. 1994; 269: 26486-26491Abstract Full Text PDF PubMed Google Scholar, 5Villoutreix B.O. Blom A.M. Dahlback B. Protein Eng. 1999; 12: 833-840Crossref PubMed Scopus (26) Google Scholar). 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 (6Murakami K. Okajima K. Uchiba M. Johno M. Nakagaki T. Okabe H. Takatsuki K. Am. J. Physiol. 1997; 2: L197-L202Google Scholar, 7Yuksel M. Okajima K. Uchiba M. Horiuchi S. Okabe H. Thromb. Haemostasis. 2002; 88: 267-273Crossref PubMed Scopus (163) Google Scholar). APC prevents lipopolysaccharide-induced pulmonary vascular injury and protects against ischemia/reperfusion-induced renal injury by inhibiting the accumulation and activation of leukocytes (6Murakami K. Okajima K. Uchiba M. Johno M. Nakagaki T. Okabe H. Takatsuki K. Am. J. Physiol. 1997; 2: L197-L202Google Scholar, 8Uchiba M. Okajima K. Murakami K. Johno M. Okabe H. Takatsuki K. Am. J. Hematol. 1997; 2: 118-123Crossref Google Scholar). In vitro, APC suppresses the nuclear factor-κB (NF-κB) pathway in both human monocytes (7Yuksel M. Okajima K. Uchiba M. Horiuchi S. Okabe H. Thromb. Haemostasis. 2002; 88: 267-273Crossref PubMed Scopus (163) Google Scholar, 9Joyce D.E. Grinnell B.W. Crit. Care Med. 2002; 30: S288-S293Crossref PubMed Scopus (182) Google Scholar) and endothelial cells (10Joyce D.E. Gelbert L. Ciaccia A. DeHoff B. Grinnell B.W. J. Biol. Chem. 2001; 276: 11199-11203Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar). APC also inhibits lipopolysaccharide-induced tumor necrosis factor-α expression in a monocytic cell line (9Joyce D.E. Grinnell B.W. Crit. Care Med. 2002; 30: S288-S293Crossref PubMed Scopus (182) Google Scholar) and inhibits endothelial cell apoptosis (11Cheng T. Liu D. Griffin J.H. Fernandez J.A. Castellino F. Rosen E.D. Fukudome K. Zlokovic B.V. Nat. Med. 2003; 9: 338-342Crossref PubMed Scopus (525) Google Scholar). The effectiveness of APC as an anticoagulant and anti-inflammatory agent is demonstrated by its efficacy as a treatment for patients with severe sepsis (12Griffin J.H. Zlokovic B. Fernandez J.A. Semin. Hematol. 2002; 39: 197-205Crossref PubMed Scopus (83) Google Scholar).APC can activate endothelial matrix metalloproteinase (MMP)-2 (13Nguyen M. Arkell J. Jackson C.J. J. Biol. Chem. 2000; 275: 9095-9098Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), 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 (14Ravanti L. Kahari V.M. Int. J. Mol. Med. 2000; 6: 391-407PubMed Google Scholar). In cultured human keratinocytes, APC enhances cell proliferation, migration, and MMP-2 activity (15Xue M. Thompson P. Kelso I. Jackson C. Exp. Cell Res. 2004; 299: 119-127Crossref PubMed Scopus (79) Google Scholar). 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 (16Jackson C.J. Xue M. Thompson P. Davey R.A. Whitmont K. Smith S. Buisson-Legendre N. Sztynda T. Furphy L.J. Cooper A. Sambrook P. March L. Wound Repair Regen. 2005; 13: 284-294Crossref PubMed Scopus (69) Google Scholar).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 (17Bikle D.D. Ng D. Tu C.L. Oda Y. Xie Z. Mol. Cell. Endocrinol. 2001; 177: 161-171Crossref PubMed Scopus (152) Google Scholar, 18Dransfield D.T. Griner R.D. Ray S. Keskintepe M. Bollag W.B. J. Investig. Dermatol. 2001; 117: 1588-1593Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). 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 (19Pivarcsi A. Bodai L. Rethi B. Kenderessy-Szabo A. Koreck A. Szell M. Beer Z. Bata-Csorgoo Z. Magocsi M. Rajnavolgyi E. Dobozy A. Kemeny L. Int. Immunol. 2003; 15: 721-730Crossref PubMed Scopus (285) Google Scholar). Keratinocytes in the basal and suprabasal layers of the epidermis express many components of the PC pathway, including thrombomodulin (20Jackson D.E. Mitchell C.A. Bird P. Salem H.H. Hayman J.A. J. Pathol. 1995; 175: 421-432Crossref PubMed Scopus (45) Google Scholar), PC inhibitor (21Krebs M. Uhrin P. Vales A. Prendes-Garcia M.J. Wojta J. Geiger M. Binder B.R. J. Investig. Dermatol. 1999; 113: 32-37Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), and EPCR (22Xue M. Campbell D. Sambrook P.N. Fukudome K. Jackson C.J. J. Investig. Dermatol. 2005; 125: 1279-1285Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). 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 ≈20 min (23Rezaie A.R. Trends Cardiovasc. Med. 2003; 13: 8-15Crossref PubMed Scopus (40) Google Scholar). 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 PROCEDURESKeratinocyte Culture and Reagents—Normal keratinocytes were isolated from neonatal foreskins (n = 30) as described previously (15Xue M. Thompson P. Kelso I. Jackson C. Exp. Cell Res. 2004; 299: 119-127Crossref PubMed Scopus (79) Google Scholar) 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 × 105 cells/well or 8-well Permanox™ 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 × 106 cells/ml in growth medium and subjected to nucleofection using the human keratinocyte Nucleofector™ kit and Amaxa Nucleofector™ 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 × 105 cells/ml) or 96-well plates (1 × 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 × 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] × 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 × 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 β-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.).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.RESULTSPC 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 (15Xue M. Thompson P. Kelso I. Jackson C. Exp. Cell Res. 2004; 299: 119-127Crossref PubMed Scopus (79) Google Scholar), 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 2PC 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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).FIGURE 3Endogenous 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 (×20) (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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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).FIGURE 4PC 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] × 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 5Blocking 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. ♣, indicates blocking both EPCR and EGFR in comparison with individual treatment; +, ♣, or *, p < 0.05; ++ or **, p < 0.01.View Large Image Figure ViewerDownload Hi-res image Download (PPT)EGFR is highly expressed in human keratinocytes in vivo and in vitro (24Fisher G.J. Kang S. Varani J. Bata-Csorgo Z. Wan Y. Datta S. Voorhees J.J. Arch. Dermatol. 2002; 138: 1462-1470Crossref PubMed Scopus (1198) Google Scholar, 25Xu Y. Tan L.J. Grachtchouk V. Voorhees J.J. Fisher G.J. J. Biol. Chem. 2005; 280: 42694-42700Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) 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.FIGURE 6EPCR 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.View La" @default.
• W2023688094 created "2016-06-24" @default.
• W2023688094 creator A5012952846 @default.
• W2023688094 creator A5025595864 @default.
• W2023688094 date "2007-05-01" @default.
• W2023688094 modified "2023-10-18" @default.
• W2023688094 title "Protein C Is an Autocrine Growth Factor for Human Skin Keratinocytes" @default.
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