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. Author manuscript; available in PMC: 2013 Sep 26.
Published in final edited form as: Wound Repair Regen. 2011 Oct 12;19(6):718–726. doi: 10.1111/j.1524-475X.2011.00734.x

Extracellular matrix-derived tripeptide proline-glycine-proline inhibits keratinocyte proliferation and migration

Yiwei Ma 1, Kyle Kleinbeck 1, W John Kao 1,2,3
PMCID: PMC3784246  NIHMSID: NIHMS429558  PMID: 22092842

Abstract

Keratinocytes are the predominant cell type in epidermis, and are primarily responsible for the epithelialization phase of wound healing. Previous studies by our group showed a positive correlation between IL-8 concentration and delayed healing of porcine cutaneous partial-thickness wounds. Interleukin-8 and collagen-breakdown product N-acetyl-Pro-Gly-Pro (PGP) are known as chemoattractant molecules for neutrophils during inflammation. The activity of both molecules is dependent on chemokine receptors CXCR1 and CXCR2. In addition to neutrophils, keratinocytes also express CXCR1 and CXCR2. Here we investigated the effects of IL-8 and PGP on keratinocyte proliferation and migration. Our results showed that IL-8 up to 100 ng/mL does not have any significant impact on keratinocyte proliferation or migration. ECM-derived tripeptide PGP chemotactically attracts neutrophils but not keratinocytes. PGP strongly inhibits keratinocyte proliferation and migration in a cell-type specific manner. Thus, collagen breakdown product PGP plays a key role in modulating both the inflammatory and epithelialization phases of wound healing.

INTRODUCTION

Wound healing is a dynamic and complex process that includes inflammation, cell proliferation, and extracellular matrix remodeling. During the reepithelialization phase of cutaneous wound healing, keratinocytes migrate across the wound site and proliferate at the margin for the closure of a wound. From our previous studies, healing of cutaneous partial-thickness wounds in pigs in response to clinical and experimental dressings indicated a strong positive correlation between IL-8 concentration and delayed healing.1 For example, at postoperative day 7, IL-8 concentration in the remodeling dermal tissue of treated wounds was 25.2-fold higher than unwounded dermal tissue. This relationship was not observed for other chemokines tested, including IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p70, IFN-γ, and TNF-α. Others had shown that IL-8 levels increased significantly in unhealed wounds compared with healed wounds or normal skin in a human model.2 Previous in vitro studies showed significant chemotactic migration of normal human keratinocytes toward IL-83,4 and a modest inhibitory effect of IL-8 on keratinocyte proliferation.2 These work suggested a potential novel regulatory role of IL-8 on keratinocyte migration and/or proliferation during the wound healing process.

Interleukin-8, a crucial member of the ELR subfamily of chemokines, is well known to induce neutrophil infiltration into the site of infection during inflammation. Neutralization of IL-8 with a monoclonal antibody in rabbit models resulted in suppression of neutrophil-mediated inflammatory responses.5–7 There are two G-protein coupled receptors for IL-8, CXCR1 and CXCR2, which are expressed at similar levels in neutrophils.8,9 Human CXCR1 binds three chemokines, CXCL6 (granulocyte chemotactic protein-2),10 CXCL7 (neutrophil activating peptide-2), and CXCL8 (interleukin-8) with high affinity, whereas CXCR2 is more promiscuous and also interacts with CXCL1 (GRO-α), CXCL2 (GRO-β), CXCL3 (GRO-γ), and CXCL5 (epithelial cell-derived neutrophil-activating peptide-78).11 Inhibition of CXCR2 by a small molecule antagonist SB 225002 was sufficient to prevent IL-8-induced neutrophil chemotaxis in vitro and sequestration in vivo.12 In addition to the above-mentioned chemokines, the collagen-breakdown product N-acetyl-proline-glycine-proline (N-acetyl PGP) is also chemoattractant for neutrophils due to a structural homology to CXC chemokines.13 The activity of PGP is dependent on CXCR1 and CXCR2, as pretreatment of human neutrophils with receptor antibodies significantly suppressed the chemotactic response.14 PGP-treated Cxcr2−/− mice did not accumulate neutrophils in the airway in contrast to wild-type mice.14,15

Apart from neutrophils and monocytes, numerous cell types have been shown to express CXCR1 and CXCR2, including neurons, various cancer cells, endothelial, and epithelial cells.16,17 There is also evidence that IL-8 acts on keratinocytes through interaction with CXCR1 and CXCR2.18 In this study, we investigated the impact of IL-8 and PGP on keratinocytes. We found that exogenous IL-8 was not a regulator of keratinocyte proliferation or migration; however, PGP significantly inhibited both responses, and this effect was specific to keratinocytes and not fibroblasts or neutrophils. The reversal role that PGP plays on neutrophils and keratinocytes provides a strong mechanistic evidence of how inflammation and reepithelialization would be modulated throughout the time course of wound healing.

MATERIALS AND METHOD

Synthesis of Pro-Gly-Pro

Tripeptide PGP was synthesized and purified by a standard solid phase peptide synthesis protocol.19 Briefly, piperidine (20% in DMF) was used to remove the Fmoc protecting group to expose the α-amino group for the reaction. Product identity and purity was confirmed by matrix-assisted laser desorption/ionization time-of-flight. Theoretical mass for the “+H” and “+Na” peak is 269.153 and 291.135 Da, respectively, and observed mass was 269.163 Da and 291.144 Da, respectively. The purity of synthesized PGP was analyzed by reversed-phase HPLC on a C18-analytical column equilibrated at a flow rate of 1 mL/minute and eluted with a linear gradient from 30% to 70 acetonitrile in water in 6 minutes.

Proliferation assay of keratinocytes

Neo-neonatal normal epidermal human keratinocytes (NHEK) in passage number 2–4 (Lonza, Basel, Switzerland) were plated into 96-well plates at a density of 5,000 cells per well in keratinocyte growth media (Genlantis, San Deigo, CA) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA). A dose range (0–100 ng/mL) of recombinant human IL-8 (Pierce, Rockford, IL) with or without 5 mg/mL PGP was added after 8-hour incubation. The concentration range of IL-8 and PGP (or N-acetyl PGP) tested in this experiment was consistent with the literature.2,20 Cells were incu bated for 1 to 7 days at 37 °C humidified condition with 5% CO2; fresh media with IL-8 and PGP was replaced every 24 hours up to 7 days. To quantify cell proliferation rate, bromodeoxyuridine (Fisher Scientific, Pittsburgh, PA) was added 20 hours prior to the end of assay, and the amount of incorporated BrdU was analyzed by an established protocol.21

Cell migration assay

Migration of keratinocytes in the presence of varying concentrations of IL-8 and other exogenous ligands (including GGG control, SB 225002, PGP, and N-α-PGP) was tested by a standard scratch assay reported by Lee et al.22 Briefly, NHEK passage number 2–4 cells were seeded into a 48-well plate (1.2 × 105 per well) and incubated in serum-containing KGM for 20 hours until >90% confluency. Gaps were created by scratching the center of the wells with sterile micropipette tips. The culture solution contained different concentrations of IL-8 (0–50 ng/mL) in the presence or absence of 5 mg/mL PGP, 0.5 mM N-acetyl PGP (AnaSpec, Fremont, CA), 5 mg/mL GGG (Sigma, St. Louis, MO) as negative control peptide or 100 nM of CXCR2-inhibitor SB 225002 (Enzo Life Science, Plymouth Meeting, PA). Ten micrograms per millimeter of mitomycin-C (Fisher Scientific) was added to inhibit cell growth as comparison.22 Cells were incubated for 4 days; medium containing fresh ligands was changed every 24 hours up to 4 days. The entire area within the border of the gap was manually traced using a video analysis system coupled to an inverted light microscope and quantified with ImageJ. Migration rate was measured by calculating percent decrease in the area of the gap as a ratio to the gap area created at the onset of the assay (t = 0 hour).

Human acute monocytic leukemia cell line THP-1 that is naturally deficient of the CXCR1 receptor was a kind gift of Prof. Kreeger (University of Wisconsin-Madison) and was explored for comparison. Cells were cultured in RPMI 1640 medium (Lonza) supplemented with 10% FBS and 10 mM HEPES buffer and seeded into 48-well plates at a density of 2 × 105 cells/well. Ten nanograms per milliliter PMA was added to the medium for 48-hour incubation, allowing THP-1 cells to adhere. At >90% confluency, migration assays were performed following procedure described above. Adult human dermal fibroblasts (Lonza) in passage number 3–4 were cultured in fibroblast basal medium (Lonza) supplemented with 10% fetal bovine serum. The dose effect of IL-8 on fibroblast migration was analyzed as for keratinocytes.

Chemotaxis assay of keratinocytes and neutrophils

The chemotactic response of keratinocytes and neutrophils toward known concentrations of IL-8 and PGP was determined using an established, commercially available 96-well chemotaxis assay with 8 μm pore size polycarbonate membranes (Millipore, Billerica, MA). Briefly, serum-free medium containing varying concentrations of IL-8 (0–50 ng/mL) and/or PGP (0–5 mg/mL) was added to the bottom plate. NHEK passage 2–4 cells that had been serum-starved for 20 hours were seeded onto the top plate at a density of 5 × 104 cells per well. Human blood-derived neutrophils were isolated by a standard protocol20 and seeded at 10 × 104 cells per well in RPMI 1640 medium. After 4-hour incubation at 37 °C humidified condition plus 5% CO2, migrated cells on the bottom surface of the insert membrane were disassociated from the membrane by incubating with vendor-provided cell detachment solution. The cells were subsequently lysed and detected by a specific green fluorescent dye that exhibits strong fluorescence enhancement when bound to cellular nucleic acids.23,24 The number of migrated cells was proportional to the strength of fluorescent signals (per vendor’s instruction). The chemotactic response was reported as migration index, which represents the percentage change compared with migration toward medium alone.

Statistical analysis

All experiments were repeated at least in triplicates, and each experiment was independently repeated three times (n = 3) or twice (n = 2, fibroblast scratch assay, THP-1 scratch assay, chemotaxis assay). The data were expressed as mean ± SEM. Analysis of the differences between means was performed by ANOVA F test and Student’s t test; a p-value < 0.05 was considered statistically significant.

RESULTS

Effect of IL-8 and PGP as ligands on keratinocyte proliferation and migration

The concentration effect of IL-8 on keratinocyte proliferation was quantified with BrdU incorporation assay. Proliferation rate did not show a dose-response relationship (Figure 1). When cells had grown for 96 hours or longer, elevated IL-8 level significantly decreased cell proliferative rate (p-value <0.05). However, the difference in proliferation between 0.1 and 100 nM IL-8 was only minimal (less than 40%). Because normal physiological IL-8 concentration is in the pM range, this result indicates that IL-8, even at an extremely high concentration, does not strongly impact keratinocyte proliferation. Collagen-breakdown product N-acetyl PGP is known to induce cell polarization and chemotactic response of human neutrophils with EC50 = 0.5 mM.20 When the N-terminus acetyl group was deleted, PGP could still exert the same effect on neutrophils but with a weaker binding affinity (reported EC50 = 15 mM = 4 mg/mL).20 When keratinocytes were treated with 5 mg/mL PGP, proliferative responses in terms of BrdU uptake were almost completely inhibited independent of the IL-8 concentration at all culturing time (Figure 1).

Figure 1.

Figure 1

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Keratinocyte proliferation in the presence of different doses of IL-8 as determined by BrdU assay (logarithm scale). Keratinocytes were incubated for 24 hours, 48 hours, 96 hours, and 7 days before BrdU was added. The concentration effect of IL-8 on cell proliferation was quantified in terms of BrdU incorporation. In order to minimize any experimental variances, the BrdU signals were normalized as the percentage of the BrdU signal without IL-8, which had values of 0.713, 0.642, 0.589, and 1.034, respectively, for 24-hour, 48-hour, 96-hour, and 7-day incubation time. The BrdU signal with 5 mg/mL PGP and without IL-8 was 13.8, 0.45, 9.24, and 0.43%, respectively, for 24-hour, 48-hour, 96-hour, and 7-day incubation time. Data are expressed as mean ± SEM; n = 3 in triplicates. ∎, 7 days; ◻, 7 days with PGP; ▴, 96 hours; Δ, 96 hours with PGP; ●, 48 hours; 엯, 48 hours with PGP; ◆, 24 hours; ◇, 24 hours with PGP. GGG, glycine-glycine-glycine; PGP, proline-glycine-proline.

In the scratch assay, the gap gradually became repopulated with cells due to growth and/or migration from the surrounding areas. When mitomycin-C was added to the culture medium, cell proliferation was inhibited, thus the closing of the gap was primarily due to cell migration. IL-8 or negative control peptide GGG did not have a significant impact on keratinocyte migration regardless of their concentration (Figure 2). The presence of PGP or N-acetyl PGP significantly reduced migration rate independent of IL-8 concentration. Without mitomycin-C, keratinocytes were able to proliferate, thus gap healing was a result of both migration and proliferation. As expected, IL-8 concentration did not play a significant role in this process (Figure S1 in supplemental materials). By 48 hours, cells repopulated the entire area of the gaps for all IL-8 levels tested. The control peptide GGG did not have any effect on cell migration and proliferation; but PGP significantly inhibited both activities—decrease in the area of the gap was less than 30% even after 96-hour incubation (Figure S2). Inhibition of keratinocyte migration and proliferation by PGP showed a positive relationship with its concentration: the higher the PGP concentration, the stronger the inhibitory effect (Figure 3).

Figure 2.

Figure 2

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Keratinocyte migration in the presence of different doses of IL-8 as determined by scratch assay. Mitomycin-C was supplemented in the culture medium to inhibit cell proliferation. The areas of the gaps were quantified after 24- (A), 48- (B), and 96-hour (C) incubation. Migration rate was quantified as percent decrease in the area of the gap compared to the area at 0 hour. Data are expressed as mean ± SEM, n = 3 in triplicates. Inline graphic, no treatment; Inline graphic, GGG; Inline graphic, SB 225002; Inline graphic, PGP; Inline graphic, N-acetyl-PGP. BrdU, bromodeoxyuridine; PGP, prolineglycine-proline.

Figure 3.

Figure 3

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The effect of PGP on keratinocyte migration as determined by scratch assay. Migration rate was measured by percent decrease in the area of the gap after 24- or 48-hour incubation with 10 μg/mL mitomycin-C in the medium. Data are expressed as mean ± SEM, n = 3 in triplicates. ◻, IL-8 = 0, 24 hours; 엯, IL-8 = 0, 48 hours; ∎, IL-8 = 50 ng/mL, 24 hours; ●, IL-8 = 50 ng/mL, 48 hours. GGG, glycineglycine-glycine; PGP, proline-glycineproline.

For comparison, fibroblast migration assay was performed to test whether the inhibitory effect of PGP on cell proliferation/migration was keratinocyte specific. In general, fibroblasts migrated much faster than keratinocytes. More than 50% area of a gap was repopulated with fibroblasts after 24-hour incubation (Figure 4) when cell proliferation was inhibited by mitomycin-C (compared with ~10% gap healing of keratinocyte with the same seeding density, shown in Figure 2). Without mitomycin-C (Figure S3 in supplemental materials), the gaps had mostly disappeared after 24 hour when confluency was observed. No differences were observed in migration rate for all experimental groups. This suggests that PGP impacts keratinocyte proliferation and migration, but not fibroblasts in a cell type-specific manner. Fibroblast migration was not influenced by SB 225002, either, indicating that the CXCR2 receptor was not involved in regulation of cell migration or proliferation (Figure 4).

Figure 4.

Figure 4

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Fibroblast migration in the presence of different doses of IL-8 as determined by scratch assay. Migration rate was measured by percent decrease in the area of the gap after 24-hour incubation with 10 μg/mL mitomycin-C in the medium. Data are expressed as mean ± SEM, n = 2 in triplicates. Inline graphic, no treatment; Inline graphic, GGG; Inline graphic, SB 225002; Inline graphic, PGP. GGG, glycine-glycine-glycine; PGP, proline-glycine-proline.

In addition to the scratch assay, we also analyzed chemotactic migration of keratinocytes and neutrophils toward IL-8 and PGP using a Boyden chamber ((Figure 5). We found that PGP attracts neutrophils in a concentration-dependent manner as expected. Increasing PGP concentration up to 5 mg/mL significantly increased the chemotactic response (p-value < 0.05). This observation is consistent with literature findings.15,16 In contrast, PGP is not a chemoattractant for keratinocytes: the concentration of PGP did not have any significant effect (p-value > 0.05) on the number of migrated keratinocytes. As expected, IL-8 induced neutrophil chemotaxis, and optimum response was reached at a concentration of 0.1–1 ng/mL (Figure 6B). However, this chemotactic effect of IL-8 on neutrophils is less than what was reported in the literature (Lin et al.25). It is possible that the polycarbonate filters used with 8 μm pore size might be large for neutrophils, although it is validated for keratinocytes. Combining PGP and IL-8 did not further promote chemotactic migration of neutrophils, possibly due to competitive binding to the CXCR receptors. In Figure 6A, there is a chemoattractant effect of IL-8 on keratinocytes with maximum chemotaxis observed at a concentration of 1 ng/mL, although this trend was only marginally statistically significant (p-value = 0.05).

Figure 5.

Figure 5

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Chemotactic migration of keratinocytes and neutrophils toward PGP. Chemotaxis is reported as Migration Index, which represents percent number of migrated cells compared to migration toward medium alone. Data are expressed as mean ± SEM, n = 2 in triplicates. Inline graphic, PMN; Inline graphic, keratinocytes.

Figure 6.

Figure 6

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Chemotactic migration of keratinocytes and neutrophils toward IL-8 and the effect of PGP on this process. Chemotaxis toward a dose range of IL-8 in the presence or absence of PGP is reported as Migration Index. 6A: keratinocytes; 6B: PMNs. Data are expressed as mean ± SEM, n = 2 in triplicates. Inline graphic and Inline graphic, no PGP; Inline graphic and Inline graphic, with 5 mg/mL PGP. PGP, proline-glycine-proline.

Role of CXCR1 and CXCR2 on keratinocyte migration

SB 225002, discovered by White et al., is a nonpeptide antagonist specific of the CXCR2 receptor.12 In vitro, it potently inhibited human neutrophil chemotaxis induced by IL-8 with IC50 = 22 vs. 0.25 nM of IL-8.12 In this study, the presence of 100 nM SB 225002 marginally retarded keratinocyte migration rate (Figure 2). In the absence of mitomycin-C, gap healing rate was approximately the same for SB225002-treated samples as for the no treatment control group. Therefore, inhibiting CXCR2 had no significant effect on keratinocyte proliferative and migrative responses.

Other than CXCR2, IL-8 also binds to CXCR1, which is a key receptor mediating the chemotactic response of neutrophils. Due to the lack of CXCR1-specific antagonist and the potentially low siRNA transfection efficiency in primary keratinocytes, we studied THP-1 cells that are naturally deficient of the CXCR1 receptor as a comparison. As shown in Table 1, CXCR1-deficient THP-1 cells migrate and proliferate very slowly. The presence of PGP did not promote or inhibit proliferation and migration of THP-1 cell. Although conclusion cannot be extrapolated to keratinocytes, it is possible that the CXCR1 receptor plays a major role in IL-8 and PGP-regulated cell signaling pathways that lead to proliferation and migration. Treatment with SB 225002 had no effect on proliferative or migrative ability of CXCR1-depleted THP-1 cells either, which was expected because SB 225002 is >150-fold selective for CXCR2 than CXCR1.

Table 1.

Semi-quantified scratch assay with CXCR1-deficient THP-1 cells

IL-8 conc.
(ng/mL)		 No treatment PGP Ac-PGP GGG SB 225002
Incubated for 0 hours, without mitomycin-C
0 1.5 ± 0.5 1 ±0 1±0 1±0 1± 0
0.1 1 ±0 1±0 1± 0 1.5 ± 0.5 1 ± 0
1 1± 0 1.5 ± 0.5 1 ±0 1± 0 1.5 ± 0.5
50 1 ±0 1±0 1± 0 1.5 ± 0.5 1 ± 0
Incubated for 24 hours, without mitomycin-C
0 5± 1 5.5 ± 0.5 4.5 ± 0.5 5.5 ± 0.5 5.5 ± 0.5
0.1 4 ±0 5± 0 5.5 ± 0.5 5 ± 1 5.5 ± 0.5
1 5.5 ± 0.5 4.75 ± 0.25 5 ± 1 5.5 ± 0.5 4.5 ± 0.5
50 5.5 ± 0.5 5 ± 0 5.5 ± 0.5 5.25 ± 0.25 5 ± 1
Incubated for 48 hours, without mitomycin-C
0 5.5 ± 0.5 5 ± 1 6.5 ± 0.5 5 ±1 6± 1
0.1 6.5 ± 0.5 5.5 ± 0.5 5.5 ± 0.5 6.5 ± 0.5 5.5 ± 0.5
1 6± 1 4.5 ± 0.5 6 ±0 6± 0 6.5 ± 0.5
50 5.5 ± 0.5 5.5 ± 0.5 5.5 ± 0.5 5 ±1 6± 0
Incubated for 0 hours, with mitomycin-C
0 1± 0 1.5 ± 0.5 1.5 ± 0.5 2 ± 0 1.5 ± 0.5
0.1 1 ±0 2± 1 1.5 ± 0.5 1.5 ± 0.5 1.5 ± 0.5
1 1.5 ± 0.5 1.5 ± 0.5 1.5 ± 0.5 1.5 ± 0.5 1.5 ± 0.5
50 1 ± 0 1.5 ± 0.5 2 ±1 2± 1 1.5 ± 0.5
Incubated for 24 hours, with mitomycin-C
0 4.5 ± 0.5 3.5 ± 0.5 3 ± 1 3.5 ± 0.5 4 ± 1
0.1 3 ±1 3±0 3±1 3± 0 3.5 ± 0.5
1 3±0 3± 0 3.5 ± 0.5 3 ±0 3± 1
50 3.5 ± 0.5 3.5 ± 0.5 2.5 ± 0.5 2.5 ± 0.5 3 ± 1
Incubated for 48 hours, with mitomycin-C
0 5.5 ± 0.5 6.5 ± 0.5 6 ±0 5± 1 5.5 ± 0.5
0.1 5 ±0 6± 1 5.5 ± 0.5 4.5 ± 0.5 5 ± 1
1 5.5 ± 0.5 5.5 ± 0.5 4.5 ± 0.5 4.5 ± 0.5 5 ± 1
50 5 ± 0 5.5 ± 0.5 5.5 ± 0.5 4.5 ± 0.5 5 ± 0
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The area within the entire gap was examined based on a semi-quantified scale instead of direct measurement as in the other data sets because THP-1 cells are much smaller in size and do not spread out as well as keratinocytes. Thus, direct measurement using video analysis coupled microscopy will introduce a high level of variable. Statistical analysis was not carried out because the scale did not have any apparent trend. The semi-quantified scale is as follows: 1, no cells in the gap; 2, 5–10 cells in the gap; 3, 20–30 cells; 4, 50–80 cells; 5, many cells, gap boundary blurred; 6, ~20% of the gap was covered with cells; 7, ~50% covered; 8, ~75% covered; 9, gap almost disappeared except for a few empty zones in the center; 10, gap completely disappeared and cells were confluent. Data are expressed as mean ± SEM, n = 2 in triplicate. GGG, glycine-glycine-glycine; PGP, proline-glycine-proline.

DISCUSSION

Both IL-8 and collagen-breakdown product PGP are known to be chemoattractant for neutrophils in vivo and in vitro. Conversely, our data show that neither molecule induces pro-chemotactic or pro-proliferative responses in keratinocytes. Others had reported a significant inhibitory effect of IL-8 (p < 0.001, ANCOVA) on the proliferation of human skin-derived keratinocyte by performing direct cell counting.2 In that study, the difference in cell number between 1 and 100 ng/mL of IL-8 was 21% after 7–9 days incubation, similar to what we obtained from the BrdU proliferation assays. However, this difference in keratinocyte proliferation rate was not sufficient to account for the impairment in cutaneous wound healing in vivo as observed by Kleinbeck et al.1 We conclude that the role of IL-8 on keratinocyte proliferation is not directly causal. There are likely other unknown mechanisms underlying the correlative relationship between IL-8 accumulation and delayed wound healing. Results from our scratch assay showed that IL-8does not have a dose-dependent effect on keratinocyte migration. Previous studies reported that HaCaT keratinocytes migrated toward IL-8 by performing a chemotaxis assay, and the optimal chemotactic concentration was between 0.1 and 1 nM.3,4 We reproduced this result in a similar manner: Figure 6A shows a potential trend of keratinocyte chemotaxis toward IL-8. It suggests that the chemotaxis and scratch assays are measuring different cellular properties—chemotactic response toward a ligand does not necessarily mean promoted migration in the presence of that ligand.

Collagen-breakdown product PGP at a concentration that is sufficient to produce 50% neutrophil chemotaxis20 significantly inhibits keratinocyte proliferation and migration. The BrdU assay showed a strong inhibitory effect of PGP on keratinocyte proliferation. The scratch assay without mitomycin-C added mimics simplified wound healing process, where keratinocytes migrate from a region of higher cell density to a gap and proliferate at the edge of the gap. Therefore, a high concentration of PGP in the ECM is likely to have a delay effect on reepithelialization by inhibiting both proliferation and migration of keratinocytes at the margin. Taken together, PGP plays a unique role in the wound healing process: it facilitates neutrophil recruitment as an integral part of the inflammatory response while it suppresses keratinocyte migration and proliferation into the site of the wound during that phase of the wound healing response.

Some natural ligands are known to show divergent effects on different cell types. For example, retinoic acid inhibits proliferation of cancer cells but promotes survival of HaCaT keratinocytes. The opposite responses are mediated by two different signaling pathways: inhibition of cell growth is a result of transcriptional activation of retinoic acid receptor, whereas the proliferative activities are regulated by nuclear receptor PPARβ/δ.26 In addition, FGF2 can elicit opposing cellular responses of a single cell type at different exposure concentrations. Low and high concentrations of FGF2 stimulate survival and differentiation of NIH3T3 fibroblasts, whereas intermediate concentrations of FGF2 stimulate proliferation in the presence of serum but apoptosis in its absence.27 Thus, it is possible that PGP has divergent or even opposite effects on neutrophils and keratinocytes. Virtually nothing is known about PGP signaling mechanism, especially that in keratinocytes. Recently, the exact identity of the binding receptor for PGP has been challenged. Although there are evidences that the chemotactic effects of PGP are absent when cells are treated with CXCR1 or CXCR2 antibodies, de Kruijf recently discovered that PGP did not displace [125I]IL-8 binding to either receptor.28 In addition, PGP has no effect on G protein signaling or β-arrestin2 recruitment.28 Our data are likely to support the hypothesis that PGP does not directly activate or inhibit CXCR2-mediated signaling events, since treatment with the CXCR2 antagonist SB 225002 does not have any impact on keratinocyte proliferation or migration. The results with CXCR1-deficient THP-1 cells indicate that the inhibitory effect of PGP on cell growth and migration is mediated by the CXCR1 receptor. Elucidating downstream effect of PGP in keratinocytes and neutrophils would provide additional information on the balance and the progression of key stages during wound healing.

Supplementary Material

Supplementary Figures

ACKNOWLEDGMENTS

The authors are grateful to Professor Pamela Kreeger in the biomedical engineering department of UW-Madison for providing us CXCR1-deficient THP-1 cells and NIH Grant EB006613 for financial support.

Glossary

BrdU

Bromodeoxyuridine

CXC

CXC chemokine receptor

ECM

Extracellular matrix

GGG

Glycine-glycine-glycine

IL-8

Interleukin-8

PGP

Proline-glycine-proline

THP-1

Human acute monocytic leukemia cell line

Footnotes

Supporting Information Additional Supporting Information may be found in the online version of this article:

Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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