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Department of Dermatology, University of California, Davis, California, USA
1Correspondence: Department of Dermatology, University of California Davis, TB 192, One Shields Ave, Davis, CA 95616, USA. E-mail: cepullar{at}ucdavis.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: wound re-epithelialization • ERK activation • keratinocyte migration • focal adhesions
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, only recently has their functional significance in this tissue been recognized. The ß2-AR subtype is the only subtype of ß-ARs expressed on membranes of the major cell types in skin: keratinocytes (2
3
4)
, fibroblasts (5)
, and melanocytes (6)
. Cutaneous keratinocytes also actively synthesize catecholamine ligands for these receptors (7
, 8)
, thus creating a self-contained hormonal mediator network. Keratinocyte-generated catecholamines have recently been demonstrated to regulate skin melanogenesis, thus providing one of the first clues as to the homeostatic regulatory function of this cutaneous paracrine signaling network (6)
. Aberrations in either keratinocyte ß2-AR function or density have also been associated with cutaneous disease. Keratinocytes derived from patients with atopic eczema display a point mutation in the ß2-AR gene and a low ß2-AR density (8)
. In psoriasis, keratinocytes within the psoriatic lesions demonstrate a low cAMP response to ß2-AR activation (9)
. These findings point to a role for the cutaneous ß2-AR network in maintaining epidermal function and integrity. Here, we provide data to support a role for the ß2-AR network in regulating cutaneous wound repair as well.
Cutaneous wound healing is a complex and well-orchestrated biological process requiring the coordinated migration and proliferation of both keratinocytes and fibroblasts, as well as other cell types. Wounding the epidermis generates cytokines, growth factors, proteases and initiates the synthesis of extracellular matrix components, all of which can regulate the processes of keratinocyte migration and proliferation essential for re-epithelialization (10
, 11)
. The first clues to a biological function for ß2-AR in wound repair came from an early study demonstrating that ß2-AR agonists delay skin wound healing in newt limbs (12)
. Subsequent studies in other epithelia, however, have yielded conflicting results. For example, ß-AR antagonists have been reported to either delay (13
, 14)
or enhance (15)
corneal epithelial wound healing. Recently, Denda et al. reported that the ß-AR could modulate epidermal barrier permeability by altering transepidermal water loss (16)
.
Work from our laboratory has focused on the effects of ß-AR agonists on the constituent cells of human skin. We found that ß-AR agonists decrease keratinocyte migration in vitro (17
, 18)
. Unlike other cell types studied, where ß-AR agonist binding activates ERK (19
20
21
22
23
24
25)
, in keratinocytes ß-AR agonists reduce ERK phosphorylation, notably in a cAMP-independent (17)
and PP2A-dependent manner (18)
. Since ERK phosphorylation is activated upon mechanical injury of keratinocytes (26)
and is also required for keratinocyte migration (27)
and proliferation (28)
, these findings suggest that ß2-adrenergic signaling could affect wound repair by modulating ERK phosphorylation, thus regulating the dual critical processes of keratinocyte migration and proliferation.
To this end, we have further extended our investigations to observe the effects of ß2-AR activation on keratinocyte polarization, migration, and ERK phosphorylation at the wound edge, cytoskeletal organization, phospho-ERK intracellular localization, proliferation, human skin wound re-epithelialization, wound-induced ERK phosphorylation, and murine skin wound healing.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and cultured using a modification of the method of Rheinwald and Green (30)
. Cells were grown in keratinocyte growth medium (KGM) (Epilife, 0.06 mM Ca2+), supplemented with human keratinocyte growth supplement (0.2 ng/mL EGF, 5 µg/mL insulin, 5 µg/mL transferrin, 0.18 µg/mL hydrocortisone, and 0.2% bovine pituitary extract) (Cascade Biologics, Inc., Portland, OR, USA) and antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin) (Gemini Bio-Products, Inc., Calabasas, CA, USA) at 37°C in a humidified atmosphere of 5% CO2. Cell strains isolated from at least two different foreskins were used and experiments were performed with subcultured cells between passages 4–7.
Scratch assay
Cells were grown to confluence in KGM on 35 mm culture dishes or glass coverslips (Fisher Scientific, Pittsburgh, PA, USA) coated with 60 µg/mL Collagen I (Vitrogen 100, Collagen Corp., Palo Alto, CA, USA). Cells were either untreated (control), treated with ß-AR agonist (1 µM) (isoproterenol, Calbiochem, San Diego, CA, USA) in KGM at time 0, or pretreated with okadaic acid (OA, Calbiochem) (10 nM) for 30 min before addition of OA alone or both OA and ß-AR agonist in KGM at time 0. A sterile pipette tip was used to scratch a 1 mm-wide wound along the center of the dish or coverslip and a demarcated area of the wound was photographed on an inverted Nikon Diaphot microscope at the time of wounding (time 0) and at various times after wounding up to wound healing (31)
at 10x or 20x magnification.
To perform immunofluorescence studies of the wound edges, cells plated on collagen-coated coverslips were either fixed immediately after wounding (time 0) or incubated in KGM for 5–30 min at 37°C in the presence or absence of 1 µM ß-AR agonist, 10 nM OA, or both prior to fixing. Phospho-ERK immunostaining was performed as described in the immunofluorescent staining method.
Immunofluorescent staining and microscopy
Sterile glass coverslips were transferred into 12-well dishes and collagen-coated with 60 µg/mL collagen I in KGM for 1 h at 37°C. Coverslips were washed three times with KGM and 3 x 104 cells were added per well and allowed to attach overnight. Cells were untreated, treated with 1 µM ß-AR agonist for 15 min, OA (10 nM) for 45 min, or pretreated with OA (10 nM) for 30 min before addition of 1 µM ß-AR agonist for 15 min. Coverslips were processed at room temperature unless otherwise noted. Coverslips were washed twice in PBS and fixed for 10 min in 4% paraformaldehyde. Coverslips were washed twice in PBS between each step. Cells were permeabilized for 5 min with 0.1% Triton-X-100/PBS, blocked with 5% goat serum/PBS for 20 min, and primary monoclonal anti-vinculin antibody (Sigma, St. Louis, MO, USA) or anti-phospho-ERK antibody (Cell Signaling Technology, Beverly, MA, USA) was added drop-wise in 1% goat serum/PBS (1:100) and incubated for 1 h at 37°C. A goat anti-mouse cy3 (Jackson labs, West Grove, PA, USA) (1:100) antibody was then added in 1% goat serum/PBS for 1 h at 37°C. Alexa 488-phalloidin (Molecular Probes, Eugene, OR, USA) (1:40) in PBS was added to the vinculin-stained coverslips for 20 min. Standard controls were performed. Coverslips were incubated with the primary antibody alone or the secondary antibody alone to ensure specificity. Finally, Prolong anti-fade reagent (Molecular Probes) was used according to manufacturer’s instructions to mount the coverslips onto glass microscope slides. Slides were viewed on an inverted fluorescent Nikon Diaphot microscope using a 40x pan fluor objective. Images were captured using Q-imaging Retiga-EX cameras (Burnaby, BC, Canada) and pseudo-colored green for Alexa 488 Phalloidin staining (actin), red for Cy3 staining (vinculin), or visualized in gray scale for phospho-ERK staining using Improvision Openlab software (Lexington, MA, USA). To observe the colocalization of vinculin and phospho-ERK, confocal immunofluorescence microscopy was conducted using a Leica TCS SP confocal microscope with a 100x 1.4NA objective (Leica, Wetzlar, Germany), an argon 488 nm excitation laser to observe fluorescein staining, and a krypton 568 nm excitation laser to observe cy3 staining.
Proliferation assay
Keratinocytes were released from the tissue culture plate by treatment with 0.25% trypsin/EDTA (Gibco, Grand Island, NY, USA), resuspended in KGM, and counted using a hemocytometer. 5 x 104 cells were plated per well in a 12-well plate in triplicate and allowed to settle and attach to the plate for 2 h. Cells were either untreated or pretreated with OA (10 nM) for 30 min prior to ß-AR agonist (1 µM) addition. Cells were then cultured in the presence or absence of 1 µM ß-AR agonist, 10 nM OA, or both. Triplicate wells were harvested and counted on days 2, 4, 6, and 8. The medium was changed every day. Significance was taken as P < 0.01, using Student’s t test (unpaired) to compare the means of the cell populations.
Human skin wound healing assay
We adapted a wound healing model developed by Kratz (32)
. Normal human skin was obtained from routine breast reductions or abdominoplasties under an approved exemption granted by the Internal Review Board at University of California, Davis. Under sterile conditions, excess subcutaneous fat was trimmed from 6" x 3" sections of skin prior to stretching and pinning onto sterile cork board. A 3 mm punch (Sklar Instruments, West Chester, PA, USA) was used to make wounds in the epidermis and into the superficial dermis and the 3 mm discs of skin were excised using sterile scissors. Skin discs (6 mm), with the 3 mm epidermal wound in the center, were excised using a 6 mm biopsy punch (SMS Inc., Columbia, MD, USA). The skin samples were immediately transferred to a 12-well dish and submerged in 2 mL of FM (Dulbecco’s modified Eagle’s medium (Gibco) containing 10% fetal bovine serum (Tissue Culture Biologicals, Tulare, CA, USA) and antibiotics in the presence or absence of 10 µM ß-AR agonist. The 12-well dishes were incubated at 37°C in a humidified atmosphere of 5% CO2. The medium was changed every day. Three biopsies were fixed in 4% neutral buffered formaldehyde every day for 5 days. The formaldehyde-fixed biopsies were dehydrated through an ethanol-xylene series and embedded in paraffin. Cross sections, 5 µM thick, taken from the center of the wound, were stained using the hematoxylin-eosin technique. Re-epithelialization was determined using light microscopy. A (+) score was given to a healed wound and a (–) score to any unhealed wounds. Slides were viewed on an inverted Nikon Diaphot microscope using a 10x objective. Images were captured using Q-imaging Retiga-EX cameras (Burnaby, BC, Canada). Specimens that were damaged in the histologic process or otherwise noninterpretable were excluded from the study. Significance was taken as P < 0.01, using the 2-tailed Fisher’s exact test to compare the number of wounds healed vs. unhealed in the absence or presence of ß-AR agonist. Measuring the linear distance covered by new epithelium and dividing that by the linear distance between the original wound edges determined the percentage of re-epithelialization. The new epithelium was clearly differentiated from the epithelial wound margin by the presence of a fully stratified epithelium and fully formed stratum corneum in the latter. Significance was taken as P < 0.05, using Student’s t test (unpaired) to compare the means of the % re-epithelialization of the control and ß-AR agonist-treated wounds each day.
Protein extraction from human skin
To determine the phosphorylation state of ERK in wounded skin, the excised 3 mm epidermal discs were preincubated in FM for 30 or 60 min in the presence or absence of 10 µM ß-AR agonist prior to freezing or placed immediately into 500 µL of 1xLaemmli sample buffer (62.5 mM Tris-HCL, pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol, 50 mM dithiothreitol) and snap frozen in liquid nitrogen prior to storing at –80°C. Two 3 mm skin pieces were frozen per tube. Each tube was thawed for 10 min at 100°C, then centrifuged at 14,000 rpm for 10 min at 4°C. Protein concentrations were estimated by A280, and equal microgram amounts were separated on 10% polyacrylamide Tris-HCl gels (Bio-Rad, Hercules, CA, USA). Proteins were transferred to Immobilon membranes (Bio-Rad) and immunoblotted with either an anti-ERK antibody (#91)2) or an anti-phospho-ERK antibody (#9101) (Cell Signaling Technology, Beverly, MA, USA). The immunoblots were developed by enhanced chemiluminescence according to the manufacturer’s instructions and within the linear range of the film (Amersham Pharmacia Biotech, Piscataway, NJ, USA). Densitometry was performed on scanned images from three separate experiments using NIH Image 1.6 and the phospho-ERK signal was normalized to total ERK, averaged, statistically analyzed and represented graphically. Significance was taken as P < 0.01, using Student’s t test (unpaired) to compare the means of the band intensities.
Murine tail-clip wounding experiments
A group of six male 8-wk-old C57Bl6 mice (Jackson labs, Bar Harbor, ME, USA) were used for this study. All experiments were performed under an animal protocol approved by the Animal Care and Use Committee Review Board at UCDavis. For wound healing measurements animals were anesthetized with ketamine-xylazine 50 µg/g via intraperitoneal injection, then tails were clipped with a single stroke of a scalpel blade 2 cm from the terminal in anesthetized animals (33)
. Mice were then treated with PBS (control n=3) or 0.2 mg/kg clenbuterol (n=3) in PBS every other day by intraperitoneal injection. Seven days postwounding an additional 5 mm of tail was removed and fixed in 3.7% formalin overnight. The formaldehyde-fixed biopsies were decalcified for 3 days in Easycut Decal solution (Master*Tech Scientific Co., Lodi, CA, USA), then dehydrated through an ethanol-xylene series and embedded in paraffin. Cross sections, 5 µM thick, were stained using the hematoxylin-eosin technique. Re-epithelialization was determined using light microscopy. Slides were viewed on an inverted Nikon Diaphot microscope using a 10x objective. Representative photographs of sections traversing the tissue in its midportion were captured using Q-imaging Retiga-EX cameras.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. To investigate the process further, we observed the edge of scratch wounds within the first few hours postwounding. In untreated cultures, keratinocytes at the edge of the wound polarize within 2 h of wounding. Broad lamellipodia can be observed extending into the denuded area. Detachment from the wound edge was observed as early as 3 h postwounding (Fig. 1
A). In contrast, ß2-AR activation prevents the initial keratinocyte polarization and migration into the denuded area (Fig. 1A
). In cultures treated with the PP2A inhibitor okadaic acid (OA) at a concentration highly selective for PP2A (10 nM) (34)
, the initial keratinocytes polarization and migration into the wound were indistinguishable from untreated cells (results not shown). Pretreatment with OA prior to ß2-AR activation restores keratinocyte polarization and migration into the wound (Fig. 1A
), demonstrating that the mechanism for the ß2-AR-mediated attenuation of wound-induced polarization and migration is PP2A dependent.
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ERK activation, at the edge of a wound in confluent rat keratinocyte cultures, is required for injury site closure (35)
. To determine whether there is an increase in ERK phosphorylation at the edge of a scratch wound in confluent human keratinocyte cultures, we fixed wounded cultures immediately after wounding or at 5–30 min postwounding and immunostained for phospho-ERK. Unwounded areas of fixed keratinocyte cultures provided an internal background control for the wound-induced phospho-ERK signal. An increase in ERK phosphorylation at the wound edge could be observed within 5 min of wounding, diminishing to background levels within 1 h (Fig. 1B
). The fluorescence intensity decreased dramatically as cells became more distant from the wound edge and was so low in unwounded areas that no fluorescence could be detected. As we had determined that ß2-AR activation decreases ERK phosphorylation and scratch wound healing (18)
, we wondered whether it would also decrease the wound-induced increase in ERK phosphorylation at the wound edge required for injury repair. Indeed, ß2-AR activation attenuates the wound-induced increase in ERK phosphorylation at the wound edge, likely contributing to the observed delay in scratch wound healing (Fig. 1B
). OA treatment has no effect on the wound-induced increase in ERK phosphorylation (results not shown). OA pretreatment prior to ß2-AR activation restores the wound-induced increase in ERK phosphorylation at the wound edge (Fig. 1B
), demonstrating that the mechanism for the ß2-AR-mediated decrease in wound edge ERK phosphorylation is PP2A dependent.
ß2-AR agonists alter keratinocyte cytoskeletal organization and OA prevents the ß2-AR mediated changes in cytoskeletal organization
Actin remodeling plays an important role in cell polarization (36)
and motility (37)
. Actin filaments terminate in focal adhesions (FAs), where several proteins, including vinculin, mediate interactions with the actin cytoskeleton (38)
. FAs mediate the mechanical attachment of cells to the extracellular matrix (39)
and act as signaling centers capable of regulating gene expression, cell growth, and survival (40)
. Small, nascent FAs have been associated with actively migrating cells (41)
. Since we have demonstrated that ß2-AR activation prevents polarization of keratinocytes at a wound edge and delays migration into the denuded area, we reasoned that these effects might involve alterations in the actin cytoskeleton.
Cells plated in the absence of ß-AR agonist are polarized and crescent shaped with a broad lamellipodium (Fig. 2
A), characteristic of the migratory phenotype (36)
. Actin and vinculin staining reveals that the majority of the untreated keratinocytes have fine actin-rich lamellipodia containing multiple small linear vinculin-containing FAs (Fig. 2A
). Pretreating with ß-AR agonist for 15 min markedly alters the keratinocyte morphology. Cells are now rounded with no apparent polarization. There is a marked increase in cortical actin stress fibers localized at the internal borders of the cells and an increase in the number and size of vinculin-rich FAs, which are no longer localized to the lammellipodium (Fig. 2B
).
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To determine whether the ß-AR agonist-mediated alteration in the cyto-architecture of actin stress fibers and vinculin-rich FAs was similarly mediated by PP2A, we pretreated keratinocytes with the PP2A-specific inhibitor OA prior to exposure to ß-AR agonist. OA treatment alone has no effect on the cytoskeletal organization, with cells displaying a normal migratory phenotype (Fig. 2C
). However, pretreating keratinocytes with OA prior to adding ß-AR agonists prevents the ß2-AR-mediated change in cytoskeletal organization (Fig. 2D
). OA pretreatment restores the migratory phenotype observed in untreated keratinocytes, confirming that the mechanism for the ß2-AR-mediated alteration of cytoskeletal organization is PP2A dependent (Fig. 2D
).
ß2-AR agonists disrupt the phosphorylation and intracellular localization of phospho-ERK, while OA preserves its localization to the leading edge of the keratinocyte lamellipodium
ERK activation plays an important role in cell migration (42)
and specifically keratinocyte migration (27)
. To determine whether ß2-AR activation alters the cellular localization of phospho-ERK in keratinocytes, we immunolocalized phospho-ERK in the presence and absence of ß-AR agonist.
In untreated keratinocytes, phospho-ERK is localized to the leading edge of the lamellipodia, a novel finding in keratinocytes (Fig. 3
A). We also observed robust nuclear and perinuclear phospho-ERK staining. ß2-AR activation prevents the localization of ERK to the perimeter of the cell and decreases the nuclear/perinuclear staining (Fig. 3B
).
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We have shown that the effects of ß2-AR activation on keratinocyte polarization, migration, and cytoskeletal organization are all PP2A dependent (Figs. 1
, 2)
; thus, we investigated whether the ß2-AR activation induced alteration in phospho-ERK localization has a similar PP2A dependence. Treatment of cells with OA has no effect on the level of ERK phosphorylation or its localization within keratinocytes (Fig. 3C
). However, OA pretreatment prevents both the ß2-AR-mediated loss of lamellipodial localization of phospho-ERK and its decrease in nuclear/perinuclear areas, confirming that the ß2-AR-mediated alteration in phospho-ERK localization is also PP2A dependent (Fig. 3D
).
Phospho-ERK is colocalized with vinculin in focal adhesions at the leading edge of the keratinocyte lamellipodia
ERK is translocated to focal contact sites at the lamellipodial edge during pancreatic carcinoma cell migration (43)
. We have demonstrated that phospho-ERK is localized to the leading edge of migrating keratinocytes (Fig. 3)
that contain multiple small linear FAs (Fig. 2)
. To determine whether the phospho-ERK observed at the edge of the lamellipodium is localized within FAs, we examined the lamellipodial edge of migrating keratinocytes that were fixed and immunostained with antibodies to both vinculin (Fig. 4
A) (as a focal adhesion marker; 38) and phospho-ERK (Fig. 4B
) using confocal microscopy. Indeed, phospho-ERK and vinculin colocalize within focal adhesion structures at the leading edge of the migrating keratinocyte lamellipodium (Fig. 4C
). No colocalization of phospho-ERK and vinculin is observed upon ß2-AR activation due to both the ß2-AR-mediated loss of polarized morphology (Fig. 2)
and absence of phosphorylated ERK at the keratinocyte perimeter (Fig. 3)
(results not shown).
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ß2-AR agonists reduce keratinocyte proliferation and OA reverses the agonist-mediated reduction in proliferative capacity
Since keratinocyte proliferation behind the migrating epithelial tongue is essential for effective re-epithelialization (11)
, it was important to determine the effect of ß2-AR activation on human keratinocyte proliferation. Therefore, human keratinocytes were grown in the presence or absence of ß-AR agonist. ß2-AR activation significantly decreases keratinocyte proliferation (Fig. 5
A). The ability of OA to restore normal migration in ß2-AR agonist-treated cells prompted us to investigate whether it could also prevent the ß2-AR-mediated decrease in keratinocyte proliferation. Indeed, OA alone has no effect on keratinocyte proliferation, but completely prevents the ß2-AR-mediated decrease in proliferation (Fig. 5B
). It therefore appears that the ß2-AR-mediated decrease in proliferation is also mediated by a PP2A-dependent mechanism in human keratinocytes.
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ß2-AR activation delays the re-epithelialization of human skin wounds
Since ß2-AR activation is both anti-motogenic and anti-mitogenic in human keratinocytes, we hypothesized that wound re-epithelialization, essential for wound healing (10)
, could be impaired by ß-AR agonists. Human skin was wounded and the wounds allowed to re-epithelialize in explant culture. Addition of ß-AR agonist to the healing wound significantly delays healing by 24 h. All control, untreated wounds are healed completely by day 4, whereas ß2-AR agonist-treated wounds heal by day 5 at the earliest (Fig. 6
A, *P<0.01). Hematoxylin and eosin-stained sections from control and ß2-AR agonist-treated wounds, days 1–5, are shown in Fig. 6B
. Due to variations in wound shape and the site within the wound from which sections were cut, leading to the variation in healing we observed on days 1–5, the percentage of re-epithelialization was calculated for each wound. ß-AR agonist treatment significantly decreases the wound re-epithelialization by 34% and 58% after 3 and 4 days in culture, respectively (Fig. 6C
, *P<0.05). These results provide confirmation that ß2-AR activation delays wound re-epithelialization in normal human skin.
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ß2-AR activation decreases the epidermal wound-induced phosphorylation of ERK
ERK activation is known to play a role in wound healing. Mechanical injury of confluent keratinocyte cultures activates ERK (26)
and, conversely, inhibition of ERK causes a delay in rabbit corneal epithelial wound healing (28)
. We therefore postulated that the mechanism for the ß2-AR-mediated delay in re-epithelialization could involve decreased ERK activation in the wounded epidermis. To study the activation state of ERK in wounded human skin, levels of phospho-ERK were assessed in periwound epidermis. Within 60 min of wounding, the phosphorlyation of ERK increases 2-fold in the periwound epidermis, while the total level of ERK remains unchanged (Fig. 7
A). ß2-AR activation decreases the wound-induced phosphorylation of ERK, so that at 30 min after ß-agonist addition levels of phospho-ERK are significantly lower than detected immediately after wounding untreated epidermis. ß-Agonist treatment decreases the wound-induced increase in phosphorylation of ERK by 80% 60 min postwounding (Fig. 7B
), providing convincing evidence that the ß2-AR activation-induced delay in human skin re-epithelialization is associated with a decrease in wound-induced epidermal ERK phosphorylation, necessary for efficient wound closure.
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ß2-AR activation delays the re-epithelialization of murine tail-clip wounds
We have demonstrated a ß2-AR-mediated delay in the re-epithelialization of human skin wounds in organ culture. To determine whether a similar delay would occur in vivo, we compared the healing rates after partial tail amputation in control and ß-AR agonist-treated mice (Fig. 8
). Control animals achieve complete re-epithelialization by day 7 postwounding, whereas ß-AR agonist-treated animals failed to re-epithelialize the wounds within the same period (Fig. 8)
, providing additional confirmation that ß2-AR activation delays wound re-epithelialization in both human and murine skin.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Here we demonstrate that these effects translate into a significantly diminished capacity for ß-AR agonist-treated human and murine skin to re-epithelialize a wound.
In vitro, ß2-AR activation prevents the polarization and initial migration of keratinocytes from a wound edge and decreases the wound-induced increase in ERK phosphorylation in keratinocytes adjacent to the denuded area. ß2-AR activation remodels the keratinocyte actin cytoskeleton from that of an actively migratory cell to that of a statically adherent one, with a dense network of cortical actin fibers just beneath the plasma membrane and abundant large vinculin-rich focal adhesions. It also prevents localization of phospho-ERK to focal adhesions at the leading edge of the keratinocyte lamellipodium and decreases keratinocyte proliferation. All the ß-AR agonist-induced alterations in morphology, intracellular signaling, migration, and proliferation are reversed when the cells are pretreated with OA at a concentration highly selective for PP2A (10 nM) (34)
, demonstrating that these events are all PP2A dependent.
Since keratinocyte polarization, migration, and proliferation are required for cutaneous wound repair; we reasoned that human and murine skin wounds treated with ß-AR agonists would heal poorly. Indeed, here we report a significant delay in the re-epithelialization of human skin wounds and murine tail-clip wounds treated with ß-AR agonists. Thus, this work documents specific ß2-AR-mediated changes in keratinocyte biology and the resultant impairment in the process of wound healing. We believe this is the first work to implicate the ß2-AR signaling pathway as a regulator of human cutaneous wound repair.
Efficient cell migration, required for wound repair, is dependent on temporally and spatially controlled reorganization of the actin cytoskeleton (37)
. Within hours of injury, skin wound keratinocytes undergo phenotypic alterations, including the formation of a fine and diffuse actin network at the advancing lammellipodium to allow for cell migration (44
, 45)
. Integrin receptors within focal adhesions stabilize the lamellipodia (46)
, allowing the migrating keratinocytes to interact with the variety of extracellular matrices (ECMs) found in the wound site, including fibronectin, vitronectin, stromal type I collagen, and fibrin (47)
. ß-AR agonist treatment markedly alters the cytoskeletal organization from that of actively migrating cells to a morphology characteristic of nonmotile cells (41)
. The migratory phenotype is restored by OA pretreatment, indicating that the ß2-AR-mediated alterations in the keratinocyte cytoskeletal organization are PP2A dependent. PP2A has many substrates in addition to ERK that either reside in FAs or play a role in migration including, but not limited to, FAK, paxillin (48)
, ß1 Integrin (49)
, Akt (50)
, and shc (51)
. It is highly likely that ß2-AR-mediated PP2A activation is altering numerous migration and adhesion pathways that we have only begun to explore.
Here we describe the novel finding of colocalization of phospho-ERK and vinculin at the leading edge of the lamellipodium in migrating keratinocytes. This colocalization is disrupted by ß2-AR activation and restored by OA, indicative of a PP2A-dependent mechanism. Although the exact function of phospho-ERK at the lamellipodial edge remains to be elucidated, direct interactions between ERK and ß integrins (52)
or paxillin (53)
suggest an important role for ERK in integrating cell adhesion and receptor-mediated signaling in the control of cell migration. Again, ß2-AR activation appears to disrupt an event that plays a part in normal cell migration.
Perhaps the strongest evidence for the role of ß2-AR in wound repair is the direct demonstration that activation of ß2-AR receptors in excised, wounded human skin (Fig. 6)
and in mice with amputated tail wounds (Fig. 8)
significantly delays skin re-epithelialization. Using tissue confers the advantages of a normal ECM and the 3-dimensional geometry of the healing wound not found in scratch assays or other assays using cultured cells. Additionally, we demonstrate that ß2-AR activation decreases ERK phosphorylation within the wounded epidermis. Since ERK is activated upon mechanical injury of confluent keratinocyte (26)
and MDCK cultures (54)
and inhibition of ERK delays rabbit corneal epithelial wound healing (28)
, it is likely that the ß2-AR-mediated decrease in ERK phosphorylation plays a role in the ß2-AR-mediated delay observed in re-epithelialization.
Although ß-AR agonists and antagonists are widely used drugs in treating asthma and cardiologic disease, respectively, there have been no specific observations regarding the ability of patients using these agents to heal wounds. However a number of reports, in addition to the work presented here, support the notion that both endogenous and exogenous ß-AR agonists and antagonists alter wound healing. Psychological stress, a condition that elevates systemic catecholamine levels (55)
, is associated with delayed skin wound healing (56)
. Denda et al. have demonstrated that emotional stress results in an impaired skin permeability barrier (57)
and, conversely, that topical application of ß-AR antagonists can accelerate skin barrier recovery after barrier disruption (16)
. ß-AR antagonists are widely used in the postburn wound recovery period, and a retrospective outcome analysis by Arbabi et al. has demonstrated a shorter time for burn wound healing in a cohort of patients that received ß-AR antagonists during their hospital stay (58)
. Thus, there are suggestions in the literature that both the endogenous ß2-AR signaling network and exogenously supplied ß-AR agonists or antagonists may modulate wound repair in the clinical setting. More systematic analysis of wound healing in cohorts of patients treated with ß-AR agonists and antagonists are warranted, and such a study is under way at our institution.
Impaired wound healing is a growing clinical problem, most evident in the remarkable numbers of chronic wounds in our aging population: 6.5 million have chronic skin ulcers caused by pressure, venous stasis or diabetes mellitus (59)
, costing the U.S. health care system a staggering $9 billion annually (60)
. Defining pathways that regulate the wound healing process provides the potential for developing new therapeutic approaches. The current finding, that ß2-AR activation significantly delays wound re-epithelialization and decreases the wound-induced increase in epidermal phospho-ERK now brings mechanistic support for the regulatory role of the ß2-adrenergic hormonal network in the wound repair process. Clearly, further investigation of this hormonal network in skin will improve our understanding of the wound healing process and hopefully lead to the development of therapies to enhance wound repair.
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ACKNOWLEDGMENTS |
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Received for publication June 6, 2005. Accepted for publication September 13, 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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 Y. Yamaguchi, T. Passeron, T. Hoashi, H. Watabe, F. Rouzaud, K.-i. Yasumoto, T. Hara, C. Tohyama, I. Katayama, T. Miki, et al. Dickkopf 1 (DKK1) regulates skin pigmentation and thickness by affecting Wnt/{beta}-catenin signaling in keratinocytes FASEB J, April 1, 2008; 22(4): 1009 - 1020. [Abstract] [Full Text] [PDF]
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S. Taboubi, J. Milanini, E. Delamarre, F. Parat, F. Garrouste, G. Pommier, J. Takasaki, J.-C. Hubaud, H. Kovacic, and M. Lehmann G{alpha}(q/11)-coupled P2Y2 nucleotide receptor inhibits human keratinocyte spreading and migration FASEB J, December 1, 2007; 21(14): 4047 - 4058. [Abstract] [Full Text] [PDF]
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), or pretreated with OA (10 nM) for 30 min before addition of OA alone or both OA and ß-AR agonist (B), (OA (X), OA/ß-AR agonist (
). Cells were harvested and counted on days 2, 4, 6, 8. The data are representative of 3 independent experiments with at least 3 different keratinocyte strains. Values plotted are means ±SE. *P < 0.01 between ß-AR agonist and other conditions.
