1 Department of Dermatology, University of Rochester Medical Center, Rochester, New York 14642; and 2 Drug Discovery Enterprises, Hughes Institute, St. Louis, Missouri 55113
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ABSTRACT |
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PGE2 levels are altered in human epidermis after in vivo wounding; however, mechanisms modulating PGE2 production in activated keratinocytes are unclear. In previous studies, we showed that PGE2 is a growth-promoting autacoid in human primary keratinocyte cultures, and its production is modulated by plating density, suggesting that regulated PGE2 synthesis is an important component of wound healing. Here, we examine the role of phospholipase A2 (PLA2) and cyclooxygenase (COX) enzymes in modulation of PGE2 production. We report that the increased PGE2 production that occurs in keratinocytes grown in nonconfluent conditions is also observed after in vitro wounding, indicating that similar mechanisms are involved. This increase was associated with coordinate upregulation of both COX-2 and secretory PLA2 (sPLA2) proteins. Increased sPLA2 activity was also observed. By RT-PCR, we identified the presence of type IIA and type V sPLA2, along with the M-type sPLA2 receptor. Thus the coordinate expression of sPLA2 and COX-2 may be responsible for the increased prostaglandin synthesis in activated keratinocytes during wound repair.
wound healing; primary human keratinocytes; arachidonic acid; prostaglandin E2; secretory phospholipase A2 receptor
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INTRODUCTION |
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AFTER MECHANICAL WOUNDING to the stratified epidermis occurs, both keratinocyte migration and cellular proliferation, followed by differentiation, are necessary to repopulate the wound bed and restore epidermal barrier function (43). For these events to occur, keratinocytes must be activated, that is, undergo a transition from a relatively sedentary and metabolically inactive cell type to a migratory and mitotically active phenotype (23). Keratinocyte activation is complex and involves the spatiotemporal modulation of a vast array of structural and regulatory proteins and signaling molecules (see review, Ref. 13). Previously, it has been reported that changes in fatty acid metabolism and prostaglandin production can modulate keratinocyte proliferation (37, 38, 58). Thus regulation of prostaglandin synthesis may be an important factor in the wound healing response (39, 58). The object of this study is to examine prostaglandin biosynthesis and mechanism(s) involved in the regulation of prostaglandin production in keratinocytes in an in vitro wounding model. This model is intended as a tool to dissect the importance of prostaglandin production in the regulated epithelial wound healing response.
Prostaglandins are produced by the sequential release of arachidonic acid (AA) from membrane phospholipids by cellular phospholipases, followed by the enzymatic peroxidation and cyclooxygenation by PGH synthases, also known as cyclooxygenases (COXs). In human epidermis, AA release can occur via action of phospholipase A2s (PLA2) (2, 22) or indirectly through the action of phospholipase C or D by the sequential hydrolysis of diacylglycerol (DAG) by DAG lipase and monoacylglyceride lipase (7). Mammalian tissues and cells generally contain more than one PLA2 enzyme and a growing superfamily has now been identified and categorized (see review, Ref. 16). Several types of PLA2s have been associated with fatty acid release, including an 85-kDa Ca2+-dependent cytosolic PLA2 (cPLA2) and several subtypes of secretory PLA2s (sPLA2). cPLA2, also called type IV PLA2, is considered to be ubiquitously expressed and has distinct substrate preference for sn2-AA-containing phosphatidylcholine, indicating its critical role in further eicosanoid synthesis (see review, Ref. 33). Modulation of cPLA2 activity is tightly regulated by postreceptor signal-transduction pathways linking its activity to rapid, yet transient, fatty acid release. Overexpression of human cPLA2 protein in HEL-30 cells, a murine keratinocyte cell line, was shown to enhance AA release, PGE2 production, and proliferation capacity (37).
sPLA2s are low-molecular-weight (~14 kDa) proteins that are found both extracellularly and on cellular membranes. They require millimolar Ca2+ levels for catalytic activity and have a broad range of substrate specificity with respect to head group or sn2 fatty acid when assayed in vitro (67). sPLA2s identified to date are distinguished mostly by their molecular sequence, disulfide bonds, and substrate preferences. Type I sPLA2 is abundantly expressed in pancreas, where it functions as a digestive enzyme (48, 53). It is also present, although in small amounts, in several nondigestive tissues (48, 53). Type IIA sPLA2 has been detected in spleen, liver, thymus, and intestine as well as several inflammatory cells, including macrophages and mast cells (see review, Ref. 48). It is also prominent in sites of active inflammation such as the synovial fluid from rheumatoid arthritis patients (65). Therefore, type IIA sPLA2 is thought to play a significant role in inflammatory responses, host defense, and proliferation. Thus it is interesting that transgenic mice expressing human type IIA sPLA2 exhibited chronic epidermal hyperplasia (21).
A recently identified subtype, type V sPLA2, is highly expressed in heart, placenta, and, to a lesser extent, in lung and liver (4, 12). Type V sPLA2 has also been shown to be involved in AA mobilization in mouse immune cells (4, 62), a role previously assigned solely to type IIA. Recent studies have demonstrated that a functional redundancy between type IIA and type V sPLA2 exists with respect to their fatty acid-releasing activity (50, 62). Type X, another recently identified member of the sPLA2 subfamily, has been identified in human immune tissues (14). In addition to their catalytic functions, several subtypes of sPLA2 have been shown to modulate cellular events, including eicosanoid production, cell proliferation, and cell motility via binding to a 180-kDa M-type sPLA2 receptor (48, 53). With this PLA2 superfamily rapidly growing and sPLA2 receptor-mediated pathways being further elucidated, the question of how multiple PLA2 subtypes function and interact within a cell or tissue needs to be addressed.
PLA2 activity has been described in human epidermis and is associated with multiple normal skin functions, including cell growth and differentiation, formation of the permeability barrier, wound repair, and bacterial host defense (8, 19, 22, 41, 44, 72). A variety of agents that cause skin damage, including ultraviolet (UV) irradiation and chemical irritants, induce PLA2-mediated fatty acid release in vitro (22, 28, 29, 36, 37, 42). Also, differences in PLA2 activities have been reported among epidermal layers (8) and between the cytosolic and membrane fractions of mouse keratinocytes (36), suggesting that regulated expression of multiple forms of PLA2s in keratinocytes may influence differentiation and barrier formation.
Keratinocytes are known to express both COX isoenzymes (COX-1 and COX-2) (64). These isoenzymes differ primarily in that COX-1 is generally considered to be constitutively expressed, whereas COX-2 expression is rapidly induced upon stimulation. Mitogens and proinflammatory stimuli have been shown to modulate gene expression, posttranscriptional regulation, and protein synthesis of COX-2 in keratinocytes (38, 46, 56, 64). Functional coupling between PLA2 and COX enzymes is thought to mediate agonist-induced, temporal production of prostaglandins. Several studies reported that coupled activation of cPLA2 and COX-1 are responsible for immediate prostaglandin synthesis, whereas coordinate induction of sPLA2 and COX-2 protein expression is involved in cytokine-induced delayed PGE2 production (3, 5, 9, 49, 51, 66). Other studies suggest that a functional coupling between sPLA2 and COX-1 is responsible for early prostaglandin synthesis (60, 61); this can occur transcellularly (61) and involve the sPLA2 receptor and activation of cPLA2 (24). It is notable that the nature of these cross talk pathways is cell-type dependent and requires more investigation in human keratinocytes.
Previous data from our laboratory have shown an increase in PGE2 synthesis in human primary keratinocytes grown under nonconfluent conditions, which gradually decreased as the density of the cultures increased (55, 58). Endogenous PGE2 is necessary for the high mitotic activity observed during nonconfluent keratinocyte growth (58) and this effect is mediated through activation of PGE receptor subtypes EP2 and/or EP4, coupled to adenylate cyclase activity (32). Persistent high levels of exogenous PGE2 are growth-inhibitory, possibly due to downregulation of EP2 or EP4 receptors. These findings indicate that modulation of PGE2 production in the activated keratinocyte is essential to the growth and migration of keratinocytes during wound healing.
In this study, we examined PGE2 production in proliferating keratinocytes in more detail, evaluating the roles PLA2 and COX enzymes play in the modulation of PGE2 synthesis. Because little information exists about sPLA2 subtypes and sPLA2 receptor in human epidermis, we examined whether both type IIA and type V sPLA2, along with the M-type sPLA2 receptor, were present in human keratinocytes. We found that similarities exist between primary human nonconfluent cultures and keratinocytes wounded in vitro. We report that a coordinate increase of both COX-2 and sPLA2 protein expression occurs in nonconfluent keratinocyte cultures, indicating the importance of both enzymes in modulation of PGE2 production in human keratinocytes.
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METHODS |
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Reagents. DMEM with high glucose, L-glutamine, and without sodium pyruvate, Hanks' buffered salt solution (HBSS), PBS, and fetal bovine serum (FBS) were obtained from BioWhittaker (Walkersvillle, MD). Sterile PBS, Taq DNA polymerase, TRIzol reagent, RNase H, penicillin, and streptomycin were purchased from GIBCO BRL (Gaithersburg, MD). Collagen (Vitrogen 100) from Collagen (Palo Alto, CA) was used at 1:6 dilution with 1× PBS to coat tissue culture plates (Falcon). Fatty acid-free BSA was obtained from Calbiochem (San Diego, CA). Hexane and glycerol were obtained from EM Science (Gibbstown, NJ). AA was purchased from Nu-Chek Prep (Elysian, MN). Random hexanucleotides mixture, AMV reverse transcriptase, protease inhibitors, Pefabloc SC, and leupeptin were obtained from Boehringer Mannheim (Indianapolis, IN). Reagents for protein assays and SDS-PAGE were purchased from Bio-Rad (Hercules, CA). PGE2 ELISA reagents and deuterated fatty acids were from Cayman Chemicals (Ann Arbor, MI). Pentafluorobenzyl bromide was obtained from Pierce (Rockford, IL). Gas chromatography-HPLC grade methanol and methylene chloride were purchased from Mallinckrodt Chemical (Paris, KY). Tetramethylammonium hydroxide, N,N-dimethyl-acetamide (DMA), aprotinin, pepstatin A, soybean trypsin inhibitor, and most other chemicals were obtained from Sigma (St. Louis, MO) and were of highest commercial grade available. The human embryonic kidney (HEK-293) cell lines overexpressing type IIA (RAS2FB) and type V (H101D) sPLA2s were kind gifts from Jay A. Tischfield and Michelle V. Winstead (Indiana University).
Isolation of primary human keratinocyte and cell cultures. Primary adult human keratinocyte cultures were prepared from human epidermis removed during reductive mammoplasties and panniculectomies, as previously described (58). Cells were plated onto collagen-coated tissue culture dishes at a density of 7.5 × 105 cells/cm2 (confluent) and 1.25 × 105 cell/cm2 (nonconfluent) and maintained in DMEM containing 5% FBS, 1.4 mM calcium chloride, penicillin (100 IU/ml), streptomycin (100 mg/ml), and 25 mM HEPES buffer, pH 7.4 (growth medium) in 95% air-5% CO2 at 37°C for 6 days. Nonpassaged cells were used in experiments when confluent cultures were 2 days postconfluence and nonconfluent cultures were at 15-30% cell density by visual inspection. HaCaTs, a spontaneously immortalized human keratinocyte cell line (10), was grown under the same conditions, but on uncoated plastic. RAS2FB and H101D cells were plated on uncoated dishes in growth medium containing 10% FBS and 200 µg/ml hygromycin B (GIBCO BRL) and maintained under the same conditions as keratinocytes.
Wounding of primary keratinocyte cultures. Confluent cultures grown on six-well plates were thoroughly washed and then scraped with the tip of a sterile glass rod; four wounds (2 × 30 mm) were made over a 96-mm2 surface area. The cells were washed twice with medium to remove debris, and supernatants from wounded and nonwounded cultures were taken at indicated times for PGE2 analysis.
PGE2 measurements and determination of COX activity.
Confluent and nonconfluent cultures were thoroughly washed with
prewarmed medium before the incubation period. At indicated times,
cultures were put on ice to stop reaction and supernatants were
removed, centrifuged at 1,000 g at 4°C to pellet cellular debris, and stored at 20°C until PGE2
determinations were performed. On thawing, the levels of
PGE2 in supernatants were determined by ELISA using
specific anti-PGE2 monoclonal antibody; the limit of
detection was 30 pg/ml. COX activity was determined by incubating cultures at 37°C for 15 min with exogenous 30 µM AA, prepared in
prewarmed, serum-free DMEM, before removal of supernatants for
PGE2 determination (22).
Basal fatty acid release: derivatization and measurement of fatty acids by gas chromatography-mass spectrometry. Confluent and nonconfluent keratinocytes grown in six-well plates were washed three times with HBSS, pH 7.4, containing penicillin, streptomycin, and HEPES in the same concentrations as used in growth medium, together with 0.01% fatty acid-free BSA and incubated in 1 ml HBSS solution for 10 min at 37°C to remove endogenous free fatty acids. Cultures were then incubated with fresh HBSS for 30-60 min. Aliquots of supernatants were taken from triplicate cultures for each condition and pooled. Deuterated D8 AA and D4 linoleic acid (LA) were added to each pooled sample as internal standards. Cell cultures were washed thoroughly with ice-cold PBS and scraped into Eppendorf tubes for protein analysis. Pooled supernatants were acidified to ~pH 3.0 with 1 N HCl and applied to conditioned Baker Bond Spec octadecyl C18 columns (J. T. Baker). The columns were then washed with H2O, followed by a 10% methanol wash. Samples were eluted in 100% methanol, evaporated to dryness, then derivatized using pentafluorobenzyl esterification. This procedure is a modification of one used previously (57). Briefly, the samples were treated with 50 µl tetramethylammonium hydroxide in DMA/methanol solution and 100 µl of a 35% pentafluorobenzyl bromide in DMA for 15 min at room temperature. The reactions were evaporated to dryness and washed twice with H2O/methylene chloride (1:4) and resuspended in hexane. The samples were then evaluated by gas chromatography/mass spectrometry using an HP-1 (cross-linked methyl silicone) column. Monitoring was in the negative ion mode for the fatty acid of interest. Mass spectrometric measurements of free fatty acid were not subjected to the problems of distribution or metabolism of label that occur with prelabeling of cellular lipids, and therefore reflect total released product. These products were normalized to the estimated protein amount present in each culture condition through the use of the Bio-Rad Dc Protein Assay method using known BSA standards.
Western blot analysis of cPLA2, sPLA2,
COX-1, and COX-2.
Confluent and nonconfluent cultures of human primary keratinocytes
grown in 100-mm petri dishes were cooled at 4°C for 10 min and
rinsed twice with ice-cold PBS containing the following protease and
phosphatase inhibitors: 5 µg/ml leupeptin, 1 µg/ml pepstatin A, 50 µg/ml soybean trypsin inhibitor, 1 mM Pefabloc SC, and 50 µM each
of sodium orthovanadate, sodium fluoride, and sodium phosphate. Gel
loading buffer (1 × GLB) containing 50 mM Tris · HCl, pH 6.8, 2% SDS, 10% glycerol, and the
above-mentioned inhibitors was then added to each culture dish and
incubated on ice for 20 min with rocking. Cells were scraped in
Eppendorf tubes, sonicated, and centrifuged at 10,000 g for 10 min at 4°C to remove particulates. The soluble portion (total cell
lysate) was isolated and stored at 20°C until further
assessment. Supernatants from confluent and nonconfluent cultures were
collected after 24 h of incubation in fresh medium and centrifuged at
4°C for 5 min at 1,000 g to remove cellular debris.
Supernatants were then concentrated by ultrafiltration using Centriprep
concentrators (Amicon) and diluted with 3× GLB containing
protease and phosphatase inhibitors and stored at
20°C.
Protein amounts were estimated as previously mentioned. Equivalent
protein amounts of cell lysates and supernatants were electrophoresed
on 8% SDS-PAGE gels (15% for sPLA2 detection) and
transferred to nitrocellulose membranes (Amersham). The membranes were
then blocked overnight at 4°C with 5% nonfat dry milk in Tris-buffered saline (TBS). For Western blotting, recommended manufacturer's procedures for using human anti-cPLA2
antibodies (Santa Cruz) were followed with these alterations. For
cPLA2 detection, membranes were incubated with human
anti-cPLA2 antibody (1:75) followed by an incubation with
rabbit anti-mouse IgG:horseradish peroxidase (Dako) at 1:1,000
dilution. To detect sPLA2, membranes were incubated with
human anti-sPLA2 (1:1,000, Cayman) and then treated with
goat anti-rabbit IgG:horseradish peroxidase (1:1,000). To detect COX-1
and COX-2, membranes were incubated with human anti-COX-1 (1:100;
Oxford) or anti-COX-2 (1:1,000, Oxford); both blots were then treated
with protein A-horseradish peroxidase (1:2,000; Amersham).
Visualization of proteins was done by enhanced chemiluminescence (Amersham).
Isolation of RNA and detection of type IIA sPLA2, type
V sPLA2, and M-type sPLA2 receptor by RT-PCR.
Total RNA from RAS2FB, H101D, HaCaT cell cultures, and primary
keratinocytes at both nonconfluent and confluent growth densities was
isolated using TRIzol reagent (GIBCO BRL) following the manufacturer's protocols with two additional phenol:chloroform (1:1) extractions added
before isopropanol precipitation. First-strand RT was performed using
~1 µg of RNA, random hexanucleotide mixture, and AMV reverse transcriptase (Boehringer Mannheim) according to the instructions of
the supplier. After the RT reactions, the samples were RNase-treated using 1 unit RNase H (GIBCO BRL) per reaction at 37°C for 20 min; cDNA products were stored at 20°C until used in PCR
reactions. For identification of type IIA and type V sPLA2,
two rounds of PCR amplification were performed for 30 and 25 cycles,
respectively, in a 50-µl reaction containing 1.5 mM
MgCl2, 200 µM dNTPs, 20 mM Tris · HCl
(pH 8.4), 50 mM KCl, 2.5 units Taq polymerase, and 4 µl of
cDNA product. First round PCR products were diluted 1:1,000 before
second round amplification reactions. For type IIA sPLA2, the first round forward and reverse primers were 5'
GGCCGGGGCAGAAGTTGAGAC 3' and 5' AGGGTAGGGAGGGAGGGTATGAGAG
3'. The conditions used for this PCR reaction were as follows:
94°C for 1 min, 56°C for 1 min, and 72°C for 1 min with an
additional 5 min at 72°C after final cycle. Type IIA
sPLA2 nested, second round forward and reverse primers were
5' CTTACCATGAAGACCCTCCTACTGTTGGCA 3' and
5'GAGGGGACTCAGCAACGAGGGGTGCT3' and PCR reaction conditions
were 94°C for 1 min, 56°C for 20 s, and 72°C for 30 s with
an additional 5 min at 72°C after final cycle. For type V
sPLA2, the first round forward and reverse primers were
5'CCAGAGATGAAAGGCCTCCTCCCACTGGGCTTG3' and
5'GAGGCCTAGGAGCAGAGGATGTTGGGAA3', and the reaction
conditions used were identical to the first round type IIA PCR
reactions. Type V sPLA2 nested, second round forward and
reverse primers were 5'GTTCCTGGCTTGTAGTGTGC3' and
5'GGATGTTGGGA- AAGTATTGG3'. The conditions were the same
as those used for nested type IIA sPLA2 primers except that
the annealing temperature was lowered to 47°C. To identify the
M-type sPLA2 receptor in human keratinocytes, primers
identical to those used to identify the sPLA2 receptor in
human gestational tissues were used (45), with some modifications to
the published PCR reaction conditions. Briefly, PCR amplifications in
the same reaction mixture as mentioned above were done using the
following conditions: 95°C for 1 min, 65°C for 1 min, and
72°C for 1 min with an additional 5 min at 72°C after final
cycle. PCR products were resolved by 1.5% agarose gel electrophoresis
and visualized with Gel Star (FMC BioProducts, Rockland, ME). PCR
products were then subjected to restriction analysis to partially
confirm their identity. Major and minor PCR product bands were excised
from gels and purified using a Qiaex II agarose gel extraction kit
(Qiagen) before sequencing, which was performed by the Nucleic Acid
Core Facility at the University of Rochester. Sequences of PCR products
from keratinocyte and overexpressing cell lines were compared with
published sequences for human type IIA SPLA2 (65), type V
sPLA2 (12), or M-type sPLA2 receptor (1).
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RESULTS |
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Transient increase in PGE2 levels in wound closure in
vitro.
As previously reported, PGE2 production in human primary
keratinocytes is modulated by their density in cell culture (58). Cells
grown at low densities (15-30% confluence) produced 27-fold more
PGE2 than in 2-day postconfluent cultures (4.46 ± 1.64 vs. 0.164 ± 0.108 ng · ml1 · 24 h
1; mean ± SD; n = 3). Elevated
PGE2 production was also observed in in vitro wound closure
experiments (Fig. 1). After
wounding confluent monolayers, dramatic changes in PGE2
levels were observed in wounded cultures compared with unwounded
confluent cultures. The rise in PGE2 levels was rapid yet
transient. A significant increase in PGE2 accumulation
within the 1st h after wounding was observed. PGE2 levels
were highest at 10 h after wounding and then gradually decreased as the
wound edge closed. No keratinocyte growth into the wounded area was
visually observed until 2-3 h after wounding. Although actual
PGE2 levels and degree of wound closure at 48 h varied
between individual cell preparations (n = 3), the pattern and
time course of PGE2 production after wounding was almost
identical in each experiment.
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Morphology of human primary keratinocytes grown at low densities is
similar to that of activated keratinocyte on wounding edge.
In morphological studies of full-thickness injury to the skin, it was
observed that keratinocytes at the wound edge undergo phenotypic
changes during the initial phase of wound healing (13, 52). Migrating
keratinocytes, particularly those originating from the
early-differentiating compartment of wounded epidermis, are reported to
adopt a polarized, elongated appearance, with some cells having ruffled
edges and cytoplasmic processes. We observed similar morphological
changes in keratinocytes on the wound edge in wounding experiments in
vitro (data not shown). Primary human keratinocyte cultures grown at
confluent and nonconfluent densities exhibit morphological differences
similar to unaffected and activated keratinocytes in a wound,
respectively. Keratinocytes at 2 days postconfluence are polygonal in
shape, with cytoplasms appearing full of dense, filamentous material
(Fig. 2A). In contrast, nonconfluent cultures (15-30% confluent; Fig. 2B) grow
clonally with small central areas of stratified cells and outer edges
of flattened, elongated cells. These outer cells have ruffled edges and
the occasional pseudopodia indicative of motility, which was confirmed
by time-lapse videomicroscopy. Long processes similar in appearance to
dendrites are a distinct feature of keratinocytes grown in nonconfluent
cultures. We have found that the presence of these processes and the
flattened, elongated morphology of keratinocytes appears concurrently
with increased PGE2 production in nonconfluent cultures.
Rhodamine phalloidin immunohistochemical staining of nonconfluent cells
showed dense staining for actin within these processes (data not
shown). Because other epidermal cell types that form dendritic-like
processes (melanocytes and Langerhans cells) are present in small
amounts in primary keratinocyte cultures, we costained with
melanocyte and Langerhans cell markers (Mel-5 and MHC class II antigen,
respectively). We found no correlation between these markers and the
processes of interest (data not shown), thus we concluded that these
processes are keratinocyte-derived.
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Increase in COX activity in nonconfluent human primary keratinocyte
cultures.
First, we examined whether COX activity was altered in nonconfluent
cultures. In Fig. 3, it is shown that
nonconfluent keratinocyte cultures synthesized ~25-fold greater basal
concentrations of PGE2 than confluent cultures (4.28 ± 0.45 vs. 0.171 ± 0.040 ng · ml1 · 15 min
1; mean ± SD). However, this 25-fold
difference in basal PGE2 synthesis between confluent and
nonconfluent keratinocytes could reflect limiting substrate due to
differences in phospholipase and/or COX activity. To determine whether
the increased production of PGE2 was dependent on increased
COX activity, saturating levels of exogenous AA (30 µM) were added at
the beginning of the incubation. It was found that nonconfluent
keratinocytes still produced ~12-fold greater PGE2 than
confluent keratinocytes (Fig. 3; 18.43 ± .77 vs. 1.59 ng · ml
1 · 15 min
1; mean ± SD). These results demonstrated that
nonconfluent keratinocytes exhibit markedly increased COX activity when
compared with confluent cultures.
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Increased expression of COX-2 protein levels in nonconfluent human
primary keratinocyte cultures.
We then examined whether the increased COX activity was due to
increased expression of COX-2 and/or COX-1 isoforms. In Western blotting of total cellular lysates prepared from paired nonconfluent and confluent primary keratinocyte cultures, COX-1 protein levels were
detected in both confluent and nonconfluent culture conditions. It is
interesting that COX-1 protein levels varied between individual skin
preparations, with some having undetectable levels (Fig. 4A) and others having substantially
higher levels. In the latter case, we observed a modest increase in
COX-1 levels in nonconfluent conditions (Fig. 4B). In all
individual skin samples examined, a substantial amount of COX-2 protein
was detected only in nonconfluent conditions (Fig. 4). No COX-2 protein
was detected in any confluent cultures. These results indicate a
distinct induction of COX-2 protein expression in nonconfluent culture
conditions, with some variability in COX-1 protein levels between
individuals.
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Increase in fatty acid release in nonconfluent human primary
keratinocyte cultures.
The availability of substrate is another rate-limiting step in
eicosanoid biosynthesis. Because both AA and LA are substrates in this
pathway, we measured the basal levels of these fatty acids released
from nonconfluent and confluent cultures over a 30-min incubation
period by gas chromatography-mass spectrometry (Table 1). Accumulation of free fatty acids in
cell supernatants was maximal at 30 min, after which it was at
equilibrium with fatty acid reuptake (data not shown). There was
tremendous inter-individual variability in absolute concentrations of
free fatty acids monitored in each experiment. However, the ratio of
nonconfluent-to-confluent (NC/C) fatty acid release clearly showed that
basal AA and LA levels were higher in nonconfluent cultures compared
with confluent cultures with mean values of 9.7 ± 7.6 and
6.8 ± 3.6 for AA and LA, respectively. Most experiments showed that
AA was the prevalent fatty acid released; although in one case
(expt 3), the levels of LA were higher in both growth
conditions. Thus these data suggest that a non-substrate-specific
phospholipase activity commonly associated with sPLA2 is
likely to be involved in the increased fatty acid release in
nonconfluent cultures.
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Increased expression of sPLA2 but not cPLA2
protein levels in nonconfluent human primary keratinocyte cultures.
Because AA is the predominant fatty acid released, we examined whether
increased cPLA2 protein expression contributed to increased fatty acid release in nonconfluent cultures. We performed Western blot
analysis on total cellular homogenates of both confluent and
nonconfluent keratinocytes to examine cPLA2 expression
(Fig. 5). The levels of cPLA2
in both conditions were very low but clearly detectable. Nonconfluent
keratinocytes showed no increase in total cellular cPLA2
protein compared with confluent keratinocytes, suggesting that the
increase in free AA release was not due to upregulation of
cPLA2 expression. In contrast, there was a dramatic increase in sPLA2 expression in total cellular homogenates
from nonconfluent cultures compared with confluent cultures. Identical results were seen in supernatants, although the levels of
sPLA2 protein detected in supernatants were much lower. It
should be noted that the sPLA2 antibodies used in these
studies can detect both type IIA sPLA2 and type V
sPLA2; therefore, we cannot conclude which
sPLA2 subtypes were upregulated from this experiment.
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Identification of type IIA and type V sPLA2 subtypes in
human primary keratinocyte cultures by RT-PCR.
With recent reevaluation of the role of sPLA2 subtypes in
AA metabolism, we found it important to assess which of these
sPLA2s were present in human keratinocytes. We used RT-PCR
to detect the presence of both type IIA and V sPLA2
transcripts in nonconfluent and confluent human primary keratinocyte
cultures (Fig. 6). RNA from two HEK-293
cell lines, RAS2FB and H101D, which overexpress type IIA
sPLA2 and type V sPLA2, respectively, were
included as positive controls. Low abundance of message necessitated
two rounds of PCR with nested primers to detect products in
keratinocytes. Major bands corresponding to the predicted sizes (448 bp
for type IIA sPLA2 and 380 bp for type V sPLA2)
were detected in nonconfluent and confluent conditions, as well as in
the appropriate overexpressing cell line (Fig. 6, A and
B). Another band, designated the minor product band (~280
bp), was also observed in type V sPLA2 RT-PCR analysis.
This additional band appeared in both nonconfluent and confluent
keratinocyte conditions but not in overexpressing cell line. Sequencing
analysis for type IIA sPLA2 PCR product showed that the
product obtained from both nonconfluent and confluent conditions are
identical to the published type IIA sPLA2 cDNA sequence
(65). Sequencing analysis of the type V sPLA2 PCR products showed that the major PCR product (380 bp) in nonconfluent and confluent conditions are identical to the published type V
sPLA2 cDNA sequence (12). The minor PCR product (~280 bp)
present in nonconfluent and confluent conditions also shared 98 and
99% homology with the published sequence and with the 380-bp product, except that it was missing bases 316-422, which encompass the 5' end of exon III as reported by Chen et al. (12).
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Identification of M-type sPLA2 receptor in human primary
keratinocyte cultures by RT-PCR.
Because the M-type sPLA2 receptor also may play a role in
mediating AA metabolism and other physiological responses such as proliferation and migration, we sought to establish whether this receptor type was present in human epidermis. Using primers that identified the presence of sPLA2 receptor in human
gestational tissues (45), we performed RT-PCR on RNA obtained from
nonconfluent and confluent keratinocytes, as described in
METHODS, through the use of the appropriate negative
controls. A band corresponding to the predicted size of 399 bp was
detected in both nonconfluent and confluent conditions (Fig.
7). The PCR product from confluent keratinocyte conditions was sequenced and found to share 99% homology with the reported human M-type SPLA2 receptor sequence (1). In both HEK-overexpressing cell lines, no PCR product was detected (data not shown). However, a PCR product of the same size was observed
in RT-PCR reactions using RNA from HaCaT cultures (data not shown),
suggesting that the sPLA2 receptor is
keratinocyte-associated.
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DISCUSSION |
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Physical separation of keratinocytes from normal skin and transfer to tissue culture constitutes an activation, or wound signal for keratinocytes (23). In subconfluent cultures, keratinocytes tend to grow in colonies and require cell migration to sustain clonal growth (6). On reaching confluence, the activated phenotype is lost and the cells begin to stratify and express various markers of keratinocyte differentiation (59). In previous studies, we have shown an inverse relationship between the level of basal PGE2 production and state of confluence in human primary keratinocyte cultures (55, 58). In this study, we observed a similar pattern of PGE2 synthesis in an in vitro wound model (Fig. 1). After the wounding of confluent cultures, PGE2 levels increased rapidly but transiently; as the wound closed and confluence was restored, the levels of PGE2 declined. Although there was some sample variability in the actual levels of PGE2 measured and in extent of wound closure by 48 h, the time course of change in PGE2 levels remained consistent (n = 3). This result was indicative of distinct modulation of the enzymes involved in PGE2 synthesis after in vitro wounding, and validated the use of the nonconfluent culture system as an injury model.
Keratinocytes grown at nonconfluent densities (15-30% confluent) adopt a distinct morphology (Fig. 2). The phenotypes of keratinocytes grown at low densities and in wounds in vitro and in vivo (52) are all strikingly similar. Moreover, these morphological similarities concur with increased PGE2 synthesis observed in nonconfluent cultures and in in vitro wounding as reported in this study and in in vivo wounds (39). Thus we consider the small clonal island of keratinocytes found in nonconfluent cultures to be akin to a 360° wound with outer cells analogous to the wound edge. We postulated that nonconfluent cultures are a good model in which to examine the modulation of enzymes involved in increased PGE2 biosynthesis in activated keratinocytes.
PGE2 biosynthesis in intact cells is modulated by the level of COX proteins present, the amount of substrate available, and the level of phospholipases present. In this study, we found that in nonconfluent keratinocyte cultures, COX-2 protein expression together with nonspecific fatty acid release and sPLA2 protein expression was increased and is most likely responsible for the increase in PGE2 production in this culture condition. First, we established that nonconfluent cultures exhibit increased COX activity compared with confluent cultures (Fig. 3). The addition of saturating levels of exogenous AA (30 µM) is a direct way of assessing COX activity in intact cells. Here, we observed that COX activity in nonconfluent cultures was still considerably higher, suggesting that COX protein levels are elevated in nonconfluent cultures. It was shown by Western blot analysis that COX-2 protein expression was induced in only nonconfluent cultures, whereas there was often little to no change in COX-1 expression between the two culture conditions. Similar results were observed in mouse epidermis after in vivo mechanical injury (64), in which the induction of COX-2 expression was time dependent. Also, regulation of COX-2 gene expression, at the transcriptional (71) and posttranscriptional levels (20, 63), can influence the COX-2 protein expression; however, these regulatory pathways have not been well examined in human keratinocytes.
The fact that the level of COX-1 expression varied between individuals was of interest because it may be related to the level of COX-2 induction. This observation was consistent with a recent report examining the compensatory PGE2 biosynthesis in lung fibroblast cells from COX-1- or COX-2-deficient mice in which the expression of remaining functional COX gene was upregulated to compensate for the lack of the other isoform (30). Therefore, additional studies of the difference in the ratio of COX-1 to inducible COX-2 between individuals are required to assess its role in individual variability in wound healing.
Second, we found that basal AA release was elevated in nonconfluent cultures compared with confluent conditions (Table 1). Thus there is more substrate available for COX enzyme metabolism in nonconfluent conditions. The finding that basal LA levels are also higher in nonconfluent cultures indicated that nonsubstrate-specific PLA2 activity is elevated in nonconfluent keratinocyte cultures. Although increased LA release has little influence on PGE2 biosynthesis in nonconfluent cultures, LA plays a direct role for establishment and maintenance of the epidermal permeability barrier (17). In addition, a 15-lipoxygenase product of LA, 13-hydroxyoctadecadienoic acid, has a potentiating effect on epidermal growth factor-dependent mitogenesis in rodent fibroblasts (18). Also, 12-hydroxyeicosatetraenoic acid, a 12-lipoxygenase product of AA, has stimulatory effects including activation of protein kinase C and cell motility in tumor cells (26, 40). Together, these reports suggest that linoleic and arachidonic lipoxygenase metabolites may also be important in the migration and/or proliferation of keratinocytes during wound healing.
In an examination of cPLA2 and sPLA2 protein expression in confluent and nonconfluent keratinocyte cultures, we found that cPLA2 levels, although low, were unchanged. In contrast, there was an increase in sPLA2 (type IIA and/or type V) protein levels in nonconfluent conditions. We observed that most of the sPLA2 protein was cell-associated rather than secreted, although a small amount was detected in supernatants from nonconfluent conditions. This is consistent with other reports in which membrane-associated sPLA2 was the predominant form in other cell types (49, 66). Therefore, upregulation of sPLA2 protein expression in coordination with induction of COX-2 expression appears to be the dominant pathway by which keratinocytes increase PGE2 production in nonconfluent cultures.
Despite the clear coordinate induction of sPLA2 and COX-2, increased activation of cPLA2 may also contribute to elevated AA release. Studies in human astrocytes (24) and rat mesangial cells (27) show that type IIA-sPLA2 caused posttranslational modification of cPLA2, probably through either a sPLA2 receptor-mediated process (24) or production of lipid metabolites (27). Finally, indirect production of AA by phospholipase C and/or D pathways may also be involved because both phospholipases are present in human keratinocytes and are activated by UV damage (11). However, AA release studies using bovine endothelial cells found no changes in phospholipase C activity from the progression of cells from the nonconfluent to confluent state (70).
Andersen et al. (2) reported that a nonpancreatic type II sPLA2 mRNA transcript and protein is present in human epidermis, with low amounts found in normal epidermis and increased levels in involved skin from patients with psoriasis. Recently, Li-Stiles et al. (36) identified a type IIA sPLA2 in mouse keratinocytes by RT-PCR, but the presence of type V sPLA2 was not evaluated. We found that both type IIA and type V sPLA2 messages were expressed in human keratinocytes. Expression of both sPLA2 transcripts appeared to be very low. Given the low abundance of messages, a more quantitative measure of differences in mRNA expression levels by Northern blot analysis was not attempted. Reports indicated that it is type V sPLA2, not type IIA sPLA2, that is involved in the AA mobilization and PGE2 production triggered by inflammatory stimuli (4, 62). To determine whether one of these sPLA2s is selectively responsible for increased fatty acid release observed in nonconfluent culture conditions is currently being investigated. Recently, Murakami et al. (50) demonstrated that these sPLA2s are functionally redundant in terms of AA release and membrane binding using overexpressing cell lines. Therefore, specific preference for different fatty acid pools, the localization of action, and modes of regulation involved may be the important determinants for distinguishing the function of various sPLA2s expressed in a cell or tissue. This needs to be further elucidated by using in vivo models.
We also identified that message for an M-type sPLA2 receptor was expressed in both nonconfluent and confluent primary keratinocyte cultures. The presence of the sPLA2 receptor may be of particular importance to the mechanism of keratinocyte activation because the receptor has been shown to mediate cell proliferation and chemotactic cell migration, as well as endotoxic shock and eicosanoid biosynthesis in other cell types (24, 48, 53, 66). The 180-kDa M-type sPLA2 receptor, originally identified in rabbit skeletal muscle (35), has been cloned and characterized from many sources (1, 25). Previously, human sPLA2 receptor has been found to be expressed in kidney, placenta, pancreas, lung, and skeletal muscle. However, a high-affinity ligand for the human receptor has not yet been identified; binding studies using recombinantly expressed human type IB and type IIA sPLA2 have shown only low-affinity binding for a cloned human sPLA2 receptor (15). Binding properties for type V and type X sPLA2s have yet to be reported. Studies showing rapid internalization of sPLA2 on binding to its receptor suggest a possible regulatory role of the sPLA2 receptor in the clearance of sPLA2 from the extracellular environment (73). In addition, sPLA2 receptor may mediate cross talk between different PLA2s. For example, several studies found that exogenous addition of a type I sPLA2 induced production and secretion of type IIA sPLA2 on receptor binding and, in turn, increased PGE2 production in rat mesangial cells (31, 47). Another study showed activation of a mitogen-activated protein kinase cascade and cPLA2 on addition of type IIA sPLA2 (24). The role of the sPLA2 receptor in keratinocytes remains to be elucidated.
Several groups have reported that a functional coupling between sPLA2 and COX-2 was responsible for the delayed, sustained prostaglandin synthesis on agonist stimulation in several cell types (5, 9, 34, 49, 51, 66). The time course of PGE2 production shown in our in vitro wounding experiments was strikingly similar to the time courses shown in the above-mentioned studies, indicating that functional coupling between COXs and PLA2 may also occur during in vitro wounding. In human keratinocytes grown at nonconfluent cell densities, our results suggested that sPLA2 and COX-2 are functionally coupled because both sPLA2 and COX-2 protein expression were coinduced under these conditions. It is not clear what mediates the coordinated induction of sPLA2 and COX-2 protein expression and what continually maintains their expression in nonconfluent cultures. Keratinocytes in nonconfluent conditions may be responding to the lack of neighboring cells and/or the mechanical stress induced by motility (54). Mechanical stress has been shown to cause the upregulation of genes, including COX-2 in human vascular endothelial cells (69).
In conclusion, nonconfluent primary keratinocyte cultures are a good model for studying keratinocyte activation because they appear to mimic functional and morphological characteristics observed during wound healing. Increased production of fatty acids and prostaglandins in nonconfluent keratinocytes appears to involve the coordinate induction of sPLA2s and COX-2 expression. With our identification of type V sPLA2 and an M-type sPLA2 receptor, another level of complexity must be considered. In doing so, we hope to further understand the mechanisms that regulate fatty acid metabolism during keratinocyte activation and in turn, affect the proliferation and migration of keratinocytes necessary for wound healing.
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ACKNOWLEDGEMENTS |
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We thank Jay A. Tischfield and Michelle V. Winstead for their kind gift of sPLA2 overexpressing cell lines and primer recommendations.
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FOOTNOTES |
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This work was supported by National Institutes of Health (NIH) Grant RO1AR-40574. K. E. Rys-Sikora is supported by NIH Individual National Research Service Award Grant ARO85020. R. L. Konger is a Wilmont Cancer Research Fellow.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. P. Pentland, Dept. of Dermatology, Univ. of Rochester Medical Center, Box 697, 601 Elmwood Ave., Rochester, NY 14642 (E-mail: Alice_Pentland{at}urmc.rochester.edu).
Received 15 July 1999; accepted in final form 1 November 1999.
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