• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Prostaglandins Leukot Essent Fatty Acids. Author manuscript; available in PMC Apr 2, 2007.
Published in final edited form as:
PMCID: PMC1847382
NIHMSID: NIHMS19239

Sequential induction of pro- and anti-inflammatory prostaglandins and peroxisome proliferators activated receptor-gamma during normal wound healing: A time course study.

Summary

Lipid mediators generated from metabolism of arachidonic acid play a crucial role in the initiating and resolution of acute inflammation by shifting from pro-inflammatory prostaglandin (PG) E2 to anti-inflammatory PGD2 and its metabolites. The changes in PG levels over time during the normal wound repair process have not, however, been reported. We determined the temporal expression of PG and their biosynthetic enzymes using the full thickness incisional model of normal wound healing in mice. We demonstrate that during normal wound repair, there is a shift in the metabolism of arachidonate from PGE2 during the acute inflammatory phase to PGD2 during the repair phase. This shift is mediated by temporal changes in the expression of cyclooxygenases (COX) and microsomal PGES (mPGES)-1. Inducible COX (COX-2) expression is sustained throughout the initiation and repair process, but mPGES-1 is increased only during the acute inflammatory phase and its disappearance coincides with increased PGD2. PGD2 and its degradation products are known to mediate their anti-inflammatory effects by binding to peroxisome proliferators activated receptor gamma (PPARγ). In this study, we show that PPARγ is upregulated during the resolution phase of wound repair concomitant with the shift to PGD2, and may be responsible for initiating endogenous mechanism resulting in healing/resolution.

Introduction

Normal wound repair is a multifactorial pathophysiological process involving complex interactions of soluble mediators, growth factors and cytokines which help repair the injured tissue [1]. Extensive work has been carried out in order to elucidate the underlying mechanisms which initiate the acute inflammatory response in a wound repair process, but little attention has been focused towards the endogenous mediators which may help resolve the inflammatory episode during the time course of a normal wound healing process. Recent studies in other models of acute inflammation have indicated that prostaglandins (PGs) generated from the metabolism of arachidonic acid (AA) may play a crucial role in not only initiating the inflammation but also in the resolution of inflammation. The PG profile in these models shifts from the predominantly pro-inflammatory prostaglandin (PG)E2 to the anti-inflammatory PGD2 during the time course of acute inflammation [24]. This temporal pattern of AA metabolism during the time course of normal wound repair process has not previously been reported.

During tissue injury, PGs are formed by metabolism of AA by cyclooxygenases (COX) to generate an intermediate substrate PGH2 which is further metabolized by terminal synthases to generate specific PGs [46]. These terminal synthases include PGE synthases (PGES) for PGE2, PGDS for PGD2, PGFS for PGF and PGIS for PGI2 (prostacyclin) respectively [7]. COX exists in two isoforms, COX-1 and COX-2. A splice variant of COX-1 has also been reported in some tissues but its biological relevance is unclear [8, 9],[10]. During wound repair, COX-1 is constitutively expressed while elevated levels of COX-2 are observed during the early inflammatory phase of wound repair [11]. However, recent studies in animal models of acute inflammation suggest that COX-2 and COX-2 derived PGs may have anti-inflammatory properties [2, 3]. The majority of the wound healing studies that have focused on PG metabolism have explored only the pro-inflammatory role of COX-2. No report to date has evaluated the potential role of COX-2 and its derived PGs towards resolution of inflammation in a normal wound repair process.

Among terminal PG synthases, PGES specifically catalyzes the conversion of PGH2 to PGE2 [1113]. At least three forms of PGES have been cloned and characterized, and are termed as cytosolic PGES (cPGES), microsomal PGES (mPGES)-1, and mPGES-2 [1214]. mPGES-1 is a glutathione-dependent enzyme that shows coordinated induction with COX-2 by inflammatory stimuli in various cells and tissues [15, 16]. Since it is an inducible enzyme, it has been of great interest to investigate the role of mPGES-1 in inflammatory processes. cPGES is constitutively expressed in the cytosol and is functionally coupled with COX-1 under basal conditions [13]. mPGES-2 is also constitutively expressed in various cells and tissues [17]. No report has elucidated the contribution of mPGES-1, cPGES and mPGES-2 towards the process of normal wound healing.

PGDS is the key terminal synthase involved in the conversion of PGH2 to PGD2. Two district forms of PGDS, lipocalin PGDS (L-PGDS) and hematopoietic PGDS (H-PGDS) have been cloned and characterized [18, 19]. L-PGDS is mainly involved in central nervous system including sleep induction, body temperature, and analgesia [18]. H-PGDS is widely distributed in peripheral tissues [19] and is a key enzyme for the readily undergoes dehydration in vitro and in vivo to yield production of PGD2. PGD2 biologically active PGs of the J2 series [20, 21]. J2 series PGs are characterized by the presence of a reactive, β-unsaturated ketone in the cyclopentenone ring and therefore referred as cyclopentenone PGs [22]. Cyclopentenone PGs are actively incorporated into cells and accumulate in the nucleus and form adducts with various proteins and transcription factors [23]. PGD2 and its J-series degradation product, 15-deoxy-Δ12, 14-PJG2 (15dPGJ2) have been shown to exert anti-inflammatory effects on peripheral inflammation and promote resolution via inhibition of various pro-inflammatory signaling molecules including cytokines, inducible nitric oxide synthase (iNOS) and nuclear factor kappa B (NFκB) [2, 2430]. The majority of the anti-inflammatory mechanisms mediated by PGD2 and its dehydrated product 15dPGJ2 are now thought to be mediated by peroxisome proliferator activated receptor gamma (PPARγ) [31]. This is based on the fact that 15dPGJ2 is an endogenous ligand for PPARγ and its activation results in upregulation of mechanisms involved in the resolution of inflammation [27, 28, 31, 32]. In vitro and in vivo studies using endogenous and synthetic ligands of PPARγ have been shown to inhibit broad range of pro-inflammatory immune mediators including interleukin (IL)-1β, tumour necrosis factor (TNF)-α and NFκB [33, 34]. Animal studies using rosiglitazone (a potent synthetic ligand of PPARγ) also suggest that rosiglitazone has the ability to limit inflammation through a PPARγ dependent pathway [3537]. No report has demonstrated the role of H-PGDS, PGD2 and PPARγ in the resolution of a normal wound healing process.

We hypothesized that analogous to other inflammatory processes, the initiation and resolution phases of normal wound healing would be associated with characteristic patterns of biosynthetic enzymes and product expression. Since it is likely that anti-inflammatory mechanisms result from the engagement of PGs such as PGD2 with PPARγ and its downstream targets, we studied PPARγ in parallel. To elucidate the role of these endogenous mediators during the process of normal wound repair, it was vital to determine their temporal profiles during the complete time course of a normal wound repair. The aims of this study were to determine the temporal expression profiles of COX-1, COX-2, mPGES-1, mPGES-2, cPGES, hPGDS, PGIS; temporal production profiles of major AA metabolites including PGE2, PGD2, 6-keto-PGF; and the temporal expression profile of PPARγ during the inflammatory and resolution phases of normal wound healing process. To address these questions we used a well established full thickness incisional model of normal wound healing in mice.

Materials and Methods

Materials

Female DBA/1 Lac J mice were purchased from Jackson laboratories (Bar Harbor, Maine), Rabbit anti-human mPGES-1 antiserum was gifted from Dr. Per-Johan Jakobsson (Karolinska Institute, Stockholm). Rabbit anti-mouse COX-2 polyclonal antibody, rabbit anti-human mPGES-2 polyclonal antibody, rabbit anti-human cPGES polyclonal antibody, rabbit anti-mouse PGI synthase (PGIS) polyclonal antibody, rabbit anti-mouse hematopoietic PGD synthase (H-PGDS) polyclonal antibody, rabbit anti-mouse COX-1 polyclonal antibody, enzyme-linked immunosorbent assay (ELISA) kits for PGE2, 6-keto-PGF and PGD2 were all purchased from Cayman Chemical Co. (Ann Arbor, MI). Mouse monoclonal PPARγ primary antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-human beta-actin monoclonal antibody was obtained from Sigma Aldrich (St. Louis, MO). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG were obtained from Jackson ImmunoResearch (West Grove, PA). TRIpure was purchased from Roche Diagnostics (Indianapolis, IN). The polyvinylidene difluoride (PVDF) membrane and enhanced chemiluminescence (ECL) reagent were purchased from Amersham Pharmacia Biotech (Buckinghamshire, UK).

Animals

Female DBA/1 Lac J mice (25–30g) were housed at 25°C and communally kept on a 14 hour light/10h dark cycle. Standard mouse diet and water were available ad libitum. All mice were allowed to acclimatize for at least three days before shaving/wounding. Following surgery, animals were housed individually at 25°C and kept on a 14 hour light/10h dark cycle.

Wound model

Twenty four hours prior to wounding, animals were anaesthetized with ketamine 80–100 mg/kg and xylazine 8–10 mg/kg (mixed) injected IP using ½ inch 26 gauge needles, the backs were shaved and incision points marked on the dorsum. Twenty four hours after shaving the animals were anaesthetized again and 1cm full dermal thickness incisional wounds were made 3 and 8 cm (using a sterile surgical scalpel blade) from the base of the neck and 2 cm from the spine to give a total of 4 wounds per animal. Wounds were left uncovered; the animals were allowed to recover from anesthesia and then housed individually.

Tissue collection

Immediately after euthanasia, the tissue samples were harvested from the wounded regions of the mice on days 0, 1, 3, 7, 14 and 21, and were subsequently used to perform immunohistochemistry, western blotting, reverse transcription-polymerase chain reaction (RT-PCR) and enzyme immunoassays (EIA).

Reverse transcription-polymerase chain reaction

RNA from the isolated wound tissues was extracted with TRIpure reagent according to the manufacturer’s instructions. Reverse transcription was performed according to the manufacturer’s instructions using a SuperScript preamplification system (Invitrogen, Carlsbad, CA) with 1 μg of total RNA from the tissues as a template. Subsequent amplifications of the cDNA fragments by PCR with HotStarTaq polymerase (Qiagen, Valencia, CA) were performed using 0.5 μl of the reverse-transcribed mixture as a template with specific oligonucleotide primers and cycle number as follows: mouse mPGES-1 (28 cycles), sense 5′-CAC ACT GCT GGT CAT CAA GA-3′ and antisense 5′-ACA CCA AGT CCG CAA GTT C-3′; mouse COX-2 (25 cycles), sense 5′-GGG CCC TTC CTC CAG TAG CAG A-3′ and antisense 5′-CAT CAG ACC AGG CAC CAG ACC AA-3′; mouse mPGES-2 (25 cycles), sense 5′-GGT GGC CCA GGA AGG AGA CAG C-3′ and antisense 5′-GCA GCC GCG CCC ACA TAC TTG-3′; mouse cPGES (30 cycles), sense 5′-CCC GCC CAC CCG TTT GTC-3′ and antisense 5′-TCT GGC ATC TTT TCA TCA TCA CTG-3′; mouse PGIS (30 cycles), sense 5′-TTC TGG CTC CTT CTT TTC CTC CTC-3′ and antisense 5′-CTT CAG CCG TTT CCC ATC TTT GTA -3′; mouse H-PGDS (31 cycles), sense 5′-ATG CCT AAC TAC AAA CTG CTT-3′ and antisense 5′-CTA GAG TTT TGT CTG TGG CCT-3′; mouse COX-1 (30 cycles), sense 5′-CCC CAG CCC TCC GAC CTA CAA-3′ and antisense 5′-CCC CGG AAG CAA CCC AAA CAC-3′; mouse PPARγ ( 31 cycles), sense 5′-CCT CTC CGT GAT GGA AGA CC-3′ and antisense 5′-GCA TTG TGA GAC ATC CCC AC-3′; mouse GAPDH (20 cycles), sense 5′-GGG GTG AGG CCG GTG CTG AGT AT-3′ and antisense 5′-CAT TGG GGG TAG GAA CAC GGA AGG-3′. After initial denaturation at 95°C for 15 min, PCR involved amplification cycles of 30 sec at 95°C, 30 sec at 56°C and 45 sec at 72°C, followed by elongation for 5 min at 72°C. The amplified cDNA fragments were resolved by electrophoresis on 2% (w/v) agarose gel and were visualized under UV light using a BioRad Chemidoc Apparatus (Hercules, CA) after staining of the gel with ethidium bromide.

Western blotting

Wound tissues were homogenized in 50 mM Tris-buffered saline (TBS) containing 0.1% sodium dodecyl sulfate (SDS) and the protein content was determined using bicinchoninic acid (BCA) protein assay reagent (Pierce, Rockford, IL) with bovine serum albumin as the standard. Wound homogenates were adjusted to equal equivalents of protein and then were applied to SDS-polyacrylamide gels (10–20%) for electrophoresis. Next, the proteins were electroblotted onto polyvinylidene difluoride membranes. After the membranes were blocked in 10 mM TBS containing 0.1% Tween-20 (TBS-T) and 5% skim milk, the membranes were probed for 1.5 hrs with the respective antibodies (1:1000 for mPGES-1, COX-2, mPGES-2, cPGES, PGIS, H-PGDS, COX-1, PPARγ and beta-actin) in TBS-T for 1.5 hrs. After washing the membranes with TBS-T, the membranes were incubated with HRP-conjugated anti-rabbit (for mPGES-1, COX-2, mPGES-2, cPGES, PGIS, H-PGDS and COX-1) or HRP-conjugated anti-mouse (for PPARγ and beta-actin) IgG (1:10,000 dilution in TBS-T containing 5% skim milk) for overnight at 4°C. After further washing with TBS-T, protein bands were visualized with an ECL Western blot analysis system using a BioRad Chemidoc Apparatus (BioRad, Hercules, CA).

Enzyme Immunoassay

The concentrations of PGE2, 6-keto-PGF (a stable metabolite of PGI2) and PGD2 in the wounded skin tissue were measured by ELISA (Cayman Chemicals, Ann Arbor MI). Assays were performed according to the manufacturer’s recommendation.

Immunohistochemistry

Frozen wound tissues were embedded in optimal cutting temperature (OCT) compound and 0.5 μm sections were cut using a cryostat (Leica, Bannockburn, IL) and collected on poly-l-lysine-coated slides. Immunolabeling of COX-2, mPGES-1 and PPARγ were performed using the DakoCytomation LSAB+ System-HRP kit. Immunohistochemical procedures were performed according to the manufacturer’s recommendations. Briefly, endogenous peroxidase was blocked using 0.5% H2O2 in methanol for 5 minutes. Non-specific IgG binding was blocked by incubating sections with bovine serum albumin (0.1%) in PBS for 1 hour and then incubated with primary antibodies for COX-2, mPGES-1 and PPARγ (1:100) in a humidified chamber and left overnight at 4°C. Next, sections were incubated with biotynylated link for 30 minutes followed by incubation with streptavidin for 30 minutes. The chromogen diaminobenzidine tetrahydrochloride (DAB), was then added till sufficient color development and sections counterstained with Harris’s hematoxylin.

Statistical analysis

The data are expressed as mean ± SEM. Statistical analysis was performed using Student’s t-test. P<0.05 was considered statistically significant.

Results

Differential induction pattern of COX-2 and mPGES-1 during inflammatory phase (days 1 and 3) and resolution phase (days 7, 14 and 21) of normal wound healing

Protein and mRNA expression profiles of COX-2 and mPGES-1 were determined by western blotting and RT-PCR, respectively. Our results show that, COX-2 and mPGES-1 were co-induced during early inflammatory phase (days 1 and 3 postwounding) of wound healing process. Peak protein (Fig. 1) and mRNA (Fig. 2) expression levels for both COX-2 and mPGES-1 were detected on day 3 postwounding (P<0.05). However, during the resolution phase of normal wound repair process, a differential expression profile for COX-2 and mPGES-1 was detected. Following peak on day 3 postwounding, mPGES-1 protein and mRNA expression levels rapidly depleted and were undetectable on day 21 postwounding. For COX-2, the expression levels (mRNA and protein) on day 7 reduced to a lesser extent compared to day 3 postwounding and sustained expression levels for COX-2 were maintained till day 21 postwounding (P<0.05 for days 7, 14 and 21). Thus, COX-2 in comparison to mPGES-1 was upregulated not only during inflammatory phase but also during the resolution phase of wound healing process.

Figure 1
Temporal protein expression profile of COX-2 and mPGES-1 during the time course of normal wound healing
Figure 2
Temporal mRNA expression profile of COX-2 and mPGES-1 during the time course of normal wound healing

Localization of COX-2 and mPGES-1 during normal wound repair process

COX-2 and mPGES-1 localization in the wound tissue was detected by immunohistochemistry. Results show that peak expression levels of COX-2 (Fig. 3a) and mPGES-1 (Fig. 3b) were observed on day 3 postwounding in concordance with the protein and mRNA expression profiles. Immunohistochemical staining of wound sections showed that both COX-2 and mPGES-1 were expressed in the epidermal and dermal regions of the wounded skin. However, COX-2 expression levels were most intense in the dermis area at day 3 postwounding, whereas lower levels of COX-2 were observed in the epidermal region of the wounded skin. Contrastingly, high levels of mPGES-1 were detected in the epidermis of day 3 wound sections whereas low levels were detected in the dermis region of the wounded skin. We also determined the localization of H-PGDS in days 3 and 7 wounds. However, we were unable to localize H-PGDS in the epidermal and dermal regions of the wounded skin (data not shown). This may be due to very low levels of H-PGDS expressed during wound repair response.

Figure 3
Localization of COX-2 and mPGES-1 in the day 3 wounds

Constitutive expression of COX-1, mPGES-2, cPGES, H-PGDS and PGIS during the inflammatory and resolution phases of normal wound healing process

Our results show that COX-1, mPGES-2, cPGES, H-PGDS and PGIS protein (Fig. 4a) and mRNA (Fig. 4b) expressions were detectable on day 0 (unwounded skin), suggesting the presence of these enzymes in the skin under unwounded state. However, no change in the expression levels of COX-1, mPGES-2, cPGES, H-PGDS and PGIS were detected during early inflammatory as well as the resolution phases of normal wound healing process. Thus, these enzymes were constitutively expressed throughout the time course of during normal wound repair process.

Figure 4Figure 4
Temporal protein (a) and mRNA (b) expression profiles of COX-1, mPGES-2, cPGES, H-PGDS and PGIS during the time course of normal wound healing

Differential production pattern of PGE2 and PGD2 during inflammatory and resolution phases of wound healing process

Levels of PGE2 and PGD2 (Fig 5) in the wound tissues were determined by EIAs. Low levels of PGE2 were detected in the day 0 unwounded control skin. A significant increase in the levels of PGE2 were observed on day 1 (P<0.05) and 3 (P<0.05) postwounding, peaking on day 3 and subsequently levels returned to normal basal levels by days 7, 14 and 21 postwounding.

Figure 5
Temporal production profile of PGE2 and PGD2 during the time course of normal wound healing

Low levels of PGD2 were observed on day 0 and day 1 postwounding. However, a significant increase in the levels of PGD2 were observed on 7 (P<0.05), 14 (P<0.05) and 21 (P<0.05) post wounding, peaking on day 7. We also determined the temporal production levels of 6-keto-PGF during the time course of wound repair. Very low basal levels of 6-keto-PGF were observed before wounding and during the complete time course of normal wound repair process (data not shown). Thus, PGE2 and PGD2 production profiles showed a differential production pattern with peak levels of PGE2 observed during early inflammatory phase of normal wound repair process. However during the resolution phase of normal wound repair, there was a switch in the metabolism towards PGD2 which was the predominant PG product.

Increased expression of PPARγ during the resolution phase of normal wound repair process

PPARγ expression (protein and mRNA) were detectable in day 0 unwounded control skin (Fig 6a and 6b). Low levels of PPARγ (protein and mRNA) were observed during early inflammatory phase of wound repair process. However, a significant increase in the expression levels of PPARγ both at protein and mRNA levels were observed during the resolution phase (day 7, 14 and 21 postwounding), peaking at day 14 (P<0.05) for protein expression and day 21 (P<0.05) for mRNA levels. Immunohistochemistry results on day 14 wound sections show that PPARγ was predominantly expressed in the epidermal region of the wounded tissue whereas low levels of PPARγ were detected in the dermis (Fig 6c).

Figure 6Figure 6Figure 6
Temporal expression of PPARγ during the time course of normal wound healing

Discussion

This study for the first time reports a distinct temporal pattern of AA-derived PGs during inflammatory and resolution phases of normal wound repair. Using a well established full thickness incisional model of normal wound healing in mice we show that during inflammatory phase of wound repair, PGE2 is the predominant AA-derived metabolite. However, as the inflammation subsides and wound repair process approaches resolution there is a switch in the AA metabolism from pro-inflammatory PGE2 towards anti-inflammatory PGD2. This temporal switch may play a key role in initiating early inflammation and subsequent transition towards resolution of inflammation during a normal wound repair process.

Vast literature suggests that PGE2 is a major pro-inflammatory PG product produced during inflammation though some anti-inflammatory and immunosuppressive effects of PGE2 have also been reported [8, 38]. We and others have previously shown that COX-2 and mPGES-1 are key enzymes involved in the production of PGE2 during inflammation [11, 39, 40]. In the present study we demonstrate that mPGES-1 along with COX-2 are upregulated during early inflammatory phase of wound repair process with no change in the levels of other metabolic enzymes including COX-1, mPGES-2, cPGES, H-PGDS and PGIS. The expression of mPGES-1 drops rapidly to basal levels during the resolution phase; however, expression levels of COX-2 remain elevated till day 21 postwounding. Interestingly, levels of PGE2 mirrored the temporal expression profile of mPGES-1 exhibiting elevation in the levels upon wounding with a peak on day 3 and thereafter returning to baseline during the beginning of resolution phase of wound repair. This confirms previous observations that production of PGE2 during inflammation requires induction of COX-2 and mPGES-1 [11, 39, 40]. Recently, using embryonic fibroblasts isolated from mice deficient with mPGES-1, we reported that mPGES-1 is essential for induced PGE2 production [40]. Results presented in this study also suggest that increased mPGES-1 expression along with induction of COX-2 coincide with the high levels of PGE2 produced during wound repair. However, there remains the possibility of involvement of COX-1 for the generation of PGE2 during wound repair. The fact that COX-2 is highly expressed in the dermis while mPGES-1 is highly expressed in the epidermis suggests that COX-1 could be a source for PGH2 used by mPGES-1 to generate PGE2 during wound repair. A previous study showed a tight coupling of COX-1 expression with PGE2 and PGD2 biosynthesis in the region of wounds instead of COX-2 during wound repair [41]. However, this study used a full thickness excisional wound healing model in BALB/C and C57BLKS mice in comparison to full thickness incisional wound healing model used in the present study using DBA/1 Lac J mice.

In this study we also observed that when PGE2 levels were elevated during early inflammatory phase of wound repair, PGD2 levels were low. Simultaneously, as the PGE2 levels dropped, there was a significant elevation in the levels of PGD2 which remained elevated during the resolution phase of normal wound healing process. This transition in the PG production pattern during the resolution phase of wound repair may in part be due to the ability of COX enzymes to convert AA to PGH2 and subsequently PGD2 in the absence of mPGES-1. It also vital to note that no change in the levels of H-PGDS were observed during inflammatory and resolution phases of wound healing process but constitutive H-PGDS levels may in fact be sufficient to convert PGH2 to PGD2 in the absence of mPGES-1.

Upregulation of PGD2 during the resolution phase of wound healing process observed in this study provides further support for recently reported anti-inflammatory/resolution mechanisms mediated by PGD2 [2, 8, 24, 25, 30, 42]. PGD2 and its degradation product, 15dPGJ2 exhibit anti-inflammatory effects and induce crucial events in the resolution of inflammation, including inhibition of cytokines such as TNF-α, IL-1β and IL-6 and inhibition of NF-κB [5,1925,34]. More recently, PGD2 and its degradation product 15dPGJ2 were shown to initiate resolution of acute inflammation in rat pleurisy by causing neutrophil and macrophage apoptosis [43]. On the contrary, studies have also reported pro-inflammatory effects associated with PGD2. PGD2 has been shown to play a key pro-inflammatory role in allergic diseases such as allergic rhinitis and asthma [44]. Antagonists of the PGD2 receptors have also shown promise for the treatment of asthma and rhinitis [45].

Recent studies suggest that the mechanisms by which PGD2 and its metabolites mediate their anti-inflammatory effects are due in part to their action as PPARγ ligands. We therefore determined the temporal profile of PPARγ during the complete time course of normal wound healing. Our results show that during the resolution phase of wound repair a significant increase in the expression of PPARγ mirrored the temporal profile of PGD2. Furthermore, using immunohistochemical technique we show that PPARγ expression was predominantly localized in the epidermal region of the wounded skin. This is the first report demonstrating that PPARγ is upregulated during the resolution phase of wound repair. Based on the fact that PPARγ expression mirrored the profile of PGD2 during wound repair process, and that PGD2-derived metabolites are important PPARγ ligands, we hypothesize that PPARγ and PGD2 may cooperate to achieve resolution of inflammation during wound repair.

In summary, we have shown that during the time course of wound repair, there is a gradual shift in the metabolism of AA from pro-inflammatory PGE2 (during inflammatory phase) to anti-inflammatory PGD2 (during the resolution phase). This shift in the PG production profile may in part be regulated by temporal changes in the expression levels of mPGES-1 enzyme and COX-2 during early inflammatory and resolution phases of wound repair. This study further shows the contribution of COX-2 may not be limited to the early inflammation associated with wounding and may well contribute to the resolution of inflammation by generating PGD2 during later stages of repair. In addition, we show that PPARγ is upregulated during the resolution phase of wound repair process concomitant with PGD2 and may in part be responsible for initiating endogenous mechanism resulting in healing/resolution.

Footnotes

Sources of Support: Arthritis Foundation Biomedical Grant and NIH/NIAMS AR49010

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

1. Kapoor M, Kojima F, Appleton I, Kawai S, Crofford LJ. Major enzymatic pathways in dermal wound healing: current understanding and future therapeutic targets. Curr Opin Investig Drugs. 2006;7:418–422. [PubMed]
2. Gilroy DW, Colville-Nash PR, Willis D, Chivers J, Paul-Clark MJ, Willoughby DA. Inducible cyclooxygenase may have anti-inflammatory properties. Nat Med. 1999:698–701. [PubMed]
3. Ianaro A, Ialenti A, Maffia P, Pisano B, Di Rosa M. Role of cyclopentenone prostaglandins in rat carrageenin pleurisy. FEBS Lett. 2001;508:61–66. [PubMed]
4. Kapoor M, Clarkson AN, Sutherland BA, Appleton I. The role of antioxidants in models of inflammation: emphasis on L-arginine and arachidonic acid metabolism. Inflammopharmacology. 2005;12:505–519. [PubMed]
5. Crofford LJ. Prostaglandin biology. Gastroenterol Clin North Am. 2001;30:863–876. [PubMed]
6. Srinivasan DM, Kapoor M, Kojima F, Crofford LJ. Growth factor receptors: implications in tumor biology. Curr Opin Investig Drugs. 2005;6:1246–1249. [PubMed]
7. Kojima F, Kato S, Kawai S. Prostaglandin E synthase in the pathophysiology of arthritis. Fundam Clin Pharmacol. 2005;19:255–261. [PubMed]
8. Kapoor M, Shaw O, Appleton I. Possible anti-inflammatory role of COX-2-derived prostaglandins: implications for inflammation research. Curr Opin Investig Drugs. 2005;6:461–466. [PubMed]
9. Chandrasekharan NV, Dai H, Roos KL, Evanson NK, Tomsik J, Elton TS, Simmons DL. COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression. Proc Natl Acad Sci U S A. 2002;99:13926–13931. [PMC free article] [PubMed]
10. Dinchuk JE, Liu RQ, Trzaskos JM. COX-3: in the wrong frame in mind. Immunol Lett. 2003;86:121. [PubMed]
11. Kapoor M, Howard R, Hall I, Appleton I. Effects of epicatechin gallate on wound healing and scar formation in a full thickness incisional wound healing model in rats. Am J Pathol. 2004;165:299–307. [PMC free article] [PubMed]
12. Jakobsson PJ, Thoren S, Morgenstern R, Samuelsson B. Identification of human prostaglandin E synthase: a microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target. Proc Natl Acad Sci U S A. 1999;96:7220–7225. [PMC free article] [PubMed]
13. Tanioka T, Nakatani Y, Semmyo N, Murakami M, Kudo I. Molecular identification of cytosolic prostaglandin E2 synthase that is functionally coupled with cyclooxygenase-1 in immediate prostaglandin E2 biosynthesis. J Biol Chem. 2000;275:32775–32782. [PubMed]
14. Tanikawa N, Ohmiya Y, Ohkubo H, et al. Identification and characterization of a novel type of membrane-associated prostaglandin E synthase. Biochem Biophys Res Commun. 2002;291:884–889. [PubMed]
15. Murakami M, Naraba H, Tanioka T, et al. Regulation of prostaglandin E2 biosynthesis by inducible membrane-associated prostaglandin E2 synthase that acts in concert with cyclooxygenase-2. J Biol Chem. 2000;275:32783–32792. [PubMed]
16. Kojima F, Naraba H, Sasaki Y, Okamoto R, Koshino T, Kawai S. Coexpression of microsomal prostaglandin E synthase with cyclooxygenase-2 in human rheumatoid synovial cells. J Rheumatol. 2002;29:1836–1842. [PubMed]
17. Murakami M, Nakashima K, Kamei D, et al. Cellular prostaglandin E2 production by membrane-bound prostaglandin E synthase-2 via both cyclooxygenases-1 and -2. J Biol Chem. 2003;278:37937–37947. [PubMed]
18. Urade Y, Fujimoto N, Hayaishi O. Purification and characterization of rat brain prostaglandin D synthetase. J BiolChem. 1985;260:12410–12415. [PubMed]
19. Kanaoka Y, Ago H, Inagaki E, et al. Cloning and crystal structure of hematopoietic prostaglandin D synthase. Cell. 1997;90:1085–1095. [PubMed]
20. Kikawa Y, Narumiya S, Fukushima M, Wakatsuka H, Hayaishi O. 9-Deoxy-delta 9, delta 12–13,14-dihydroprostaglandin D2, a metabolite of prostaglandin D2 formed in human plasma. Proc Natl Acad Sci U S A. 1984;81:1317–1321. [PMC free article] [PubMed]
21. Hirata Y, Hayashi H, Ito S, et al. Occurrence of 9-deoxy-delta 9,delta 12–13,14-dihydroprostaglandin D2 in human urine. J Biol Chem. 1988;263:16619–16625. [PubMed]
22. Fukushima M. Biological activities and mechanisms of action of PGJ2 and related compounds: an update. Prostaglandins Leukot Essent Fatty Acids. 1992;47:1–12. [PubMed]
23. Narumiya S, Fukushima M. Site and mechanism of growth inhibition by prostaglandins. I. Active transport and intracellular accumulation of cyclopentenone prostaglandins, a reaction leading to growth inhibition. J Pharmacol Exp Ther. 1986;239:500–505. [PubMed]
24. Ando M, Murakami Y, Kojima F, et al. Retrovirally introduced prostaglandin D2 synthase suppresses lung injury induced by bleomycin. Am J Respir Cell Mol Biol. 2003;28:582–591. [PubMed]
25. Murakami Y, Akahoshi T, Hayashi I, et al. Inhibition of monosodium urate monohydrate crystal-induced acute inflammation by retrovirally transfected prostaglandin D synthase. Arthritis Rheum. 2003;48:2931–2941. [PubMed]
26. Ji JD, Cheon H, Jun JB, et al. Effects of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) on the expression of inflammatory cytokines and apoptosis induction in rheumatoid synovial fibroblasts and monocytes. J Autoimmun. 2001;17:215–221. [PubMed]
27. Farrajota K, Cheng S, Martel-Pelletier J, et al. Inhibition of interleukin-1beta-induced cyclooxygenase 2 expression in human synovial fibroblasts by 15-deoxy-Delta12,14-prostaglandin J2 through a histone deacetylase-independent mechanism. Arthritis Rheum. 2005;52:94–104. [PubMed]
28. Kawahito Y, Kondo M, Tsubouchi Y, et al. 15-deoxy-delta(12,14)-PGJ(2) induces synoviocyte apoptosis and suppresses adjuvant-induced arthritis in rats. J Clin Invest. 2000;106:189–197. [PMC free article] [PubMed]
29. Cuzzocrea S, Wayman NS, Mazzon E, et al. The cyclopentenone prostaglandin 15-deoxy-Delta(12,14)-prostaglandin J(2) attenuates the development of acute and chronic inflammation. Mol Pharmacol. 2002;61:997–1007. [PubMed]
30. Willoughby DA, Moore AR, Colville-Nash PR, Gilroy D. Resolution of inflammation. Int J Immunopharmacol. 2000;22:1131–1135. [PubMed]
31. Ricote M, Li AC, Willson TM, Kelly CJ, Glass CK. The peroxisome proliferator-activated receptor-gamma is a negative regulator of macrophage activation. Nature. 1998;391:79–82. [PubMed]
32. Kliewer SA, Lenhard JM, Willson TM, Patel I, Morris DC, Lehmann JM. A prostaglandin J2 metabolite binds peroxisome proliferator-activated receptor gamma and promotes adipocyte differentiation. Cell. 1995;83:813–819. [PubMed]
33. Ricote M, Huang JT, Welch JS, Glass CK. The peroxisome proliferator-activated receptor (PPARgamma) as a regulator of monocyte/macrophage function. J Leukoc Biol. 1999;66:733–739. [PubMed]
34. Fahmi H, Di Battista JA, Pelletier JP, Mineau F, Ranger P, Martel-Pelletier J. Peroxisome proliferator--activated receptor gamma activators inhibit interleukin-1beta-induced nitric oxide and matrix metalloproteinase 13 production in human chondrocytes. Arthritis Rheum. 2001;44:595–607. [PubMed]
35. Cuzzocrea S, Mazzon E, Dugo L, et al. Reduction in the evolution of murine type II collagen-induced arthritis by treatment with rosiglitazone, a ligand of the peroxisome proliferator-activated receptor gamma. Arthritis Rheum. 2003;48:3544–3556. [PubMed]
36. Shiojiri T, Wada K, Nakajima A, et al. PPAR gamma ligands inhibit nitrotyrosine formation and inflammatory mediator expressions in adjuvant-induced rheumatoid arthritis mice. Eur J Pharmacol. 2002;448:231–238. [PubMed]
37. Sanchez-Hidalgo M, Martin AR, Villegas I, Alarcon De La Lastra C. Rosiglitazone, an agonist of peroxisome proliferator-activated receptor gamma, reduces chronic colonic inflammation in rats. Biochem Pharmacol. 2005;69:1733–1744. [PubMed]
38. Sampey AV, Monrad S, Crofford LJ. Microsomal prostaglandin E synthase-1: the inducible synthase for prostaglandin E2. Arthritis Res Ther. 2005;7:114–117. [PMC free article] [PubMed]
39. Kojima F, Naraba H, Miyamoto S, Beppu M, Aoki H, Kawai S. Membrane-associated prostaglandin E synthase-1 is upregulated by proinflammatory cytokines in chondrocytes from patients with osteoarthritis. Arthritis Res Ther. 2004;6:R355–365. [PMC free article] [PubMed]
40. Kapoor M, Kojima F, Qian M, Yang L, Crofford LJ. Shunting of prostanoid biosynthesis in microsomal prostaglandin E synthase-1 null embryo fibroblasts: regulatory effects on inducible nitric oxide synthase expression and nitrite synthesis. Faseb J. In Press. [PubMed]
41. Kampfer H, Brautigam L, Geisslinger G, et al. Cyclooxygenase-1-coupled prostaglandin biosynthesis constitutes an essential prerequisite for skin repair. J Invest Dermatol. 2003;120:880–890. [PubMed]
42. Kohno S, Endo H, Hashimoto A, et al. Inhibition of skin sclerosis by 15deoxy Delta12,14-prostaglandin J2 and retrovirally transfected prostaglandin D synthase in a mouse model of bleomycin-induced scleroderma. Biomed Pharmacother. 2006;60:18–25. [PubMed]
43. Gilroy DW, Colville-Nash PR, McMaster S, Sawatzky DA, Willoughby DA, Lawrence T. Inducible cyclooxygenase-derived 15-deoxy(Delta)12–14PGJ2 brings about acute inflammatory resolution in rat pleurisy by inducing neutrophil and macrophage apoptosis. Faseb J. 2003;17:2269–2271. [PubMed]
44. Miadonna A, Tedeschi A, Brasca C, et al. Mediator release after endobronchial antigen challenge in patients with respiratory allergy. J Allergy Clin Immunol. 1990;85:906–913. [PubMed]
45. Ulven T, Kostenis E. Targeting the prostaglandin D2 receptors DP and CRTH2 for treatment of inflammation. Curr Top Med Chem. 2006;6:1427–1444. [PubMed]
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...