| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 280, Issue 18, 18163-18170, May 6, 2005
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1-induced Changes in Epidermal Peroxisome Proliferator-activated Receptor /
Expression Dictate Wound Repair Kinetics*
From the Center for Integrative Genomics, National Center of Competence in Research Frontiers in Genetics, University of Lausanne, Lausanne CH-1015, Switzerland
Received for publication, November 12, 2004 , and in revised form, January 18, 2005.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
/
occupies a unique position at the intersection of diverse inflammatory or anti-inflammatory signals that influence wound repair. This study shows how changes in PPAR/ expression have a profound effect on wound healing. Using two different in vivo models based on topical application of recombinant transforming growth factor (TGF)-1 and ablation of the Smad3 gene, we show that prolonged expression and activity of PPAR/ accelerate wound closure. The results reveal a dual role of TGF-1 as a chemoattractant of inflammatory cells and repressor of inflammation-induced PPAR/ expression. Also, they provide insight into the so far reported paradoxical effects of the application of exogenous TGF-1 at wound sites. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
11 has the broadest effects, influencing nearly every aspect of tissue repair (1). TGF-1 signals through a membrane receptor complex that recruits and phosphorylates Smad2 and 3 proteins. Once phosphorylated, they associate with Smad4 and translocate into the nucleus to control gene transcription in several ways (2, 3). Smad3-null mice, whose TGF-1 signaling pathway is impaired, show accelerated wound healing characterized by enhanced reepithelialization, increased keratinocyte proliferation, and reduced immune cell infiltration (4). Interestingly, TGF-1-null mice display either delayed or accelerated wound healing depending on the model analyzed (5, 6). Equally complex is the effect of exogenous TGF-1 on a wound because the healing rate was found to be dependent on the delivery vehicle, dose of the growth factor, its application schedule, and wound model (7–9). In a rabbit ear dermal ulcer model, a single application of TGF-1 at the time of wounding had a beneficial effect equal to that of multiple doses, whereas application 24 h after wounding did not improve healing (10). Doses of TGF-1 used to treat wounds topically have varied over a 1,000-fold concentration range from a few ng to several µg, depending on delivery vehicle and wound models (8, 9, 11, 12). All studies have shown a bell-shaped dose-dependent response to TGF-1, with either no or decreasing effects at lower or higher than optimal concentrations. The reason for such diverse responses to exogenous TGF-1 is part of the present investigation.
The nuclear receptor PPAR/ plays critical roles in the keratinocyte response to inflammation signals produced immediately after a skin injury. Its inflammation-induced increase in activity at the wound edge maintains a sufficient number of viable keratinocytes for reepithelialization (13–15). Using exogenous TGF-1 application as well as Smad3 and PPAR/ single- and double-knock-out mice, we provide evidence for an in vivo cross-talk between TGF-1 and PPAR/ signaling.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
), anti-phospho-Akt-Thr308, anti-phospho-Akt-Ser473, and an in vitro Akt kinase activity assay kit were from Cell Signaling. Anti-keratin 6 (K6), anti-filaggrin, anti-involucrin, and anti-Ki67 were from Babco. Anti-PPAR/ was from Affinity Bioreagent. Anti--smooth muscle actin (-SMA) and anti--SMA-fluorescein isothiocyanate were from Sigma. Recombinant TGF-1 was from PeproTech EC. The specificity of the anti-PPAR/ antibody was verified as shown in Fig. S4.
Animals and Wounding Experiments—PPAR/+/-Smad3+/- mice were obtained by breeding male Smad3-/- with female PPAR/-/- mice. Smad3+/- and PPAR/-/-Smad3+/- mice were obtained by interbreeding PPAR/+/-Smad3+/- littermates. Females and males homozygous for the Smad3 deletion are sterile and hypofertile, respectively. Hence, Smad3+/- littermates were intercrossed to obtain the wild type, Smad3+/-, and Smad3-/- mice, whereas the PPAR/-/-, PPAR/-/-Smad3+/-, and PPAR/-/-Smad3-/- mice were obtained by PPAR/-/-Smad3+/- breeding. All mice used in this study are in C57BL/6 background and were individually caged, housed in a temperature-controlled room (23 °C) on a 10 h dark/14 h light cycle, and fed with the standard mouse chow diet.
Wounding and healing analyses were performed on 6-week-old females as described previously (6, 14). Briefly, the hair follicle cycle of each mouse was synchronized by shaving the back of the animal 2 weeks before starting the experiment. Mice were then anesthetized, shaved, and a full thickness middorsal wound (0.5 cm2, square-shaped) was created by excising the skin and the underlying panniculus carnosus. Wound closure was measured daily in a double-blinded fashion until complete wound closure. A subset of wild type animals was treated topically with either vehicle (3% methylcellulose in phosphate-buffered saline and 4 mM HCl) or 100 ng of TGF-1. Treatments were rotated to avoid site bias. At the indicated days postwounding, the entire wound including a 5-mm margin was excised. Wounds were dissected for immunohistochemistry, RNA, and protein analyses, as described previously (16). Immunohistochemistry was performed on 8-µm cryosections of wound biopsies with the following antibody dilutions: K6, involucrin, 1:1,000; filaggrin, Akt1-phosphoserine 473, 1:500; -SMA-fluorescein isothiocyanate, 1:400; Ki67, Akt1-phosphothreonine 308, 1:200; PPAR/, Mac3, 1:100. The signal was detected either by immunofluorescence or amplified using the ABC-peroxidase method (Vector Laboratories) and revealed using 3,3'-diaminobenzidine with (dark blue coloration) or without (brown coloration) metal enhancer. Apoptotic cells were detected by TUNEL assay (Roche Applied Science).
|
/, L27, ILK, and/or PDK1, using RNA isolated from wound biopsies of various mutant mice excised at the indicated postinjury time points. The measured expression levels were normalized against the mRNA levels of the ribosomal protein L27. The normalized value of unwounded (day 0) skin biopsies was assigned a value of 1. |
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
1 on Wound Healing1 on wound closure depend on numerous factors (1). Recently, we discovered a cross-talk between PPAR/ and TGF-1/Smad3 signaling in primary keratinocytes (16), but the effects of exogenous TGF-1 at injury site, in vivo, were not investigated. Here, we studied these effects on PPAR/ expression and wound closure. Recombinant TGF-1 was applied either immediately upon (TGF-1(day 0)) or 2 days after injury (TGF-1(day 2)).
TGF-1(Day 0) Treatment—The TGF-1(day 0) treatment accelerated wound repair with a complete closure at days 13–14 compared with the vehicle-treated wounds, which closed at day 17 (Figs. 1A and S1A). Wound biopsy analyses showed that the expression of PPAR/ was already elevated 24 h after application, an accelerated response compared with vehicle-treated wounds (Figs. 1B and S2A). Although PPAR/ levels did not reach the peak observed in vehicle-treated wounds, they remained elevated for up to 5 days after injury. As expected, the expression patterns of two PPAR/ target genes, ILK and PDK1, and hence Akt1 activity, were changed according to the altered PPAR/ expression profile (Figs. 1B and S2A) (13).
Wound contraction also contributes to wound closure and takes place when the granulation tissue is populated with myofibroblasts. Because TGF-1 is a major cytokine of myofibroblast differentiation (17), we next examined the presence of these cells in wound biopsies using -SMA as a specific marker. Consistent with other reports, the expression of -SMA peaked between days 7 and 10 after injury (Figs. 1B and S2A) (18, 19). In TGF-1(day 0)-treated wounds, a higher expression of -SMA was already detected at day 5. Subsequently, it peaked and declined similarly to vehicle-treated wounds (Figs. 1B, S2A, and S3).
|
/ mRNA expression (Fig. 1B), more PPAR/-positive keratinocytes were detected in TGF-1(day 0)-than in vehicle-treated wound biopsies (TGF-1 versus vehicle, 94.6 ± 7.5 versus 36.5 ± 3.2/section) (Fig. 1C). Because TGF-1 is one of the most potent chemoattractants for macrophages and neutrophils at the injury site (20), we suspected that an early recruitment of a higher number of inflammatory cells would accelerate the release of cytokines, such as tumor necrosis factor-, which up-regulate PPAR/ expression and activity (15, 21). Indeed, 24 h post-treatment, a higher number of Mac3-positive macrophages infiltrated the TGF-1(day 0)-treated compared with vehicle-treated control wounds (TGF-1 versus vehicle, 109.6 ± 15.2 versus 34.2 ± 7.7/section) (Fig. 1C).
We propose that these early recruited macrophages produce enough inflammatory cytokines to overcome the inhibitory action of TGF-1 on PPAR/ (16) and to up-regulate its expression. Indeed, the chemotactic dose response to TGF-1 follows a bell-shaped curve (2). Consistent with other reports showing that the infiltration of macrophages begins 48 h postinjury (22, 23), the optimal response occurs around days 2–4 in the vehicle-treated mice, when the expression of PPAR/ is high and that of -SMA low (Fig. 1D). In TGF-1(day 0)-treated wound, the infiltration of macrophages occurs earlier, between days 1 and 2 (Fig. 1D), producing a sufficient amount of cytokines to overcome the inhibitory action of TGF-1 on PPAR/. Accordingly, a different outcome with a later application of the TGF-1 treatment is expected.
TGF-1(Day 2) Treatment—A later exposure to TGF-1, 48 h after injury (TGF-1(day 2) treatment), resulted in a significant, but transient delay in reepithelialization, with a wound closure time similar to that of the vehicle-treated wound (Figs. 2A and S1B). Interestingly, the peak of increased PPAR/ expression typically observed in vehicle-treated wound biopsies was strongly repressed by the TGF-1(day 2) treatment (Figs. 2B and S2B). The diminished PPAR/ expression also resulted in reduced ILK and PDK expression as well as reduced Akt1 activity (Figs. 2B and S2B). In contrast, an early increase in -SMA expression was detected already at day 3 and peaked between days 5 and 10 (Figs. 2B, S2B, and S3).
Immunohistochemistry of day 3 postwound biopsies, i.e. 24 h after TGF-1 treatment, revealed fewer PPAR/-positive keratinocytes (TGF-1 versus vehicle, 25.6 ± 3.4 versus 86.6 ± 2.5/section). In contrast to TGF-1(day 0) treatment, there was no difference in the amount of infiltrating macrophages between vehicle-treated and TGF-1(day 2)-treated samples (Fig. 2C). The TGF-1(day 2) treatment also shifted the chemotactic response to an earlier time point (Fig. 2D). But in contrast to the TGF-1(day 0) treatment, we speculate that this delayed administration of TGF-1, added to already high endogenous TGF-1, exceeds the optimal TGF-1 dose for chemotaxis. Consequently, no further increase in infiltrating inflammatory cells was observed (Fig. 2C), and thus the TGF-1 repression on inflammation-stimulated expression of PPAR/ was not overcome (16).
|
1, similar experiments were performed on Smad3-null mice. Regardless of the application schedule, TGF-1(day 0) or TGF-1(day 2), no significant increase in the number of Mac3-positive macrophages was detected in 24-h post-treatment biopsies compared with vehicle-treated wounds (Fig. S5). This suggests that Smad3 is critical for TGF-1-mediated chemotaxis, which is consistent with earlier reports showing that topical applications of TGF-1 immediately before wounding did not influence inflammatory cell recruitment in Smad3-deficient mice (4, 24).
Taken together, these results underscore a finely tuned temporal balance between inflammatory signals and TGF-1inthe regulation of PPAR/ expression and hence of wound repair. They also suggest that prolonged PPAR/ expression promotes rapid wound closure.
Prolonged PPAR/ Activity in Smad3-/- Mice
To understand better the effect of TGF-1 on the PPAR/ expression profile, we examined full thickness excisional wound repair using Smad3-/- mice. As reported earlier (5), we also observed an accelerated wound closure rate in the Smad3-/- mice compared with their wild type (WT) littermates (Fig. 3A; Smad3-/- versus WT; closure completed at day 10 versus 17, respectively). Wound biopsies from the WT mice showed a peak of PPAR/ mRNA expression 3 days postinjury. Interestingly, in Smad3 mutant mice (Smad3+/- and Smad3-/-), a premature maximal increase in PPAR/ expression was already detected 24 h postwounding, which remained high throughout the following 6 days (Figs. 3B and S6A). This sustained PPAR/ expression resulted in similar prolonged expression levels of ILK and PDK1 (Figs. 3B, and S6, A and B) and, consequently of Akt1 kinase activity (Figs. 3C and S6B). These results emphasize the role of Smad3 in mediating the down-regulation of inflammation-induced PPAR/ by TGF-1. Very interestingly, these profiles are reminiscent of those observed after TGF-1(day 0) treatment (see Fig. 1B).
To evaluate the effect of the prolonged PPAR/ expression on reepithelialization, we performed immunohistochemistry on wound biopsies at day 5, when the expression levels of PPAR/, PDK1, ILK, and the activity of Akt1 were dramatically higher in Smad3-/- wounds. Compared with WT mice, Smad3-/- mice exhibited a thicker hyperproliferative and more extended epithelial tongue at the wound site, as revealed by K6 staining (Fig. 4A). Consistent with the sustained PPAR/ mRNA and Akt1 activity levels, a higher number of PPAR/-positive keratinocytes was detected in Smad3-/- biopsies (WT versus Smad3-/-, 32.4 ± 3.1 versus 68.4 ± 2.1/section; Fig. S4), and more phosphorylated Akt1 was detected at the leading wound edges and in the basal wound keratinocytes (Fig. 4B).
|
/ and Smad3 play critical roles in the balance between keratinocyte proliferation and apoptosis (15, 16). In Smad3-/- mice, a higher number of proliferating Ki67-positive keratinocytes was detected in the lower layers of the neoepithelium (Smad3-/- versus WT, 209.8 ± 4.6 versus 77.8 ± 2.3/section), a region where phosphorylated Akt1 was more abundant (Fig. 4B). In parallel, the number of apoptotic TUNEL-positive cells that are restricted to the upper layers was reduced in Smad3-/- wound biopsies compared with their WT littermates (Fig. 4B; Smad3-/- versus WT, 11.2 ± 1.3 versus 36.1 ± 4.9/section).
Together, these results provide evidence for prolonged epidermal PPAR/ expression in Smad3-/- mice, which produces a prolonged Akt1 activity, higher keratinocyte proliferation, and lower apoptosis rates in the regenerating epithelium. Most importantly, these events are associated with much faster healing in Smad3-/- mice.
Sustained PPAR/ Expression and Activity Are Required for Rapid Wound Closure
To demonstrate further that the prolonged PPAR/ is responsible for the accelerated wound closure in the Smad3 mutant mice, we examined wound healing in PPAR/ and Smad3 double-knock-out animals (PPAR/-/-Smad3-/-), which were growth retarded (Table I). Similar growth retardation was seen in PPAR/-/- and Smad3-/- single-null animals albeit with reduced severity. Consistent with the role of PPAR/ in hair follicle development (25) and keratinocyte differentiation (21), the PPAR/-/-Smad3-/- animals also showed a transient delay in neonatal hair growth (Fig. 5A) as well as altered filaggrin and involucrin expression as revealed by immunofluorescence (Fig. 5B). However, between weeks 6 and 9, at the time of the wounding experiments, no apparent aberrant skin phenotype was observed. ILK/PDK1 expression and Akt1 activity were similar in PPAR/-/- and PPAR/-/-Smad3-/- wound biopsies (Fig. 6A). However, compared with the WT and Smad3-/- biopsies, both ILK/PDK expression and Akt1 activity were reduced (Figs. 3B, 6A, and S6). Although TGF-1 has been implicated in phosphoinositide 3-kinase/Akt1 signaling in other cell types (26, 27), our results clearly underscore a prominent role of PPAR/ in modulating Akt1 activity during skin wound healing.
|
/-deficient day 5 wound biopsies, a 15–20-fold increase in the number of apoptotic keratinocytes was observed (PPAR/-/- versus WT, 391.1 ± 10.2 versus 36.1 ± 4.9/section; PPAR/-/-Smad3-/- versus Smad3-/-, 424.5 ± 8.6 versus 21.5 ± 2.2/section). Furthermore, in contrast to WT and Smad3-/- biopsies, apoptotic keratinocytes were detected throughout the entire wound epidermis in the PPAR/-deficient mice (Figs. 4B and 6B). This dramatic increase in apoptotic keratinocytes, as a result of PPAR/ deficiency, significantly delayed wound closure by 7–14 days compared with Smad3-/- and WT mice (Figs. 3A and 6C). Both PPAR/-/- and PPAR/-/-Smad3-/- mice had a lower number of proliferating keratinocytes than the WT mice (WT, PPAR/-/-, and PPAR/-/-Smad3-/-: 74.3 ± 6.8, 53.0 ± 6.1, and 43.9 ± 3.8/section, respectively), which further delayed wound closure in these animals (Figs. 4B and 6B).
These results reveal that in vivo PPAR/ and Smad3 coordinate the balance between keratinocyte apoptosis and proliferation. Importantly again, a prolonged elevated PPAR/ expression profile favors a rapid wound closure.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
1 or genetic ablation of the Smad3 gene, a prolonged elevated expression and activity of PPAR/ dictates an accelerated wound closure. This prolonged PPAR/ activity allows for extended Akt1 activity that reduces apoptosis and increases proliferation of keratinocytes at the wound site, and hence promotes faster healing.
The differential effects of exogenous TGF-1 on wound closure underscore the complexity of the wound repair mechanism. The TGF-1(day 0) treatment accelerates, whereas the TGF-1(day 2) application transiently delays wound healing. The balance between promotion and slowdown of healing depends on doses and the application schedule of TGF-1. Analyses of these parameters suggest that the described paradoxical actions of exogenous TGF-1 on wound healing most likely reside in its dual role. As a chemoattractant of immune cells, it stimulates PPAR/ expression via the production of inflammatory cytokines (28). As a repressor, it reduces the inflammation-induced PPAR/ expression via Smad3 signaling. Early TGF-1-mediated recruitment of macrophages, achieved via a timely appropriate level of TGF-1 as illustrated by the TGF-1(day 0) treatment, can overcome the TGF-1-repressive action on inflammation-induced PPAR/ and promotes rapid wound closure. Conversely, delayed TGF-1 application, as in the TGF-1(day 2) treatment, most likely resulting in a higher TGF-1 level, fails to recruit increased amounts of macrophages and to overcome its repression effect on PPAR/ expression. In this model, an early very high dose of TGF-1 which exceeds the concentration range for TGF-1-mediated chemotaxis should also have a negative effect on wound healing. In agreement with this proposition, Beck et al. (10) showed positive effects of TGF-1 on wound healing, at doses of 5–100 ng, which was lost at higher doses. Further investigation of the dual role of TGF-1 would benefit from the availability of conditional epidermal Smad3-deficient mice.
|
1(day 0)-treated animals. Notably, the TGF-1(day 0) treatment influenced the PPAR/ expression profile such that it was almost identical to that of Smad3 mutant mice, which explains the similarities of the healing kinetics. Despite a similar outcome, the means by which it is reached in the two different experimental models are different. In the TGF-1(day 0) protocol, the chemoattractant effect of exogenous TGF-1 triggers an early infiltration of immune cells, which produce inflammatory cytokines that up-regulate PPAR/ expression (15). In the Smad3-/- mice, despite an impaired local inflammatory response (4), the enhanced and sustained expression of PPAR/ results from a loss of the suppressive effect of Smad3 on the expression of PPAR/ (17).
In the TGF-1(day 2) protocol, despite the strong suppression of inflammation-induced PPAR/ expression and a transient healing delay, a similar time point of complete wound closure was observed compared with vehicle-treated mice. This apparent discrepancy with our earlier reports describing a delayed wound closure in PPAR/-deficient mice (14, 28) is most likely for two reasons. First, the TGF-1(day 2) treatment resulted in only a local suppression of PPAR/ expression compared with the absence of the receptor in the entire PPAR/-deficient animal. Second, an enhanced wound contraction can also contribute to wound closure. Indeed, the TGF-1(day 2) protocol produced an earlier and robust up-regulation of -SMA, a marker of myofibroblasts, than vehicle and, surprisingly, TGF-1(day 0)-treated biopsies. The different effects seen in the two TGF-1 treatments suggest the involvement of additional cytokines at different time points. Indeed, it was shown recently that -SMA expression is antagonistically regulated by TGF-1 and interleukin-1. The latter is present abundantly during the early inflammation phase of wound repair (29). Of interest is the inverse expression pattern between PPAR/ and Akt1 activity on the one hand and -SMA on the other hand. This is consistent with earlier studies (3, 16, 30, 31), which showed that elevated phosphorylated Akt may inhibit the transcriptional activity of Smad3, which is crucial to the up-regulation of -SMA expression (17, 32).
|
/ ligands to be considered as potential wound repair drugs, several factors including application schedule, bioavailability, and dose have to be taken into account. The narrow peak of increase in PPAR/ expression after injury corresponds to a small time window for effective ligand activation of the receptor. A timely biochemically controlled attenuation of TGF-1/Smad3 signaling, which leads to a prolonged PPAR/ expression and also reduces fibrotic tissue (4, 33), coupled with an increase in PPAR/ activity by an agonist, should result in an accelerated rate of wound repair with minimal scarring.
Our results suggest that monitoring changes in the PPAR/ expression profile may serve as a very useful indicator of the differential effects of exogenous TGF-1 or alternative treatments on wound closure. Such insights into how incoming extracellular signals regulate PPAR/, which in turn coordinates their action on wound healing, may aid in the development of better treatments for ulcers and chronic wound disorders.
|
FOOTNOTES |
|---|
The on-line version of this article (available at http://www.jbc.org) contains Figs. S1–S6.
To whom correspondence should be addressed. Tel.: 41-21-692-4110; Fax: 41-21-692-4115; E-mail: walter.wahli{at}unil.ch.
1 The abbreviations used are: TGF-1, transforming growth factor-1; ILK, integrin-linked kinase; K6, keratin 6; PDK1, 3-phosphoinositide-dependent kinase-1; PPAR, peroxisome proliferator-activated receptor; -SMA, -smooth muscle actin; TUNEL, termnal nucelotidyl transferase-mediated UTP nick end labeling; WT, wild type.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  H. J. Kim, S. A. Ham, S. U. Kim, J.-Y. Hwang, J.-H. Kim, K. C. Chang, C. Yabe-Nishimura, J.-H. Kim, and H. G. Seo Transforming Growth Factor-{beta}1 Is a Molecular Target for the Peroxisome Proliferator-Activated Receptor {delta} Circ. Res., February 1, 2008; 102(2): 193 - 200. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. S. Tan, G. Icre, A. Montagner, B. B.-t. Heggeler, W. Wahli, and L. Michalik The Nuclear Hormone Receptor Peroxisome Proliferator-Activated Receptor {beta}/{delta} Potentiates Cell Chemotactism, Polarization, and Migration Mol. Cell. Biol., October 15, 2007; 27(20): 7161 - 7175. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||
