- Email: [email protected]
δ in skin wound healing
International Congress Series 1302 (2007) 45 – 52
www.ics-elsevier.com
Roles of the peroxisome proliferator-activated receptor (PPAR) α and β/δ in skin wound healing Liliane Michalik ⁎, Walter Wahli ⁎ Center for Integrative Genomics, National Research Center Frontiers in Genetics, University of Lausanne, Le Génopode, CH-1015 Lausanne, Switzerland
Abstract. The peroxisome proliferator-activated receptors (PPARxα, PPARβ/δ, called PPARβ below, and PPARγ) are ligand-activated transcription factors. They belong to the nuclear hormone receptor superfamily and are sensors of physiological levels of fatty acids and fatty acid derivatives. In the healthy skin of adult mice, they are expressed at very low levels in interfollicular epidermis keratinocytes, and are found at high levels in hair follicle keratinocytes. After a skin injury, the expression of PPARα and PPARβ is stimulated at the site of the wound. A detailed analysis of PPARβ revealed a spatiotemporal control program that first stimulates PPARβ expression immediately after the injury. Second, as the wound heals, this expression is gradually repressed to reach background levels after completion of wound closure. This regulation depends on opposite effects of TNF-α, which stimulates, and TGF-β1, which down-regulates the activity of the PPARβ gene. © 2007 Elsevier B.V. All rights reserved. Keywords: PPAR; TNF-α; TGF-β1; Keratinocyte; Skin repair
1. Introduction There are three PPAR isotypes that have been identified in rodents, frogs, fish and humans, named PPARα (NR1C1), PPARβ/δ (NR1C2, called PPARβ herein) and PPARγ (NR1C3) [1–3,31]. They are ligand-induced transcription factors, which are members of the nuclear hormone receptor (NHR) family. PPARs can be seen as sensors for fatty acids, especially for polyunsaturated fatty acids (PUFAs), and diverse fatty acid derivatives [4–7]. In addition, several synthetic compounds and marketed drugs are PPAR ligands. For instance, the fibrates used to treat dyslipidemia ⁎ Corresponding authors. Tel.: +41 21 692 4110; fax: +41 21 692 4115. E-mail addresses: [email protected] (L. Michalik), [email protected] (W. Wahli). 0531-5131/ © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2006.10.023
46
L. Michalik, W. Wahli / International Congress Series 1302 (2007) 45–52
act through PPARα activation, and the antidiabetic thiazolidinediones (TZD) activate PPARγ [8]. PPARs display the characteristic structural organization of nuclear receptors. The N-terminal A/B domain contains a putative ligand-independent transactivation function (AF-1) and is flanked by the C domain (DBD) that binds DNA. Then, a short hinge domain (D) links the DBD to the C-terminal ligand-binding domain (LBD) that contains the ligand-dependent transactivation function 2 (AF-2). In addition to their activation by a ligand, PPARα and γ can also be regulated by phosphorylation via activation of the mitogen-activated protein kinase (MAPK) pathway [9,10]. Binding of a ligand to the LBD induces a conformational change in the binding pocket, which promotes interactions with coactivators forming a complex that results in the stimulation of specific target genes. In addition, PPARs can repress gene activity using various mechanisms discussed recently [11]. PPARα is mainly expressed in the liver and brown adipose tissue, and is also found at lower levels in the gut, muscle, and kidney and other organs [12]. It is involved in lipid catabolism and exerts anti-inflammatory effects [13–18]. PPARγ is mostly expressed in adipose tissues, but is also found in the gut, the immune system and other organs at lower levels. Interestingly, it is induced in the liver by a high fat diet or conditions resulting in liver steatosis [18]. PPARβ is expressed ubiquitously, often at levels that are higher than those of PPARα and PPARγ [19]. The reactivation of PPARα and PPARβ in the murine interfollicular epidermis after an injury is especially important for a timely repair of the wound [20,21]. Their roles are discussed below. 2. The keratinocyte response to skin injury: roles of PPARα and PPARβ The skin is a barrier that protects the organism from various insults. Due to its peripheral localization, it is prone to be wounded. The first response to an injury is an inflammatory response that is followed by re-epithelialization and remodeling of the scar. This repair process is a crucial survival reaction that involves activation of many cells and the interaction of some of them with the extracellular matrix (ECM) to restore the integrity of the wounded area [22]. This life-saving process is initiated immediately after the injury with disruption of blood vessels, platelet activation and the release of growth factors and cytokines. Immune cells invade the blood clot and secrete inflammatory cytokines [23]. The release of growth factors and cytokines induces keratinocyte proliferation and migration. Fibroblast activation, collagen deposition, angiogenesis and wound contraction are also important steps in the regeneration of the damaged skin. Finally, remodeling of the scar completes the wound repair process. PPARα and PPARβ participate in this healing process. PPARα is up-regulated transiently during the inflammatory phase, while PPARβ re-expression lasts over the entire healing period. Invalidation of PPARα in PPARα-null mice transiently delays repair, whereas invalidation of PPARβ in PPARβ-null mice retards the completion of wound closure for 2 to 3 days [21]. The whole wound closure time is not prolonged in PPARα−/− mice. However, a transient delay coinciding with the inflammatory phase is observed during the first 4 days following the injury. Once the inflammatory response is over, wound healing efficiency is restored.
L. Michalik, W. Wahli / International Congress Series 1302 (2007) 45–52
47
During this phase, an impaired recruitment of neutrophils and monocytes/macrophages to the wound bed in PPARα−/− animals was reported [21]. Interestingly, transgenic mice expressing a dominant-negative form of PPARα in keratinocytes displayed the same pattern in wound closure as the PPARα−/− mice, with a transient delay in repair overlapping with the inflammatory phase, too [24]. This observation indicates that the detected impairment is the consequence of the genetic ablation of PPARα in keratinocytes and not in immune cells and fibroblasts. In fact, no defect in immune cell recruitment was observed in these transgenic mice, but the inflammatory reaction was exacerbated as revealed by an increase in the expression of TNF-α. The up-regulation of PPARβ expression during wound healing is concomitant with keratinocyte proliferation and migration on the extracellular matrix (ECM) to re-epithelialize the damaged region. Therefore, it is not surprising that the wound repair delay in PPARβ−/− affects the entire process. Following the release of pro-inflammatory cytokines such as TNF-α, the stress associated protein kinase pathway is activated in keratinocytes. This process results in both the stimulation of PPARβ expression through the recruitment of AP-1 to its promoter, and the production of an endogenous ligand for PPARβ [20]. Activated PPARβ promotes cell survival through an inhibition of apoptosis. This occurs via stimulation of the genes coding for 3-phosphoinositide-dependent kinase-1 (PDK1) and integrin-linked kinase (ILK), which causes activation the PKB/Akt kinase, which controls
Fig. 1. PPARβ as a regulator of cell survival and migration during wound healing. Upon injury, TNF-α is produced by the keratinocytes and the infiltrating immune cells. It contributes to PPARβ re-expression in the interfollicular epidermis via an AP-1 response element in the PPARβ promoter [20]. Activated PPARβ stimulates the expression of PDK1 and ILK, which activate PKB/Akt1. Activated PKB/Akt1 then protects keratinocytes from apoptosis [25]. During wound repair progression (re-epithelialization and remodeling of the scar), both wound fibroblasts and immune cells produce TGF-β1. In turn, TGF-β1 activates the TGF-β1/Smad3 pathway, which antagonizes the TNF-α effect. The repression mechanism involves an inhibition of the binding of c-JUN–p300 on the AP-1 site in the PPARβ promoter [26]. The same PPARβ activation pathway is used during hair follicle development during which hepatocyte growth factor (HGF) produced by the dermal papilla induces the production of PPARβ ligands [30].
48
L. Michalik, W. Wahli / International Congress Series 1302 (2007) 45–52
the cell survival pathway (Fig. 1) [25]. Furthermore, PPARβ promotes cell adhesion and migration, which are affected in primary cultures of PPARβ-null keratinocytes [21]. Further work identified TGF-β1 as the cytokine that down-regulates the TNF-α-induced PPARβ expression in keratinocytes [26]. The mechanism of this repression is based on the interaction of c-JUN with Smad3 (a downstream effector of TGF-β1 signalling), preventing the binding of AP-1 to the PPARβ promoter. Interestingly, the same AP-1 response element in the PPARβ promoter is used by both TNF-α induction of PPARβ expression, and its down-regulation by TGF-β1. This mechanism is confirmed indirectly by the prolonged expression of PPARβ observed after genetic ablation of Smad3 (Smad3−/−) in the whole animal [27], or via treatment of the wound with TGF-β1 immediately after the injury, which both correlate with a prolonged increase in PKB/Akt1 activity and an accelerated skin wound closure [28]. TGF-β1 is a well known chemo-attractant for macrophages and neutrophils [29]. In accord with this role, an increase in the number of recruited macrophages at the wound site was observed in the animals treated with TGF-β1 on the day of injury [28]. These data suggest that early recruited macrophages produce a sufficient amount of inflammatory cytokines to overcome the inhibition of the PPARβ gene by TGF-β1, and thus, its expression is up-regulated as previously described [20]. In summary, it is the spatial and temporal action of the two cytokines, which modulates the response of keratinocytes after an injury. TNF-α and TGF-β1, which are mainly produced by the cells populating the wound site, attract and activate surrounding keratinocytes. They then proliferate and migrate through the provisional matrix temporarily deposited in the clot. In the early phase of the repair process, activated keratinocytes produce TNF-α that stimulates the expression of PPARβ via the SAP–kinase pathway. In later phases of healing, TGF-β1 antagonizes this effect and down-regulates PPARβ expression. We propose that this temporal regulation is central for the regenerating epithelium. 3. Conclusion The expression pattern of PPARα and PPARβ in mouse epidermis is intriguing. It suggests a dual role for both of them in skin development and in healing after an insult, on which we have concentrated herein. While they remain undetectable in the normal adult mouse interfollicular epidermis, they are constitutively expressed in hair follicles. In contrast, they are expressed in all parts of the embryonic epidermis. We found that a full thickness injury of the adult mouse skin induces a moderate and strong stimulation of PPARα and PPARβ expression in interfollicular keratinocytes, respectively. Different mutant mice, PPARα−/− , PPARαΔ13 (dominant-negative PPARα), and PPARβ−/−, were very valuable for deciphering the roles of PPARα and PPARβ in wound healing. In PPARα−/− and PPARαΔ13 mice, there was a transient retard in wound closure during the inflammatory phase of repair. In PPARα−/− mice, the recruitment of inflammatory cells to the wound bed is impaired, an effect which is most probably due to a keratinocyte-dependent defect, since delayed healing was also observed in PPARαΔ13 mice that produce increased amounts of TNF-α after an injury. We conclude on an impaired inflammatory response in both PPARα mutant animals. These data lend support to the notion that PPARα controls the inflammatory phase of skin repair, which confirms its major role in the control of inflammatory responses [14]. TNF-α whose levels increase at the
L. Michalik, W. Wahli / International Congress Series 1302 (2007) 45–52
49
wound site immediately after the injury was found to be responsible for the inflammationinduced expression and activation of PPARβ in the wounded epithelium. The underlying mechanism comprises both the activation of the AP-1 transcription factor complex that binds to a specific element in the PPARβ promoter, and the production of an endogenous, so far unidentified PPARβ ligand. As healing proceeds, TGF-β1 represses binding of AP-1 to the PPARβ promoter, leading to a gradual decrease in PPARβ expression during the reepithelialization and remodeling phases. During the PPARβ expression window, which is determined by the balanced action of TNF-α and TGF-β1, keratinocytes are protected from cell death via the activation of the PKB/Akt1 pathway. In conclusion, PPARα and PPARβ are key regulators of skin repair. They control the whole process through PKB/Akt1 that occupies a key position in the modulation of cell survival, proliferation and migration, all essential for wound healing. Acknowledgments The work done in the authors' laboratory was supported by grants from the Swiss National Science Foundation and the Etat de Vaud. The authors thank Nathalie Constantin for her help in preparing the manuscript. References [1] B. Desvergne, W. Wahli, Peroxisome proliferator-activated receptors: nuclear control of metabolism, Endocr. Rev. 20 (1999) 649–688. [2] C. Dreyer, et al., Control of the peroxisomal beta-oxidation pathway by a novel family of nuclear hormone receptors, Cell 68 (1992) 879–887. [3] I. Issemann, S. Green, Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators, Nature 347 (1990) 645–650. [4] S. Kersten, W. Wahli, Peroxisome proliferator activated receptor agonists, EXS 89 (2000) 141–151. [5] G. Krey, et al., Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay, Mol. Endocrinol. 11 (1997) 779–791. [6] S.A. Kliewer, et al., Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors alpha and gamma, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 4318–4323. [7] B.M. Forman, J. Chen, R.M. Evans, Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors alpha and delta, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 4312–4317. [8] B. Desvergne, L. Michalik, W. Wahli, Be fit or be sick: peroxisome proliferator-activated receptors are down the road, Mol. Endocrinol. 18 (2004) 1321–1332. [9] L. Gelman, et al., Kinase signaling cascades that modulate peroxisome proliferator-activated receptors, Curr. Opin. Cell Biol. 17 (2005) 216–222. [10] C. Diradourian, J. Girard, J.P. Pegorier, Phosphorylation of PPARs: from molecular characterization to physiological relevance, Biochimie 87 (2005) 33–38. [11] J.N. Feige, et al., From molecular action to physiological outputs: peroxisome proliferator-activated receptors are nuclear receptors at the crossroads of key cellular functions, Prog. Lipid Res. 45 (2006) 120–159. [12] O. Braissant, et al., Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat, Endocrinology 137 (1996) 354–366. [13] B. Staels, et al., Activation of human aortic smooth-muscle cells is inhibited by PPARalpha but not by PPARgamma activators, Nature 393 (1998) 790–793.
50
L. Michalik, W. Wahli / International Congress Series 1302 (2007) 45–52
[14] P.R. Devchand, et al., The PPARalpha–leukotriene B4 pathway to inflammation control, Nature 384 (1996) 39–43. [15] R. Kostadinova, W. Wahli, L. Michalik, PPARs in diseases: control of mechanisms of inflammation, Curr. Med. Chem. 12 (2005) 2413–2446. [16] R. Genolet, W. Wahli, L. Michalik, PPARs as drug targets to modulate inflammatory responses? Curr. Drug Targets Inflamm. Allergy 3 (2004) 361–375. [17] P. Delerive, J.C. Fruchart, B. Staels, Peroxisome proliferator-activated receptors in inflammation control, J. Endocrinol. 169 (2001) 453–459. [18] S. Kersten, B. Desvergne, W. Wahli, Roles of PPARs in health and disease, Nature 405 (2000) 421–424. [19] P. Escher, et al., Rat PPARs: quantitative analysis in adult rat tissues and regulation in fasting and refeeding, Endocrinology 142 (2001) 4195–4202. [20] N.S. Tan, et al., Critical roles of PPAR beta/delta in keratinocyte response to inflammation, Genes Dev. 15 (2001) 3263–3277. [21] L. Michalik, et al., Impaired skin wound healing in peroxisome proliferator-activated receptor (PPAR)alpha and PPARbeta mutant mice, J. Cell Biol. 154 (2001) 799–814. [22] K.S. Midwood, L.V. Williams, J.E. Schwarzbauer, Tissue repair and the dynamics of the extracellular matrix, Int. J. Biochem. Cell Biol. 36 (2004) 1031–1037. [23] S. Werner, R. Grose, Regulation of wound healing by growth factors and cytokines, Physiol. Rev. 83 (2003) 835–870. [24] L. Michalik, et al., Selective expression of a dominant-negative form of peroxisome proliferator-activated receptor in keratinocytes leads to impaired epidermal healing, Mol. Endocrinol. 19 (2005) 2335–2348. [25] N. Di-Poi, et al., Antiapoptotic role of PPARbeta in keratinocytes via transcriptional control of the Akt1 signaling pathway, Mol. Cell 10 (2002) 721–733. [26] N.S. Tan, et al., Essential role of Smad3 in the inhibition of inflammation-induced PPARbeta/delta expression, EMBO J. 23 (2004) 4211–4221. [27] G.S. Ashcroft, et al., Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response, Nat. Cell Biol. 1 (1999) 260–266. [28] N.S. Tan, et al., Genetic- or transforming growth factor-beta 1-induced changes in epidermal peroxisome proliferator-activated receptor beta/delta expression dictate wound repair kinetics, J. Biol. Chem. 280 (2005) 18163–18170. [29] S.M. Wahl, et al., Transforming growth factor type beta induces monocyte chemotaxis and growth factor production, Proc. Natl. Acad. Sci. U. S. A. 84 (1987) 5788–5792. [30] N. Di-Poi, et al., Epithelium–mesenchyme interactions control the activity of peroxisome proliferatoractivated receptor beta/delta during hair follicle development, Mol. Cell. Biol. 25 (2005) 1696–1712. [31] M.J. Leaver, et al., Three peroxisome proliferator-activated receptor isotypes from each of two species of marine fish, Endocrinology 146 (2005) 3150–3162.
Discussion Maria Sibilia Have you checked if your PPAR beta knockout keratinocytes express levels of the EGF receptor equal to the controls? They do not really respond to EGF. Walter Wahli Yes, this has been done. The expression is normal. Jeffrey Davidson There’s no doubt from your data that there’s a striking keratinocyte phenotype, but if one looks at the wound closure parameters, there are actually marked differences in the first two days of closure when one wouldn’t expect very much keratinocyte participation at all. So what is going on in the connective tissue in the fibroblasts of these knockout animals?
L. Michalik, W. Wahli / International Congress Series 1302 (2007) 45–52
51
Walter Wahli This is a crucial question and the interaction between fibroblasts and keratinocytes is something we are working on. What we’d like to do is to co-culture fibroblasts knocked out for PPAR beta with wild type keratinocytes, for instance. Preliminary experiments indicate that signals are coming from the fibroblasts Reinhard Fässler You proposed that ILK which is considered as a kinase that binds to the cytoplasmic tail of integrins, was phosphorylating Akt. I guess that the integrin community is thinking that ILK is a pseudo-kinase, and you could very nicely test this by replenishing your PPAR beta deficient keratinocytes with PDK1 and ILK, and see whether this gives normal Akt activity. Did you do such an experiment? Walter Wahli No, but if you have the plasmids, we would be happy to do the experiment. Reinhard Fässler We have all the plasmids. People have also suggested that ILK phosphorylates gsk3 beta. Did you also connect ILK with gsk, because you do connect it with Akt, but not with gsk. That’s kind of surprising. Walter Wahli Well, it’s along the same line of experiment you have mentioned. One would have to dissect in detail the pathway and certainly, this would be worth doing systematically, but probably in collaboration with other laboratories, because it is a lot of work to check each of the different members of these pathways individually. Reinhard Fässler Do you know how the turnover of integrins is controlled by PPAR at the molecular level? Walter Wahli ILK may be important. In the knockout cells, there is much less ILK. We have verified this on the skin sections as well as in cultured keratinocytes. An open question is whether PPAR beta is involved in endosome trafficking within the cells. And this would be fascinating to study. We think that it could potentially happen, because PPAR-beta is also involved in Paneth cell differentiation in the gut, and one of the defects we see in these cells after PPAR beta invalidation is that the secretion granules are not correctly formed. Paolo Dotto Are you doing work on the biological clocks in PPARs? Walter Wahli Yes. The expression of PPARs is very dependent on the time of the day. In mice, for example, PPAR alpha is high in the afternoon. It is when mice are starving, so it’s a normal reaction. In contrast, PPAR beta is high at four o’clock in the morning, but we don’t yet know why. Paolo Dotto In all tissues? Walter Wahli We have especially looked in the liver. So far we haven’t looked in the skin.
52
L. Michalik, W. Wahli / International Congress Series 1302 (2007) 45–52
Paolo Dotto It has been suggested that PPAR could also function as an indirect mechanism to control transcription by blocking AP-1. Is it so? Walter Wahli It’s not yet totally clear how it works. In some situations you get interaction between the PPARs and subunits of, for instance, NF-kappaB; with AP-1 less is known, but PPAR gamma has been reported to antagonize the activity of AP-1. The experiments addressing these questions are very often cell culture experiments where different factors are overexpressed and therefore, one has to be critical about the results obtained, because overexpression artifacts are possible. Paolo Dotto If you use your knockout model, all evidence is pointing to a function which is always due to direct control of transcription, or not? Walter Wahli The direct control of transcription occurs especially for stimulation. In contrast, the down-regulation of PTEN, which I have presented, is via an indirect mechanism requiring protein de novo synthesis. It’s probably the product of one of the target genes of PPAR-beta, which is involved in the repression, but we haven’t found which one it is yet. Martin Schwab You said that PPAR-beta is quite ubiquitous. How ubiquitous is the function you described in terms of actin cytoskeleton regulation and migration, for instance, in neuronal growth cones or fibroblasts? Walter Wahli This has not yet been done. We have observed the effect on the actin cytoskeleton quite recently. In addition, we know that integrin recycling is also affected. We tend to think that the mechanism we are studying is probably general; we have one or two additional examples, for instance proximal tubular cells, but not many more; and for cytoskeleton, it’s a first description.
www.ics-elsevier.com
Roles of the peroxisome proliferator-activated receptor (PPAR) α and β/δ in skin wound healing Liliane Michalik ⁎, Walter Wahli ⁎ Center for Integrative Genomics, National Research Center Frontiers in Genetics, University of Lausanne, Le Génopode, CH-1015 Lausanne, Switzerland
Abstract. The peroxisome proliferator-activated receptors (PPARxα, PPARβ/δ, called PPARβ below, and PPARγ) are ligand-activated transcription factors. They belong to the nuclear hormone receptor superfamily and are sensors of physiological levels of fatty acids and fatty acid derivatives. In the healthy skin of adult mice, they are expressed at very low levels in interfollicular epidermis keratinocytes, and are found at high levels in hair follicle keratinocytes. After a skin injury, the expression of PPARα and PPARβ is stimulated at the site of the wound. A detailed analysis of PPARβ revealed a spatiotemporal control program that first stimulates PPARβ expression immediately after the injury. Second, as the wound heals, this expression is gradually repressed to reach background levels after completion of wound closure. This regulation depends on opposite effects of TNF-α, which stimulates, and TGF-β1, which down-regulates the activity of the PPARβ gene. © 2007 Elsevier B.V. All rights reserved. Keywords: PPAR; TNF-α; TGF-β1; Keratinocyte; Skin repair
1. Introduction There are three PPAR isotypes that have been identified in rodents, frogs, fish and humans, named PPARα (NR1C1), PPARβ/δ (NR1C2, called PPARβ herein) and PPARγ (NR1C3) [1–3,31]. They are ligand-induced transcription factors, which are members of the nuclear hormone receptor (NHR) family. PPARs can be seen as sensors for fatty acids, especially for polyunsaturated fatty acids (PUFAs), and diverse fatty acid derivatives [4–7]. In addition, several synthetic compounds and marketed drugs are PPAR ligands. For instance, the fibrates used to treat dyslipidemia ⁎ Corresponding authors. Tel.: +41 21 692 4110; fax: +41 21 692 4115. E-mail addresses: [email protected] (L. Michalik), [email protected] (W. Wahli). 0531-5131/ © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2006.10.023
46
L. Michalik, W. Wahli / International Congress Series 1302 (2007) 45–52
act through PPARα activation, and the antidiabetic thiazolidinediones (TZD) activate PPARγ [8]. PPARs display the characteristic structural organization of nuclear receptors. The N-terminal A/B domain contains a putative ligand-independent transactivation function (AF-1) and is flanked by the C domain (DBD) that binds DNA. Then, a short hinge domain (D) links the DBD to the C-terminal ligand-binding domain (LBD) that contains the ligand-dependent transactivation function 2 (AF-2). In addition to their activation by a ligand, PPARα and γ can also be regulated by phosphorylation via activation of the mitogen-activated protein kinase (MAPK) pathway [9,10]. Binding of a ligand to the LBD induces a conformational change in the binding pocket, which promotes interactions with coactivators forming a complex that results in the stimulation of specific target genes. In addition, PPARs can repress gene activity using various mechanisms discussed recently [11]. PPARα is mainly expressed in the liver and brown adipose tissue, and is also found at lower levels in the gut, muscle, and kidney and other organs [12]. It is involved in lipid catabolism and exerts anti-inflammatory effects [13–18]. PPARγ is mostly expressed in adipose tissues, but is also found in the gut, the immune system and other organs at lower levels. Interestingly, it is induced in the liver by a high fat diet or conditions resulting in liver steatosis [18]. PPARβ is expressed ubiquitously, often at levels that are higher than those of PPARα and PPARγ [19]. The reactivation of PPARα and PPARβ in the murine interfollicular epidermis after an injury is especially important for a timely repair of the wound [20,21]. Their roles are discussed below. 2. The keratinocyte response to skin injury: roles of PPARα and PPARβ The skin is a barrier that protects the organism from various insults. Due to its peripheral localization, it is prone to be wounded. The first response to an injury is an inflammatory response that is followed by re-epithelialization and remodeling of the scar. This repair process is a crucial survival reaction that involves activation of many cells and the interaction of some of them with the extracellular matrix (ECM) to restore the integrity of the wounded area [22]. This life-saving process is initiated immediately after the injury with disruption of blood vessels, platelet activation and the release of growth factors and cytokines. Immune cells invade the blood clot and secrete inflammatory cytokines [23]. The release of growth factors and cytokines induces keratinocyte proliferation and migration. Fibroblast activation, collagen deposition, angiogenesis and wound contraction are also important steps in the regeneration of the damaged skin. Finally, remodeling of the scar completes the wound repair process. PPARα and PPARβ participate in this healing process. PPARα is up-regulated transiently during the inflammatory phase, while PPARβ re-expression lasts over the entire healing period. Invalidation of PPARα in PPARα-null mice transiently delays repair, whereas invalidation of PPARβ in PPARβ-null mice retards the completion of wound closure for 2 to 3 days [21]. The whole wound closure time is not prolonged in PPARα−/− mice. However, a transient delay coinciding with the inflammatory phase is observed during the first 4 days following the injury. Once the inflammatory response is over, wound healing efficiency is restored.
L. Michalik, W. Wahli / International Congress Series 1302 (2007) 45–52
47
During this phase, an impaired recruitment of neutrophils and monocytes/macrophages to the wound bed in PPARα−/− animals was reported [21]. Interestingly, transgenic mice expressing a dominant-negative form of PPARα in keratinocytes displayed the same pattern in wound closure as the PPARα−/− mice, with a transient delay in repair overlapping with the inflammatory phase, too [24]. This observation indicates that the detected impairment is the consequence of the genetic ablation of PPARα in keratinocytes and not in immune cells and fibroblasts. In fact, no defect in immune cell recruitment was observed in these transgenic mice, but the inflammatory reaction was exacerbated as revealed by an increase in the expression of TNF-α. The up-regulation of PPARβ expression during wound healing is concomitant with keratinocyte proliferation and migration on the extracellular matrix (ECM) to re-epithelialize the damaged region. Therefore, it is not surprising that the wound repair delay in PPARβ−/− affects the entire process. Following the release of pro-inflammatory cytokines such as TNF-α, the stress associated protein kinase pathway is activated in keratinocytes. This process results in both the stimulation of PPARβ expression through the recruitment of AP-1 to its promoter, and the production of an endogenous ligand for PPARβ [20]. Activated PPARβ promotes cell survival through an inhibition of apoptosis. This occurs via stimulation of the genes coding for 3-phosphoinositide-dependent kinase-1 (PDK1) and integrin-linked kinase (ILK), which causes activation the PKB/Akt kinase, which controls
Fig. 1. PPARβ as a regulator of cell survival and migration during wound healing. Upon injury, TNF-α is produced by the keratinocytes and the infiltrating immune cells. It contributes to PPARβ re-expression in the interfollicular epidermis via an AP-1 response element in the PPARβ promoter [20]. Activated PPARβ stimulates the expression of PDK1 and ILK, which activate PKB/Akt1. Activated PKB/Akt1 then protects keratinocytes from apoptosis [25]. During wound repair progression (re-epithelialization and remodeling of the scar), both wound fibroblasts and immune cells produce TGF-β1. In turn, TGF-β1 activates the TGF-β1/Smad3 pathway, which antagonizes the TNF-α effect. The repression mechanism involves an inhibition of the binding of c-JUN–p300 on the AP-1 site in the PPARβ promoter [26]. The same PPARβ activation pathway is used during hair follicle development during which hepatocyte growth factor (HGF) produced by the dermal papilla induces the production of PPARβ ligands [30].
48
L. Michalik, W. Wahli / International Congress Series 1302 (2007) 45–52
the cell survival pathway (Fig. 1) [25]. Furthermore, PPARβ promotes cell adhesion and migration, which are affected in primary cultures of PPARβ-null keratinocytes [21]. Further work identified TGF-β1 as the cytokine that down-regulates the TNF-α-induced PPARβ expression in keratinocytes [26]. The mechanism of this repression is based on the interaction of c-JUN with Smad3 (a downstream effector of TGF-β1 signalling), preventing the binding of AP-1 to the PPARβ promoter. Interestingly, the same AP-1 response element in the PPARβ promoter is used by both TNF-α induction of PPARβ expression, and its down-regulation by TGF-β1. This mechanism is confirmed indirectly by the prolonged expression of PPARβ observed after genetic ablation of Smad3 (Smad3−/−) in the whole animal [27], or via treatment of the wound with TGF-β1 immediately after the injury, which both correlate with a prolonged increase in PKB/Akt1 activity and an accelerated skin wound closure [28]. TGF-β1 is a well known chemo-attractant for macrophages and neutrophils [29]. In accord with this role, an increase in the number of recruited macrophages at the wound site was observed in the animals treated with TGF-β1 on the day of injury [28]. These data suggest that early recruited macrophages produce a sufficient amount of inflammatory cytokines to overcome the inhibition of the PPARβ gene by TGF-β1, and thus, its expression is up-regulated as previously described [20]. In summary, it is the spatial and temporal action of the two cytokines, which modulates the response of keratinocytes after an injury. TNF-α and TGF-β1, which are mainly produced by the cells populating the wound site, attract and activate surrounding keratinocytes. They then proliferate and migrate through the provisional matrix temporarily deposited in the clot. In the early phase of the repair process, activated keratinocytes produce TNF-α that stimulates the expression of PPARβ via the SAP–kinase pathway. In later phases of healing, TGF-β1 antagonizes this effect and down-regulates PPARβ expression. We propose that this temporal regulation is central for the regenerating epithelium. 3. Conclusion The expression pattern of PPARα and PPARβ in mouse epidermis is intriguing. It suggests a dual role for both of them in skin development and in healing after an insult, on which we have concentrated herein. While they remain undetectable in the normal adult mouse interfollicular epidermis, they are constitutively expressed in hair follicles. In contrast, they are expressed in all parts of the embryonic epidermis. We found that a full thickness injury of the adult mouse skin induces a moderate and strong stimulation of PPARα and PPARβ expression in interfollicular keratinocytes, respectively. Different mutant mice, PPARα−/− , PPARαΔ13 (dominant-negative PPARα), and PPARβ−/−, were very valuable for deciphering the roles of PPARα and PPARβ in wound healing. In PPARα−/− and PPARαΔ13 mice, there was a transient retard in wound closure during the inflammatory phase of repair. In PPARα−/− mice, the recruitment of inflammatory cells to the wound bed is impaired, an effect which is most probably due to a keratinocyte-dependent defect, since delayed healing was also observed in PPARαΔ13 mice that produce increased amounts of TNF-α after an injury. We conclude on an impaired inflammatory response in both PPARα mutant animals. These data lend support to the notion that PPARα controls the inflammatory phase of skin repair, which confirms its major role in the control of inflammatory responses [14]. TNF-α whose levels increase at the
L. Michalik, W. Wahli / International Congress Series 1302 (2007) 45–52
49
wound site immediately after the injury was found to be responsible for the inflammationinduced expression and activation of PPARβ in the wounded epithelium. The underlying mechanism comprises both the activation of the AP-1 transcription factor complex that binds to a specific element in the PPARβ promoter, and the production of an endogenous, so far unidentified PPARβ ligand. As healing proceeds, TGF-β1 represses binding of AP-1 to the PPARβ promoter, leading to a gradual decrease in PPARβ expression during the reepithelialization and remodeling phases. During the PPARβ expression window, which is determined by the balanced action of TNF-α and TGF-β1, keratinocytes are protected from cell death via the activation of the PKB/Akt1 pathway. In conclusion, PPARα and PPARβ are key regulators of skin repair. They control the whole process through PKB/Akt1 that occupies a key position in the modulation of cell survival, proliferation and migration, all essential for wound healing. Acknowledgments The work done in the authors' laboratory was supported by grants from the Swiss National Science Foundation and the Etat de Vaud. The authors thank Nathalie Constantin for her help in preparing the manuscript. References [1] B. Desvergne, W. Wahli, Peroxisome proliferator-activated receptors: nuclear control of metabolism, Endocr. Rev. 20 (1999) 649–688. [2] C. Dreyer, et al., Control of the peroxisomal beta-oxidation pathway by a novel family of nuclear hormone receptors, Cell 68 (1992) 879–887. [3] I. Issemann, S. Green, Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators, Nature 347 (1990) 645–650. [4] S. Kersten, W. Wahli, Peroxisome proliferator activated receptor agonists, EXS 89 (2000) 141–151. [5] G. Krey, et al., Fatty acids, eicosanoids, and hypolipidemic agents identified as ligands of peroxisome proliferator-activated receptors by coactivator-dependent receptor ligand assay, Mol. Endocrinol. 11 (1997) 779–791. [6] S.A. Kliewer, et al., Fatty acids and eicosanoids regulate gene expression through direct interactions with peroxisome proliferator-activated receptors alpha and gamma, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 4318–4323. [7] B.M. Forman, J. Chen, R.M. Evans, Hypolipidemic drugs, polyunsaturated fatty acids, and eicosanoids are ligands for peroxisome proliferator-activated receptors alpha and delta, Proc. Natl. Acad. Sci. U. S. A. 94 (1997) 4312–4317. [8] B. Desvergne, L. Michalik, W. Wahli, Be fit or be sick: peroxisome proliferator-activated receptors are down the road, Mol. Endocrinol. 18 (2004) 1321–1332. [9] L. Gelman, et al., Kinase signaling cascades that modulate peroxisome proliferator-activated receptors, Curr. Opin. Cell Biol. 17 (2005) 216–222. [10] C. Diradourian, J. Girard, J.P. Pegorier, Phosphorylation of PPARs: from molecular characterization to physiological relevance, Biochimie 87 (2005) 33–38. [11] J.N. Feige, et al., From molecular action to physiological outputs: peroxisome proliferator-activated receptors are nuclear receptors at the crossroads of key cellular functions, Prog. Lipid Res. 45 (2006) 120–159. [12] O. Braissant, et al., Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat, Endocrinology 137 (1996) 354–366. [13] B. Staels, et al., Activation of human aortic smooth-muscle cells is inhibited by PPARalpha but not by PPARgamma activators, Nature 393 (1998) 790–793.
50
L. Michalik, W. Wahli / International Congress Series 1302 (2007) 45–52
[14] P.R. Devchand, et al., The PPARalpha–leukotriene B4 pathway to inflammation control, Nature 384 (1996) 39–43. [15] R. Kostadinova, W. Wahli, L. Michalik, PPARs in diseases: control of mechanisms of inflammation, Curr. Med. Chem. 12 (2005) 2413–2446. [16] R. Genolet, W. Wahli, L. Michalik, PPARs as drug targets to modulate inflammatory responses? Curr. Drug Targets Inflamm. Allergy 3 (2004) 361–375. [17] P. Delerive, J.C. Fruchart, B. Staels, Peroxisome proliferator-activated receptors in inflammation control, J. Endocrinol. 169 (2001) 453–459. [18] S. Kersten, B. Desvergne, W. Wahli, Roles of PPARs in health and disease, Nature 405 (2000) 421–424. [19] P. Escher, et al., Rat PPARs: quantitative analysis in adult rat tissues and regulation in fasting and refeeding, Endocrinology 142 (2001) 4195–4202. [20] N.S. Tan, et al., Critical roles of PPAR beta/delta in keratinocyte response to inflammation, Genes Dev. 15 (2001) 3263–3277. [21] L. Michalik, et al., Impaired skin wound healing in peroxisome proliferator-activated receptor (PPAR)alpha and PPARbeta mutant mice, J. Cell Biol. 154 (2001) 799–814. [22] K.S. Midwood, L.V. Williams, J.E. Schwarzbauer, Tissue repair and the dynamics of the extracellular matrix, Int. J. Biochem. Cell Biol. 36 (2004) 1031–1037. [23] S. Werner, R. Grose, Regulation of wound healing by growth factors and cytokines, Physiol. Rev. 83 (2003) 835–870. [24] L. Michalik, et al., Selective expression of a dominant-negative form of peroxisome proliferator-activated receptor in keratinocytes leads to impaired epidermal healing, Mol. Endocrinol. 19 (2005) 2335–2348. [25] N. Di-Poi, et al., Antiapoptotic role of PPARbeta in keratinocytes via transcriptional control of the Akt1 signaling pathway, Mol. Cell 10 (2002) 721–733. [26] N.S. Tan, et al., Essential role of Smad3 in the inhibition of inflammation-induced PPARbeta/delta expression, EMBO J. 23 (2004) 4211–4221. [27] G.S. Ashcroft, et al., Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response, Nat. Cell Biol. 1 (1999) 260–266. [28] N.S. Tan, et al., Genetic- or transforming growth factor-beta 1-induced changes in epidermal peroxisome proliferator-activated receptor beta/delta expression dictate wound repair kinetics, J. Biol. Chem. 280 (2005) 18163–18170. [29] S.M. Wahl, et al., Transforming growth factor type beta induces monocyte chemotaxis and growth factor production, Proc. Natl. Acad. Sci. U. S. A. 84 (1987) 5788–5792. [30] N. Di-Poi, et al., Epithelium–mesenchyme interactions control the activity of peroxisome proliferatoractivated receptor beta/delta during hair follicle development, Mol. Cell. Biol. 25 (2005) 1696–1712. [31] M.J. Leaver, et al., Three peroxisome proliferator-activated receptor isotypes from each of two species of marine fish, Endocrinology 146 (2005) 3150–3162.
Discussion Maria Sibilia Have you checked if your PPAR beta knockout keratinocytes express levels of the EGF receptor equal to the controls? They do not really respond to EGF. Walter Wahli Yes, this has been done. The expression is normal. Jeffrey Davidson There’s no doubt from your data that there’s a striking keratinocyte phenotype, but if one looks at the wound closure parameters, there are actually marked differences in the first two days of closure when one wouldn’t expect very much keratinocyte participation at all. So what is going on in the connective tissue in the fibroblasts of these knockout animals?
L. Michalik, W. Wahli / International Congress Series 1302 (2007) 45–52
51
Walter Wahli This is a crucial question and the interaction between fibroblasts and keratinocytes is something we are working on. What we’d like to do is to co-culture fibroblasts knocked out for PPAR beta with wild type keratinocytes, for instance. Preliminary experiments indicate that signals are coming from the fibroblasts Reinhard Fässler You proposed that ILK which is considered as a kinase that binds to the cytoplasmic tail of integrins, was phosphorylating Akt. I guess that the integrin community is thinking that ILK is a pseudo-kinase, and you could very nicely test this by replenishing your PPAR beta deficient keratinocytes with PDK1 and ILK, and see whether this gives normal Akt activity. Did you do such an experiment? Walter Wahli No, but if you have the plasmids, we would be happy to do the experiment. Reinhard Fässler We have all the plasmids. People have also suggested that ILK phosphorylates gsk3 beta. Did you also connect ILK with gsk, because you do connect it with Akt, but not with gsk. That’s kind of surprising. Walter Wahli Well, it’s along the same line of experiment you have mentioned. One would have to dissect in detail the pathway and certainly, this would be worth doing systematically, but probably in collaboration with other laboratories, because it is a lot of work to check each of the different members of these pathways individually. Reinhard Fässler Do you know how the turnover of integrins is controlled by PPAR at the molecular level? Walter Wahli ILK may be important. In the knockout cells, there is much less ILK. We have verified this on the skin sections as well as in cultured keratinocytes. An open question is whether PPAR beta is involved in endosome trafficking within the cells. And this would be fascinating to study. We think that it could potentially happen, because PPAR-beta is also involved in Paneth cell differentiation in the gut, and one of the defects we see in these cells after PPAR beta invalidation is that the secretion granules are not correctly formed. Paolo Dotto Are you doing work on the biological clocks in PPARs? Walter Wahli Yes. The expression of PPARs is very dependent on the time of the day. In mice, for example, PPAR alpha is high in the afternoon. It is when mice are starving, so it’s a normal reaction. In contrast, PPAR beta is high at four o’clock in the morning, but we don’t yet know why. Paolo Dotto In all tissues? Walter Wahli We have especially looked in the liver. So far we haven’t looked in the skin.
52
L. Michalik, W. Wahli / International Congress Series 1302 (2007) 45–52
Paolo Dotto It has been suggested that PPAR could also function as an indirect mechanism to control transcription by blocking AP-1. Is it so? Walter Wahli It’s not yet totally clear how it works. In some situations you get interaction between the PPARs and subunits of, for instance, NF-kappaB; with AP-1 less is known, but PPAR gamma has been reported to antagonize the activity of AP-1. The experiments addressing these questions are very often cell culture experiments where different factors are overexpressed and therefore, one has to be critical about the results obtained, because overexpression artifacts are possible. Paolo Dotto If you use your knockout model, all evidence is pointing to a function which is always due to direct control of transcription, or not? Walter Wahli The direct control of transcription occurs especially for stimulation. In contrast, the down-regulation of PTEN, which I have presented, is via an indirect mechanism requiring protein de novo synthesis. It’s probably the product of one of the target genes of PPAR-beta, which is involved in the repression, but we haven’t found which one it is yet. Martin Schwab You said that PPAR-beta is quite ubiquitous. How ubiquitous is the function you described in terms of actin cytoskeleton regulation and migration, for instance, in neuronal growth cones or fibroblasts? Walter Wahli This has not yet been done. We have observed the effect on the actin cytoskeleton quite recently. In addition, we know that integrin recycling is also affected. We tend to think that the mechanism we are studying is probably general; we have one or two additional examples, for instance proximal tubular cells, but not many more; and for cytoskeleton, it’s a first description.