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Potentiates Cell Chemotactism, Polarization, and Migration
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Guillaume Icre,
Alexandra Montagner,
Béatrice Bordier-ten Heggeler,
Walter Wahli, and
Liliane Michalik*
Center for Integrative Genomics, National Research Center Frontiers in Genetics, University of Lausanne, CH-1015 Lausanne, Switzerland
Received 14 March 2007/ Returned for modification 17 April 2007/ Accepted 17 July 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(PPARß/) enhances two phosphatidylinositol 3-kinase-dependent pathways, namely, the Akt and the Rho-GTPase pathways. This PPARß/ activity amplifies the response of keratinocytes to a chemotactic signal, promotes integrin recycling and remodeling of the actin cytoskeleton, and thereby favors cell migration. Using three-dimensional wound reconstructions, we demonstrate that these defects have a strong impact on in vivo skin healing, since PPARß/–/– mice show an unexpected and rare epithelialization phenotype. Our findings demonstrate that nuclear hormone receptors not only regulate intercellular communication at the organism level but also participate in cell responses to a chemotactic signal. The implications of our findings may be far-reaching, considering that the mechanisms described here are important in many physiological and pathological situations. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(PKB), a kinase which contains a pleckstrin homology (PH) domain that binds with high affinity to the PIP3 product of PI3K. Although recent data suggest that its function may depend on the cell type and context (44-46), Akt1 is thought to promote migration by inhibition of glycogen synthase kinase 3ß (GSK-3ß) (27, 31). A second pathway involves the Rho small GTPase family (17, 29). Among the members of this family of proteins, Rac1 plays a role in the protrusion of lamellipodia and in forward movements, whereas cdc42 maintains cell polarity, including lamellipodium activity at the leading wound edge. Interestingly, Rac1 normal activity is required for efficient wound repair in vivo (40). Furthermore, both Rac1 and cdc42 participate in a positive-feedback loop that increases the PIP3/PIP2 ratio at the leading edge (8, 25), thereby further enhancing localized Akt activity.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3 integrin (Santa Cruz Biotechnology); anti-Rac1 and antitubulin (Signal Transduction); anti-phospho-PAK2(Ser20), anti-phospho-PAK1(Ser144)/PAK2(Ser141), anti-PAK1, anti-PAK2, anti-phospho-LIMK1(Thr508)/LIMK2(Thr505), anti-LIMK1, anti-histone H3, and anti-ILK1 (Cell Signaling); antihemagglutinin (anti-HA) tag (Sigma); rhodamine-phalloidin (Molecular Probes); DAPI (4',6'-diamidino-2-phenylindole) and Vectashield mounting medium (Vector Laboratories); bromodeoxyuridine (BrdU) detection kit and anti-c-myc tag (Roche); biotinylated goat anti-rabbit antibody (Vector Laboratories); streptavidin-tagged Alexa 488 and Alexa 350 antibodies (Molecular Probes); mouse EGF (Sigma); porous membrane inserts (BD Biosciences; Falcon); and the Akt kinase assay kit (Cell Signaling). cDNA clones for the dominant-negative and constitutively active mutants of human Rac1 and cdc42 and for the HA-tagged wild-type (wt) Rac1 and cdc42 are from the Guthrie cDNA Resource Center (http://www.cdna.org). Keratinocyte and skin explant cultures. Primary mouse keratinocytes (passage 3, except for Fig. 1D, passage 0) were cultured and transfected as previously described (37), with dominant-negative or constitutively active Rac1/cdc42, HA-wt Rac1, HA-wt cdc42, or Myc-tagged wt PPARß, or PH-Akt-green fluorescent protein (GFP) expression vectors. Transfection efficiency ranged between 60 and 80%. At 16 h posttransfection, the cells were replated on a glass-bottomed chamber slide and cultured for 6 h. Directional sensing of the EGF signal was performed using PH-Akt-GFP-transfected live cells, imaged before and after stimulation by a point source of EGF (15 nM) at indicated times (34). Akt1 activity was determined using an in vitro kinase assay on GSK-3ß as a substrate, before and after treatment of the cells with EGF and according to the provider's instructions (Cell Signaling). Pseudopodium extension and protein isolation were performed as described in reference 7. The separation of pseudopodia from cell bodies for further analysis was controlled using labeling of the nuclear histone H3. The scraping wound experiments were performed as previously described (26). Fluorescent staining assays were performed as follows. Cells were fixed in 4% formaldehyde-2% sucrose-phosphate-buffered saline (PBS) for 15 min and permeabilized with 0.02% Triton X-100-PBS for 5 min. Blocking was performed by incubating the samples in either 5% normal goat serum-PBS or normal rabbit serum-PBS for 1 h. Actin was stained with rhodamine-phalloidin added at a 1:100 dilution. Nuclei were counterstained with DAPI. Pictures were taken using a Zeiss MT510 confocal microscope.
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GTPase activation assay for cdc42 and Rac1. GTPase activation assays were carried out as previously described with some modifications (33). Cells were treated with mouse EGF (2 ng/ml; 0.3 nM), harvested at the indicated time, washed in ice-cold PBS, incubated for 5 min on ice in a lysis buffer (50 mM Tris-HCl, pH 7.4, 2 mM MgCl2, 1% NP-40, 10% glycerol, 100 mM NaCl, 1 µg/ml leupeptin and pepstatin), and centrifuged for 5 min at 21,000 x g and 4°C. Aliquots were taken from the supernatant to compare protein amounts and Western blotting for total Rac1 or cdc42. Amounts of total and active Rac1 and cdc42 were determined in the same cell lysates. Total Rac1 and cdc42 were quantified using anti-Rac1 or anti-cdc42. GTP-bound active Rac1 and cdc42 were examined using the p21 binding domain of p21-activated kinase (PAK) (glutathione S-transferase [GST]-PAK-cdc42 Rac interacting domain [CRID]), which binds to active Rac1-GTP and cdc42-GTP. The supernatant was incubated with a bacterially produced p21 binding domain of PAK (GST-PAK-CRID fusion protein), bound to glutathione-coupled Sepharose beads at 4°C for 25 min. After washing, the beads and proteins bound to the fusion protein were eluted in Laemmli buffer. Immunoblotting using anti-Rac1 or anti-cdc42 antibody revealed bound active Rac1 or cdc42 proteins. wt keratinocytes were incubated with either vehicle (dimethyl sulfoxide) or L165041 (PPARß ligand; 5 µM) 6 h before EGF treatment. Cells were exposed to PI3K inhibitors (LY294002 [50 µM] or wortmannin [100 µM]) 1 h before EGF treatment.
The level of activity of Rac1 and cdc42 after reexpression of wt PPARß in PPARß–/– keratinocytes was quantified following the same protocol, with the following modifications. Ectopic expression of PPARß was achieved by transfection of Myc-tagged wt PPARß in PPARß-null keratinocytes; the cells were cotransfected with either HA-tagged Rac1 or HA-tagged cdc42. Immunoblotting was performed using anti-HA tag antibody to reveal bound active HA-Rac1 or HA-cdc42 proteins.
Wound experiments. PPARß+/+, PPARß–/–, and Smad3–/– mice were wounded as previously described (26, 37). Wounded mice were allowed to heal for 4 days prior to tissue harvesting. Wound biopsy specimens for immunohistochemistry were excised and cryopreserved in a freezing medium (Tissue Tek O.C.T. compound; Sakura). Ten-micrometer sections were stained with hematoxylin prior to photography. Pictures of wound biopsy sections stained with hematoxylin were taken using a Leica Metalux microscope, Leica DC200 digital camera, and Leica IM50 software. Quantification of the thickness, length, and area of the migration tongue was performed using ImageJ software (see Fig. 6). At least five nonadjacent sections of three animals (minimum of 15 sections in total) per genotype were analyzed, on both sides of the wounds. The migration length was measured as the distance between the initial wound edge (as assessed by the morphology of the epidermis, dermis, and hypodermis) and the migratory front; the area represents the entire surface of the hyperproliferative epidermis, from the initial wound edge to the migratory front and from the epidermal-dermal junction to the uppermost keratinocyte layer; the thickness was calculated as the average thickness of the hyperproliferative epidermis, from the initial wound edge to the migratory front.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Upon directional stimulation with EGF supplied locally using a nearby micropipette, transfected PH-Akt-GFP was recruited to a single site of the keratinocyte surface closest to the stimulus, indicating an increase in the PIP3/PIP2 ratio at this site (Fig. 1A). In contrast to the single pseudopodium observed in wt cells, PPARß–/– keratinocytes presented delayed translocation of PH-Akt-GFP and often exhibited several smaller protrusions. Consistent with previously shown reduced phosphorylation of Akt1 (11), in vitro kinase assays showed that the PPARß-null keratinocytes exhibited lower Akt1 activity than the wt cells did, illustrating a deficient Akt1 pathway in the PPARß-null cells at the basal level of activity (Fig. 1B, left). Upon stimulation with EGF, the wt cells exhibited a rapid and sustained increase in Akt1 activity compared to a transient and weaker increase in the PPARß–/– cells (Fig. 1B, right). The defect in cell polarization towards the EGF chemotactic signal was efficiently rescued by transfection of the PPARß–/– keratinocytes with a wt PPARß cDNA (Fig. 1D, top). These observations suggest a reduced sensing response to EGF in PPARß–/– cells, most likely because of an altered internal PIP3 accumulation at the leading edge and, as a consequence, reduced recruitment and activation of Akt1 (Fig. 1A).
GSK-3ß is a target of Akt1 known to phosphorylate KLCs and, thus, to negatively regulate kinesin-based motility (27) and integrin recycling (31). Phosphorylation of GSK-3ß by Akt1 inhibits its activity, thereby promoting migration in several cell types (14, 16, 19). Concurrently with the reduced Akt1 activity, a lower level of GSK-3ß phosphorylation, corresponding to a higher kinase activity, was observed in PPARß–/– cells (Fig. 1C; see also Fig. 9). As shown in Fig. 1C, KLC2 was hyperphosphorylated in PPARß–/– cells, which suggests that integrin recycling may be impaired in the PPARß–/– keratinocytes (see below).
PPARß–/– cells show reduced activation of the Rho GTPases and of downstream effectors. The small GTPases of the Rho family are effectors of the PI3K/PIP3 pathway. Among them, Rac1 and cdc42, but not RhoA, were shown to play important roles in directional sensing and migration by driving EGF-induced chemotaxis (17, 29). They activate downstream effectors such as the PAKs, which in turn activate LIM kinases (LIMKs) (21). LIMKs inhibit cofilin, a member of the actin depolymerization factor family (3, 18) (see Fig. 9). Both LIMKs and cofilin are important in processes requiring fast actin reorganization. In the vehicle-treated primary keratinocytes, no difference was seen in the levels of active Rac1 and cdc42 (Fig. 1B, left). wt and PPARß–/– keratinocytes responded differently to EGF by a sustained and transient activation of two GTPases, Rac1 and cdc42, respectively (Fig. 1B, right). Rescue experiments using ectopic expression of PPARß in the PPARß-null keratinocytes efficiently restored the activation of Rac1 and cdc42 (Fig. 1D, bottom). Compared to the PPARß–/– cells, the PPARß–/– keratinocytes expressing ectopic PPARß exhibited a twofold-higher basal Rac1 activity than did the nontransfected cells. When stimulated with EGF, the PPARß-expressing cells showed a significantly higher increase in Rac1 and cdc42 activity. The level of phosphorylation of PAKs and LIMKs was examined in PPARß–/– epidermis, as well as in primary keratinocytes treated with either vehicle or EGF. In both cases, PPARß deficiency led to a reduced phosphorylation of PAK1 (Thr423 and Ser144) and reduced expression and phosphorylation of PAK2 (Ser141 and Ser20) (Fig. 1C). Reduced phosphorylation and hence activity of PAK1 and PAK2 in PPARß–/– cells were translated into a reduced phosphorylation of LIMK1 and LIMK2 (Fig. 1C). This should impact on the actin cytoskeleton plasticity via cofilin, a hypothesis which will be addressed later in this work.
In summary, the absence of PPARß causes reduced response to EGF, as exemplified by reduced PIP3 accumulation at the membrane and impaired formation of protrusions. Two target pathways of PI3K/PIP3, the Akt1 and the Rho GTPase pathways, as well as their respective downstream effectors GSK-3ß and PAKs/LIMKs, are affected in the PPARß–/– keratinocytes.
Ligand-activated PPARß potentiates the Rac1/cdc42 pathway. Since the absence of PPARß reduces Rac1 and cdc42 activities, activation of PPARß with a selective ligand (L165041) should increase EGF-dependent Rho GTPase activities. Upon exposure to EGF, wt keratinocytes showed a sustained activation of Rac1 and cdc42 (Fig. 2A). The combined treatment with EGF and the PPARß ligand led to a faster Rac1 and cdc42 activation (Fig. 2A). Consistent with the role of PI3K in the amplification of the internal gradient required to establish cell polarity, this stimulation was neutralized by the PI3K inhibitors LY294002 and wortmannin (Fig. 2B).
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Delayed formation of protrusions correlates with decreased actin nucleation and elongation activity in the PPARß–/– keratinocytes. The above conclusions led us to further analyze cell polarization, the second event in chemotaxis (9). In this, active Rac1 and cdc42 are recruited to the pseudopodium at the leading edge of the cell, where they stimulate PAKs, which in turn leads to the stabilization and growth of the pseudopodium formed de novo. We examined whether PPARß modulates the formation of this structure using pseudopodium growth through a porous membrane in response to EGF.
wt keratinocytes exposed to EGF from below the membrane extended pseudopodia through the pores of the membrane (Fig. 3A). These pseudopodia were detected as early as 30 min after exposure to EGF, and they were still growing 3 hours later. In contrast, in PPARß–/– cells, the formation of pseudopodia could be detected only after 3 h of incubation (Fig. 3A).
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2-fold lower than that in wt cells (Fig. 3B). Interestingly, only the overexpression of both constitutively active Rac1 (Rac1G12V) and cdc42 (cdc42G12V) in the PPARß–/– keratinocytes resulted in significant pseudopodium extension (Fig. 3C). These results revealed that Rac1 and cdc42 fulfill nonredundant complementary functions in pseudopodium growth and that both functions are altered in PPARß–/– keratinocytes. Growth of a pseudopodium requires the recruitment of Rac1/cdc42 effectors involved in the nucleation and the elongation of actin filaments such as the Wiskott-Aldrich syndrome protein (N-WASP) and WASP family verprolin-homologous proteins (WAVEs) and the downstream Arp2/3 complex (17) (see Fig. 9). While the amounts of N-WASP and Arp2 proteins were similar in both the cell body and pseudopodium fractions from either wt or PPARß–/– keratinocytes, coimmunoprecipitation indicated an increase in the effector complex N-WASP/Arp2 in the wt compared with PPARß–/– pseudopodia (Fig. 3B). Similarly, more WAVE2 was recruited to the extending pseudopodia of PPARß+/+ than to those of PPARß–/– cells (Fig. 3B).
These results show that the reduced/delayed activation of Rac1/cdc42 in the PPARß–/– keratinocytes is associated with reduced N-WASP/Arp2 interaction and WAVE recruitment. These alterations are consistent with the altered actin cytoskeleton dynamic and reduced formation of pseudopodia.
The organization of actin stress fibers and subsequent migration are impaired in PPARß–/– keratinocytes. Following activation of the cascade of events detailed above, cell movement depends on the coordinated rearrangement of actin filaments (6). All our data converge towards the hypothesis that actin cytoskeleton organization is impaired in PPARß–/– keratinocytes. Therefore, the organization of actin filaments and the migration of primary keratinocytes were further studied. In vitro wounds were created by scraping primary keratinocyte monolayers. Striking differences were observed between the PPARß wt and null keratinocytes in the organization of the cell monolayer and of the actin cytoskeleton. The percentage of keratinocytes that had detached from the wound edge 3 h postscraping in order to colonize the empty space was significantly lower in the PPARß–/– than the PPARß+/+ primary cultures (20.6% versus 43.8% of the edge keratinocytes, respectively). At 3 and 16 h after scraping, the edge formed by the PPARß–/– cells remained mostly blunt, with close contacts between cells (Fig. 4, bottom). Moreover, the actin cytoskeleton was cortical and failed to organize into fibers or lamellipodia directed towards the empty space (Fig. 4, bottom, arrowheads). In contrast, the faster-migrating PPARß wt keratinocytes lost contacts with neighboring cells, showed well-organized stress fibers, and formed lamellipodia as early as 3 h after scraping (Fig. 4, top, white arrows). Consistently, the in vitro kinetic of healing of the scraping wounds in PPARß–/– keratinocyte cultures was delayed compared to that of PPARß+/+ cultures (70% versus 15% of closure of the empty space by the wt and null keratinocytes, respectively, 16 h after scraping; see also reference 26).
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These data indicated that the sensing and migration defects resulting from the absence of PPARß, described using cell cultures and skin explants, also occur in vivo. Especially striking is the formation of two migratory fronts at the wound edge in the early phase of healing in the PPARß–/– animals.
Protein recruitment to the integrin migratory complex is impaired in vivo at the wound edge.
Using the same model, we also addressed the consequences of altered signaling in PPARß–/– keratinocytes by studying the localization of 3 integrin and of the actin binding protein ILK (integrin-linked kinase). The integrin receptor 3ß1 was shown to be an important actor in keratinocyte migration (28). As shown above (Fig. 1C), the phosphorylation levels of GSK-3ß and KLC2 suggested that integrin recycling might be impaired in the PPARß–/– keratinocytes. Upon activation, 3ß1 recruits actin stress fibers through adaptor proteins, among which ILK, a PPARß direct target product (11), is a major player (see Fig. 9). A similar intensity of the labeling suggested that the expression level of ILK was equivalent in the unwounded epidermis of PPARß–/– and PPARß+/+ animals (Fig. 7A, a' and b'), whereas it was reduced in the migratory part of the regenerating epidermis 4 days after the wounding of the PPARß–/– compared with the PPARß+/+ animals (Fig. 7A, a'' and b''). The level of expression (data not shown) and the localization (Fig. 7B, Hs) of the 3 integrin subunit were similar in the PPARß+/+ and PPARß–/– healthy skin. As expected for PPARß+/+ skin sections, 3 integrin staining became restricted to the basal plasma membrane of basal keratinocytes located near the initial wound edge and in the migratory layer (Fig. 7B, arrows in panels a' and a''). However, the 3 integrin staining was weaker and diffuse and was seen all around the plasma membrane of the basal PPARß–/– keratinocytes (Fig. 7B, arrows in panels b' and b''). This is consistent with the impaired localization of the 3 integrin subunit at the leading edge of PPARß–/– keratinocytes migrating out of skin explant cultures (data not shown).
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3ß1 integrin and ILK appears to show alterations in the PPARß–/– animals. 3D reconstruction of PPARß+/+ and PPARß–/– in vivo wounds. In order to strengthen the above in vivo observations, we decided to obtain a 3D view of the injured region through imaging and 3D reconstruction. Pictures of serial sections encompassing complete wounds of PPARß+/+ and PPARß–/– mice (n = 5) at day 4 after wounding were piled up to reconstruct 3D views of wound edges (Fig. 8). These reconstructions revealed striking differences between PPARß+/+ and PPARß–/– wound edges. The PPARß+/+ epidermal migratory layer was thin as it progressed over the wound bed (Fig. 8a and b; black arrowhead and brown area). No significant receding under the healthy tissue was observed (Fig. 8a and b). In contrast, the PPARß–/– migratory epidermis showed high variability in its morphology, displaying a receding layer (Fig. 8c and d, asterisk) and thickening of the migratory epidermis (Fig. 8c and d, brown area). These data showed that the impaired migration of the hyperproliferative/migrating epidermis is not only a local random alteration but is also distributed over the entire healing wound edge in the PPARß–/– mice. Together, these results indicated that the migration defect significantly contributes to the delayed healing of skin wounds observed in the PPARß mutant mice.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We have demonstrated previously that the healing of a skin wound is slower in PPARß-null mice (26). We further showed that this phenotype is partially due to increased apoptosis of the PPARß-null keratinocytes (26). Herein we demonstrate the involvement of PPARß in keratinocyte directional sensing, as well as in the regulation of two PI3K/PIP3 downstream pathways that relay sensing to cell movement, namely, the Rho-GTPase and the Akt pathways. We show that cell directional sensing and activation of these pathways are impaired in PPARß–/– keratinocytes. Consistently, we demonstrate that the plasticity of actin cytoskeleton is altered in the absence of PPARß. These defects contribute significantly to affected epithelialization and to delayed healing of skin wounds in the absence of PPARß in vivo.
The nuclear hormone receptor PPARß is an important modulator of cell directional sensing and movement. Nuclear hormone receptors are usually best characterized by their functions in embryogenesis, body growth, reproduction, and energy homeostasis. So far, only the estrogen receptor has been shown to be involved in the regulation of actin cytoskeleton plasticity and cell movement (35). The findings presented herein provide an important new insight into nuclear hormone receptor functions. We show that PPARß, otherwise involved in lipid catabolism, potentiates the establishment of the internal signal required for directional sensing towards a cue such as EGF, as well as the activity of Akt1/GSK-3ß and Rac1/cdc42 and their downstream effectors (Fig. 9). PPARß acts on several steps of the Akt1/GSK-3ß and Rac1/cdc42 pathways (Fig. 9). We previously demonstrated that PPARß decreases the expression of PTEN while directly increasing the expression of PDK1 (11). We also showed that, by downregulating the expression of PTEN, PPARß potentiates the production of PIP3 (11). Therefore, the coordinated action of PPARß on PTEN/PIP3 production and PDK1 expression indirectly results in increased Akt1, PAK, and Rac1/cdc42 activity and in increased directional sensing efficiency, as well as enhancement of the EGF signaling (3, 20). Finally, we showed that PPARß directly activates the expression of ILK (11), thereby potentiating integrin receptor-dependent signaling. These defects have significant consequences for actin cytoskeleton reorganization and integrin recycling (see below), with consequences for cell movement in cell culture and, importantly, also in vivo.
Because impaired PI3K signaling in PPARß-null keratinocytes is due to increased expression of PTEN, with consequently reduced PIP3 levels (11), this defect concerns all signals that act on the PI3K pathway upstream of PTEN and, therefore, is not specific to the EGF response. In line with this, we have shown that a similar defect has also been observed in response to insulin-like growth factor (data not shown) and that cultured primary keratinocytes show increased PTEN expression and a reduced amount of PIP3 in the absence of any challenge (11). Therefore, PPARß-null keratinocytes have impaired basal PI3K signaling, a defect which is exacerbated in challenging situations, such as the response to a chemotactic signal, and further influences cell migration efficiency.
PPARß modulates cell polarization and migration. The downstream consequence of cell directional sensing at the leading edge of a wound is the recruitment of specialized proteins required for actin filament plasticity and pseudopodium projection. This includes the Rho-GTPase effectors WAVE-WASP-Arp2/3 complexes, involved in de novo actin filament nucleation and elongation, and PAKs/LIMK/cofilin, required for fast actin reorganization (17, 18). PPARß–/– keratinocytes showed reduced active Rac1/cdc42, which translated to less-active Arp2/3 and LIMK. We demonstrated that the decreased activity of these proteins led to the delayed polarization and formation of pseudopodia in PPARß–/– primary keratinocytes.
PPARß also plays a role in the localization of integrin receptors at the cell membrane, which is crucial for cell adhesion and migration and for relaying signals between the extracellular matrix and the actin cytoskeleton (41). As discussed above, the absence of PPARß results in lower activity of Akt1 and consequently in increased activity of GSK-3ß. Consistent with the role of GSK-3ß in inhibiting integrin recycling via phosphorylation of KLC (27, 31), the localization of the 3 integrin subunit is impaired in PPARß–/– cells. Importantly, the PPARß direct target gene product ILK is part of the complex implicated in integrin interaction with actin (4, 11), and it was recently shown to be required for epidermal morphogenesis, keratinocyte adhesion, and directional migration (23). ILK expression is decreased in PPARß–/– keratinocytes, which most probably affects integrin-actin interactions. It is interesting that the active Rac1/cdc42 profile observed in PPARß–/– keratinocytes is similar to that reported for keratinocytes with impaired 6ß4 integrin expression (32). Recently, we have shown that PPARß-stimulated Akt1 signaling resulted in both a profound redistribution of integrins in human proximal tubular epithelial cells (HK-2) and protection against apoptosis during ischemic acute renal failure (22). Labeling of primary or explant keratinocytes also showed that in PPARß–/– cells, actin tends to locate at the periphery of the cells, suggesting that the actin filaments remain associated with cell-cell junctions rather than being reorganized in order to allow cell movement. Thus, the impaired migration of PPARß–/– keratinocytes is most likely the consequence of altered actin cytoskeleton dynamics, decreased ILK activity, and disturbed integrin localization and functions.
The absence of PPARß has severe consequences for the epithelialization stage of skin wound healing.
PPARß modulates keratinocyte directional sensing, as well as migration via actin cytoskeleton plasticity and integrin function. Decreased expression of ILK and impaired localization of the 3 integrin subunit were observed in vivo, which strongly suggests that these pathways are affected in vivo in the absence of PPARß. The consequence of impaired chemotaxis and migration in vivo is the formation of two migration fronts in skin wounds of PPARß–/– mice. One of them recedes towards the unwounded tissue, and the other, although correctly oriented towards the wound bed, is twice as short as that in wt animals, with stacking of keratinocytes in a thicker migratory tongue. As in the PPARß-null mice, impaired epithelialization usually leads to delayed closure of the wounded area. However, migration of keratinocytes away from the wound bed is a peculiar phenotype that has been reported only once so far, during the healing process of skin wounds in the fibrinogen-null mouse (13). In the PPARß-null mice, this phenotype was particularly striking when comparing 3D reconstructions of whole-skin wounds from PPARß wt and PPARß–/– animals. Importantly, receding epithelial tongues were not seen in Smad3–/– animals, and the slower migration of PPARß keratinocytes was still dramatic in skin explants after inhibition of proliferation. This indicates that the phenotype observed in the PPARß–/– wounds is not primarily due to the increased proliferation of the wound epidermis that was observed previously (37) but to the sensing and migration defect in the PPARß–/– cells.
Like most of the other nuclear hormone receptors, PPARß is activated through binding to an agonist. We previously showed that treatment of primary keratinocytes with inflammatory cytokines not only induced the upregulation of PPARß expression but also stimulated the production of an endogenous ligand, unidentified so far (37). In vivo, the injury-triggered release of inflammatory cytokines reactivated the expression of PPARß (26) and probably generated the production of the physiological agonists necessary for PPARß activation in the keratinocytes at the wound edges. Therefore, the activity of PPARß results from both its increased expression and the production of ligands. Although the nature of such ligands remains to be identified, it is tempting to hypothesize that COX2 derivatives may be involved in PPARß activation. COX2 expression is known to be increased in inflammatory conditions, and we demonstrated that it generates PPARß agonists during hair follicle maturation in mouse pups (10). However, this does not rule out the involvement of other pathways, and further work is needed to isolate the physiological agonists that trigger PPARß activity in the epidermis.
Our results do not exclude the possibility of other confounding defects in keratinocyte migration or in the production of chemoattractants per se. In addition, the above-mentioned defect in the control of keratinocyte proliferation, the underlying mechanism of which is presently under investigation, certainly participates in the delayed reepithelialization observed in PPARß–/– animals (26). The main finding of this work is a newly reported function of PPARß, a member of the nuclear hormone receptor family, in modulating keratinocyte chemotactic response and migration. PPARß acts via transcriptional up- and downregulation of gene expression (ILK, PDK1, and PTEN) and consequently via the activation of the PI3K/PIP3 pathway and of two of its downstream routes that involve Akt1/GSK-3ß, Rac1/cdc42 GTPases, and PAKs. Thus, PPARß actively participates in the reorganization of the actin cytoskeleton of keratinocytes at a wound edge and is directly involved in the early response of these cells to injury-induced stimuli. The sensing defect due to the absence of PPARß in vivo causes an unusual epithelialization impairment in the early phase of healing, with the formation of a second migratory front moving away from the wound bed. The role of PPARß as a regulator of chemotactic response is probably more widespread, since PPARß is ubiquitously expressed (5), as are the other players of cell migration studied herein. In addition, the question remains open as to whether other nuclear hormone receptors may also fulfill similar functions. Looked at from this viewpoint, the findings presented here may be far-reaching, considering that cell adhesion and movement are fundamental processes involved in a wide range of both physiological and pathological cellular processes, such as morphogenesis in embryonic development, fibrosis, metastasis of tumor cells, and atherosclerosis (1, 38, 39).
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ACKNOWLEDGMENTS |
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This work was supported by grants from the Swiss National Science Foundation to W.W., from the Etat de Vaud, and from the Fondation pour la Recherche Médicale to A.M.
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FOOTNOTES |
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Published ahead of print on 6 August 2007.
Supplemental material for this article may be found at http://mcb.asm.org/.
Present address: School of Biological Sciences, College of Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 673551, Singapore.
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B (
S, as negative and positive controls, respectively. The ratio of active GTP-bound Rac1 and cdc42 to the total Rac1 and cdc42 protein was quantified in the same protein lysates. Numbers below the Western blots represent the changes in active Rac1 and cdc42 relative to basal active Rac1 and cdc42 from at least six independent experiments. The data shown in panels A and B were obtained in the same experiment; therefore, all the panels can be compared with each other. DMSO, dimethyl sulfoxide. (C) Western blot analysis of total and phosphorylated PAK1, PAK2, and LIMK of wt keratinocyte extracts, treated as described for panel A. Protein expression in vehicle (PBS)-treated cells was normalized to that of ß-tubulin and assigned a value of 1. Numbers represent changes (n-fold) in normalized protein expression relative to basal level in vehicle (PBS)-treated cells from six independent experiments. The multiple bands detected for the phospho-PAKs correspond to various phosphorylated forms. p(Thr508)-LIMK1 and p(Thr505)-LIMK2 cannot be distinguished by the antibody and appear as a single band. The standard deviations of the changes (n-fold) given in the panels were below 8.6% (highest value observed).
