The nuclear hormone receptor peroxisome proliferator-activated receptor beta/delta potentiates cell chemotactism, polarization, and migration

Abstract

After an injury, keratinocytes acquire the plasticity necessary for the reepithelialization of the wound. Here, we identify a novel pathway by which a nuclear hormone receptor, until now better known for its metabolic functions, potentiates cell migration. We show that peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) enhances two phosphatidylinositol 3-kinase-dependent pathways, namely, the Akt and the Rho-GTPase pathways. This PPARbeta/delta 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 PPARbeta/delta-/- 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.

FIG. 1.

PPARβ-deficient keratinocytes show impaired Rac1/cdc42-mediated translocation of PH-Akt-GFP to the plasma membrane. (A) PH-Akt-GFP-expressing wt or PPARβ^−/− keratinocytes were directionally stimulated with EGF. White and blue arrows show recruitment of PH-Akt-GFP to the cell membrane at the leading edge and the retraction of the trailing end, respectively; the asterisks show the point sources of EGF; 96% of transfected PPARβ^−/− keratinocytes showed delayed recruitment of PH-Akt-GFP to the plasma membrane. Bars, 20 μm. (B) Western blot analysis of active Rac1, cdc42, and Akt1 in wt and PPARβ^−/− keratinocytes exposed for the indicated time periods (min) with EGF or PBS alone. Numbers below the Western blots represent the changes in active relative to basal Rac1, cdc42, and Akt1 levels from at least four independent experiments. (C) Western blot analysis of total and phosphorylated PAK1, PAK2, LIMK, GSK-3β, and KLC2 in wt and PPARβ^−/− epidermis (left panel; samples from two animals for each genotype are shown) or keratinocytes (right panel) treated for 20 min with either vehicle (V) or EGF (E). Protein levels were normalized to total PAK1, LIMK1, or GSK-3β, and the value 1 was assigned to wt epidermis or vehicle-treated wt keratinocytes. Numbers represent changes (n-fold) relative to corresponding controls from at least four 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. In panels B and C, the standard deviations of the changes (n-fold) registered were below 7.2% (highest value observed). (D) (Top) PPARβ^−/− or PPARβ^−/− keratinocytes expressing ectopic Myc-tagged wt PPARβ (PPARβ^−/−/Rescue) were transfected with PH-Akt-GFP cDNA and directionally stimulated with EGF. White arrows show recruitment of PH-Akt-GFP to the cell membrane at the leading edge; blue arrows show retraction of the trailing end; the asterisks show the point sources of EGF. Bars, 10 μm. (Bottom) Western blot analysis of active HA-tagged Rac1 and cdc42 in PPARβ^−/− keratinocytes and in PPARβ^−/− keratinocytes expressing ectopic Myc-tagged wt PPARβ (PPARβ^−/−/Rescue), exposed for the indicated time periods (min) to EGF. Numbers below the Western blots represent the changes relative to basal Rac1 and cdc42 levels in the PPARβ^−/− cells.

FIG. 2.

Ligand-activated PPARβ sustains the activation of Rac1 and cdc42 and of their downstream effectors, PAKs and LIMK. (A and B) Shown are results of Western blot analysis of total and active Rac1 and cdc42 after stimulation with either EGF (0.3 nM), PPARβ ligand L165041 (5 μM), or both (A), with or without EGF in the presence or absence of two PI3K inhibitors, LY294002 (50 μM) and wortmannin (100 μM) (B). Keratinocyte lysates were treated with either excess GDP or nonhydrolyzable GTP-γS, 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 β-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).

FIG. 3.

Pseudopodium extension by wt and PPARβ^−/− keratinocytes. (A) Concentrations of PPARβ^+/+ (left panel) and PPARβ^−/− (right panel) pseudopodium proteins on the underside of a porous membrane were examined for indicated times in the absence (NT) or presence of EGF in the bottom or top compartment or both compartments. Each point represents the mean ± standard error of the mean of six triplicate membranes obtained from three independent experiments. (B) Western blot analysis of wt (left panel) and PPARβ^−/− (right panel) cell body and pseudopodium protein (pseudo P) extracts isolated at indicated times after exposure to EGF in the bottom compartment. Numbers represent the changes in either active Rac1 and cdc42 or protein expression in the cell body and pseudopodial fractions, relative to basal (NT) Rac1 and cdc42 or protein level, respectively. Numbers under each profile represent the means of six triplicate membranes obtained from three independent experiments. (C) wt and PPARβ^−/− keratinocytes transfected with dominant-negative Rac1 (Rac1T17N) and cdc42 (cdc42T17N) and with constitutively active Rac1 (Rac1G12V) and cdc42 (cdc42G12V), respectively, were examined for pseudopodium formation toward an EGF gradient for 60 min as described for panel A. Each bar represents the mean ± standard error of the mean of three triplicate membranes from three independent experiments. An aliquot of cells used for the pseudopodium assay was also lysed and Western blotted for exogenous Rac and cdc42 expression. Myc-His-tagged mutant Rac1 and cdc42 showed reduced mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis relative to endogenous proteins. Standard deviations of the changes (n-fold) given in the panels were below 10.6% (highest observed value). O.D., optical density.

FIG. 4.

Impaired migration and actin organization in PPARβ^−/− primary keratinocytes after in vitro wounding of a cell monolayer. Shown is fluorescent labeling of the actin cytoskeleton of PPARβ wt (PPARβ^+/+) and PPARβ^−/− primary keratinocytes at the edge of an in vitro wound created by scraping of a cell monolayer 3 h and 16 h after scraping, respectively. A minimum of 450 cells were counted in three independent experiments. Nuclei were counterstained using DAPI. White arrows point to lamellipodia visible in the PPARβ wt keratinocytes; white arrowheads point to the cortical organization of the actin cytoskeleton in PPARβ^−/− keratinocytes. Bars, 50 μm.

FIG. 5.

Impaired keratinocyte outgrowth from PPARβ^−/− skin explants. (A) Representative pictures of keratinocyte outgrowth from PPARβ^+/+ (top) and PPARβ^−/− (bottom) skin explants after 2 or 6 days of culture, stained by colorimetric development with diaminobenzidine after immunohistochemistry with an anti-keratin 6 antibody. (B) Quantification of the outgrowth surface (mm²) of keratinocytes migrating from PPARβ wt (blue lines) and PPARβ^−/− (red lines) skin explants, in the absence (solid line; control) or the presence (dashed line) of mitomycin C to block proliferation.

FIG. 6.

Quantification of the thickness, area, and length of the hyperproliferative and migrating wounded epidermis in PPARβ^+/+ and PPARβ^−/− mice. (A) Keratin 6 staining of the hyperproliferative and migrating epidermis of PPARβ^+/+ (top) and PPARβ^−/− (bottom) in vivo wounded skin, at day 4 after the injury. The defects in epidermis migration were quantified based on the following parameters: the thickness and the area occupied by the hyperproliferative healing epidermis, the total length of the hyperproliferative epidermis (white triangles), the distance between the initial wound edge and the migratory front (black triangles), and the distance between the initial wound edge and the receding front towards the unwounded tissue (asterisks). HF, hair follicles. (B) Quantification of the thickness of the hyperproliferative epithelium in PPARβ^+/+ (blue bar) and PPARβ^−/− (brown bar) wounded epidermis, at day 4 after the injury. (C) Quantification of the total area of the hyperproliferative epithelium in PPARβ^+/+ (blue bar) and PPARβ^−/− (brown bar) wounded epidermis at day 4 after the injury. (D) Quantification of the length of the migrating epithelium in PPARβ^+/+ and PPARβ^−/− wounded epidermis, at day 4 after the injury. Black triangles, length of the epithelial tongue migrating towards the wound bed; white triangles, length of the total epithelial migrating tongue; asterisks, length of the epithelial tongue receding towards the unwounded tissue. Quantification was performed on three animals of each genotype, on a minimum of five sections per animal. Triangles and asterisks show the values obtained from individual animals; horizontal bars show the average values obtained for each genotype.

FIG. 7.

Reduced expression of ILK and impaired localization of α3 integrin in the keratinocytes of a skin wound in PPARβ^−/− mice. (A) ILK localization during wound healing. Immunolabeling of ILK was performed on wound biopsy specimens from PPARβ^+/+ (a to a″) and PPARβ^−/− animals (b to b″) at day 4 after the injury. Neg, negative controls without primary antibody. Higher magnifications are shown in boxes a′ to a″ (PPARβ^+/+ samples) and b′ to b″ (PPARβ^−/− samples). Black arrowheads indicate the initial wound edge at day 0. Bars, 100 μm (top) and 50 μm (bottom). Immunostaining was performed on three animals of each genotype. HF, hair follicles. (B) α3 integrin localization during wound healing. Immunolabeling of α3 integrin was performed on wound biopsy specimens from PPARβ^+/+ (a to a″) and PPARβ^−/− animals (b to b″) at day 4 after the injury. Higher magnifications are shown in boxes a′ to a" (PPARβ^+/+ samples) and b′ to b″ (PPARβ^−/− samples). Black arrowheads indicate the initial wound edge at day 0. Black arrows in boxes a′, a″, b′, and b″ point to a representative distribution of the α3 integrin subunit. Neg, negative controls without primary antibody; Hs, α3 integrin staining of healthy skin. Immunostaining was performed on three animals of each genotype.

FIG. 8.

3D reconstruction of PPARβ^+/+ and PPARβ^−/− wounded skin. (a and b) Two different rotation views of the same 3D reconstruction of the wound edge of a PPARβ^+/+ animal at day 4 after the injury. (c and d) Two different rotation views of the same 3D reconstruction of the wound edge of a PPARβ^−/− animal at day 4 after the injury. Black arrowheads indicate the initial wound edge at day 0. Blue, unwounded epidermis; brown, hyperproliferative/migrating epidermis. Dermis is not shown on the figure. Black arrows, direction of the migration towards the wound bed; asterisk, receding hyperproliferative epidermis. 3D reconstruction was performed on three animals of each genotype.

FIG. 9.

Proposed model of the role of PPARβ in keratinocyte directional sensing, polarization, and migration. The exposure of cells to growth factors triggers PI3K activity. Its lipid product PIP₃ accumulates to the plasma membrane at the leading edge of the keratinocytes and allows for a polarized recruitment and activation of PH-containing proteins such as Akt1 and PDK1. Active Rac1 and cdc42 participate in a positive feedback mechanism that amplifies the internal signal (PIP₃). Active Rac1 and cdc42 also activate the PAKs, which participate in the regulation of actin plasticity via LIMK and cofilin and the Arp2/3 complex via WAVEs/N-WASP, which enhances actin polymerization. The action of Rac1/cdc42 and PAK on the actin cytoskeleton is coordinated with the redistribution of integrins via various adaptor proteins, including ILK, an adaptor for integrin β1 and β3 subunits. The redistribution of integrins requires active Akt1 and the inhibition of GSK-3β. This pathway is augmented by PPARβ at several levels. PPARβ expression and activity stimulate the expression of ILK and of PDK1 and reduce the expression of PTEN. The downregulation of PTEN promotes the accumulation of PIP₃, which otherwise would be rapidly dephosphorylated to PIP₂. PDK1 phosphorylates threonine 423 of PAK1, maintaining its full catalytic activity towards its substrates, and participates in the activation of Akt1. Akt1 participates in the modulation of cell motility and also stimulates the production of MMP-9 via activation of NF-κB (11). This remarkable coordination of numerous integrated events is crucial for chemotaxis and migration.