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. 2001 Aug 20;154(4):799-814.
doi: 10.1083/jcb.200011148.

Impaired skin wound healing in peroxisome proliferator-activated receptor (PPAR)alpha and PPARbeta mutant mice

L Michalik  1 , B Desvergne, N S Tan, S Basu-Modak, P Escher, J Rieusset, J M Peters, G Kaya, F J Gonzalez, J Zakany, D Metzger, P Chambon, D Duboule, W Wahli
Affiliations

Impaired skin wound healing in peroxisome proliferator-activated receptor (PPAR)alpha and PPARbeta mutant mice

L Michalik et al. J Cell Biol. .

Abstract

We show here that the alpha, beta, and gamma isotypes of peroxisome proliferator-activated receptor (PPAR) are expressed in the mouse epidermis during fetal development and that they disappear progressively from the interfollicular epithelium after birth. Interestingly, PPARalpha and beta expression is reactivated in the adult epidermis after various stimuli, resulting in keratinocyte proliferation and differentiation such as tetradecanoylphorbol acetate topical application, hair plucking, or skin wound healing. Using PPARalpha, beta, and gamma mutant mice, we demonstrate that PPARalpha and beta are important for the rapid epithelialization of a skin wound and that each of them plays a specific role in this process. PPARalpha is mainly involved in the early inflammation phase of the healing, whereas PPARbeta is implicated in the control of keratinocyte proliferation. In addition and very interestingly, PPARbeta mutant primary keratinocytes show impaired adhesion and migration properties. Thus, the findings presented here reveal unpredicted roles for PPARalpha and beta in adult mouse epidermal repair.

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Figures

Figure 1.
Figure 1.
Differential expression of the PPARs in mouse epidermis. (A) RNase protection analysis of PPAR mRNA during mouse skin development. Total lysates of skin from day 15.5 to 18.5 embryos (E15.5–E18.5), newborn (NB), 5- and 9-d-old (+5, +9), and adult mice were hybridized with radiolabeled probes specific for PPARα, PPARβ, PPARγ, and L27 mRNA (a). The amount of mRNA for each PPAR isotype was quantified based on the L27 mRNA amount and on the specific activity of each probe (b; n = 3–6). (B) In situ hybridization analysis of PPAR expression during fetal development. Cryosections of mouse skin from embryonic day 13.5–18.5 (E13.5–18.5) were hematoxilin and eosin stained (HE) or hybridized with specific antisense digoxygenin–labeled riboprobes (PPARα, β, or γ). (C) In situ hybridization analysis of PPAR expression during post natal growth. Cryosections of mouse skin from newborn (NB), 5- or 9-d-old pups (+5, +9) and adult animals were hematoxilin/eosin stained (HE) or hybridized with specific antisense digoxygenin-labeled riboprobes (PPARα, β, or γ). Arrows indicate the epidermis/dermis interface. For both B and C, a similar pattern of expression was observed for each time point in five different mice from independent litters. Bars, 80 μm.
Figure 1.
Figure 1.
Differential expression of the PPARs in mouse epidermis. (A) RNase protection analysis of PPAR mRNA during mouse skin development. Total lysates of skin from day 15.5 to 18.5 embryos (E15.5–E18.5), newborn (NB), 5- and 9-d-old (+5, +9), and adult mice were hybridized with radiolabeled probes specific for PPARα, PPARβ, PPARγ, and L27 mRNA (a). The amount of mRNA for each PPAR isotype was quantified based on the L27 mRNA amount and on the specific activity of each probe (b; n = 3–6). (B) In situ hybridization analysis of PPAR expression during fetal development. Cryosections of mouse skin from embryonic day 13.5–18.5 (E13.5–18.5) were hematoxilin and eosin stained (HE) or hybridized with specific antisense digoxygenin–labeled riboprobes (PPARα, β, or γ). (C) In situ hybridization analysis of PPAR expression during post natal growth. Cryosections of mouse skin from newborn (NB), 5- or 9-d-old pups (+5, +9) and adult animals were hematoxilin/eosin stained (HE) or hybridized with specific antisense digoxygenin-labeled riboprobes (PPARα, β, or γ). Arrows indicate the epidermis/dermis interface. For both B and C, a similar pattern of expression was observed for each time point in five different mice from independent litters. Bars, 80 μm.
Figure 1.
Figure 1.
Differential expression of the PPARs in mouse epidermis. (A) RNase protection analysis of PPAR mRNA during mouse skin development. Total lysates of skin from day 15.5 to 18.5 embryos (E15.5–E18.5), newborn (NB), 5- and 9-d-old (+5, +9), and adult mice were hybridized with radiolabeled probes specific for PPARα, PPARβ, PPARγ, and L27 mRNA (a). The amount of mRNA for each PPAR isotype was quantified based on the L27 mRNA amount and on the specific activity of each probe (b; n = 3–6). (B) In situ hybridization analysis of PPAR expression during fetal development. Cryosections of mouse skin from embryonic day 13.5–18.5 (E13.5–18.5) were hematoxilin and eosin stained (HE) or hybridized with specific antisense digoxygenin–labeled riboprobes (PPARα, β, or γ). (C) In situ hybridization analysis of PPAR expression during post natal growth. Cryosections of mouse skin from newborn (NB), 5- or 9-d-old pups (+5, +9) and adult animals were hematoxilin/eosin stained (HE) or hybridized with specific antisense digoxygenin-labeled riboprobes (PPARα, β, or γ). Arrows indicate the epidermis/dermis interface. For both B and C, a similar pattern of expression was observed for each time point in five different mice from independent litters. Bars, 80 μm.
Figure 1.
Figure 1.
Differential expression of the PPARs in mouse epidermis. (A) RNase protection analysis of PPAR mRNA during mouse skin development. Total lysates of skin from day 15.5 to 18.5 embryos (E15.5–E18.5), newborn (NB), 5- and 9-d-old (+5, +9), and adult mice were hybridized with radiolabeled probes specific for PPARα, PPARβ, PPARγ, and L27 mRNA (a). The amount of mRNA for each PPAR isotype was quantified based on the L27 mRNA amount and on the specific activity of each probe (b; n = 3–6). (B) In situ hybridization analysis of PPAR expression during fetal development. Cryosections of mouse skin from embryonic day 13.5–18.5 (E13.5–18.5) were hematoxilin and eosin stained (HE) or hybridized with specific antisense digoxygenin–labeled riboprobes (PPARα, β, or γ). (C) In situ hybridization analysis of PPAR expression during post natal growth. Cryosections of mouse skin from newborn (NB), 5- or 9-d-old pups (+5, +9) and adult animals were hematoxilin/eosin stained (HE) or hybridized with specific antisense digoxygenin-labeled riboprobes (PPARα, β, or γ). Arrows indicate the epidermis/dermis interface. For both B and C, a similar pattern of expression was observed for each time point in five different mice from independent litters. Bars, 80 μm.
Figure 2.
Figure 2.
PPARβ expression is upregulated in SV129 adult mouse epidermis upon keratinocyte proliferation stimulation. TPA topical application. Vehicle- (a–f) or TPA-treated (g–l) dorsal skin. Hematoxilin/eosin (HE) staining (a and g); Keratin 6 (b and h) and Ki67 (c and i) immunolabeling; in situ hybridization with PPARα (d and j), PPARβ (e and k), and PPARγ (f and l) antisense probes (ASense); in situ hybridization of TPA-treated samples with sense control probes are shown (m–o). (B) Hair plucking. Unplucked (a–f) or plucked (g–l) dorsal skin. Hematoxilin/eosin (HE) staining (a and g); Keratin 6 (b and h) and Ki67 (c and i) immunolabeling; in situ hybridization with PPARα (d and j), PPARβ (e and k), and PPARγ (f and l) antisense probes (ASense); in situ hybridization of plucked samples with sense control probes are shown (m–o). Arrows indicate the epidermis/dermis interface. For both A and B, similar results were observed in six SV129 mice from independent litters. Bars, 80 μm.
Figure 2.
Figure 2.
PPARβ expression is upregulated in SV129 adult mouse epidermis upon keratinocyte proliferation stimulation. TPA topical application. Vehicle- (a–f) or TPA-treated (g–l) dorsal skin. Hematoxilin/eosin (HE) staining (a and g); Keratin 6 (b and h) and Ki67 (c and i) immunolabeling; in situ hybridization with PPARα (d and j), PPARβ (e and k), and PPARγ (f and l) antisense probes (ASense); in situ hybridization of TPA-treated samples with sense control probes are shown (m–o). (B) Hair plucking. Unplucked (a–f) or plucked (g–l) dorsal skin. Hematoxilin/eosin (HE) staining (a and g); Keratin 6 (b and h) and Ki67 (c and i) immunolabeling; in situ hybridization with PPARα (d and j), PPARβ (e and k), and PPARγ (f and l) antisense probes (ASense); in situ hybridization of plucked samples with sense control probes are shown (m–o). Arrows indicate the epidermis/dermis interface. For both A and B, similar results were observed in six SV129 mice from independent litters. Bars, 80 μm.
Figure 3.
Figure 3.
Enhanced keratinocyte proliferative response in PPARβ1/− upon stimulation. (A) TPA topical application. (a–f) PPARβ+/+ vehicle (a–c) or TPA-treated (d–f) dorsal epidermis, hematoxilin/eosin staining (HE) (a and d), and after keratin 6 (b and e) or Ki67 (c and f) immunostaining. (g–l) PPARβ+/− vehicle– (g–i) or TPA-treated (j–l) dorsal epidermis, hematoxilin/eosin staining (HE) (g and j), and after keratin 6 (h and k) or Ki67 (i and l) immunostaining. (B) Hair plucking. (a–f) PPARβ+/+ unplucked (a–c) or plucked (d–f) dorsal epidermis, hematoxilin/eosin staining (HE) (a and d), and after keratin 6 (b and e) or Ki67 (c and f) immunostaining. (g–l) PPARβ+/− unplucked (g–i) or plucked (j–l) dorsal epidermis, hematoxilin/eosin staining (HE) (g and j), and after keratin 6 (h and k) or Ki67 (i and l) immunostaining. Arrows indicate the epidermis/dermis interface. Bars, 80 μm.
Figure 3.
Figure 3.
Enhanced keratinocyte proliferative response in PPARβ1/− upon stimulation. (A) TPA topical application. (a–f) PPARβ+/+ vehicle (a–c) or TPA-treated (d–f) dorsal epidermis, hematoxilin/eosin staining (HE) (a and d), and after keratin 6 (b and e) or Ki67 (c and f) immunostaining. (g–l) PPARβ+/− vehicle– (g–i) or TPA-treated (j–l) dorsal epidermis, hematoxilin/eosin staining (HE) (g and j), and after keratin 6 (h and k) or Ki67 (i and l) immunostaining. (B) Hair plucking. (a–f) PPARβ+/+ unplucked (a–c) or plucked (d–f) dorsal epidermis, hematoxilin/eosin staining (HE) (a and d), and after keratin 6 (b and e) or Ki67 (c and f) immunostaining. (g–l) PPARβ+/− unplucked (g–i) or plucked (j–l) dorsal epidermis, hematoxilin/eosin staining (HE) (g and j), and after keratin 6 (h and k) or Ki67 (i and l) immunostaining. Arrows indicate the epidermis/dermis interface. Bars, 80 μm.
Figure 4.
Figure 4.
Normal keratinocyte-terminal differentiation in PPARβ1/− skin. (a–f) PPARβ+/+ fetal (E18.5) (a–c) or adult (d–f) dorsal epidermis, hematoxilin/eosin staining (HE) (a and d), and after involucrin (b and e) or loricrin (c and f) immunostaining. (g–l) PPARβ+/− fetal (E18.5) (g–i) or adult (j–l) dorsal epidermis, hematoxilin/eosin staining (HE) (g and j), and after involucrin (h and k) or loricrin (i and l) immunostaining. Arrows indicate the epidermis/dermis interface. Bars, 40 μm.
Figure 5.
Figure 5.
Differential expression of PPAR in adult mouse epidermis during cutaneous wound closure. Cryosections of mouse skin from day 1 to 10 (+1 to +10) after the excision of a full thickness dorsal skin biopsy were hematoxilin/eosin stained (HE) or hybridized with specific antisense digoxygenin-labeled riboprobes (PPARα, β, or γ). Arrows indicate the epidermis/dermis interface. A similar pattern of expression was observed for each time point in six different mice from independent litters. Bar, 80 μm.
Figure 6.
Figure 6.
PPARα and β mutant mice are unable to sustain normal wound healing. (A–C) After excision of a full thickness skin biopsy, the surfaces of the healing wounds were measured over time on wild-type (+/+), heterozygous (+/−), or null (−/−) mice: PPARγ (A), PPARα (B), and PPARβ (C). The surfaces are plotted as a percentage of the surface of the wound at day zero (± SEM, n = 8–10). Asterisks indicate that the difference is statistically significant (asterisk, P < 0.05; double asterisk, P < 0.01). Arrows indicate the mean time for complete healing of the wild-type control mice (black lozenge) or transgenic mice (grey square).
Figure 6.
Figure 6.
PPARα and β mutant mice are unable to sustain normal wound healing. (A–C) After excision of a full thickness skin biopsy, the surfaces of the healing wounds were measured over time on wild-type (+/+), heterozygous (+/−), or null (−/−) mice: PPARγ (A), PPARα (B), and PPARβ (C). The surfaces are plotted as a percentage of the surface of the wound at day zero (± SEM, n = 8–10). Asterisks indicate that the difference is statistically significant (asterisk, P < 0.05; double asterisk, P < 0.01). Arrows indicate the mean time for complete healing of the wild-type control mice (black lozenge) or transgenic mice (grey square).
Figure 6.
Figure 6.
PPARα and β mutant mice are unable to sustain normal wound healing. (A–C) After excision of a full thickness skin biopsy, the surfaces of the healing wounds were measured over time on wild-type (+/+), heterozygous (+/−), or null (−/−) mice: PPARγ (A), PPARα (B), and PPARβ (C). The surfaces are plotted as a percentage of the surface of the wound at day zero (± SEM, n = 8–10). Asterisks indicate that the difference is statistically significant (asterisk, P < 0.05; double asterisk, P < 0.01). Arrows indicate the mean time for complete healing of the wild-type control mice (black lozenge) or transgenic mice (grey square).
Figure 7.
Figure 7.
PPAR α and β expression and respective mice phenotypes compared with the major phases of skin wound healing. (A) Summary of time sequence of the major overlapping phases of skin wound healing. (B) Plain lines indicate the expression of PPARα and β in the healing epidermis; dotted lines indicate the duration of the observed phenotype on PPAR mutant mice.
Figure 8.
Figure 8.
Elastin and collagen deposit is not altered in the dermis of PPARβ1/− mice. PPARβ+/+ (a–d) or PPARβ+/− (e–h) dorsal epidermis, elastin (a and e), or collagen (b–d and f–h) staining. (a, b, e, and f) Unchallenged adult dorsal skin. (c, d, g, and h) Mouse skin at day 10 and 20 (+10 and +20) after the excision of a full thickness dorsal skin biopsy. Arrows indicate the epidermis/dermis interface. Bar, 80 μm.
Figure 9.
Figure 9.
PPARβ1/− primary keratinocytes show impaired adhesion and migration properties. Primary keratinocytes isolated from skin of PPARβ+/+ (a–d) or PPARβ+/− (e–h) newborn pups. (a) Wild-type keratinocytes 24 h after plating; (e) PPARβ+/− keratinocytes 3 d after plating. (b–d and f–h) Wounded cultures of primary keratinocytes: scrape wounds were made at day 0 (day 0 corresponds to the obtention of 70–80% confluent cell culture) (b and f). Panels c and g and d and h represent the wounds at day 2 and 4 after scraping, respectively. Bar: (a, b, e, f, and insets) 100 μm; (c, d, g, and h) 200 μm.
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