First published online 12 May 2009
doi: 10.1242/jcs.047274
Journal of Cell Science 122, 1823-1833 (2009)
Published by The Company of Biologists 2009
Loss of dioxin-receptor expression accelerates wound healing in vivo by a mechanism involving TGFβ
Jose M. Carvajal-Gonzalez1,
Angel Carlos Roman1,
M. Isabel Cerezo-Guisado1,*,
Eva M. Rico-Leo1,
Gervasio Martin-Partido2 and
Pedro M. Fernandez-Salguero1,
1 Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Extremadura, Avenida de Elvas s/n, 06080-Badajoz, Spain
2 Departamento de Biologia Celular, Facultad de Ciencias, Universidad de Extremadura, Avenida de Elvas s/n, 06080-Badajoz, Spain
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Fig. 1. Wound healing is accelerated in Ahr–/– mice. Full-thickness 4 mm wounds were made in the dorsal skin of Ahr+/+ and Ahr–/– mice and healing followed for up to 7 days. Wounded tissue was dissected, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). The left panel shows representative H&E stained sections from Ahr+/+ and Ahr–/– mice. Arrows indicate the position of the epithelium at both sides of the wound. The right panel includes a quantification of the progression of the neo-epithelium (upper) or the granulation tissue (lower) in wounds of each genotype. Six wounds were performed in three mice of each genotype for each time point. Scale bars: 200 µm. Data are shown as mean ± s.e.m. The P values for statistical comparison between genotypes are indicated.
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Fig. 2. Ahr–/– wounds have increased re-epithelialization but similar proliferation rates. Wounds were made and processed in Ahr+/+ and Ahr–/– mice as indicated in the legend for Fig. 1. (A) Hematoxylin and eosin staining was performed and the length of the neo-epithelium measured and quantified. Left and right arrows indicate the wound site and its margin, respectively. (B) PCNA immunostaining was used to determine proliferation rates in the epithelial layer. Data were quantified using ImageJ software. The analysis was performed in at least eight wounds isolated from four Ahr+/+ and Ahr–/– mice. The neo-epithelial layer covering the area between the wound site and the margin of the regenerating tissue is marked by red dotted lines. Scale bars: 50 µm (A) and 100 µm (B). Data are shown as mean ± s.e.m. The P values for statistical comparison between genotypes are indicated.
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Fig. 3. Keratinocytes lacking AhR have increased migration in tissue explants and in primary culture. (A) Explants were obtained from Ahr+/+ and Ahr–/– mouse dorsal skin and placed in culture. Emigration of the keratinocytes from the explants was measured for up to 6 days and the results obtained plotted against time. Migration increased with time in both genotypes with a kinetic that could be adjusted to a linear equation (R2=0.9768 and R2=0.9915 for Ahr+/+ and Ahr–/–, respectively). The difference in slopes between Ahr+/+ and Ahr–/– explants was statistically significant at P=0.00214. At least five wounds from three different Ahr+/+ and Ahr–/– mice were used. (B) Primary keratinocyte cultures were obtained from Ahr+/+ and Ahr–/– newborn mice, plated on collagen- or fibronectin-coated plates and grown to confluence. Wounds of the same size were made and migration measured after 15 hours in serum-free medium. Data were quantified and are represented in the right panel. The experiments were performed in triplicate using primary cultures from four independent mice of each genotype. Scale bars: 50 µm (A and B). Data are shown as mean ± s.e.m. The P values for statistical comparison between genotypes are indicated. The equations for the linear regression of the data are shown in A.
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Fig. 4. Inflammatory response and fibroblast recruitment in Ahr+/+ and Ahr–/– wounds. Wounds were made and processed as indicated in the legend for Fig. 1A,B. Content of macrophages and neutrophils in the granulation tissue was determined by immunohistochemistry using F4/80 and Ly-6G antibodies, respectively. (C) Fibroblast recruitment was analyzed by immunohistochemistry after staining with vimentin. Fibroblast content in the wounds was also estimated by western immunoblotting using 15 µg total proteins and a vimentin specific antibody. The expression of β-actin was used as loading control. Three sections were analyzed from four wounds corresponding to four different animals of each genotype. Cell counting for each marker and mouse genotype was referred to the same tissue area. Data are shown as mean ± s.e.m. The P values for statistical comparison between genotypes are indicated.
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Fig. 6. Loss of AhR expression increases active TGFβ levels and TGFβ-dependent signaling. (A) Biopsies of basal skin and wound tissue were taken from three different Ahr+/+ and Ahr–/– mice. Aliquots of 15 µg total cell extracts were prepared and analyzed for TGFβ expression by western immunoblotting using a specific antibody. β-actin was used to normalize TGFβ expression as indicated on the right panel. (B) Primary dermal fibroblasts and primary keratinocytes were cultured from the skin of newborn Ahr+/+ and Ahr–/– mice. Each cell type was cultured for 72 hours and conditioned medium (CM-DF and CM-Ker) obtained for every genotype. CM-DF and CM-Ker from Ahr+/+ and Ahr–/– mice were used to quantify total and active TGFβ levels by ELISA. Measurements were done in triplicate and four primary cultures were prepared from different Ahr+/+ and Ahr–/– mice. (C) TGFβ signaling was analyzed in non-wounded skin and wounds from Ahr+/+ and Ahr–/– mice by quantifying the number of Smad2-P-positive cells (p-Smad2; arrowheads) with respect to tissue area. Sections were analyzed from four wounds of individual mice of each genotype. Scale bars: 100 µm. Data are shown as mean ± s.e.m. The P values for statistical comparison between genotypes are indicated.
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Fig. 7. TGFβ activity secreted by Ahr–/– dermal fibroblasts regulates keratinocyte migration. (A) The effect of self-secreted molecules on keratinocyte migration was determined in Ahr+/+ and Ahr–/– keratinocytes treated with conditioned medium from the same or the opposite genotype (CM-Ker). Wounds were performed and analyzed as indicated in the legend for Fig. 3B. (B) The paracrine effect of secreted molecules on keratinocyte migration was analyzed in Ahr+/+ and Ahr–/– keratinocyte cultures treated with medium conditioned by Ahr+/+ or Ahr–/– dermal fibroblasts (CM-DF). Wounds were performed and keratinocyte migration calculated as indicated in the legend for Fig. 3B. (C) Ahr+/+ and Ahr–/– keratinocytes were treated with conditioned medium from Ahr+/+ or Ahr–/– dermal fibroblasts (CM-DF). To address the role of TGFβ in the phenotype, experiments were performed using conditioned medium from Ahr+/+ DF plus 10 ng/ml recombinant TGFβ or conditioned medium from Ahr–/– DF plus 1 µg/ml neutralizing anti-TGFβ antibody. The experiments were performed in four independent primary keratinocyte cultures of each genotype and using conditioned medium from the same keratinocyte preparations or from three cultures of primary dermal fibroblasts. Data are shown as mean ± s.e.m. The P values for statistical comparison between genotypes are indicated.
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Fig. 8. TGFβ overexpression in Ahr–/– mice underlines accelerated keratinocyte migration and re-epithelialization. (A) The effect of TGFβ on keratinocyte migration from skin explants of Ahr–/– mice was analyzed by treatment with 1 µg/ml neutralizing anti-TGFβ antibody. Six explants from at least three different mice of each genotype were used. (B) Wounds were performed in the dorsal skin of Ahr–/– mice and, at day 3, those on one flank treated with three doses of 50 µl anti-TGFβ antibody at 50 µg/ml concentration. Wounds on the other flank were treated under the same conditions with PBS. Tissues were collected and processed for hematoxylin and eosin staining. Progression of the neo-epithelium is indicated by arrows. Six wounds from three different Ahr+/+ and Ahr–/– mice were analyzed. Scale bars: 180 µm. Data are shown as mean ± s.e.m. The P values for statistical comparison between genotypes are indicated.
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Fig. 9. AhR downregulation in Ahr+/+ mice accelerates wound healing and increases TGFβ-dependent signaling. (A) AhR was downregulated in Ahr+/+ skin wounds at day 3 by in vivo administration of an antisense oligonucleotides. Control sense oligonucleotide was used under the same experimental conditions as negative control. Antisense oligonucleotides were applied to the wounds in one flank whereas sense oligonucleotides were applied to the wounds on the opposite flank. AhR expression level was analyzed by immunofluorescence using an AhR specific primary antibody and an Alexa Fluor 488-labeled secondary antibody. Downmodulation of AhR expression in presence of antisense oligonucleotides was also determined by western immunoblotting using 20 µg protein and an AhR-specific antibody. Experiments were done in duplicate using two wounds for each experimental condition. (B) Sense and antisense oligonucleotide-treated skin wounds were dissected and processed for hematoxylin and eosin staining. Progression of the regenerating epithelium was measured as indicated in the legend for Fig. 1. (C) The number of keratinocytes that responded to TGFβ-dependent signaling (arrows) was quantified by immunohistochemistry as Smad2-P-positive cells/area. At least six wounds from three different Ahr+/+ and Ahr–/– mice were analyzed. Scale bars: 100 µm (A), 40 µm (B and C). Data are shown as mean ± s.e.m. The P values for statistical comparison between genotypes are indicated.
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© The Company of Biologists Ltd 2009
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