1823Research Article

IntroductionInefficient wound healing represents an important health problem

for individuals suffering accidental, surgical or chronic skin lesions

and has special relevance for the elderly and for immunosuppressed,

diabetic or cancer patients (Reed and Clark, 1985; Schafer and

Werner, 2008). Tissue repair is a complex process that involves new

blood vessel formation and the recruitment to the wound of

inflammatory cells, such as macrophages and neutrophils, to

eliminate contaminants and bacteria and fibroblasts that produce

extracellular matrix (ECM) components (e.g. collagen and

fibronectin). In parallel to the formation of granulation tissue,

increased proliferation and migration of keratinocytes takes place

to repair the damaged epidermis (Eckes et al., 1999; Martin, 1997;

Schafer and Werner, 2008; Singer and Clark, 1999). The

intermediate molecules involved in adult skin repair are only

partially known. Nevertheless, analysis of overexpression and

knockout animal models has revealed the important contribution

of adhesion molecules such as β1 integrins (Grose et al., 2002; White

et al., 2004), growth factors such as FGF and HGF (Werner and

Grose, 2003), ECM constituents such as osteopontin (Mori et al.,

2008) and cytokines such as TGFβ (Ashcroft et al., 1997; Singer

and Clark, 1999).

TGFβ is one of the best characterized profibrogenic molecules

(Bauer and Schuppan, 2001; Kanzler et al., 1999) and alterations

in its synthesis, secretion and intracellular signaling are associated

with many pathological states including cancer and tissue fibrosis

(Blobe et al., 2000; Corchero et al., 2004; Massague, 2000;

Massague and Chen, 2000). Despite the fact that exogenous TGFβ3

administration is currently under clinical trial as a novel anti-scarring

agent (Shah et al., 1995), the effect of this cytokine on tissue repair

remains controversial. Some studies suggest that TGFβ regulates

wound healing because its expression is increased by platelets,

inflammatory cells and fibroblasts located at the site of injury

(Amendt et al., 2002; Leibovich and Ross, 1975; Schafer and

Werner, 2007). Genetic manipulation of TGFβ or its receptors,

however, can both promote and inhibit tissue regeneration (Amendt

et al., 2002; Brown et al., 1995; Crowe et al., 2000; Shah et al.,

1999). Furthermore, additional work has revealed that depending

on the level of TGFβ activity and/or the experimental model used,

this cytokine has been shown not to affect (Leask et al., 2008), to

inhibit (Hosokawa et al., 2005; Yang et al., 2001) or to promote

skin re-epithelialization (Gailit et al., 1994; Reynolds et al., 2005;

Reynolds et al., 2008). TGFβ-dependent signaling is mediated by

its binding and activation of plasma membrane serine-threonine

kinase receptors that will phosphorylate and activate intracellular

intermediates of the Smad family of proteins (Smad2, Smad3 and

Smad4). Activated Smads will heterodimerize and enter the cell

nucleus where they activate target gene expression (Massague, 2000;

Siegel and Massague, 2003).

The aryl hydrocarbon (dioxin) receptor (AhR) is a member of

the class VII of basic-helix-loop-helix-PAS (bHLH-PAS) family of

transcription factors. AhRs regulate gene expression through

heterodimerization with the nuclear protein aryl hydrocarbon

receptor nuclear translocator ARNT (Furness et al., 2007). In

Delayed wound healing caused by inefficient re-epithelialization

underlines chronic skin lesions such as those found in diabetes.

The dioxin receptor (AhR) modulates cell plasticity and

migration and its activation by occupational polycyclic aromatic

hydrocarbons (PAHs) results in severe skin lesions such as

contact hypersensitivity, dermatitis and chloracne. Using wild-

type (Ahr+/+) and AhR-null (Ahr–/–) mouse primary keratinocyte

cultures and tissue explants, we show that lack of AhR increases

keratinocyte migration and accelerates skin re-epithelialization

without affecting cell proliferation or recruitment of

inflammatory cells. Wounds in Ahr–/– animals had elevated

numbers of fibroblasts and increased collagen content in their

granulation tissue. Importantly, Ahr–/– dermal fibroblasts

secreted higher levels of active TGFβ that increased keratinocyte

migration in culture and that could account for over-activation

of the TGFβ pathway and for faster wound healing in the AhR-

null neo-epithelium. Consistently, a TGFβ neutralizing antibody

decreased keratinocyte migration in culture and halted re-

epithelialization in Ahr–/– mice. Moreover, in vivo treatment with

an antisense oligonucleotide for AhR increased TGFβ signaling

and improved re-epithelialization in wounds of wild-type mice.

These data indicate that AhR is relevant for wound repair and

suggest that AhR downmodulation might be a potential new

tool for the treatment of chronic, surgical or accidental wounds.

Supplementary material available online at

http://jcs.biologists.org/cgi/content/full/122/11/1823/DC1

Key words: Dioxin receptor, TGFβ, Wound healing

Summary

Loss of dioxin-receptor expression accelerates woundhealing in vivo by a mechanism involving TGFβJose M. Carvajal-Gonzalez1, Angel Carlos Roman1, M. Isabel Cerezo-Guisado1,*, Eva M. Rico-Leo1,Gervasio Martin-Partido2 and Pedro M. Fernandez-Salguero1,‡

1Departamento de Bioquímica y Biología Molecular and 2Departamento de Biologia Celular, Facultad de Ciencias, Universidad de Extremadura,Avenida de Elvas s/n, 06080-Badajoz, Spain*Present address: Departamento de Inmunologia y Oncologia, Centro Nacional de Biotecnologia-CNB, C/ Darwin 3, 28049-Madrid, Spain‡Author for correspondence (e-mail: pmfersal@unex.es)

Accepted 25 February 2009Journal of Cell Science 122, 1823-1833 Published by The Company of Biologists 2009doi:10.1242/jcs.047274

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addition to its relevant role in xenobiotic-induced toxicity and

carcinogenesis (Fernandez-Salguero et al., 1996; Mimura et al.,

1997; Nebert et al., 2004; Shimizu et al., 2000), AhR is gaining

considerable interest because of its contribution to the control of

cell proliferation, differentiation and tissue homeostasis (Barouki

et al., 2007; Gomez-Duran et al., 2008b; Puga et al., 2002). Among

the different cell functions requiring AhR, the control of TGFβactivation appears particularly relevant. In cell culture systems, AhR

activity has been functionally related to increased secretion and

activation of TGFβ in primary hepatocytes (Zaher et al., 1998) and

mouse embryo fibroblasts (Elizondo et al., 2000; Gomez-Duran et

al., 2008a; Gomez-Duran et al., 2006; Santiago-Josefat et al., 2004)

and, consistently, with diminished cell proliferation and increased

apoptosis. In mice, knockdown of AhR expression results in

fibrotic lesions in the liver (Corchero et al., 2004; Fernandez-

Salguero et al., 1995; Peterson et al., 2000), heart and skin

(Fernandez-Salguero et al., 1997), which, at least for the hepatic

tissue, colocalized to the portal areas with increased levels of TGFβ(Corchero et al., 2004). Collectively, these studies offer the

possibility that changes in AhR expression could affect tissue repair

by controlling TGFβ activity.

The involvement of AhR in the control of skin disease and tissue

remodeling is only just beginning to emerge. A recent report showed

that constitutive activation of AhR and increased expression of

Cyp1a1, Gsta1, Fos and TGFA are underlying factors in the

development of chloracne in human subjects occupationally or

accidentally exposed to significant doses of polycyclic aromatic

hydrocarbons (Imamura et al., 2007; Tang et al., 2008). Regeneration

studies in adult zebra fish have revealed that AhR2 activation by

acute exposure to dioxin (TCDD, 2,3,7,8-tetrachlorodibenzo-[p]-

dioxin) impairs caudal (tail) fin regeneration (Andreasen et al.,

2007), which suggests that AhR has an inhibitory role in tissue

remodeling. Finally, transgenic mice expressing a constitutively

activated form of AhR in their keratinocytes develop severe skin

lesions with itching and inflammation that resembled atypical atopic

dermatitis (Tauchi et al., 2005). These results indicate that AhR

activation impairs skin wound healing and suggest that maintained

receptor activity by chronic exposure to occupational and

environmental xenobiotics could exacerbate an inflammatory

response eventually affecting tissue repair.

In this work, we have analyzed whether the lack of AhR

expression can accelerate wound healing in vivo and whether TGFβhas a role in such putative AhR-dependent process. Using wild-

type and AhR-null mice, primary keratinocyte cultures and tissue

explants, and by the modulation of TGFβ activity and AhR

expression, we report that loss of AhR increases keratinocyte

migration and enhances the efficiency of wound healing in vivo

and that such mechanisms require TGFβ activity. Since

downregulation of AhR at the wound site by antisense

oligonucleotides significantly accelerated wound healing in Ahr+/+

mice, this study provides a potential new tool that could be used

to improve re-epithelialization in diseased skin wounds.

ResultsLoss of AhR expression improves wound healing byaccelerating re-epithelializationConsidering previous studies showing that AhR activation in mouse

causes inflammatory skin lesions (Tauchi et al., 2005) and impairs

fin regeneration in zebra fish (Andreasen et al., 2007), and since

humans exposed to dioxin develop the skin disease chloracne

associated with an increase in AhR-dependent transcription (Tang

et al., 2008), we first analyzed whether AhR expression modulates

wound healing in vivo. Histological analyses of wounds performed

in the dorsal skin of Ahr+/+ and Ahr–/– mice revealed that AhR-null

mice closed their wounds significantly faster than wild-type mice,

this effect being more pronounced between days 3 and 5 (wound

diameter was measured as the distance between both flanks of the

regenerating neo-epithelium or granulation tissue) (Fig. 1, arrows).

At 7 days after wounding, Ahr+/+ and Ahr–/– mice had completed

closure of the skin. Further analysis of the healing process allowed

us to determine that the neo-epithelium progressed faster in Ahr–/–

than in Ahr+/+ mice. By contrast, the diameter of the granulation

tissue did not significantly vary during wound healing, suggesting

that the epidermal layer was a main target for AhR-dependent re-

epithelialization (Fig. 1). Additionally, macroscopic analysis of the

wounds confirmed that Ahr–/– mice were more efficient in the

healing reaction than wild-type mice (data not shown). The largest

differences in wound healing were observed at day 5; however, we

performed the in vivo experiments at day 3 because the almost

complete healing in Ahr–/– mice at day 5 could distort comparison

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Fig. 1. Wound healing is accelerated inAhr–/– mice. Full-thickness 4 mm woundswere made in the dorsal skin of Ahr+/+ andAhr–/– mice and healing followed for up to 7days. Wounded tissue was dissected,embedded in paraffin, sectioned and stainedwith hematoxylin and eosin (H&E). The leftpanel shows representative H&E stainedsections from Ahr+/+ and Ahr–/– mice.Arrows indicate the position of theepithelium at both sides of the wound. Theright panel includes a quantification of theprogression of the neo-epithelium (upper) orthe granulation tissue (lower) in wounds ofeach genotype. Six wounds were performedin three mice of each genotype for each timepoint. Scale bars: 200 μm. Data are shown asmean ± s.e.m. The P values for statisticalcomparison between genotypes areindicated.

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1825AhR in wound healing through TGFβ

with wild-type mice regarding relevant parameters such as

keratinocyte proliferation and migration and TGFβ response (over

90% of the Ahr–/– wounds were totally closed at day 5). In addition,

AhR expression did not significantly vary during the wound-healing

process, or with respect to basal skin, suggesting that delayed re-

epithelialization in Ahr+/+ mice was not due to alterations in AhR

protein levels (supplementary material Fig. S1).

To further analyze the role of the epithelium in the AhR-

dependent wound-healing phenotype, we measured the length of

the neo-epithelial layer covering the area between the wound site

and the margin of the regenerating tissue (Fig. 2A, arrows and red

dotted line). It can be seen that the length of the neo-epithelium (e)

was significantly larger in Ahr–/– mice than in Ahr+/+ mice (Fig. 2A,

right). Since re-epithelialization requires an increase in keratinocyte

proliferation (Schafer and Werner, 2007; Schafer and Werner, 2008),

we next analyzed whether differences in keratinocyte proliferation

could help explain the more efficient wound healing in Ahr–/– mice.

PCNA immunostaining showed that proliferation rates were similar

in Ahr+/+ and Ahr–/– wounds (Fig. 2B), which suggested the

involvement of additional mechanisms.

Keratinocytes lacking AhR have increased migrationCell migration is an important parameter that markedly affects the

quality of tissue repair (Schafer and Werner, 2007; Schafer and

Werner, 2008). Our results prompted us to study whether an increase

in keratinocyte migration could be important for the Ahr–/–

phenotype. An ex vivo approach useful for analysis of changes in

cell migration consists of culturing skin explants under conditions

that favor keratinocyte emigration but inhibit cell proliferation in

absence of serum (Guasch et al., 2007). In preliminary experiments,

immunofluorescence staining for the keratinocyte-specific marker

cytokeratin 14 confirmed the epithelial phenotype of the emigrating

cells (supplementary material Fig. S2). To avoid interference due

to cell proliferation, keratinocyte migration experiments (from tissue

explants or primary cells) were performed in absence of serum. As

Fig. 2. Ahr–/– wounds have increased re-epithelialization but similarproliferation rates. Wounds were made and processed in Ahr+/+ and Ahr–/–

mice as indicated in the legend for Fig. 1. (A) Hematoxylin and eosin stainingwas 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 theepithelial layer. Data were quantified using ImageJ software. The analysis wasperformed in at least eight wounds isolated from four Ahr+/+ and Ahr–/– mice.The neo-epithelial layer covering the area between the wound site and themargin 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 forstatistical comparison between genotypes are indicated.

Fig. 3. Keratinocytes lacking AhR have increased migration in tissue explantsand in primary culture. (A) Explants were obtained from Ahr+/+ and Ahr–/–

mouse dorsal skin and placed in culture. Emigration of the keratinocytes fromthe explants was measured for up to 6 days and the results obtained plottedagainst time. Migration increased with time in both genotypes with a kineticthat could be adjusted to a linear equation (R2=0.9768 and R2=0.9915 forAhr+/+ and Ahr–/–, respectively). The difference in slopes between Ahr+/+ andAhr–/– explants was statistically significant at P=0.00214. At least five woundsfrom three different Ahr+/+ and Ahr–/– mice were used. (B) Primarykeratinocyte 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 inserum-free medium. Data were quantified and are represented in the rightpanel. The experiments were performed in triplicate using primary culturesfrom 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 comparisonbetween genotypes are indicated. The equations for the linear regression of thedata are shown in A.

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shown in Fig. 3A, keratinocytes from Ahr–/– explants had increased

migration rates over time (slope 145.96) than keratinocytes from

Ahr+/+ explants (slope 68.55). Statistical analysis revealed that the

difference in slopes between Ahr+/+ and Ahr–/– explants was highly

significant at P=0.00214. Thus, migration appears to be relevant

for the increased wound healing observed in absence of AhR in

vivo. This hypothesis was further confirmed by establishing primary

keratinocyte cultures from Ahr+/+ and Ahr–/– newborn mice. Primary

keratinocytes were placed in collagen- or fibronectin-coated culture

plates and grown to confluence to halt cell proliferation. Wounds

were performed and cell migration measured 15 hours later as the

distance between margins (Fig. 3B, left). Quantification of the data

revealed that Ahr–/– primary keratinocytes migrated significantly

faster than Ahr+/+ keratinocytes in both ECM components collagen

and fibronectin. Altogether, these results indicate that keratinocytes

lacking AhR have increased migration rates and suggest that this

cellular characteristic could underline the higher efficiency of Ahr–/–

mice in re-epithelialization and wound healing.

Ahr–/– wounds have increased fibroblasts and collagen butsimilar recruitment of inflammatory cellsAt the early stages of wound healing (e.g. 3 days), an inflammatory

response takes place that recruits inflammatory cells such as

macrophages and neutrophils to the granulation tissue (Schafer and

Werner, 2008). To determine whether differences in inflammation

could contribute to increased wound healing in Ahr–/– mice, we

analyzed by immunohistochemistry the content of macrophages

(staining for F4/80) and neutrophils (Ly-6G antigen) in the

granulation tissue. AhR expression did not significantly alter the

ability of the wounded tissue to recruit those inflammatory cells,

because their numbers were very similar between Ahr+/+ and Ahr–/–

mice (Fig. 4A,B). Additionally, under basal conditions, the skin of

Ahr+/+ and Ahr–/– mice had a very low content of macrophages and

neutrophils (data not shown). Fibroblast cells also accumulate in

the wounded tissue and promote re-epithelialization by the

production of ECM proteins such as collagen and by the secretion

of growth factors (Schafer and Werner, 2008). The marker protein

vimentin, although expressed by endothelial cells, can be used to

estimate the presence of fibroblasts. Immunohistochemistry for

vimentin revealed that Ahr–/– wounds had a moderate, although

consistent, increase in fibroblast numbers with respect to Ahr+/+

wounds (Fig. 4C). Western blot analyses also showed elevated

vimentin levels in AhR-null wounds (Fig. 4C). Stromal fibroblasts

can differentiate into α-smooth muscle actin (α-SMA)-expressing

myofibroblasts in certain pathologies such as cancer (Elenbaas and

Weinberg, 2001; Ronnov-Jessen et al., 1996). We analyzed such a

possibility by measuring the expression of the myofibroblast-

specific marker α-SMA in Ahr–/– mice wounds. As shown in Fig.

5A, α-SMA was found at similar levels in Ahr–/– and Ahr+/+ wounds,

indicating that lack of AhR did not significantly affect myofibroblast

differentiation during re-epithelialization. A moderate increase in

fibroblast numbers could enhance matrix deposition in the

granulation tissue. To address this issue, we performed Sirius red

and fast green staining, and found that wounds from Ahr–/– mice

had a significant increase in collagen content in their granulation

tissue compared with Ahr+/+ wounds (Fig. 5B). To further support

this observation, hydroxyproline levels were also measured to more

accurately quantify collagen deposition. Consistently, Ahr–/– wounds

showed a significant elevation in hydroxyproline content that was

indicative of elevated collagen deposition (Fig. 5C). Thus,

accelerated wound healing in untreated Ahr–/– mice does not seem

to involve an exacerbated inflammatory response, although it

requires increased fibroblast recruitment and enhanced collagen

deposition in the ECM.

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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 westernimmunoblotting using 15 μg total proteins and a vimentin specific antibody. The expression of β-actin was used as loading control. Three sections were analyzedfrom four wounds corresponding to four different animals of each genotype. Cell counting for each marker and mouse genotype was referred to the same tissuearea. Data are shown as mean ± s.e.m. The P values for statistical comparison between genotypes are indicated.

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1827AhR in wound healing through TGFβ

TGFβ signaling is increased in Ahr–/– woundsThe higher collagen content in the granulation tissue of Ahr–/– mice

suggested the possibility that an increase in growth factor secretion

could modulate wound healing. In addition, AhR has a role in TGFβactivation because AhR-null mice produce increased levels of active

cytokine (Corchero et al., 2004; Elizondo et al., 2000; Gomez-Duran

et al., 2008a; Gomez-Duran et al., 2008b; Gomez-Duran et al., 2006;

Santiago-Josefat et al., 2004). Based on this information, we

performed experiments aimed to analyze the contribution of TGFβsignaling in the AhR-dependent wound-healing phenotype. Protein

expression in whole cell extracts from basal skin showed that Ahr–/–

mice expressed higher levels of total TGFβ than did Ahr+/+ mice.

Wounding induced an increase in total TGFβ protein in both mouse

genotypes that normalized the differences present in basal skin (Fig.

6A). Since active, rather than latent TGFβ accounts for TGFβ-

dependent signaling (Massague, 2000; Massague and Gomis, 2006),

we determined by ELISA whether the lack of AhR expression

affected the secretion of active cytokine by primary keratinocytes

(as target cells for re-epithelialization) and dermal fibroblasts (as a

source for active TGFβ). Ahr–/– dermal fibroblasts secreted higher

levels of active TGFβ (measured as the active:total cytokine ratio)

than did wild-type cells (Fig. 6B, CM-DF). By contrast, similar

amounts of active cytokine were secreted by primary keratinocytes

Fig. 5. Myofibroblasts and collagen content in Ahr+/+ and Ahr–/– wounds.(A) Western immunoblotting for the myofibroblast-specific marker α-smoothmuscle actin (α-SMA) was used to analyze the presence of such cells in thewounds. Aliquots of 15 μg protein were separated. The expression of β-actinserved as loading control. Three wounds obtained from three different mice ofeach genotype were used. (B) Collagen content in the granulation tissue belowthe epithelium was analyzed by Sirius red and fast green staining. Pictureswere also processed in pseudo-color using ImageJ software. Sirius red stainingwas referred to the same area of tissue. At least eight wounds from fourdifferent Ahr+/+ and Ahr–/– mice were analyzed. Scale bar: 20 μm. (C) Collagencontent was also analyzed by determining the amount of hydroxyprolinepresent in Ahr+/+ and Ahr–/– wounds. The results are represented as fold changein wounded tissue with respect to normal skin. Measurements were done induplicate in three wounds from three different mice of each genotype. Data areshown as mean ± s.e.m. The P values for statistical comparison betweengenotypes are indicated.

Fig. 6. Loss of AhR expression increases active TGFβ levels and TGFβ-dependent signaling. (A) Biopsies of basal skin and wound tissue were takenfrom three different Ahr+/+ and Ahr–/– mice. Aliquots of 15 μg total cellextracts were prepared and analyzed for TGFβ expression by westernimmunoblotting using a specific antibody. β-actin was used to normalizeTGFβ expression as indicated on the right panel. (B) Primary dermalfibroblasts and primary keratinocytes were cultured from the skin of newbornAhr+/+ and Ahr–/– mice. Each cell type was cultured for 72 hours andconditioned 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 andactive TGFβ levels by ELISA. Measurements were done in triplicate and fourprimary cultures were prepared from different Ahr+/+ and Ahr–/– mice.(C) TGFβ signaling was analyzed in non-wounded skin and wounds fromAhr+/+ and Ahr–/– mice by quantifying the number of Smad2-P-positive cells(p-Smad2; arrowheads) with respect to tissue area. Sections were analyzedfrom 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 comparisonbetween genotypes are indicated.

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in both experimental groups (Fig. 6B, CM-Ker). Increased

production of active TGFβ by Ahr–/– dermal fibroblasts might be

relevant for progression of the neo-epithelium during wound

healing. Analysis of TGFβ-dependent signaling revealed that the

number of keratinocytes activating the TGFβ pathway (determined

as the ratio of Smad2-P-positive cells/area) was significantly higher

in Ahr–/– than in Ahr+/+ wounds (Fig. 6C). Thus, although wounding

increases total TGFβ to similar levels in Ahr+/+ and Ahr–/– mice,

the increased secretion of active cytokine by AhR-null dermal

fibroblasts could enhance TGFβ signaling and re-epithelialization

in vivo.

Modulation of TGFβ levels alters Ahr+/+ and Ahr–/– keratinocytemigration and rescues the Ahr–/– wound-healing phenotypeAltogether, the data obtained led to the hypothesis that accelerated

wound healing in Ahr–/– mice could be due, at least in part, to an

increase in keratinocyte migration induced by overproduction of

active TGFβ by dermal fibroblasts. Nevertheless, the fact that AhR-

null primary keratinocytes had intrinsic differences in migration

(Fig. 3B), suggests that these cells could secrete molecules

modulating motility in a cell-type-autonomous fashion. To test this

possibility, migration of Ahr+/+ and Ahr–/– keratinocytes was

determined in the presence of their own conditioned medium or

medium conditioned by the opposite phenotype (Fig. 7A). Ahr–/–

keratinocytes in their conditioned medium migrated faster than

Ahr+/+ keratinocytes growing in their own medium. Addition of

conditioned medium from Ahr–/– keratinocytes (CM-Ker Ahr–/–)marginally increased migration of Ahr+/+ cells (P=0.125) whereas

medium from Ahr+/+ cells (CM-Ker Ahr+/+) slightly decreased

migration of Ahr–/– keratinocytes (P=0.069). Thus, although

keratinocytes could modulate their own migration autonomously,

and these observations are of potential interest, we focused our study

on dermal fibroblasts based on the functional relationship between

AhR and TGFβ in fibroblast cells. We first analyzed the effect of

conditioned medium from dermal fibroblasts (CM-DF) on

keratinocyte migration in culture. Growth of wild-type keratinocytes

with medium from AhR-null dermal fibroblasts increased their

migration rates above levels obtained by the addition of wild-type

conditioned medium (Fig. 7B). Consistently, culture of Ahr–/–

keratinocytes with conditioned medium from wild-type dermal

fibroblasts inhibited their migration rates to levels below those

produced by AhR-null CM-DF (Fig. 7B). Since these results

suggested that dermal fibroblasts secreted a molecule(s) affecting

keratinocyte migration, and considering the AhR-TGFβ relationship,

as well as the relevance of TGFβ in wound healing, we performed

further experiments designed to modulate keratinocyte migration

by adjusting TGFβ activity. Ahr–/– keratinocytes, growing in CM-

DF Ahr+/+ medium and treated with 10 ng/ml recombinant TGFβ,

exhibited a significant increase in migration compared with the same

experimental conditions without TGFβ addition (Fig. 7C, compare

bars 2 and 4). Accordingly, Ahr–/– keratinocytes, cultured in CM-

DF Ahr–/– medium and treated with 1 μg/ml of neutralizing anti-

TGFβ antibody, decreased their migration to levels close to those

induced by CM-DF Ahr+/+ medium (Fig. 7C, compare bars 6 and

8 with 2). Ahr+/+ keratinocytes had a similar response, and their

culture in CM-DF Ahr+/+ medium supplemented with 10 ng/ml

recombinant TGFβ also increased migration compared with results

obtained with CM-DF Ahr+/+ alone (Fig. 7C, compare bars 3 and

1). Moreover, Ahr+/+ keratinocytes cultured in CM-DF Ahr–/–

migrated faster than the same cells in presence of CM-DF Ahr+/+

(Fig. 7C, compare bars 5 and 1), whereas addition of 1 μg/ml of

neutralizing anti-TGFβ antibody to CM-DF Ahr–/– significantly

decreased migration of Ahr+/+ keratinocytes (Fig. 7C, compare bars

7 and 5). Therefore, increasing TGFβ levels in medium conditioned

by Ahr+/+ or Ahr–/– dermal fibroblasts increased Ahr–/– and Ahr+/+

keratinocytes migration, whereas lowering TGFβ activity in medium

conditioned by Ahr–/– or Ahr+/+ dermal fibroblasts decreased

migration of wild-type and AhR-null keratinocytes. The neutralizing

activity of the anti-TGFβ antibody has been previously determined

(IC50=0.06 μg/ml to inhibit the proliferation of Mv1Lu cells by 50%)

(Santiago-Josefat et al., 2004).

To determine whether an increase in TGFβ activity is relevant

to stimulate more efficient keratinocyte migration and improved

wound healing in Ahr–/– mice, we first cultured skin explants from

Ahr–/– mice in the presence of 1 μg/ml of neutralizing anti-TGFβ

Journal of Cell Science 122 (11)

Fig. 7. TGFβ activity secreted by Ahr–/– dermal fibroblasts regulateskeratinocyte migration. (A) The effect of self-secreted molecules onkeratinocyte migration was determined in Ahr+/+ and Ahr–/– keratinocytestreated with conditioned medium from the same or the opposite genotype(CM-Ker). Wounds were performed and analyzed as indicated in the legendfor Fig. 3B. (B) The paracrine effect of secreted molecules on keratinocytemigration was analyzed in Ahr+/+ and Ahr–/– keratinocyte cultures treated withmedium conditioned by Ahr+/+ or Ahr–/– dermal fibroblasts (CM-DF). Woundswere performed and keratinocyte migration calculated as indicated in thelegend for Fig. 3B. (C) Ahr+/+ and Ahr–/– keratinocytes were treated withconditioned medium from Ahr+/+ or Ahr–/– dermal fibroblasts (CM-DF). Toaddress the role of TGFβ in the phenotype, experiments were performed usingconditioned medium from Ahr+/+ DF plus 10 ng/ml recombinant TGFβ orconditioned medium from Ahr–/– DF plus 1 μg/ml neutralizing anti-TGFβantibody. The experiments were performed in four independent primarykeratinocyte cultures of each genotype and using conditioned medium fromthe same keratinocyte preparations or from three cultures of primary dermalfibroblasts. Data are shown as mean ± s.e.m. The P values for statisticalcomparison between genotypes are indicated.

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1829AhR in wound healing through TGFβ

antibody. We found that neutralizing TGFβ activity reduced

migration of Ahr–/– keratinocytes to a level similar to that observed

in explants from Ahr+/+ mice (Fig. 8A). In agreement with our

hypothesis, neutralization of TGFβ activity in vivo also inhibited

wound healing in Ahr–/– mice to values that were similar to those

observed in untreated Ahr+/+ wounds (Fig. 8B). In agreement with

the data presented in Fig. 1, wound-healing inhibition by the anti-

TGFβ antibody blocked migration of the epithelial layer without a

significant effect on the progression of the granulation tissue (Fig.

8B). It is interesting to note that increased TGFβ response in Ahr–/–

keratinocytes might not only involve higher levels of active cytokine

but also changes in TGFBR1 and TGFBR2 receptors. Real-time

RT-PCR analyses of Tgfbr1 and Tgfbr2 mRNA expression showed

that although mRNA levels for the type 1 receptor did not

significantly vary between Ahr+/+ and Ahr–/– keratinocytes, the

expression of the cytokine-binding TGFBR2 receptor was

moderately increased in AhR-null cells (supplementary material Fig.

S3). Thus, increased TGFβ signaling in Ahr–/– wounds could involve

a complex mechanism of cytokine overactivation and TGFBR2

overexpression.

AhR downmodulation by antisense oligonucleotide mimics theAhr–/– wound-healing phenotype and increases TGFβ-dependent signaling in the neo-epitheliumCollectively, our data indicate that AhR has a causal role in

modulating the efficiency of wound healing and that such a process

involves the regulation of TGFβ activity. To further demonstrate

the role of AhR in wound healing, we applied antisense

oligonucleotide to wounds in Ahr+/+ mice and quantified differences

in re-epithelialization and in the activation of the TGFβ pathway.

Addition of a gel containing antisense oligonucleotides decreased

AhR levels in the granulation tissue of Ahr+/+ wounds whereas a

control sense oligonucleotide did not show a significant effect, as

determined by immunofluorescence (Fig. 9A, area below dotted

line) or western immunoblotting (Fig. 9A). Remarkably, antisense

oligonucleotides significantly increased re-epithelialization and

accelerated wound healing in Ahr+/+ mice (Fig. 9B), which supported

a causal role for AhR in epithelial regeneration. Furthermore, in

agreement to the regulatory role of TGFβ activity in wound healing

in Ahr–/– mice, treatment with antisense oligonucleotides for AhR

also increased the number of keratinocytes activating TGFβ-

dependent signaling in the neo-epithelial layer (quantified as the

ratio of Smad2-P-positive cells/area in Fig. 9C). Thus,

downregulation of AhR expression accelerates wound healing in

vivo through a mechanism involving increased TGFβ activity.

DiscussionThe cellular functions of the dioxin receptor AhR appear far more

complex than those related to the regulation of xenobiotic

metabolism (Barouki et al., 2007; Gomez-Duran et al., 2008b; Puga

et al., 2005), and different studies have demonstrated its role in cell

proliferation, differentiation and apoptosis (Barouki et al., 2007;

Furness et al., 2007; Nebert and Dalton, 2006). Interestingly, early

reports already indicated that AhR had a role in epithelial cell

adhesion because suspension of human keratinocytes activated this

receptor in the absence of xenobiotics (Sadek and Allen-Hoffmann,

1994). Later work on the keratinocyte cell line HaCaT showed that

AhR regulates the expression of the epithelial-to-mesenchymal

marker Slug and that AhR becomes activated in cells located at the

leading edge in wound healing in vitro (Ikuta and Kawajiri, 2006).

Despite these studies in cultured cells, the involvement of AhR in

the control of epithelial cell migration in vivo is mostly unknown.

In this study, we used AhR-null mice, skin explants and primary

keratinocytes cultures to demonstrate that lack of AhR expression

accelerates wound healing and re-epithelialization by increasing

keratinocyte migration, and that such a phenotype is dependent on

elevated levels of active TGFβ.

Although AhR activation in cultured epithelial cells has been

associated with increased cell migration (Ikuta and Kawajiri, 2006;

Sadek and Allen-Hoffmann, 1994), we found that genetic knockout

of AhR expression accelerates wound healing in vivo, therefore

suggesting that skin wound repair could be hampered under

conditions of maintained AhR activation. This initial result has in

fact been confirmed in a different AhR-null mouse line in which a

preliminary macroscopic examination of skin wounds also revealed

that AhR-null mice had a faster wound-healing response (Ikuta et

al., 2008). Interestingly, the accelerated wound healing that we found

Fig. 8. TGFβ overexpression in Ahr–/– mice underlines acceleratedkeratinocyte migration and re-epithelialization. (A) The effect of TGFβ onkeratinocyte migration from skin explants of Ahr–/– mice was analyzed bytreatment with 1 μg/ml neutralizing anti-TGFβ antibody. Six explants from atleast three different mice of each genotype were used. (B) Wounds wereperformed in the dorsal skin of Ahr–/– mice and, at day 3, those on one flanktreated with three doses of 50 μl anti-TGFβ antibody at 50 μg/mlconcentration. Wounds on the other flank were treated under the sameconditions with PBS. Tissues were collected and processed for hematoxylinand eosin staining. Progression of the neo-epithelium is indicated by arrows.Six wounds from three different Ahr+/+ and Ahr–/– mice were analyzed. Scalebars: 180 μm. Data are shown as mean ± s.e.m. The P values for statisticalcomparison between genotypes are indicated.

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in Ahr–/– mice involved a larger progression of the epithelial layer

rather than changes in the progression of the granulation tissue.

However, the increased length of the neo-epithelium in Ahr–/–

wounds did not result from higher proliferation rates of their

keratinocytes, which suggested that additional parameters, such as

increased cell migration and/or augmented inflammatory reaction,

are involved. Interestingly, although TGFβ inhibits cell proliferation,

Ahr–/– wounds had only a marginal decrease in keratinocyte

proliferation with respect to Ahr+/+ wounds, suggesting that TGFβcould affect proliferation and migration of keratinocytes with

differing sensitivity. Future experiments will be required to

determine whether TGFβ secreted by dermal fibroblasts

differentially affects cell proliferation and migration of Ahr+/+ and

Ahr–/– keratinocytes. This hypothesis is particularly interesting

considering that keratinocytes lacking AhR overexpress the

cytokine-interacting TGFβR-2 receptor, which could cooperate with

TGFβ to exert different effects on epithelial cell proliferation and

migration. Recruitment of inflammatory cells important for wound

healing, such as macrophages and neutrophils, was similar in Ahr+/+

and Ahr–/– wounds, indicating that a difference in the inflammatory

response is not a critical factor in the AhR-null phenotype. In

agreement with a delaying activity of AhR in wound healing, a

previous study has shown that transgenic mice expressing a

constitutively active form of the receptor in their keratinocytes

developed skin lesions that resembled atypical atopic dermatitis and

that involved a significant inflammatory reaction (Tauchi et al.,

2005). Thus, although Ahr–/– wounds develop a normal

inflammatory response, inflammation becomes aggravated in the

skin lesions of mice overexpressing a constitutively activated AhR.

Taken together, these studies suggest that a reduction in the

physiological levels of AhR can increase the efficiency of wound

healing in vivo without a major local inflammatory reaction.

Our results prompted us to analyze whether accelerated wound

healing in Ahr–/– mice was the result of increased keratinocyte

migration. Experiments performed in skin explants ex vivo and in

primary keratinocyte cultures clearly indicated that lack of AhR

expression significantly increased epithelial cell migration. It is well

known that many cytokines and growth factors regulate the

efficiency of wound healing (Grose and Werner, 2003; Scheid et

al., 2000; Singer and Clark, 1999). We focused our attention on

TGFβ for several reasons: (1) mouse models with targeted

inactivation of the genes encoding β3 integrin (Reynolds et al., 2005)

and Dpr2 (Meng et al., 2008) showed accelerated re-epithelialization

and an enhanced response to TGFβ, whereas decreased TGFβactivity correlated with impaired wound healing in PKCε-null mice

(Leask et al., 2008) and (2) we have extensively shown that absence

of AhR expression results in increased TGFβ activity in certain cell

types such as fibroblasts and hepatocytes (Corchero et al., 2004;

Elizondo et al., 2000; Gomez-Duran et al., 2008a; Gomez-Duran

et al., 2006; Santiago-Josefat et al., 2004). Fibroblasts produce many

components of the ECM and are also a relevant source of TGFβ.

After synthesis and secretion, TGFβ is first linked to the ECM via

LTBP, from which the cytokine will be released and activated by

extracellular proteases (Annes et al., 2003; Gomez-Duran et al.,

2006). Ahr–/– wounds had a moderate increase in fibroblast numbers

in their granulation tissue that correlated with a significant

accumulation of collagen in the ECM. Since increased collagen

content colocalized with elevated TGFβ levels in Ahr–/– liver

(Corchero et al., 2004), we analyzed whether AhR-null wounds

produced higher levels of TGFβ. Wounding increased total TGFβ

Journal of Cell Science 122 (11)

Fig. 9. AhR downregulation in Ahr+/+ mice accelerates woundhealing and increases TGFβ-dependent signaling. (A) AhR wasdownregulated in Ahr+/+ skin wounds at day 3 by in vivoadministration of an antisense oligonucleotides. Control senseoligonucleotide was used under the same experimental conditionsas negative control. Antisense oligonucleotides were applied tothe wounds in one flank whereas sense oligonucleotides wereapplied to the wounds on the opposite flank. AhR expressionlevel was analyzed by immunofluorescence using an AhRspecific primary antibody and an Alexa Fluor 488-labeledsecondary antibody. Downmodulation of AhR expression inpresence of antisense oligonucleotides was also determined bywestern immunoblotting using 20 μg protein and an AhR-specificantibody. Experiments were done in duplicate using two woundsfor each experimental condition. (B) Sense and antisenseoligonucleotide-treated skin wounds were dissected andprocessed for hematoxylin and eosin staining. Progression of theregenerating epithelium was measured as indicated in the legendfor Fig. 1. (C) The number of keratinocytes that responded toTGFβ-dependent signaling (arrows) was quantified byimmunohistochemistry as Smad2-P-positive cells/area. At leastsix wounds from three different Ahr+/+ and Ahr–/– mice wereanalyzed. Scale bars: 100 μm (A), 40 μm (B and C). Data areshown as mean ± s.e.m. The P values for statistical comparisonbetween genotypes are indicated.

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content to a similar extent in Ahr+/+ and Ahr–/– mouse skin,

normalizing the differences present in non-wounded skin.

Regardless the amount of total TGFβ secreted, this cytokine can

only initiate signaling through its receptors when released in its

active form (Massague, 2000; Massague and Gomis, 2006;

Massague and Wotton, 2000). Therefore, we determined the amount

of active TGFβ secreted by dermal fibroblasts and keratinocytes.

Interestingly, Ahr–/– dermal fibroblasts, but not keratinocytes,

produced significantly more active TGFβ than did wild-type cells,

indicating that the granulation tissue of AhR-null wounds could

contain higher amounts of active TGFβ. In agreement with such a

possibility, TGFβ-dependent signaling, measured as the number of

Smad2-P-positive cells, was increased in Ahr–/– neo-epithelium. We

hypothesize that, in the absence of AhR expression, increased TGFβactivity by dermal fibroblasts could overactivate the TGFβ pathway

in keratinocytes, promoting their migration and accelerating re-

epithelialization.

Different sets of experimental results support our hypothesis.

First, although conditioned medium from Ahr–/– dermal fibroblasts

increased migration of Ahr+/+ keratinocytes, the opposite was also

true and medium conditioned by Ahr+/+ dermal fibroblasts decreased

migration of Ahr–/– keratinocytes. Second, exogenous TGFβincreased migration of Ahr+/+ keratinocytes, whereas a neutralizing

anti-TGFβ antibody inhibited Ahr–/– keratinocyte migration. Finally,

in vivo treatment of Ahr–/– wounds with the neutralizing anti-TGFβantibody inhibited re-epithelialization and wound healing to a degree

of closure that was similar to that found in Ahr+/+ wounds.

Therefore, active TGFβ secreted by dermal fibroblasts can modulate

keratinocyte migration and wound healing by a mechanism

involving AhR. Despite this experimentally supported mechanism,

the fact that media conditioned by wild-type or AhR-null

keratinocytes seems to modulate, to some extent, keratinocyte

migration in culture, led us to suggest that a cell-autonomous

mechanism could cooperate with fibroblast-secreted TGFβ-

dependent signaling. Importantly, AhR was required for TGFβ-

dependent wound healing, because in vivo downregulation of AhR

expression by antisense oligonucleotides significantly improved

wound healing in Ahr+/+ mice as well as increasing TGFβ-

responsiveness in their neo-epithelium.

In summary, we report here that lack of AhR expression in mouse

skin accelerates wound healing by enhancing re-epithelialization.

Consistent with the already known role of AhR in modulating TGFβactivity, our mechanism proposes that increased production of active

TGFβ by dermal fibroblasts can exert a paracrine effect on the

keratinocytes of the neo-epithelium that will result in increased

migration along the regenerating wound. Remarkably, the fact that

in vivo administration of an antisense oligonucleotide against AhR

increased wound healing in wild-type mice, offers the potential for

AhR knockdown to be useful in the treatment of accidental,

surgical or chronic skin wounds. Therefore, AhR, as earlier

suggested for osteopontin (Mori et al., 2008), could be a novel

therapeutic target to improve the quality of skin repair.

Materials and MethodsAntibodies and reagentsProliferating cell nuclear antigen (PCNA) antibody was obtained from Neomarkers.The antibody to detect mouse AhR was from ABR. Antibodies against Smad2-P andvimentin were purchased from Cell Signaling and Anacrom Diagnostics, respectively.The macrophage marker F4/80 and the neutrophil antigen Ly-6G were detected usingspecific antibodies from Serotec and Transduction Laboratories, respectively. Anti-TGFβ neutralizing antibody (clone 1D11) was from R&D. Antibody against α-smoothmuscle actin, recombinant TGFβ protein and β-actin antibody were obtained fromSigma. TRICT-labeled anti-cytokeratin 14 (AF 64) was purchased from Covance.

MiceAhr+/+ and Ahr–/– mice were produced by homologous recombination in embryonicstem cells as described (Fernandez-Salguero et al., 1995). All the experimentationinvolving animals were performed following the guidelines established by the AnimalCare and Use Committee of the University of Extremadura. Adult male mice wereused at 9-12 weeks of age and had free access to water and rodent chow. Beforeperforming surgical procedures, animals were anesthetized by an 300 μl i.p. injectionof 2.5% avertin (100% stock prepared by mixing 10 g tribromoethyl alcohol in 10ml tertiary amyl alcohol).

Wound-healing assaysAhr+/+ and Ahr–/– mice were anesthetized using avertin and their dorsal skin shavedand sterilized by topical application of povidone. Typically, two wounds of 4 mm indiameter were performed in each flank of each mouse (total of four wounds in eachanimal) for experiments requiring treatments. Untreated mice received a single woundin each dorsal flank. Experiments were performed using 3-4 mice of each genotype.Skin biopsies containing both dermis and epidermis were taken from each wound atdays 3, 5, and 7 after surgery. Tissues were fixed at 4°C in 4% paraformaldehydeand processed for immunohistochemistry as described below. In some experiments,wounds were treated in vivo with anti-TGFβ neutralizing antibody or with AhR-antisense (ODN-As) or AhR-sense (ODN-Se) oligonucleotides as detailed below.Sections were routinely obtained at 8 μm.

Whole-skin explant cultureWhole-skin biopsies of 4 mm in diameter were obtained from the dorsal area ofshaved Ahr+/+ and Ahr–/– mice. These tissues were flattened with their dermis downon tissue culture plates previously treated with 5 μg/ml collagen or 15 μg/mlfibronectin. Experiments were performed in the absence of serum to prevent cellproliferation. To obtain a kinetic of keratinocytes migration from the explants, pictureswere taken every 24 hours for 7 days using a NIKON TE2000U microscope. Migrationat each time point was quantified using the ImageJ software as the distance from theedge of the skin explant to the border of the keratinocyte monolayer.

Primary keratinocytes and dermal fibroblast culturePrimary keratinocytes and dermal fibroblasts were obtained from Ahr+/+ and Ahr–/–

newborn mice at 2-3 days of age. After sterilization in povidone solution, mice werewashed in sterile water and rinsed in 70% ethanol in PBS (137 mM NaCl, 2.7 mMKCl, 4.3 mM PO4HNa2, 1.5 mM PO4H2K pH 7.2). All four legs and the tail wereremoved and the complete skin dissected using forceps. The resulting skins were floateddermis down in sterile culture dishes containing 0.25% trypsin for 16-18 hours at 4°C.Next, dermis and epidermis were separated and individually minced in 2-3 ml/mouseof plating medium (E-MEM containing 4% fetal bovine serum pre-treated with chelexand 0.2 mM Ca2+ and gentamycin as antibiotic). Tissues were further digested byincubation for 45 minutes at 4°C with gentle agitation. For the preparation ofkeratinocytes, the digested epidermis was filtered through a 140 μm mesh to removeaggregates and undigested tissue and the cell suspension was seeded at a density of2�106 cells in 60 mm culture plates pre-treated with 5 μg/ml collagen or 15 μg/mlfibronectin. After 24 hours, keratinocytes were washed with PBS and grown inmaintenance culture medium (plating medium supplemented with 0.05 mM Ca2+) topromote proliferation and to inhibit differentiation. Dermal fibroblasts were obtainedfrom mouse skin following the protocol used in our laboratory to isolate mouse embryofibroblasts (Santiago-Josefat et al., 2001). Conditioned medium was produced byculturing Ahr+/+ and Ahr–/– keratinocytes and Ahr+/+ and Ahr–/– dermal fibroblasts for72 hours in E-MEM or OptiMEM, respectively. For keratinocyte wound-closure assays,cells were allowed to reach confluence in serum-containing medium and wounds wereperformed with the aid of a pipette tip. After incubation for 15 hours in serum-freemedium, culture plates were photographed in a NIKON TE2000U microscope.

ImmunohistochemistryTissue sections were deparaffinized and gradually rehydrated to PBS. Endogenousperoxidase activity was blocked by treatment with H2O2 for 45 minutes at roomtemperature (1% H2O2 diluted in PBS-T: PBS containing 0.05% Triton X-100). Afterrinsing in PBS-T, non-specific epitopes were blocked by incubation for 1 hour inPBS-T containing 2 mg/ml gelatin and 0.1 M lysine. Sections were then incubatedovernight at 4°C with the corresponding primary antibodies diluted in PBS-T-gelatin.Following extensive washing in PBS-T-gelatin, sections were incubated for 1 hourat room temperature with the appropriate biotinylated secondary antibody. Afteradditional washing, tissues were incubated with peroxidase-conjugated streptavidinand color developed using a diaminobenzidine (DAB) solution (0.025% DAB w/v,0.06% H2O2 v/v in PBS). Sections were dehydrated, mounted and visualized usinga NIKON TE2000U microscope. PCNA, Smad2-P, Ly-6G and F4/80-reactive cellswere quantified using ImageJ software. Immunofluorescence for AhR in whole skinsections was performed as described above using a mouse anti-AhR primary antibodyand an Alexa Fluor 488-labeled secondary antibody.

Hematoxylin and eosin stainingDeparaffinized and rehydrated sections of skin wounds were incubated with Harrishematoxylin for 3 minutes at room temperature. After washing with tap water, eosin

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solution was added for 1 minute. A final washing step was performed and the tissueswere dehydrated, mounted and observed in a NIKON TE2000U microscope.

Sirius red and fast green staining, and hydroxyproline enzymaticassayThe presence of total collagen in the granulation tissue was analyzed using Siriusred and fast green staining as previously described (Gascon-Barre et al., 1989; Lopez-De Leon and Rojkind, 1985; Peterson, 1993). Briefly, tissue sections were incubatedin 0.04% fast green in saturated picric acid for 15 minutes at room temperature.Sections were then washed with distilled water and further incubated for 30 minutesat room temperature in 0.04% fast green containing 0.1% Sirius red in picric acid(Sigma). After washing in distilled water, sections were mounted and observed in aNIKON TE2000U microscope. Collagen content was also measured in the woundsby quantifying hydroxyproline levels as described previously (Sauzeau et al., 2007).

In vivo treatment with a TGFβ antibody and with AhR antisenseoligonucleotideTo analyze how blockade of TGFβ activity affects wound healing in vivo, aneutralizing antibody for this cytokine was used. The two wounds on one flank ofthe dorsal skin of Ahr–/– mice were injected in their marginal area with three dosesof 50 μl of neutralizing anti-TGFβ antibody at 50 μg/ml concentration. The twowounds on the opposite flank in each mouse were injected under the same conditionswith sterile PBS. Wounds were dissected and processed for histology andimmunohistochemistry as indicated above.

To determine how AhR downmodulation affects re-epithelialization in vivo, anAhR antisense oligonucleotide was applied to Ahr+/+ wounds. Both AhR antisenseoligonucleotide and a negative control sense oligonucleotide were synthesized asdescribed (Peters and Wiley, 1995). To prevent degradation, oligonucleotides weremodified by the addition of phosphorothiolated linkages at their 5� and 3� ends. Thesequence of the antisense oligonucleotide is fully complementary to that of the murineAhR mRNA between nucleotides 39 and 59. Sequences used were: antisense, 5�-GGGGATGGGCTTTACTGTTT-3� and sense, 5�-AACCTTGGGTTTGGGTTTGG-3�. Before use, oligonucleotides were mixed with 30% pluronic F127 (Sigma) insterile PBS to obtain a soft gel. The two wounds on one flank of each Ahr+/+ micewere treated with 50 μl antisense oligonucleotide whereas the two wounds on theopposite flank were treated with the same amount of control sense oligonucleotide,as described (Mori et al., 2008; Reynolds et al., 2008). Wound tissue was dissectedand analyzed by histology and immunohistochemistry as indicated above.

SDS-PAGE and western immunoblottingSDS-PAGE and western immunoblotting for TGFβ in whole-skin cell extracts wereperformed essentially as described (Mulero-Navarro et al., 2005).

Statistical analysesData are shown as mean ± s.e.m. Statistical comparison between experimentalconditions was done using GraphPad Prism 4.0 software (GraphPad). Comparisonsbetween conditions were made using unpaired Student’s t-test.

We are very grateful to Francisco Javier Martin-Romero for assistancewith microscopy. A detailed protocol for TGFβ treatment in vivo waskindly provided by Louise E. Reynolds and Kairbaan M. Hodivala-Dilke. This work was supported by Grants from the Spanish Ministryof Education and Sciences (SAF2005-00130 and SAF2008-00462),from the Junta de Extremadura (2PR04A060) and from the RedTemática de Investigación Cooperativa en Cáncer (RTICC)(RD06/0020/1016, Fondo de Investigaciones Sanitarias (FIS), CarlosIII Institute, Spanish Ministry of Health) (to P.M.F.-S.). A.C.R. andJ.M.C.-G. were supported by fellowships from the Spanish Ministryof Education and Sciences and Junta de Extremadura, respectively. AllSpanish funding is co-sponsored by the European Union FEDERprogram.

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