In Utero Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin Causes Accelerated Terminal Differentiation in Fetal Mouse Skin

Jennifer A. Loertscher*, Tien-Min Lin{dagger}, Richard E. Peterson*,{dagger} and B. Lynn Allen-Hoffmann*,{ddagger},1

* Molecular and Environmental Toxicology Center and {dagger} School of Pharmacy, University of Wisconsin, Madison, Madison, Wisconsin 53706; {ddagger} Department of Pathology, University of Wisconsin Medical School, Madison, Wisconsin 53706

Received September 7, 2001; accepted April 1, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD), a ubiquitous environmental toxin, has been shown to cause a human skin pathology called chloracne. The majority of laboratory mouse strains, with the exception of mice bearing a mutation in thehairless gene, fail to display overt signs of chloracne upon exposure to TCDD. As a result, only minimal data exist on the effects of TCDD in adult haired mice and no data exist on the effects of TCDD in developing mouse skin. Here we report that TCDD affects the temporal expression of protein markers of keratinocyte terminal differentiation during murine skin morphogenesis. Immunohistochemical analysis of E16 mice reveals accelerated expression of the intermediate filament-associated protein filaggrin in response to TCDD. At a later developmental time and after birth, expression of filaggrin and loricrin is indistinguishable between treatment and control groups. At E16 expression of keratins 5, 6, and 10 are unaltered in TCDD-exposed individuals and TUNEL analysis shows no apoptotic cells in the basal and spinous layers of either treatment or control groups. At E16, immunohistochemical analysis of AhR-null mouse skin reveals accelerated filaggrin expression in both vehicle and TCDD exposed animals. We therefore hypothesize that AhR acts as a modulator of late stage keratinocyte terminal differentiation.

Key Words: TCDD; skin; AhR; filaggrin; mouse.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is one congener of a family of halogenated aromatic hydrocarbons (HAH) known as dioxins (reviewed in Safe, 1990Go). Dioxins are ubiquitous environmental toxins whose chemical stability and lipophilicity make them highly persistent in the environment and biological systems. Exposure of mammals to TCDD produces an array of pathological manifestations including teratogenesis, dermatopathology, hepatotoxicty, and wasting syndrome (reviewed in Poland and Knutson, 1982Go). Most, if not all, TCDD-induced pathologies are mediated via TCDD binding to the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor (reviewed in Schmidt and Bradfield, 1996Go). Although the effects of dioxin are diverse and tissue-specific, a growing body of research has shown that common features among the effects relate to dioxin’s ability to alter epithelial morphogenesis and homeostasis.

Chloracne, a hyperkeratotic skin disorder affecting the hair follicle and interfollicular epidermis, has historically been the clinical hallmark of TCDD exposure in humans. Clinical examination of exposed patients 30 to 60 days after exposure revealed cysts and open comedones. Histological analysis of the skin of patients with clinical chloracne reveals hyperkeratosis of the follicular and interfollicular epidermis and a variety of morphological abnormalities related to the sebaceous and eccrine glands (Caputo et al., 1988Go; Moses and Prioleau, 1985Go).

Attempts to understand the mechanistic basis of chloracne have been hindered by the lack of an adequate animal model. Inbred laboratory mouse strains express either one of two AhR alleles: Ahb, the aromatic hydrocarbon responsive allele, which binds ligand with high affinity, or Ahd, the nonresponsive allele, which binds ligand with low affinity (Thomas et al., 1972Go). Genetic experiments using mice homozygous for either allele showed a greater, dose-dependent induction of xenobiotic-metabolizing enzyme activity in animals expressing the high-affinity receptor (Poland and Glover, 1975Go). Despite the fact that mice bearing the high affinity Ahb allele develop an array of pathologies in response to TCDD, most strains of laboratory mice, including those with high affinity receptor, do not develop the severe, TCDD-induced dermatopathology observed in humans and nonhuman primates. As a result, studies of TCDD effects on squamous differentiation have been limited to mice with a mutation in the hairless gene locus, which do display chloracne upon TCDD exposure (Knutson and Poland, 1982Go). Although the hairless mutation may provide a genetically sensitized background that reveals TCDD-induced dermatopathologies, haired murine skin is not refractory to TCDD and merits further examination. Prior to studies presented here, assessment of TCDD effects on murine skin has relied primarily on histological analysis and enzymatic activity assays (Poland et al., 1984Go; Puhvel and Sakamoto, 1988Go). Examination of terminal differentiation marker expression has been limited to the keratins (Panteleyev et al., 1997Go). In these studies, we take a new approach and present evidence that TCDD affects expression of filaggrin, a late stage marker of keratinocyte terminal differentiation, while leaving keratin expression unaffected.

Skin is a continually renewing tissue and acts as a defense system against environmental insult by providing a barrier function for the body. Skin is composed of two layers, the dermis, a mesenchymal tissue and the epidermis, a stratified squamous epithelium. The function of skin as a barrier depends in part on the ability of keratinocytes within the epidermis to undergo a maturation process known as terminal differentiation. During the process of terminal differentiation, keratinocytes undergo dramatic and predictable changes in morphology and protein expression. Ultimately, keratin fibrils are organized into bundles and encased by the cornified envelope, a durable outer shell composed of highly cross-linked proteins (reviewed in Eckert et al., 1997aGo,bGo). Barrier function depends not only on coordinated expression of keratins and other differentiation-specific proteins including filaggrin and loricrin, but also on production and secretion of lipids resulting in a cornified layer that is not only mechanically strong, but also watertight (Downing, 1992Go; Nemes and Steinert, 1999Go).

In this study we present data describing the effects of TCDD on developing skin of the C57Bl/6J strain, which possesses the Ahb allele. Examination of mouse fetal skin at E16, a time of dramatic changes in murine development, reveals accelerated expression of the differentiation-specific protein filaggrin in individuals exposed in utero to TCDD at E13. Keratin expression is not affected at this time and ultimate formation of a morphologically normal epidermis is not impaired. Surprisingly, AhR-null fetuses at E16 express filaggrin in the absence of TCDD. We therefore conclude that AhR has an endogenous role in modulation of some aspects of keratinocyte terminal differentiation during mouse skin morphogenesis and that this function may be disrupted in the presence of an AhR ligand, such as TCDD.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Treatment of animals.
Mice heterozygous for the exon II knockout of AhR were a kind gift of Dr. Christopher A. Bradfield (Schmidt et al., 1996Go). A colony of these mice was established in the University of Wisconsin School of Pharmacy Animal Facility. Dams were kept individually in clear plastic cages and maintained on a 0600–1800 h light:dark cycle in a temperature controlled room (24 ± 1°C) with 35 ± 4% relative humidity. The mice were provided food (Purina 5015) and tap water ad libitum. Animals used in this study have been backcrossed onto the C57Bl/6J background for 11–13 generations. Heterozygote matings were performed for all experiments generating pups of all possible genotypes (AhR +/+, +/-, -/-) in each litter. Mating pairs were placed together in the evening and the following morning (if dams were sperm plug positive) was considered E0. Pregnant dams were treated by po gavage on E13 with 5 µg/kg TCDD in oil and fetuses harvested on E16 and E18 and pups on postnatal day 2 (PND 2).

Genotyping of animals.
Fetal tail tissue (2 mm) was lysed in 25 µl buffer (50 mM Tris-HCl, 20 mM NaCl, 1 mM EDTA, and 1% SDS, pH 8.0) and 1 µl of 20 mg/ml Proteinase K. Digestion was done at 65°C for 30 min with 15 s vortexes after 15 and 30 min of incubation. The final volume was brought up to 275 µl with Milli-Q water and the mixture was boiled for 5 min. PCR was performed using 1 µl of the mixture. PCR mixture (20 µl) consists of 1x Amplitaq buffer, 0.2 mM dNTPs, 5% DMSO, 0.5 u AmpliTaq, and 4 primers (2 µM NeoF [ttg ggt gga gag gct att cg], 2 µM NeoR [cca ttt tcc acc atg ata ttc g], 2.5 µM AhRI3F [tct tgg gct cga tct tgt gtc a], 2.5 µM AhRX2RB [ttg act taa ttc ctt cag cgg]). The expected wild-type PCR product is 670 bp in length while the mutant product is 560 bp in length. Conditions for PCR were as follows: 940C for 2 min initial denaturation, 35 cycles of 94°C for 5 s, 60°C for 30 s, and 72°C for 1 min, with a final extension at 72°C for 2 min.

Histology.
Skin was removed from the back of fetuses and pups, pinned flat to a foam surface, and fixed in 4% paraformaldehyde in PBS overnight. Samples were paraffin embedded, sectioned, and stained with hematoxylin and eosin. Sections were examined using an Olympus IX-70 microscope.

Immunohistochemical analysis.
Polyclonal primary antibodies used were antimouse filaggrin, antimouse loricrin, antimouse keratin 5, antimouse keratin 6, and antimouse keratin 10 (Babco/Covance, Richmond, CA). For filaggrin, loricrin, keratin 5, and keratin 10 immunohistochemistry, paraffin-embedded skins were serially sectioned (5 µm), mounted on glass slides and deparaffinized in xylene followed by an ethanol series. For keratin 6 immunohistochemistry cryopreserved skin sections were used. Skin was removed from fetuses, fixed for 2 h in 1% paraformaldehyde in PBS, and equilibrated in 20% sucrose in PBS overnight. Prepared skins were frozen in Tissue-Tek O.C.T. Compound (Sakura Finetec Inc., Torrance, CA), sectioned with a cryostat, mounted on glass slides and fixed for 5 min in ice cold acetone.

Subsequent to the above-described procedures, frozen and paraffin-embedded sections were handled identically. Tissue sections were blocked for 1 h at room temperature using 5% normal goat serum (Sigma, St. Louis, MO) in PBS. Sections were incubated with primary antibody (1:1000 antifilaggrin, 1:1000 antikeratin 5, 1:1000 antikeratin 6, 1:500 antikeratin 10, 1:250 antiloricrin) for 1 h in a humidified chamber at room temperature. Sections were then washed in PBS and incubated with secondary antibodies, a 1:500 dilution of Alexa 594-conjugated goat antirabbit IgG (Molecular Probes, Eugene, OR). All sections were counterstained with 5 µg/ml Hoechst 33258. Samples were viewed with an IX-70 inverted fluorescence microscope (Olympus) equipped with Hoechst (462 ± 1.5 nm), and Texas Red (627 nm ± 2 nm) band pass filters. Digital images were captured by an Optronics DEI-750 CE camera (Goleta, CA) using ImagePro Plus software (Media Cybernetics, Silver Spring, MD).

TUNEL analysis.
Free 3' ends of DNA were detected in paraffin-embedded mouse back skin sections (5 µm) using the APOPTAG Fluorescein in situ Apoptosis Detection kit (Intergen, Purchase, NY). Sections were deparaffinized and protein digested with 20 µg/ml proteinase K for 15 min in water. Terminal deoxynucleotidal transferase (TdT) in sample buffer was applied. Samples were covered with plastic coverslips and incubated for 1 h at 37°C. Slides were placed in 37°C stop buffer for 30 min. Antidigoxin fluor, diluted in block buffer, was applied. Samples were covered with plastic coverslips and incubated 1 h at room temperature. Samples were counterstained with Hoechst 33258. Sections were viewed with an IX-70 inverted fluorescence microscope (Olympus) equipped with FITC (462 ± 1.5 nm) and Hoechst (525 ± 20 nm) band pass filters. Digital images were captured by an Optronics DEI-750 CE camera (Goleta, CA) using ImagePro Plus software (Media Cybernetics, Silver Spring, MD).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TCDD Alters the Expression Pattern of Terminal Differentiation-Associated Protein Markers in E16 Mouse Fetuses
In order to assess the impact of TCDD on mouse skin development, epidermal morphology and protein expression were examined in E16 fetuses of dams exposed to 5 µg/kg TCDD at E13. More than 20 fetuses from at least 6 different litters were examined for each treatment group. Morphological examination of skin collected from the back of mouse fetuses revealed a well-organized, normal epidermis in both TCDD- and vehicle-exposed individuals. Basal, spinous, and granular compartments are all visible in hematoxylin and eosin stained sections (Figs. 1A and 1BGo).



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FIG. 1. Exposure of mouse fetuses to TCDD causes premature expression of filaggrin at E16. Fetuses were exposed to either oil (A, C, and E) or TCDD (B, D, and F) at E13 and harvested at E16. Back skins were fixed, sectioned, and stained with hematoxylin and eosin (A and B), antifilaggrin antibody (C and D), or antiloricrin antibody (E and F). Original magnification x400.

 
Immunohistochemical examination of E16 skin revealed observable differences in expression of differentiation-specific protein markers between TCDD- and vehicle-exposed groups. The most striking effect of TCDD exposure is accelerated onset of filaggrin expression. Filaggrin, an intermediate filament-associated protein, is responsible for aggregating keratin filaments into the well-organized bundles of fibrils characteristic of the cornified layer (reviewed in Dale et al., 1994Go). In developing mouse skin filaggrin expression is first observable at E17 (Bickenbach et al., 1995Go). As expected based on previous reports of filaggrin’s temporal expression, we observed little to no filaggrin expression at E16 in the back skin of vehicle control fetuses (Fig. 1CGo). In contrast, greatly expanded filaggrin expression is visible in the back skin of TCDD-exposed individuals at E16 (Fig. 1DGo). Although the level of expression is premature, the punctate pattern of expression in the granular layer is apparent. Filaggrin protein is synthesized as a precursor, profilaggrin. Dephosphorylation and proteolytic processing of profilaggrin releases mature filaggrin, which is able to carry out its function of keratin filament aggregation (Dale et al., 1997Go; Resing et al., 1985Go). The antifilaggrin antibody employed in these studies detects both profilaggrin and mature filaggrin (Yuspa et al., 1989Go).

We next investigated whether expression of other terminal differentiation-associated protein markers was altered by TCDD exposure. Loricrin, a substrate for keratinocyte-specific transglutaminase, is one of the primary components of the mature cornified envelope (Mehrel et al., 1990Go). Loricrin expression begins in the granular layer at E16 (Yoneda and Steinert, 1993Go) where it is found in keratohyalin granules with profilaggrin. Ultimately, loricrin is cross-linked by transglutaminase and localizes to the cell periphery in the cornified layer (Mehrel et al., 1990Go; Yoneda et al., 1992Go). We observed loricrin expression in the granular layers of back skin obtained from both TCDD-treated and vehicle control animals at E16. Staining was modestly more intense in TCDD-exposed individuals (Figs. 1E and 1FGo).

Expression of specific keratin family members also varies according to differentiation status of keratinocytes. Basal keratinocytes express the K5/K14 pair of keratin proteins, whereas keratinocytes committed to terminal differentiation express the K1/K10 pair (reviewed in Fuchs, 1995Go). K5 mRNA expression is detected early in epidermal development at E9.5 in specific zones of the embryo and by E12.5, K5 is detected throughout the stratified ectoderm (Byrne et al., 1994Go). Expression of the differentiation-specific keratins K1 and K10 is first detectable in specific areas at E13.5, but exhibits a dramatic increase at E15.5. We observed no change in differentiation-specific keratin protein expression at E16 in response to TCDD. K5 was detected in the basal and immediately suprabasal layers (Figs. 2A and 2BGo) and K10 (Figs. 2C and 2DGo) was appropriately expressed in all suprabasal keratinocytes regardless of treatment conditions. Staining patterns were indistinguishable between TCDD and vehicle-exposed animals. A third keratin family member, keratin 6 (K6) is normally not expressed in adult interfollicular epidermis, but rather is specifically expressed in hyperproliferative disease states and hair follicles in mature skin and in the periderm in developing skin (Bickenbach et al., 1995Go; Mazzalupo and Coulombe, 2001Go). In developing skin K6 is expressed as the periderm, a single cellular layer that covers the epidermis, until E18 when it is shed and replaced by the fully matured cornified layer (Bickenbach et al., 1995Go; Byrne et al., 1994Go; Yoneda and Steinert, 1993Go). Immunohistochemical analysis of TCDD- and vehicle-exposed E16 embryos reveals K6 protein expression in the outermost layer of both groups, indicating that at E16 periderm is present regardless of treatment (Figs. 2E and 2FGo).



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FIG. 2. TCDD exposure does not affect keratin protein expression at GD16. Fetuses were exposed to either oil (A, C, and E) or TCDD (B, D, and F) at E13 and harvested at E16. Back skin was fixed, sectioned, and stained with antikeratin 5 (A and B), antikeratin 10 (C and D), or antikeratin 6 (E and F) antibodies. Original magnification x300 (K5) or x400 (K10 and K6).

 
TCDD Exposure Does Not Perturb Ultimate Epidermal Morphogenesis
No histological differences were detected in skin collected from the backs of TCDD- and vehicle-exposed E18 mouse fetuses and PND 2 neonatal mice (at least 3 individuals from each treatment group and time point were examined). At E18, skin is fully stratified with a prominent cornified layer (Figs. 3A and 3BGo). In both treatment and control groups the periderm has been shed as evidenced by lack of K6 staining at the outermost surface of the skin (data not shown). Immunohistochemical analysis of E18 skin reveals plentiful, punctate expression of filaggrin in the granular layers of both TCDD- and vehicle-exposed animals (Figs. 3C and 3DGo). Furthermore, the expression pattern of loricrin is appropriate and indistinguishable between TCDD-treated and control groups (Figs. 3E and 3FGo). Skin morphology is normal in both TCDD-treated and control 2-day-old mouse neonates (Figs. 3G and 3HGo). Because initial analysis of skin at E18 and PND2 revealed no histological or immunohistochemical differences between TCDD- and vehicle-exposed groups, we terminated experiments at those time points after examination of a relatively small number of individuals and focused resources into analysis of E16 fetuses. Based on the samples analyzed, we conclude that although TCDD causes alterations in protein expression at specific points during skin development, it does not interfere with the ability to ultimately form a histologically normal epidermis.



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FIG. 3. TCDD exposure does not preclude filaggrin expression and formation of an intact epidermis as seen at E18 and PND 2. Fetuses were exposed to either oil (A, C, E, and G) or TCDD (B, D, F, and H) at E13. At E18 (A–F) and PND 2 (G and H) animals were harvested, back skin was fixed, sectioned, and stained with hematoxylin and eosin (A, B, G, H), antifilaggrin antibody (C and D), or antiloricrin antibody (E and F). Original magnification x400.

 
In Utero TCDD Exposure Does Not Induce Apoptosis in Developing Mouse Skin
TCDD has been shown to impact apoptotic cascades in a variety of systems including thymus and developing palate (Abbott and Birnbaum, 1989Go; Kamath et al., 1997Go). Furthermore, some skin pathologies, such as psoriasis, have been linked to abnormal epidermal apoptosis (Karasek, 1999Go). In order to confirm that TCDD exposure in utero specifically affects keratinocyte terminal differentiation without inducing aberrant apoptotic events, we performed a TUNEL assay to identify free 3' ends of DNA in tissue sections obtained from the backs of TCDD- or vehicle-exposed animals. In both cases we observed no TUNEL-positive cells in the basal or spinous layers (Fig. 4Go). This is in agreement with observations made of TCDD-treated organotypic cultures of human keratinocytes (Loertscher et al., 2001Go). Keratinocytes grown in organotypic culture and exposed to radiation or cycloheximide exhibited many labeled cells and acted a positive control for the assay (data not shown). We therefore conclude that TCDD’s ability to induce apoptosis during development is a tissue-specific phenomenon.



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FIG. 4. TCDD does not induce apoptosis in fetal mouse skin at E16. Fetuses were exposed to either oil (A and B) or TCDD (C and D) at E13. In situ DNA 3' end-labeling was performed and the fluorescent label visualized (A and C). Sections were counterstained with Hoechst to visualize the nuclei (B and D). Original magnification x300.

 
AhR Acts as a Modulator of Filaggrin Expression
Many, if not all, of the known effects of TCDD on animals are mediated through AhR. In order to verify that the observed skin effects are AhR-dependent, we examined the expression of a differentiation-specific protein in AhR-null E16 fetuses. Seven vehicle-exposed AhR-null individuals from 4 different litters and 11 TCDD-exposed AhR-null individuals from 4 different litters were examined. To our surprise, vehicle-exposed AhR-null animals exhibited greatly elevated filaggrin protein expression as compared to wild type animals (Figs. 5A and 5CGo). A range of filaggrin expression was observed at E16 in vehicle-exposed, AhR-null animals, with most skin sections resembling expression seen in TCDD-exposed wild type animals. Filaggrin expression in TCDD-exposed, AhR-null individuals was slightly less than those observed in TCDD-exposed wild type embryos (Figs. 5B and 5DGo).



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FIG. 5. AhR is a modulator of terminal differentiation in embryo mouse skin. At E16 AhR wild type and AhR-null fetuses were harvested. Back skin was fixed, sectioned, and stained with antifilaggrin antibody. Panels A and B depict filaggrin expression in AhR-null individuals exposed to either vehicle (A) or TCDD (B). Filaggrin expression in wild type individuals exposed to either vehicle (C) or TCDD (D) is depicted. Original magnification x400.

 
Because only representative images of filaggrin expression in each genotype/treatment group could be reproduced here, a graphical summary of relative filaggrin expression in all E16 fetuses analyzed based on calculation of weighted averages for each AhR genotype and treatment group is shown in Figure 6Go. Weighted averages were calculated by assigning a quantitative value (0–3) to a qualitative level of filaggrin expression observed in sections of E16 mouse skin stained with an antifilaggrin antibody. A value of 0 was assigned when no expression was observed; 1 when discontinuous, patchy expression was observed; 2 when a thin, continuous band of expression was observed; and 3 when a broad, continuous band of expression was observed. These values were then summed and divided by the total number of individuals analyzed in each AhR genotype and treatment group.



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FIG. 6. Summary of relative levels of filaggrin expression as determined by calculation of weighted averages. Weighted averages were calculated by attributing numerical values to the relative level of filaggrin expression in each E16 fetus examined (0 = no stain visible, 1 = discontinuous patches of stain, 2 = thin band of continuous stain, 3 = broad band of continuous stain), summing these numbers, and dividing by the total number of individuals in each group. This graph illustrates an acceleration of filaggrin expression both in response to TCDD and in the absence of AhR. AhR wild type, vehicle-exposed (WT Veh), AhR wild type, TCDD-exposed (WT TCDD), AhR-null, vehicle-exposed (KO Veh), AhR-null, and TCDD-exposed (KO TCDD) are presented.

 
The above-described observations suggest that, in the absence of xenobiotics, AhR influences the temporal expression of some, but not all, keratinocyte terminal differentiation-associated proteins, as evidenced by premature filaggrin expression in AhR-null fetuses. Furthermore, based on the fact that TCDD-exposure of wild type fetuses mimics the effects observed when AhR is absent, we conclude that TCDD most likely exerts its effects on skin by diverting AhR from its endogenous function in keratinocyte terminal differentiation.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In utero exposure of mice to TCDD is known to be teratogenic resulting in a spectrum of pathologies including cleft palate and hydronephrosis (Abbott and Birnbaum, 1989Go; Bryant et al., 1997Go). Although the cell biological effects of TCDD on developing palate and urinary tract epithelia have been extensively studied, the impact of TCDD on developing epidermis has not been explored.

In this study of the effects of TCDD on murine epidermal morphogenesis, we found that exposure of mouse fetuses to TCDD in utero results in alterations in keratinocyte terminal differentiation. These findings provide evidence that TCDD has an impact on murine skin development in strains not bearing the hairless mutation. Furthermore, examination of AhR-null animals also reveals alterations in keratinocyte terminal differentiation in the absence of an exogenous ligand, suggesting that AhR functions as an endogenous modulator of late stage keratinocyte terminal differentiation. We therefore hypothesize that TCDD may exert its effects on developing skin through disruption of endogenous AhR function.

The toxic effects of TCDD are highly variable among species. As a result, development of an appropriate animal model for the TCDD-induced human dermatopathology, chloracne, has been challenging. Primates and rabbits develop the gross clinical and histological characteristics observed in human chloracne upon TCDD exposure, whereas standard laboratory mouse strains do not. However, pioneering rodent studies performed by Poland and coworkers showed that TCDD treatment of mice mutant at the hairless gene locus causes severe dermatopathology similar to human chloracne. The effects include a dose- and time-dependent development of epidermal hyperplasia and hyperkeratosis, metaplasia of the sebaceous gland, and keratinization of dermal cysts (remnants of hair follicles; Knutson and Poland, 1982Go; Poland et al., 1984Go). Subsequent biochemical and histological analysis of the effects of TCDD on haired murine skin revealed that a mutation in the hairless gene is not absolutely required for TCDD to exert effects on this tissue. Puhvel and Sakamoto observed sebaceous gland involution and increased transglutaminase activity in the skin of haired mice following TCDD exposure, but did not observe other pathologies characteristic of chloracne such as epidermal hyperplasia and an intense inflammatory response (Puhvel and Sakamoto, 1988Go). Similarly we found that TCDD alters aspects of keratinocyte terminal differentiation during epidermal morphogenesis in the skin of mouse embryos that are normal at the hairless gene locus.

Examination of AhR knockout animals has begun to define an endogenous role for AhR in tissue morphogenesis (Schmidt et al., 1996Go). Physiological investigation of liver defects in AhR-deficient mice conducted by Lahvis and coworkers suggests that AhR mediates the formation of three-dimensional structures during development. In this study, decreased liver size could be directly linked to a reduction in hepatocyte size, which subsequently was found to be due to a vasculature defect causing massive portosystemic shunting. Vascular anomalies were also found in the kidney and eye (Lahvis et al., 2000Go). The involvement of AhR in the formation of three-dimensional vascular structures fits well into an emerging body of evidence in Drosophila, which indicates that many PAS family members respond to environmental signals by directing the formation of three-dimensional structures (reviewed in Crews, 1998Go). Our investigation of the AhR dependency of TCDD-induced changes in skin morphogenesis revealed that AhR-null, untreated embryos exhibit premature filaggrin expression. Based on these observations, it is tempting to speculate that in stratified squamous epithelia, AhR may participate in signaling necessary for establishment of the functional three-dimensional structure of skin.

The process of keratinocyte terminal differentiation relies on cell type-specific and proper spatio-temporal modulation of the expression of a spectrum of terminal differentiation-specific proteins. Although AhR is not required for skin development or stratification, it could be acting in combination with other transcription factors, such as AP-1 family members, to modulate expression of terminal differentiation-specific proteins by directly binding to regulatory elements in the DNA. An AhR-responsive gene we have identified is filaggrin. Little 5'-regulatory sequence is available for the mouse filaggrin gene. However, 3600 bp upstream of the transcriptional start site have been sequenced in the human profilaggrin gene (Markova et al., 1993Go). TCDD-treatment of organotypic cultures of human keratinocytes and dermal fibroblasts results in temporal and spatial acceleration of characteristics of terminal differentiation, including filaggrin expression (Loertscher et al., 2001Go). These results in human cells mirror those reported here and suggest that human and mouse profilaggrin genes respond similarly to TCDD. Analysis of the available human profilaggrin sequence using MatInspector (Quandt et al., 1995Go) revealed 2 putative XREs (GCGTG core sequence) at 1340 and 1947 bp upstream of the transcriptional start site, suggesting that TCDD may have a direct, AhR-dependent impact on transcription of the filaggrin gene. This assertion is supported not only by data presented here, but also by recent examination of skin-specific ablation of Arnt, the dimerization partner of AhR. Mice lacking Arnt specifically in skin die shortly after birth due to severely compromised barrier function (Takagi et al., 2001Go).

Although the most dramatic effects reported here pertain to changes in filaggrin expression, it is possible that TCDD-induced or AhR-dependent differences in expression of other differentiation-specific proteins were not observed because we chose to look too late in gestation. Filaggrin expression in the untreated mouse fetus normally begins at E17, 1 day after our earliest time point, E16. As a result we definitively observed an acceleration of filaggrin expression in response to TCDD because it was present 1 day earlier than expected. In contrast, TCDD-induced changes in loricrin expression at E16 are subtle and are characterized by a broadening of the region of loricrin expression in TCDD-exposed animals. By E18 this difference is no longer observable, indicating that the increased expression observed at E16 is due to an acceleration of loricrin expression, an observation that may have been more dramatic if we had chosen to look at E15, 1 day before loricrin expression would be expected. In this study no differences in expression of any keratins were observed in response to TCDD. Because keratins are expressed earlier in epidermal morphogenesis than filaggrin, we simply may have failed to detect TCDD-induced accelerated keratin expression because of our choice of time points. However, studies conducted in adult HRS/J hairless mouse skin suggest that this is not the case. Panteleyev and coworkers also observed no changes in interfollicular expression of K1 (the dimerization partner of K10) mRNA in response to TCDD treatment (Panteleyev et al., 1997Go). Although additional information about the influence of TCDD on keratin expression during development may be obtained by examining earlier gestational times, we find it likely that TCDD alters expression of some terminal differentiation-associated proteins (filaggrin and loricrin) during epidermal morphogenesis, while leaving expression of differentiation-associated keratin proteins unaffected (keratins).

In conclusion, we have observed TCDD-induced alterations in aspects of keratinocyte terminal differentiation in developing mouse skin. The most dramatic effect at the times analyzed is acceleration of filaggrin protein expression observed at E16. Additionally analysis of AhR-null embryos revealed accelerated expression of filaggrin in AhR-null animals in developing skin. We therefore hypothesize that AhR participates in modulating keratinocyte terminal differentiation and that TCDD may exert its toxic effects on skin by disrupting this function. Further studies are underway to investigate other late stage differentiated functions such as lipid synthesis and processing required for establishment of barrier function.


    ACKNOWLEDGMENTS
 
Many thanks to the staff at the McArdle Laboratories histology facility for processing of skin samples. Thanks also to Ulla Simanainen for assistance in treatment of mice. We would also like to acknowledge Allen Comer for his intellectual contributions. Research supported by NIH-AR-42853 (L.A.H.), NIH-AR-40284 (L.A.H.), NIH ES01332 (R.E.P.), and the University of Wisconsin Industrial and Economic Development Research Fund (L.A.H.). J.A.L. was supported by grant number T32 ES07015 from the National Institute of Environmental Health Sciences (NIEHS), NIH. Contribution 336, Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, WI 53706.


    NOTES
 
1 To whom correspondence should be addressed at 5605 Medical Sciences Center, 1300 University Avenue, Madison, WI 53706. Fax: (608) 265-3058. E-mail: blallenh{at}facstaff.wisc.edu. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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