* Molecular and Environmental Toxicology Center and
School of Pharmacy, University of Wisconsin, Madison, Madison, Wisconsin 53706;
Department of Pathology, University of Wisconsin Medical School, Madison, Wisconsin 53706
Received September 7, 2001; accepted April 1, 2002
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ABSTRACT |
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Key Words: TCDD; skin; AhR; filaggrin; mouse.
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INTRODUCTION |
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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., 1988; Moses and Prioleau, 1985
).
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., 1972). 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, 1975
). 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, 1982
). 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., 1984
; Puhvel and Sakamoto, 1988
). Examination of terminal differentiation marker expression has been limited to the keratins (Panteleyev et al., 1997
). 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., 1997a,b
). 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, 1992
; Nemes and Steinert, 1999
).
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.
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MATERIALS AND METHODS |
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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).
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RESULTS |
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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., 1990). Loricrin expression begins in the granular layer at E16 (Yoneda and Steinert, 1993
) 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., 1990
; Yoneda et al., 1992
). 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 1F
).
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, 1995). 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., 1994
). 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 2B
) and K10 (Figs. 2C and 2D
) 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., 1995
; Mazzalupo and Coulombe, 2001
). 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., 1995
; Byrne et al., 1994
; Yoneda and Steinert, 1993
). 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 2F
).
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DISCUSSION |
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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, 1982; Poland et al., 1984
). 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, 1988
). 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., 1996). 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., 2000
). 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, 1998
). 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., 1993). 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., 2001
). 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., 1995
) 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., 2001
).
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., 1997). 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.
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ACKNOWLEDGMENTS |
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NOTES |
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