Article |
Address correspondence to Walter Wahli, Institut de Biologie Animale, Université de Lausanne, Bâtiment de Biologie, CH-1015 Lausanne, Switzerland. Tel.: (41) 21-692-41-10. Fax: (41) 21-692-41-15. E-mail: walter.wahli{at}iba.unil.ch
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Abstract |
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Key Words: mouse keratinocytes; PPAR gene expression; PPAR gene targeted disruption; skin wound healing; nuclear hormone receptors
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Introduction |
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Among the various factors that influence skin maturation and development, many nuclear hormone receptors have been implicated. After binding of their respective ligands, nuclear hormone receptors activate the transcription of specific target genes. Various ligands for several members of the nuclear hormone receptor family are known to influence epidermis differentiation. Thyroid hormones and glucocorticoids accelerate the permeability barrier maturation of rat skin in vivo and in vitro (Aszterbaum et al., 1993; Hanley et al., 1996a, 1997a). Estrogen accelerates the skin barrier formation, whereas testosterone delays the process (Hanley et al., 1996b). Retinoids also influence the differentiation of the epidermis, even though the description of their functions are different when assayed in vitro or in vivo (Imakado et al., 1995; Saitou et al., 1995; Li et al., 2000, 2001). More recently, peroxisome proliferatoractivated receptor (PPAR)* and farnesol Xactivated receptor ligands were shown to accelerate epidermal development when added to fetal rat skin explants, whereas PPAR ligands have no effect (Hanley et al., 1997b, 1998). The situation seems to be different in human keratinocytes, since only a PPARß-selective ligand, but not PPAR
or
ligands, induced the expression of keratinocyte differentiation markers (Westergaard et al., 2001). In addition, recent results suggested that a cross talk exists between the PPAR and the cholesterol metabolism pathways in the epidermis (Hanley et al., 2000).
The subfamily formed by the PPARs binds fatty acids and their derivatives as well as hypolipidemic and antidiabetic agents and plays important roles in energy homeostasis. Three isotypes have been identified (PPAR, ß/
or FAAR or NUC1, and
; NR1C1, NR1C2, NR1C3, respectively; Nuclear Receptor Nomenclature Committee, 1999) in various species (Xenopus laevis, rodents, human), each of them having a specific pattern of expression (for review see Desvergne and Wahli, 1999).
Consistent with a potential role of PPAR ligands in epidermis maturation, PPARs are expressed both in rat skin and human keratinocytes (Braissant et al., 1996; Braissant and Wahli, 1998; Rivier et al., 1998). In skin, RNase protection assay and in situ hybridization reveals that PPAR and PPARß are both expressed in the epidermis during embryogenesis. However, no major skin defect has been described in PPAR
null mice, suggesting that PPAR
is not essential for skin maturation in rodents (Lee et al., 1995). In contrast, we show in this study that PPAR
and PPARß are crucial for rapid skin repair in the adult animal.
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Results |
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Thus, in situ hybridization revealed that the expression of the PPARs in the adult skin, measured by ribonuclease protection assay (RPA), is mainly due to their presence in the hair follicles, whereas they are undetectable in the interfollicular epidermis. The pattern of expression of PPAR in the epidermis at embryonic and adult stage suggests that the presence of the PPARs in the keratinocytes is related to proliferation and/or differentiation during development, rather than to the normal adult epidermis renewal.
PPARß expression is upregulated in vivo upon stimulation of keratinocyte proliferation
To address the hypothesis that the expression of PPARs in the epidermis is related to keratinocyte proliferation, we looked at their expression in the adult mouse epidermis after stimulation of keratinocyte proliferation either by topical application of tetradecanoylphorbol acetate (TPA) or by hair plucking. If PPARs are involved in keratinocyte proliferation and/or differentiation, their expression might be reactivated by these stimuli.
TPA applied on the dorsal skin of SV129 mice induced thickening of the epidermis within 48 h, whereas no change was observed on the vehicle-treated control samples. Histological staining of the TPA-treated skin showed a typical increase in keratinocyte stratification compared with the control (Fig. 2
A, and Table I). As markers for keratinocyte proliferation, we used the expression of both keratin 6 (K6) cytoskeletal protein (Navarro et al., 1995) and the Ki67 nuclear antigen. As shown in Fig. 2 A, K6 immunolabeling remained negative in the ethanol-treated control epidermis, whereas high levels were detected in the epidermis after TPA application, confirming that this agent induced the expected proliferation of the keratinocytes. Consistent with this, the number of Ki67-positive cells in the basal layer was also increased in the TPA-treated samples (Fig. 2 A, and Table I). In situ hybridization with PPAR-, ß-, and
-specific probes revealed that PPARß expression was significantly upregulated in the TPA-treated epidermis, whereas only a faint signal was detected for PPAR
and no signal for PPAR
(Fig. 2 A). Consistent with the results shown in Fig. 1 C, none of the three PPAR isotypes was detected in the interfollicular epidermis of the control sample.
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Thus, the marked increase in PPARß expression under conditions inducing keratinocyte proliferation and stratification provides a strong indication that PPARß might be directly implicated in these processes.
Generation of PPARß and mutant mice
To study each PPAR function in the skin in vivo, we used PPAR mutant mice. The PPAR null mouse has been described previously (Lee et al., 1995) and we generated PPARß and PPAR
mutant animals (unpublished data). Early embryonic lethality of PPAR
null mutants was observed, as also reported recently by others (Barak et al., 1999; Kubota et al., 1999). Similarly, due to incomplete but very high penetrance of a lethal phenotype, only few PPARß null mice could be obtained but no null mice line could be established so far. Similar difficulties in generating PPARß homozygous null animals was also described recently by Peters et al. (2000); although, in that case, a PPARß knock out line was finally obtained on a different genetic background. Therefore, and due to the above-mentioned difficulties in obtaining homozygous null mice, we used heterozygous PPARß and PPAR
mice in our experiments.
For each mouse line, the PPAR mRNA and protein levels were analyzed by RNase protection assay and Western blot. The amounts of PPARß and PPAR mRNA and protein are decreased by half in the PPARß and PPAR
+/- mice, respectively, with no compensation by the other PPAR isotypes (Table II and data not shown).
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These observations suggest that one functional PPARß allele is sufficient to maintain normal fetal and postnatal epidermal development in the PPARß mutant mice, or that PPARs may have redundant functions in terms of epithelial differentiation.
PPAR gene expression in a wounded epidermis
In addition to the link between PPARß and keratinocyte proliferation, the expression patterns of the three PPAR isotypes suggest that they are most likely associated with epidermal fetal maturation. Thus, we studied PPAR expression during skin wound healing, an extreme situation in which the adult skin has to regenerate a neoepidermis. This epidermal repair includes both hyperproliferation of the keratinocytes and differentiation of a new epithelium upon closure of the wound.
A half centimeter square, full thickness skin biopsy was excised from the back of adult mice, and PPAR expression was assessed by in situ hybridization at several time points after the injury at the site of the wound (Fig. 5)
. In situ hybridization revealed that PPAR and PPARß are both upregulated in the epidermis of the wound edges, compared with a normal adult epidermis where they cannot be detected. PPARß reactivation was detected as early as 24 h after the injury. Furthermore, it remained expressed in the epidermis of the wound edges and in the neoepithelium during the whole healing process. After closure of the wound, PPARß was downregulated and it became undetectable 20 d after injury. In contrast, PPAR
expression was observed during a short period of time only,
3 d after the injury, and was not observed thereafter. The third isotype, PPAR
, was hardly detectable, which suggests modest or no implication of this isotype in the skin woundhealing process.
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PPAR-/- and ß+/-, but not PPAR
mutant mice, exhibit altered skin wound healing
The respective roles of PPAR, ß, or
during cutaneous wound healing were assessed by measuring the efficiency of the healing of a full thickness wound in PPAR
, ß, or
mutant mice.
As described above, a dorsal skin biopsy was excised on adult mice, the surfaces of the wounds were measured until complete healing, and the effect of either the PPAR, ß, or
mutation was addressed by comparing the kinetics of wound healing in transgenic and normal littermates. In all the mice, either transgenic or wild-type controls, the reepithelialization was preceded by the formation of a scab, decrease of the wound surface, and loss of the scab upon healing of the wound. A significant degree of strain variability was observed in the wound closure kinetics of the control animals of the three strains of mice. This observation is in agreement with reports on healing of skin wounds, in which initial closure (1 d after the biopsy) of wild-type control animals varied from 1060%, depending on the mouse strain (Kaya et al., 1997; Crowe et al., 2000; Gallucci et al., 2000; Streit et al., 2000; Echtermeyer et al., 2001). Due to this variation, only the differences in wound healing between mutant and their wild-type counterparts of each strain has been analyzed.
The PPAR+/- heterozygous mice were not different from wild-type littermates in their wound-healing process (Fig. 6
A), which is consistent with PPAR
being hardly detectable after injury.
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Discussion |
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Differential expression of PPARs in the developing epidermis and during postnatal stages
Here we show that the three PPAR isotypes are expressed during embryonic epidermal development, starting before stratification and differentiation of the epidermis, and in early postnatal stage, whereas they are below detection levels in the adult interfollicular epidermis. PPAR and ß were also found to be present at low levels in the fetal dermis. This distribution strongly suggests that PPARs are not required, or only at very low levels, for normal skin homeostasis in the adult, but participate in the fetal maturation and differentiation of the skin. The reexpression of PPARs during wound healing, with a similar overall pattern as seen during development, provides a valuable experimental model. Indeed, it implies that wound healing reactivates processes that are normally part of the developmental program rather than those involved in normal adult skin renewal.
Development of a functional barrier during late embryonic development (Hardman et al., 1998) correlates with both changes in the organization and the abundance of the extracellular lipid lamellar structures (Aszterbaum et al., 1992; Hardman et al., 1998). This is in part reflected with fetal keratohyalin granules, which contain large lipid-like droplets (DuBrul, 1972). In the adult, the epidermis is continuously renewed through proliferation of the cells forming the basal layer, and the daughter cells differentiate along migration to the upper cornified layer. No large lipid-like droplets are seen, suggesting a difference in the lipid content and organization with respect to the fetal skin. However, at the molecular level the difference in the mechanisms involved in the fetal and adult epidermis are poorly understood. Thus, the different PPAR expression patterns might be of high interest with respect to the molecular events underlying the differences between fetal and adult epidermis.
Genetic analysis of specific roles of PPAR, ß, and
in skin maturation
PPAR activators accelerate rat epidermal development (Hanley et al., 1997b) and can increase the level of expression of several keratinocyte terminal differentiation markers in rat keratinocyte culture (Hanley et al., 1998). However, under normal conditions, no altered skin phenotype has been detected so far in PPAR null mice (Lee et al., 1995). Similarly, no skin alteration was reported for the PPAR
null mouse, born after placental rescue using tetraploid cells (Barak et al., 1999), and PPAR
+/- mice appear normal as well (herein and Kubota et al. [1999]). Finally, as mentioned above, the PPARß null mice that we and others (Peters et al., 2000) have obtained exhibit no obvious skin defect, and the PPARß+/- mice appear normal as well. At this point, it is of interest to note that most of the previous mutant mouse lines, resulting from targeted disruption of nuclear hormone receptors genes, failed to exhibit a skin phenotype even though there is evidence for a role of these nuclear hormone receptors in the skin. The absence of a skin phenotype at birth in the various PPAR mutant mice might be due to a functional redundancy of the three isotypes. Alternatively, as with many processes during development, gene expression in skin formation may depend on a regulatory network rather than on a linear cascade, allowing for adaptation to take place during in utero development. In adult skin, the absence or below in situ hybridization detection level of PPAR expression in interfollicular epidermis is consistent with the absence of an altered skin in the mutant mice. However, a careful histological observation and Ki67 quantification indicated a small but reproducible amount of proliferative cells in PPARß+/- mice, suggesting that even in unchallenged condition, skin homeostasis is slightly altered in the PPARß+/- mice.
PPAR and the inflammation stage in wound healing
Skin wound healing can be divided into three major overlapping and interacting phases which follow a defined time sequence: inflammation, new tissue formation, including the differentiation of a neoepithelium, and remodeling (Fig. 7). The initial inflammatory phase after an injury is a beneficial step that precedes normal repair of the wound. It allows clot formation and control of infectious agents, favors vascularization, and allows local afflux of growth factors. Attracted by chemotactic factors and chemokines, neutrophils accumulate first in the wound bed and serve as an initial line of defense and source of proinflammatory cytokines. After neutrophils, monocytes/macrophages are recruited and, in addition to providing an immune response, release large amounts of growth factors and cytokines. If not controlled, inflammation can contribute to pathological healing, such as extensive scarring or fibrosis, which underscore the importance of a tight control of this early phase of the healing process. The pattern of PPAR expression, mainly in the very first days after the injury nicely overlaps the timing of the inflammation stage. Accordingly, PPAR
null mice exhibit a transient but significant delay in the healing process in the early phase. Since we showed that PPAR
null mice exhibit normal keratinocyte proliferation after TPA treatment of the dorsal epidermis, the delay in the skin healing process of the PPAR
null mice is unlikely to be due to an uncontrolled proliferation. Moreover, the quantification of the inflammatory infiltration shows that the recruitment of the neutrophils and monocytes/macrophages to the wound bed are both impaired in the PPAR
-/- mice during the very early inflammatory phase. This strongly suggests that the transient delay of healing observed in the PPAR
null mice is due to uncontrolled inflammation at the wound site. The normal recruitment of immune cells is then restored in the PPAR
-/- mice, which reflects the ability of these mice to finally reestablish appropriate inflammation control and, consequently, normal resolution of the healing process. These data correlate with previous observations that PPAR
participates in the control of an inflammatory response (Devchand et al., 1996; Staels et al., 1998). At the molecular level, specific quantification of chemotactic factors and chemokines released in the wound bed will help in deciphering the mechanism, leading to altered recruitment of immune cells in the PPAR
null mice. Of particular interest will be the measurement of the levels of IL-1
released at the wound site. Indeed, several reports indicate that keratinocytes, after a skin injury, may participate in the early inflammatory phase by secreting large amounts of preformed active IL-1
(Kupper, 1990; Kupper and Groves, 1995). In this context, it is certainly noteworthy that our results demonstrate that PPAR
expression is upregulated in the keratinocytes at the wound edges during the inflammatory phase of skin wound healing.
In contrast, PPAR seems not to be involved in skin inflammation as it is not expressed in any of the models tested in our study.
PPARß expression and keratinocyte proliferation, adhesion, and migration
In the second step of wound healing, which begins within hours of a skin injury, the keratinocytes will start to migrate from the wound edges and proliferate to cover the wound. The final stage then consists of stratification and differentiation of the neoepidermis and colonization of the epithelium by the nonkeratinocyte cells (e.g., immune cells). Upregulation of PPARß in the keratinocytes takes place during all these successive processes. In addition, the reexpression of PPARß in two models of intense keratinocyte proliferation (TPA and hair plucking) strengthens the link between PPARß and the control of cell proliferation. A role of PPARß in the control of epithelial cell proliferation has also been recently described in colon cancer cells (He et al., 1999), whereas Matsuura et al. (1999) associated an increased PPARß expression to induced human keratinocyte differentiation in vitro and in vivo. Interestingly, our PPARß mutant mice exhibit an altered control of keratinocyte proliferation, characterized by a hyperproliferative reaction, in response to TPA stimulation and hair plucking. No defect is observed in PPARß+/- keratinocyteterminal differentiation, as suggested by the expression of keratinocyte differentiation markers. Consistent with these observations, an enhanced hyperplastic response, associated to a higher expression of cell cycle proteins but normal expression of differentiation markers, was reported recently in the epidermis of PPARß null mice (Peters et al., 2000). In addition to this observation, and very importantly, we also report a defect in PPARß mutant keratinocyte adhesion and migration capacities, as observed in primary keratinocyte cultures. This result strongly suggests that a keratinocyte migration defect is at least partially responsible for the delay in the healing process in the PPARß+/- mice. In the whole animal, indeed, quantification of the cells at the edges of a wound indicates that the keratinocytes are slightly more numerous in the PPARß mutant wounds, consistent with an increased proliferation and slower migration of these cells. Thus, although we cannot rule out a more general implication of PPARß in the skin, our data, together with the recently reported phenotype of the PPARß null mice, provide evidence for the necessity of an increased PPARß expression to control a well balanced proliferation/differentiation process and efficient keratinocyte migration required for nonpathological wound healing.
In conclusion, our observations reveal an important role of PPAR and PPARß in epidermis repair. In addition and very importantly, despite sharing many characteristics these two isotypes clearly do not have redundant functions. Their respective expression and function are complementary and cover the different phases of skin woundhealing processes. Thus, elucidation of the molecular mechanisms responsible for the differential expression of PPAR
in the various inflammation models, as well as those leading to PPARß activation in the proliferating stage and cessation of its activity upon completion of healing, should be very informative with respect to the potential use of PPAR
and PPARß agonists, or antagonists when available, as therapeutic tools in skin affections.
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Materials and methods |
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In situ hybridization
Mouse PPAR, ß, and
specific sense (S) and antisense (AS) digoxygeninlabeled riboprobes were obtained by in vitro transcription, using mouse PPAR
, ß, or
A/B domain cDNA as a template (size of the probes: PPAR
AS, 230 b; PPAR
S, 230 b; PPARß AS, 200 b; PPARß S, 170 b; PPAR
AS, 230 b; PPAR
S, 222 b). The digoxygenin incorporation and the specificity of the probes were tested on slot-blot hybridizations. In situ hybridization was processed as described previously (Braissant et al., 1996).
Immunofluorescence
The keratin 6, 10, and 14 cytoskeletal protein (BabCo), loricrin (BabCo), involucrin (BabCo), and the Ki67 nuclear antigen (Novo Castra) were detected using rabbit polyclonal primary antibodies. For the Ki67 labeling, an antigen-unmasking step was performed (citrate buffer, pH 6, 100°C, 10 mn). The slides were then processed as followed: 1 x PBS, 0.1% BSA/30 mn/RT; primary antibodies in 1 x PBS buffer, 0.1% BSA/2 h/RT; washings in 1 x PBS buffer; FITC-conjugated goat antirabbit IgG secondary antibody (Sigma-Aldrich) in 1 x PBS buffer, 0.1% BSA/1 h/RT. The slides were subsequently washed and mounted before microscopic observation.
RNase protection assays
Mouse PPAR, ß and
specific antisense riboprobes were obtained by in vitro transcription with the T7 or SP6 RNA polymerase, using mouse PPAR
(A/B domain), ß or
(A/B/C domain) cDNA subcloned in the pGEM3Zf(+) (Promega) as templates. The 228-bases PPAR
probe, the 272-bases PPARß probe, and the 331-bases PPAR
probe resulted in digested fragments of 192, 254, and 295 bp, for PPAR
, PPARß, and PPAR
, respectively. The L27 probe has been described previously (Lemberger et al., 1994). For all PPAR probes, a ratio of 1:1 of
32-PUTP to cold UTP was used, whereas a 1:20 ratio of L27 probe was used. Incorporation and specific activity of each probe was determined after purification via Rneasy Clean-Up (QIAGEN).
Direct lysate RPA was carried out as described by the manufacturer (Ambion) with some modifications. In brief, tissues were lysed in Lysis/Denaturation solution (2 mg/ml) and clarified by centrifugation (Qiashredder; QIAGEN). 20 µL of lysate was hybridized to 1 ng of specific PPAR probes (109 cpm/µg) and 10 ng of L27 probe (108 cpm/µg). RNase digestion (10 U/ml RNaseA; 400 U/ml RNaseT1) was carried out for all probes at 37°C/20 min. The products of RPA were resolved in a 6% electrolyte-gradient denaturing polyacrylamide gel. Gels were dried and exposed on phosphor screen of a StormImager 840 (Molecular Dynamics). Quantitative analysis was performed by using IQuant 2.5 software. PPAR mRNA expression was normalized to the previously calculated specific activity of the probe and to L27 mRNA expression. The PPAR/L27 ratio was further normalized to the UTP content of each PPAR probe.
Wound-healing experiments
The hair follicle cycle of each mouse was synchronized by shaving the back of the animal 2 wk before starting the experiment. Control and transgenic mice were then anesthetized, shaved, and a full thickness middorsal wound (0.5-cm2 surface, square shaped) was created by excising the skin and the underlying panniculus carnosus. The wounds were then allowed to dry and form a scab. Wound closure was measured daily in a double-blinded fashion, on young (68 wk) and old (1218 mo) animals until complete healing of both control and transgenic mice. The surfaces of the wounds were measured by a single individual by covering each wound with a transparent plastic sheet and tracing the wound area on anesthetized animals (Gross et al., 1995; Streit et al., 2000). Wound areas were quantified (Sigma-Scan; Sigma-Aldrich) and were standardized and expressed as a percentage of the initial wound size (100%). The mean values (n = 810 animals) were plotted for each time point, ± SEM. A Student's t test was used for comparison of the control and PPAR mutant groups.
To examine PPAR expression at the site of the injury, the mouse was sacrificed and an area including the scab and the complete epithelial edges of the wounds was excised at each time point. For each mouse, a control of normal dorsal skin was taken at distance from the wounded tissue.
Inflammatory infiltration
Neutrophil and monocyte/macrophage infiltration was quantified on tissue sections of PPAR+/+ and -/- mice. Sections from wounds at day 1, 3, and 5 postwounding were hematoxilin/eosin stained. The density of neutrophils and monocytes/macrophages was determined by manually counting the infiltrating immune cells based on morphological criteria in five standardized microscopic fields (400x magnification) for each wound. Statistical analysis of the data is based on the Student's t test.
Keratinocyte proliferation assays
TPA application
5 nmoles of TPA in 200 µl ethanol was topically applied on the shaved left part of the dorsal epidermis of control and transgenic animals. As a control, the right part of the dorsal epidermis, away from the TPA-treated part, was treated with vehicle only. Samples were harvested 2 d after the TPA application.
Hair plucking
For synchronization purposes, hair was plucked a first time on a 0.5-cm2 dorsal surface of control and transgenic mice. After a period of 10 d, the same surface was plucked a second time and the treated region was dissected 2 d later. As a control, skin samples were taken at a distance from the plucked surface.
The dissected tissues were then processed as described above and used for histological staining, immunolabeling, or in situ hybridization. Quantitative analysis of the mean epidermal thickening and the Ki67-positive cells was performed using the Object Image software.
Primary keratinocyte culture and in vitro scraping wound
Mouse keratinocytes were isolated from epidermis as reported by Hager et al. (1999) with the following modifications: the epidermis was separated from the dermis after overnight incubation at 4°C in 2.5 U/ml of Dispase. The epidermis was placed in a 50-ml centrifuge tube with 10 ml of keratinocyte serumfree medium and the tube was given 50 firm shakes. Keratinocytes were resuspended in keratinocyte serumfree medium containing 0.05 mM Ca2+ and 0.1 ng/ml epidermal growth factor, and seeded at 2 x 105 cells/cm2 for the wild-type keratinocytes, and at 46 x 105 cells/cm2 for the mutant keratinocytes. Keratinocytes were used after 23 passages.
For the scraping experiment, keratinocytes were cultured in a 60-mm diameter tissue culture dish. At 7080% confluency, a scrape (1.52 mm) was made (day 0) across the diameter using a cell scraper. At the indicated time, pictures of the cells near the edges were taken until complete closure of the scrape wound. In total, the cells were maintained in culture for 23 wk, depending on their genotype.
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Footnotes |
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Jeffrey Peters' present address is Department of Veterinary Science, Center for Molecular Toxicology, Pennsylvania State University, 226 Fenske Laboratory, University Park, PA 16802.
Sharmila Basu-Modak's present address is Department of Pharmacy and Pharmacology, University of Bath, BA2 7AY Bath, United Kingdom.
* Abbreviations used in this paper: AS, antisense; K6, keratin 6; PPAR, peroxisome proliferatoractivated receptor; RPA, ribonuclease protection assay; S, sense; TPA, tetradecanoylphorbol acetate.
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Acknowledgments |
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This work was supported by the Swiss National Science Foundation (grants to Walter Wahli and to Béatrice Desvergne), by the Etat de Vaud, by the Human Frontier Science Program Organization, and by Parke-Davis Pharmaceutical Research.
Submitted: 29 November 2000
Accepted: 16 July 2001
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References |
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