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Originally published In Press as doi:10.1074/jbc.M411522200 on November 4, 2004

J. Biol. Chem., Vol. 280, Issue 4, 3012-3021, January 28, 2005
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Dominant-negative Retinoic Acid Receptors Elicit Epidermal Defects through a Non-canonical Pathway*

Chang Feng Chen{ddagger}§ and David Lohnes, A chercheur bourcier (Senior) of the Fonds de la Recherches en Sante de Quebec{ddagger}§||**

From the {ddagger}Division of Experimental Medicine, Department of Medicine, McGill University, the §Clinical Research Institute of Montreal, Montreal, Quebec H2W 1R7, and the ||Department of Molecular Biology, University of Montreal, Montreal K1H 8M5, Canada

Received for publication, October 8, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous work has shown that a dominant-negative retinoic acid receptor {alpha} (dnRAR{alpha}), expressed under the K14 promoter, causes severe epidermal defects. Similar defects are, however, not seen in RAR{alpha}{gamma} double null mutant mice, which lack the entire complement of RARs expressed in the epidermis. To investigate the mechanism of action of these dominant-negative receptors, dnRAR{alpha} or a DNA binding-deficient variant, dnRAR{alpha}DBD, were targeted to the basal epidermis. Expression of either receptor type led to similar epidermal phenotypes suggesting that both RAR mutants acted through a common mechanism. The epidermal phenotype was reminiscent of defects seen in p63-/- mice. Consistent with this, reduced p63 expression was observed in transgenic offspring expressing either RAR mutant, suggesting that down-regulation of p63 might underlie the effects of these receptors on epidermal development. By contrast, expression of p63 in the epidermis of RAR{alpha}{gamma}-/- offspring was unaffected, indicating that RARs were not essential for p63 expression. These findings suggest that dnRARs may impact on epidermal development through one or more non-canonical pathways, which are independent of receptor-DNA interaction.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Retinoic acid (RA)1 has long been known to modulate cell growth and differentiation in many epithelial tissues, including the epidermis (1-8). Topical administration of pharmacological levels of RA causes epidermal hyperplasia (5, 9, 10) and impairs epidermal barrier function (11). Conversely, deprivation of dietary vitamin A, the precursor of RA, generally leads to epidermal hyperkeratosis (12, 13).

The biological functions of RA are mediated by two groups of nuclear receptors, retinoic acid receptors (RAR{alpha}, -{beta}, and -{gamma} and their isoforms) and retinoid X receptors (RXR{alpha}, -{beta}, and -{gamma} and their isoforms). RA target genes are regulated by heterodimers between RARs and RXRs, which impact on gene expression by binding to cis-acting regulatory sequences (RAREs). RAREs usually consist of direct repeats of the consensus PuG(G/T)TCA with five nucleotides intervening the repeats (DR5), although a number of variant motifs have been described (2, 8, 14, 15). Unliganded RXR/RAR heterodimers can recruit transcriptional co-repressor (CoR) complexes to RAREs. These CoR complexes are associated with histone deacetylase activity, which results in chromatin condensation and repression of transcription. Ligand binding to the RAR moiety leads to conformational changes in the ligand-binding domain of the receptor, causing co-repressor release and recruitment of co-activators. Histone acetylation mediated by acetyltransferase associated with such co-activator complexes results in chromatin decondensation that facilitates gene transcription (2, 15-17).

The epidermis expresses RAR{alpha}, RAR{gamma}, RXR{alpha}, and RXR{beta}, with RAR{gamma} and RXR{alpha} being the predominant receptor types (5, 18, 19). Although RA has strong effects on epidermal homeostasis under pharmacological conditions (5, 9-13), the role of the RARs in epidermal development and homeostasis under physiological conditions is less clear. Mice double null for RAR{alpha} and RAR{gamma} exhibit minor epidermal abnormalities affecting the granular layer (9, 20). In marked contrast, transgenic mice expressing a dominant-negative RAR{alpha} in basal keratinocytes display severe skin defects suggestive of an early block in epidermal differentiation (21). These disparate outcomes are not due to functional rescue by RAR{beta} in the knockout animals, because RAR{beta} remains undetectable in the epidermis of RAR{alpha}{gamma}-/- offspring (9). Rather it has been suggested that the dnRAR{alpha} might impact on epidermal development via a non-canonical pathway (22).

The dnRAR{alpha} used in the above transgenic experiments contained one amino acid mutation in the ligand-binding domain (G303E), which significantly reduces ligand binding without affecting heterodimerization or DNA binding. Because of this, RA is likely to be compromised in its ability to dissociate CoR complexes associated with the dnRAR{alpha}, leading to "constitutive" repression of RA target genes (23-25); it is conceivable that such non-physiological repression could result in the epidermal phenotype observed in the transgenic offspring. Alternatively, other mechanisms, such as titration of co-factors used by multiple signaling pathways, may contribute to the transgenic phenotype. To investigate these potential mechanisms of action, we compared the skin phenotypes of transgenic offspring expressing either a dnRAR{alpha} or an identical dominant-negative receptor incapable of DNA binding (designated dnRAR{alpha}DBD) as well as RAR{alpha}{gamma} double null mutants. Consistent with prior work (21), dnRAR{alpha} transgenic mice exhibited severe epidermal abnormalities, including reduced suprabasal layers, underdeveloped hair follicles, and abnormal expression of several differentiation markers. These histological and molecular defects were recapitulated in transgenic offspring expressing dnRAR{alpha}DBD, suggesting a mechanism of action that does not require receptor-DNA interaction. In addition, because the RAR{alpha}{gamma} double null mice did not exhibit similar defects, it is likely that these receptors affect epidermal differentiation in a manner not related to canonical retinoid signaling.

The phenotype of the dnRAR transgenics bore similarity to the defects exhibited by p63 null mutants. p63, a homolog of p53, is essential for development and maintenance of a number of epithelial tissues, including the epidermis (26-29). Consistent with a relationship between p63 and dnRAR function, immunohistochemistry revealed that p63 expression was severely compromised in both dnRAR{alpha} and dnRAR{alpha}DBD offspring. By contrast, RAR{alpha}{gamma}-/- epidermis, which can be considered as a pan-RAR null material (9), was histologically indistinguishable from wild-type littermates and exhibited normal expression of p63. These data suggest that interference with expression of p63 may underlie the epidermal defects elicited by dnRARs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation and Characterization of Mutant RARs—A cysteine to alanine mutation in codon 88 (C88A) or a glycine to glutamic acid mutation in codon 303 (G303E) were introduced into the murine RAR{alpha}1 cDNA using the QuikChangeTM site-directed mutagenesis kit (Stratagene) and confirmed by sequencing. These mutations were predicted to disrupt DNA binding and to generate a dnRAR{alpha}, respectively, based on prior work (25, 30). A PstI-SmaI fragment from the RAR{alpha}1 (G303E) mutant (designated dnRAR{alpha}) was used to replace the same region from RAR{alpha}1 (C88A), giving rise to a DNA binding-deficient variant of dnRAR{alpha}, denoted dnRARDBD.

DNA binding of the RAR mutants was assessed by electrophoretic mobility shift assay using a canonical RARE as previously described (19, 31). Transcriptional potency of the mutant receptors was assessed by transient transfection assays in P19 embryocarcinoma cells using a 3x{beta}RARE reporter vector as previously detailed (19, 31).

Generation of Mice—The dnRAR{alpha} and dnRARDBD cDNAs were excised from the parental vector by digestion with EcoRI, blunted with Klenow fragment, and inserted into pGEM3Z-K14 (32), which had been linearized with BamHI and blunted with Klenow fragment. Transgenes were excised by digestion with EcoRI and HindIII, isolated by preparative gel electrophoresis, and injected into fertilized mouse eggs.

F0 transgenic offspring were recovered at embryonic day (E)18.5 by cesarean section and identified by Southern blot analysis using DNA prepared from tail biopsies. Dorsal skin from transgenic and control littermates was recovered and either fixed in Bouin's solution, for histological or immunohistochemistry analysis, or frozen at -80 °C.

Transgene expression was assessed by RT-PCR. Briefly, first strand cDNA was synthesized from 5 µg of total RNA isolated from epidermal samples using the TRIzol reagent (Invitrogen) by reverse transcription with Moloney murine leukemia virus reverse transcriptase (Invitrogen). Primers specific for dnRAR{alpha} (CGCATCTACAAGCCTTG and CAAGTCGGTGAGGGGCT) or for dnRAR{alpha}DBD (CGCATCTACAAGCCTGC and CAAGTCGGTGAGGGGCT) were used to amplify the relevant cDNAs by PCR. As a control, expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also monitored by RT-PCR using the primers TCGGTGTGAACGGATTTG and ATTCTCGGCCTTGACTGT. RAR{alpha}{gamma}-/- mutants were generated by RAR{alpha}{gamma}+/- intercrosses, recovered by cesarean section at E18.5 and identified by PCR as described previously (33, 34).

Histological and Immunohistochemical Analysis—Dorsal skin samples from E18.5 offspring were fixed in Bouin's solution, embedded in paraffin, and sectioned at 7 µm. Immunohistochemistry was performed as described previously (23, 35). Briefly, after deparaffination and rehydration, sections were blocked with 10% serum (Sigma) in phosphate-buffered saline/0.2% Tween 20 at room temperature for 1 h, and subsequently incubated with primary antibodies (K5 (Covance), 1:500 dilution; K10 (Abcam), 1:25 dilution; or p63 (Santa Cruz Biotechnology), 1:25 dilution) at 4 °C overnight. Slides were then washed three times with phosphate-buffered saline/0.2% Tween 20, re-blocked with 10% serum, and incubated with biotinylated secondary antibodies (1:150 dilution, Vector Laboratories) for 1 h. Samples were subsequently washed and incubated with horseradish peroxidase-coupled streptavidin (1:1000, PerkinElmer Life Sciences), and reactivity revealed by incubation with a diamino benzidine (Sigma; 0.05%; imidazole, 0.01 M; NiCl2, 0.064%; H2O2, 0.009%) solution in Tris-buffered saline for 3-10 min. Specimens were counterstained with methyl green (Sigma) before mounting.

Transient Transfection—Keratinocytes were cultured in S-minimum essential medium (Invitrogen) with 10% Chelex-treated fetal calf serum (Invitrogen, 0.5 mM final calcium concentration), insulin (5 µg/ml), hydrocortisone (0.5 µM), MgCl2 (1.5 mM), cholera toxin (1.2 x 10-11 M), adenine (24 µg/ml), gentamycin (10 µg/ml), and epidermal growth factor (10 ng/ml). Cells were transfected using Lipofect-Ace (Invitrogen) as previously described (19, 31). DNA used for transfections included {Delta}N-p63 promoter-luc (36), 3x{beta}RARE-luc (19, 31), pLuc-CYP4A-Z (PPARE-luc) (37), and VDRE-lacZ (a gift from J. White) and pSG5-hPPAR{alpha} (a gift from W. Wahli). pSG5-RAR{alpha}, pSG5-dnRAR{alpha}, pSG5-dnRAR{alpha}DBD, and pSG5-RAR{gamma} expression vectors were derived by sub-cloning the appropriate cDNAs into pSG5 (Stratagene). pSG5 was added, where needed, to normalize the total DNA amount for each transfection.

After transfection, the cells were washed twice with phosphate-buffered saline and treated with all-trans-RA (10-6 M, in Me2SO), 1{alpha},25-dihydroxyvitamin D3 (10-8 M, in ethanol), or WY-14,643 (50 µM, in Me2SO) for various times, as noted. Monolayer cultures were subsequently treated with lysis buffer (Nonidet P-40, 10%; Tris (pH 8.0), 0.1 M; dithiothreitol, 1 mM), and luciferase activity was measured using an LB953 Autolumat luminometer (Berthold). Luciferase activity was normalized for {beta}-galactosidase activity as described (19, 31) and expressed as the mean ± S.D. of independent triplicate transfections.

For {beta}-galactosidase activity assays, lysates were incubated with 2-nitrophenyl {beta}-D-galactopyranoside (Sigma; in NaH2PO4, 23 nM; Na2HPO4, 77 nM; MnCl2, 0.1 mM; MgSO4, 2 mM; {beta}-mercaptoethanol, 40 mM), and enzyme activity was measured as a function of optical density at 405 nM.

Analysis of p63 Expression in Transfected Keratinocytes—Keratinocytes were grown on 10-cm tissue culture dishes (Nunc) and transfected with the relevant RAR expression vector (pSG5-RAR{alpha}, dnRAR{alpha}, or dnRAR{alpha}DBD, 20 µg/transfection) together with a pEGFP-C1 construct (Clontech, 10 µg/transfection) as described above. EGFP-positive cells were subsequently isolated using a MoFlo cell sorter (Cytomation), and total RNA was extracted using the TRIzol reagent (Invitrogen). RT-PCR was performed using cDNA synthesized as described above, and PCR was carried out using primers specific for {Delta}N-p63 as done previously (38).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characterization of dnRAR{alpha} and dnRAR{alpha}DBD—The dnRAR{alpha} used in the present study was generated as previously described (25). As expected, binding of this dnRAR{alpha} to a consensus DR-5 RARE did not differ from that of the wild-type receptor as assessed by electrophoretic mobility shift assay (Fig. 1A). In transfection assays in P19 embryocarcinoma cells, dnRAR{alpha} strongly inhibited transactivation mediated by endogenous RARs, attenuating greater than 90% of the activity of the wild-type receptors at the lowest concentration tested (Fig. 1B). An identical dominant-negative effect was also observed in wild-type keratinocytes.



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  		FIG. 1.
Characterization of dominant-negative RARs. A, dnRAR-DNA association. Electrophoretic mobility shift assay was performed using nuclear proteins extracted from COS-7 cells that were transfected with either empty vector (lane 1), pSG5-RAR{alpha} (lanes 3-5), pSG5-dnRAR{alpha} (lanes 6-8), or pSG5-dnRAR{alpha}DBD (lanes 9-11). The relative amount of protein used in each assay is noted above each lane. Lane 2 was equivalent to lane 3 except that the incubation included a 20-fold excess of unlabeled DR-5 oligonucleotide as competitor. B, transactivation assay. P19 cells were transfected with a 3x{beta}RARE reporter plasmid (0.8 µg) and either pSG5-dnRAR{alpha} or pSG5-dnRAR{alpha}DBD (0.5, 1, or 2 µg/transfection). Cells were subsequently treated with RA (1 µM) or vehicle for 24 h following which lysates were assessed for luciferase activity. An expression vector encoding {beta}-galactosidase (0.5 µg) was included in all transfections and used to normalize for transfection efficiency. Results were expressed as -fold induction by RA relative to vehicle and as the mean ± S.D. of independent triplicate transfections.

 
To generate a DNA binding-deficient RAR{alpha}, we mutated codon 88 (in the first zinc finger) from cysteine to alanine. This mutation was then shuttled into dnRAR{alpha} to generate dnRAR{alpha}DBD. As expected, dnRAR{alpha}DBD did not exhibit detectable DNA binding to canonical RAREs from the RAR{beta} promoter as assessed by electrophoretic mobility shift assay (Fig. 1A; compare lanes 9-11 with lanes 3-8). In transfection assays, dnRAR{alpha}DBD also failed to inhibit target gene transcription mediated by endogenous or co-transfected RARs in either P19 cells or keratinocytes (Fig. 1B and data not shown). This double mutant, therefore, did not appear to impact on DNA binding-dependent transactivation functions of wild-type RARs.

Skin Phenotypes Elicited by dnRAR{alpha} and dnRAR{alpha}DBD— RAR{alpha}{gamma}-/- newborns lack the entire complement of RARs normally expressed in the epidermis, but did not exhibit any significant epidermal phenotype upon gross examination, apart from a slightly "shiny" skin as previously described (Fig. 2A) (9, 20). Overt histological abnormalities were likewise absent (Fig. 2B), although minor defects in the granular layer of RAR{gamma}-/- and RAR{alpha}{gamma}-/- offspring have been reported (7). The keratinocyte terminal differentiation program was also not perturbed in the RAR{alpha}{gamma} null offspring, because expression of K5, K14, K1, K10, fillagrin, and involucrin was comparable between mutant and control littermates (9, 20).



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  		FIG. 2.
Histological assessment of RAR{alpha}{gamma}-/- epidermis. A, RAR{alpha}{gamma} null mutant offspring were procured by cesarean section at term (E18.5). Note that the RAR{alpha}{gamma}-/- skin was essentially normal, although shiner than that of the littermate control. B, histological analysis. Dorsal skin samples were fixed, sectioned, and stained with hematoxylin and eosin. No overt histological defects were observed in the RAR{alpha}{gamma}-/- skin sample. B, basal layer; S, spinous layer; G, granular layer; HF, hair follicles; SB, suprabasal layers; SC, stratum corneum; D, dermis; E, epidermis; arrows indicated the dermal/epidermal junctions.

 
The dnRARs were expressed in basal keratinocytes using the human K14 promoter (Fig. 3A) (32). Approximately 15% of the pups recovered at E18.5 were identified as transgenics for dnRAR{alpha} (19/127) or dnRAR{alpha}DBD (24/169), respectively, as assessed by genomic Southern blot (Fig. 3B and data not shown). A similar percentage of offspring from either the dnRAR{alpha} or the dnRAR{alpha}DBD transgenes exhibited severe skin abnormalities. In all such cases, the affected pups expressed the mutant RARs in the epidermis, as assessed by transgene-specific PCR (Fig. 3C and data not shown). In these offspring, the skin appeared shiny and sticky and was extremely thin and fragile (Fig. 4A; compare transgenics with littermate controls). Transgene expression was not detectable in unaffected littermates (data not shown).



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  		FIG. 3.
Generation of transgenic offspring. A, schematic depiction of the transgenic constructs and the region used as a probe for Southern blot analysis. The insert comprising the K14 promoter, dnRAR open reading frame, and polyadenylation signal was released by digestion with EcoRI and HindIII, gel-purified, and used for the generation of transgenic offspring. B, Southern blot analysis of offspring. Genomic DNA (10 µg) from tail biopsies was digested with EcoRV and assessed for transgene integration by Southern blot analysis using a probe derived from the sequences noted in A. C, RT-PCR analysis of epidermal transgene expression. Total RNA was extracted from the epidermis of the offspring and used for RT-PCR. Amplifications were performed using primers specific either for the mutant RARs or for GAPDH, which was used as an internal control. Products were resolved by agarose gel electrophoresis.

 



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  		FIG. 4.
Epidermal phenotype of dnRAR transgenic offspring. Transgenic offspring were procured by cesarean section at term (E18.5). A, transgenic offspring were photographed with non-transgenic littermates. Note the overt epidermal defects in both dnRAR{alpha} and dnRAR{alpha}DBD offspring. B, histological assessment. Dorsal skin samples were fixed, sectioned, and stained with hematoxylin and eosin. Relative to the non-transgenic offspring (B, upper panel), transgenic offspring exhibited poorly developed suprabasal (SB) layers and markedly fewer hair follicles (HF). Note that similar defects were evident in offspring from both transgenes. B, basal layer; S, spinous layer; G, granular layer; SC, stratum corneum; D, dermis; E, epidermis; arrows indicated the dermal/epidermal junctions.

 
Histologically, the skin from affected transgenic offspring contained an intact basal layer. However, in all the cases, the suprabasal layers were greatly reduced, and there was also a marked decline in the number of hair follicles, which, when present, appeared to be arrested at early stages of development (Fig. 4B). These effects were similar to those elicited by a dn-hRAR{alpha} transgenic construct expressed from the K14 promoter (21).

Offspring derived from either transgene also exhibited perturbed expression patterns for several markers of epidermal differentiation. Affected offspring from either dnRAR{alpha} or dnRAR{alpha}DBD constructs expressed K5, a basal epidermal marker, but at a reduced level when compared with non-transgenic littermates (Fig. 5, B and C; compare with A). This differed somewhat from previous observations from K14-dn-hRAR{alpha} transgenic pups, which exhibited strong ectopic K5 expression in the suprabasal layers (21). The basis for this discrepancy is presently unknown but might be related to the different hybrid transgenic backgrounds between these two studies. In addition to a reduction in K5 expression, both dnRAR{alpha} and dnRAR{alpha}DBD transgenic offspring exhibited a marked reduction in the expression of K10, an early epidermal differentiation marker (Fig. 5, E and F, compare with D). These striking phenotypic similarities between dnRAR{alpha} and dnRAR{alpha}DBD transgenics suggested that these RAR mutants affected epidermal development through a common mechanism that was independent of receptor-DNA association.



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  		FIG. 5.
Keratin expression in transgenic offspring. Immunostaining for K5 (A-C) and K10 (D-F) was performed on sections of dorsal skin from E18.5 offspring. Arrows indicated the dermal/epidermal junction.

 
Loss of p63 Expression in Transgenic Epidermis—p63 is a homolog of the tumor suppressor p53. p63-/- offspring exhibit severe epidermal defects (26, 27) suggestive of an essential role in epidermal development (26-29, 39, 40). A number of these defects, including blocked suprabasal differentiation, lack of hair follicles, and loss of differentiation markers such as K10 (26, 41), were recapitulated in the dnRAR transgenic offspring in the present study.

Consistent with a potential relationship between dnRAR function and p63, immunohistochemistry revealed that offspring from either transgenic background were essentially devoid of p63 expression, which was normally evident in the nuclei of the basal epidermis and the outer root sheath of hair follicles of the controls (Fig. 6A, compare B and C with A). This observation suggests that loss of p63 may, at least in part, underlies the epidermal defects observed in the dnRAR transgenics.



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  		FIG. 6.
Dominant-negative RARs inhibited p63 expression. A, p63 expression. Immunostaining for p63 was performed using sections of dorsal skin samples from E18.5 offspring. Note the near-complete loss of expression in the transgenic offspring. Arrows indicated the dermal/epidermal junction. B, RT-PCR analysis of p63 expression. RAR expression vectors (20 µg/transfection, pSG5-RAR{alpha}, pSG5-dnRAR{alpha}, or pSG5-dnRAR{alpha}DBD) were co-transfected with the EGFP expression plasmid, pEGFP-C1 (10 µg/transfection). Total RNA was extracted from EGFP-positive cells isolated by fluorescence-activated cell sorting. Amplification was performed using primers specific either for {Delta}N-p63 or for GAPDH, which was used as an internal control. Products were resolved by agarose electrophoresis. Note that both dnRAR{alpha} (lane 4) and dnRAR{alpha}DBD (lane 5) significantly attenuated {Delta}N-p63 expression, whereas wild type RAR{alpha} had no significant effect (lane 3). C and D, effects of RARs on expression from the p63 promoter. A reporter plasmid driven by the {Delta}N-p63 promoter (0.8 µg/transfection) was co-transfected with RAR expression vectors as noted (in C: 0.1, 0.2, 0.5, 1.0, or 1.5 µg/transfection; in D: 0.2 or 0.5 µg/transfection). Following transfection, cells were treated with RA (10-6 M) or vehicle, and luciferase activity was measured 24 h post-treatment. Luciferase activities were expressed in arbitrary units and as the mean ± S.D. (C) or as the mean of -fold induction by RA relative to vehicle (D) from independent triplicate transfections in both cases. Note that RA induced the {Delta}N-p63 promoter in cells overexpressing wild-type RAR{alpha}, but not in cells overexpressing wild-type RAR{gamma}.

 
The effect of the RAR mutants on p63 expression was also tested in vitro. {Delta}N-p63 is the predominant p63 isoform expressed in virtually all epithelia, including the epidermis (26-29, 39, 40). Consistent with in vivo findings, both dnRAR types elicited a comparable reduction of endogenous {Delta}N-p63 expression in wild-type keratinocytes in culture (Fig. 6B, compare lanes 4 and 5 with lane 2). By contrast, overexpression of wild-type RAR{alpha} in the absence of RA had only a modest inhibitory effect on {Delta}N-p63 expression (Fig. 6B, compare lane 3 with lane 2). This finding is also consistent with previous work showing that overexpression of wild-type RAR{alpha} from the K14 promoter does not affect epidermal development (21).

Transfection assays using RAR{alpha}{gamma} null keratinocytes revealed that RA could induce expression from a {Delta}N-p63-responsive reporter (36) in the presence of exogenous RAR{alpha}, and that both basal, as well as RA-induced expression, was inhibited by both dnRAR types in this assay (Fig. 6C; see also below). This finding was consistent with the effects of these mutant receptors on expression of endogenous {Delta}N-p63 in cultured keratinocytes and suggests an effect of the dnRARs on the level of {Delta}N-p63 transcription.

As noted above, RA induced {Delta}N-p63 expression in RAR{alpha}{gamma} null keratinocytes in the presence of exogenous RAR{alpha}. By contrast, no such induction was observed in wild-type keratinocytes (data not shown), which express RAR{alpha} and RAR{gamma} (5, 18, 19). However, induction was observed upon co-transfection with RAR{alpha}, but not RAR{gamma} (Fig. 6D). The differential effect of RAR subtypes in terms of their ability to mediate gene activation has been reported in many cell types, including keratinocytes (31, 42). This suggests that RA may be able to regulate {Delta}N-p63 in the context of elevated levels of RAR{alpha}. Despite this observation, {Delta}N-p63 may not be a direct retinoid target gene, because computer analysis has not identified any RARE-like elements in the {Delta}N-p63 promoter fragment used in these studies and there is no appreciable loss of p63 expression in any RAR null background examined to date (see below).

RAR Ablation Does Not Affect p63 Expression—In contrast to the transgenic fetuses, the epidermis from RAR{alpha}{gamma}-/- offspring appeared histologically normal (Fig. 2) and did not exhibit altered p63 expression compared with wild-type controls (Fig. 7A). Consistent with this, loss of these RARs did not impact on expression of {Delta}N-p63 in keratinocyte cultures (Fig. 7B, compare lanes 4 and 5 with lanes 2 and 3). These findings suggest that RARs are not essential for p63 expression both in vivo and in vitro.



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  		FIG. 7.
Loss of RARs does not affect p63 expression in vivo. A, immunohistochemical analysis of p63 expression. Immunostaining for p63 was performed using sections of dorsal skin samples from E18.5 offspring. Note that p63 expression was normal in the RAR{alpha}{gamma}-/- sample. Arrows indicated the dermal/epidermal junction. B, RT-PCR assays of p63 expression. Total RNA was extracted from wild type or RAR{alpha}{gamma} null keratinocytes as noted. Amplifications were performed using primers specific either for {Delta}N-p63 or for GAPDH, which was used as an internal control. Products were resolved by agarose electrophoresis. Loss of RARs did not overtly affect {Delta}N-p63 expression (compare lanes 4 and 5 with lanes 2 and 3), and RA did not induce {Delta}N-p63 in wild-type (Wt) keratinocytes (compare lane 3 with lane 2).

 
Potential Mechanisms of Action of dnRARs—RXRs are essential heterodimeric partners for a number of nuclear receptors, including the RARs, vitamin D3 receptor (VDR), peroxisome proliferator-activated receptors (PPARs), and thyroid hormone receptors (TRs), among others (2, 43, 44). Because both dnRAR{alpha} and dnRAR{alpha}DBD elicited comparable skin phenotypes, one conceivable mechanism of action could be through sequestration of RXRs by dnRARs from such partners. In this regard, another dominant-negative RAR, RAR{alpha}403, elicits epidermal barrier defects when expressed in the suprabasal epidermis. It has been proposed that RAR{alpha}403 elicits this effect by interference with PPAR signaling, potentially via RXR sequestration, and thus to compromise lipid metabolism essential for epidermal barrier function (45, 46). To test whether the dnRAR{alpha} used in this study was also able to affect other nuclear receptor-mediated signaling, we examined the effect of overexpression of either dnRAR{alpha} or dnRAR{alpha}DBD on VDR- or PPAR-mediated signaling, both of which have been shown to impact on epidermal development (47-53).

In transfection assays using RAR{alpha}{gamma} null keratinocytes, 1{alpha},25-dihydroxyvitamin D3 treatment induced expression of a vitamin D reporter, and co-transfection with either RAR{alpha} or dnRAR{alpha} inhibited this transactivation. By contrast, dnRAR{alpha}DBD showed no such inhibitory effects (Fig. 8A).



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  		FIG. 8.
The effect of dnRARs on VDR- and PPAR-mediated signaling. Reporter plasmids driven by a VDRE (A) or a PPARE (B) (1 µg/transfection) were co-transfected with the noted RAR expression (0.1, 0.2, 0.5, 1.0, or 1.5 µg/transfection). After transfection, the cells were treated with the relevant ligands (VD3; 10-8 M, WY-14,643; 50 µM) or vehicle. Cell lysates were collected 24 h post-treatment and assessed for {beta}-galactosidase and luciferase activity. Data were expressed as -fold induction relative to vehicle and are the mean ± S.D. of independent triplicate samples. Overexpression of either wild-type RAR{alpha} or dnRAR{alpha} repressed transactivation from the VDRE (A)- or the PPARE (B)-driven reporters, whereas overexpression of dnRAR{alpha}DBD had no significant effect.

 
PPAR-mediated induction of a cognate reporter was found to be low in keratinocyte cultures (data not shown and Ref. 46), necessitating the inclusion of exogenous PPAR in transfection assays. As was observed for VDR-mediated transactivation, overexpression of either wild-type RAR{alpha} or dnRAR{alpha} reduced expression from a PPARE-driven reporter, whereas overexpression of dnRAR{alpha}DBD had no significant effect (Fig. 8B). These data suggest that dnRAR{alpha} and dnRAR{alpha}DBD exhibit differential abilities to interfere with other RXR-dependent pathways and that such a mechanism is unlikely to underlie the effects of these mutant receptors on epidermal development seen in the transgenic studies.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Dominant-negative RARs Impact on Epidermal Development through a Non-canonical Pathway—The dominant-negative RARs used in the present study were created by modeling an inherited mutation (G347E) in the ligand-binding domain of TR{beta} identified in patients with generalized thyroid hormone resistance (25, 54). These mutant receptors are believed to suppress target gene expression due to compromised ligand binding and enhanced affinity to CoRs. Therefore, competition for DNA binding at RAREs may underlie the dominant-negative effects of dnRARs in terms of gene transactivation (24). This is consistent with the finding that the dnRAR{alpha} used in this study competed for transactivation from a canonical RARE, whereas loss of receptor DNA binding abolished this effect.

As with prior work (21), the dnRAR{alpha} used in the present study evoked epidermal defects when expressed from the K14 promoter. However, it is unlikely that this outcome was the result of attenuation of target gene expression, because a similar phenotype was also elicited by the dnRAR{alpha}DBD, which could not associate with RAREs in vitro and which did not interfere with wild-type RARs in transfection assays. Thus, it would appear that these mutant receptors impact on epidermal development through a non-canonical pathway.

The nature of the pathway affected by these dnRARs is presently unknown. As one possibility, it is conceivable that the mutant receptors could sequester cofactors involved in other signaling pathways. However, such ancillary factors would not likely be widely used among nuclear receptors, because expression of either a dnTR{beta} (TR{beta}-Glu347) or overexpression of wild-type RAR{alpha} in basal keratinocytes does not impact on epidermal development (21). Alternatively, the dnRARs could conceivably titrate out RXRs, thus impacting other nuclear receptor pathways involved in epidermal development such as the VDR or PPARs. In this regard, expression of the dominant-negative RAR{alpha}403 to suprabasal keratinocytes causes epidermal abnormalities in loss of barrier function due to abnormal lipid metabolism. This defect has been proposed to be a result of interference with PPAR signaling by RXR sequestration (45, 46). In this regard, the epidermis expresses RXR{alpha} and RXR{beta}, with RXR{alpha} being the predominant form (5, 56, 57). Conditional knockout of RXR{alpha} in the skin does not cause overt developmental defects in the epidermis (58), although the mutant mice develop skin abnormalities during adult life (58, 59). This observation does not exclude a critical role for RXR signaling in epidermal development, as functional redundancy between RXR{alpha} and RXR{beta} has been suggested (59). However, only dnRAR{alpha}, but not dnRAR{alpha}DBD, appeared capable of interfering with VDR- or PPAR-dependent signaling, both of which are RXR-dependent. Interference with RXR therefore does not appear to be the mechanism by which these dnRARs impact on epidermal development.

In addition to the above mechanisms of action dnRARs may also lead to aberrant gene expression through augmented gene repression (60). Indeed, repression of target genes has been proposed to be an important mechanism in events related to osteogenesis and head development (61, 62). However, this mechanism is unlikely to underlie the epidermal defects observed in the present study, because the dnRAR{alpha}DBD, which is DNA binding-deficient, would not be anticipated to affect repression at target loci.

Loss of p63 Expression and Epidermal Development—p63 is a homolog of the tumor suppressor p53 and is essential for the maintenance of epithelial stem cells in a number of tissues, including the epidermis (26-30, 37). Loss of one p63 allele underlies human EEC (ectrodactyly, ectodermal dysplasia, and facial clefts) syndrome, which also involves the skin (58, 59). Disruption of p63 also supports a pivotal role for this protein in skin. p63 null mice present with a single layer of basal cells, with all suprabasal layers, as well as hair follicles, being greatly reduced or absent. The epidermis of p63-/- mice does not express K1, filaggrin, loricrin, or K6 and exhibits reduced expression of K14 (26), consistent with a block in differentiation. The effect of p63 loss of function, however, can also be influenced by genetic background, which in some cases results in null offspring displaying patches of epidermal keratinocytes that do express markers of differentiation (27).

Both dnRAR{alpha} and dnRAR{alpha}DBD offspring exhibited defects similar to those observed in p63-/- mice (26), although the transgenics appeared less affected. Notably, transgenic skin contained an intact basal layer with greatly reduced suprabasal layers and hair follicles, a reduction in expression of K5 and K10, and an absence of K6 (data not shown). Although the stratum corneum was formed, squames appeared fragmented. Transgenic offspring also exhibited a marked reduction in p63 immunoreactivity, in line with the noted phenotypic similarities. In this regard, the less severe defects elicited by the transgenics, relative to p63 null offspring, might be attributed either to residual expression of p63 or to the differences in the genetic backgrounds used between these studies.

The mechanism by which the dnRAR{alpha} transgenes inhibited p63 expression is unclear. Although RA could induce a p63-responsive promoter in tissue culture, this effect only occurred in the presence of exogenous RAR{alpha}. By contrast, RA did not affect {Delta}N-p63 expression, the predominant p63 isoform expressed in the epidermis, in the absence of exogenous RAR{alpha}, nor was p63 reduced in the epidermis of RAR{alpha}{gamma} double null offspring. These findings are also consistent with prior work showing that overexpression of wild-type RAR{alpha} does not elicit any epidermal defects (21) and suggest that p63 is not normally affected by retinoid signaling in the skin.

The above findings are consistent with a non-canonical mechanism of action by which the dnRARs attenuate {Delta}N-p63 expression in vivo, leading to the observed skin phenotypes. As these mutant receptors reduced the expression of both endogenous {Delta}N-p63 in keratinocyte cultures, and attenuated expression of a p63-responsive promoter, it is likely that the dnRARs impacted on expression at the level of transcription. The regulation of p63 is poorly understood, although a number of pathways, including p53 (65), Bmp (66), and EGF (67, 55) have been implicated. Although it is conceivable that the dnRARs could influence these pathways with subsequent impact on p63, we have not yet been able to establish a relationship between any of these pathways and the dnRARs. Our present work does, however, suggest that at least certain of the results obtained using such dominant-negative reagents may be caused by unforeseen mechanisms of action that are not related to normal retinoid signaling.


   FOOTNOTES
 
* This work was supported in part by the National Cancer Institute of Canada with funds from the Canadian Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Supported by a scholarship from the Cancer Research Society, Inc. Back

** To whom correspondence should be addressed: Dept. of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Rd., Ontario K1H 8M5, Canada. Tel.: 613-562-5800 (ext. 8684); Fax: 613-562-5434; E-mail: dlohnes{at}uottawa.ca.

1 The abbreviations used are: RA, all-trans-retinoic acid; RAR, retinoic acid receptor; RXR, retinoid X receptor; RARE, retinoic acid-responsive element; CoR, transcription co-repressor; DBD, DNA-binding domain; GAPDH, glyceraldehydes-3-phosphate dehydrogenase; PPAR, peroxisome proliferators-activated receptor; TR, thyroid hormone receptor; VD3, 1{alpha},25-dihydroxyvitamin D3; VDR, vitamin D3 receptor; EEC, ectrodactyly, ectodermal dysplasia, and facial clefts syndrome; EGF, epidermal growth factor; EGFP, enhanced green fluorescent protein. Back


   ACKNOWLEDGMENTS
 
We thank Qinzhang Zhu and Michel Robillard for generating the transgenics, Claudia Toulouse and Jean-Rene Sylvestre for animal husbandry, Duhaime Johanne for computer analysis of the {Delta}N-p63 promoter, Annie Vallée for sectioning, and H. Lienard and C. Charbonneau for assistance with preparation of the figures. We also express our gratitude to E. Fuchs, M. Dobbelstein, E. Johnson, J. White, and W. Wahli for kindly providing with reagents.



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