dominant-negative rars elicit epidermal defects through a ... chang feng chen 1,2 and david lohnes...

Chen and Lohnes RARs and epidermal development

Dominant-negative RARs elicit epidermal defects through a non-canonical

pathway

Chang Feng Chen 1,2 and David Lohnes 1,2,3*

1Division of Experimental Medicine, Department of Medicine, McGill University,

Canada 2Clinical Research Institute of Montreal, Montreal, Quebec, Canada

3Department of Molecular Biology, University of Montreal, Montreal, Canada

*Author for correspondence:

David Lohnes, Ph.D. Department of Cellular and Molecular Medicine University of Ottawa 451 Smyth Road K1H 8M5 Canada

Tel. (613) 562-5800 ext. 8684 Email: [email protected] Fax: (613) 562-5434

Running title: RARs and epidermal development Keywords: RA, RAR, epidermis, p63, keratinocyte, keratin.

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JBC Papers in Press. Published on November 4, 2004 as Manuscript M411522200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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Abstract

Previous work has shown that a dominant-negative RAR dnRAR

expressed under the K14 promoter, causes severe epidermal defects. Similar

defects are, however, not seen in RAR double null mutant mice, which lack the

entire complement of RARs expressed in the epidermis. To investigate the

mechanism of action of such dominant-negative receptors, dnRAR or a DNA-

binding deficient variant, dnRAR 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 downregulation of p63 might

underlie the effects of these receptors on epidermal development. By contrast,

expression of p63 in the epidermis of RAR -/- offspring was unaffected,

indicating that RARs were not essential for p63 expression. These findings

suggest that dnRARs may impact on epidermal development through a non-

canonical pathway(s) which is independent of receptor-DNA interaction.

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Introduction

Retinoic acid (RA) 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 , and and their isoforms) and retinoid

X receptors (RXR , and 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 5 nucleotides intervening the

repeats (DR-5), 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 (HDAC) 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 (LBD) of the

receptor, causing co-repressor release and recruitment of co-activators (CoA).

Histone acetylation mediated by acetyltransferase (HAT) associated with such

co-activator complexes results in chromatin decondensation that facilitates gene

transcription (2;15-17).

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The epidermis expresses RAR , RAR , RXR , and RXR with RAR and

RXR 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 and RAR exhibit minor

epidermal abnormalities affecting the granular layer (9;20). In marked contrast,

transgenic mice expressing a dominant-negative RAR 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 in the knockout animals, as RAR remains undetectable in the

epidermis of RAR -/- offspring (9). Rather it has been suggested that the

dnRAR might impact on epidermal development via a non-canonical pathway

(22).

The dnRAR used in the above transgenic experiments contained one

amino acid mutation in the LBD (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 , 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 signalling pathways,

may contribute to the transgenic phenotype. To investigate these potential

mechanisms of action, we compared the skin phenotypes of transgenic offspring

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expressing either a dnRAR or an identical dominant-negative receptor

incapable of DNA binding (designated dnRAR DBD) as well as RAR double null

mutants. Consistent with prior work (21), dnRAR transgenic mice exhibited

severe epidermal abnormalities, including reduced suprabasal layers, under-

developed hair follicles and abnormal expression of several differentiation

markers. These histological and molecular defects were recapitulated in

transgenic offspring expressing dnRAR DBD, suggesting a mechanism of action

that does not require receptor-DNA interaction. In addition, as the RAR 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 signalling.

The phenotype of the dnRAR transgenics bore similarity to the defects

exhibited by p63 null mutants. p63, a homolog of p53, is essential for the

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 and dnRAR DBD offspring. By contrast, RAR -/-

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.

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Materials and Methods

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 1

cDNA using the QuickChangeTM Site-Directed Mutagenesis Kit (Stratagene), and

confirmed by sequencing. These mutations were predicted to disrupt DNA

binding and to generate a dnRAR , respectively, based on prior work (25;30). A

PstI-SmaI fragment from the RAR 1 (G303E) mutant (designated dnRAR ) was

used to replace the same region from RAR 1 (C88A), giving rise to a DNA-

binding deficient variant of dnRAR denoted dnRARDBD.

DNA-binding of the RAR mutants was assessed by electrophoresis

mobility shift assay (EMSA) 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 RARE

reporter vector as previously detailed (19;31).

Generation of mice.

The dnRAR 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,

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isolated by preparative gel electrophoresis, and injected into fertilized mouse

eggs.

F0 transgenic offspring were recovered at embryonic day (E)18.5 by

Caesarean 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 –80oC.

Transgene expression was assessed by RT-PCR. Briefly, first strand

cDNA was synthesized from 5 g of total RNA isolated from epidermal samples

using the TRIzol reagent (Invitrogen) by reverse transcription with M-MLV

reverse transcriptase (Invitrogen). Primers specific for dnRAR

(CGCATCTACAAGCCTTG and CAAGTCGGTGAGGGGCT) or for dnRAR 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 -/- mutants were generated by RAR +/- 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 m. Immunohistochemistry was

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performed as described previously (23;35). Briefly, after deparaffination and

rehydration, sections were blocked with 10% serum (Sigma) in PBS/0.2%

Tween-20 at room temperature for 1 hour, and subsequently incubated with

primary antibodies (K5 (Covance), 1:500 dilution; K10 (Abcam), 1:25 dilution; or

p63 (Santa Cruz), 1:25 dilution) at 4oC overnight. Slides were then washed three

times with PBS/0.2% Tween-20, re-blocked with 10% serum and incubated with

biotinylated secondary antibodies (1:150 dilution, Vector Laboratories) for 1 hour.

Samples were subsequently washed and incubated with horseradish peroxidase-

coupled streptavidin (1:1000, NEN Life Sciences), and reactivity revealed by

incubation with a diamino benzidine (DAB, Sigma; 0.05%; imidazole, 0.01M;

NiCl2, 0.064%; H2O2, 0.009%) solution in TBS for 3-10 minutes. Specimens were

counterstained with methyl green (Sigma) before mounting.

Transient transfection.

Keratinocytes were cultured in S-MEM (Invitrogen) with 10% chelex-

treated FCS (Invitrogen; 0.5 mM final calcium concentration), insulin (5 g/ml),

hydrocortisone (0.5 M), MgCl2 (1.5 mM), cholera toxin (1.2x10-11M), adenine (24

g/ml), gentimycin (10 g/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 N-p63 promoter-luc (36), 3x RARE-luc (19;31),

pLuc-CYP4A-Z (PPARE-luc) (37), and VDRE-lacZ (a gift from J. White) and

pSG5-hPPAR (a gift from W. Wahli). pSG5-RAR , pSG5-dnRAR , pSG5-

dnRAR DBD and pSG5-RAR expression vectors were derived by subcloning the

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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 PBS, and treated with

all-trans RA (10-6 M, in DMSO), 1 , 25-dihydroxyvitamin D3 (10-8 M, in ethanol),

or WY-14,643 (50 M, in DMSO) for various times, as noted. Monolayer cultures

were subsequently treated with lysis buffer (Nonidet P-40, 10%; Tris (pH8.0), 0.1

M; dithiothreitol, 1 mM), and luciferase activity was measured using an LB953

Autolumat luminometer (Berthold). Luciferase activity was normalized for -

galactosidase activity as described (19;31), and expressed as the mean +/- the

standard deviation (S.D.) of independent triplicate transfections.

For -galactosidase activity assays, lysates were incubated with 2-

nitrophenyl -D-galactopyranoside (Sigma; in NaH2PO4, 23 nM; Na2HPO4, 77

nM; MnCl2, 0.1 mM; MgSO4, 2 mM; -mercaptoethanol, 40 mM), and enzyme

activity 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 , dnRAR , or

dnRAR DBD, 20 g/transfection) together with a pEGFP-C1 construct (Clontech,

10 g/transfection) as described above. EGFP positive cells were subsequently

isolated using a MoFlo cell sorter (Cytomation), and total RNA extracted using

the TRIzol reagent (Invitrogen). RT-PCR was performed using cDNA synthesized

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as described above, and PCR carried out using primers specific for N-p63 as

done previously (38).

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Results

Characterization of dnRAR and dnRAR DBD.

The dnRAR used in the present study was generated as previously

described (25). As expected, binding of this dnRAR to a consensus DR-5 RARE

did not differ from that of the wild-type receptor as assessed by EMSA (Fig. 1A).

In transfection assays in P19 embryocarcinoma cells, dnRAR 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.

In order to generate a DNA-binding deficient RAR , we mutated codon 88

(in the first zinc finger) from cysteine to alanine. This mutation was then shuttled

into dnRAR to generate dnRAR DBD. As expected, dnRAR DBD did not exhibit

detectable DNA-binding to canonical RAREs from the RAR promoter as

assessed by EMSA (Figure 1A; compare lanes 9-11 with lanes 3-8). In

transfection assays, dnRAR DBD also failed to inhibit target gene transcription

mediated by endogenous or co-transfected RARs in either P19 cells or

keratinocytes (Figure 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 and dnRAR DBD.

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RAR -/- 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 -/-

and RAR -/- offspring have been reported (7). The keratinocyte terminal

differentiation program was also not perturbed in the RAR null offspring, as

expression of K5, K14, K1, K10, fillagrin, and involucrin was comparable between

mutant and control littermates (9;20).

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 (19/127) or dnRAR 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 or the

dnRAR 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).

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,

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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 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 or dnRAR DBD constructs expressed K5, a basal

epidermal marker, but at a reduced level when compared to non-transgenic

littermates (Figure 5B and 5C; compare with 5A). This differed somewhat from

previous observations from K14-dn-hRAR 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 and dnRAR DBD transgenic offspring exhibited a

marked reduction in the expression of K10, an early epidermal differentiation

marker (Fig. 5E and 5F, compare with 5D).

These striking phenotypic similarities between dnRAR and dnRAR DBD

transgenics suggested that these RAR mutants affected epidermal development

through a common mechanism that was independent of receptor-DNA

association.

Loss of p63 expression in transgenic epidermis.

p63 is a homolog of the tumour suppressor p53. p63-/- offspring exhibit

severe epidermal defects (26;27) suggestive of an essential role in epidermal

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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, underlie the epidermal defects observed in the

dnRAR transgenics.

The effect of the RAR mutants on p63 expression was also tested in vitro.

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 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 in the absence of RA had only a

modest inhibitory effect on 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 from the K14 promoter does not affect epidermal development

(21).

Transfection assays using RAR null keratinocytes revealed that RA

could induce expression from a N-p63-responsive reporter (36) in the presence

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of exogenous RAR , 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 N-p63 in cultured keratinocytes and suggests an effect of the

dnRARs on the level of N-p63 transcription.

As noted above, RA induced N-p63 expression in RAR null

keratinocytes in the presence of exogenous RAR . By contrast, no such

induction was observed in wild-type keratinocytes (data not shown), which

express RAR and RAR (5;18;19). However, induction was observed upon co-

transfection with RAR , but not RAR (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 N-p63 in the context of elevated levels of RAR . Despite this

observation, N-p63 may not be a direct retinoid target gene, as computer

analysis has not identified any RARE-like elements in the 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 -/- offspring

appeared histologically normal (Fig. 2) and did not exhibit altered p63 expression

compared to wild-type controls (Figure 7A). Consistent with this, loss of these

RARs did not impact on expression of N-p63 in keratinocyte cultures (Fig. 7B,

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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.

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). As both dnRAR and dnRAR 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 403, elicits epidermal barrier defects when

expressed in the suprabasal epidermis. It has been proposed that RAR 403

elicits this effect by interference with PPAR signalling, potentially via RXR

sequestration, and thus to compromise lipid metabolism essential for epidermal

barrier function (45;46).

To test whether the dnRAR used in this study was also able to affect

other nuclear receptor-mediated signalling, we examined the effect of

overexpression of either dnRAR or dnRAR DBD on VDR- or PPAR-mediated

signalling, both of which have been shown to impact on epidermal development

(47-53).

In transfection assays using RAR null keratinocytes, 1 ,25-

dihydroxyvitamin D3 treatment induced expression of a vitamin D reporter, and

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co-transfection with either RAR or dnRAR inhibited this transactivation. By

contrast, dnRAR DBD showed no such inhibitory effects (Fig. 8A).

PPAR-mediated induction of a cognate reporter was found to be low in

keratinocyte cultures (data not shown; 46), necessitating the inclusion of

exogenous PPAR in transfection assays. As was observed for VDR-mediated

transactivation, overexpression of either wild-type RAR or dnRAR reduced

expression from a PPARE driven reporter, while overexpression of dnRAR DBD

had no significant effect (Fig. 8B). These data suggest that dnRAR and

dnRAR 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.

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Discussion

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 LBD of TR 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 used in

this study competed for transactivation from a canonical RARE, while loss of

receptor DNA-binding abolished this effect.

As with prior work (21), the dnRAR 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 since a

similar phenotype was also elicited by the dnRAR 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 co-factors involved in other signalling pathways. However, such

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ancillary factors would not likely be widely used among nuclear receptors, since

expression of either a dnTR (TR -E347) or overexpression of wild-type RAR in

basal keratinocytes does not impact on epidermal development (21).

Alternatively, the dnRARs could conceivably titrate out RXRs, thus impacting on

other nuclear receptor pathways involved in epidermal development such as the

VDR or PPARs. In this regard, expression of the dominant-negative RAR 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 signalling by RXR sequestration (45;55). In

this regard, the epidermis expresses RXR and RXR , with RXR being the

predominant form (5;56;57). Conditional knockout of RXR 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 signalling in epidermal development, as

functional redundancy between RXR and RXR has been suggested (59).

However, only dnRAR , but not dnRAR DBD, appeared capable of interfering

with VDR- or PPAR-dependent signalling, 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

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mechanism is unlikely to underlie the epidermal defects observed in the present

study, as the dnRAR 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 tumour 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 or loricrin or K6, and exhibits reduced

expression of K14 (26), consistent with a block in differentiation. The effect of

p63 loss of function, however, may also be influenced by genetic background,

which in some cases results in null offspring displaying patches of epidermal

keratinocytes which do express markers of differentiation (27).

Both dnRAR and dnRAR 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

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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 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 . By

contrast, RA did not affect N-p63 expression, the predominant p63 isoform

expressed in the epidermis, in the absence of exogenous RAR , nor was p63

reduced in the epidermis of RAR double null offspring. These findings are also

consistent with prior work showing that overexpression of wild-type RAR does

not elicit any epidermal defects (21), and suggest that p63 is not normally

affected by retinoid signalling in the skin.

These above findings are consistent with a non-canonical mechanism of

action by which the dnRARs attenuate N-p63 expression in vivo, leading to the

observed skin phenotypes. As these mutant receptors reduced the expression of

both endogenous 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;68) have been

implicated. While 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

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work does, however, suggest that at least certain of the results obtained using

such dominant-negative reagents may be due to unforeseen mechanisms of

action that are not related to normal retinoid signalling.

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Figure legends

Figure 1. Characterization of dominant-negative RARs. A. dnRAR-DNA

association. EMSA was performed using nuclear proteins extracted from Cos-7

cells that were transfected with either empty vector (lane 1), pSG5-RAR (lanes

3-5), pSG5-dnRAR (lanes 6-8) or pSG5-dnRAR 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

unlabelled DR-5 oligonucleotide as competitor. B. Transactivation assay. P19

cells were transfected with a 3x RARE reporter plasmid (0.8 g) and either

pSG5-dnRAR or pSG5-dnRAR DBD (0.5, 1 or 2 g/transfection). Cells were

subsequently treated with RA (1 M) or vehicle for 24 hours following which

lysates were assessed for luciferase activity. An expression vector encoding -

galactosidase (0.5 g) 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.

Figure 2. Histological assessment of RAR -/- epidermis. A. RAR null

mutant offspring were procured by Caesarean section at term (E18.5). Note that

the RAR -/- skin was essentially normal, although shiner than that of the

littermate control. B. Histological analysis. Dorsal skin samples were fixed,

sectioned and stained with haematoxylin and eosin. No overt histological defects

were observed in the RAR -/- skin sample. B, basal layer; S, spinous layer; G,

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granular layer; HF, hair follicles; SB, suprabasal layers; SC, stratum corneum; D,

dermis; E, epidermis; arrows indicated the dermal/epidermal junctions.

Figure 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 were released by digestion with EcoRI and Hind III, gel

purified, and used for the generation of transgenic offspring. B. Southern blot

analysis of offspring. Genomic DNA (10 g) 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.

Figure 4. Epidermal phenotype of dnRAR transgenic offspring. Transgenic

offspring were procured by Caesarean section at term (E18.5) A. Transgenic

offspring were photographed with non-transgenic littermates. Note the overt

epidermal defects in both dnRAR and dnRAR DBD offspring. B. Histological

assessment. Dorsal skin samples were fixed, sectioned and stained with

haematoxylin and eosin. Relative to the non-transgenic offspring (4B, upper

panel), transgenic offspring exhibited poorly developed suprabasal (SB) layers

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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.

Figure 5. Keratin expression in transgenic offspring. Immunostaining for K5

(A, B, and C) and K10 (D, E, and F) was performed on sections of dorsal skin

from E18.5 offspring. Arrows indicated the dermal/epidermal junction.

Figure 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 g/transfection, pSG5-

RAR , pSG5-dnRAR , or pSG5-dnRAR DBD) were co-transfected with the

EGFP-expression plasmid, pEGFP-C1 (10 g/transfection). Total RNA was

extracted from EGFP-positive cells isolated by FACS. Amplification was

performed using primers specific either for N-p63 or for GAPDH, which was

used as an internal control. Products were resolved by agarose electrophoresis.

Note that both dnRAR (lane 4) and dnRAR DBD (lane 5) significantly attenuated

N-p63 expression, while wild type RAR had no significant effect (lane 3). C.

and D. Effects of RARs on expression from the p63 promoter. A reporter plasmid

driven by the N-p63 promoter (0.8 g/transfection) was co-transfected with RAR

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expression vectors as noted (in C., 0.1, 0.2, 0.5, 1.0, or 1.5 g/transfection; in D.,

0.2 or 0.5 g/transfection). Following transfection, cells were treated with RA (10-

6 M) or vehicle and luciferase activity measured 24 hours post-treatment.

Luciferase activities were expressed in arbitrary unit 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 N-

p63 promoter in cells overexpressing wild-type RAR , but not in cells

overexpressing wild-type RAR .

Figure 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 -/- sample. Arrows indicated the

dermal/epidermal junction. B. RT-PCR assays of p63 expression. Total RNA was

extracted from wild-type or RAR null keratinocytes as noted. Amplifications

were performed using primers specific either for 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 N-p63 expression (compare

lanes 4 and 5 with lanes 2 and 3), and RA did not induce N-p63 in wild-type

(Wt) keratinocytes (compare lane 3 with lane 2).

Figure 8. The effect of dnRARs on VDR- and PPAR-mediated signalling.

Reporter plasmids driven by a VDRE (A) or a PPARE (B) (1 g/transfection)

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were co-transfected with the noted RAR expression (0.1, 0.2, 0.5, 1.0, or 1.5

g/transfection). After transfection, the cells were treated with the relevant

ligands (VD3; 10-8 M, WY-14,643; 50 M) or vehicle. Cell lysates were collected

24 hours post-treatment, and assessed for -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 or dnRAR repressed transactivation from the VDRE (A) or the

PPARE (B) –driven reporters, while overexpression of dnRAR DBD had no

significant effect.

Abbreviations. CoA, transcription co-activator CoR; transcription co-repressor;

DBD, DNA-binding domain; GAPDH, glyceraldehydes-3-phosphate

dehydrogenase; LBD, ligand-binding domain; PPAR, peroxisome proliferator-

activated receptor; RA, all-trans retinoic acid; RAR, retinoic acid receptor; RXR,

retinoid X receptor; RARE, retinoic acid responsive element; TR, thyroid

hormone receptor; VD3, 1 , 25-dihydroxyvitamin D3; VDR, Vitamin D3 receptor.

Acknowledgements. The authors would like to 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

N-p63 promoter, Annie Vallée for sectioning and H. Lienard and C.

Charbonneau for assistance with preparation of the figures. The authors also

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express their gratitude to E. Fuchs, M. Dobbelstein, E. Johnson, J. White, and W.

Wahli for kindly providing with reagents. This work was supported by the National

Cancer Institute of Canada with funds from the Canadian Cancer Society. C.F.C

was supported by a scholarship from the Cancer Research Society, Inc. D.L. is a

chercheur bourcier (Senior) of the Fonds de la Recherches en Sante de Quebec.

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Moc

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C. F. Chen and David L. LohnesDominant negative RARs elicit epidermal defects through a non-canonical pathway

published online November 4, 2004J. Biol. Chem.

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http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M411522200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2004/11/04/jbc.M411522200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2004/11/04/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2004/11/04/jbc.M411522200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

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