Departments of Cell Biology (P.S.T., S.I., J.R.B., D.R.R.),
Dermatology (J.R.B., D.R.R.) and Center for Comparative Medicine
(M.M.), Baylor College of Medicine, Houston, Texas 77030,
The Dows Institute (P.W.W.), College of Dentistry,
University of Iowa, Iowa City, Iowa 52242
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ABSTRACT |
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INTRODUCTION |
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Retinoid action is mediated through the retinoic acid receptors
(RARs), and the retinoid X receptors (RXRs) (6). Both RARs and RXRs are
subgroups of the steroid hormone, nuclear receptor superfamily. Steroid
hormone receptors activate upon intracellular ligand binding, followed
by dimerization to another steroid receptor and translocation to the
nucleus. Once inside the nucleus, these dimers can bind to their
respective response elements and modify target gene expression. The RAR
family consists of three isoforms: RAR, RARß, and RAR
, whose
natural ligand is all-trans-RA. The RXR subfamily also
consists of three isoforms, RXR
, RXRß, and RXR
. The RXRs are
not activated by all-trans-retinoic acid (RA), but instead
by one of its naturally occurring metabolites, 9-cis-RA.
RARs require heterodimerization with the RXRs for signal transduction;
however, RXRs can function independently of RARs via homodimerization
(for reviews see Refs. 7 and 8). Some nuclear receptors, including the
RARs, can be influenced in the degree of their activation or repression
by cofactors (9, 10), such as N-CoR (nuclear corepressor) (11, 12) and
SMRT [silencing mediator (corepressor) for retinoid and
thyroid-hormone receptors (13)]. These cofactors can repress
transcription by blocking sites on the receptor that recruit
transcription factor machinery or on the ligand-binding regions. It is
additionally known that, at least in the case of N-CoR, ligand binding
by the receptor can release the corepressor, thereby relieving its
transcriptional repression (11).
Even excluding the new complications of corepressors, study of
individual steroid receptors was difficult because of cross-talk and
redundancy between receptors. This redundancy became apparent when
several steroid receptors were knocked-out via recombinant stem cell
technology. For example, targeted disruption of the RAR or RAR
genes showed that the loss of a single receptor type affected only a
few selected epithelia. In most other tissues, including the epidermis,
in which these RARs are the major isoforms, the absence of a single
receptor type was overcome through redundant signaling, probably by
other RAR family members (14, 15, 16). The knockout of the RXR
gene
resulted in embryonic lethality between embryonic days 13.5 (E13.5) and
E16.5 (17), but no information about the effect on epidermal
development was recovered because functional maturation of the
epidermis does not occur until just before birth, E18. Because of this
functional redundancy in epidermis, our laboratory set out to explore
the role of retinoids in epidermal development using a
dominant-negative approach. The dominant-negative receptor RAR
403 is
a truncated form of RAR
. It can bind ligand, heterodimerize with the
RXRs, and bind to DNA response elements, but it cannot transactivate
(18). It has also recently been shown that the RAR
403 has a higher
affinity for binding to the SMRT corepressor (4-fold greater than the
wild type RAR receptor), thus potentially providing an explanation for
its potent activity as a negative regulator. By expressing this
RAR
403 dominant-negative under the control of the human keratin 1
(HK1) promoter, which restricts expression of the transgene primarily
to the suprabasal layers of the epidermis, and is not expressed during
development until E15, we were able to avoid embryonic lethality seen
in other studies. The shiny, red skin observed in the RAR
403
transgenic mice resulted from a failure to form lipid multilamellar
structures in the stratum corneum (SC), despite the fact that the
lamellar bodies that give rise to them appeared to form and fuse to the
cell membrane properly (19).
In the epidermis, lipids and some of the lipid-processing
hydrolytic enzymes are packaged together in the lamellar bodies as
keratinocytes differentiate (20, 21, 22). In the upper granular layer, the
lamellar bodies migrate to the apical end of the cell, and the bounding
membrane of the lamellar body fuses into the cell plasma membrane. The
contents of the lamellar bodies are then exocytosed into the
intercellular space. As this extruded lipid-rich mixture enters the
space between the granular layer and the SC, phospholipases act upon
the remaining phospholipids, and glycosidases deglycosylate the
monohexosylceramides, which results in a lipid mixture in the SC
consisting mainly of ceramides, cholesterol, and FFA (23, 24). In
murine epidermal SC, only small proportions of phospholipids and
glycolipids are present in the SC. The physical form of the lipid is
also modified as it passes into the cornified layer. The material
initially extruded from the lamellar bodies appears to consist of short
stacks of lamellae, but on passage into the stratum corneum this is
processed into broad multilamellar sheets that fill most of the
intercellular space (25). In view of the altered multilamellar
structures in the RAR403 mouse and the fact that these intercellular
lipid-composed structures determine the permeability of the skin, it
was not surprising that transepidermal water loss (TEWL) was elevated
3-fold in severely phenotypic mice (19).
The initial characterization of the RAR403 phenotype left many
unanswered questions about the specific nature of the barrier defect
and how this defect is causing lethality in neonates. In this paper, we
have examined the biochemistry of the multilamellar structures more
closely, with specific attention being given to their lipid
composition. We verify previous in vitro data by
demonstrating that the epidermis of severe RAR
403 neonates is
impaired in its ability to activate target genes in response to topical
application of RA, while also showing that the RAR
403 transgene does
not interfere with signaling via the vitamin D receptor (VDR), another
RXR heterodimerization partner. We show that neonatal lethality may be
a result of decreased body temperature and provide evidence that some
very severely affected mice can survive and develop a scaling phenotype
consistent with the hyperkeratosis observed upon depletion of
retinoids. This latest information provides more insight into the
phenotypes associated with inhibition of the retinoid-signaling pathway
and its affect on epidermal barrier function.
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RESULTS |
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DISCUSSION |
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Induction of the Vitamin D3-Regulated Gene
25-Hydroxyvitamin D3-Hydroxylase Provides
Insight into the in Vivo Action of the RAR403
Transgene
As mentioned earlier, RARs require heterodimerization with RXRs to
transactivate target genes. We have shown previously that when primary
keratinocytes are transfected in vitro with the RAR403
transgene, they had a reduced ability to transactivate an
RARE-containing luciferase reporter. We also showed that the RAR
403
inhibited signaling from a peroxisome proliferator response element
reporter construct and postulated that this inhibition was due to
RAR
403 sequestering the available RXR away from its other
heterodimerization partners (19). If RXR is being sequestered, then the
epidermal phenotype seen in the RAR
403 pups might be due to loss of
signaling from any of the partners of RXR [including RAR, PPAR,
thyroid hormone receptor (TR), or VDR] although a previous TR
dominant-negative was expressed in the epidermis under a different
promoter and showed no phenotype (29).
To test this hypothesis in vivo, VDR responsiveness was
measured by topical vitamin D3 application to RAR403
neonate skin. To our surprise, we observed no attenuation of VDR
responsiveness. Several potential explanations for the difference
observed between PPAR- and VDR-signaling ability exist: the first and
perhaps most straightforward explanation is that PPAR and VDR are
present in different concentrations in epidermal cells. If, for
example, VDR is expressed 10-fold higher than PPAR, there is an
increased chance that VDR will be able to compete for binding with RXR
when signaling is necessary. Although the levels of RARs and RXRs have
previously been quantitated in the human epidermis (30), no one has yet
examined the relative levels of other epidermal receptors.
Another difference between PPAR and VDR signaling may result from
different affinities for RXR. If VDR has a higher affinity for RXR than
PPAR, VDR could compete effectively for the RXR even if PPARs were
present in greater quantities. Receptor-binding affinities are very
difficult to quantitate in vivo, making this hypothesis
difficult to test. Of course, it is possible that the promoter for
25-hydroxyvitamin D3 hydroxylase is unique in some way that
allows it full responsiven-ess despite the presence of the
dominant-negative RAR403. To resolve these questions it will be
necessary to test other VDRE- containing genes, as well as to measure
the in vivo signaling of the PPAR pathway in RAR
403
neonates to determine whether it is the same as previously observed
in vitro (19). RAR
403 neonates could be treated topically
with clofibric acid, a ligand for PPAR, and the levels of a
PPAR-responsive gene such as the
-hydroxylase CYP4A6 (31), could be
measured. Regardless of the outcome of this experiment, the full
responsiveness of a VDRE-containing gene and the lack of phenotype from
the dominant-negative TR mice (29) provide strong evidence that the
barrier defect in our RAR
403 transgenic line is not the result of
deficient VDR signaling via global sequestration of RXR.
The RAR403 4-Day Phenotype Mimics Vitamin A-Deprived Skin
The scaling phenotype observed in some very severely affected
transgenic mice correlates well with previous in vivo
studies that show hyperkeratosis and flaking when skin is deprived of
retinoids. It suggests that the scaling observed under these conditions
may have resulted from the failure to form proper multilamellar
structures, thereby linking them to specific enzymatic processing
defects (32, 33, 34). The multilamellar structures formed between
corneocytes not only contribute to the epidermal water barrier, but
also form the "mortar" in the bricks and mortar model of the SC
structure (35). Because the RAR403 mice are unable to form fully
matured multilamellar structures, it is not surprising that this
deficient mortar leads to excessive flaking and general loosening of SC
integrity. It is interesting that the RAR
403 mice are not born with
a scaling phenotype as might be expected from previous studies of
vitamin A deficiency; however, vitamin A withdrawal studies represent a
gradual decline in retinoid levels. This is a very different situation
from that experienced by the developing RAR
403 mice who experience
acute inhibition of the retinoid-signaling pathway with the onset of
transgene expression that occurs coincident with the initial stages of
epidermal maturation (E15).
The barrier defect in the RAR403 mice is reminiscent of the barrier
defect seen in the Gaucher model mice, which are deficient in the
lipid-processing enzyme ß-glucocerebrosidase (33). This enzyme
catalyzes the hydrolysis of glucosylceramides to ceramides and, when
deficient, leads to an ichthyotic phenotype in mice (33) and the
ichthyoisoform skin abnormality observed in severely affected type 2
Gaucher patients (36). At an ultrastructural level, the Gaucher
mice show some multilamellar structures, although they are frequently
discontinuous and loosely packed (36). The fact that some multilamellar
structures do form in the Gaucher mice, whereas the RAR
403 mice are
completely deficient, suggests that the RAR
403 barrier disruption
either affects multiple enzyme pathways or effects enzymes upstream of
ß-glucocerebrosidase.
Decreased Mean Body Temperature May Cause RAR403 Neonatal
Lethality
The failure to form an intact multilamellar structure leads to a
dramatic 4-degree drop in mean body temperature. It is important to
point out that this measurement is from a pup immediately after removal
from under its mother. We initially attempted to measure decreased body
temperature over time but found that not only was there great
variability in rates of decrease between severe pups, but also that
their temperatures would literally plummet after only a few minutes
away from the warmth of their mothers and littermates. This pronounced
temperature drop probably contributed greatly to the poor survivability
of these animals. It may also explain why some severe mice can survive
to adulthood. A good mother will keep her pups clustered together where
warmth from other littermates and from the mother help to maintain body
temperature. We have also previously observed a gradual reduction in
epidermal phenotypic severity, perhaps due to an adaptive response to
restore tissue homeostasis, as transgenic mice develop into adulthood
(37). Therefore, a pup that survives the first, most critical, days has
a good probability of living to adulthood. Poor mothers spend little
time nursing and warming their pups, thus accelerating the
transgenics loss of body temperature and, ultimately, their
death.
The dominant-negative RAR403 is a potent transcription silencer due,
in part, to its tight association (4-fold higher than endogenous RAR)
with SMRT, a corepressor of transcription. It has been shown in
vitro that high concentrations of RA can cause dissociation of
SMRT from the RAR
403 mutant, thereby increasing transcription rates
(13). Thus, in an effort to prolong the life of transgenic pups, we
attempted topical RA treatments. No correlation could be found between
survivability and recovery of barrier function as measured by body
temperature (data not shown); however, it is likely that the lack of
correlation resulted from the technical difficulty of the experiment.
Topical ligand applications are straightforward, but maternal rejection
in such cases was high. Also, it cannot be ruled out that the vehicle,
acetone, might have caused further damage to the SC barrier. For this
reason we have considered attempting in utero rescue
experiments using systemic retinoids or RAR isoform-specific
chemical analogs. Because the RAR
403 transgene does not become
active until E15, which is relatively late in mouse
development, it may be possible to give a dose of systemic retinoids
that could rescue, at least transiently, the RAR
403
barrier defect phenotype, without causing major teratogenic
changes.
Lipid Analysis Points to a Defect in Lipid Processing After
Lamellar Granule Fusion
Biochemical analysis of the major lipid components of the SC have
revealed that the glycosylceramides and phospholipids are elevated in
the SC of the phenotypically severe RAR403 mice. Previous evidence
indicates that the actions of both phospholipases (34) and
glucocerebrosidase (33) are required in the conversion of the initially
extruded lamellar granule contents into the broad multilamellar sheets
found in the intercellular spaces of normal SC. Given these enzymatic
requirements for formation of the permeability barrier, the present
results on lipid composition are in accord with the previous
ultrastructural observation that SC does not contain normal
multilamellar arrays in phenotypically severe RAR
403 mice (19). This
is also consistent with the decreased barrier function indicated by
increased TEWL.
Reductions in the levels of phospholipase and glycosidase action could reflect altered gene transcription or altered levels of activators, inhibitors, or cofactors. In porcine epidermal SC, phospholipids and glycolipids are absent, whereas in the porcine keratinizing oral epithelium, significant levels of phospholipids and glycolipids survive into the SC (38). Furthermore, the permeability of the keratinized oral mucosa is 1 order of magnitude greater than that of the skin, and the oral SC contains poorly formed lamellar structures. These differences may reflect differences in both enzyme activity levels and more rapid cell transit in the oral epithelium.
The Nile Red staining was undertaken to verify the biochemical findings
of increased polar lipids in the SC, as well as to assess lipid changes
in other areas of the epidermis. The dye revealed that not only were
there more polar lipids in the SC, but also that the origin of those
polar lipids was likely to be the SG-SC interface. It is well
established that the SG-SC interface is the location at which lamellar
bodies fuse with the cell membrane and the multilamellar structure
forms, both of which are critical for epidermal barrier function. As
previously mentioned, the RAR403 mice fail to form proper
multilamellar structures. This ultrastructural defect may be the result
of defective lipid processing that should occur after extrusion of
lipids into the intercellular space. Since both Nile Red staining and
biochemical lipid analysis indicate elevated phospholipids in severe
animals, and since the polar lipid staining seems to persist into the
SC, the defect in proper barrier function may be attributable to
deficient phospholipase activity.
Because of the large number of known phospholipases, it is difficult to
be certain which specific enzymes might be defective in the RAR403
epidermis (39). However, it has recently been shown that chemical
inhibition of the group I phospholipase A2
(PLA2-I) interferes with the formation of the broad
multilamellar sheets of the SC and appears to reduce the amount of
neutral lipid in the SC as judged by Nile Red staining (34). When this
information is combined with our own data, it is tempting to speculate
that the RAR
403 barrier deficiency might be attributable to a
PLA2-I defect. To test such a hypothesis, it would be
necessary to test the protein levels of PLA2-I, as well as
to measure its activity, since altered expression of activators or
inhibitors (due to the RAR
403 transgene) could impact the enzymes
function.
Changes in the lipid composition of the SC, either as a result of altered synthesis or processing, can produce scaling disorders. Such changes have been documented to occur as a result of inborn errors in lipid metabolism or as side effects from hypolipidemic drugs (40). The underlying mechanism causing similar side effects from retinoids is not known; however, our current results strongly suggest an effect on lipid metabolism. Further investigation of this transgenic mouse model should provide new insights into the role of the retinoid-signaling pathway in modulating epidermal lipid processing and may suggest novel strategies to minimize these side effects.
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MATERIALS AND METHODS |
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RNA Isolation from the Epidermis
RNA from neonatal epidermis was isolated using RNazol B
(Tel-Test Inc., Friendsworth, TX), according to standard protocols.
Briefly, separated epidermis was frozen in liquid nitrogen and mixed
with RNazol B, which also freezes in liquid nitrogen. The sample was
ground to a powder with the frozen RNazol, then allowed to thaw to room
temperature. The liquid slurry was pipetted out and 100 µl of
chloroform were added. Samples were centrifuged, the upper aqueous
layer was removed and incubated with ethanol overnight, and RNA was
pelleted by centrifugation and resuspended in 50 µl 1 mM
EDTA.
Topical Ligand Treatment
Neonates were taken within 1 h of birth and treated
topically with either 20 µl 0.5% all-trans-RA in acetone,
or 0.5% vitamin D3 in acetone. To cover the entire body,
the 20 µl were applied in four, 5-µl aliquots to the back, stomach,
and left and right sides. No occlusion or protective coating was used
to prevent loss of the applied ligand; however, the neonates were kept
under a yellow light to prevent degradation of these light-sensitive
chemicals. After 14 h the animals were killed and RNA was isolated
from the epidermis as described above.
Ribonuclease (RNase) Protection
RNase protection was performed using the RNase II kit (Ambion
Inc. Austin TX), according to the supplied protocol. Briefly, antisense
RNA probes to CRABPII, 25-hydroxyvitamin D3-hydroxylase,
and glyceraldehyde-3-phosphate dehydrogenase were mixed with RNA from
either normal or severe littermates and denatured at 95 C for 5 min,
and then incubated at 42 C overnight. A mixture of RNase A/T1 was then
added to each reaction tube and incubated for 1 h at 37 C. Samples
were then precipitated and resuspended in loading dye, followed by
heating to 95 C for 2 min and immediate loading on a 6% denaturing
polyacrylamide gel. Gels were run at 300 V for 2.5 h. All RNase
protection assays were performed using glyceraldehyde-3-phosphate
dehydrogenase as an internal control for total RNA quantity.
Lipid Extraction
Isolated SC was placed in tared glass culture tubes,
lyophilized, and weighed. To ensure a clean separation of SC from the
living layers, random SC samples were sectioned and stained with
hematoxylin and eosin to verify that no nucleated cells were present.
Lipids were then extracted for 2 h each with chloroform-methanol
2:1, 1:1, and 1:2 at room temperature. The combined extracts from each
sample were dried under a gentle stream of nitrogen. Each extract was
then dissolved in 5 ml chloroform-methanol (2:1) and washed with 1 ml 2
M KCl. After centrifugation, the lower phase was
transferred to a clean culture tube and dried under nitrogen.
TLC
Twenty x 20-cm glass plates coated with 0.25-mm-thick
silica gel G (Adsorbosil-plus-1; Alltech Associates; Deerfield IL) were
washed with chloroform-methanol (2:1) and activated in a 110 C oven,
and the adsorbent was scored into 6-mm-wide lanes. Calibrated glass
capillaries were used to apply samples 23 cm from the bottom edge of
the plate, and the chromatograms were developed. To resolve nonpolar
lipids, chromatograms were developed to 20 cm with hexane followed by
development to 20 cm with toluene followed by development to 11 cm with
hexane-ethyl ether-acetic acid, 70:30:1. After development,
chromatograms were air dried, sprayed with 50% sulfuric acid, and
slowly heated to 220 C on an aluminum slab on a hot plate. After 2
h, charring was complete and the chromatogram was quantitated by
photodensitometry. Standards used for identification of lipids as well
as for quantification included cholesterol oleate, tripalmitin, stearic
acid, cholesterol, sphingomyelin, phosphatidylethanolamine,
cerebroside, and ceramide (Sigma Chemical Company, St. Louis MO). To
establish standard curves, standard amounts were varied between 0.1 and
25 mg.
Nile Red Staining
Tissue TekII OCT-embedded (Lab-Tek Products, Naperville, IL),
6-mm frozen sections were stained with 0.15 mg Nile Red stain in 1 ml
75% glycerol. After 2 min, slides were coverslipped, sealed with nail
polish, and photographed.
Body Temperature Measurement
Mean body temperature was measured using an ear probe
thermometer (model VTTS-1000, Exergen Corp., Newton MA). Pups were kept
with their mothers until temperatures were taken. Each pup was
individually removed from its mother and the thermometer placed under
the left armpit for approximately 5 sec, as indicated by the
instrument. Temperatures were taken only once per mouse because the
body temperature of the severely affected animals rapidly declined once
the animals were removed from their mothers.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Current Address: Tokyo University Hospital, Department of Dermatology,
73-1, Hongo, Bunkyo-ku, Tokyo, 113, Japan.
This work was supported in part by NIH Awards AR-40240 and HD-25479 (to D.R.R.) and DE-10516 (to P.W.W.).
Received for publication February 21, 1997. Accepted for publication March 18, 1997.
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REFERENCES |
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