Inhibition of Retinoid Signaling in Transgenic Mice Alters Lipid Processing and Disrupts Epidermal Barrier Function

Paul S. Attar, Philip W. Wertz, Mark McArthur, Sumihisa Imakado1, Jackie R. Bickenbach and Dennis R. Roop

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To explore the role of retinoids in epidermal development, we recently targeted expression of a dominant-negative, retinoic acid receptor mutant (RAR{alpha}403) in the epidermis of transgenic mice and observed an unexpected loss of barrier function. In this paper, we demonstrate that transgenic mice expressing the RAR{alpha}403 transgene show attenuated responsiveness to topical application of all-trans retinoic acid, in agreement with our previous in vitro data. We also show that the vitamin D3 receptor is unaffected in its ability to transactivate in the presence of the dominant-negative RAR{alpha}403 transgene, indicating that the RAR{alpha}403 is unlikely to be functioning through a global sequestration of retinoid X receptors. Additionally, we show that the disruption of epidermal barrier function results in a dramatic 4 C drop in mean body surface temperature, probably accounting for the extremely high incidence of neonatal mortality in severely phenotypic pups. Some severely affected pups do survive and show a pronounced hyperkeratosis at postpartum day 4, consistent with previously documented effects of vitamin A deficiency. Biochemical analysis of the severely phenotypic neonates indicates elevated phospholipids and glycosylceramides in the stratum corneum, which results from altered lipid processing. Taken together with previous studies, these data provide strong evidence linking the retinoid-signaling pathway with modulation of lipid processing required for formation of epidermal barrier function.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Retinoids are chemical derivatives and synthetic analogs of vitamin A, retinol. These compounds have undergone a dramatic resurgence of interest in recent years because of their ability to influence proliferation and differentiation of epithelial tissues, which permits them to be used therapeutically for everything from acne to keratinization disorders (1). Additionally, it is now well established that retinoids have tremendous potential as anti-cancer agents both in the treatment of preneoplastic lesions and established cancers (2, 3). Unfortunately, the therapeutic benefits that retinoids impart are reduced due to the many side effects that often require discontinuation of treatment, or at least reduction of dosage, within weeks or months of initiating therapy. The most pronounced side effects of retinoid treatment are scaling and/or desquamation of the skin and drying of the mucous membranes, resulting in painful cracking of the nasal and esophageal regions (4, 5). The large number of therapeutic uses for retinoids makes understanding their cellular mechanisms of action paramount to maximizing their therapeutic benefits, while also minimizing their harmful side effects.

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{alpha}, RARß, and RAR{gamma}, whose natural ligand is all-trans-RA. The RXR subfamily also consists of three isoforms, RXR{alpha}, RXRß, and RXR{gamma}. 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{alpha} or RAR{gamma} 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{alpha} 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{alpha}403 is a truncated form of RAR{alpha}. 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{alpha}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{alpha}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{alpha}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 RAR{alpha}403 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 RAR{alpha}403 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{alpha}403 neonates is impaired in its ability to activate target genes in response to topical application of RA, while also showing that the RAR{alpha}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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RAR{alpha}403 Neonates Show an Attenuated Response to Topical RA
We have previously shown that the dominant-negative RAR{alpha}403 can inhibit signaling from a retinoic acid response element (RARE) in primary keratinocytes in culture (19). Additionally, we demonstrated that the dominant-negative could block signaling through a peroxisome proliferator response element, presumably through the sequestration of the RXR receptors. To confirm that signaling via the retinoid pathway was also inhibited in vivo, we assessed epidermal responsiveness to topical all-trans-RA treatment on normal and severe RAR{alpha}403 neonates. The CRABPII gene was chosen because it is a commonly used marker for measuring retinoid responsiveness and is known to contain an RARE in its promoter. Fourteen hours after RA application, normal animals showed a 9- to 10-fold increase in CRABPII expression. Severe animals treated with RA showed an attenuated increase in CRABPII expression of only 3- to 4-fold (Fig. 1Go). However, basal transcription of CRABPII in severe RAR{alpha}403 epidermis is elevated nearly 2-fold over basal transcription levels in normal littermates.



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Figure 1. CRABPII Induction by RA Is Inhibited in RAR{alpha}403 Neonates

Normal neonates showed a 10-fold induction of CRABPII expression with topical all-trans-RA. Severely phenotypic pups had an attenuated response of only 4-fold. Interestingly, CRABPII basal expression is 2-fold higher in RAR{alpha}403 pups than controls, suggesting competition between the RAR{alpha}403 and endogenous receptors for transcriptional corepressors. Each bar represents an average from six independent animals.

 
VDR Signal Transduction Is Not Inhibited by the RAR{alpha}403 Transgene
Because it is known that VDR requires heterodimerization with RXR to transactivate, we were interested in determining whether the presence of the RAR{alpha}403 dominant-negative (which can bind to RXR) would impair VDR signal transduction in vivo, as it did peroxisome proliferator-activated receptor (PPAR) signaling in vitro. The gene 25-hydroxyvitamin D3-hydroxylase encodes an enzyme that catalyzes the breakdown of vitamin D3. It is known to possess a VDRE and has a measurable basal expression in epidermal keratinocytes. Topical application of vitamin D3 to neonates induced 25-hydroxyvitamin D3-hydroxylase gene expression approximately 5-fold in normal animals. Unlike the in vitro PPAR repression, the severe RAR{alpha}403 animals showed an identical 5-fold induction, indicating that the presence of the RAR{alpha}403 does not affect signaling from this VDRE-containing promoter, either at the basal level or after induction with vitamin D3 (Fig. 2Go).



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Figure 2. Vitamin D3 Induction of 25-Hydroxyvitamin D3-Hydroxylase Is Not Inhibited in RAR{alpha}403 Neonates

normal and severely phenotypic pups showed identical responsiveness to topical vitamin D3, arguing against involvement of the vitamin D3 pathway in the lamellar processing defect. Basal levels of expression were also identical. Each bar represents an average from six independent animals.

 
RAR{alpha}403 Neonates Show Decreased Mean Body Temperature
All transgenic pups have a defect in barrier formation, which leads to increased TEWL, which is most pronounced in high transgene expressors, i.e. severely affected animals (19). As might be expected, increased TEWL results in a drop in mean body temperature (Table 1Go). Normal neonates had an average surface temperature of 29.8 C, while severely affected littermates showed a 4.1 C lower body temperature (25.7 C). Moderate transgene expressors could be identified by their less pronounced phenotype and showed a 2 C decrease in surface temperature (27.8 C). The direct correlation between transgene expression and mean body temperature shows how closely linked the barrier defect is to the cellular levels of the dominant-negative receptor.


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Table 1. Mean Body Surface Temperature1

 
Transgenic Pups Develop Hyperkeratosis
In our original report, we described the epidermal phenotype of the RAR{alpha}403 mice, and in the initial studies all severely affected pups died, suggesting that the severe phenotype caused lethality within 36 h of birth (19). Since that time, we have examined many more RAR{alpha}403 neonates and discovered that some severely affected neonates can survive to adulthood. This survival is probably due to several environmental factors, including maternal care. The surviving severe pups began life with the red, shiny skin previously described, but this phenotype changed with time. By 4 days after birth, some very severe pups developed a gross scaling phenotype, indicative of severe hyperkeratosis (Fig. 3Go).



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Figure 3. Scaling Phenotype Exhibited by Surviving 4-Day-Old RAR{alpha}403 Pups

Some severely phenotypic pups lose their initial shiny appearance and develop a hyperkeratotic, scaly appearance that resembles the effects of classic vitamin A deficiency.

 
Biochemical Analysis Shows Elevated Phospholipids and Glycosylceramides in SC of RAR{alpha}403 Mice
Due to the correlation between transgene expression and loss of barrier function, we compared the lipid composition of severe RAR{alpha}403 epidermis with that of normal littermates. TLC analyses demonstrated that all of the lipid classes found in normal mice were also synthesized in phenotypic mice, excluding a defect in lipid synthesis (data not shown). However, the SC of the phenotypically severe pups contained proportions of phospholipids and glycosylceramides that were higher than in normal SC. The proportions of phospholipids, including both sphingomyelin and phosphoglyceramides, and glycosylceramides were both 2- to 3-fold higher than in control SC (Table 2Go). These data most likely reflect incomplete processing of these lipids by the hydrolytic enzymes that normally process polar lipids during maturation of the permeability barrier in the epidermis.


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Table 2. Stratum Corneum Lipids (µg/mg dry tissue)1

 
Nile Red Stain Shows Elevated Polar Lipids in SC of RAR{alpha}403 Mice
To verify the lipid biochemical measurements and to determine whether the location of the phospholipids could be visualized, Nile Red staining was performed on severe and normal skin. Nile Red is a fluorescent dye that stains polar lipids red and nonpolar lipids green (26). In normal animals, the staining pattern showed minimal diffuse red stain throughout the entire epidermis, indicating very low levels of polar lipids (Fig. 4CGo), with intense red staining at the stratum granulosum-stratum corneum (SG-SC) interface. This area showed a thin line of highly localized polar lipids that appeared sometimes diffuse and sometimes punctuate (Fig. 4AGo). Confirming the biochemical data, the severe RAR{alpha}403 animals showed both increased polar lipid staining at the SG-SC interface, and intense polar lipid staining extending several cell layers into the SC (Fig. 4Go, B and D). No change in nonpolar lipid staining pattern was observed (data not shown). The Nile Red data, along with the biochemical analysis, gives strong evidence for a link between lipid processing and cellular responsiveness to retinoids.



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Figure 4. Nile Red Stain of Polar Lipids in Normal and Severe RAR{alpha}403 Skin

A and C (Normal skin), Polar lipids (red) stain intensely at the SG-SC interface with minimal, diffuse staining in the SC and spinous layer. B and D (RAR{alpha}403 skin), Note increased staining of polar lipids with intense staining that extends several cell layers into the SC (D), unlike in normals (C). Panels A and B, magnification 20x; panels C and D, 40x; arrows mark SG-SC interface.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
CRABPII-Altered Expression May Be Due to RAR{alpha}403’s Interactions with Corepressors
CRABPII has been shown in previous reports to be up-regulated by RA and to possess an RARE (27, 28). Thus, as expected, the presence of the dominant-negative RAR{alpha}403 significantly attenuated the induction of CRABPII upon topical ligand treatment. The increase in basal transcription of CRABPII in the severe animals is not entirely unexpected. The RAR{alpha}403 mutant receptor has a 4-fold increased affinity for binding the corepressor SMRT, even when the mutant receptor is not bound to DNA (13). With this in mind, it is possible that the high RAR{alpha}403 protein levels are sequestering endogenous SMRT away from other potential interactions with wild type RAR receptors, effectively causing a higher basal transcription rate from all genes containing RAREs. An alternate hypothesis is that the cells have some unknown mechanism for "sensing" their own retinoid responsiveness. In this scenario, the cells attempt to compensate for their RA insensitivity by up-regulating CRABPII. As a protein that binds RA, higher levels of CRABPII in a cell could increase the effective concentration of RA within its own cytoplasm. The problem with the latter hypothesis is that the proposed function for CRABPII is as a disposal system for excessive cellular retinoids. Although the specific mechanism for its action is unknown, it is postulated to decrease cellular response to retinoids by binding intracellular all-trans-RA and enhancing its catabolism (8). The CRABPII data demonstrate a clear loss of responsiveness to retinoid-induced gene expression and also suggest that other genes containing RAREs may be similarly affected by the presence of the RAR{alpha}403. If the barrier defect is a result of altered lipid-processing enzyme levels, it may be possible that the change in enzyme levels originates from decreased transcription, thereby directly linking RA responsiveness to maturation of the SC lipid barrier.

Induction of the Vitamin D3-Regulated Gene 25-Hydroxyvitamin D3-Hydroxylase Provides Insight into the in Vivo Action of the RAR{alpha}403 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 RAR{alpha}403 transgene, they had a reduced ability to transactivate an RARE-containing luciferase reporter. We also showed that the RAR{alpha}403 inhibited signaling from a peroxisome proliferator response element reporter construct and postulated that this inhibition was due to RAR{alpha}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{alpha}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 RAR{alpha}403 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 RAR{alpha}403. 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{alpha}403 neonates to determine whether it is the same as previously observed in vitro (19). RAR{alpha}403 neonates could be treated topically with clofibric acid, a ligand for PPAR, and the levels of a PPAR-responsive gene such as the {omega}-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{alpha}403 transgenic line is not the result of deficient VDR signaling via global sequestration of RXR.

The RAR{alpha}403 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 RAR{alpha}403 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{alpha}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{alpha}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 RAR{alpha}403 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{alpha}403 mice are completely deficient, suggests that the RAR{alpha}403 barrier disruption either affects multiple enzyme pathways or effects enzymes upstream of ß-glucocerebrosidase.

Decreased Mean Body Temperature May Cause RAR{alpha}403 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 RAR{alpha}403 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{alpha}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{alpha}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{alpha}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 RAR{alpha}403 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{alpha}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 RAR{alpha}403 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 RAR{alpha}403 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{alpha}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{alpha}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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Separation of Epidermis and Dermis
To separate the epidermis from the underlying dermis, neonatal skins were floated on 0.25% trypsin and 1 mM EDTA, at 4 C for 4 h, after which the epidermis could be pulled away from the underlying dermis with forceps.

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 2–3 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.


    ACKNOWLEDGMENTS
 
The authors would like to thank Drs. Peter Davies and Sophia Tsai for comments on the manuscript and N. J. Laminack for preparation of the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Dennis R. Roop, Ph.D., Department of Cell Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030.

1 Current Address: Tokyo University Hospital, Department of Dermatology, 7–3-1, Hongo, Bunkyo-ku, Tokyo, 113, Japan. Back

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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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