ARTICLE |
Correspondence to: Denis Salomon, Clinique de Dermatologie, Hôpital Cantonal Universitaire, CH-1211 Genève 4, Switzerland.
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Summary |
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After 14 weeks of topical application of 0.1% all-trans-retinoic acid to the napes of volunteers, we observed a 2.5-fold increase in the thickness of epidermis, owing to an increase (p<0.001) in the number and size of keratinocytes and the induction of keratin 6. These changes in the differentiation of epidermal keratinocytes were paralleled by an increase in the amount of Cx43, a connexin that is normally expressed in human epidermis, and by the massive induction of Cx26, which is barely detectable in normal interfollicular epidermis, as judged at both the transcript (Northern blotting) and the protein level (immunolabeling). In contrast, retinoic acid treatment did not alter the morphology and connexin pattern of hair follicles or of sebaceous and sweat glands, and did not induce the expression of other connexins (C32, Cx37, Cx40) in either skin adnexae or epidermis. These observations suggest that the expression of two distinct connexins by interfollicular keratinocytes is related to selective changes in the differentiation program of epidermis that are induced by retinoic acid. (J Histochem Cytochem 45:1207-1215, 1997)
Key Words: gap junctions, connexins, all-trans-retinoic acid, epidermis, keratinocytes
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Introduction |
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Keratinocytes of human skin are connected by gap junctions (
This regulation is dependent on the interplay of a number of growth factors and morphogenetic molecules, among which all-trans-retinoic acid (RA) plays a prominent role (
The gap junctions of human keratinocytes comprise at least two distinct connexins. Cx43 is abundantly expressed within the interfollicular epidermis (
A first objective of this study was therefore to investigate whether the two connexins that are normally expressed in normal skin are similarly affected by an RA treatment that markedly alters the differentiation program of human keratinocytes. Because such an alteration is known to be associated with the appearance of proteins that are not natively expressed by interfollicular keratinocytes, e.g., keratin K6 (
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Materials and Methods |
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RA Treatment and Tissue Sampling
The in vivo treatment was performed in agreement with the guidelines of our institutional committee for clinical investigation and with the informed consent of seven volunteers (four women and three men, 35-52 years old).
Each volunteer applied once a day, over 14 weeks, 0.2 ml of vehicle solution to an area of approximatively 6 cm2 on one side of the nape and a similar volume of a 0.1% all-trans-retinoic acid solution (Cilag AG Pharma; Schaffhausen, Switzerland) on the other side. Skin biopsies were taken in the center of treated and control areas under intra-dermal anesthesia with 1% lidocaine and 0.5 UI/ml Ornipressine (Sandoz; Basel, Switzerland). Samples were rapidly frozen by immersion in 2-methylbutane (Merck; Basel, Switzerland), which was cooled in liquid nitrogen, and stored at -80C until cryostat sectioning (for immunolabeling), or fixed at room temperature in 4% paraformaldehyde and 0.1% glutaraldehyde in PBS (for morphometric measurements).
For Northern blot analysis, three of the volunteers (one woman and two men, 35-39 years old) applied RA as indicated above on their buttock. This region was chosen because of its low density in hair follicles and the possibility of obtaining dermatome skin samples of constant thickness (0.3 mm) without esthetic prejudice.
Immunostaining
For immunofluorescence, sections about 5-µm thick were cut with a cryomicrotome (1720 digital MGW; Leitz, Ober-kochen, Germany), collected on gelatin-coated slides, and fixed for 3 min in -80C acetone. All slides were then rinsed in cold (4C) PBS containing 0.5% bovine serum albumin and were processed for indirect immunofluorescence staining.
For localization of gap junction proteins, sections were first incubated for 2 hr at room temperature with one of the following antibodies: (a) affinity-purified rabbit polyclonal antibodies against liver Cx32, diluted 1:100 (
A similar protocol was followed to localize keratin 6, using either an affinity-purified rabbit polyclonal serum, diluted 1:200, (kindly provided by Dr. E. Fuchs) or mouse monoclonal antibody D5/16B4 (Boehringer; Mannheim, Germany), diluted 1:50. Accordingly, the second incubation was performed with fluorescein-conjugated anti-rabbit or anti-mouse antibodies, diluted 1:200.
In all immunostaining experiments, negative controls included exposure of sections during the first incubation to either pre-immune serum, normal rabbit or mouse serum, or fluorescein-conjugated anti-rabbit or anti-mouse antibodies, whichever applicable. Cryosections of rat liver, pancreas, and heart and of mouse skin were incubated in parallel with the human samples and used as positive controls for the different anti-connexin antibodies tested.
Morphometric Evaluation
Sections of RA- and vehicle-treated samples from each volunteer were incubated with an identical batch of affinity-purified antibodies against either Cx43 or Cx26, to quantitate immunostaining within the interfollicular epidermis. To this end, color slides of each section, taken at a magnification of x157, were projected at a final magnification of x2440 on a graphics tablet connected to an IBM personal computer for area measurements and event counting. In each slide, we scored the number of keratinocytes and immuno-fluorescent spots and measured the area of epidermis. Data were collected both cumulatively for the entire epidermis (excluding the stratum corneum, which never showed specific immunolabeling) and, separately, in the basal layer of the tissue, in the uppermost layer of the granular region, and in one layer in the middle of the spinous region. Controls showed that the latter layer was representative of the entire spinous region, as far as Cx labeling was concerned (not shown). On the basis of these data, we calculated the number of fluorescent spots per layer of keratinocytes at different stages of differentiation.
Aldehyde-fixed skin from RA- and vehicle-treated samples was embedded in Lowicryl K4M resin and sectioned at about 1-µm thickness using a Reichert OM10 ultramicrotome. Sections were stained with methylene blue and photographed as described above. Color slides of each section were projected at a final magnification of x1840 and were used to evaluate the number of keratinocytes per epidermal field (defined as the area of epidermis photographed in each slide) and the mean area of keratinocyte profiles in the basal, spinous, and granular layers of interfollicular epidermis. All areas were measured by planimetry.
Data were analyzed by analysis of variance and compared by the median, the Kolmogorov-Smirnov, and the Mann-Whitney tests, using programs ANOVA and NPAR TESTS of the SPSS-X statistics package (SPSS; Chicago, IL).
Plasmid Constructions
Human connexin cDNAs for Cx43 (
Identification of Connexin Transcript
Samples were homogenized in 2.5 ml 0.1 M Tris-HCl, pH 7.4, containing 1.3 M ß-mercaptoethanol and 4 M guani-dium thiocyanate. After addition of solid CsCl (0.4 g/ml), the homogenate was layered on a 2-ml cushion of 5.7 M CsCl-0.1 M EDTA (pH 7.4) and centrifuged at 35,000 rpm at 20C for 20 hr. Pelleted RNA was resuspended in 300 ml 10 mM Tris-HCl, pH 8.1, suppplemented with 5 mM EDTA and 0.1% SDS, extracted twice with phenol-chloroform, precipated in ethanol, and resuspended in water. For Northern blots, total cellular RNA was denatured by 1 M glyoxal in 0.01 M phosphate buffer containing 50% DMSO, electrophoresed in a 1% agarose gel (5-10 µg total cellular RNA/lane), and transferred overnight onto nylon membranes (Hybond N; Amersham International, Poole, UK). Filters were exposed for 30 sec to 302-nm light and stained with methylene blue. Radiolabeled cRNAs were synthezised by in vitro DNA transcription in a Tris buffer supplemented with 0.05 mg/ml BSA, 16 mM DTT, 1 U RNAse inhibitor, 3 NTP with RNA polymerase. Filters were prehybridized for 2 hr at 65C in a 0.05 M Pipes-buffered (pH 6.8) solution of 50% formamide which was supplemented with 2 mM EDTA, 0.1% SDS, 0.1 mg/ml salmon sperm DNA, 0.8 M NaCl, and 2.5 x Denhardt's solution. Filters were then hybridized for 18 hr at 65C with 2.106 cpm/ml solution 32P-labeled probe, washed twice at 65C in 3 x SSC and 2 x Denhardt's solution, followed by three washings at 70C in 0.2 x SSC, 0.1% SDS, and 0.1% sodium pyrophosphate. Filters were then exposed to film (XAR-5; Eastman Kodak, Rochester, NY) between intensifying screens at -80C for 1 to 4 days.
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Results |
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RA Treatment Affected the Differentiation of Interfollicular Keratinocytes
Compared to controls, all samples of RA-treated skin showed a striking increase in epidermal thickness, which was on average 2.5-fold (p<0.0001), as evaluated by epidermal area in both Lowicryl-embedded (Figure 1) and frozen skin samples (Table 1). Additional measurements showed that this change was due to an increase (p<0.0001) in both the number of keratinocytes per epidermal field and in the average area of their profiles (Table 1). These changes were observed in all layers of the epidermis except for the basal layer, and were prominent in the spinous layers. Results were similar in all samples irrespective of the age and sex of volunteers. By contrast, the morphology of epidermal adnexae appeared unaffected by the RA treatment. In full agreement with previous observations (
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RA Treatment Modulated the Expression of Cx43
Immunostaining of cryosections for Cx43, using one affinity-purified and one monoclonal antibody, revealed this gap junction protein in the interfollicular epidermis of control and RA-treated samples (Figure 2). Qualitatively, the distribution of Cx43 appeared similar in all cases. In both control and RA-treated skin, immunolabeling of Cx43 was weak in the basal layer of keratinocytes, strong throughout the spinous layers, weak again in the granular layers, and absent in the stratum corneum (Figure 2). Quantitative comparison of sections that had all been incubated with the same affinity-purified serum (f in the list reported in the Material and Methods section), revealed a significant (p<0.05) increase in the total amount of Cx43 immunofluorescent spots (Figure 2). This change was essentially due to a larger (p<0.05) number of these spots over the keratinocytes of spinous layers (Figure 2). Northern blot analysis of comparable samples confirmed that these changes were paralleled by increased levels of Cx43 mRNA (Figure 4).
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Analysis of skin adnexae failed to detect changes in their immunolabeling for Cx43. This connexin was detected in hair follicles, sebaceous glands, and ducts of eccrine sweat glands in both control and RA-treated skin samples (Table 2).
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RA Treatment Induced the Expression of Cx26
Immunostaining of cryosections of control skin, using three different antibodies against Cx26, usually failed to detect this protein in interfollicular epidermis (Figure 3), except in some keratinocytes forming the upper granular layers. In contrast, when applied to sections of RA-treated skin, the same antibodies resulted in an abundant punctate staining of interfollicular keratinocytes of spinous and granular layers (Figure 3). Quantitative comparison of sections that had all been incubated with the same affinity-purified serum against Cx26 (d in the list reported in the Material and Methods section) revealed that this change predominated in keratinocytes of spinous and granular layers (p<0.0001) and was more modest in those of the basal layer (Figure 3). Northern blot analysis of comparable samples confirmed that the changes in Cx26 immunofluorescence were paralleled by a marked increase in the expression of Cx26 mRNA (Figure 4). Hence, whereas this transcript was not detectable in dermatome samples of normal skin, it was readily detected in samples of RA-treated skin from all the volunteers tested.
Contrasting with the major alteration observed within the interfollicular epidermis, no change in Cx26 immunolabeling was detected in skin adnexae. In both control and RA-treated skin, this connexin was readily detected in hair follicles and ducts of sweat glands, but not in sebaceous glands (Table 2).
RA Treatment Did not Induce Expression of Cx32, Cx37, and Cx40
Antibodies that identified Cx32, Cx37, and Cx40 in other tissues (human liver for Cx32, human heart for Cx37 and Cx40) failed to detect these gap junction proteins in the interfollicular epidermis of both control and RA-treated human skin (Table 2).
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Discussion |
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Human keratinocytes are connected by many gap junctions (
We have first confirmed that, in normal volunteers, this treatment induces a thickening of interfollicular epidermis (
Under these conditions of altered differentiation, we have first found that interfollicular keratinocytes increase by about 1.7-fold the expression of Cx43, a connexin that they natively express under control conditions. This increase was paralleled by moderate upregulation in the expression of Cx43 mRNA, analogous to that previously reported for another protocol of topical retinoid treatment (
This finding shows that keratinocytes may express different connexins, not only depending on the species and the region of the epidermis but also as a function of the differentiation program of the tissue. Under the conditions studied here, differentiating keratinocytes of spinous and granular layers shifted their connexin pattern in vivo from the virtually sole expression of Cx43 to an increased expression of both Cx43 and Cx26. Clearly, the levels of these two connexins were not modified to the same extent. Hence, multiple connexins of a same keratinocyte subpopulation may be differentially regulated in vivo, as has been shown in vitro (
As yet, the signals crossing gap junctions in vivo remain undetermined, as is the biological function of their cell-to-cell passage. However, the present experiments indicate that a relationship should exist between the control of connexin expression and that of a specific differentiated phenotype. Therefore, chronic exposure to RA, which induces the keratinocytes of spinous and granular layers to enlarge and to express a keratin that is usually retricted to keratinocytes of skin adnexae (
The mechanism by which retinoic acid modulates the differentiation of the epidermis is still uncertain but appears to be related to selective changes in connexin expression (
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Acknowledgments |
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Supported by grants from the Swiss National Science Foundation (32-42539.94 and 32-34086.95), the European Union (BMH4-CT96-1427), and the Research and Inventive Technologies Interstate Commission, Generalitat de Catalunya, Spain (BE 91-134 and BE 92-24).
We thank P. Carraux, A. Charollais, F. Cogne, J.-P. Gerber and E. Sutter for excellent technical assistance. We also thank Drs J. Goliger, Y. Shibata, O. Traub, and E. Beyer for providing us with anticonnexin antibodies, and Drs G. Fishman and S. Lee for human connexin cDNAs.
Received for publication January 2, 1997; accepted March 7, 1997.
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