Background: Discovery of the significant impact of filaggrin (FLG) mutations on the genetic predisposition to atopic dermatitis (AD) focused attention on the 1q21 locus, where not only FLG but also other epidermal genes are located. In the present study, we compared 1q21 gene expression in lesional versus nonlesional AD skin. Methods: A real-time quantitative PCR analysis of 10 1q21 genes, selected on the basis of a previous microarray study, was performed in skin biopsies from 33 individuals with AD. Three alternative pathway keratins were also evaluated. Results: In chronic AD skin lesions, we observed an increase in RNA encoding involucrin, S100 calcium-binding proteins A2 and A7–A9 and small proline-rich region (SPRR) proteins 1A and 2C, with fold changes ranging from 2.0 for S100A2 to 15.4 for S100A8 (p < 0.001, Bonferroni corrected), in parallel to the overexpression of the alternative pathway keratins 6A, 6B and 16. The loricrin (LOR) expression level was significantly decreased in lesional AD skin (fold change 0.5; p < 0.01). The expression of the majority of 1q21 genes and alternative keratins was closely correlated; however, for SPRR1A (and SPRR2C) in lesional skin, the correlation with other genes was lost. Conclusions: We hypothesize that the deregulated increase in SPRR1A expression in chronic atopic skin lesions reflects an insufficient rise in SPRR transcripts, unable to compensate for the lack of LOR and thus contributing to the persistence of chronic AD skin lesions. Turning off the stress response in the skin may be regarded as a goal in the treatment of AD skin lesions, and SPRR genes might be targets for such an approach.

In recent years, much attention has been paid to the function of the epidermal skin barrier and to the molecular determinants of its deregulation in atopic dermatitis (AD). The cornified envelope (CE) is a key mechanism in barrier development, and disturbances in CE differentiation are central to the pathogenesis of AD skin lesions [1, 2]. In AD, the impaired function of the CE is genetically predetermined by loss-of-function mutations in the filaggrin (FLG) gene, seen in approximately half of European patients [3, 4]. This gene is located on chromosome 1q21, within the human epidermal differentiation complex (EDC). At the protein level, FLG expression is very distinctly decreased or even obliterated in FLG mutation carriers [5], while mRNA levels may be less distinctly impaired [6]. Howell et al. [7] recently documented a decrease in FLG transcript and protein in acute AD lesions and related it to the influence of IL-4 and IL-13.

The EDC comprises several gene families [8], not only the family of fused genes encoding FLG itself and FLG-like proteins, among them FLG 2, repetin, trichohyalin, cornulin and hornerin. Short tandem peptide repeats in the central region characterize another gene family, which contains loricrin (LOR), involucrin (IVL) and a small proline-rich region (SPRR) gene cluster with not less than 11 genes [9, 10]. Another group is composed of S100 family genes, with at least 14 transcripts encoding calcium-binding proteins with EF hand domains (11 of them are expressed in human epidermis). Also, 3 clusters of late envelope protein genes should be mentioned. During the physiological formation of the CE, several genes from each family are expressed in a coordinated way and the epidermal barrier activity is acquired in a stepwise manner [11], firstly with IVL expression, followed by the expression of S100 proteins as well as SPRR proteins and, finally, LOR and FLG as late differentiation markers. A large number of CE proteins are transglutaminase substrates, used to assemble a cross-linked scaffold beneath the keratinocyte plasma membrane [12]. In normal epidermis, the major CE protein is LOR, which constitutes up to 70–80% of the total cross-linked protein mass and is cross-linked by SPRR proteins [1]. FLG, produced by post-translational proteolysis of the precursor protein, profilaggrin, plays a unique and pivotal role in cornification by promoting the formation of disulfide bonds and, as a result, aggregation of keratin filaments. Later on, FLG amino acid residues are modified and the molecule is degraded; this is instrumental in maintaining epidermal hydration and flexibility.

While the expression of FLG in AD has been studied extensively at both the RNA and protein level [5, 7], data concerning the expression of other CE genes are far less consistent [5, 13, 14]. We decided to study RNA expression because it has been proven that EDC genes are regulated mainly at the transcriptional level [15, 16]. Importantly, a large-scale DNA microarray analysis performed in AD skin biopsies by Sugiura et al. [6] indicated that several 1q21 locus genes responsible for the protective functions of the CE exhibited profound changes in their transcript expression. The authors concluded that a key abnormality in AD might be deterioration of epidermal differentiation associated with altered expression of genes located on 1q21. However, the reported level of significance was not always sufficient and, even more essentially, no validation quantitative real-time reverse transcription PCR (QPCR) study has followed until now.

In our analysis, we evaluated the expression of the 1q21 genes identified by the microarray study mentioned above and compared it to that of the alternative keratinization pathway genes by applying a well-controlled QPCR approach in lesional and nonlesional skin of AD patients.

Patients

Thirty-three individuals (20 men and 13 women; age 18–45 years, median 24 years) with AD, diagnosed according to Hanifin and Rajka criteria, were included in the study after giving informed consent. The duration of AD ranged between 5 and 45 years. Atopic family history was positive in 45.5% of these patients (15/33), while personal history was positive in 85% (28/33). Standard skin prick tests (Allergopharma, Germany) were positive in all but 5 patients. Serum IgE (Allergopharma) was increased above 100 kU/l in 26 patients. The severity of AD was assessed by the SCORAD index [17]. No patients were treated with systemic corticosteroids or other immunosuppressive drugs. Systemic antihistamine drugs and/or local treatment were discontinued for at least 4 weeks before skin biopsy.

Full-thickness 4-mm punch skin biopsies (Stiefel Laboratories) of 3-mm depth were taken and immediately placed in RNAlater solution (Qiagen). Only chronic AD lesions were biopsied, as confirmed by histopathological assessment. The location of the skin biopsies was determined to avoid cosmetic consequences and to fulfill the condition of symmetry between lesional versus nonlesional areas, as well as keeping at least a 10-cm distance between nonlesional biopsy locations and other AD skin lesions. Arm skin was biopsied in 24 subjects, forearm skin or thigh skin in 3 and hand skin or trunk skin in 1. The study was approved by the local medical ethics committee and carried out according to Declaration of Helsinki regulations.

Isolation of RNA and cDNA Synthesis

Samples were stored at –80°C. Tissue was homogenized using a FastPrep FP120 instrument (lysing matrix D, Qbiogene). Total RNA was extracted using RNeasy Mini kits (Qiagen), including an on-column DNAse digestion step, according to the manufacturer’s instructions. RNA was quantified using a NanoDrop ND-1000 spectrophotometer, and sample quality was assessed by an Agilent 2100 Bioanalyzer using an RNA 6000 Nano Assay (Agilent Technologies). RNA qualtiy, assessed by the RIN (RNA integrity) index, was within the 6.3–9.7 range. Paired samples from one individual did not exhibit significant differences in RNA quality (median difference in RIN between samples in pairs was 0.4). cDNA was synthesized from 200 ng of total RNA using an Omniscript cDNA synthesis kit (Qiagen), a mixture of oligo-dT primers (1 µM) and random nonamer primers (4 µM; Sigma) and 10 units of RNase inhibitor (Sigma) per reaction (37°C for 1 h).

Quantitative Real-Time Reverse Transcription PCR

Nine EDC genes selected by Sugiura et al. [6] in their gene expression profiling study were investigated; we also evaluated expression of the repetin gene. Three alternative pathway keratins, identified previously by the above-mentioned microarray study, were studied in parallel for the evaluation of the stress response of the epidermis. QPCR assay was performed using locked nucleic acid 5′-nuclease fluorescent probes (Universal Probe Library, Roche). Amplicons were designed using a Web-based application (www.roche-applied-science.com/sis/rtpcr/upl; for primer sequences, see table 1); intron-spanning amplicons were designed where possible (table 1). QPCR was carried out using an ABI Prism 7700 Sequence Detection System (Applied Biosystems) and a 96-well optical reaction plate. Five microliters of template cDNA (equivalent to 5 ng of total RNA) were added to 15 µl of PCR reaction mix containing 10 µl of TaqMan Universal PCR Master Mix (Applied Biosystems), 1 µl of forward and reverse primers (200 nM), 1 µl of probe (100 nM) and water. Thermal cycling conditions were as follows: 50°C for 2 min, then 95°C for 10 min followed by 40 cycles of PCR (95°C for 15 s and 60°C for 1 min). Each sample was examined in duplicate; the duplicates were assayed in independent runs. The mean coefficient of variability between duplicates was 3.03%; it ranged from 1.6 to 4.5% for different genes. A standard curve used throughout the experiments (serial dilutions of 200, 100, 40, 20, 8, 2, 0.4 and 0.04 ng, assayed in triplicate) was prepared from Human Reference RNA (Stratagene, used for reference genes) or normal skin RNA, mixed from 3 individual donors (used for the studied EDC genes). The efficiency for each gene was calculated from the standard curve slope by a modified delta delta Ct method (table 1), according to the formula given by Pfaffl [18, 19]; linearity of the obtained standard curves was verified by the R2 coefficient. Gene expression values were obtained relative to calibrator samples (the same as the samples used to prepare the standard curve, 200 ng) run in triplicate on each plate.

Table 1

Amplicons used for QPCR measurement of analyzed genes

Amplicons used for QPCR measurement of analyzed genes
Amplicons used for QPCR measurement of analyzed genes

Gene Expression Normalization

To normalize the results obtained by QPCR, we analyzed the expression of 5 housekeeping genes (HADHA, UBE2D2, EIF5 EIF3S10 and ATP6V1E). The chosen genes showed no differences in expression between lesional and nonlesional skin. To obtain the reference index (based on the geometric mean of the reference genes’ expression values), geNorm software was used (fig. 1) [20]. All obtained results were normalized to this reference index, which ranged between 0.19 and 1.03. The most stable reference genes were HADHA and EIF5. The reference index obtained by combining more than 2 genes had increased stability.

Fig. 1

Assessment of the reference genes used to normalize QPCR data. a Stability of the selected genes. b Pairwise differences between reference indexes based on different number of genes. Analysis was performed using geNorm software [20].

Fig. 1

Assessment of the reference genes used to normalize QPCR data. a Stability of the selected genes. b Pairwise differences between reference indexes based on different number of genes. Analysis was performed using geNorm software [20].

Close modal

Statistical Analysis

To determine between-group differences for paired samples, we used the Wilcoxon signed-rank nonparametric test, with exact computation of p values.p values were corrected for multiple comparisons by the Bonferroni method, using the number of genes tested. Results significant at a p value of 0.05 after Bonferroni correction were deemed significant. Correlations were assessed by the Pearson coefficient on log10-transformed data; they were interpreted as significant at p < 0.001 (only results with p < 0.05 are shown). Hierarchical clustering was performed by average linkage and Pearson correlation as a distance metric. SSPS 13 (SPSS, Chicago, Ill., USA) was used for all statistical calculations.

Expression of S100 Genes

S100A2, S100A7, S100A8 and S100A9 exhibited a strong and very significant upregulation in lesional AD skin, with fold changes ranging from 2.0 for S100A2 to 15.4 for S100A8 (table 2). There was almost absolute correlation between S100A8 and S100A9 levels (R = 0.95 for nonlesional and 0.98 for lesional skin, p < 0.001; table 3). Close correlations were observed for S100A7 and S100A9 levels, and S100A7 and S100A8 levels, both in nonlesional and lesional skin, while the expression of S100A2 exhibited a weak correlation with S100A7 and A9, observed only in nonlesional skin.

Table 2

Gene expression comparison in nonlesional and lesional skin from individuals with AD

Gene expression comparison in nonlesional and lesional skin from individuals with AD
Gene expression comparison in nonlesional and lesional skin from individuals with AD
Table 3

Correlation between individual transcript levels in skin biopsies from patients with AD

Correlation between individual transcript levels in skin biopsies from patients with AD
Correlation between individual transcript levels in skin biopsies from patients with AD

Expression of SPRR Genes

Similarly to S100 genes, the investigated SPRR transcripts were also distinctly increased in lesional AD skin, with an approximately 3-fold change (table 2). They also resembled S100 genes in their lack of correlation between lesional and nonlesional skin levels (table 2). In nonlesional skin, the SPRR genes showed significant correlations with the S100 gene expression levels, the strongest being for SPRR2C/S100A7 (R = 0.84, p < 0.001; table 3). For SPRR1A, correlations with LOR, FLG, SPRR2C and keratin 16, significant at p < 0.001 in nonlesional skin, were absent in AD skin lesions, while 2 new correlations appeared, with S100A2 and IVL (although the latter did not fully meet our criterion of statistical significance at p = 0.005).

Expression of Other CE Genes: LOR, IVL, FLG and Repetin

IVL expression was distinctly upregulated in lesional AD skin (fold change 2.2 between lesional and nonlesional skin; table 2). In contrast, LOR expression showed downregulation, with a significant fold change of 0.5. Median FLG mRNA content was only slightly decreased in AD skin lesions, with an uncorrected p value of 0.048 (0.9-fold change between lesional and nonlesional skin), and appeared nonsignificant after Bonferroni correction for multiple comparisons. Repetin mRNA, which encoded an FLG-like peptide, also did not show any significant change.

The expression of IVL, LOR and FLG showed a significant correlation between nonlesional and lesional skin from the same individual (p < 0.001; table 2), the closest correlation being for IVL (R = 0.82). FLG RNA correlated with LOR in both AD lesions and intact skin (table 3), with similar correlation coefficients (R = 0.83 and 0.79, respectively, p <0.001). However, this correlation was much weaker at the low expression levels of both genes, seen more often in lesional skin (fig 2a). An inverse correlation with the LOR transcript level was noticed for S100A8 and A9 both in nonlesional and lesional skin; for S100A7 and SPRR1A, such a correlation was noticed in nonlesional skin only.

Fig. 2

Correlations between expression levels of LOR and FLG (a) and LOR and keratin 16 (b).

Fig. 2

Correlations between expression levels of LOR and FLG (a) and LOR and keratin 16 (b).

Close modal

Expression of Alternative Pathway Keratins

Keratins 6A, 6B and 16 showed a very distinct increase in mRNA in lesional skin (fold changes of 4.8, 2.1 and 13.5, respectively; table 2). Only keratin 6A expression was significantly correlated between nonlesional and lesional skin (R = 0.64, p < 0.001). In both nonlesional and lesional skin, keratin 16 was correlated with S100A7–A9 (the strongest correlation was noted for S100A8: R = 0.77, p < 0.001) and inversely correlated with LOR and FLG (fig. 2b, table 3); only for SPRR1A was the correlation lost in lesional skin. For keratin 6B, correlations were more significant in lesional skin, and 2 of them reached the level of p < 0.001.

Hierarchical Clustering of Gene Expression Values in Lesional and Nonlesional Skin

Hierarchical clustering was carried out to analyze the pattern of similarity in gene expression in lesional and nonlesional skin and thus to identify gene clusters. In nonlesional AD skin, we found coexpression of S100A7–A9, SPRR, IVL and alternative pathway keratin genes (fig. 3). A moderate similarity was observed between S100A2 and repetin. On the other hand, LOR and FLG transcripts formed a distinctly different cluster.

Fig. 3

Hierarchical clustering of genes in nonlesional (left panel) and lesional (right panel) skin. Note the changed position of SPRR1A and SPRR2C. KRT = Keratin; RPTN = repetin.

Fig. 3

Hierarchical clustering of genes in nonlesional (left panel) and lesional (right panel) skin. Note the changed position of SPRR1A and SPRR2C. KRT = Keratin; RPTN = repetin.

Close modal

A quite different pattern was observed in lesional skin; SPRR genes lost the strong association with S100 and keratin genes seen in nonlesional skin and appeared to be coregulated with S100A2 (SPRR1A) and repetin (SPRR2C). Coexpression of FLG and LOR was retained in lesional skin.

In the present study, we compared the expression pattern of EDC genes in lesional skin biopsies to that found in biopsies of nonlesional skin from the same AD patients, all taken contralaterally at the same location, at a sufficient distance from other lesions. Recently, it has been shown that a defective skin barrier, caused by loss-of-function mutations in the FLG gene, significantly predisposes individuals to AD. Unfortunately, the FLG genotype of our patients was unknown; thus, we were unable to assess the downstream effects of FLG loss-of-function mutations. Instead, we focused on other 1q21 genes which had previously been found to exhibit changes in expression by an AD microarray study [6], in order to validate the results by an independent approach and to investigate in a more detailed manner their expression pattern, so far uncharacterized. In chronic AD skin lesions, we observed a distinct rise in RNA encoding IVL and S100 calcium-binding proteins A2, A7, A8 and A9, as well as SPRR proteins 1A and 2C. The rise occurred in parallel to the overexpression of alternative pathway keratins 6A, 6B and 16. Among the closely correlated genes, a deregulation of SPRR1A was noticed; of 9 significant correlation changes (table 3), 6 affected this transcript. Also, a distinct change in the hierarchical clustering tree was observed for SPRR1A and, to a lesser extent, SPRR2C (fig. 3).

In intact human epidermis, the SPRR1 and 2 proteins are cross-linked to LOR, together with keratins 1, 2 and 10 as well as FLG and elafin [2]. Upregulation of SPRR1A and 2C mRNA, found in AD lesions in our study, might partially compensate for the observed LOR transcript deficiency. This was seen in studies using LOR-deficient mice [2, 21], and was also accompanied by augmented IVL expression, similar to our human study. The induction of SPRR1 and 2 gene expression in inflammatory skin diseases has been shown previously [22] and was interpreted as part of the response to conditions that require rapid epidermal regeneration. Expression of SPRR genes is believed to be of major importance for CE biomechanical properties [16, 23]. SPRR2C protein was previously described to be much more intensely upregulated in psoriasis than in AD [24] and is regarded as an epithelial host defense protein [6]. These changes need to be further verified at the protein level. Sugiura et al. [6] also found changes in other SPRR family transcripts, i.e. upregulation of SPRR1B and downregulation of SPRR3. Unfortunately, these transcripts were not included in our panel of studied genes.

Contrary to the conclusions of Sugiura et al. [6], we noticed a significant upregulation of IVL RNA in AD skin lesions. At the protein level, a decrease in IVL expression in AD skin lesions was reported in other studies [5, 14]. However, our results are consistent with those of Hirao et al. [13], who described a distinct but heterogenous rise in IVL-containing immature CE in stratum corneum cells of lesional AD skin. IVL is regarded as a human keratinocyte differentiation marker [25], and its rise is an early event in normal epidermal differentiation. We conclude that this step of EDC differentiation is not impaired in AD. However, the IVL RNA increase was not accompanied by an increase in LOR RNA, as would be expected in normal skin; on the contrary, a significant decrease in LOR RNA was observed in our study. LOR is the major component of the cornified cell envelope. It is expressed in a keratinocyte- and differentiation-specific manner and is present only in terminally differentiated keratinocytes. Immunohistochemistry and Western blot analysis showed LOR expression to be upregulated 3-fold in both nonlesional and lesional AD skin when compared to normal controls [14]. In the same study, there was no difference in LOR transcript copy number at the mRNA level when lesional AD skin was compared to nonlesional AD skin. Our observation was different and remains in agreement with the cited microarray data [6]. It is also consistent with the downregulation of LOR described by Hohl [26], who also noticed the similarity between FLG and LOR expression changes. This change may be important in light of the well-known strict regulation of the LOR gene at the transcriptional level [15, 27].In situ hybridization analyses have shown that LOR transcripts are localized only in the upper spinous and granular layers of the normal epidermis [28]. In vitro data [15] suggest a complex interplay between transcription factors of the Sp1, CREB and AP1/AP2 families in the regulation of the LOR gene. Until now, there have been no data regarding whether these mechanisms do operate in AD lesional skin. On the other hand, the observed decrease might be secondary to the high level of protein product [14].

Other CE genes, activated during normal development before LOR or simultaneously with its expression, were distinctly upregulated in lesional AD skin. The coordinated upregulation of S100A7–A9 reported in our study confirms the data obtained by Broome at al. [29 ]in inflammatory disorders of the skin. The expression of S100A7–A9 is low in normal epidermis. Their overexpression is typical for psoriasis; however, S100A7 has been found in many other epidermal inflammatory diseases including AD and was proposed to function as a keratinocyte-derived chemotactic agent [9]. Multiple immunological functions have been attributed to S100 proteins, as well as a role in epidermal wound repair, differentiation and the response to stress [30, 31].

We did not observe significant changes in FLG RNA in chronic AD skin lesions; however, our conclusion is weakened by the fact that our choice of primers for this transcript was suboptimal – both primers were located within exon 3 and led to the amplification of 2 amplicons of identical size. Nevertheless, the possible variability in the number of repeats in the FLG gene should not influence the comparison of FLG gene expression between affected and unaffected skin, as an equal number of repeats is expected in both specimens from one individual. As already mentioned, nothing is known in relation to the crucial question whether the changes in 1q21 gene expression observed in our study differ depending on the presence of germline FLG mutations predisposing to AD. However, it should be considered that the mutations in the FLG gene responsible for the development of AD in a significant number of patients may not be reflected by a decrease in FLG transcript itself. These mutations nearly always occur in the last exon of the gene, and the mutated transcript escapes the non-sense-mediated mRNA degradation pathway, while the decrease in protein expression is very distinct and related to the loss of translation or instability of the protein product [3, 4, 32].

Our study comprised only AD patients, without a healthy control group. Thus, the only comparison possible was the comparison between nonlesional and lesional AD skin. This model may potentially miss certain changes seen in AD patients even in nonlesional skin. It is well known that in AD patients, the FLG content of nonlesional skin is lower than in healthy persons; the same variation has been described for LOR and IVL [5, 14, 26]. These genes exhibited a strong correlation of expression level between nonlesional and lesional skin, indicating indirectly that they were influenced by a genetic predisposition.

In light of the observed increase in the expression of alternative keratins, the changes seen in chronic AD skin lesions may be interpreted as the stress response of the epidermis in the context of high regeneration requirements; keratins 6 and 16 are markers of an alternative pathway of keratinocyte differentiation, a response to various conditions that require rapid epidermal regeneration and protective gene expression [33]. Sugiura et al. [6] were the first to document their upregulation in AD, concurrently with changes in the locus 1q21 genes.

An important finding of our study is the deregulated increase in SPRR expression in chronic atopic skin lesions; SPRR1A and (to a lesser extent) SPRR2C lose their coexpression with S100 genes and other 1q21 transcripts. We hypothesize that this altered pattern reflects an insufficient rise in SPRR expression, unable to compensate for the lack of LOR and thus contributing to the persistence of chronic AD skin changes. According to Segre [16], it is necessary to elucidate the multiple ways in which keratinocytes signal a barrier disruption in AD lesions and maintain a stress response. Turning off the stress response in the skin may be regarded as a goal in the treatment of AD skin lesions, and SPRR genes might be the new targets of such an approach.

We are grateful to Ms. Teresa Prabucka for her excellent technical assistance and to Dr. Aleksander Sochanik, PhD, for thorough language revision of the manuscript.

This study was supported by an internal grant of the Silesian Medical University.

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