HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 17/13/1969 03-0148fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by WOLF, R. Articles by RUZICKA, T. Search for Related Content PubMed PubMed Citation Articles by WOLF, R. Articles by RUZICKA, T.

Molecular cloning and characterization of alternatively spliced mRNA isoforms from psoriatic skin encoding a novel member of the S100 family¹

RONALD WOLF^*,2, ALIREZA MIRMOHAMMADSADEGH^*, MARKUS WALZ^*, BARBORA LYSA^,*, ULRIKE TARTLER^*, RALPH REMUS, ULRICH HENGGE^*, GÜNTER MICHEL^* and THOMAS RUZICKA^*

^* Department of Dermatology and
Institute of Human Genetics, Heinrich-Heine University, Düsseldorf, Germany; and
Department of Dermatology, Charles University, Prague, Czech Republic

²Correspondence: Department of Dermatology, Heinrich-Heine University Düsseldorf, Moorenstrasse 5, 40225 Düsseldorf, Germany. E-mail: Ronald.Wolf{at}med.uni-duesseldorf.de

SPECIFIC AIMS

Psoriasis is a chronic, inflammatory disease of widespread occurrence, characterized by increased proliferation and altered differentiation of keratinocytes, possibly caused by altered gene expression. To identify and clone psoriasis-associated genes, we compared gene expression in normal and psoriatic skin using the differential-display reverse transcriptase-polymerase chain reaction (RT-PCR) analysis.

PRINCIPAL FINDINGS

1. Identification of a novel human S100 cDNA
By differential-display RT-PCR analysis, a fragment of 650 bp (clone 110) was detected, which was highly up-regulated in lesional skin. Sequence analysis revealed homology to a noncoding cDNA (NICE-2). By using clone 110 as a probe for Northern blot hybridization, a transcript of 4.4 kb was detected in RNA of psoriatic skin (Fig. 1 A). Multiple genomic analyses predicted a particular coding region 4 kb upstream of clone 110 consisting of two exons (clone RP1-128L15, acc. no. AL591704). To prove if clone 110 and the open reading frame (ORF) predicted are part of the 4.4-kb transcript detected, PCR primers specific for the 3'-untranslated region (UTR) and the putative coding region were designed and used for cDNA amplification of psoriatic skin. The cDNA clones obtained completed the 3'-UTR (clone 110/NICE-2) and consisted of a 5'-UTR and the coding region, resulting in a total transcript of 4.351 kb in size (Fig. 1A , S100A15-L, acc. no. AY189118).

View larger version (45K): [in this window] [in a new window]   Figure 1. Differential expression of the S100A15 isoforms in normal and psoriatic skin. A) Northern blot analysis: Individual, total RNA samples from normal (four), nonlesional (three), and lesional (three) skin were hybridized with probes specific for the long and short isoform of the S100A15. As a result of the homology to S100A7, probes were restricted to less than 250 nt in length to obtain isoform-specific detection. B) Semiquantitative RT-PCR analysis: Comparative expression analysis of the short and the long S100A15 transcript in pooled samples (n=5) of normal, nonlesional, and lesional skin performed in two independent experiments. Note: Different RNA samples were used for Northern and semiquantitative RT-PCR analysis. *, Significant differences (P<0.01) among indicated bars.

2. Characterization of the S100A15
Subsequent ORF analysis predicted a characteristic sequence of 101 amino acids for a S100 protein, designated as S100A15, with a calculated molecular weight of 11,305 Da and an isoelectric point of pH 7.57. The deduced protein sequence of the S100A15 contains two helix–loop–helix structural motifs corresponding to EF-hand Ca²⁺-binding sites: a variant, S100-specific motif at the N terminus (aa 12–39), and a canonical site at the C terminus (aa 54–82), which is a highly conserved domain among the S100 members.

The predicted S100A15 protein is highly related to psoriasin (S100A7), sharing 93% sequence identity. The main differences between these two proteins concern particular amino acids at the N terminus, which are thought to be important for the structure of the EF-hand domain (G for R at aa position 22; G for D at position 25) or in the Ca²⁺-binding domain (E for D at position 28). In contrast to S100A7, analysis of the amino acid sequence led to prediction of an N-terminal S100 Ca²⁺-binding domain (EF-hand) for the S100A15. As for the human and bovine S100A7, the peptide sequence and the three amino acids gap in the nonconserved N terminus of the S100A15 assign its position as a divergent member within the S100 family (34% identity to S100A11; 29% identity to S100A8).

3. Identification of an alternatively spliced isoform of the S100A15
The high homology between the S100A15 and the S100A7 gene suggested a similar genomic organization. As a small transcript of 437 nt was described for the S100A7, we assumed the existence of a small mRNA of similar size for the S100A15. The differences between the corresponding genomic sequences upstream of the coding regions permitted PCR cloning of a short transcript of 491 nt in length (S100A15-S) from psoriatic lesional skin. As shown for its long counterpart, the S100A15-S contains the same coding region (140–445 nt) but differs in length and composition of the untranslated regions (Fig. 2 , S100A15-S, acc. no. AY189119). Northern blot analysis of RNA from psoriatic skin using a specific probe for the short S100A15 mRNA revealed a band at 0.5 kb (Fig. 1A ).

View larger version (14K): [in this window] [in a new window]   Figure 2. Genomic structure of the human S100A15. Schematic representation of the genomic (middle) and exon/intron organization for the two mRNA splice variants. Boxes represent exons, hatched regions indicate the coding sequence, and intervening lines denote introns.

4. Chromosomal localization and genomic organization of the alternatively spliced S100A15 isoforms
The S100A15 is located within the epidermal differentiation complex on chromosome 1q21. The S100A15 gene (acc. no. AY189117) is organized in three exons and two introns (Fig. 2) , and all exon/intron boundaries follow the GT-AG rule. Exon 1 is untranslated, and exon 2 contains the translation initiation codon and encodes for the variant N-terminal Ca²⁺-binding domain, whereas exon 3 harbors the canonical Ca²⁺-binding motif at the C terminus. It is interesting that the noncoding exon 1 is restricted to the long isoform, whereas for the short transcript, an additional sequence enlarging the 5'-UTR of exon 2 is found (Fig. 2) . However, despite the differences in composition and length of their UTRs, both S100A15 isoforms share the same coding region.

5. Expression and distributional analysis of the alternatively spliced S100A15 isoforms
Northern blot analysis revealed an increased hybridization for both S100A15 transcripts in nonlesional and lesional psoriatic skin compared with normal skin with comparable expression levels between individual samples (Fig. 1A ). These data were verified by semiquantitative RT-PCR, analyzing additional samples of normal and psoriatic skin, summarized in Figure 1B . In addition, we found that the two isoforms showed different expressional character in lesional skin, with an increase of more than 20-fold for the short S100A15 and more than 70-fold for the long isoform compared with healthy tissue, respectively.

In situ hybridization of human normal skin indicated that the expression of S100A15 transcripts restricted to the epidermis. In line with differential-display RT-PCR analysis and Northern blot experiments, the results showed that elevated levels of S100A15 mRNAs are present in psoriatic epidermis as compared with its normal counterpart (Fig. 3 ). Within the faint staining of healthy skin, a granular pattern was present in the basal epidermal layer (Fig. 3 , arrows), whereas the suprabasal layers of the epidermis appeared to be more strongly stained in lesional skin.

View larger version (62K): [in this window] [in a new window]   Figure 3. In situ hybridization of S100A15 in normal and psoriatic skin. Detection of the short isoform of the S100A15 in normal and psoriatic lesional skin. In normal skin, the basal cell layer showing a granular staining is marked by arrows. Representative results from one of three independent experiments are shown (original magnification: 100-fold).

CONCLUSION AND SIGNIFICANCE

In the present study, we have cloned and characterized a novel member of the human S100 family based on differential-display RT-PCR analysis and subsequent PCR cloning using RNA from psoriatic skin.

The differences between both proteins led to a prediction of a functional N-terminal S100 Ca²⁺-binding domain (EF-hand) for the S100A15 but not for the S100A7. The prediction for the S100A7 protein is in agreement with structural analysis reporting a low Ca²⁺-binding capacity of the protein.

In general, S100 genes code for a single transcript consisting of three exons and two introns, and the first exon is short and untranslated and the second and third, encoding an EF-hand motif each. For the S100A15, however, we detected two transcripts of 0.5 and 4.4 kb in size resulting from the different noncoding regions. Alternative splicing has been reported only for the S100A4 so far, resulting in small transcripts of similar size (507 and 512 nt). Whereas the diversity of the S100A4 transcripts is defined by addition of a 5'-noncoding exon, for the S100A15 transcripts, we found the use of a first noncoding exon (S100A15-L) or an extended first coding exon (S100A15-S; Fig. 2 ). In contrast to the S100A4 variants reported, additional diversity of the S100A15 is caused by the large 3'-UTR only present in the S100A15-L, which extended size is rather exceptional for members of the S100 family. The organization of the S100A15 transcripts suggests that in addition to the 5' alternative splicing, the use of two different polyadenylation signals may result in 3'-UTRs of different lengths. For a number of genes, it was shown that the composition and length of the splice variants influence mRNA transcription-processing efficiency and half-life. It is possible that one of these mechanisms could explain the different levels of the S100A15-spliced isoforms in psoriatic skin (Figs. 1 and 3) .

We found the expression of S100A15 restricted to the proliferative basal cell layer of normal skin (Fig. 3) . In psoriatic skin, the altered epidermal calcium distribution might be responsible for the up-regulation of the S100A15 in the upper epidermis, as has also been shown for other Ca²⁺-binding proteins. The expression of the S100A15 in proliferating keratinocytes of normal skin and hyperplastic psoriatic epidermis is indicative of the involvement of this protein in Ca²⁺-dependent proliferation processes. Furthermore, the increased S100A15 expression could be linked to the enhanced psoriasis-associated inflammation or immune activation in analogy to the related S100A7 and other S100 Ca²⁺-binding proteins. It is interesting that the expression of S100A7 has been shown to be associated with abnormal epithelial differentiation and tumor development. As Ca²⁺ binding regulates the activity of S100 proteins, further studies have to show if S100A15 confers an altered state of activation or additional functions based on an enhanced Ca²⁺-binding capacity compared with the S100A7. Therefore, it will be of great interest to discriminate the expression of both genes in different diseases.

Our study reports the discovery and characterization of the novel psoriasis-associated gene S100A15. Its unique genomic organization within the S100 family, leading to alternative splice variants, promises further insight into the regulation of S100 genes. The novel S100A15 gene, encoding for a protein with two Ca²⁺-binding EF-hand motifs, is likely to participate in the calcium homeostasis in the skin and might thereby be an important factor for proliferation and differentiation in psoriasis and other diseases.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0148fje; doi: 10.1096/fj.03-0148fje

This article has been cited by other articles:

				R. Wolf, O. M. Z. Howard, H.-F. Dong, C. Voscopoulos, K. Boeshans, J. Winston, R. Divi, M. Gunsior, P. Goldsmith, B. Ahvazi, et al. Chemotactic Activity of S100A7 (Psoriasin) Is Mediated by the Receptor for Advanced Glycation End Products and Potentiates Inflammation with Highly Homologous but Functionally Distinct S100A15 J. Immunol., July 15, 2008; 181(2): 1499 - 1506. [Abstract] [Full Text] [PDF]

				A. Abtin, L. Eckhart, M. Mildner, F. Gruber, J.-M. Schroder, and E. Tschachler Flagellin is the principal inducer of the antimicrobial peptide S100A7c (psoriasin) in human epidermal keratinocytes exposed to Escherichia coli FASEB J, July 1, 2008; 22(7): 2168 - 2176. [Abstract] [Full Text] [PDF]

				Y. Hahn, S. Jeong, and B. Lee Inactivation of MOXD2 and S100A15A by Exon Deletion during Human Evolution Mol. Biol. Evol., October 1, 2007; 24(10): 2203 - 2212. [Abstract] [Full Text] [PDF]

				C. W. Heizmann S100B Protein in Clinical Diagnostics: Assay Specificity Clin. Chem., January 1, 2004; 50(1): 249 - 251. [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 17/13/1969 03-0148fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by WOLF, R. Articles by RUZICKA, T. Search for Related Content PubMed PubMed Citation Articles by WOLF, R. Articles by RUZICKA, T.