Letters to the Editor / Journal of Dermatological Science 61 (2011) 129–150 131

[2] Levi K, Dauskardt RH. Application of substrate curvature method to differenti-ate drying stresses in topical coatings and human stratum corneum. Int JCosmet Sci 2010;32:294–8.

[3] Wu KS, Stefik MM, Ananthapadmanabhan KP, Dauskardt RH. Graded delami-nation behavior of human stratum corneum. Biomaterials 2006;27:5861–70.

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different amounts of lipids. Acta Derm Venereol 1992;327–30.[6] Ghadially R, Halkier-Sorensen L, Elias PM. Effects of petrolatum on stratum

corneum structure and function. J Am Acad Dermatol 1992;26:387–96.[7] Levi K, Kwan A, Rhines AS, Gorcea M, Moore DJ. Emollient molecule effects on

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Kemal LeviAlice Kwan

Allison S. RhinesDepartment of Materials Science and Engineering,

Stanford University, Stanford, CA, USA

Table 1SCCE haplotypes distribution in 120 AD and 203 controls. Haplotypes have been created u

controls has been performed. Remaining corresponds to the pooling of haplotypes with

SNPtag software. Those are at positions 3224, 5713 and 5874 respectively. ‘‘AACCins58

grouped together. Pc, corrected p-value.

SCCE haplotypes (C3224G-G5713C-AACC/AACCins5874) Control

C-G-AACC 168

C-G-AACCins5874 76

C-C-AACCins5874 21

G-G-AACC 59

G-G-AACCins5874 38

G-C-AACCins5874 32

AACCins5874-all together 167

Remaining 12

Total 406

Mihaela GorceaDavid J. Moore

International Specialty Products, Wayne, NJ, USA

Reinhold H. Dauskardt*Department of Materials Science and Engineering,

Stanford University, Stanford, CA, USAs

*Corresponding author at: Department of Materials Science andEngineering, 496 Lomita Mall, Durand Bldg., Rm. 121, Stanford

University, Stanford, CA 94305-2205, USA.Tel.: +1 650 725 0679; fax: +1 650 725 4034

E-mail address: dauskardt@stanford.edu (R.H. Dauskardt)

18 November 2010

doi:10.1016/j.jdermsci.2010.11.011

Letter to the Editor

The 30-UTR AACCins5874 in the stratum corneum chymotrypticenzyme gene (SCCE/KLK7), associated with atopic dermatitis;causes an increased mRNA expression without altering itsstability

Atopic dermatitis (AD) is a chronic inflammatory skin diseaseassociated with cutaneous hyper-reactivity to environmentaltriggers innocuous to normal, non-atopic individuals, causedmainly by changes in the cutaneous and systemic immuneresponses [1]. However increasing evidence highlight the specu-lation of a primary genetic defect in the skin barrier which couldaffect the levels of protease activation within the skin, leading tothe development of AD [2]. The stratum corneum chymotrypticenzyme (SCCE/KLK7) is believed to be an important player inregulating the epidermal homeostasis of the normal skin barrier[3,4]. We previously reported a 4-bp insertion (AACCins5874) atthe 30-UTR of the SCCE gene to be significantly associated with AD[5].

In this study we analysed five more SNP’s throughout SCCE

gene (Fig. S1). All SNPs were analyzed by PCR-RFLP (Table S1)in 120 AD patients and 203 matched-controls [5]. Allelicdistribution analysis showed no significant difference betweencases and controls. However, a dose effect was detected for SCCE

AACCins5874 [ORHeterozygote, odds ratio (OR) = 1.09 (0.62,1.92) andORHomozygote = 2.73 (1.52,4.91) respectively]. Therefore, a x2-test

trend was carried out for SCCE-AACCins5874 insertion [OR = 2.62(1.58–4.35); Pc = 0.009], suggesting that SCCE-AACCins5874 alleleconfers more than 2–fold risk for disease in dose-dependentmanner under a recessive model of inheritance (Table S2).Haplotype analysis using the programs EHPLUS [6] and SNPtagger(Table S3) confirmed the single-marker analysis (Table 1). SCCE

C3224-5713C-AACCins5874 haplotype showed a strong associa-tion with AD (Pc = 0.00001). Moreover, haplotypes containingAACCins5874 when grouped together also showed significantassociation with disease (Pc = 0.0001). In contrast, a weak but signi-ficant negative association was revealed with the SCCE 3224G-G5713-AACC haplotype (Pc = 0.02), suggesting a protector effect.

As variations in the 30-UTR of genes may affect the stability and/or translation of the mRNA [7,8], we analysed the effect ofAACCins5874 in the 30-UTR of SCCE on gene expression andstability using a destabilised luciferase reporter vector. The latterwas based on the pGL3-Basic Vector (Promega, Madison, WI)modified so that the luc-SV40poly(A) fragment was replaced with afragment, downstream of the synthetic poly(A) signal site,encompassing the IL-8 promoter, the destabilised luciferase gene(luc-PEST) and the IL-1b 30-UTR sequence. The IL-8 promoter wasused to induce the production of the luciferase-PEST RNA byactivation upon IL-1b stimulation through the Nfkb pathway.Addition of the synthetic fragment encoding the proteolytic ‘‘PEST’’signal to the firefly luciferase coding sequence aimed to destabilise

sing EH program and the comparison of frequencies of each haplotypes in cases and

frequency less than 5% in the same group. Three SNPs have been selected by the

74-all together’’ means that all haplotypes, which contains AACCin5874 have been

Freq AD cases Freq Pc

0.413 81 0.338 0.432

0.188 37 0.155 0.228

0.052 40 0.165 0.00001

0.146 16 0.068 0.02

0.093 37 0.155 0.162

0.079 21 0.088 0.558

0.411 135 0.563 0.0001

0.029 8 0.031 –

1.00 240 1.00 –

[()TD$FIG]

Fig. 1. (a) Schematic diagram of the SCCE 30UTR AACC/AACCins5874 variants subcloned downstream of the luc gene at the BamHI/ApaI of the pGL3-IL8-LucPest destabilised

luciferase reporter vector (the box contains the SCCE 30UTR sequence where the AACC insertion is shown in bold and the AU-rich element, ATTTA, is underlined). (Note: PEST

destabilises the luciferase protein.) The figure is not drawn to scale. (b) Luciferase relative activity among the SCCE reporter constructs (SCCE-AACC, Allele 1; SCCE-

AACCins5874, Allele 2) in COS7 and HeLa cells. COS7 and HeLa cells were transfected with pGL3-IL8-LucPest containing SCCE 30UTR with either AACC or AACCins5874 allele.

Cells were stimulated with IL-1b (3 ng/ml) 24 h after transfection, for a time-course of 5 h. Luciferase activities (firefly, Renilla) were measured with the Dual-luciferase assay

and the normalized ratio of firefly/Renilla luciferase at 1, 2, 3, 4 and 5 h time points are reported in HeLa and COS-7 cells. Results shown are a representative experiment (one

out of three) conducted in quadruplicate (mean � SD). After 4 h of stimulation the luciferase activity of SCCE-AACC decreased in both COS-7 and HeLa cells (1.25-fold and 1.33-fold

respectively), whereas the luciferase activity of SCCE-AACCins5874 variant was slightly increased (1.18-fold in COS-7 cells and 1.03-fold in HeLa cells, respectively). (c) Comparison

of the rate of the mRNA decay among SCCE 30UTR variants (SCCE-AACC, Allele 1; SCCE-AACCins5874, Allele 2). COS7 and HeLa cells were transfected with pGL3-IL8-LucPest

containing SCCE 30UTR with either AACC or AACCins5874 allele. 24 h after transfection, cells were stimulated with IL-1b (3 ng/ml) for 4 h. Luciferase activities (firefly, Renilla) were

Letters to the Editor / Journal of Dermatological Science 61 (2011) 129–150132

Letters to the Editor / Journal of Dermatological Science 61 (2011) 129–150 133

the luciferase protein by reducing its functional half-life from t1/

2 = 3.68 to t1/2 = 0.84 [9]. The SCCE cDNA was initially isolated fromthe SCCE image clone (Accession number: BC032005; IMAGE:4750452; UK-HGMP). The pCR2.1-SCCE30UtrAACC plasmid wasused as template for mutagenesis using the QuickChange Site-directed Mutagenesis kit (Stratagene, La Jolla, CA) to create theAACCins5874 variant construct. The two SCCE 30-UTR plasmidswere then sub-cloned downstream of the luc gene at the BamHI/ApaI site of the pGL3-IL8-LucPest vector (Fig. 1a). The pGL3-IL8-LucPest-SV40latePoly(A) plasmid was constructed by replacing theIL1b 30-UTR with the SV40latePoly(A) for positive control. The pRL-TK vector (Promega) was used as an internal control for normal-isation of transfection efficiency.

At 70–80% confluency, COS-7 and HeLa cells maintained inDMEM (Dulbecco’s modified Eagle’s medium), supplemented with10% FCS (Foetal calf serum), 100 U/ml penicillin, 100 mg/mlstreptomycin and 2 mM L-Glutamine, were transiently co-trans-fected using the Polyfect transfection reagent, under instructionsby the manufacturer (Qiagen, Valencia, CA). At 24 h aftertransfection cells were washed with PBS and stimulated withhuman IL-1b (3 ng/ml) (R&D Systems, Abingdon, UK) for 4 h. Thecells were then washed with PBS and treated with Actinomycin D(10 mg/ml; Sigma) for 4 h in a humidified 5% CO2 atmosphere at37 8C. After inhibition of transcription cells were lysed with cell-culture Lysis reagent (Promega) and whole-cell extracts were usedto measure firefly and renilla luciferase activities in a Fusionuniversal microtitre analyser (Packard-Bioscience, Illinois, USA)using the Dual-Luciferase1 Reporter Assay System (Promega).After normalisation of the firefly luciferase activity by the renilla

luciferase activity, data were expressed as RLU and are themean � SD of four independent transfection samples. Statisticalsignificance was tested with Mann–Whitney U test.

The results demonstrated that the level of the luciferase activityfor both SCCE-AACC and SCCE-AACCins5874 alleles was increasedthrough the 4 h time-course (Fig. 1b). There is a relative increase ofSCCE-AACCins5874 compare to SCCE-AACC starting to be detectedat 2 h in both cell lines but this difference seems to disappearquickly in COS-7 (3 h), whereas the difference persists betweenSCCE-AACCins5874 and SCCE-AACC in HeLa cells at 3 h anddisappear at 4 h. This could be due to a quicker turn-over ofSCCE-AACCins5874 mRNA than SCCE-AACC one. 1 h later afterstopping stimulation (5 h) the luciferase activity of SCCE-AACCdecreased in both COS-7 and HeLa cells (1.25-fold and 1.33-foldrespectively), whereas SCCE-AACCins5874 variant did not showany significant change in both cell lines (a small increase has beenobserved in the case of COS-7), suggesting that the mRNA and/orprotein of SCCE-AACCins5874 variant has a lower decay rate thanSCCE-AACC. Therefore, the level of luciferase activity at 0, 2 and 4 hafter transcription inhibition was determined using the Dual-luciferase assay. The mRNA content was set to 1 by means of theaverage of the normalised luciferase activity at the time point of0 h and the ratios of the mRNA decay for each sample of each allelicconstruct at each time point (i.e. 2 and 4 h) were calculated(Fig. 1c). There was no significant difference in the rate of themRNA decay between the SCCE 30-UTR variants using both COS-7(P2h = 0.271; P4h = 0.079) and HeLa (P2h = 0.24; P4h = 0.1).

In conclusion our evidence suggests that the AACCins5874insertion in the 30UTR of the SCCE gene cause an over-expression inCOS-7 and HeLa cells but does not have an effect on mRNA stability.Thus AACCins5874 insertion could be involved in post-transcrip-tional modification of the SCCE transcript by an alteration in itstranslation efficiency, through modulation of the binding of

measured with the Dual-luciferase assay after cells were treated with 10 mg/ml actinomyc

inhibition of mRNA transcription activity by actinomycin D. Ratios of mRNA decay for each sa

for the ratios calculated for three independent experiments conducted in quadruplicate (n

nuclear factors to this particular sequence variant or neighboringelement such as the AUUUA motif [8,10], something that warrantsfurther experiments to be confirmed.

Acknowledgements

We would like to thank the patients and their families who tookpart in this study. This work was supported by funding from RTA’sDC7559 and The University of Sheffield.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at doi:10.1016/j.jdermsci.2010.11.013.

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[6] Zhao JH, Curtis D, Sham PC. Model-free analysis and permutation tests forallelic associations. Hum Hered 2000;50:133–9.

[7] Day DA, Tuite MF. Post-transcriptional gene regulatory mechanisms in eukar-yotes: an overview. J Endocrinol 1998;157:361–71.

[8] Di Paola R, Frittitta L, Miscio G, Bozzali M, Baratta R, Centra M, et al. A variationin 30UTR of hPTP1B increases specific gene expression and associates withinsulin resistance. Am J Hum Genet 2002;70:806–12.

[9] Leclerc GM, Boockfor FR, Faught WJ, Frawley LS. Development of a destabilisedfirefly luciferase enzyme for measurement of gene expression. BioTechniques2000;29:590–601.

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Yiannis Vasilopoulos*Nazar Sharaf

Franco di GiovineThe Medical School, University of Sheffield, Sheffield, UK

Michel SimonCNRS-University of Toulouse III, Toulouse, France

Michael J. CorkGordon W. Duff

Rachid Tazi-AhniniThe Medical School, University of Sheffield, Sheffield, UK

*Corresponding author. Current address: Department ofBiochemistry and Biotechnology, University of Thessaly, Ploutonos

26 and Aiolou str, 41221, Larissa, Greece.Tel.: +30 2410 565262; fax: +30 2410 565290

E-mail address: iovasilopoulos@bio.uth.gr(Y. Vasilopoulos)

15 October 2010

doi:10.1016/j.jdermsci.2010.11.013

in D. Normalised ratio of the firefly/Renilla luciferase is reported at 0, 2 and 4 h after

mple of each allelic construct at each time point were calculated. Results are mean � SD

= 12 for COS7 and n = 4 for HeLa, for each time point).