nickel binding has not been reported.Unfortunately, SC protein extractionmust be performed under denaturingconditions, and the presence of residentproteins that need their secondary ortertiary structure for nickel bindingcould not be evaluated. Moreover, asthe cornified envelope resists breakdowninto its protein constituents, they areinaccessible for analysis. Finally, thesetup did not allow for assessment ofnickel binding by free amino acids.In summary, we demonstrate thatfilaggrin derived from both SC and fullepidermis binds nickel. Other epidermalproteins may bind nickel, but filaggrin isa strong, denaturation-resistant chelator,and the relevance of the other proteins,e.g., for the accumulation of nickelin the SC, remains unclear. As filaggrinnull mutations are associated with anincreased risk of allergic nickel der-matitis in European populations, thisstudy provides a possible link bet-ween genetics, protein expression, andfunction.

CONFLICT OF INTERESTThe authors state no conflict of interest.

ACKNOWLEDGMENTSWe thank the Copenhagen County ResearchFoundation, the Aage Bang Foundation, and theDepartment of Plastic Surgery, CopenhagenUniversity Hospital Herlev.

Katrine Ross-Hansen1,Ole Østergaard2, Julia T. Tanassi2,Jacob P. Thyssen1, Jeanne D. Johansen1,Torkil Menne1 andNiels H.H. Heegaard2

1National Allergy Research Centre, Departmentof Dermato-Allergology, CopenhagenUniversity Hospital Gentofte, Hellerup,Denmark and 2Department of ClinicalBiochemistry, Immunology and Genetics,Statens Serum Institut, Copenhagen, DenmarkE-mail: katrine.ross-hansen@regionh.dk

This work was done in Hellerup and Copenhagen,Denmark.

SUPPLEMENTARY MATERIAL

Supplementary material is linked to the onlineversion of the paper at http://www.nature.com/jid

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Candi E, Schmidt R, Melino G (2005) The cornifiedenvelope: a model of cell death in the skin.Nat Rev Mol Cell Biol 6:328–40

Kezic S, Kemperman PMJH, Koster ES et al. (2008)Loss-of-function mutations in the filaggringene lead to reduced level of natural moistur-izing factor in the stratum corneum. J InvestDermatol 128:2117–9

Kubo A, Ishizaki I, Kubo A et al. (2013) Thestratum corneum comprises three layers withdistinct metal-ion barrier properties. Sci Rep25:1731

McKinley-Grant LJ, Idler WW, Bernstein IA et al.(1989) Characterization of a cDNA cloneencoding human filaggrin and localizationof the gene to chromosome region 1q21.Proc Natl Acad Sci USA 86:4848–52

Novak N, Baurecht H, Schafer T et al. (2007) Loss-of-function mutations in the filaggrin gene andallergic contact sensitization to nickel. J InvestDermatol 128:1430–5

Palmer CNA, Irvine AD, Terron-Kwiatkowski Aet al. (2006) Common loss-of-function var-iants of the epidermal barrier protein filaggrinare a major predisposing factor for atopicdermatitis. Nat Genet 38:441–6

Ross-Hansen K, Menne T, Johansen JD et al. (2011)Nickel reactivity and filaggrin null mutations—evaluation of the filaggrin bypass theory in ageneral population. Contact Dermatitis 64:24–31

Sandilands A, Sutherland C, Irvine AD et al. (2009)Filaggrin in the frontline: role in skin barrierfunction and disease. J Cell Sci 122:1285–94

Simon M, Haftek M, Sebbag M et al. (1996)Evidence that filaggrin is a component ofcornified cell envelopes in human plantarepidermis. Biochem J 317:173–7

Smith FJD, Irvine AD, Terron-Kwiatkowski A et al.(2006) Loss-of-function mutations in the geneencoding filaggrin cause ichthyosis vulgaris.Nat Genet 38:337–42

Steinert PM, Marekov LN (1995) The proteinselafin, filaggrin, keratin intermediate fila-ments, loricrin, and small proline-rich proteins1 and 2 are isodipeptide cross-linked compo-nents of the human epidermal cornified cellenvelope. J Biol Chem 270:17702–11

Thulin CD, Taylor JA, Walsh KA (1996) Micro-heterogeneity of human filaggrin: analysis of acomplex peptide mixture using mass spectro-metry. Protein Sci 5:1157–64

Thyssen JP, Menne T (2010) Metal allergy—areview on exposures, penetration, genetics,prevalence, and clinical implications. ChemRes Toxicol 23:309–18

Wells GC (1956) Effects of nickel on the skin. Br JDermatol 68:237–42

Evidence for an Alternatively Spliced MITF Exon 2 VariantJournal of Investigative Dermatology (2014) 134, 1166–1168; doi:10.1038/jid.2013.426; published online 14 November 2013

TO THE EDITORAlternative splicing of exon 1 in themicrophthalmia-associated transcriptionfactor (MITF) gene gives rise to a familyof transcription factors that differ only inexon 1 sequence (Steingrimsson et al.,2004). Each MITF isoform is associatedwith a specific promoter and eachunique promoter/isoform combinationresults in MITF expression in a differentcell lineage. In the melanocytic celllineage, MITF is expressed from the

M-promoter, leading to a transcript con-taining exon 1 M; therefore, this isoformis termed MITF-M. The amplification ofMITF-M occurs in 10–20% of all mela-noma cases, and it has been suggestedthat MITF-M may be a prognostic mar-ker for poor survival in melanoma(Garraway et al., 2005; Ugurel et al.,2007). Recently, a germline mutation inMITF (E318K) has also been shown tocontribute to increased susceptibility tomelanoma (Bertolotto et al., 2011;

Yokoyama et al., 2011). The MITFsignaling pathway has further beenshown to contribute to melanomasusceptibility. A recent study identifiedfrequent somatic mutations in bothMITF and its regulator SOX10, with14% of primary and 20% of metastaticmelanoma containing mutations inthese genes (Cronin et al., 2009).

Recently, an isoform of MITF-M that isalternatively spliced at exons 2 and 6has been reported. This shortened iso-form, termed MITF-MDel, was expressedat a detectable level only in melanocytesAccepted article preview online 14 October 2013; published online 14 November 2013

JL Simmons et al.MITF Exon 2 Variant

1166 Journal of Investigative Dermatology (2014), Volume 134

and melanoma cells and was predictedto be a prognostic biomarker for mela-noma (Wang et al., 2010). The functio-nal significance of MITF-MDel in eithermelanocyte or melanoma developmentis still unknown and debated.

Exon 2 of MITF-M contains a ‘‘crypticsplice donor site’’, which divides theexon into 2 A (60 bp) and 2B (168 bp).MITF-MDel was found to be lackingexon 2B (Hallsson et al., 2000; Wanget al., 2010). Mice carrying a homozy-gous germline deletion of exon 2B(MITFmi-bws) have a mild pigmentationphenotype, whereas others have shownthat exon 2B is dispensable for melano-cyte development (Hallsson et al., 2000;Bismuth et al., 2005; Bauer et al., 2009).Phosphorylation of a conserved serineresidue (S73) contained within exon 2Bhas been proposed to both enhance thetranscriptional activity and decrease thestability of the protein (Wu et al., 2000).It is therefore possible that the absence

of S73 could contribute to the pigmen-tation defects observed in MITFmi-bws

mice and to any functional conseque-nces of MITF-MDel expression.

While cloning MITF-M from culturedhuman melanocytes, we found a thirdshortened transcript that we sequencedand aligned against both MITF-M andMITF-MDel (Figure 1a). This revealed a75 bp in-frame deletion in exon 2 com-mencing at the cryptic splice donor siteat the start of exon 2B; here, we call thisalternatively spliced isoform MITF-M2C.Alignment of the predicted proteinsequence for MITF-M2C with MITF-Mand MITF-MDel showed a deletion of25 amino acids with those outsideof the deletion remaining unaffected(Figure 1b). No previously identifiedfunctional protein components are con-tained within the deletion, and it istherefore possible that this transcriptdoes not encode a protein with uniquefunction. Importantly, the key residue

for phosphorylation of S73 remainsencoded in the MITF-M2C transcript.We cannot, however, rule out anychange in the secondary structure and,therefore, potential function in theMITF-M2C protein.

To determine whether MITF-M2C waswidely expressed, we performed quan-titative real-time reverse-transcriptase–PCR (qRT–PCR) to measure each ofthe exon 2 variants across a panel ofcell lines and patient tumor specimens.The PCR experiment was designedwith one common forward primer andthree separate reverse primers, whichdifferentiated between the transcriptson the basis of the sequence acrossthe exon 2 splice junction (Supple-mentary information online). Analysisof 19 human cell lines, including 18metastatic melanoma, one primarymelanoma (MM200) and normal humanmelanocytes, revealed a low-levelexpression of MITF-M2C across all celllines tested (Figure 2a). Metastatic mel-anoma samples representing brain, skin(regional lymph node), and kidneymetastases derived from 6 patientssimilarly showed a low-level expressionof MITF-M2C in all specimens(Figure 2b). Sequencing of the MITF-M2C PCR product confirmed specificamplification and shows the MITF-M2Csplice junction (Figure 2c). Western blotanalysis provides some evidence thatthe protein may be expressed in bothmelanocytes and melanoma cell lines(Supplementary Figure S1 online). Wewere able to detect minor bands of asmaller apparent molecular weight thanMITF-M. As the antibody used to detectMITF was raised against the N-terminalregion, which is common to all threeisoforms, we are unable to definitivelyidentify and distinguish MITF-M2C andMITF-MDel. However, the relativeabundance of the detected proteinswas consistent with levels detectedby qRT-PCR, supporting our tentativeidentification of these isoforms.

The results presented here show thatthis previously unreported splice variantof MITF-M is expressed at a lower levelin a variety of metastatic melanoma celllines and tumors. These results alsoindicate that MITF-M2C may be a com-mon feature of cells and tissues thatexpress MITF-M. On the basis of the

a

b

Figure 1. Microphthalmia-associated transcription factor (MITF)-M isoform alignments. (a) Alignment of

exons 1–3 of MITF-M (NM_000248.3) with MITF-Mdel (GU355676.1) and MITF-M2C. Alternative exons

are indicated in blue and deleted bases with a dash (� ). (b) MITF-M2C protein prediction aligned with

MITF-M (NM_000239.1) and MITF-Mdel (ABD90411). Alignment corresponds to exons 1–3, and

alternative exons are indicated in blue and deleted amino acids with a dash.

JL Simmons et al.MITF Exon 2 Variant

www.jidonline.org 1167

current understanding, we feel that theremay be little physiological significanceof MITF-M2C expression in either mel-anogenesis or melanoma development.However, given the low expressionlevel of this transcript, it will be inter-esting to see whether MITF-M2C isdetected in high-throughput sequencingstudies in melanoma tumor samples.

CONFLICT OF INTERESTThe authors state no conflict of interest.

ACKNOWLEDGMENTSThis work was supported by the grants from theNational Health and Medical Research Council ofAustralia (APP1045650) and the Cancer CouncilQueensland (APP1027728).

Jacinta L. Simmons1, Carly J. Pierce1

and Glen M. Boyle1

1Cancer Drug Mechanisms Group, Departmentof Cell and Molecular Biology, QIMR BerghoferMedical Research Institute, Herston,Queensland, AustraliaE-mail: Glen.Boyle@qimrberghofer.edu.au

SUPPLEMENTARY MATERIAL

Supplementary material is linked to the onlineversion of the paper at http://www.nature.com/jid

REFERENCES

Bauer GL, Praetorius C, Bergsteinsdottir K et al.(2009) The role of MITF phosphorylation sitesduring coat color and eye development inmice analyzed by bacterial artificial chro-mosome transgene rescue. Genetics 183:581–94

Bertolotto C, Lesueur F, Giuliano S et al. (2011) ASUMOylation-defective MITF germline muta-tion predisposes to melanoma and renalcarcinoma. Nature 480:94–U259

Bismuth K, Maric D, Arnheiter H (2005) MITF andcell proliferation: the role of alternative spliceforms. Pigment Cell Res 18:349–59

Cronin JC, Wunderlich J, Loftus SK et al. (2009) Fre-quent mutations in the MITF pathway in melan-oma. Pigment Cell Melanoma Res 22:435–44

Garraway LA, Widlund HR, Rubin MA et al. (2005)Integrative genomic analyses identify MITF asa lineage survival oncogene amplified inmalignant melanoma. Nature 436:117–22

Hallsson JH, Favor J, Hodgkinson C et al. (2000)Genomic, transcriptional and mutational ana-lysis of the mouse microphthalmia locus.Genetics 155:291–300

Steingrimsson E, Copeland NG, Jenkins NA (2004)Melanocytes and the Microphthalmia tran-scription factor network. Ann Rev Genet38:365–411

Ugurel S, Houben R, Schrama D et al. (2007)Microphthalmia-associated transcription fac-tor gene amplification in metastatic mela-noma is a prognostic marker for patientsurvival, but not a predictive marker forchemosensitivity and chemotherapy response.Clin Cancer Res 13:6344–50

Wang Y, Radfar S, Liu S et al. (2010) Mitf-Mdel, anovel melanocyte/melanoma-specific isoformof microphthalmia-associated transcriptionfactor-M, as a candidate biomarker for mela-noma. BMC Med 8:14

Wu M, Hemesath TJ, Takemoto CM et al. (2000)c-Kit triggers dual phosphorylations, which cou-ple activation and degradation of the essentialmelanocyte factor Mi. Genes Dev 14:301–12

Yokoyama S, Woods SL, Boyle GM et al. (2011) Anovel recurrent mutation in MITF predisposesto familial and sporadic melanoma. Nature480:99–103

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Figure 2. Detection of microphthalmia-associated transcription factor (MITF)-M isoforms in human

melanocytes and melanoma cell lines. Quantitative reverse transcription PCR for MITF-M (white), MITF-

Mdel (gray), and MITF-M2C (black), normalized to glyceraldehyde-3-phosphate dehydrogenase. (a)

Human melanoma and melanocyte cell lines. MM200 was derived from a primary tumor. (b) Human

metastatic melanoma tumors isolated from the brain (Br), skin/lymph node (Sk), and kidney (Ki). Error bars

represent standard deviation, n¼X2. (c) An MITF-M2C reverse transcription PCR product was sequenced

to confirm specific amplification of this isoform. The chromatogram shows the splice boundary, the

location of the reverse PCR primer is underlined in black, and the bases at the splice junction are boxed

in red.

JL Simmons et al.MITF Exon 2 Variant

1168 Journal of Investigative Dermatology (2014), Volume 134