RP Hobbs and KJ Green Desmoplakin Regulates Hyperadhesion

Kallel Sellami M, Zitouni M, Tombari W et al. Peng Z, Rasic N, Liu Y et al. (2002) Mosquito REFERENCES (2007) Anti--1 antibodies are pre- saliva-specific IgE and IgG antibodies in Amagai M (2010) Autoimmune and infections skin valent in Tunisian patients with hydatidosis 1059 blood donors. J Allergy Clin Immunol diseases that target . Proc Jpn and leishmaniasis. Br J Dermatol 156:591–3 110:816–7 Acad Ser B Phys Biol Sci 86:524–37 Kamhawi S (2000) The biological and immuno- Remoue F, Alix E, Cornelie S et al. (2007) IgE Aoki V, Millikan RC, Rivitti EA et al. (2004) modulatory properties of sand fly saliva and and IgG4 antibody responses to Aedes Environmental risk factors in endemic pem- its role in the establishment of Leishmania saliva in African children. Acta Trop 104: phigus foliaceus (fogo selvagem). J Investig infections. Microbes Infect 2:1765–73 108–15 Dermatol Symp Proc 9:34–40 Lombardi C, Borges PC, Chaul A et al. (1992) Reunala T, Brummer-Korvenkontio H, Palosuo K Bastuji-Garin S, Turki H, Mokhtar I et al. (2002) Environmental risk factors in endemic et al. (1994a) Frequent occurrence of IgE Possible relation of Tunisian pemphigus with pemphigus foliaceus (fogo selvagem). J Invest and IgG4 antibodies against saliva of traditional cosmetics: a multicenter case- Dermatol 98:847–50 Aedes communis and Aedes aegypti mosqui- control study. Am J Epidemiol 155:249–56 Marzouki S, Ben Ahmed M, Boussoffara T et al. toes in children. Int Arch Allergy Immunol Brummer-Korvenkontio H, Lappalainen P, Reunala (2011) Characterization of the antibody 104:366–71 T et al. (1994) Detection of mosquito saliva- response to the saliva of Phlebotomus papa- Reunala T, Brummer-Korvenkontio H, Palosuo T specific IgE and IgG4 antibodies by immuno- tasi in people living in endemic areas of (1994b) Are we really allergic to mosquito blotting. J Allergy Clin Immunol 93:551–5 cutaneaous leishmaniasis. Am J Trop Med bites? Ann Med 26:301–6 Culton DA, Qian Y, Li N et al. (2008) Advances in Hyg 84:653–61 Rohousova I, Ozensoy S, Ozbel Y et al. (2005) pemphigus and its endemic pemphigus foliaceus Mbow ML, Bleyenberg JA, Hall LR et al. (1998) Detection of species-specific antibody res- (Fogo Selvagem) phenotype: a paradigm of Phlebotomus papatasi sand fly salivary ponse of humans and mice bitten by sand human authoimmunity. J Autoimmun 31:311–24 gland lysate down-regulates a Th1, but up- flies. Parasitology 130:493–9 Diaz LA, Arteaga LA, Hilario-Vargas J et al. (2004) regulates a Th2, response in mice infected Shan EZ, Taniguchi Y, Shimizu M et al. Anti-desmoglein-1 antibodies in onchocer- with Leishmania major. J Immunol (1995) Immunoglobulins specific to ciasis, leishmaniasis and Chagas disease 161:5571–7 mosquito salivary gland in the sera suggest a possible etiological link to fogo Muller UR (2005) Bee venom allergy in bee- of persons with common or hypersensitive selvagem. J Invest Dermatol 123:1045–51 keepers and their family members. Curr Opin reactions to mosquito bites. J Dermatol 22: Kallel Sellami M, Ben Ayed M, Mouquet H et al. Allergy Clin Immunol 5:343–7 411–8 (2004) Anti-desmoglein 1 antibodies in Tu- Palosuo K, Brummer-Korvenkontio H, Mikkola J Valenzuela JG, Belkaid Y, Garfield MK et al. nisian healthy subjects: arguments for the et al. (1997) Seasonal increase in human IgE (2001) Toward a defined anti-Leishmania role of environmental factors in the occur- and IgG4 antisaliva antibodies to Aedes vaccine targeting vector antigens: character- rence of Tunisian pemphigus foliaceus.. Clin mosquito bites. Int Arch Allergy Immunol ization of a protective salivary . J Exp Exp Immunol 137:195–200 114:367–72 Med 194:331–42

Desmoplakin Regulates Hyperadhesion

Journal of Investigative Dermatology (2012) 132, 482–485; doi:10.1038/jid.2011.318; published online 13 October 2011

TO THE EDITOR Desmosome assembly is a calcium- intercellular adhesive strength termed The skin is subjected to continuous dependent process, where major cyto- hyperadhesion (Cirillo et al., 2010; physical stress. Keratinocytes resist plasmic plaque components of the Thomason et al., 2010). It has been mechanical stress by tethering the desmosome (e.g., DP, ) coalesce hypothesized that the acquisition of tension-bearing keratin intermediate with other desmosomal proteins (i.e., hyperadhesion is essential for the epi- filament to sites of inter- , armadillo proteins) at sites dermis to resist the perpetual mechan- cellular contact known as desmo- of nascent junction formation (Pasdar ical stress to which it is subjected somes (Green and Simpson, 2007; et al., 1991). Desmosomal proteins (Garrod and Kimura, 2008). Garrod and Chidgey, 2008). The accumulate at cell–cell borders over The underlying signaling and struc- protein desmoplakin (DP) is time to form robust intercellular junc- tural properties that govern desmosome an obligate desmosomal constituent tions capable of resisting mechanical maturation and acquisition of hyperad- necessary for keratin anchorage at stress. More recently, it has been hesion have only recently begun to be cell–cell contacts. Establishing and reported that as mature studied in greater detail. The data maintaining the DP–keratin association over the course of several days, they no suggest that activation of protein kinase is essential for regulating desmosomal longer require calcium to maintain C (PKC) in hyperadhesive epithelial adhesive strength in both developing intercellular adhesion (Wallis et al., sheets of Madin-Darby canine kidney and adult stratified tissue 2000; Cirillo et al., 2010). Calcium- or HaCaT cells is sufficient to convert (Vasioukhin et al., 2001; Huen et al., independent desmosomes have been desmosomes from calcium-indepen- 2002). associated with a state of enhanced dent to calcium-dependent (Wallis et al., 2000; Cirillo et al., 2010). Like- Abbreviations: DP, desmoplakin; Gly, ; PKC, protein kinase C; Ser, wise, inhibition of PKC signaling is

482 Journal of Investigative Dermatology (2012), Volume 132 RP Hobbs and KJ Green Desmoplakin Regulates Hyperadhesion

DMSO PMA 2 Days 6 Days 2 Days 6 Days

Wild-type DP expressing

2 Days 6 Days 2 Days 6 Days

DP S2849G expressing

100 Wild-type DP * 90 DP Ser2849Gly Wild-type DP DP S2849G expressing expressing 80 70 * 60 siDP ––+ + ––+ + * Dox ––++ ––++ 50 40

No. of fragments No. 30 ** 20 IB: DP (NW6) 10 0 Day 2Day 6Day 2Day 6 DMSO PMA

Figure 1. DP Ser2849Gly promotes hyperadhesion. (a, left) DMSO-treated A431 epithelial sheets resist mechanical stress following 6 days at confluency, but not at 2 days. (a, right) Protein kinase C (PKC) stimulation is sufficient to weaken the adhesive strength of day 2 and day 6 sheets. (b, left) A431 sheets expressing desmoplakin (DP) Ser2849Gly exhibit enhanced stress resistance at day 2, and (b, right) do not respond to phorbol 12-myristate 13-acetate (PMA)-induced fragmentation. (c) Western blot of A431 monolayers collected in urea sample buffer at day 6 and probed for DP (NW6) showing doxycycline (Dox)-induced expression of exogenous DP in the absence of endogenous DP (siDP lanes). (d) Quantification of fragments under each condition in a and b. Graph represents three independent experiments, performed in triplicate. Bars ¼ mean±SEM. *Po0.01 (Bonferroni-corrected two-factor analysis of variance (ANOVA) with replication, a ¼ 0.0125) for the interaction between the indicated groups of data. **Po0.02 (Bonferroni-corrected single-factor ANOVA, a ¼ 0.0125) between the indicated data sets. IB, immunoblot.

adequate to promote calcium-indepen- hyperadhesive cells may undergo a DP–keratin anchorage is critical for dence and hyperadhesion acquisition structural reorganization on wounding maintaining intercellular adhesive (Wallis et al., 2000). Additionally, (Garrod et al., 2005). Altogether, these strength in epithelial sheets (Huen hyperadhesive epithelial sheets become findings suggest an ‘‘inside-out’’ signal, et al., 2002). Thus, we hypothesized calcium-dependent on wounding, and whereby PKC-driven modification of that regulation of the DP–keratin asso- PKCa has been reported to localize to a desmosomal component may induce ciation might contribute to the desmo- the desmosome at the leading edge of a structural rearrangement within some maturation process necessary wounded epithelial cell sheets (Wallis the desmosome to facilitate the loss for establishing calcium-independence et al., 2000; Garrod et al., 2005). of hyperadhesion and the formation and robust resistance to mechanical Furthermore, a transmission electron of calcium-dependent desmosomes. stress. To test our prediction that microscopy and crystallographic mod- However, the events downstream of enhancing the DP–keratin interaction eling study reported that the ectodo- PKC signaling have yet to be eluci- would promote the acquisition of hyper- mains of desmosomal cadherins in dated. adhesion in epithelial sheets, we took

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Maturation SCC12f cells constitutively expressing Assembled desmosome wild-type DP or mutant DP (Supple- mentary Figure S1b online). Impor- Assembly tantly, these results were obtained in PKCα Intercellular PKC stimulation the background of endogenous DP activity adhesive strength depletion using small interfering RNA oligos targeting the DSP 30 untranslated region, which ensured that the induci- Modulation of desmoplakin–keratin DP Ser2849Gly bly expressed DP transgene would be association the predominant form of DP expressed during the assay (Figure 1c). Altogether, our data indicate that Desmosome Mature assembly desmosomes Hyperadhesion DP Ser2849 is a key mediator in the acquisition of desmosomal hyper- Figure 2. Model depicting the roles of PKC signaling and DP–keratin association in regulating adhesion. As DP Ser2849 resides within desmosome assembly and maturation. (Left, assembly) Stimulation of protein kinase C (PKC) signaling a PKC consensus sequence, the data promotes desmosome assembly, in part through regulation of the desmoplakin (DP)–keratin interaction raise the possibility that PKC-mediated in a Ser2849-dependent manner (Godsel et al., 2005; Bass-Zubek, 2008). (Right, maturation) Once assembled, desmosomes provide intercellular adhesive strength, which increases over time as of DP Ser2849 may desmosomes mature and ultimately reach a state of hyperadhesion. PKC stimulation induces the serve as a molecular mechanism to reversion of hyperadhesion, but not in epithelial sheets expressing the DP Ser2849Gly point mutant, tune the adhesive strength of epithelia which is unresponsive to PKC stimulation and promotes hyperadhesion. during wound healing and morpho- genesis (Figure 2). Given the recent advantage of a previously engineered We observed that 6-day-old sheets report of hyperadhesion protecting DP point mutation (Ser2849Gly) that acquired a state of calcium-indepen- epithelial sheets from pemphigus- enhances binding dence and robustly resisted fragmen- induced loss of adhesion (Cirillo et al., by 9-fold compared with wild-type DP tation following the application of me- 2010), identifying underlying mecha- (Meng et al., 1997). This enhanced chanical stress, whereas 2-day-old sheets nisms that regulate the acquisition of binding results in sequestration of the did not exhibit this level of stress hyperadhesion may lead to new ther- mutant DP on keratin filaments, thus resistance (Figure 1a and d). Pharmaco- apeutic strategies for treating skin blis- altering its dynamic properties and logical stimulation of PKC signaling tering disorders. delaying its assembly into newly form- (15 minutes treatment with 1 mM phorbol ing desmosomes. However, when al- 12-myristate 13-acetate (PMA)) prior to CONFLICT OF INTEREST lowed time to accumulate at cell–cell incubation of sheets in low-calcium The authors state no conflict of interest. borders, the DP Ser2849Gly protein is media induced greater fragmentation capable of localizing to sites of cell–cell for both 2- and 6-day sheets (Figure 1a ACKNOWLEDGMENTS contact (Godsel et al., 2005). and d). We observed similar findings This work was supported by an American Heart These previous findings led us to posit for sheets of normal human epidermal Association fellowship (0810061Z) and NIH training grant (T32 CA009560) to RPH and NIH that incorporation of the DP Ser2849Gly keratinocytes and SCC12f cells (Supple- R37 AR043380 to KJG. KJG is supported in part mutant into the junction would promote mentary Figure S1 online). by the Joseph L. Mayberry Endowment. the acquisition of hyperadhesion due to As hypothesized, mutant DP express- 1 the enhanced DP–keratin association at ing sheets appreciably resisted frag- Ryan P. Hobbs and 1,2 cell borders. To address this hypothesis, mentation compared with wild-type Kathleen J. Green A431 epithelial cells inducibly expres- DP-expressing sheets (Figure 1b and d). 1Department of Pathology, Northwestern sing wild-type DP or DP Ser2849Gly Additionally, mutant DP-expressing University, Chicago, Illinois, USA and 2Department of Dermatology, Northwestern (mutant DP; Godsel et al., 2005) were sheets were no longer responsive to University, Chicago, Illinois, USA grown 2–6 days past confluency and the PMA-induced reversion of hyper- E-mail: [email protected] treated with low-calcium growth media adhesion (Figure 1b and d), indicating (DMEM, 10% fetal bovine serum, 1% that DP Ser2849 can control the res- 2 þ SUPPLEMENTARY MATERIAL penicillin/streptomycin, 0.05 mM Ca ) ponse of hyperadhesive epithelial for 45 minutes to preserve only calcium- sheets to PKC signaling. Furthermore, Supplementary material is linked to the online version of the paper at http://www.nature.com/jid independent desmosomes. Intact epi- mutant DP-expressing sheets exhibited thelial sheets were lifted off the surface reduced fragmentation at 6 days com- of the dish using dispase II treat- pared with 2 days (Figure 1d), suggest- REFERENCES ment (30–45minutes; 2.4 U ml1,Roche, ing that robust keratin anchorage over Bass-Zubek AE, Hobbs RP, Amargo EV et al. #04942078001 (Mannheim, Germany)) the course of desmosome maturation (2008) 2: a critical scaffold for PKC alpha that regulates intercellular junc- and subjected to rotational mechanical may be a driving factor in governing the tion assembly. J Cell Biol 181:605–13 stress (e.g., 150 r.p.m. for 5 minutes) to acquisition of desmosome hyperadhe- Cirillo N, Lanza A, Prime SS (2010) Induction induce fragmentation. sion. Parallel results were observed in of hyper-adhesion attenuates autoimmune-

484 Journal of Investigative Dermatology (2012), Volume 132 MH Law et al. TERT–CLPTM1L Locus and Reduced Melanoma Risk

induced keratinocyte cell-cell detachment sions: multiple phases differentially regulated by Pasdar M, Krzeminski KA, Nelson WJ (1991) and processing of adhesion molecules via intermediate filaments and . JCellBiol Regulation of desmosome assembly in mechanisms that involve PKC. Exp Cell Res 171:1045–59 MDCK epithelial cells: coordination of mem- 316:580–92 Green KJ, Simpson CL (2007) Desmosomes: new brane core and cytoplasmic plaque domain Garrod D, Chidgey M (2008) Desmosome struc- perspectives on a classic. J Invest Dermatol assembly at the plasma membrane. J Cell Biol ture, composition and function. Biochim 127:2499–515 113:645–55 Biophys Acta 1778:572–87 Huen AC, Park JK, Godsel LM et al. (2002) Thomason HA, Scothern A, McHarg S et al. (2010) Garrod D, Kimura TE (2008) Hyper-adhesion: a Intermediate filament-membrane attachments Desmosomes: adhesive strength and signalling new concept in cell-cell adhesion. Biochem function synergistically with actin-dependent in health and disease. Biochem J 429:419–33 Soc Trans 36:195–201 contacts to regulate intercellular adhesive Vasioukhin V, Bowers E, Bauer C et al. (2001) Garrod DR, Berika MY, Bardsley WF et al. (2005) strength. J Cell Biol 159:1005–17 Desmoplakin is essential in epidermal sheet Hyper-adhesion in desmosomes: its regula- Meng JJ, Bornslaeger EA, Green KJ et al. (1997) formation. Nat Cell Biol 3:1076–85 tion in wound healing and possible relation- Two-hybrid analysis reveals fundamental Wallis S, Lloyd S, Wise I et al. (2000) The alpha ship to crystal structure. J Cell Sci differences in direct interactions between isoform of protein kinase C is involved in 118:5743–54 desmoplakin and cell type-specific inter- signaling the response of desmosomes Godsel LM, Hsieh SN, Amargo EV et al. (2005) mediate filaments. J Biol Chem 272: to wounding in cultured epithelial cells. Desmoplakin assembly dynamics in four dimen- 21495–503 Mol Biol Cell 11:1077–92

Meta-Analysis Combining New and Existing Data Sets Confirms that the TERT–CLPTM1L Locus Influences Melanoma Risk

Journal of Investigative Dermatology (2012) 132, 485–487; doi:10.1038/jid.2011.322; published online 13 October 2011

TO THE EDITOR present here unpublished Australian melanocyte proliferation, and nevus A number of genome-wide association data and rationalize the findings. count is positively associated with mel- studies have observed an association The 5p15.33 SNPs are located within anoma risk. Longer telomeres have between single-nucleotide polymor- or adjacent to two in strong linkage been associated with increased nevus phisms (SNPs) located in 5p15.33 and disequilibrium (LD), encoding telomer- count and size, as well as with a non- an increased risk for a range of cancers, ase reverse transcriptase (TERT,MIM: significant increase in melanoma risk including some non-melanoma skin 187270) and CLPTM1-like protein (OR ¼ 1.85, 95% CI 0.99–3.44; Han cancers (Baird, 2010). Contrary to the (CRR9p; CLPTM1L, MIM: 612585). et al., 2009). Nan et al. (2011) reported increased risk observed for other cancers, CLPTM1L was identified as upregulated a marginal association between the the peak variant, rs401681 C allele, has in cisplatin-resistant cancer cells (Yama- rs401681 C allele and shorter telomere been associated with a decreased risk moto et al., 2001) and, although a role length, an intriguing result given their for melanoma (odds ratio (OR) ¼ 0.86, for CLPTM1L should not be excluded, earlier observation of decreased nevus 95% confidence interval (CI) 0.81–0.91, little is known about its function. TERT count in those with shorter telomere P ¼ 5.0 108;Staceyet al., 2009). is a striking candidate, as it encodes the length (Han et al., 2009). Specifically, There have been two attempts at inde- catalytic subunit of telomerase. Incom- rs401681 C may associate with reduced pendent replication. Nan et al. (2011) plete replacement of telomere repeat melanoma incidence via shortened telo- observed a similar direction of effect in sequences by telomerase following their mere-mediated inhibition of nevus growth. a small sample (OR ¼ 0.73, 95% CI loss during S phase is a likely cause However a far larger study observed no 0.59–0.91). However an additional of cell senescence (Shawi and Autexier, association between rs401681 and telo- replication study observed no evidence 2008). Although TERT expression is mere length (Pooley et al., 2010). for association between rs401681 C generally absent in adult tissues, it is We recently conducted a large mela- allele and melanoma (OR ¼ 1.01, 95% enhanced in most, but not all, cancerous noma genome-wide association study in CI 0.87–1.19) (Pooley et al., 2010). As cells (Engelhardt et al., 1997; Kolquist a Caucasian population by combining replication has been inconsistent, we et al., 1998). Nevi (moles) result from 2,168 cases from the Q-MEGA (Queens- land study of Melanoma: Environment and Genetic Associations; Baxter et al., Abbreviations: AMFS, Australian Melanoma Family Study; CLPTM1L, CLPTM1-like protein; Q-MEGA, Queensland study of Melanoma: Environment and Genetic Associations; TERT, telomerase reverse 2008) and AMFS (Australian Melanoma transcriptase Family Study; Cust et al., 2009) studies

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