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Epigen Transgenic Mice Develop Enlarged Sebaceous Glands

Journal of Investigative Dermatology (2010) 130, 623–626; doi:10.1038/jid.2009.251; published online 20 August 2009

TO THE EDITOR integration sites of the transgene were (Strachan et al., 2001; Kochupurakkal Epidermal receptor obtained. They were visibly smaller et al., 2005). Interestingly, we observed (EGFR) activity is essential for skin and than their non-transgenic littermates that epigen is expressed both in un- hair follicle homeostasis and its dereg- and development of the first coat was differentiated and in differentiated se- ulation results in a series of disorders, obviously delayed (Figure 1a). Once bocytes in EPGN-tg mice, but only in including psoriasis and cancer (Sibilia hair became visible, it grew in a patchy differentiated sebocytes in control lit- et al., 2007; Pastore et al., 2008; pattern and appeared greasy (Figure termates (Supplementary Figure 1b). Schneider et al., 2008b). Further, in- 1b,c). Overall, the number of epigen-positive flammatory rash, dry skin, and hair and Although EPGN-tg mice were not cells in EPGN-tg mice was significantly periungual alterations are common sterile, they generated small litters and higher (Supplementary Figure 1). Since side-effects in cancer patients treated consistently failed to produce trans- mouse and human epigen with EGFR inhibitors genic descendants (except for one sequences are highly conserved (82% and anti-EGFR monoclonal antibodies founder, which produced a litter with identity), we assume that this observa- (Lacouture, 2006). The EGFR can be a single transgenic pup). EPGN-tg tion reflects the detection of both activated by a family of seven peptide founder mice were therefore sacrificed, endogenous (mouse) and transgenic growth factors (Schneider and Wolf, and expression of the transgene in the (human) epigen in EPGN-tg mice. 2009). Upon ligand binding, EGFR skin was confirmed by Northern blot- We next investigated the mechanism homodimers or heterodimers (with the ting (Figure 1d). Histological examina- underlying the sebaceous gland related ERBB receptors) are formed, tion of hematoxylin-eosin-stained back enlargement. Cell counting revealed a resulting in the activation of multiple skin and tail skin sections (Figure 1e) 2.5-fold increase in the average cell signaling cascades (Yarden and Shilo, and immunofluorescence against the number/gland in EPGN-tg mice com- 2007). EGFR ligands are not redundant, lipid-associated protein adipophilin pared to controls (Figure 2a). BrdU but rather exert distinctive and specific (Figure 1f) demonstrated strongly en- incorporation studies showed that the biological functions. For example, larged sebaceous glands. Quantitative number of proliferating cells was strongly alterations of the expression levels of analysis revealed a significant increase increased in EPGN-tgmicecomparedto different EGFR ligands in the skin in the area occupied by sebaceous non-transgenic littermates (Figure 2b). resulted in very diverse cutaneous glands in the skin of EPGN-tg mice This finding, together with expression of phenotypes (reviewed in (Schneider compared to non-transgenic littermates transgene-derived epigen in undifferen- et al., 2008b). (Figure 1g), further suggesting that the tiated sebocytes in EPGN-tg mice, sug- Epigen is the newest and least greasy fur of the mice was a conse- gests that the enlargement of sebaceous characterized EGFR ligand (Strachan quence of increased sebum production. glands is attributable to expansion of the et al., 2001; Kochupurakkal et al., However, since measurements of epi- basal, undifferentiated cells in the gland 2005). In spite of its much lower affinity dermal sebum levels or quantification in response to epigen. It will be interest- for the EGFR, epigen is more mitogenic of sebum production could not be ing to determine if Blimp1-expressing than EGF due to less effective receptor- performed, it cannot be excluded that unipotent sebocyte progenitors (Horsley mediated ligand depletion and a rather the greasy aspect of the coat is a et al., 2006) are affected by the increased inefficient receptor ubiquitylation and consequence of structural changes in epigen expression. down-regulation (Kochupurakkal et al., the hair shaft. The absence of overt Epidermal thickness was visibly in- 2005). To study its biological activities, phenotypical alterations in other organs creased in transgenic mice, in particu- we generated transgenic mice over- from EPGN-tg mice (data not shown) lar in the tail skin (Figure 1e). This could expressing human epigen under the indicates that the skin is particularly be due to different levels of transgene control of the CMV enhancer/chicken sensitive to increased levels of epigen. expression in the back versus tail skin beta-actin promoter. This promoter can Immunofluorescence localized a few or to different responsiveness of tail and drive expression of transgenes in the epigen-positive cells in the interfollicu- back skin keratinocytes to the increased pilosebaceous unit and the interfollicu- lar epidermis of tail skin and in the epigen levels. Terminal differentiation lar epidermis, among many other tis- outer root sheath of the hair follicle of of interfollicular keratinocytes was not sues (Schneider et al., 2008a). Six non-transgenic animals (Supplementary obviously affected as revealed by the founder mice (EPGN-tg) with different Figure 1a), confirming a previous report appropriate expression of the differen- tiation markers keratin (K) 10, K14, and Abbreviations: BrdU, bromodeoxyuridine; CMV, cytomegalovirus; EGFR, epidermal ; loricin (Figure 2c). In addition, inter- EPGN, epigen; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; TGF-a, transforming growth factor-alpha follicular expression of K6, a marker of

www.jidonline.org 623 M Dahlhoff et al. Epigen Transgenic Mice

Co Transgenic founders * * Epigen * Gapdh * Co Adipophilin * Hoechst

Co Co Tg

) 2.0 2 Tg Tg ** 1.5

1.0

0.5

Sebaceous gland area (mm 0.0 Control EPGN-tg

Figure 1. Phenotypic alterations in transgenic mice overexpressing Epigen. (a-c) Macroscopic appearance of control and EPGN-tg mice (identified by asterisks) at the ages of 2 (a), 6 (b), and 12 (c) weeks of age. (d) Northern blot showing expression of the transgene in the skin of transgenic mice. Hybridization with a Gapdh probe served as a loading control. (e) H&E staining of the back skin (left) and tail skin (right) showing the normal size of the sebaceous glands in an adult control mouse (top panel, arrows) and the remarkable hyperplasia of the sebaceous glands in EPGN-tg mice (low panel). (f) Immunofluorescence staining against adipophilin showing large amounts of lipids in the skin of EPGN-tg mice. (g) Quantitative evaluation of sebaceous gland area in EPGN-tg mice (n ¼ 5) as compared to aged-matched, non transgenic mice (n ¼ 4). Bars represent 100 mm (e,f); ** in (g) indicate statistical significance, Po0.01.

abnormal differentiation and hyperpro- pression of resulted in a TGF-a resulted in a hyperproliferative, liferation, was not observed (Figure 2c). psoriasis-like phenotype (Cook et al., tumor-prone skin (Vassar and Fuchs, The prominent sebaceous gland 1997, 2004), increased levels of EGF 1991; Dominey et al., 1993). Thus, our phenotype observed in EPGN-tg mice caused hyperproliferation of basal cells findings support the concept that EGFR is unexpected and differs considerably and hair follicle developmental arrest ligands are not redundant, but exert from the changes elicited by other (Mak and Chan, 2003), distinct and specific functions. The EGFR ligands in transgenic mouse transgenic animals showed a relatively enlarged sebaceous glands of EPGN-tg models. Its presence in six founder mild skin phenotype, including delayed mice, together with the observation of mice demonstrates that the effect is hair cycle induction and increased endogenous epigen expression in this specific for epigen and does not result angiogenesis at wound sites (Schneider appendage, suggest a role for epigen in from insertional mutagenesis. Overex- et al., 2008a), and overexpression of sebocyte physiology and a potential

624 Journal of Investigative Dermatology (2010), Volume 130 M Dahlhoff et al. Epigen Transgenic Mice

30 Control Transgenic ***

20

10 Cells/sebaceous gland K6 K6 0 Control EPGN-tg Hoechst Hoechst

Co Tg

K10 K14 K10 K14 Hoechst Hoechst 3 ***

2 gland

cells/sebaceous 1 +

BrdU Loricrin Loricrin 0 Hoechst Hoechst Control EPGN-tg

Figure 2. Cell proliferation and differentiation in transgenic mice overexpressing Epigen. (a) Evaluation of the number of cells in sebaceous glands in EPGN-tg mice and control littermates (n ¼ 4 mice/group). (b) Immunohistochemistry staining against BrdU and quantitative evaluation (n ¼ 4 mice/group) showing a significant increase in the number of BrdU-positive cells within the sebaceous glands of EPGN-tg mice. (c) The distribution of the differentiation markers K6, K10, K14, and loricrin (color-coded) was not altered in the tail skin of EPGN-tg animals as compared to control mice. Bars represent 50 mm (b,c)*** indicate statistical significance, Po0.001.

application of this growth factor for the 1Institute of Molecular Animal Breeding and expression of the human amphiregulin modulation of sebum production. Gen- Biotechnology, and Laboratory for Functional induces a psoriasis-like phenotype. J Clin Genome Analysis (LAFUGA), Gene Center, Invest 100:2286–94 eration of transgenic mouse lines with LMU Munich, Munich, Germany and Dominey AM, Wang XJ, King LE Jr, Nanney LB, 2 skin-specific overexpression of epigen Department of Biology, Institute of Cell Gagne TA, Sellheyer K et al. (1993) Targeted will be required to further study the Biology, ETH Zurich, Zurich, Switzerland overexpression of transforming growth factor influence of epigen on the appendages E-mail: [email protected] alpha in the epidermis of transgenic mice elicits hyperplasia, hyperkeratosis, and spon- and the interfollicular epidermis. taneous, squamous papillomas. Cell Growth SUPPLEMENTARY MATERIAL Differ 4:1071–82 CONFLICT OF INTEREST Supplementary material is linked to the online Horsley V, O’Carroll D, Tooze R, Ohinata Y, The authors state no conflict of interest. version of the paper at http://www.nature.com/jid Saitou M, Obukhanych T et al. (2006) Blimp1 defines a progenitor population that governs cellular input to the sebaceous gland. Cell REFERENCES 126:597–609 ACKNOWLEDGMENTS Cook PW, Brown JR, Cornell KA, Pittelkow MR We thank Professor Yosef Yarden (The Weizmann Kochupurakkal BS, Harari D, Di Segni A, Maik- (2004) Suprabasal expression of human am- Institute of Science, Rehovot, Israel) for the human Rachline G, Lyass L, Gur G et al. (2005) phiregulin in the epidermis of transgenic mice EPGN cDNA and Dr. Ingrid Renner-Mu¨ller and Epigen, the last ligand of ErbB receptors, Petra Renner (Gene Center, LMU Munich) for induces a severe, early-onset, psoriasis-like reveals intricate relationships between affi- excellent animal care. skin pathology: expression of amphiregulin in nity and mitogenicity. J Biol Chem 1 the basal epidermis is also associated with 280:8503–12 Maik Dahlhoff , Anna-Katharina synovitis. Exp Dermatol 13:347–56 2 1 Lacouture ME (2006) Mechanisms of cutaneous Mu¨ller , Eckhard Wolf , Sabine Cook PW, Piepkorn M, Clegg CH, Plowman GD, toxicities to EGFR inhibitors. Nat Rev Cancer 2 1 Werner and Marlon R. Schneider DeMay JM, Brown JR et al. (1997) Transgenic 6:803–12

www.jidonline.org 625 LE Rhodes et al. PLE Prevalence in the European Population

Mak KK, Chan SY (2003) Schneider MR, Werner S, Paus R, Wolf E (2008b) Strachan L, Murison JG, Prestidge RL, as a biologic switch in hair growth cycle. Beyond wavy hairs: the epidermal growth Sleeman MA, Watson JD, Kumble KD J Biol Chem 278:26120–6 factor receptor and its ligands in skin biology (2001) Cloning and biological activity of Pastore S, Mascia F, Mariani V, Girolomoni G and pathology. Am J Pathol 173:14–24 epigen, a novel member of the epidermal growth factor superfamily. J Biol Chem (2008) The epidermal growth factor receptor Schneider MR, Wolf E (2009) The epidermal system in skin repair and inflammation. 276:18265–71 growth factor receptor ligands at a glance. J Invest Dermatol 128:1365–74 Vassar R, Fuchs E (1991) Transgenic mice J Cell Physiol 218:460–6 Schneider MR, Antsiferova M, Feldmeyer L, Dahl- provide new insights into the role of Sibilia M, Kroismayr R, Lichtenberger BM, Natar- hoff M, Bugnon P, Hasse S et al. (2008a) TGF-alpha during epidermal development Betacellulin regulates hair follicle develop- ajan A, Hecking M, Holcmann M (2007) The and differentiation. Dev 5: ment and hair cycle induction and enhances epidermal growth factor receptor: from de- 714–27 angiogenesis in wounded skin. Journal of velopment to tumorigenesis. Differentiation Yarden Y, Shilo BZ (2007) SnapShot: EGFR Investigative Dermatology 128:1256–65 75:770–87 Signaling Pathway. Cell 131:1018

Polymorphic Light Eruption Occurs in 18% of Europeans and Does Not Show Higher Prevalence with Increasing Latitude: Multicenter Survey of 6,895 Individuals Residing from the Mediterranean to Scandinavia

Journal of Investigative Dermatology (2010) 130, 626–628; doi:10.1038/jid.2009.250; published online 20 August 2009

TO THE EDITOR 471), Heidelberg (Germany, 49.51), accuracy±2%, a sample size of 1,000 Polymorphic light eruption (PLE) is Leiden (The Netherlands, 521), Man- was required. Statistical analyses were acknowledged to be the most common chester (UK, 53.51), and Turku (Finland, carried out in Heidelberg using SAS 9.1 idiopathic photodermatosis, but epide- 60.51). All subjects were indoor work- Windows (Cary, NC). miological data are sparse. From sur- ers, predominantly hospital staff, The interviewed cohorts are charac- veys in 550 people in Perth and Ballarat whereas factory workers were included terized in Table 1a and the subgroups (Australia) and London (UK), PLE pre- in Heidelberg. Interviewees completed with PLE features in Table 1b. A valence was estimated at 5.2, 3.6, and a questionnaire under the supervision response rate of 490% was achieved 14.8%, respectively (Pao et al., 1994), of an investigator at each center. A at each center, with an overall total of whereas a postal questionnaire of 397 short (one-page) questionnaire was de- 6,895 respondents (62.8% women, Swedish subjects showed symptoms of veloped to encourage high response 37.2% men). Of 6,836 subjects with PLE in 21% (Ros and Wennersten, (Figure S1). The first part of this ques- complete questionnaires, 1,235 gave a 1986), and from 271 Boston (USA) tionnaire asked questions regarding history consistent with PLE, giving a residents the prevalence of PLE was skin type, age, and sex, together with suspected lifetime prevalence of PLE of estimated at 11% (Morison and Stern, the central question, ‘‘Do you get an 18% in the study population (18.2% 1982). These investigations led to the itching rash/abnormal skin after short after age and sex standardization). A assumption that PLE prevalence is sun exposures, which you do not regard similar age and sex-standardized pre- lower in countries nearer the equator, as sunburn?’’ Interviewees answering valence (range 17–19.5%), was found whereas latitudes with higher seasonal ‘‘yes’’ continued the questionnaire, among centers, apart from a low pre- UV radiation modulation may predis- including questions on occurrence valence of 13.6% in Turku. pose to the condition. However, reli- during springtime and sunny holidays, PLE rashes were reported to occur able data concerning this hypothesis age at first appearance, timing of during springtime by equal proportions are lacking. In our large-scale cross- appearance after sun exposure, and of the total interviewed population per sectional study, we assessed PLE pre- duration. Ethics approval was gained country (between 7 and 10%), without valence in the adult population of six at participating institutions, informed indication of a latitudinal gradient European countries located from the consent was obtained, and the study (Table 1b). Frequency of PLE during Mediterranean to Scandinavia. adhered to the Declaration of Helsinki sunny holidays in the total population Participating centers were university Principles. Data were collected was strikingly low among Finnish parti- dermatology departments in Athens from March 2003 to April 2004. To cipants (9%), compared with other (Greece, NL 381), Besanc¸on (France, estimate a prevalence of 10% with countries (14–20%), explaining the overall low prevalence of PLE in Fin- Abbreviations: PLE, polymorphic light eruption land. Among people with PLE, the

626 Journal of Investigative Dermatology (2010), Volume 130