Ultraviolet B irradiation increases the expression of cornulin and retepin in human skin xenotransplants

Ultraviolet B irradiation increases the expression of cornulin and retepin in human skin xenotransplants | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Ultraviolet B irradiation increases the expression of cornulin and retepin in human skin xenotransplants Teruhiko Makino, Megumi Mizawa, Keita Takemoto, Tadamichi Shimizu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3852080/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 May, 2024 Read the published version in Experimental Dermatology → Version 1 posted You are reading this latest preprint version Abstract Cornulin (CRNN) and repetin (RPTN) belong to the fused-type S100 protein family. Although these proteins have been reported to be expressed in the granular layer of the epidermis and are suggested to be associated with barrier formation in the epidermis, the exact function of these proteins remains unclear. The present study examined the effects of UVB irradiation on the CRNN and RPTN expression in human skin xenotransplantation. The expression of CRNN increased in the granular layer of UVB-irradiated skin two days after UVB irradiation in comparison to sham-irradiated skin. Interestingly, CRNN signals were observed not only in the cytoplasm but also in the peripheral region of the granular keratinocytes. In contrast, RPTN was rarely expressed in sham-irradiated skin; however, RPTN signals were markedly increased in the granular layer of UVB-irradiated skin. The number of RPTN-positive keratinocytes on day 2 after UVB irradiation was significantly higher than that in the sham-irradiated skin. Accordingly, the present study demonstrated that CRNN and RPTN are novel proteins whose expression can be increased by UVB irradiation. In addition, we speculate that the role of CRNN and RPTN in barrier formation of the epidermis may differ according to the expression pattern of these proteins in UVB-irradiated skin. cornulin repetin ultraviolet B epidermis skin barrier keratinocyte Figures Figure 1 Figure 2 Introduction The epidermal differentiation complex (EDC) at chromosome band 1q21.3 contains a family of proteins described as fused-type S100 proteins, which include filaggrin (FLG), trichohyalin, repetin (RPTN), cornulin (CRNN), filaggrin-2, hornerin (HRNR) and trichohyalin-like 1 [ 1 ]. Among these proteins, CRNN is a 495-amino-acid protein that is mainly observed in the upper spinous and granular layers of the epidermis and is suggested to be involved in cornified envelope (CE) formation [ 2 ]. RPTN is a 784-amino-acid protein and is also observed in the granular layer of the epidermis [ 3 ]. The expression of RPTN seems to be upregulated upon alteration of the epidermal barrier, although it is rarely expressed in the homeostatic epidermis [ 3 ]. Therefore, these proteins may play unique roles in the epidermis. Ultraviolet (UV) irradiation exerts various effects on the epidermis, including suntan, sunburn, immunosuppression, photoaging or carcinogenesis [ 4 ]. Previous studies have reported that ultraviolet-B (UVB) irradiation affects the proliferation and differentiation of the epidermis [ 4 – 7 ]. Bino et al. previously demonstrated that human skin xenotransplanted into nude mice is a valuable tool for investigating the effects of UVB on early and late epidermal differentiation [ 8 ]. We also reported that UVB irradiation affects the expression of HRNR and TCHHL1 in human skin xenotransplants [ 9 , 10 ]. In the present study, we examined the effects of UVB irradiation on the expression of CRNN and RPTN in human skin xenotransplants to explore the role of these proteins in the epidermis. Materials and Methods Clinical materials: Human skin samples were obtained from the abdominal skin of four healthy Japanese volunteers with informed consent. All of them had type III skin. Skin samples were transplanted onto the backs of nude mice, KSN/Slc mice (Nippon SLC, Hamamatsu, Japan). Four mice in each experimental group (eight mice in total) were used for the study. This study was performed in compliance with the Principles of the Declaration of Helsinki and was approved by the Medical Ethics Committee of the University of Toyama. Ultraviolet irradiation: The UVB light source was a fluorescent lamp (GL40SE; Sankyo Denki Co, Kanagawa, Japan) that emitted 0.1 mW/cm 2 of UV light between 280 and 315 nm (peak: 306 nm) at a distance of 40 cm, as measured by a UV radiometer (EKO Instruments Co., Tokyo, Japan). Two months after transplantation, the grafted skin was exposed to 500 mJ/cm 2 UVB, which is the minimal dose necessary to induce pyrimidine dimers, sun-burn cells, and apoptotic keratinocytes [ 8 , 9 ]. Skin tissues were excised two days after UVB irradiation and analyzed using immunofluorescence. Immunohistochemistry: Skin specimens were directly dipped into OCT compound and rapidly frozen in liquid nitrogen. Sections were blocked with Protein Block Serum-Free (Dako, Carpinteria, CA, USA) and incubated with primary antibodies, such as anti-human CRNN antibodies (11799-1-AP, Proteintech, Curanbry, NJ, USA), anti-human RPTN antibodies (HPA030483, Atlas Antibodies AB, Bromma, Sweden), and anti-human FLG antibodies (Abcam, St. Louis, MA, USA), followed by incubation with anti-rabbit IgG Alexa Fluor 488 or anti-mouse IgG Alexa Fluor 555 (Molecular Probes, Eugene, OR, USA) secondary antibodies. Tissue sections were observed under a fluorescence microscope (Olympus IX71; Olympus Co., Tokyo, Japan) or a confocal laser microscope, LSM780 (Carl Zeiss, Oberkochen, Germany). The number of CRNN-positive or RPTN-positive keratinocytes in the epidermis was counted in each 500 µm field for a total of 5.0 mm length. Statistical analysis: Data are shown as mean ± S.D. Statistical significance was assesed using the Mann-Whitney U test. Statistical significance was set at P < 0.05. Results The expression of CRNN in UVB-irradiated skin We first examined the expression of CRNN in UVB-irradiated skin. CRNN was irregularly detected in the granular layer close to the corny layer of the epidermis in sham-irradiated skins ( Fig. 1 a, left panels) ; however, on day 2 after irradiation, CRNN signals were observed in the upper spinous and granular layers, and were colocalized with those of FLG ( Fig. 1 a right panels) . The number of CRNN-positive keratinocytes on day 2 after irradiation was significantly higher than that in the sham-irradiated skin ( Fig. 1 b ) . At high magnification, although the signals of CRNN and FLG were colocalized in the cytoplasm of the granular keratinocytes in a granular pattern, CRNN signals were also observed in the peripheral region of the granular keratinocytes ( Fig. 1 c ). The expression of RPTN in UVB-irradiated skin Next, we examined the expression of RPTN in UVB-irradiated skin. RPTN was rarely detected in the sham-irradiated skin; however, its expression was markedly increased in the granular keratinocytes of UVB-irradiated skin on day 2 ( Fig. 2 a, left panels) . Most RPTN-positive keratinocytes colocalized with FLG-positive keratinocytes ( Fig. 2 a, right panels) . The number of RPTN-positive keratinocytes on day 2 after irradiation was significantly higher than that in the sham-irradiated skin ( Fig. 2 b ) . At high magnification, although the signals of RPTN were observed in a granular pattern in the cytoplasm of the granular keratinocytes, and were almost colocalized with those of FLG, RPTN-positive granules were also irregularly detected in a different distribution from those expressing FLG at two days after UVB irradiation ( Fig. 2 c ) . Discussion The present study showed that the expression of CRNN and RPTN in xenotransplanted human skin was markedly increased after UVB irradiation (500 mJ/cm 2 ). The expression of CRNN increased in parallel with that of FLG in UVB-irradiated epidermis. A previous study described the association of SNP rs941934 in CRNN with the development of atopic dermatitis (AD) and the mRNA level of CRNN was decreased in skin lesions of AD [ 11 ]. In addition, CRNN is known to be reduced in human oral and esophageal squamous cell carcinoma [ 12 , 13 ]. This suggests that CRNN may be associated with terminal differentiation or the barrier formation in keratinocytes. However, CRNN, but not FLG, was observed in the peripheral region, in addition to the cytoplasm of granular keratinocytes. Therefore, we hypothesized that CRNN may play a different role than FLG in UVB-irradiated skin. In contrast, the expression of RPTN was markedly induced by UVB irradiation, although RPTN signals were rarely detected in the sham-irradiated skin. Previous studies have demonstrated that the increased expression of RPTN was observed in Kruppel-like factor 4-null mice or loricrin-deficient mice, which are characterized by an impaired epidermal barrier function [ 14 , 15 ]. Furthermore, the mRNA level of RPTN is increased in skin lesions of AD, in which the barrier function of the epidermis is reduced [ 4 ]. We previously reported that the expression of HRNR, another member of the S100 fused-protein family, may be induced in relation to the reconstruction of epidermal barrier dysfunction induced by UVB irradiation. Therefore, the overexpression of RPTN in UVB-irradiated skin may also be involved in compensatory mechanisms to repair the altered epidermal barrier. In conclusion, the present study demonstrated that CRNN and RPTN are novel proteins whose expression can be increased by UVB irradiation, and suggest markers of acute UV damage to the skin. Furthermore, we hypothesized that the role in the barrier formation may differ among CRNN, RPTN and FLG, according to the expression pattern of these proteins in UVB-irradiated skin. Further studies are necessary to clarify the exact mechanism underlying the role of CRNN and RPTN in UVB-irradiated skin. However, the present study will contribute to extending the knowledge of the S100 fused protein family. Declarations Author contribution: T.M. and T.S. involved in conceptualization. T.M., M.M. and K.T. involved in formal analysis. T.M. involved in funding acquisition. T.M., M.M. and K.T. involved in data analysis. T.M. and K.T. involved in visualization. T.M. and M.M. involved in writing original draft preparation. All authors involved in writing review and editing. Acknowledgements: We thank Mr. Kenji Matsunaga for his valuable technical support. Fundings: This work was supported by JSPS KAKENHI grant numbers JP21K08296 and JP18K08265 (to TM). Conflict of Interest: The authors declare no conflicts of interest in association with the present study. Ethical approval: All patients provided written informed consent for participation in the present study, which complied with the principles of the Declaration of Helsinki. This study was approved by the Medical Ethic Committee of the University of Toyama, Toyama, Japan. References Kypriotou M, Huber M, Hohl D (2012) The human epidermal differentiation complex: cornified envelope precursors, S100 proteins and the 'fused genes' family. Exp Dermatol 21: 643-649. doi: 10.1111/j.1600-0625.2012.01472.x. Contzler R, Favre B, Huber M, Hohl D (2005). Cornulin, a new member of the "fused gene" family, is expressed during epidermal differentiation. J Invest Dermatol 124: 990-997. doi: 10.1111/j.0022-202X.2005.23694.x. Huber M, Siegenthaler G, Mirancea N, Marenholz I, Nizetic D, Breitkreutz D, Mischke D, Hohl D (2005) Isolation and characterization of human repetin, a member of the fused gene family of the epidermal differentiation complex. J Invest Dermatol 24: 998-1007. doi: 10.1111/j.0022-202X.2005.23675.x. Ichihashi M, Ueda M, Budiyanto A, Bito T, Oka M, Fukunaga M, Tsuru K, Horikawa T (2003) UV-induced skin damage. Toxicology 189: 21-39. doi: 10.1016/s0300-483x(03)00150-1. Moll I, Bohnert E, Treib U, Jung EG (1994) Effects of ultraviolet B radiation on cytoskeletal and adhesion molecules in human epidermis. Photodermatol Photoimmunol Photomed 10: 26-32. Smith MD, Rees JL (1994) Wavelength-specific upregulation of keratin mRNA expression in response to ultraviolet radiation. J Invest Dermatol 1994; 102: 433-439. doi: 10.1111/1523-1747.ep12372958. Bernerd F, Asselineau D (1997) Successive alteration and recovery of epidermal differentiation and morphogenesis after specific UVB-damages in skin reconstructed in vitro. Dev Biol 183: 123-138. doi: 10.1006/dbio.1996.8465. Del Bino S, Vioux C, Rossio-Pasquier P, Jomard A, Demarchez M, Asselineau D, Bernerd F (2004) Ultraviolet B induces hyperproliferation and modification of epidermal differentiation in normal human skin grafted on to nude mice. Br J Dermatol 150: 658-667. doi: 10.1111/j.0007-0963.2004.05886.x. Makino T, Yamakoshi T, Mizawa M, Shimizu T (2014) Ultraviolet B irradiation induces the expression of hornerin in xenotransplanted human skin. Acta Histochem 116: 20-24. doi: 10.1016/j.acthis.2013.05.001. Makino T, Mizawa M, Yoshihisa Y, Shimizu T (2019) Ultraviolet B irradiation increases the expression of trichohyalin-like 1 protein in human skin xenotransplants. Clin Exp Dermatol 44: 773-776. doi: 10.1111/ced.13904. Trzeciak M, Sakowicz-Burkiewicz M, Wesserling M, Gleń J, Dobaczewska D, Bandurski T, Nowicki R, Pawelczyk T (2017) Altered Expression of Genes Encoding Cornulin and Repetin in Atopic Dermatitis. Int Arch Allergy Immunol 172: 11-19. doi: 10.1159/000453452. Chen K, Li Y, Dai Y, Li J, Qin Y, Zhu Y, Zeng T, Ban X, Fu L, Guan XY (2013) Characterization of tumor suppressive function of cornulin in esophageal squamous cell carcinoma. PLoS One 8: e68838. doi: 10.1371/journal.pone.0068838. Govindaraj PK, Kallarakkal TG, Mohd Zain R, Tilakaratne WM, Lew HL (2021) Expression of Ki-67, Cornulin and ISG15 in non-involved mucosal surgical margins as predictive markers for relapse in oral squamous cell carcinoma (OSCC). PLoS One 16: e0261575. doi: 10.1371/journal.pone.0261575. Segre JA, Bauer C, Fuchs E (1999) Klf4 is a transcription factor required for establishing the barrier function of the skin. Nat Genet 22: 356-360. doi: 10.1038/11926. Koch PJ, de Viragh PA, Scharer E, Bundman D, Longley MA, Bickenbach J, Kawachi Y, Suga Y, Zhou Z, Huber M, Hohl D, Kartasova T, Jarnik M, Steven AC, Roop DR (2000) Lessons from loricrin-deficient mice: compensatory mechanisms maintaining skin barrier function in the absence of a major cornified envelope protein. J Cell Biol 151: 389-400. doi: 10.1083/jcb.151.2.389. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 01 May, 2024 Read the published version in Experimental Dermatology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3852080","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":266419504,"identity":"69310b8f-9886-4fa6-b744-85bbf14f2a7d","order_by":0,"name":"Teruhiko Makino","email":"data:image/png;base64,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","orcid":"","institution":"University of Toyama","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Teruhiko","middleName":"","lastName":"Makino","suffix":""},{"id":266419505,"identity":"0491208a-8e39-44df-80f3-52d0bc53e177","order_by":1,"name":"Megumi Mizawa","email":"","orcid":"","institution":"University of Toyama","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Megumi","middleName":"","lastName":"Mizawa","suffix":""},{"id":266419506,"identity":"e85ec950-bbc2-4d25-8321-c743280ce6f5","order_by":2,"name":"Keita Takemoto","email":"","orcid":"","institution":"University of Toyama","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Keita","middleName":"","lastName":"Takemoto","suffix":""},{"id":266419507,"identity":"897fc1f2-31b8-4004-81e3-8415bc760804","order_by":3,"name":"Tadamichi Shimizu","email":"","orcid":"","institution":"University of Toyama","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tadamichi","middleName":"","lastName":"Shimizu","suffix":""}],"badges":[],"createdAt":"2024-01-11 02:59:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3852080/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3852080/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1111/exd.15109","type":"published","date":"2024-05-01T05:58:11+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":49532232,"identity":"841963c4-7811-4a97-a40e-67191fd887e2","added_by":"auto","created_at":"2024-01-12 14:07:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2877811,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of CRNN in UVB-irradiated skin.\u003c/p\u003e\n\u003cp\u003e(a) Double staining of CRNN (green) and FLG (red) in sham-irradiated skin and UVB-irradiated skin was removed two days after exposure to UVB (500 mJ/cm\u003csup\u003e2\u003c/sup\u003e). DNA staining by 4’, 6-diamidine-2’-phenylindole dihydrochloride appears in blue. The broken lines indicate the basement membrane. (b) The numbers of CRNN-positive cells was counted in each 500 nm field for a total of 5.0 mm length in the sham-irradiated skin and UVB-irradiated skin removed two days after exposure to UVB. The graph shows the mean values ± S.D. (c) Observation of CRNN (green) and FLG (red) in the granular layers using a confocal lasar microscope (LSM780). DNA staining by 4’, 6-diamidine-2’-phenylindole dihydrochloride appears in blue.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3852080/v1/a0b2c20b4f5e0409c06282e0.png"},{"id":49532577,"identity":"92b029fb-e0f4-498e-931d-f91619b9a9ec","added_by":"auto","created_at":"2024-01-12 14:15:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2908153,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of RPTN in UVB-irradiated skin.\u003c/p\u003e\n\u003cp\u003e(a) Double staining for RPTN (green) and FLG (red) in the sham-irradiated skin and UVB-irradiated skin was removed two days after exposure to UVB (500 mJ/cm\u003csup\u003e2\u003c/sup\u003e). DNA staining by 4’, 6-diamidine-2’-phenylindole dihydrochloride appears in blue. The broken lines indicate the basement membrane. (b) The numbers of RPTN-positive cells was counted in each 500 nm field for a total of 5.0 mm length in the sham-irradiated skin and UVB-irradiated skin removed two days after exposure to UVB. The graph shows the mean values ± S.D. (c) Observation of RPTN (green) and FLG (red) in the granular layers using a confocal laser microscope (LSM780). DNA staining by 4’, 6-diamidine-2’-phenylindole dihydrochloride appears in blue.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3852080/v1/35cb9861225c06c65a32ef55.png"},{"id":57320163,"identity":"6e876d6f-e2b4-4727-90d9-23bb21f23c15","added_by":"auto","created_at":"2024-05-29 05:58:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7141026,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3852080/v1/083ac340-dd4c-4a1b-952a-dc5181493274.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ultraviolet B irradiation increases the expression of cornulin and retepin in human skin xenotransplants","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe epidermal differentiation complex (EDC) at chromosome band 1q21.3 contains a family of proteins described as fused-type S100 proteins, which include filaggrin (FLG), trichohyalin, repetin (RPTN), cornulin (CRNN), filaggrin-2, hornerin (HRNR) and trichohyalin-like 1 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Among these proteins, CRNN is a 495-amino-acid protein that is mainly observed in the upper spinous and granular layers of the epidermis and is suggested to be involved in cornified envelope (CE) formation [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. RPTN is a 784-amino-acid protein and is also observed in the granular layer of the epidermis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The expression of RPTN seems to be upregulated upon alteration of the epidermal barrier, although it is rarely expressed in the homeostatic epidermis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, these proteins may play unique roles in the epidermis.\u003c/p\u003e \u003cp\u003eUltraviolet (UV) irradiation exerts various effects on the epidermis, including suntan, sunburn, immunosuppression, photoaging or carcinogenesis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Previous studies have reported that ultraviolet-B (UVB) irradiation affects the proliferation and differentiation of the epidermis [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Bino et al. previously demonstrated that human skin xenotransplanted into nude mice is a valuable tool for investigating the effects of UVB on early and late epidermal differentiation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. We also reported that UVB irradiation affects the expression of HRNR and TCHHL1 in human skin xenotransplants [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In the present study, we examined the effects of UVB irradiation on the expression of CRNN and RPTN in human skin xenotransplants to explore the role of these proteins in the epidermis.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003eClinical materials: Human skin samples were obtained from the abdominal skin of four healthy Japanese volunteers with informed consent. All of them had type III skin. Skin samples were transplanted onto the backs of nude mice, KSN/Slc mice (Nippon SLC, Hamamatsu, Japan). Four mice in each experimental group (eight mice in total) were used for the study. This study was performed in compliance with the Principles of the Declaration of Helsinki and was approved by the Medical Ethics Committee of the University of Toyama.\u003c/p\u003e \u003cp\u003eUltraviolet irradiation: The UVB light source was a fluorescent lamp (GL40SE; Sankyo Denki Co, Kanagawa, Japan) that emitted 0.1 mW/cm\u003csup\u003e2\u003c/sup\u003e of UV light between 280 and 315 nm (peak: 306 nm) at a distance of 40 cm, as measured by a UV radiometer (EKO Instruments Co., Tokyo, Japan). Two months after transplantation, the grafted skin was exposed to 500 mJ/cm\u003csup\u003e2\u003c/sup\u003e UVB, which is the minimal dose necessary to induce pyrimidine dimers, sun-burn cells, and apoptotic keratinocytes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Skin tissues were excised two days after UVB irradiation and analyzed using immunofluorescence.\u003c/p\u003e \u003cp\u003eImmunohistochemistry: Skin specimens were directly dipped into OCT compound and rapidly frozen in liquid nitrogen. Sections were blocked with Protein Block Serum-Free (Dako, Carpinteria, CA, USA) and incubated with primary antibodies, such as anti-human CRNN antibodies (11799-1-AP, Proteintech, Curanbry, NJ, USA), anti-human RPTN antibodies (HPA030483, Atlas Antibodies AB, Bromma, Sweden), and anti-human FLG antibodies (Abcam, St. Louis, MA, USA), followed by incubation with anti-rabbit IgG Alexa Fluor 488 or anti-mouse IgG Alexa Fluor 555 (Molecular Probes, Eugene, OR, USA) secondary antibodies. Tissue sections were observed under a fluorescence microscope (Olympus IX71; Olympus Co., Tokyo, Japan) or a confocal laser microscope, LSM780 (Carl Zeiss, Oberkochen, Germany). The number of CRNN-positive or RPTN-positive keratinocytes in the epidermis was counted in each 500 \u0026micro;m field for a total of 5.0 mm length.\u003c/p\u003e \u003cp\u003eStatistical analysis: Data are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.D. Statistical significance was assesed using the Mann-Whitney U test. Statistical significance was set at P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eThe expression of CRNN in UVB-irradiated skin\u003c/h2\u003e \u003cp\u003eWe first examined the expression of CRNN in UVB-irradiated skin. CRNN was irregularly detected in the granular layer close to the corny layer of the epidermis in sham-irradiated skins \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, \u003cb\u003eleft panels)\u003c/b\u003e; however, on day 2 after irradiation, CRNN signals were observed in the upper spinous and granular layers, and were colocalized with those of FLG \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea \u003cb\u003eright panels)\u003c/b\u003e. The number of CRNN-positive keratinocytes on day 2 after irradiation was significantly higher than that in the sham-irradiated skin \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. At high magnification, although the signals of CRNN and FLG were colocalized in the cytoplasm of the granular keratinocytes in a granular pattern, CRNN signals were also observed in the peripheral region of the granular keratinocytes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eThe expression of RPTN in UVB-irradiated skin\u003c/h2\u003e \u003cp\u003eNext, we examined the expression of RPTN in UVB-irradiated skin. RPTN was rarely detected in the sham-irradiated skin; however, its expression was markedly increased in the granular keratinocytes of UVB-irradiated skin on day 2\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, \u003cb\u003eleft panels)\u003c/b\u003e. Most RPTN-positive keratinocytes colocalized with FLG-positive keratinocytes \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, \u003cb\u003eright panels)\u003c/b\u003e. The number of RPTN-positive keratinocytes on day 2 after irradiation was significantly higher than that in the sham-irradiated skin \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e. At high magnification, although the signals of RPTN were observed in a granular pattern in the cytoplasm of the granular keratinocytes, and were almost colocalized with those of FLG, RPTN-positive granules were also irregularly detected in a different distribution from those expressing FLG at two days after UVB irradiation \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study showed that the expression of CRNN and RPTN in xenotransplanted human skin was markedly increased after UVB irradiation (500 mJ/cm\u003csup\u003e2\u003c/sup\u003e). The expression of CRNN increased in parallel with that of FLG in UVB-irradiated epidermis. A previous study described the association of SNP rs941934 in CRNN with the development of atopic dermatitis (AD) and the mRNA level of CRNN was decreased in skin lesions of AD [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In addition, CRNN is known to be reduced in human oral and esophageal squamous cell carcinoma [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This suggests that CRNN may be associated with terminal differentiation or the barrier formation in keratinocytes. However, CRNN, but not FLG, was observed in the peripheral region, in addition to the cytoplasm of granular keratinocytes. Therefore, we hypothesized that CRNN may play a different role than FLG in UVB-irradiated skin. In contrast, the expression of RPTN was markedly induced by UVB irradiation, although RPTN signals were rarely detected in the sham-irradiated skin. Previous studies have demonstrated that the increased expression of RPTN was observed in Kruppel-like factor 4-null mice or loricrin-deficient mice, which are characterized by an impaired epidermal barrier function [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Furthermore, the mRNA level of RPTN is increased in skin lesions of AD, in which the barrier function of the epidermis is reduced [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. We previously reported that the expression of HRNR, another member of the S100 fused-protein family, may be induced in relation to the reconstruction of epidermal barrier dysfunction induced by UVB irradiation. Therefore, the overexpression of RPTN in UVB-irradiated skin may also be involved in compensatory mechanisms to repair the altered epidermal barrier.\u003c/p\u003e \u003cp\u003eIn conclusion, the present study demonstrated that CRNN and RPTN are novel proteins whose expression can be increased by UVB irradiation, and suggest markers of acute UV damage to the skin. Furthermore, we hypothesized that the role in the barrier formation may differ among CRNN, RPTN and FLG, according to the expression pattern of these proteins in UVB-irradiated skin. Further studies are necessary to clarify the exact mechanism underlying the role of CRNN and RPTN in UVB-irradiated skin. However, the present study will contribute to extending the knowledge of the S100 fused protein family.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eT.M. and T.S. involved in conceptualization. T.M., M.M. and K.T. involved in formal analysis. T.M. involved in funding acquisition. T.M., M.M. and K.T. involved in data analysis. T.M. and K.T. involved in visualization. T.M. and M.M. involved in writing original draft preparation. All authors involved in writing review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Mr. Kenji Matsunaga for his valuable technical support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by JSPS KAKENHI grant numbers JP21K08296 and JP18K08265 (to TM).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest in association with the present study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll patients provided written informed consent for participation in the present study, which complied with the principles of the Declaration of Helsinki. This study was approved by the Medical Ethic Committee of the University of Toyama, Toyama, Japan.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKypriotou M, Huber M, Hohl D (2012) The human epidermal differentiation complex: cornified envelope precursors, S100 proteins and the \u0026apos;fused genes\u0026apos; family. Exp Dermatol 21: 643-649. doi: 10.1111/j.1600-0625.2012.01472.x.\u003c/li\u003e\n\u003cli\u003eContzler R, Favre B, Huber M, Hohl D (2005). Cornulin, a new member of the \u0026quot;fused gene\u0026quot; family, is expressed during epidermal differentiation. J Invest Dermatol 124: 990-997. doi: 10.1111/j.0022-202X.2005.23694.x.\u003c/li\u003e\n\u003cli\u003eHuber M, Siegenthaler G, Mirancea N, Marenholz I, Nizetic D, Breitkreutz D, Mischke D, Hohl D (2005) Isolation and characterization of human repetin, a member of the fused gene family of the epidermal differentiation complex. J Invest Dermatol 24: 998-1007. doi: 10.1111/j.0022-202X.2005.23675.x.\u003c/li\u003e\n\u003cli\u003eIchihashi M, Ueda M, Budiyanto A, Bito T, Oka M, Fukunaga M, Tsuru K, Horikawa T (2003) UV-induced skin damage. Toxicology 189: 21-39. doi: 10.1016/s0300-483x(03)00150-1.\u003c/li\u003e\n\u003cli\u003eMoll I, Bohnert E, Treib U, Jung EG (1994) Effects of ultraviolet B radiation on cytoskeletal and adhesion molecules in human epidermis. Photodermatol Photoimmunol Photomed 10: 26-32.\u003c/li\u003e\n\u003cli\u003eSmith MD, Rees JL (1994) Wavelength-specific upregulation of keratin mRNA expression in response to ultraviolet radiation. J Invest Dermatol 1994; 102: 433-439. doi: 10.1111/1523-1747.ep12372958.\u003c/li\u003e\n\u003cli\u003eBernerd F, Asselineau D (1997) Successive alteration and recovery of epidermal differentiation and morphogenesis after specific UVB-damages in skin reconstructed in vitro. Dev Biol 183: 123-138. doi: 10.1006/dbio.1996.8465.\u003c/li\u003e\n\u003cli\u003eDel Bino S, Vioux C, Rossio-Pasquier P, Jomard A, Demarchez M, Asselineau D, Bernerd F (2004) Ultraviolet B induces hyperproliferation and modification of epidermal differentiation in normal human skin grafted on to nude mice. Br J Dermatol 150: 658-667. doi: 10.1111/j.0007-0963.2004.05886.x.\u003c/li\u003e\n\u003cli\u003eMakino T, Yamakoshi T, Mizawa M, Shimizu T (2014) Ultraviolet B irradiation induces the expression of hornerin in xenotransplanted human skin. Acta Histochem 116: 20-24. doi: 10.1016/j.acthis.2013.05.001.\u003c/li\u003e\n\u003cli\u003eMakino T, Mizawa M, Yoshihisa Y, Shimizu T (2019) Ultraviolet B irradiation increases the expression of trichohyalin-like 1 protein in human skin xenotransplants. Clin Exp Dermatol 44: 773-776. doi: 10.1111/ced.13904.\u003c/li\u003e\n\u003cli\u003eTrzeciak M, Sakowicz-Burkiewicz M, Wesserling M, Gleń J, Dobaczewska D, Bandurski T, Nowicki R, Pawelczyk T (2017) Altered Expression of Genes Encoding Cornulin and Repetin in Atopic Dermatitis. Int Arch Allergy Immunol 172: 11-19. doi: 10.1159/000453452.\u003c/li\u003e\n\u003cli\u003eChen K, Li Y, Dai Y, Li J, Qin Y, Zhu Y, Zeng T, Ban X, Fu L, Guan XY (2013) Characterization of tumor suppressive function of cornulin in esophageal squamous cell carcinoma. PLoS One 8: e68838. doi: 10.1371/journal.pone.0068838.\u003c/li\u003e\n\u003cli\u003eGovindaraj PK, Kallarakkal TG, Mohd Zain R, Tilakaratne WM, Lew HL (2021) Expression of Ki-67, Cornulin and ISG15 in non-involved mucosal surgical margins as predictive markers for relapse in oral squamous cell carcinoma (OSCC). PLoS One 16: e0261575. doi: 10.1371/journal.pone.0261575.\u003c/li\u003e\n\u003cli\u003eSegre JA, Bauer C, Fuchs E (1999) Klf4 is a transcription factor required for establishing the barrier function of the skin. Nat Genet 22: 356-360. doi: 10.1038/11926.\u003c/li\u003e\n\u003cli\u003eKoch PJ, de Viragh PA, Scharer E, Bundman D, Longley MA, Bickenbach J, Kawachi Y, Suga Y, Zhou Z, Huber M, Hohl D, Kartasova T, Jarnik M, Steven AC, Roop DR (2000) Lessons from loricrin-deficient mice: compensatory mechanisms maintaining skin barrier function in the absence of a major cornified envelope protein. J Cell Biol 151: 389-400. doi: 10.1083/jcb.151.2.389.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"cornulin, repetin, ultraviolet B, epidermis, skin barrier, keratinocyte","lastPublishedDoi":"10.21203/rs.3.rs-3852080/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3852080/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCornulin (CRNN) and repetin (RPTN) belong to the fused-type S100 protein family. Although these proteins have been reported to be expressed in the granular layer of the epidermis and are suggested to be associated with barrier formation in the epidermis, the exact function of these proteins remains unclear. The present study examined the effects of UVB irradiation on the CRNN and RPTN expression in human skin xenotransplantation. The expression of CRNN increased in the granular layer of UVB-irradiated skin two days after UVB irradiation in comparison to sham-irradiated skin. Interestingly, CRNN signals were observed not only in the cytoplasm but also in the peripheral region of the granular keratinocytes. In contrast, RPTN was rarely expressed in sham-irradiated skin; however, RPTN signals were markedly increased in the granular layer of UVB-irradiated skin. The number of RPTN-positive keratinocytes on day 2 after UVB irradiation was significantly higher than that in the sham-irradiated skin. Accordingly, the present study demonstrated that CRNN and RPTN are novel proteins whose expression can be increased by UVB irradiation. In addition, we speculate that the role of CRNN and RPTN in barrier formation of the epidermis may differ according to the expression pattern of these proteins in UVB-irradiated skin.\u003c/p\u003e","manuscriptTitle":"Ultraviolet B irradiation increases the expression of cornulin and retepin in human skin xenotransplants","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-12 14:07:03","doi":"10.21203/rs.3.rs-3852080/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d199e8cc-9699-4959-9f92-c25f38024e33","owner":[],"postedDate":"January 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-05-29T05:58:11+00:00","versionOfRecord":{"articleIdentity":"rs-3852080","link":"https://doi.org/10.1111/exd.15109","journal":{"identity":"experimental-dermatology","isVorOnly":true,"title":"Experimental Dermatology"},"publishedOn":"2024-05-01 05:58:11","publishedOnDateReadable":"May 1st, 2024"},"versionCreatedAt":"2024-01-12 14:07:03","video":"","vorDoi":"10.1111/exd.15109","vorDoiUrl":"https://doi.org/10.1111/exd.15109","workflowStages":[]},"version":"v1","identity":"rs-3852080","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3852080","identity":"rs-3852080","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}