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

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 have been suggested to be associated with barrier formation in the epidermis, their exact function remains unclear. This study examined the effects of ultraviolet B (UVB) irradiation on CRNN and RPTN expression in human skin xenotransplantation. The CRNN expression increased in the granular layer of UVB‐irradiated skin 2 days after UVB irradiation compared to that in sham‐irradiated skin. Interestingly, CRNN signals were observed not only in the cytoplasm, but also in the peripheral regions of granular keratinocytes. In contrast, RPTN was rarely expressed in sham‐irradiated skin; however, RPTN signals were markedly increased in the granular layer of the UVB‐irradiated skin. In addition, activation of ERK1/2 and STAT3 was observed in UVB‐irradiated skin. Accordingly, the present study demonstrated that CRNN and RPTN are novel proteins whose expression can be increased by UVB irradiation. The activation of ERK1/2 and STAT3 may be associated with the regeneration of a UVB‐damaged epidermis, and CRNN and RPTN may be induced to repair any dysfunction in the epidermal barrier during this regeneration process.

valuable tool for investigating the effects of UVB on early and late epidermal differentiation. 11We also reported that UVB irradiation affects the expression of HRNR and TCHHL1 in human skin xenotransplants. 12,13

| QUE S TIONS ADDRE SS ED
According to previous studies, CRNN and RPTN were suggested to have unique functions in barrier formation in the epidermis; however, the exact role of and mechanism underlying the expression of these proteins remains unclear.Therefore, 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 function of these proteins in the epidermis.

| Clinical materials
Human skin samples were obtained from the abdominal skin of four healthy Japanese volunteers who provided their informed consent.
All of the patients had type III skin.Skin samples were transplanted onto the backs of the KSN/Slc mice (Nippon SLC, Hamamatsu, Japan).Four mice in each experimental group (eight in total) were used for the study.
This study was performed in compliance with the Principles of the Declaration of Helsinki and approved by the Medical Ethics Committee of the University of Toyama.

| UV 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 using a UV radiometer (EKO Instruments Co., Tokyo, Japan).2][13] Skin tissues were excised 2 days after UVB irradiation and analysed by immunohistochemistry.

| Immunostaining
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.The signals were detected using ImmPRESS (Vector Laboratories; Burlingame, CA, USA), followed by staining using a Liquid DAB+ Substrate Chromogen System (Dako).For immunofluorescence observation, anti-rabbit IgG Alexa Fluor 488 or anti-mouse IgG Alexa Fluor 555 (Molecular Probes, Eugene, OR, USA) were used as secondary antibodies.After thorough washing, either anti-CRNN or RPTN antibodies labelled by FlexAble CoraLite Plus 555 Kit (proteintech, Rosemont, IL, USA) was applied for double staining.Tissue sections were observed under a fluorescence microscope (Olympus IX71; Olympus Co., Tokyo, Japan) or confocal laser microscope, LSM780 (Carl Zeiss, Oberkochen, Germany).The number of CRNN-or RPTN-positive keratinocytes in the epidermis was counted in each 500 μm field for a total length of 5.0 mm.

| Statistical analyses
Data are shown as the mean ± standard deviation, and statistical significance was assessed using the Mann-Whitney U test.Statistical significance was set at p < 0.05.

| The expression of CRNN in UVB-irradiated skin
We first examined the expression of CRNN in the UVB-irradiated skin.CRNN was irregularly detected in the granular layer close to the horny layer of the epidermis in sham-irradiated skins (Figure 1A, left panels); however, on day 2 after UVB irradiation, signals of CRNN increased (Figure 1A) and were observed in the upper spinous and granular layers (Figure 1A, right panels).The number of CRNN-positive keratinocytes on day 2 after UVB irradiation was significantly higher than that in the sham-irradiated skin (Figure 1B).FLG expression was also increased in UVB-irradiated skin compared to that in sham-irradiated skin (Figure 1E,F

| The expression of RPTN in UVB-irradiated skin
Next, we examined the expression of RPTN in UVB-irradiated skin.
RPTN was rarely detected in sham-irradiated skin; however, its expression was markedly increased in the granular keratinocytes of UVB-irradiated skin on day 2 (Figure 1C).The number of RPTN-positive keratinocytes on day 2 after UVB irradiation was significantly higher than that in the sham-irradiated skin (Figure 1D).At high magnification, RPTN signals were observed in a granular pattern in the cytoplasm of granular keratinocytes, and were almost colocalized with those of FLG.In contrast, RPTN-positive granules were irregularly detected in a different distribution from those expressing FLG in UVB-irradiated skin (Figure 2, bottom panels).

| Activation of ERK1/2 and Stat3 in UVB-irradiated skin
To clarify the underlying mechanism of CRNN and RPTN expression,

| CON CLUS I ON AND PER S PEC TIVE S
The present study showed that the expression of CRNN, RPTN and FLG markedly increased after UVB irradiation in xenotransplanted human skin.DNA damage due to UVB irradiation is known to induce epidermal hyperplasia. 14Our previous reports showed that cytokeratin 6 was strongly detected and a dramatic increase in the number of Ki67-positive cells occurred in UVB-irradiated skin. 12,13These findings suggested that UVB-irradiated epidermis is hyperproliferative, and the epidermis may enter a regenerating process after acute damage caused by UVB irradiation.Increased expression of FLG has also been reported in the regenerative epidermis. 15e present study showed a marked increase in CRNN expression in the UVB-irradiated skin.In contrast, a previous study demonstrated that the expression of CRNN decreased in the skin lesions of AD.Accordingly, CRNN expression may be induced by coordination with the FLG.Interestingly, CRNN was observed in the peripheral region in addition to the cytoplasm of granular keratinocytes.This unique distribution suggests that CRNN may play a different role from FLG, such as CE formation, in UVB-irradiated skin.
RPTN signals were rarely detected in sham-irradiated skin as reported in normal human skin. 6However, the expression of RPTN was markedly induced by UVB irradiation.Previous studies have demonstrated that RPTN expression was induced in Kruppel-like factor 4-null mice or loricrin-deficient mice, which were characterized by an impaired epidermal barrier function. 16,17Furthermore, RPTN expression increased in the lesional skin of AD, in which barrier dysfunction of the epidermis was found. 7Therefore, the overexpression of RPTN in UVB-irradiated skin may be involved in compensatory mechanisms to repair the altered epidermal barrier.
In addition, ERK1/2 and STAT3 activation was detected in the epidermis of UVB-irradiated skin.ERK1/2 is a mitogen-activated protein kinase (MAPK) 18 and is mainly associated with the cell proliferation, differentiation, and survival.A recent study suggested that activation of ERK1/2 may be associated with the pathogenesis of AD. 19 STAT3 is a transcription factor involved in cell proliferation and differentiation and the re-epithelization process lead to activation of STAT3 in keratinocytes. 20UVB irradiation was reported to activate ERK1/2 and p38 MAPK signalling pathways, which are involved in the proliferation of keratinocytes, via reactive oxygen species, 21 although activation of only ERK1/2 was observed in the present study.Furthermore, UVB induces STAT3 activation in keratinocytes through oxygen species and DNA damage. 22Accordingly, we hypothesized that the activation of ERK1/2 and STAT3 may be ).At high magnification, although the CRNN and FLG signals 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 (Figure2, top panels).
we examined the activation of MAPK signalling pathways in UVBirradiated skin.The number of phospho-ERK1/2-positive cells was markedly increased in UVB-irradiated skin compared to that in sham-irradiated skin (Figure3A,B).Phospho-ERK1/2 signals were mainly observed in the nucleus and cytoplasm of upper spinous F I G U R E 1 The expression of CRNN and RPTN in UVB-irradiated skin.(A) Staining of CRNN in sham-irradiated skin (left panel) and UVB-irradiated skin (right panel).The broken lines indicate the basement membrane.(B) The numbers of CRNN-positive cells were counted in each 500 nm field for a total length of 5.0 mm in the sham-irradiated skin and UVB-irradiated skin.The graph shows the mean values ± standard deviation.(C) Staining of RPTN in sham-irradiated skin (left panel) and UVB-irradiated skin (right panel).The broken lines indicate the basement membrane.(D) The numbers of RPTN-positive cells were counted in each 500 nm field for a total length of 5.0 mm in the sham-irradiated skin and UVB-irradiated skin.The graph shows the mean values ± standard deviation.(E) Staining of FLG in sham-irradiated skin (left panel) and UVB-irradiated skin (right panel).The broken lines indicate the basement membrane.(F) The numbers of FLG-positive cells were counted in each 500 nm field for a total length of 5.0 mm in the sham-irradiated skin and UVB-irradiated skin.The graph shows the mean values ± standard deviation.and granular keratinocytes (Figure 3B).Phosphorylation of SAPK/ JNK and p38 MAPK was not observed in the UVB-irradiated skin (Figure 3C-F).Furthermore, we examined the activation of STAT3 in the UVB-irradiated skin.The number of phospho-STAT3-positive cells markedly increased 2 days after irradiation compared to that in the sham-irradiated skin (Figure 3G,H).The most signals of phosphor-STAT3 were detected in the nucleus of the spinous and granular keratinocytes (Figure 3H).Double immunostaing revealed that most signals of CRNN and RPTN were observed in cells close to but different from phospho-ERK1/2-or phospho-STAT3-positive cells (Figure S1).
associated with epidermal regeneration after the occurrence of damage due to UVB irradiation.During this regeneration process, CRNN and RPTN may be induced to repair the dysfunction of the epidermal barrier because most signals of CRNN and RPTN were observed in cells close to but different from ERK1/2-or STAT3-activated cells.In conclusion, the present study demonstrated that CRNN and RPTN are novel proteins whose expression can be increased by UVB irradiation and are suggested to be markers of acute UV damage to the skin.In addition, we hypothesized that the role of barrier formation may differ between CRNN and RPTN, according F I G U R E 2 The distribution of CRNN and RPTN in UVB-irradiated skin.Double staining of CRNN and FLG (top panels) and RPTN and FLG (bottom panels) in the granular layers of UVB-irradiated skin using a confocal laser microscope (LSM780).DNA staining by 4′, 6-diamidine-2′phenylindole dihydrochloride is shown in blue.to the expression pattern of these proteins in UVB-irradiated skin.As one limitation associated with this study, the results of the present study did not demonstrate any causal relationship between the expression of CRNN and RPTN and the activation of ERK1/2 and STAT3.Further studies are necessary to clarify the exact mechanisms underlying the role of CRNN and RPTN in UVB-irradiated skin.However, the present study will help expand our knowledge of the S100 fused protein family.AUTH O R CO NTR I B UTI O N S T.M. and T.S. were involved in the conceptualization.T.M., M.M., and K.T. were involved in the formal analysis.T.M. was involved in funding acquisition.T.M., M.M., and K.T. were involved in the data analysis.T.M. and K.T. were involved in visualization.T.M. and M.M. wrote the original draft.All of the authors were involved in writing review and editing.