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Actinic lentigines from Japanese and European volunteers share similar impaired biological functions

Open AccessPublished:July 02, 2022DOI:https://doi.org/10.1016/j.jdermsci.2022.07.001

      Abstract

      Background

      Hyperpigmented spots develop earlier and with a higher incidence in Asian individuals compared with Europeans. Although actinic lentigines (AL) are very common, the biological events underlying their formation remain ill-defined.

      Objective

      AL from Japanese volunteers were characterized through morphological and gene expression analyses. Data were then compared with published data on European volunteers.

      Methods

      AL on hands were selected through dermoscopic imaging and pattern scoring in Japanese women. Skin biopsies of AL and adjacent non-lesional (NL) skin were processed for histology and gene expression profiling. Japanese and European studies were compared after harmonizing the data using the same mathematical and statistical methods.

      Results

      Histologically, AL from Japanese individuals revealed deep epidermal invaginations with melanin accumulation in the depth of epidermal rete ridges. Transcriptomic data identified 245 genes differentially expressed in AL versus NL skin samples, associated with the different skin compartments and multiple functional families and biological processes, such as epidermal homeostasis, extracellular matrix organization and ion binding/transmembrane transport. Strikingly, melanogenesis-related genes were not significantly modulated in AL compared with NL skin.
      Comparison of the molecular profiles of Japanese and European AL showed that a huge majority of genes were modulated in the same way, recapitulating the overall biological alterations.

      Conclusion

      AL from Japanese volunteers exhibited morphological and molecular alterations of the whole skin structure with impairment of multiple biological functions similar to that found in European women. These findings will contribute to the development of efficient treatments of AL lesions.

      Keywords

      Abbreviations:

      AL (actinic lentigines), NL (non-lesional), DEJ (dermal-epidermal junction), ECM (extracellular matrix)

      1. Introduction

      Actinic lentigines (AL) are very common hyperpigmented lesions. They develop on chronically sun-exposed skin usually after 35–40 years of age and represent a visible sign of skin ageing [
      • Bastiaens M.
      • Hoefnagel J.
      • Westendorp R.
      • Vermeer B.J.
      • Bouwes J.N.
      Bavinck, Solar lentigines are strongly related to sun exposure in contrast to ephelides.
      ]. AL correspond to brown dark spots indicating a global increase in melanin [
      • Warrick E.
      • Duval C.
      • Nouveau S.
      • Bastien P.
      • Piffaut V.
      • Chalmond B.
      • et al.
      Morphological and molecular characterization of actinic lentigos reveals alterations of the dermal extracellular matrix.
      ,
      • Barysch M.J.
      • Braun R.P.
      • Kolm I.
      • Ahlgrimm-Siesz V.
      • Hofmann-Wellenhof R.
      • Duval C.
      • et al.
      Keratinocytic malfunction as a trigger for the development of solar lentigines.
      ,
      • Andersen W.K.
      • Labadie R.R.
      • Bhawan J.
      Histopathology of solar lentigines of the face: a quantitative study.
      ,
      • Choi W.
      • Yin L.
      • Smuda C.
      • Batzer J.
      • Hearing V.J.
      • Kolbe L.
      Molecular and histological characterization of age spots.
      ]. The presence of more or less pronounced elongated rete ridges is a histological hallmark of AL. They can take the form of characteristic club-shaped extensions into the dermis [
      • Montagna W.
      • Hu F.
      • Carlisle K.
      A reinvestigation of solar lentigines.
      ,
      • Cario-Andre M.
      • Lepreux S.
      • Pain C.
      • Nizard C.
      • Noblesse E.
      • Taieb A.
      Perilesional vs. lesional skin changes in senile lentigo.
      ] and the degree of deformation of the dermal-epidermal junction (DEJ) may be an indicator of the lesion severity from “early” to “late” stages [
      • Cario-Andre M.
      • Lepreux S.
      • Pain C.
      • Nizard C.
      • Noblesse E.
      • Taieb A.
      Perilesional vs. lesional skin changes in senile lentigo.
      ,
      • Lin C.B.
      • Hu Y.
      • Rossetti D.
      • Chen N.
      • David C.
      • Slominski A.
      • et al.
      Immuno-histochemical evaluation of solar lentigines: The association of KGF/KGFR and other factors with lesion development.
      ]. Although very common, the biological mechanisms underlying AL formation remain unclear. The modulation of several molecular pathways has been reported in AL, such as KGF/KGFR [
      • Lin C.B.
      • Hu Y.
      • Rossetti D.
      • Chen N.
      • David C.
      • Slominski A.
      • et al.
      Immuno-histochemical evaluation of solar lentigines: The association of KGF/KGFR and other factors with lesion development.
      ,
      • Chen N.
      • Hu Y.
      • Li W.H.
      • Eisinger M.
      • Seiberg M.
      • Lin C.B.
      The role of keratinocyte growth factor in melanogenesis: a possible mechanism for the initiation of solar lentigines.
      ,
      • Kovacs D.
      • Cardinali G.
      • Aspite N.
      • Cota C.
      • Luzi F.
      • Bellei B.
      • et al.
      Role of fibroblast-derived growth factors in regulating hyperpigmentation of solar lentigo.
      ], SCF/c-KIT [
      • Hattori H.
      • Kawashima M.
      • Ichikawa Y.
      • Imokawa G.
      The epidermal stem cell factor is over-expressed in lentigo senilis: implication for the mechanism of hyperpigmentation.
      ], ET-1/ETBR [
      • Kadono S.
      • Manaka I.
      • Kawashima M.
      • Kobayashi T.
      • Imokawa G.
      The role of the epidermal endothelin cascade in the hyperpigmentation mechanism of lentigo senilis.
      ], the upregulation of genes involved in inflammation and fatty-acid metabolism, and the downregulation of keratinization-related genes [
      • Aoki H.
      • Moro O.
      • Tagami H.
      • Kishimoto J.
      Gene expression profiling analysis of solar lentigo in relation to immunohistochemical characteristics.
      ,
      • Goyarts E.
      • Muizzuddin N.
      • Maes D.
      • Giacomoni P.U.
      Morphological changes associated with aging: age spots and the microinflammatory model of skin aging.
      ]. However, Lin et al. [
      • Lin C.B.
      • Hu Y.
      • Rossetti D.
      • Chen N.
      • David C.
      • Slominski A.
      • et al.
      Immuno-histochemical evaluation of solar lentigines: The association of KGF/KGFR and other factors with lesion development.
      ] suggested that the molecular alterations in AL seem to be related to the lesion grade and to evolve through its progression.
      In a previous study, AL from European women displaying an elongated dermoscopy pattern were selected as advanced lesions. Increased melanin content and drastic disorganization of the whole cutaneous structure confirmed the grade of the selected lesions [
      • Warrick E.
      • Duval C.
      • Nouveau S.
      • Bastien P.
      • Piffaut V.
      • Chalmond B.
      • et al.
      Morphological and molecular characterization of actinic lentigos reveals alterations of the dermal extracellular matrix.
      ]. Transcriptomic analysis of lesional skin versus non-lesional skin revealed that most of the functional alterations were not related to melanogenesis but rather to various biological functions, such as epidermal homeostasis, inflammation, ion channels and transport and interestingly to the modulation of genes linked to extracellular matrix (ECM) and DEJ. Local modifications of the dermal structure were confirmed at the protein level especially in the sub-epidermal zone [
      • Warrick E.
      • Duval C.
      • Nouveau S.
      • Bastien P.
      • Piffaut V.
      • Chalmond B.
      • et al.
      Morphological and molecular characterization of actinic lentigos reveals alterations of the dermal extracellular matrix.
      ] thus reinforcing the role of epidermal-dermal crosstalk in such lesions [
      • Kovacs D.
      • Cardinali G.
      • Aspite N.
      • Cota C.
      • Luzi F.
      • Bellei B.
      • et al.
      Role of fibroblast-derived growth factors in regulating hyperpigmentation of solar lentigo.
      ,
      • Iriyama S.
      • Ono T.
      • Aoki H.
      • Amano S.
      Hyperpigmentation in human solar lentigo is promoted by heparanase-induced loss of heparan sulfate chains at the dermal-epidermal junction.
      ].
      AL lesions are observed in all skin colour types, but the onset, frequency or severity highly depends on the studied population, especially with respect to constitutive pigmentation and ancestry [
      • Alexis A.F.
      • Obioha J.O.
      Ethnicity and aging skin.
      ]. In particular, the prevalence of pigmented spots is a more prominent and earlier sign of skin ageing in Asian populations than in Europeans [
      • Goh S.H.
      The treatment of visible signs of senescence: the Asian experience.
      ,
      • Chua-Ty G.
      • Goh C.L.
      • Koh S.L.
      Pattern of skin diseases at the National Skin Centre (Singapore) from 1989-1990.
      ,
      • Vierkotter A.
      • Kramer U.
      • Sugiri D.
      • Morita A.
      • Yamamoto A.
      • Kaneko N.
      • et al.
      Development of lentigines in German and Japanese women correlates with variants in the SLC45A2 gene.
      ]. These differences cannot only be related to the constitutive pigmentation since Asian populations from Northern East Asian countries, such as Japan and China, correspond to Light and Intermediate groups (ITA° skin colour classification), close to the European population [
      • Del Bino S.
      • Bernerd F.
      Variations in skin colour and the biological consequences of ultraviolet radiation exposure.
      ]. Search for genetic predisposition for lentigines in Japanese and German cohorts revealed that 12 among 25 single-nucleotide polymorphisms (SNPs) relevant to melanin synthesis were differentially distributed between the 2 populations but only 1 variant of SLC24A5 gene significantly correlated with the occurrence of lentigines [
      • Vierkotter A.
      • Kramer U.
      • Sugiri D.
      • Morita A.
      • Yamamoto A.
      • Kaneko N.
      • et al.
      Development of lentigines in German and Japanese women correlates with variants in the SLC45A2 gene.
      ].
      For Asian population, there is a high demand for an effective AL treatment explaining the increasing practice of aesthetic procedures, especially laser treatments. The risk of developing post-inflammatory hyperpigmentation (PIH) after such invasive treatments, however, is high (20–30%) [
      • Negishi K.
      • Akita H.
      • Tanaka S.
      • Yokoyama Y.
      • Wakamatsu S.
      • Matsunaga K.
      Comparative study of treatment efficacy and the incidence of post-inflammatory hyperpigmentation with different degrees of irradiation using two different quality-switched lasers for removing solar lentigines on Asian skin.
      ]. Therefore, a better understanding of the physiopathological features of AL in Asian individuals is needed to propose effective and safe topical treatments.
      To identify the potential specific features of AL lesions in Asian compared with European individuals, a study was conducted in Japanese women. A homogeneous group of advanced AL lesions was selected using epiluminescence imaging and pigmented pattern scoring, as previously described [
      • Warrick E.
      • Duval C.
      • Nouveau S.
      • Bastien P.
      • Piffaut V.
      • Chalmond B.
      • et al.
      Morphological and molecular characterization of actinic lentigos reveals alterations of the dermal extracellular matrix.
      ]. Morphological analysis and gene expression profiling were performed on AL compared with adjacent non-lesional (NL) skin. Results were then compared to previous data obtained from AL lesions of European volunteers.

      2. Material and methods

      2.1 Japanese study design

      A single-centre, open, randomised prospective clinical study was performed with 20 Japanese women, aged 54–71, phototype III-IV. Volunteers provided written informed consent. The protocol complied with the Helsinki declaration and was approved by the local ethics committee (Study N°503 approved by Nagoya City University of Medicine).

      2.1.1 AL selection

      AL from the dorsal side of the hands were selected through dermoscopic imaging (X70 magnification, Fotofinder dermoscope®, Teachscreen, Germany) and pigmented pattern scoring using the previously described methodology and selection criteria [
      • Warrick E.
      • Duval C.
      • Nouveau S.
      • Bastien P.
      • Piffaut V.
      • Chalmond B.
      • et al.
      Morphological and molecular characterization of actinic lentigos reveals alterations of the dermal extracellular matrix.
      ].

      2.1.2 Processing of biopsies

      Pairs of 3-mm biopsies (AL lesion and adjacent NL skin) were obtained from each volunteer. One set of biopsies (8 volunteers) was processed for histology, Fontana-Masson staining and microphthalmia transcription factor (MITF) immunostaining. The other set (12 volunteers) was used for gene expression profiling using Affymetrix® U133A 2.0 chips (Affymetrix, USA).

      2.1.3 Bioinformatics and statistical analysis

      Raw data was normalized using the Robust Multichip Average method. Unsupervised analysis and clustering were performed using a Non-negative Matrix factorization (NMF) approach, and a supervised differential analysis was performed using a T-test for non-paired data. After fold-change and P-value cut-offs were applied (mean fold change between AL and NL ≥ 1.5 for up-regulated genes or ≤ −1.5 for down-regulated genes, adjusted P-value<0.05), results from both approaches were pooled. Raw expression data have been deposited in NCBI's Gene Expression Omnibus (GEO) database (accession number GSE192565).

      2.2 European study

      Fifteen European women aged 51–67 years, phototype II-III were included after providing written informed consent. The protocol complied with the Helsinki declaration and was approved by the local ethics committee (Comité de Protection des Personnes Sud Méditerranée V, ID 2007-A00175–48). Selection of AL lesions and processing of AL and NL biopsies for transcriptomic analysis are detailed in Warrick et al. [
      • Warrick E.
      • Duval C.
      • Nouveau S.
      • Bastien P.
      • Piffaut V.
      • Chalmond B.
      • et al.
      Morphological and molecular characterization of actinic lentigos reveals alterations of the dermal extracellular matrix.
      ]. Transcriptomic raw data were re-analysed using the same software and methodology as described in this paper, to make the comparison relevant. Raw expression data have been deposited in NCBI's Gene Expression Omnibus (GEO) database (accession number GSE192564).
      Details concerning skin biopsies, morphometric analysis, immunostainings, microarrays processing, bioinformatics and statistical analysis are provided in Appendix A (Supplementary Materials and Methods).

      3. Results and discussion

      3.1 Morphological analysis of AL from Japanese volunteers

      Histological analysis was performed on AL selected with defined dermoscopic criteria using dedicated software (area occupied by elongated patterns over 20%, see Materials and Methods and Appendix A). Compared with adjacent NL skin, AL sections showed a drastic deformation of the DEJ with deeply clubbed and budding epidermal invaginations into the dermis (Fig. 1a). The undulation index was significantly higher in AL versus NL samples confirming the high degree of DEJ deformation in AL (Fig. 1b). Dermoscopic pictures, scoring and histological illustrations for all volunteers are presented in Appendix B, Fig. B.1. Increased melanin accumulation was observed and quantified in the epidermis of AL, particularly along the basal layer and deep in epidermal rete ridges (Fig. 1c-d). MITF-immunostaining showed a correct distribution of melanocytes in the basal layer and the absence of clusters in AL and NL sections (Fig. 1e). Although the number of melanocytes per unit length of the stratum corneum was increased in AL compared with NL skin, the number of melanocytes per unit of basal layer length was constant, confirming a physiological distribution of melanocytes along the DEJ in AL (Fig. 1f). These drastic morphological alterations in AL have been associated with later stage lesions [
      • Choi W.
      • Yin L.
      • Smuda C.
      • Batzer J.
      • Hearing V.J.
      • Kolbe L.
      Molecular and histological characterization of age spots.
      ,
      • Cario-Andre M.
      • Lepreux S.
      • Pain C.
      • Nizard C.
      • Noblesse E.
      • Taieb A.
      Perilesional vs. lesional skin changes in senile lentigo.
      ,
      • Lin C.B.
      • Hu Y.
      • Rossetti D.
      • Chen N.
      • David C.
      • Slominski A.
      • et al.
      Immuno-histochemical evaluation of solar lentigines: The association of KGF/KGFR and other factors with lesion development.
      ,
      • Aoki H.
      • Moro O.
      • Tagami H.
      • Kishimoto J.
      Gene expression profiling analysis of solar lentigo in relation to immunohistochemical characteristics.
      ,
      • Noblesse E.
      • Nizard C.
      • M C.-A.
      • Lepreux S.
      • Pain C.
      • Schnebert S.
      • et al.
      Skin Ultrastructure in Senile Lentigo.
      ]. Like in our previous study on AL from European volunteers selected with the same dermoscopic criteria [
      • Warrick E.
      • Duval C.
      • Nouveau S.
      • Bastien P.
      • Piffaut V.
      • Chalmond B.
      • et al.
      Morphological and molecular characterization of actinic lentigos reveals alterations of the dermal extracellular matrix.
      ], the severe alterations with profound DEJ deformation observed in all Japanese lesions confirmed a correlation between the score of elongated dermoscopic pattern and later stage–associated histological defects. These observations strengthen (i) the robustness of the methodology to select homogenous AL lesions and (ii) the presence of similar alterations independently of the geographical origin.
      Fig. 1
      Fig. 1Morphological analysis of actinic lentigines (AL) and adjacent non-lesional skin (NL) of Japanese volunteers. (a) Representative histological features (HES staining) of NL and AL for 2 subjects (J4, J8) show the prominent deformation of the dermal epidermal junction and the presence of epidermal invaginations into the superficial dermis. (b) The undulation index (ratio between the length of the basal layer and the length of the stratum corneum) measured on 10 HES-stained sections per biopsy for each subject (n = 8) was significantly higher in AL versus control NL skin. (c) Fontana-Masson staining of AL and NL skin from the same 2 subjects illustrates the accumulation of melanin in the basal layer of the epidermis. (d) Quantification of the melanin content by image analysis in 10 FM-stained sections per biopsy for each subject (n = 8) indicates significantly increased basal melanin levels in AL versus NL. (e) Representative immunostaining of microphtalmia transcription factor (MITF) of NL and AL for the same 2 subjects show a regular positioning of melanocytes (red) along the basal layer. (f) Quantification of MITF-positive cells on sections from AL and NL (8 subjects). An increase in the number of melanocytes per unit of stratum corneum (SC) length is observed but no significant modulation of the number of melanocytes per unit of length of basal layer. This shows even distribution of melanocytes along the epidermal basal layer. The P-value is indicated when a statistical difference is observed between AL and NL groups using Wilcoxon signed rank test (P < 0,05). N.s. non-significant. (a), (c), (e): magnification X200.

      3.2 Transcriptomic analysis of AL from Japanese volunteers

      Unsupervised clustering of transcriptomic data using NMF methodology [
      • Fogel P.
      • Young S.S.
      • Hawkins D.M.
      • Ledirac N.
      Inferential, robust non-negative matrix factorization analysis of microarray data.
      ] displayed a good separation between Japanese AL and NL biopsies (P-value<10–4), thus confirming the specific molecular signature of AL (Appendix B, Fig. B.2). After application of a fold-change (FC) cut-off and the addition of probesets found differentially modulated in a complementary supervised analysis (see Appendix A), 332 probesets were found differentially expressed in AL compared with NL skin, corresponding to 245 genes, 119 upregulated (mean FC ≥ 1,5) and 126 downregulated (mean FC ≤ −1,5).
      A gene ontology study was performed using GOTM (http://bioinfo.vanderbilt.edu/gotm/) on the 332 probesets (Appendix C, Table C.1). The main biological functions were related to development and morphogenesis and linked to epidermal biology. The main cellular component corresponded to “extracellular region”, specifically ECM and basement membrane.
      Genes were classified into functional families using a targeted bibliographic analysis focused on skin biology (Fig. 2). The main represented functions were linked to Development & Morphogenesis, Epidermis, Extracellular matrix & DEJ, Transmembrane transport & Channels, Inflammation & immunity, Intercellular transport & Cytoskeleton and Metabolism.
      Fig. 2
      Fig. 2Functional families associated with the 245 genes differentiating AL from NL skin in Japanese volunteers. Biological functions associated with the 119 upregulated genes (a), and the 126 downregulated genes (b) in AL versus NL skin are shown. The number of genes related to the total number of modulated genes (%) in each family is indicated.
      Global gene expression analysis thus revealed a clear molecular signature differentiating AL from NL skin. The diversity of GO terms and functional families is consistent with the overall disorganization of the cutaneous structure in AL.

      3.3 Analysis of pigmentation-related genes in AL from Japanese volunteers

      Because AL are hyperpigmented disorders, a specific analysis was performed for pigmentation-related genes. Strikingly, none was significantly modulated in AL compared with NL skin (Appendix C, Table C.2). This result is in line with our previous report [
      • Warrick E.
      • Duval C.
      • Nouveau S.
      • Bastien P.
      • Piffaut V.
      • Chalmond B.
      • et al.
      Morphological and molecular characterization of actinic lentigos reveals alterations of the dermal extracellular matrix.
      ] although other studies report the up-regulation of melanogenesis-related genes in AL [
      • Aoki H.
      • Moro O.
      • Tagami H.
      • Kishimoto J.
      Gene expression profiling analysis of solar lentigo in relation to immunohistochemical characteristics.
      ,
      • Motokawa T.
      • Kato T.
      • Katagiri T.
      • Matsunaga J.
      • Takeuchi I.
      • Tomita Y.
      • et al.
      Messenger RNA levels of melanogenesis-associated genes in lentigo senilis lesions.
      ]. This suggests that melanocyte alterations cannot be considered mandatory in AL, but it cannot exclude the stimulation of melanogenesis earlier in lesion development. A slight overexpression of the KITLG gene was observed. KITLG/SCF, the major ligand for c-KIT receptor, is implicated in the regulation of pigmentation [
      • Yamaguchi Y.
      • Brenner M.
      • Hearing V.J.
      The regulation of skin pigmentation.
      ] but also in the inflammatory response and mast cells regulation [
      • Reber L.
      • Da Silva C.A.
      • Frossard N.
      Stem cell factor and its receptor c-Kit as targets for inflammatory diseases.
      ]. Overexpression of SCF protein and mRNA has been shown in the epidermis of lentigo senilis [
      • Imokawa G.
      Autocrine and paracrine regulation of melanocytes in human skin and in pigmentary disorders.
      ]. Besides, the slight downregulation of pro-opiomelanocortin (POMC) gene, the precursor of the propigmenting α-melanocyte-stimulating hormone (α-MSH) [
      • Yamaguchi Y.
      • Brenner M.
      • Hearing V.J.
      The regulation of skin pigmentation.
      ], suggests that there is no overproduction of α-MSH, at least in such advanced lesions.
      Together with a normal melanocyte density at the DEJ, these data highlight the absence of drastic alterations in the melanogenesis program.

      3.4 Comparison of Japanese and European AL molecular signatures

      Due to the huge similarities between data obtained for AL in Japanese volunteers and previous data generated in European subjects [
      • Warrick E.
      • Duval C.
      • Nouveau S.
      • Bastien P.
      • Piffaut V.
      • Chalmond B.
      • et al.
      Morphological and molecular characterization of actinic lentigos reveals alterations of the dermal extracellular matrix.
      ], a comparative analysis of gene expression profiles was performed. In the time interval between the 2 studies, methods and software were upgraded and became more robust, allowing for better discrimination of genes differentially expressed in AL and NL conditions. Therefore, a rigorous comparison required a re-analysis of European data using the same statistical methods as used for the Japanese study (see Appendix A). A list of 266 probesets corresponding to 196 genes differentially expressed in AL versus NL condition was thus established for the European study. Eighty per cent of the probesets in the new list were included in the first analysis and recapitulated all the functional dysregulations previously described [
      • Warrick E.
      • Duval C.
      • Nouveau S.
      • Bastien P.
      • Piffaut V.
      • Chalmond B.
      • et al.
      Morphological and molecular characterization of actinic lentigos reveals alterations of the dermal extracellular matrix.
      ].

      3.5 Common Japanese and European AL molecular signature

      Comparison of probesets with a P-value< 0.05 in at least 1 study showed that more than 90% of the probesets were modulated in the same way in European and Japanese AL (Fig. 3a). Comparison revealed that 178 probesets, corresponding to 136 genes, were significantly modulated in the same way in AL in both studies with an absolute mean FC ≥ 1.5, representing a strong signature of AL lesions, regardless of the population studied.
      Fig. 3
      Fig. 3Comparison of gene expression profiles and functional families in AL versus NL skin in Japanese and European studies. (a) Comparison of lists of probesets with a P-value< 0.05 in at least 1 study using TIBCO Spotfire (TIBCO Software, Palo Alto, USA). Fold-change values in the European study and in the Japanese study are represented on the vertical and horizontal axes, respectively. This global comparison shows that more than 90% of all probesets were modulated in the same way in European and Japanese AL. FC Eu: Fold-change European study; FC Jp: Fold-change Japanese study. Green: common probesets significantly modulated in both studies with mean FC value ≥ 1,5 (for up-regulated genes) or ≤ −1,5 (for down regulated genes) and an adjusted P-value< 0.05; Blue: probesets significantly modulated in the European study only; Red: probesets significantly modulated in the Japanese study only. ns: not significantly modulated. (b) Comparison of biological functions associated with genes differentiating AL from NL skin in European (n = 196) and Japanese (n = 245) volunteers with the biological functions associated with common genes, modulated in both studies (n = 245). Note that the same functional families are similarly represented in European and Japanese AL, and that the common signature recapitulates this biological profile.
      Because most other probesets were also modulated in the same way in both populations, probesets with an absolute mean FC ≥ 1.5 in one study and between 1.25 and 1,5 in the other (P-value<0.05) were also added to the common AL signature leading to a list of 245 common genes. Only 15 and 22 genes could then be considered as being specifically modulated in AL from European and Japanese volunteers, respectively.
      The common genes differentially expressed in both studies were distributed in functional families, and no specific unexpected function was highlighted, reproducing the signature already found in the 2 separate studies (Fig. 3b and Appendix B, Fig. B.3).
      We then focused the analysis on substantial functions, namely those related to “Development & morphogenesis/ Epidermis proliferation/differentiation”, “Transmembrane transport & Channels” and “Extracellular matrix & DEJ” (Table 1).
      Table 1Genes associated with development and morphogenesis, epidermal proliferation and differentiation, transmembrane transport and ion channels, extracellular matrix and dermal epidermal junction (DEJ), found to be significantly modulated in AL versus NL skin. Common genes from Japanese and European studies are represented here. FC: fold-change.
      FunctionGene symbolNameFC JapanFC Europe
      Development &PITX2paired-like homeodomain 25,44,42
      MorphogenesisHOXD11homeobox D115,063,61
      ZIC2Zic family member 2 (odd-paired homolog. Drosophila)4,615,13
      HOXD10homeobox D103,923,67
      HOXB7homeobox B73,132,41
      HOXD8homeobox D82,972,44
      PAX6paired box 62,672,09
      DLX1distal-less homeobox 12,522,29
      DLX2distal-less homeobox 22,161,91
      FOXF2forkhead box F21,921,69
      PLAG1pleiomorphic adenoma gene 11,912,01
      WNT3wingless-type MMTV integration site family, member 31,781,4
      ODZ2odz, odd Oz/ten-m homolog 2 (Drosophila)1,71,28
      FOXP2forkhead box P21,621,54
      ASCL2achaete-scute complex homolog 2 (Drosophila)1,621,4
      SOX6SRY (sex determining region Y)-box 61,551,54
      EN2engrailed homeobox 21,531,7
      RSPO3R-spondin 3 homolog (Xenopus laevis)1,481,6
      PITX1paired-like homeodomain 11,341,54
      NHLH2nescient helix loop helix 21,321,57
      CITED2Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2-1,25-1,6
      MYBv-myb myeloblastosis viral oncogene homolog (avian)-1,5-1,27
      ETV7ets variant 7-1,51-1,46
      FRYfurry homolog (Drosophila)-1,51-1,38
      CSRP2cysteine and glycine-rich protein 2-1,61-1,62
      DLX5distal-less homeobox 5-1,64-1,5
      TRPS1trichorhinophalangeal syndrome I-1,66-1,62
      PTPRDprotein tyrosine phosphatase. receptor type. D-2-1,85
      SP8Sp8 transcription factor-3,47-3,27
      Epidermis (differenciation,PI3peptidase inhibitor 3. skin-derived2,642,31
      proliferation)KRT15keratin 151,731,97
      CCND2cyclin D21,631,32
      KRT16keratin 161,441,52
      HALhistidine ammonia-lyase-1,31-1,56
      A2ML1alpha-2-macroglobulin-like 1-1,42-1,59
      BLMHbleomycin hydrolase-1,49-1,56
      LCE1Elate cornified envelope 1E-1,51-1,54
      CRNNcornulin-1,96-1,38
      BTCbetacellulin-2,58-2,01
      Transmembrane transport & ion channelsSLC1A3solute carrier family 1 (glial high affinity glutamate transporter). member 31,852,26
      CLIC2chloride intracellular channel 21,841,44
      KCNS1potassium voltage-gated channel. delayed-rectifier. subfamily S. member 11,711,66
      SLC39A14solute carrier family 39 (zinc transporter). member 141,551,7
      KCNG3potassium voltage-gated channel subfamily G member 31,51,28
      KCNJ2potassium inwardly-rectifying channel, subfamily J, member 21,451,54
      ABCG2ATP-binding cassette, sub-family G (WHITE), member 21,451,5
      SLC46A2solute carrier family 46, member 2-1,46-1,53
      KCTD4potassium channel tetramerisation domain containing 4-1,47-1,52
      AQP9aquaporin 9-1,51-1,38
      TMEM170Btransmembrane protein 170B-1,58-1,44
      CLCN4chloride channel 4-1,6-1,43
      CLCA4chloride channel accessory 4-1,68-1,72
      SLC45A4solute carrier family 45. member 4-1,68-1,74
      CACNA2D1calcium channel. voltage-dependent. alpha 2/delta subunit 1-1,76-1,89
      TMEM99transmembrane protein 99-1,76-1,62
      TUSC3tumour suppressor candidate 3-1,79-1,61
      SLC7A11solute carrier family 7. (cationic amino acid transporter. y + system) member 11-1,8-1,64
      ATP13A4ATPase type 13A4-1,81-1,6
      CHRNA9cholinergic receptor. nicotinic. alpha 9-1,85-1,65
      SLC6A14solute carrier family 6 (amino acid transporter). member 14-1,88-1,52
      SLC16A14solute carrier family 16. member 14 (monocarboxylic acid transporter 14)-1,99-1,63
      KCNMB4potassium large conductance calcium-activated channel. subfamily M. beta member 4-2,12-1,82
      SLC8A1solute carrier family 8 (sodium/calcium exchanger). member 1-2,15-2,07
      TRAM1L1translocation associated membrane protein 1-like 1-2,65-2,31
      SLC47A2solute carrier family 47. member 2-2,83-2,65
      TMEM20transmembrane protein 20-3,05-2,15
      SLC1A6solute carrier family 1 (high affinity aspartate/glutamate transporter). member 6-3,3-3,39
      ExtracellularFRAS1Fraser syndrome 13,482,79
      matrix & DEJPI3peptidase inhibitor 3, skin-derived2,642,31
      COL4A6collagen. type IV. alpha 61,891,83
      HMCN1hemicentin 11,831,42
      LEPREL1leprecan-like 11,751,79
      PAPLNPapilin, proteoglycan-like sulfated glycoprotein1,741,89
      LYZlysozyme1,721,53
      THBS2thrombospondin 21,671,65
      COL4A5collagen, type IV, alpha 51,641,43
      COL17A1collagen, type XVII, alpha 11,551,31
      EFEMP1EGF-containing fibulin-like extracellular matrix protein 11,531,6
      ADAMTSL3ADAMTS-like 31,521,71
      BMP2bone morphogenetic protein 21,521,4
      LAMB3laminin, beta 31,51,35
      PLAUplasminogen activator. urokinase1,471,56
      ASPNasporin1,41,66
      HS3ST6heparan sulfate (glucosamine) 3-O-sulfotransferase 6-1,5-1,45
      SPINK7serine peptidase inhibitor. Kazal type 7 (putative)-1,63-1,79
      ADAMTS3ADAM metallopeptidase with thrombospondin type 1 motif, 3-1,73-1,45
      PLOD2procollagen-lysine. 2-oxoglutarate 5-dioxygenase 2-1,89-1,72
      Modulations of representative genes from each functional family were validated using quantitative PCR analysis (qPCR) (Appendix A Supplementary Material and Methods, Appendix C Table C.3). For each volunteer, a strong correlation was found between qPCR and microarray results. Moreover, the clear consistency of results in both Japan and Europe studies confirmed the robustness of the clinical criteria used to select a homogeneous set of AL lesions.

      3.6 Genes associated with development & morphogenesis/ epidermis proliferation/differentiation modulated in Japanese and European AL

      The genes involved in development and morphogenesis and significantly modulated in AL in both studies are listed in Table 1. They may be related to complete disorganization of the global cutaneous structure observed in AL, in line with a new gene expression program driven by transcription factors involved in morphogenesis and tissue patterning such as Hox family members [
      • Hombria J.C.
      • Lovegrove B.
      Beyond homeosis--HOX function in morphogenesis and organogenesis.
      ]. Several HOX and related DLX genes which are regulators of normal skin development [
      • Stelnicki E.J.
      • Komuves L.G.
      • Kwong A.O.
      • Holmes D.
      • Klein P.
      • Rozenfeld S.
      • et al.
      HOX homeobox genes exhibit spatial and temporal changes in expression during human skin development.
      ] were upregulated. When expressed in adult cells those genes are thought to ensure a maintenance of the normal phenotype. Due to their role as master transcriptional regulators, modulation in their expression may be associated with phenotypic changes as shown in various cancers during oncogenic transformation. Unfortunately, the cutaneous function of the HOX genes modulated in this study is not known, except for HOXB7 which is implicated in squamous cell carcinoma invasion [
      • Gao D.
      • Chen H.Q.
      Specific knockdown of HOXB7 inhibits cutaneous squamous cell carcinoma cell migration and invasion while inducing apoptosis via the Wnt/beta-catenin signaling pathway.
      ]. Generally HOX proteins (except HOXB13) are considered to be activators of proliferation [
      • Komuves L.G.
      • Michael E.
      • Arbeit J.M.
      • Ma X.K.
      • Kwong A.
      • Stelnicki E.
      • et al.
      HOXB4 homeodomain protein is expressed in developing epidermis and skin disorders and modulates keratinocyte proliferation.
      ].
      In parallel, genes related to epidermal proliferation and differentiation were also identified as strong molecular markers of AL (Table 1). Among them, KRT15 and CCDN2, biomarkers for the epidermal proliferative basal layer [
      • Lloyd C.
      • Yu Q.C.
      • Cheng J.
      • Turksen K.
      • Degenstein L.
      • Hutton E.
      • et al.
      The basal keratin network of stratified squamous epithelia: defining K15 function in the absence of K14.
      ,
      • Rodriguez-Puebla M.L.
      • LaCava M.
      • Miliani De Marval P.L.
      • Jorcano J.L.
      • Richie E.R.
      • Conti C.J.
      Cyclin D2 overexpression in transgenic mice induces thymic and epidermal hyperplasia whereas cyclin D3 expression results only in epidermal hyperplasia.
      ], and KRT16 which is associated with hyper proliferative keratinocytes [
      • Freedberg I.M.
      • Tomic-Canic M.
      • Komine M.
      • Blumenberg M.
      Keratins and the keratinocyte activation cycle.
      ] were upregulated in AL. This is consistent with an increased proliferative rate of AL keratinocytes revealed by increased Ki67 staining [
      • Aoki H.
      • Moro O.
      • Tagami H.
      • Kishimoto J.
      Gene expression profiling analysis of solar lentigo in relation to immunohistochemical characteristics.
      ]. Increased KRT15 expression is also associated with the AL development process [
      • Lin C.B.
      • Hu Y.
      • Rossetti D.
      • Chen N.
      • David C.
      • Slominski A.
      • et al.
      Immuno-histochemical evaluation of solar lentigines: The association of KGF/KGFR and other factors with lesion development.
      ]. Immunostaining analysis confirmed the increase of K15 protein in the basal layer of AL versus NL skin, particularly impressive in epidermal invaginations (Fig. 4). These data suggest that basal keratinocytes with increased proliferative activity contribute to the development of epidermal rete ridges formation. In contrast genes that participate in the terminal differentiation process and cornified envelope formation, such as cornulin [
      • Lieden A.
      • Ekelund E.
      • Kuo I.C.
      • Kockum I.
      • Huang C.H.
      • Mallbris L.
      • et al.
      Cornulin, a marker of late epidermal differentiation, is down-regulated in eczema.
      ], LCE1E [
      • Marshall D.
      • Hardman M.J.
      • Nield K.M.
      • Byrne C.
      Differentially expressed late constituents of the epidermal cornified envelope.
      ] and BLMH, the filaggrin-processing enzyme [
      • Thyssen J.P.
      • Jakasa I.
      • Riethmuller C.
      • Schon M.P.
      • Braun A.
      • Haftek M.
      • et al.
      Filaggrin expression and processing deficiencies impair corneocyte surface texture and stiffness in mice.
      ], were down regulated. Simultaneous alterations in both epidermal proliferation and differentiation reinforce the fact that epidermal homeostasis is globally compromised in AL.
      Fig. 4
      Fig. 4Immunostaining of K15 protein in sections of AL and NL skin. Representative illustrations of lesions (AL) and adjacent non-lesional skin (NL) for 2 subjects from (a) the European study (E3, E15), and (b) the Japanese study (J1, J3) show the increased staining of K15 protein in the basal layer of AL versus NL, notably remarkable in epidermal invaginations (bar = 100 µm).

      3.7 Genes involved in transmembrane transport & channels modulated in Japanese and European AL

      Strikingly, almost 30 genes coding for transmembrane transporters and ion channels were differentially expressed in AL versus NL, preferentially down-regulated (Table 1). Their function in skin is mainly not described, although some genes, such as ABCG2, CHRNA9 or SLC1A3 may be related to epidermal homeostasis. The transporter ABCG2 is overexpressed in the basal layer of healing skin [
      • Chang H.M.
      • Huang W.Y.
      • Lin S.J.
      • Huang W.C.
      • Shen C.R.
      • Mao W.Y.
      • et al.
      ABCG2 deficiency in skin impairs re-epithelialization in cutaneous wound healing.
      ] and its role in stemness and proliferative status of keratinocytes [
      • Bebes A.
      • Kis K.
      • Nagy T.
      • Kurunczi A.
      • Polyanka H.
      • Bata-Csorgo Z.
      • et al.
      The expressions of ABCC4 and ABCG2 xenobiotic transporters in human keratinocytes are proliferation-related.
      ] can be linked to other markers of proliferation in AL. Cholinergic receptor α9 (CHRNA9 gene) is involved in the adhesion and motility of keratinocytes at early stages of epidermal morphogenesis [
      • Chernyavsky A.I.
      • Arredondo J.
      • Vetter D.E.
      • Grando S.A.
      Central role of alpha9 acetylcholine receptor in coordinating keratinocyte adhesion and motility at the initiation of epithelialization.
      ]. The glutamate transporter SLC1A3 may also have a role in keratinocyte differentiation orchestration, based on the essential role of glutamate in the barrier function and re-epithelialization process [
      • Fuziwara S.
      • Inoue K.
      • Denda M.
      NMDA-type glutamate receptor is associated with cutaneous barrier homeostasis.
      ]. SLC1A3 was recently identified as a marker of highly proliferative progenitor cells during skin growth [
      • Reichenbach B.
      • Classon J.
      • Aida T.
      • Tanaka K.
      • Genander M.
      • Göritz C.
      Glutamate transporter Slc1a3 mediates inter-niche stem cell activation during skin growth.
      ].
      Additionally, the downregulation of several genes associated with calcium flux (CACNA2D1, ATP13A4, SLC8A1, TMEM20/SLC35G1) strengthens the hypothesis of impaired epidermal differentiation and skin barrier given that the gradient of epidermal calcium concentration is essential in these processes [
      • Lee S.E.
      • Lee S.H.
      Skin barrier and calcium.
      ].
      No genes were related to skin pigmentation excepted the cysteine transporter SLC7A11, known to be involved in pheomelanin production [
      • Chintala S.
      • Li W.
      • Lamoreux M.L.
      • Ito S.
      • Wakamatsu K.
      • Sviderskaya E.V.
      • et al.
      Slc7a11 gene controls production of pheomelanin pigment and proliferation of cultured cells.
      ] in addition to other critical functions such as cell proliferation and oxidative stress defence.
      Modulation of these genes and other SLC transporter genes (SCL39A14, SLC6A14, SLC16A14, SLC45A4, SCL46A2) with ill-defined functions may reflect a global perturbation of cell activity and interaction with their microenvironment, in connection with the diversity of functional families and processes altered in AL.

      3.8 Extracellular matrix (ECM) and DEJ-related genes modulated in Japanese and European AL

      Table 1 shows genes coding for DEJ and ECM components, and TGFβ/BMP pathways regulation [
      • Verrecchia F.
      • Mauviel A.
      Transforming growth factor-beta signaling through the Smad pathway: role in extracellular matrix gene expression and regulation.
      ]. A huge majority of these genes (80%) are over-expressed in AL versus NL skin as highlighted in the previous European AL study [
      • Warrick E.
      • Duval C.
      • Nouveau S.
      • Bastien P.
      • Piffaut V.
      • Chalmond B.
      • et al.
      Morphological and molecular characterization of actinic lentigos reveals alterations of the dermal extracellular matrix.
      ].
      Over-expression of basement membrane genes in AL such as FRAS1 and THBS2 may be consistent with the increased length of the DEJ and with the formation of elongated epidermal rete ridges. These drastic structural modifications strongly suggest a reorganization of the DEJ and dermis during AL formation, in line with modifications of the ECM (collagens, proteoglycans) and DEJ related genes but also with genes highly involved in matrix remodeling, such as PLAU, ADAMTLS3, PI3 (elafin) or lysozyme (LYZ). Of particular interest, the two latter genes are associated with dermal photoaging and solar elastosis [
      • Muto J.
      • Kuroda K.
      • Wachi H.
      • Hirose S.
      • Tajima S.
      Accumulation of elafin in actinic elastosis of sun-damaged skin: elafin binds to elastin and prevents elastolytic degradation.
      ,
      • Seite S.
      • Zucchi H.
      • Septier D.
      • Igondjo-Tchen S.
      • Senni K.
      • Godeau G.
      Elastin changes during chronological and photo-ageing: the important role of lysozyme.
      ]. These data confirm that dermal alterations and photoaging-related markers represent key features in AL, regardless of the origin of the subjects [
      • Warrick E.
      • Duval C.
      • Nouveau S.
      • Bastien P.
      • Piffaut V.
      • Chalmond B.
      • et al.
      Morphological and molecular characterization of actinic lentigos reveals alterations of the dermal extracellular matrix.
      ,
      • Cario-Andre M.
      • Lepreux S.
      • Pain C.
      • Nizard C.
      • Noblesse E.
      • Taieb A.
      Perilesional vs. lesional skin changes in senile lentigo.
      ,
      • Iriyama S.
      • Ono T.
      • Aoki H.
      • Amano S.
      Hyperpigmentation in human solar lentigo is promoted by heparanase-induced loss of heparan sulfate chains at the dermal-epidermal junction.
      ]. Interestingly, dermal photoaging is of growing interest with regard to the physiopathology of pigmentary disorders, as recently highlighted for the melasma [
      • Passeron T.
      • Picardo M.
      Melasma, a photoaging disorder.
      ].
      Finally, the common signature of AL from Japanese and European subjects recapitulates the major functions observed separately. In the present study, however, only a small number of modulated genes represent a specific signature for each geographical origin of volunteers.

      3.9 Genes significantly modulated in AL versus NL in only one population

      Only 22 genes were specifically modulated in AL versus NL skin in Japanese volunteers but not in European ones (Table 2). These genes belong to most of the functional families already described, indicating that lesions of Japanese volunteers are not associated with particular biological functions. In addition, the number of modulated genes in the different functional families is low (between 1 and 4 genes) except for Epidermis/proliferation and differentiation function where 6 genes were specifically modulated in Japanese AL. Four genes belong to the EDC (epidermal differentiation complex), a specific chromosomic locus that contains genes such as SPRR or LCE which code for actors of epidermal terminal differentiation [
      • Oh I.Y.
      • de Guzman Strong C.
      The molecular revolution in cutaneous biology: EDC and locus control.
      ]. Interestingly SPRR2G even showed a trend to be inversely modulated in lesions from Japanese volunteers compared to European ones. Studies aiming at comparing skin barrier function depending on ethnic origin are often inconclusive and did not evidence clear differences [
      • Wesley N.O.
      • Maibach H.I.
      Racial (ethnic) differences in skin properties: the objective data.
      ]. The clinical relevance of this finding thus remains to be further investigated.
      Table 2Genes significantly modulated in AL versus NL skin in a specific group of volunteers. FC: fold-change. Numbers in bold indicate that the gene is significantly modulated in AL versus NL skin (absolute FC ≥ 1.5 and P-value<0.05).
      FunctionGene SymbolNameFC

      Europe
      FC

      Japan
      Genes significantly modulated in AL versus NL skin in Japanese but not European volunteers
      EpidermisSPRR2Bsmall proline-rich protein 2B1,322,37
      KRT33Akeratin 33A1,551,77
      SPRR3small proline-rich protein 31,191,73
      SPRR2Gsmall proline-rich protein 2 G-1,241,58
      LCE3Dlate cornified envelope 3D1,101,55
      KRT6Akeratin 6A1,431,52
      Innate immunityCD1BCD1b molecule1,051,63
      C1Scomplement component 1, s subcomponent1,211,51
      //SERPINB3//SERPINB4//serpin peptidase inhibitor, clade B (ovalbumin), member 3//member 41,291,51
      InflammationCCL13chemokine (C-C motif) ligand 131,311,65
      LCN2lipocalin 21,351,57
      Extracellular matrixCOMPcartilage oligomeric matrix protein1,402,04
      FREM2FRAS1 related extracellular matrix protein 21,291,83
      Intercellular communicationIGFBP6insulin-like growth factor binding protein 61,431,55
      SCUBE2signal peptide, CUB domain, EGF-like 21,201,52
      Oxidative stressGSTA3glutathione S-transferase alpha 3-1,04-1,51
      Intracellular communicationGPR98G protein-coupled receptor 981,071,92
      DevelopmentOSR2odd-skipped related 2 (Drosophila)1,421,57
      CytoskeletonNEXNnexilin (F actin binding protein)1,391,65
      NeuronsOMGOligodendrocyte myelin glycoprotein1,352,17
      Unknown or uncertainC9orf152chromosome 9 open reading frame 152-1,01-1,56
      //GABBR1//UBD//gamma-aminobutyric acid (GABA) B receptor, 1 // ubiquitin D1,221,61
      Genes significantly modulated in AL versus NL skin in European but not Japanese volunteers
      Intercellular communicationSCGB1D2secretoglobin, family 1D, member 21,661,01
      HTR3A5-hydroxytryptamine (serotonin) receptor 3A-1,54-1,07
      EpidermisSFRP4secreted frizzled-related protein 41,741,31
      EREGepiregulin-1,51-1,20
      Ion channelsSLC6A2solute carrier family 6 (neurotransmitter transporter, noradrenalin), member 21,591,31
      SLC13A2solute carrier family 13 (sodium-dependent dicarboxylate transporter), member 21,51-1,11
      ImmunityPTX3pentraxin 3, long1,891,08
      LAMP3lysosomal-associated membrane protein 3-1,58-1,20
      Cell cycleE2F8E2F transcription factor 8-1,55-1,31
      Intracellular communicationCHRM3cholinergic receptor, muscarinic 31,64-1,05
      CytoskeletonACTG2actin, gamma 2, smooth muscle, enteric1,601,06
      Extracellular matrixADAMTS5ADAM metallopeptidase with thrombospondin type 1 motif, 51,501,03
      ChromatinTOX3TOX high mobility group box family member 31,691,21
      UbiquitinationUSP2ubiquitin specific peptidase 2-1,56-1,19
      UnknownC1orf59chromosome 1 open reading frame 591,571,14
      Among the 15 genes specifically modulated in European but not in Japanese subjects (Table 2), no specific functional family could be identified, due to the low number of genes in each family.

      4. Conclusion

      Advanced AL lesions carefully selected according to the same clinical criteria in Japanese and European women displayed similar alterations of the whole skin structure, with the presence of elongated, melanin-loaded epidermal rete ridges. They are associated with a common gene expression profile, with genes involved in multiple biological functions and skin compartments that recapitulate the overall biological alterations of AL lesions, regardless of the origin of the subjects. Interestingly genes related to melanocyte biology are not found modulated. These lesions must therefore be considered globally, and not only through the melanocyte prism. Major modulations of genes related to epidermal homeostasis and the dermal compartment indicate dysregulation of keratinocyte proliferation/stemness, defects in terminal differentiation, and dermal matrix organization. These molecular features may explain the morphological features observed in AL. These findings open new doors for the development of effective treatment of AL aimed at restoring global tissue homeostasis.

      Funding sources

      This study was funded by L’Oréal Research and Innovation.

      Conflicts of interest

      All authors, except A. Morita, are or were employees of L’Oréal.

      Acknowledgements

      We are grateful to Dr Sophie Deret for her constant scientific support and expertise regarding the transcriptomic study and biostatistical analysis.
      The patients in this manuscript have given written informed consent to publication of their case details.

      Appendix A. Supplementary material

      References

        • Bastiaens M.
        • Hoefnagel J.
        • Westendorp R.
        • Vermeer B.J.
        • Bouwes J.N.
        Bavinck, Solar lentigines are strongly related to sun exposure in contrast to ephelides.
        Pigment Cell Res. 2004; 17: 225-229
        • Warrick E.
        • Duval C.
        • Nouveau S.
        • Bastien P.
        • Piffaut V.
        • Chalmond B.
        • et al.
        Morphological and molecular characterization of actinic lentigos reveals alterations of the dermal extracellular matrix.
        Br. J. Dermatol. 2017; 177: 1619-1632
        • Barysch M.J.
        • Braun R.P.
        • Kolm I.
        • Ahlgrimm-Siesz V.
        • Hofmann-Wellenhof R.
        • Duval C.
        • et al.
        Keratinocytic malfunction as a trigger for the development of solar lentigines.
        Dermatopathol. (Basel, Switz. ). 2019; 6: 1-11
        • Andersen W.K.
        • Labadie R.R.
        • Bhawan J.
        Histopathology of solar lentigines of the face: a quantitative study.
        J. Am. Acad. Dermatol. 1997; 36: 444-447
        • Choi W.
        • Yin L.
        • Smuda C.
        • Batzer J.
        • Hearing V.J.
        • Kolbe L.
        Molecular and histological characterization of age spots.
        Exp. Dermatol. 2017; 26: 242-248
        • Montagna W.
        • Hu F.
        • Carlisle K.
        A reinvestigation of solar lentigines.
        Arch. Dermatol. 1980; 116: 1151-1154
        • Cario-Andre M.
        • Lepreux S.
        • Pain C.
        • Nizard C.
        • Noblesse E.
        • Taieb A.
        Perilesional vs. lesional skin changes in senile lentigo.
        J. Cutan. Pathol. 2004; 31: 441-447
        • Lin C.B.
        • Hu Y.
        • Rossetti D.
        • Chen N.
        • David C.
        • Slominski A.
        • et al.
        Immuno-histochemical evaluation of solar lentigines: The association of KGF/KGFR and other factors with lesion development.
        J. Dermatol. Sci. 2010; 59: 91-97
        • Chen N.
        • Hu Y.
        • Li W.H.
        • Eisinger M.
        • Seiberg M.
        • Lin C.B.
        The role of keratinocyte growth factor in melanogenesis: a possible mechanism for the initiation of solar lentigines.
        Exp. Dermatol. 2010; 19: 865-872
        • Kovacs D.
        • Cardinali G.
        • Aspite N.
        • Cota C.
        • Luzi F.
        • Bellei B.
        • et al.
        Role of fibroblast-derived growth factors in regulating hyperpigmentation of solar lentigo.
        Br. J. Dermatol. 2010;
        • Hattori H.
        • Kawashima M.
        • Ichikawa Y.
        • Imokawa G.
        The epidermal stem cell factor is over-expressed in lentigo senilis: implication for the mechanism of hyperpigmentation.
        J. Invest Dermatol. 2004; 122: 1256-1265
        • Kadono S.
        • Manaka I.
        • Kawashima M.
        • Kobayashi T.
        • Imokawa G.
        The role of the epidermal endothelin cascade in the hyperpigmentation mechanism of lentigo senilis.
        J. Invest Dermatol. 2001; 116: 571-577
        • Aoki H.
        • Moro O.
        • Tagami H.
        • Kishimoto J.
        Gene expression profiling analysis of solar lentigo in relation to immunohistochemical characteristics.
        Br. J. Dermatol. 2007; 156: 1214-1223
        • Goyarts E.
        • Muizzuddin N.
        • Maes D.
        • Giacomoni P.U.
        Morphological changes associated with aging: age spots and the microinflammatory model of skin aging.
        Ann. N. Y. Acad. Sci. 2007; 1119: 32-39
        • Iriyama S.
        • Ono T.
        • Aoki H.
        • Amano S.
        Hyperpigmentation in human solar lentigo is promoted by heparanase-induced loss of heparan sulfate chains at the dermal-epidermal junction.
        J. Dermatol. Sci. 2011; 64: 223-228
        • Alexis A.F.
        • Obioha J.O.
        Ethnicity and aging skin.
        J. Drugs Dermatol.: JDD. 2017; 16: s77-s80
        • Goh S.H.
        The treatment of visible signs of senescence: the Asian experience.
        Br. J. Dermatol. 122. 1990; Suppl 35: 105-109
        • Chua-Ty G.
        • Goh C.L.
        • Koh S.L.
        Pattern of skin diseases at the National Skin Centre (Singapore) from 1989-1990.
        Int. J. Dermatol. 1992; 31: 555-559
        • Vierkotter A.
        • Kramer U.
        • Sugiri D.
        • Morita A.
        • Yamamoto A.
        • Kaneko N.
        • et al.
        Development of lentigines in German and Japanese women correlates with variants in the SLC45A2 gene.
        J. Invest Dermatol. 2012; 132: 733-736
        • Del Bino S.
        • Bernerd F.
        Variations in skin colour and the biological consequences of ultraviolet radiation exposure.
        Br. J. Dermatol. 169. 2013; Suppl 3: 33-40
        • Negishi K.
        • Akita H.
        • Tanaka S.
        • Yokoyama Y.
        • Wakamatsu S.
        • Matsunaga K.
        Comparative study of treatment efficacy and the incidence of post-inflammatory hyperpigmentation with different degrees of irradiation using two different quality-switched lasers for removing solar lentigines on Asian skin.
        J. Eur. Acad. Dermatol. Venereol.: JEADV. 2013; 27: 307-312
        • Noblesse E.
        • Nizard C.
        • M C.-A.
        • Lepreux S.
        • Pain C.
        • Schnebert S.
        • et al.
        Skin Ultrastructure in Senile Lentigo.
        Ski. Pharmacol. Physiol. 2006; 19: 95-100
        • Fogel P.
        • Young S.S.
        • Hawkins D.M.
        • Ledirac N.
        Inferential, robust non-negative matrix factorization analysis of microarray data.
        Bioinformatics. 2007; 23: 44-49
        • Motokawa T.
        • Kato T.
        • Katagiri T.
        • Matsunaga J.
        • Takeuchi I.
        • Tomita Y.
        • et al.
        Messenger RNA levels of melanogenesis-associated genes in lentigo senilis lesions.
        J. Dermatol. Sci. 2005; 37: 120-123
        • Yamaguchi Y.
        • Brenner M.
        • Hearing V.J.
        The regulation of skin pigmentation.
        J. Biol. Chem. 2007; 282: 27557-27561
        • Reber L.
        • Da Silva C.A.
        • Frossard N.
        Stem cell factor and its receptor c-Kit as targets for inflammatory diseases.
        Eur. J. Pharm. 2006; 533: 327-340
        • Imokawa G.
        Autocrine and paracrine regulation of melanocytes in human skin and in pigmentary disorders.
        Pigment Cell Res. 2004; 17: 96-110
        • Hombria J.C.
        • Lovegrove B.
        Beyond homeosis--HOX function in morphogenesis and organogenesis.
        Differ. ; Res. Biol. Divers. 2003; 71: 461-476
        • Stelnicki E.J.
        • Komuves L.G.
        • Kwong A.O.
        • Holmes D.
        • Klein P.
        • Rozenfeld S.
        • et al.
        HOX homeobox genes exhibit spatial and temporal changes in expression during human skin development.
        J. Invest Dermatol. 1998; 110: 110-115
        • Gao D.
        • Chen H.Q.
        Specific knockdown of HOXB7 inhibits cutaneous squamous cell carcinoma cell migration and invasion while inducing apoptosis via the Wnt/beta-catenin signaling pathway.
        Am. J. Physiol. Cell Physiol. 2018; 315 (C675-c686)
        • Komuves L.G.
        • Michael E.
        • Arbeit J.M.
        • Ma X.K.
        • Kwong A.
        • Stelnicki E.
        • et al.
        HOXB4 homeodomain protein is expressed in developing epidermis and skin disorders and modulates keratinocyte proliferation.
        Dev. Dyn.: Off. Publ. Am. Assoc. Anat. 2002; 224: 58-68
        • Lloyd C.
        • Yu Q.C.
        • Cheng J.
        • Turksen K.
        • Degenstein L.
        • Hutton E.
        • et al.
        The basal keratin network of stratified squamous epithelia: defining K15 function in the absence of K14.
        J. Cell Biol. 1995; 129: 1329-1344
        • Rodriguez-Puebla M.L.
        • LaCava M.
        • Miliani De Marval P.L.
        • Jorcano J.L.
        • Richie E.R.
        • Conti C.J.
        Cyclin D2 overexpression in transgenic mice induces thymic and epidermal hyperplasia whereas cyclin D3 expression results only in epidermal hyperplasia.
        Am. J. Pathol. 2000; 157: 1039-1050
        • Freedberg I.M.
        • Tomic-Canic M.
        • Komine M.
        • Blumenberg M.
        Keratins and the keratinocyte activation cycle.
        J. Invest Dermatol. 2001; 116: 633-640
        • Lieden A.
        • Ekelund E.
        • Kuo I.C.
        • Kockum I.
        • Huang C.H.
        • Mallbris L.
        • et al.
        Cornulin, a marker of late epidermal differentiation, is down-regulated in eczema.
        Allergy. 2009; 64: 304-311
        • Marshall D.
        • Hardman M.J.
        • Nield K.M.
        • Byrne C.
        Differentially expressed late constituents of the epidermal cornified envelope.
        Proc. Natl. Acad. Sci. USA. 2001; 98: 13031-13036
        • Thyssen J.P.
        • Jakasa I.
        • Riethmuller C.
        • Schon M.P.
        • Braun A.
        • Haftek M.
        • et al.
        Filaggrin expression and processing deficiencies impair corneocyte surface texture and stiffness in mice.
        J. Invest Dermatol. 2020; 140 (615-623.e5)
        • Chang H.M.
        • Huang W.Y.
        • Lin S.J.
        • Huang W.C.
        • Shen C.R.
        • Mao W.Y.
        • et al.
        ABCG2 deficiency in skin impairs re-epithelialization in cutaneous wound healing.
        Exp. Dermatol. 2016; 25: 355-361
        • Bebes A.
        • Kis K.
        • Nagy T.
        • Kurunczi A.
        • Polyanka H.
        • Bata-Csorgo Z.
        • et al.
        The expressions of ABCC4 and ABCG2 xenobiotic transporters in human keratinocytes are proliferation-related.
        Arch. Dermatol. Res. 2012; 304: 57-63
        • Chernyavsky A.I.
        • Arredondo J.
        • Vetter D.E.
        • Grando S.A.
        Central role of alpha9 acetylcholine receptor in coordinating keratinocyte adhesion and motility at the initiation of epithelialization.
        Exp. Cell Res. 2007; 313: 3542-3555
        • Fuziwara S.
        • Inoue K.
        • Denda M.
        NMDA-type glutamate receptor is associated with cutaneous barrier homeostasis.
        J. Invest Dermatol. 2003; 120: 1023-1029
        • Reichenbach B.
        • Classon J.
        • Aida T.
        • Tanaka K.
        • Genander M.
        • Göritz C.
        Glutamate transporter Slc1a3 mediates inter-niche stem cell activation during skin growth.
        Embo J. 2018; 37
        • Lee S.E.
        • Lee S.H.
        Skin barrier and calcium.
        Ann. Dermatol. 2018; 30: 265-275
        • Chintala S.
        • Li W.
        • Lamoreux M.L.
        • Ito S.
        • Wakamatsu K.
        • Sviderskaya E.V.
        • et al.
        Slc7a11 gene controls production of pheomelanin pigment and proliferation of cultured cells.
        Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10964-10969
        • Verrecchia F.
        • Mauviel A.
        Transforming growth factor-beta signaling through the Smad pathway: role in extracellular matrix gene expression and regulation.
        J. Invest Dermatol. 2002; 118: 211-215
        • Muto J.
        • Kuroda K.
        • Wachi H.
        • Hirose S.
        • Tajima S.
        Accumulation of elafin in actinic elastosis of sun-damaged skin: elafin binds to elastin and prevents elastolytic degradation.
        J. Invest Dermatol. 2007; 127: 1358-1366
        • Seite S.
        • Zucchi H.
        • Septier D.
        • Igondjo-Tchen S.
        • Senni K.
        • Godeau G.
        Elastin changes during chronological and photo-ageing: the important role of lysozyme.
        J. Eur. Acad. Dermatol. Venereol. 2006; 20: 980-987
        • Passeron T.
        • Picardo M.
        Melasma, a photoaging disorder.
        Pigment Cell Melanoma Res. 2018; 31: 461-465
        • Oh I.Y.
        • de Guzman Strong C.
        The molecular revolution in cutaneous biology: EDC and locus control.
        J. Invest Dermatol. 2017; 137: e101-e104
        • Wesley N.O.
        • Maibach H.I.
        Racial (ethnic) differences in skin properties: the objective data.
        Am. J. Clin. Dermatol. 2003; 4: 843-860