Perlecan expression influences the keratin 15-positive cell population fate in the epidermis of aging skin Morgan dos Santos, Anna Michopoulou, Valérie André-frei, Sophie Boulesteix, Christine Guicher, Guila Dayan, John Whitelock, Odile Damour, Patricia Rousselle To cite this version: Morgan dos Santos, Anna Michopoulou, Valérie André-frei, Sophie Boulesteix, Christine Guicher, et al.. Perlecan expression influences the keratin 15-positive cell population fate in the epidermis ofaging skin. Aging, Impact Journals, 2016, 8 (4), pp.751-768. 10.18632/aging.100928. hal-03041948 HAL Id: hal-03041948 https://hal.archives-ouvertes.fr/hal-03041948 Submitted on 5 Dec 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. www.impactaging.com AGING, April 2016, Vol 8 No 4 Research Paper Perlecan expression influences the keratin 15‐positive cell population fate in the epidermis of aging skin Morgan Dos Santos1,2,*, Anna Michopoulou1,*, Valérie André‐Frei2, Sophie Boulesteix1, Christine 1 1 3 1,4 1 Guicher , Guila Dayan , John Whitelock , Odile Damour , and Patricia Rousselle 1Laboratoire de Biologie Tissulaire et Ingénierie Thérapeutique, UMR 5305, CNRS, University Lyon 1, Lyon, France 2 BASF BCS France SAS, 69366, Lyon, France 3 Graduate School of Biomedical Engineering, University of New South Wales Sydney, Australia 4 Cell and Tissue Bank, Hôpital Edouard Herriot, Lyon, France *Equally contributed to this work Key words: basement membrane; perlecan; keratinocyte; skin aging; keratin 15, stem cell A bbreviations: DEJ, dermal‐epidermal junction; ECM, extracellular matrix; BM, basement membrane; HSPG, heparan sulfate p roteoglycan; NHK, normal human keratinocytes; SE, skin equivalent; DE, dermal equivalent Received: 01/13/16; Accepted: 02/23/16; Published: 03/17/16 Correspondence to: Patricia Rousselle, PhD; E‐mail: [email protected] Copyright: Dos Santos et al. This is an open‐access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Abstract: The epidermis is continuously renewed by stem cell proliferation and differentiation. Basal keratinocytes append the dermal‐epidermal junction, a cell surface‐associated, extracellular matrix that provides structural support and influences their behaviour. It consists of laminins, type IV collagen, nidogens, and perlecan, which are necessary for tissue organization and structural integrity. Perlecan is a heparan sulfate proteoglycan known to be involved in keratinocyte survival and differentiation. Aging affects the dermal epidermal junction resulting in decreased contact with keratinocytes, thus impacting epidermal renewal and homeostasis. We found that perlecan expression decreased during chronological skin aging. Our in vitro studies revealed reduced perlecan transcript levels in aged keratinocytes. The production of in vitro skin models revealed that aged keratinocytes formed a thin and poorly organized epidermis. Supplementing these models with purified perlecan reversed the phenomenon allowing restoration of a well‐differentiated multi‐layered epithelium. Perlecan down‐regulation in cultured keratinocytes caused depletion of the cell population that expressed keratin 15. This phenomenon depended on the perlecan heparan sulphate moieties, which suggested the involvement of a growth factor. Finally, we found defects in keratin 15 expression in the epidermis of aging skin. This study highlighted a new role for perlecan in maintaining the self‐renewal capacity of basal keratinocytes. INTRODUCTION leave the basal layer and move upward toward the surface, where they die and slough off [1]. Basal Skin constitutes the protective barrier of the body and keratinocytes append the DEJ, a cell surface-associated mediates its interaction with the environment. A highly extracellular matrix that forms as a concerted action of specialized extracellular matrix (ECM) named dermal both epidermal and dermal cells [2]. The DEJ provides epidermal junction (DEJ) separates the epidermal and both a structural support to keratinocytes and a specific dermal compartments. The epidermis, primarily made niche that mediates signals influencing their behaviour. of keratinocytes, is continuously renewed by the Similar to all basement membranes (BMs), the DEJ proliferation of stem cells and the differentiation of their primarily consists of laminins, type IV collagen, progeny, which undergo terminal differentiation as they nidogens, and the heparan sulfate proteoglycan (HSPG) www.impactaging.com 751 AGING, April 2016, Vol. 8 No.4 perlecan, all of which are necessary for tissue from young and elderly donors confirmed earlier studies organization and structural integrity. Keratinocytes showing that perlecan influences epidermal thickness committed to the differentiation program downregulate [10]. Finally, we found that perlecan down-regulation in integrins to become less adhesive, move to the keratinocytes resulted in the depletion of the cell suprabasal compartment and continue their upward population that expressed keratin 15 and ß1-integrin. movement until they are terminally differentiated and This depletion was reversed when we supplemented shed off. This produces several layers of keratinocytes, perlecan-deficient keratinocytes with purified perlecan. at different stages of differentiation that can be identified by the expression of keratins. Basal RESULTS keratinocytes express keratins (K) 5, K14 and K15, whereas differentiating keratinocytes express keratins Perlecan expression in epidermal and capillary BMs K1 and K10 [3]. in skin aging The appearance and mechanical functions of skin We performed an immunohistochemical analysis of undergo profound changes over time with both perlecan in a cohort of 38 human skin samples from increasing chronological age and cumulative exposure donors ranging in age from 22 to 73 years. We detected to external factors, such as ultraviolet radiation [4]. In perlecan expression with a monoclonal antibody (mAb) addition to an overall thinning of the epidermis and against perlecan domain III (Figure 1a). An analysis of dermis, a major change is the flattening of the DEJ the biopsies from donors that were 22 to 35 years old resulting from retraction of both epidermal papillae and revealed continuous staining at the DEJ and around microprojections of basal cells into the dermis [5,6]. In dermal capillaries (Figure 1b and c), consistent with genetically programmed aging, the progressive decrease previous studies [16,17]. Perlecan staining began to in epidermal cell renewal results from decreased decrease in biopsies from donors aged 39 to 50 years. proliferation [7,8] and the appearance of senescent Perlecan staining again decreased in both intensity and keratinocytes [9]. area in biopsies from donors aged 54 to 70 years (Figure 1b and d). This was also observed in the capillary BMs In addition to their function in the promotion of strong (Figure 1c and e). An analysis of perlecan domains I, epidermal/dermal attachment, DEJ components play IV, and V revealed that all domains were expressed crucial roles in biological events, including the along the DEJ and around dermal capillaries, similar to adhesion, migration, proliferation and differentiation of the domain III expression pattern (Figure 1f). In aged keratinocytes [2]. In particular, perlecan was shown to skin, both the DEJ and dermal capillary BM showed play a role in keratinocyte survival and terminal reduced staining of each domain; this result suggested differentiation [10] and in DEJ cohesion [11]. Perlecan that the entire perlecan molecule was subject to is a modular HSPG with broad tissue distribution and expression changes over time. multiple functions [12]. The 470-kDa perlecan core protein is made of five distinct modules, but together To characterize the perlecan expression pattern in with several O-linked oligosaccharides and heparan cultured keratinocytes, we first examined its sulfate chains, the protein can reach a molecular mass of localization in the ECM of young keratinocyte cultures over 800 kDa (Figure 1a). The various protein modules (Figure 2a). When the anti-perlecan mAb was applied to of perlecan and its heparan sulfate chains take part in confluent cells, the protein appeared to be regularly numerous interactions with partners, including growth distributed over the entire support, which suggested that factors, BM constituents such as laminin-111 or -511 perlecan was present in the underlying ECM. At the and collagen IV, as well as ß1 integrins [13,14]. Though individual cell level, we observed that the substrate both epidermal keratinocytes and dermal fibroblasts immediately adjacent to the cells was fluorescently express perlecan at the transcriptional level, DEJ stained in regularly aligned patches, resembling associated perlecan seems to be produced by adhesion contacts. Higher magnification revealed that keratinocytes [10,15]. the ends of actin cables