Synergistic Effect of Hyaluronate Fragments in Retinaldehyde-Induced Skin Hyperplasia Which Is a Cd44-Dependent Phenomenon Laurent Barnes1, Christian Tran 1 , Olivier Sorg1, Raymonde Hotz1, Denise Grand1, Pierre Carraux1, Liliane Didierjean1, Ivan Stamenkovic2, Jean-Hilaire Saurat1, Gu¨ rkan Kaya1* 1 Department of Dermatology, University of Geneva, Geneva, Switzerland, 2 Institute of Pathology, University of Lausanne, Lausanne, Switzerland Abstract Background: CD44 is a polymorphic proteoglycan and functions as the principal cell-surface receptor for hyaluronate (HA). Heparin-binding epidermal growth factor (HB-EGF) activation of keratinocyte erbB receptors has been proposed to mediate retinoid-induced epidermal hyperplasia. We have recently shown that intermediate size HA fragments (HAFi) reverse skin atrophy by a CD44-dependent mechanism. Methodology and Principal Findings: Treatment of primary mouse keratinocyte cultures with retinaldehyde (RAL) resulted in the most significant increase in keratinocyte proliferation when compared with other retinoids, retinoic acid, retinol or retinoyl palmitate. RAL and HAFi showed a more significant increase in keratinocyte proliferation than RAL or HAFi alone. No proliferation with RAL was observed in CD442/2 keratinocytes. HA synthesis inhibitor, 4-methylumbelliferone inhibited the proliferative effect of RAL. HB-EGF, erbB1, and tissue inhibitor of MMP-3 blocking antibodies abrogated the RAL- or RAL- and HAFi-induced keratinocyte proliferation. Topical application of RAL or RAL and HAFi for 3 days caused a significant epidermal hyperplasia in the back skin of wild-type mice but not in CD442/2 mice. Topical RAL and HAFi increased epidermal CD44 expression, and the epidermal and dermal HA. RAL induced the expression of active HB-EGF and erbB1. However, treatment with RAL and HAFi showed a more significant increase in pro-HB-EGF when compared to RAL or HAFi treatments alone. We then topically applied RAL and HAFi twice a day to the forearm skin of elderly dermatoporosis patients. After 1 month of treatment, we observed a significant clinical improvement. Conclusions and Significance: Our results indicate that (i) RAL-induced in vitro and in vivo keratinocyte proliferation is a CD44-dependent phenomenon and requires the presence of HA, HB-EGF, erbB1 and MMPs, (ii) RAL and HAFi show a synergy in vitro and in vivo in mouse skin, and (iii) the combination of RAL and HAFi seems to have an important therapeutic effect in dermatoporosis. Citation: Barnes L, Tran C, Sorg O, Hotz R, Grand D, et al. (2010) Synergistic Effect of Hyaluronate Fragments in Retinaldehyde-Induced Skin Hyperplasia Which Is a Cd44-Dependent Phenomenon. PLoS ONE 5(12): e14372. doi:10.1371/journal.pone.0014372 Editor: Christophe Egles, Tufts University, United States of America Received July 5, 2010; Accepted September 7, 2010; Published December 16, 2010 Copyright: 2010 Barnes 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. Funding: This work was supported by the University of Geneva Dermatology Fund, Genderm Research Program and Swiss National Science Foundation grant 310000-122322 to GK and JHS, and Swiss National Science Foundation grant 3100-65090-01 and the Molecular Oncology National Centre for Competence in Research (NCCR) to IS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: GK and JHS are among inventors in a patent held by Pierre Fabre Dermo-Cosmetique (WO/2005/082327). This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: gkaya@hcuge.ch Introduction CD44 is a facultative cell surface proteoglycan expressed as several isoforms [1,2] and the principal cell surface receptor of hyaluronate [3,4] (HA), the major component of the extracellular matrix [5]. In our previous study we have shown that CD44 is implicated in the regulation of keratinocyte proliferation and the local HA metabolism in mice [6]. We have recently shown that the epidermal hyperplasia induced by topical retinoids was accompanied by an increased expression of CD44 and hyaluronate synthases and associated with an increase in epidermal and dermal HA in mouse skin [7]. We have also shown that the decrease of the expression of CD44 and hyaluronate induced by UVA and UVB in mouse epidermis is counteracted by topical retinoids [8]. Topical application of one of these retinoids, retinaldehyde (RAL), a natural retinoid immediate precursor of retinoic acid (RA), restores the epidermal thickness and CD44 expression which are correlated with clinical improvement in lichen sclerosus et atrophicus (LSA) lesions [9], where the epidermal expression of CD44 has been shown to be decreased or absent [10]. RAL has been shown to exert biological activity in mouse and human skin [11,12]. It has been shown that the epidermal hyperplasia induced by topical retinoids was linked to a RA receptor (RAR)-dependent heparin-binding epidermal growth factor (HB-EGF) paracrine loop [13]. It has also been shown that a heparan sulfate-bearing variant of CD44 (CD44v3) recruits proteolytically active matrix metalloproteinase 7 (MMP-7), the precursor of HB-EGF (pro-HB-EGF) and its receptor, erbB4 to form a complex on the cell surface [14]. We have recently shown PLoS ONE | www.plosone.org 1 December 2010 | Volume 5 | Issue 12 | e14372