Although the increase in the protein levels of active HB-EGF was not more significant than HAFi alone, a clear synergy was observed in the induction of pro-HB-EGF expression. The production of more HB-EGF after RAL and HAFi application might prolong the proliferative effect on the epidermis by the generation of more HB-EGF over time which interacts with its receptor erbB1 which is also upregulated. In our previous study we showed the association of CD44 with erbB1 in keratinocytes in vitro and in vivo [15]. This observation is consistent with the notion that topical HAFi upregulates the molecules shown to form a complex with CD44 in mouse uterine and mammary epithelia [14], and that erbB1 blocking antibodies have the same abrogating effect as the absence of CD44 on in vitro keratinocyte proliferation. It seems that erbB4 which serves as the receptor for HB-EGF in uterine and mammary epithelia is replaced by erbB1 in keratinocytes, and that the same or similar CD44 complex is most likely present on keratinocytes as well [15]. Topically applied RAL and HAF also show a synergy in the increase of the epidermal and dermal HA content in mouse skin. RAL alone increases more HA in epidermis than HAFi alone; in contrast, HAFi alone results in more dermal HA than RAL alone. RAL and HAFi application more significantly augments HA in epidermis and dermis than with RAL or HAFi alone. We recently showed that the increase in cutaneous HA by RAL is due to the increased expression of HA-polymerizing enzymes, hyaluronate synthase (HAS)s 1, 2 and 3 [7]. The synergistic effect on epidermal and dermal HA increase is most likely due to the upregulation of HAS1, HAS2 and HAS3 also by HAFi [15]. In our previous study we demonstrated that topically applied HAFi traverse the skin, References 1. Stamenkovic I, Aruffo A, Amiot M, Seed B (1991) The hematopoietic and epithelial forms of CD44 are distinct polypeptides with different adhesion potentials for hyaluronate-bearing cells. EMBO J 10: 343–348. 2. Screaton GR, Bell MV, Jackson DG, Cornelis FB, Gerth U, et al. (1992) Genomic structure of DNA encoding the lymphocyte homing receptor CD44 reveals at least 12 alternatively spliced exons. Proc Natl Acad Sci 89: 12160–12164. 3. Aruffo A, Stamenkovic I, Melnick M, Underhill CB, Seed B (1990) CD44 is the principal cell surface receptor for hyaluronate. Cell 61: 1303–1313. 4. Miyake K, Underhill CB, Lesley J, Kincade PW (1990) Hyaluronate can function as a cell adhesion molecule and CD44 participates in hyaluronate recognition. J Exp Med 172: 69–75. 5. Laurent TC, Fraser JR (1992) Hyaluronan. FASEB J 6: 2397–2404. 6. Kaya G, Rodriguez I, Jorcano JL, Vassalli P, Stamenkovic I (1997) Selective suppression of CD44 in keratinocytes of mice bearing an antisense CD44 transgene driven by a tissue-specific promoter disrupts hyaluronate metabolism in the skin and impairs keratinocyte proliferation. Genes Dev 11: 996–1007. 7. Kaya G, Grand D, Hotz R, Augsburger E, Carraux P, et al. (2005) Upregulation of CD44 and hyaluronate synthases by topical retinoids in mouse skin. J Invest Dermatol 124: 284–287. 8. Calikoglu E, Sorg O, Tran C, Grand D, Carraux P, et al. (2006) UVA and UVB decrease the expression of CD44 and hyaluronate in mouse epidermis which is counteracted by topical retinoids. Photochem Photobiol 82: 1342–1347. 9. Kaya G, Saurat JH (2005) Restored epidermal CD44 expression in lichen sclerosus et atrophicus and clinical improvement with topical application of retinaldehyde. Br J Dermatol 152: 570–572. 10. Kaya G, Augsburger E, Stamenkovic I, Saurat JH (2000) Decrease in epidermal CD44 expression as a potential mechanism for abnormal hyaluronate accumulation in superficial dermis in lichen sclerosus et atrophicus. J Invest Dermatol 115: 1054–1058. 11. Saurat JH, Didierjean L, Masgrau E, Piletta PA, Jaconi S, et al. (1994) Topical retinaldehyde on human skin: biologic effects and tolerance. J Invest Dermatol 103: 770–774. HA-RAL Synergy in Skin which also contributes the dermal increase of HA [15]. Exogenous RAL and HAFi as well as the endogenous HA might play an important role in the activation of CD44 platform which we call hyalurosome complex which regulates keratinocyte proliferation and HA metabolism (Figure 6). Recently we have shown that topical application of HAFi restores atrophic skin lesions in humans by a CD44-dependent mechanism [15]. Topical RAL and HAFi application resulted in skin hyperplasia in dermatoporotic human skin which was more significant than the one with RAL alone and HAFi alone. This synergistic effect was observed as early as 1 month after initiation of treatment and was accompanied by a significant clinical improvement. Taken together, our observations suggest that RAL-induced in vitro and in vivo proliferative response of keratinocytes is mediated via a CD44-dependent pathway and requires the presence of HB- EGF, erbB1 and an MMP or ADAM. Therefore CD44-dependent recruitment and processing of HB-EGF and EGF receptors may provide a mechanism for cell growth modulation induced by topical retinoids. Our results also indicate that RAL and HAFi combination may be used to develop novel therapeutic strategies for skin atrophy. Author Contributions Conceived and designed the experiments: IS JHS GK. Performed the experiments: LB CT RH DG PC GK. Analyzed the data: LB CT OS LD IS JHS GK. 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Arch Dermatol Res 295: 93–101. 21. Bennett KL, Jackson DG, Simon JC, Tanczos E, Peach R, et al. (1995) CD44 isoforms containing exon V3 are responsible for the presentation of heparin- binding growth factor. J Cell Biol 128: 687–698. 22. Higashiyama S, Nanba D (2005) ADAM-mediated ectodomain shedding of HB- EGF in receptor cross-talk. Biochim Biophys Acta 1751: 110–117. PLoS ONE | www.plosone.org 9 December 2010 | Volume 5 | Issue 12 | e14372