Determination of HA synthesis in vitro Keratinocytes were cultured in six-well culture plates (Falcon, Becton Dickinson and Co.) as described above. On day 12 of culture, HAFi (100 mg/ml), a RAL (2 mM) or RAL+HAFi were added to the cultures. 48 hrs later HA synthesis was determined by the quantification of HA in the culture supernatants by an enzyme-linked binding protein assay as described above. Discussion In this study we provide evidence that RAL induces an in vitro and in vivo proliferative response of keratinocytes which is mediated by a CD44-dependent pathway and that RAL and HAFi show a synergy in vitro and in vivo with a potential therapeutic effect in dermatoporosis. In our previous study we have shown for the first time that HAFi induce an in vitro keratinocyte proliferation [15]. This proliferative response was inhibited, like for RAL, by anti-erbB1 blocking antibodies and TIMP-3, an inhibitor of MMPs and ADAMs, suggesting that the RAL- or HAFi- induced in vitro proliferative response requires erbB1 and MMPs/ADAMs. In the present study we show that RAL and HAFi have a synergistic effect on the in vitro proliferation of keratinocytes. Our previous and present observations also provide evidence that CD44 and HB-EGF are required for RAL- or HAF-mediated in vitro keratinocyte proliferation. The absence of a proliferative response of CD442/2 keratinocytes to HB-EGF is consistent with the notion that HB-EGF interaction with its receptors requires presentation by heparan sulfate side chains of CD44v3-containing isoforms which appear to constitute a major fraction of heparan HA-RAL Synergy in Skin sulfate proteoglycans expressed on the surface of normal keratinocytes [16]. Our observations show that topical application of RAL and HAFi to normal mouse skin leads to: (1) significant epidermal hyperplasia accompanied by increased cell proliferation in epidermis and dermis, (2) increase of CD44 and its CD44v3 isoform, pro- and active HB-EGF, and erbB1 expression, (3) increase of epidermal and dermal HA. Significant epidermal hyperplasia and the increased cell proliferation in epidermis and dermis observed in SKH1 hairless and DBA/1 mice with topical RAL and HAFi was superior to the one induced by RAL alone but comparable to the one resulted from HAFi alone. The comparable result between HAFi alone and RAL and HAFi combination is most likely due to the saturation of proliferative capacity of keratinocytes. Topical RAL and HAF increase the expression of CD44 at the RNA and protein levels in mouse skin. This increase is more significant then the one after RAL or HAFi alone, indicating the synergistic effect of these two molecules on CD44 expression. RAL and HAF also upregulate the CD44v3 isoform in the epidermis. However, no synergy was observed in CD44v3 expression. The synergistic effect of RAL and HAFi on keratinocyte CD44 expression might be due either to a combined effect of RAL and HAFi on CD44 gene transcription or to a presently unknown feedback mechanism in order to upregulate the cell surface CD44 for internalization and degradation of increased HA in interker- atinocyte spaces. Topical application of RAL and HAF resulted in a significant increase of pro-HB-EGF and HB-EGF protein levels in mouse epidermis as well as a slight increase in erbB1 protein expression. Figure 6. Hypothetical assembly of CD44v3/HB-EGF/erbB1/HAS/Hyal (hyalurosome) complex and the mechanism of RAL and HAFi- induced keratinocyte proliferation. Schematic representation of the assembly of the putative complex: HAFi mediates CD44v3 and heparan- sulfate-bound pro-HB-EGF aggregation, resulting in the recruitment and activation of an MMP/ADAM. RAL increases the endogenous HA production by induction of HAS which will contribute to the aggregation of CD44v3 and pro-HB-EGF and to the activation of MMP/ADAM. Pro-HB-EGF is cleaved by the MMP/ADAM and the resulting active moiety binds and activates erbB1, generating proliferation signals. Endogenous HA synthesis is controlled by HA-degrading enzyme hyaluronidase (Hyal). doi:10.1371/journal.pone.0014372.g006 PLoS ONE | www.plosone.org 8 December 2010 | Volume 5 | Issue 12 | e14372