that CD44 is colocalized with another HB-EGF receptor, erbB1 on keratinocytes [15]. We have also shown that topically applied HAF of intermediate size (HAFi) traverse the skin and induce a CD44-dependent biological effect characterized by a skin regeneration in mice and elderly human patients showing dermatoporosis, the holistic word for human skin fragility and an emerging clinical problem due to chronological aging, long- term sun exposure and chronic use of corticosteroids [15,16,17]. To see whether retinoid-induced epidermal hyperplasia via HB- EGF was a CD44-related phenomenon, we compared the effect of different retinoids on in vitro proliferation of keratinocytes DBA/1 mice. We further tested the effect of the blockade of HA synthesis, HB-EGF, erbB1 and MMPs including MMP-7 on the proliferation of the keratinocytes of SKH1 hairless, DBA/1 and CD44-deficient (CD442/2) mice. We also analyzed the effect of RAL on the epidermal hyperplasia in SKH1 hairless, DBA/1 and CD442/2 mice, and in vivo expression of CD44v3, MMP-7, HB- EGF and its receptors in SKH1 hairless mice. To address the possibility that RAL and HAFi may have a synergy on keratinocyte proliferation and epidermal hyperplasia, we examined the effect of the combination of HAFi and RAL on the mouse skin in vitro and in vivo, and on atrophic skin of elderly patients with dermatoporosis. Results RAL is the most potent retinoid to induce keratinocyte proliferation To determine the effect of different retinoids on in vitro keratinocyte proliferation, we established primary cultures of keratinocytes from DBA/1 mice. Treatment of mouse keratino- cytes with 2 mM of RAL resulted in slight but the most significant increase in keratinocyte proliferation when compared to retinoic acid (RA), retinol (ROL) or retinoyl palmitate (ROLP) (Figure 1A). RAL-induced HA synthesis is required for keratinocyte proliferation To explore the role of HA synthesized by keratinocytes on RAL-induced keratinocyte proliferation, the keratinocytes of DBA/1 mice were treated with an HA synthesis inhibitor, 4- methylumbelliferone (4-MU), or hyaluronidase. RAL-induced keratinocyte proliferation was inhibited by 4-MU added to the cultures before or in the mean time with RAL (Figure 1B). In vitro proliferation of CD44-deficient keratinocytes is defective in response to RAL To further address the importance of the presence of CD44 in this proliferative effect, we tested the ability of proliferation of keratinocytes of CD44-deficient (CD442/) mice in response to RAL. In contrast to normal keratinocytes, no proliferation was observed in CD442/2 cells (Figure 1C). Anti-HB-EGF, anti-erbB1 and TIMP-3 inhibits RAL-induced keratinocyte proliferation in vitro Since HB-EGF is thought to be involved in retinoid-induced epidermal hyperplasia and because keratinocytes normally express several CD44 isoforms, including exon v3-containing variants (CD44v3) [18], which are substituted in heparan sulfate side chains and form a complex with matrix metalloproteinase 7 (MMP-7), heparin binding epidermal growth factor (HB-EGF) precursor (pro- HB-EGF) and its receptors (erbB4, erbB1) [14,15], we first compared the ability of CD442/2 and normal keratinocytes to proliferate in response to HB-EGF. HB-EGF (50 ng/ml) stimulated the proliferation of wild-type DBA/1 and, to a lesser extent, SKH1 HA-RAL Synergy in Skin hairless keratinocytes, whereas only a slight proliferation increase was observed in CD44-deficient keratinocytes (Figure 1C). We further analyzed the requirement of HB-EGF and its receptors in RAL-induced keratinocyte proliferation. Since erbB4 is not expressed in neither mouse [19] nor human [20] keratinocytes and erbB1 (epidermal growth factor receptor [EGFR]) is the principal receptor of HB-EGF in human keratinocytes [15,20], we tested the cultured keratinocytes of SKH1 hairless and DBA/1 mice for response to RAL in the presence of anti-HB-EGF (100 ng/ml) or anti-erbB1 (100 ng/ml) neutralizing antibodies. Anti-HB-EGF significantly inhibited RAL-induced proliferative response in normal keratinocytes (Figure 1C). Blocking antibodies against amphiregulin (AR), a growth factor which fails to bind CD44v3 [21], did not inhibit keratinocyte proliferation (Figure 1D). In contrast, anti-erbB1 significantly blocked the proliferation of keratinocytes (Figure 1D). Since MMP-7 was shown to form a complex with its substrate pro-HB-EGF and CD44v3 on the surface of some cells and MMPs [14] and the related a disintegrin and metalloproteinase (ADAM) family proteases [22] activate HB-EGF, we also tested the effect of tissue inhibitor of metalloproteinase-3 (TIMP-3) which inhibits the effect of MMPs including MMP-7 and of ADAMs, on the keratinocyte proliferation induced by RAL. Incubation of the keratinocytes of SKH1 hairless and DBA/1 mice with TIMP-3 (100 ng/ml) abrogated the RAL-induced keratinocyte proliferation (Figure 1D). RAL and HAFi show a synergy in in vitro proliferative response of mouse keratinocytes which is CD44- and HB- EGF-dependent To address the effect of the combination of different retinoids and HAFi on keratinocyte proliferation, we tested the response to 100 mg/ml of HAFi and 2 mM of ROL, RAL, RA and ROLP of cultured keratinocytes from the back skin of DBA/1 mice. Exposure to HAFi and RAL resulted in a more significant increase in keratinocyte proliferation than HAFi with other retinoids (Figure 1A). HAFi and RAL combination was more efficient in the proliferation of keratinocytes from the back skin of SKH1 hairless and DBA/1 mice than RAL or HAFi alone, whereas no proliferation was observed in CD442/2 cells (Figure 1D). The proliferative response was slightly higher in DBA/1 keratinocytes (Figure 1C). Addition of blocking anti-HB-EGF antibodies (100 ng/ ml) to RAL- and HAFi-treated cultures abrogated the proliferative response as in only RAL- or HAFi-treated cultures (Figure 1D). The effect of RAL and HAFi in vivo is CD44-dependent Topical application of the combination of 0.05% RAL and 0.2% HAFi for 3 days resulted in a significant epidermal hyperplasia in the follicular (data not shown) and interfollicular epidermis in SKH1 hairless and DBA/1 mice (Figure 2A). This hyperplasia was more significant when compared to that induced by RAL alone, but it was comparable to that induced by HAFi alone (Figure 2B). In addition, topically applied RAL and HAFi significantly increased the number of Ki-67-positive cells in the epidermis and dermis (Figure 2C). The increase in epidermal Ki- 67-positive cells was comparable to that with RAL or HAFi alone (data not shown). However, the increase in dermal Ki-67-positive cells was superior to that with RAL alone but it was not different when compared with the one with HAFi alone (data not shown). In contrast, topical application of RAL and HAFi, alone or in combination, to the back skin of CD442/2 mice did not result in epidermal hyperplasia or keratinocyte proliferation (Figure 2A, B, C, data not shown). Topically applied RAL and HAFi, as HAFi alone but not RAL alone, led to an increased cellularity in the PLoS ONE | www.plosone.org 2 December 2010 | Volume 5 | Issue 12 | e14372