HA-RAL Synergy in Skin Figure 1. The effect of retinoids and HAFi in vitro. A. RAL is the most potent retinoid to induce keratinocyte proliferation. Keratinocytes from DBA/1 mice were cultured in 96-well plates. On day 2 of culture, HAFi (100 mg/ml), RAL (2 mM), RA (2 mM), ROL (2 mM), ROLP (2 mM), RAL+HAFi, RA+HAFi, ROL+HAFi or ROLP+HAFi was added to the cultures. 48 hrs later 1 mCi of [3H]thymidine was added to each well. All experiments were done in triplicate and repeated 5 times. The results are presented as the mean incorporated counts per minute 6 SEM of three wells per group. ***p,0.001(HAFi versus none; RAL versus none, RA, ROL and ROLP; RAL+HAFi versus all; ROL+HAFi versus none; RA+HAFi versus none; ROLP+HAFi versus none) (student’s t test). B. RAL-induced HA synthesis is required for keratinocyte proliferation. Keratinocytes from DBA/1 mice were cultured in 96-well plates. On day 2 of culture, RAL (2 mM), 4-MU (0.6 mM) [alone or together with RAL (RAL+4-MU) or 30 (4-MU-30+RAL) or 60 (4-MU-60+RAL) minutes before RAL] or hyaluronidase (1 U/ml) [alone or together with RAL (RAL+Hyal)] was added to the cultures. 48 hrs later 1 mCi of [3H]thymidine was added to each well. All experiments were done in triplicate and repeated 5 times. The results are presented as the mean incorporated counts per minute 6 SEM of three wells per group. ***p,0.001(RAL versus none, RAL+4-MU, 4-MU-30+RAL and 4-MU-60+RAL; RAL+4-MU, 4-MU-30+RAL and 4- MU-60+RAL versus none) (student’s t test). C. RAL induces in vitro mouse keratinocyte proliferation which is inhibited by anti-erbB1 and TIMP-3. Keratinocytes from SKH1 and DBA/1 mice were cultured in 96-well plates. On day 2 of culture, RAL (2 mM), monoclonal anti-human amphiregulin neutralizing antibody (100 ng/ml), monoclonal anti-human erbB1 neutralizing antibody (isotype IgG1) (100 ng/ml) or mouse recombinant TIMP-3 (100 ng/ml) was added to the cultures. Mouse IgG1 was used as a control of anti-erbB1. 48 hrs later 1 mCi of [3H]thymidine was added to each well. All experiments were done in triplicate and repeated 5 times. The results are presented as the mean incorporated counts per minute 6 SEM of three wells per group. ***p,0.001(RAL versus none; anti-AR+RAL versus none; anti-erbB1+RAL versus none; TIMP3+RAL versus none; mouse IgG1 versus RAL) (student’s t test). D. RAL and HAFi show a synergy in vitro which is CD44- and HB-EGF-dependent. Keratinocytes from SKH1, DBA/1 and CD44-/- mice were cultured in 96-well plates. On day 2 of culture, RAL (2 mM), HAFi (100 mg/ml), human HB-EGF (50 ng/ml) or mouse anti-human HB-EGF neutralizing antibody (100 ng/ml) was added to the cultures. 48 hrs later 1 mCi of [3H]thymidine was added to each well. All experiments were done in triplicate and repeated 5 times. The results are presented as the mean incorporated counts per minute 6 SEM of three wells per group. ***p,0.001 (HAFi, RAL+HAFi and HB-EGF versus none; anti-HB-EGF+HAFi versus HAFi; anti-HB-EGF+RAL+HAFi versus RAL+HAFi); **p,0.01 (RAL versus none; HB- EGF versus none; anti-HB-EGF+RAL versus RAL; antiHB-EGF+HAFi versus HAFi) (student’s t test). doi:10.1371/journal.pone.0014372.g001 superficial and reticular dermis in SKH1 hairless and DBA/1 but not in CD442/2 mice (data not shown). In vivo proliferative response of mouse epidermis to RAL is CD44-dependent The effect of skin CD44 expression loss on epidermal hyperplasia induced by topically-applied RAL was addressed by comparing the histology of retinoid-treated skin areas of CD442/2 and normal littermates. Topical application of 0.05% RAL for 3 days resulted in a significant epidermal hyperplasia and keratinocyte proliferation as determined by quantitation of Ki67 in the back skin of SKH1 hairless and DBA/1 mice, whereas no epidermal hyperplasia and keratinocyte proliferation was observed in CD442/2 mice. (Figure 2A, B, C). PLoS ONE | www.plosone.org 3 December 2010 | Volume 5 | Issue 12 | e14372