Imiquimod is known to exert its effects through Toll-like receptor 7 (TLR7) and/or TLR8, resulting in expression of proinflammatory cytokines and chemokines. Keratinocytes have not been reported to constitutively express TLR7 and TLR8, and the action of imiquimod is thought to be mediated by the adenine receptor, not TLR7 or TLR8. In this study, we revealed the expression of TLR7 in keratinocytes after calcium-induced differentiation. After addition of calcium to cultured keratinocytes, the immunological responses induced by imiquimod, such as activation of NF-κB and induction of TNF-α and IL-8, were more rapid and stronger. In addition, imiquimod induced the expression TLR7, and acted synergistically with calcium to induce proinflammatory cytokines. We confirmed that the responses induced by imiquimod were significantly inhibited by microRNAs suppressing TLR7 expression. These results suggest that TLR7 expressed in keratinocytes play key roles in the activation of NF-κB signaling by imiquimod, and that their modulation in keratinocytes could provide therapeutic potential for many inflammatory skin diseases.

Melanosomes are synthesized in melanocytes and transferred to neighboring keratinocytes. However, the associations of melanosome uptake with the proliferation and differentiation of keratinocytes are not fully understood. We examined the associations of melanosome uptake with keratinocyte differentiation and proliferation. SV40T-transformed human epidermal keratinocytes (SV-HEKs) were treated with isolated melanosomes. The effects of melanosome uptake on the proliferation and differentiation of the keratinocytes were analyzed by Western blotting and flow cytometry. The relationship between melanosome uptake and keratinocyte differentiation status was verified by determining the melanin content in the cells. Melanosomes reduced the proliferation of SV-HEKs in a dose-dependent manner, but did not induce differentiation. Melanosome uptake was higher in differentiating keratinocytes compared to non-differentiating keratinocytes, and inhibited significantly by PAR-2 inhibitor. Melanosomes inhibit keratinocyte proliferation. Moreover, melanosome uptake is influenced by keratinocyte differentiation status, being highest in mid-stage differentiating keratinocytes in a PAR-2 dependent manner.

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