Figure 1.
Expression of TLR7 in differentiated keratinocytes.
(A) Keratinocytes were differentiated in the presence of 1.2 mM calcium. Cells were harvested at 1, 3, 7, and 14 days. TLR7 mRNA expression were assessed by real-time PCR analysis. Relative expression level was standardized using cyclophilin as an internal control. (B) Whole-cell extracts of keratinocytes that had undergone calcium-induced differentiation were subjected to Western blot analysis. Quantification of Western bands were done using Densitometer. Data are expressed as fold induction ± SE (n = 3). *P<0.05 versus non-calcium-treated cells.
Figure 2.
Imiquimod induces TLR7 expression in keratinocytes.
(A) Keratinocytes were grown in low calcium condition (non-differentiated) or 1.2 mM calcium condition for 7 days (differentiated). Cells were treated with 100 µM imiquimod for the indicated time points, and were then assessed for TLR7 by Western blotting. Quantification of Western bands were done using Densitometer. Data are expressed as fold induction ± SE (n = 3). *P<0.05 versus non-calcium-treated cells. (B) Aldara (5% imiquimod cream) was applied to the back skin of 6 week old ICR mouse for 7 days. Paraffin section was stained with anti-TLR7 antibody. Arrows indicate the TLR7 expression is increased in upper epidermis.
Figure 3.
Imiquimod induces cytokine expression in keratinocytes.
(A) Keratinocytes grown in low- and high-calcium (day 7) media were treated with 100 µM imiquimod for the indicated time points, and were then assessed for TLR7 by Western blotting. (B) IL-8 and TNF-α were evaluated by ELISA. Data represent the mean ± SE (n = 3).
Figure 4.
Imiquimod increases NF-κB transcriptional activity in keratinocytes.
(A) Keratinocytes were stimulated with 100 µM imiquimod for the indicated time points. Cell lysates were assessed by Western blotting using an antibody against phospho-IκBα. Quantification of Western bands were done using Densitometer. Data are expressed as fold induction ± SE (n = 3). *P<0.05 versus non-calcium-treated cells. (B) Keratinocytes were transduced with NF-κB reporter adenovirus and then treated with 100 µM imiquimod. Luciferase assays were performed and data are expressed as fold induction. Data represent the mean ± SE (n = 3). *P<0.05, **P<0.01.
Figure 5.
Imiquimod induces the expression of TNF-α via TLR7.
Keratinocytes cultured in 1.2(day 7) were transduced with adenoviruses expressing miR-scr and miR-TLR7, and then treated with 100 µM imiquimod. (A) TLR7 mRNA expression were assessed by real-time PCR analysis. Relative expression level was standardized using cyclophilin as an internal control. (B) TNF-α secretion was assessed by ELISA. Data represent the mean ± SE (n = 3). *P<0.05.
Figure 6.
Imiquimod induces activation of NF-κB via TLR7.
(A) Keratinocytes cultured in 1.2 mM calcium (day 7) were transduced with adenoviruses expressing miR-scr and miR-TLR7, together with NF-κB reporter adenovirus. After treatment with 100 µM imiquimod, luciferase assays were performed. Data are expressed as fold induction. Data represent the mean ± SE (n = 3). *P<0.05, **P<0.01. (B) Nuclear extracts were used for evaluation of NF-κB binding activity by EMSA. The specific bands indicate the binding of NF-κB in nuclear extracts from cells treated with 100 µM imiquimod. (C) Cells were transduced with adenoviruses expressing miR-scr and miR-TLR7, and then treated with 100 µM imiquimod. Protein samples were assessed in terms of p-IκBα and p-IRAK expression by Western blotting.