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. Author manuscript; available in PMC: 2017 Dec 8.
Published in final edited form as: Photochem Photobiol. 2016 Dec 8;92(6):816–825. doi: 10.1111/php.12659

Figure 2. Genetic and pharmacological TLR4 antagonism attenuates UV-induced NF-κB and AP-1 stress signaling in immortalized keratinocytes.

Figure 2

(A) Mouse JB6+ cultured keratinocytes were transfected with two types of TLR4 siRNA or non-targeted control siRNA for 72hr and the level of TLR4 mRNA was quantified using quantitative real-time RT-PCR. (B) JB6+ keratinocytes which stably express an NF-κB reporter (“NF-κB keratinocytes”) were transfected as in (A) and treated with UVB (250 kJ/m2). TLR4 siRNA transfection resulted in significant inhibition of NF-κB luciferase signaling. (C) Human HaCaT keratinocytes which stably express an AP-1 luciferase reporter (“AP-1 keratinocytes”) were transfected with a mix of three TLR4-specific siRNAs or non-targeted control siRNA 72 hr prior to UVB treatment (250 kJ/m2). TLR4 siRNA significantly inhibited UV-induced AP-1 signaling in these cells. (D) NF-κB keratinocytes were stimulated with the classical TLR4 agonist LPS (0.6 µg/mL) in the presence of resatorvid (10 µM, pre and post) or vehicle (DMSO) and harvested 6hr later. Resatorvid significantly inhibited LPS-induced NF-κB-driven luciferase signaling in these cells. (E) Similarly, NF-κB keratinocytes were treated with UVB +/− 10 µM resatorvid (or vehicle) and harvested 6 hr later. Resatorvid significantly inhibited UV-induced NF-κB luciferase signaling in keratinocytes. (F) AP-1 keratinocytes also showed significant inhibition of UVB-induced AP-1 luciferase signaling due to pre and post-treatment with 5 µM or 10 µM of resatorvid. (G and H) Likewise, additional pharmacological inhibitors of the TLR4 signaling pathway are shown to significantly inhibit UVB-induced signaling in NF-κB or AP-1 luciferase reporter assays, respectively. ST2825 (10 µM) is an inhibitor of the TLR4 signaling cofactor MyD88. Ibudilast (25 µM) is a direct inhibitor of TLR4 activity. Carbenoxolone (40 µM) is an inhibitor of HMGB1 activity. (I) HaCaT keratinocytes were pre- and post-treated with resatorvid (10 µM), exposed to SSL (40 kJ/m2 UVA/2.6 kJ/m2 UVB) and harvested for Western blot analysis at the indicated timepoints. (J) JB6+ mouse keratinocytes were pre- and post-treated with 10 µM resatorvid or vehicle and SSL (same dose as in I). Cells were harvested 24 hr later for analysis of IRF3 gene expression using quantitative RT-PCR.