Resatorvid-based Pharmacological Antagonism of Cutaneous TLR4 Blocks UV-induced NF-κB and AP-1 Signaling in Keratinocytes and Mouse Skin

Abstract

Cutaneous exposure to solar ultraviolet (UV) radiation is a major causative factor in skin carcinogenesis, and improved molecular strategies for efficacious chemoprevention of nonmelanoma skin cancer (NMSC) are urgently needed. Toll-like receptor 4 (TLR4) signaling has been shown to drive skin inflammation, photoimmunosuppression, and chemical carcinogenesis. Here we have examined the feasibility of genetic and pharmacological antagonism targeting cutaneous TLR4 for the suppression of UV-induced NF-κB and AP-1 signaling in keratinocytes and mouse skin. Using immunohistochemical and proteomic microarray analysis of human skin, we demonstrate for the first time that a significant increase in expression of TLR4 occurs in keratinocytes during the progression from normal skin to actinic keratosis, also detectible during further progression to squamous cell carcinoma. Next, we demonstrate that siRNA-based genetic TLR4 inhibition blocks UV-induced stress signaling in cultured keratinocytes. Importantly, we observed that resatorvid (TAK-242), a molecularly targeted clinical TLR4 antagonist, blocks UV-induced NF-κB and MAP kinase/AP-1 activity and cytokine expression (Il-6, Il-8, and Il-10) in cultured keratinocytes and in topically treated murine skin. Taken together, our data reveal that pharmacological TLR4 antagonism can suppress UV-induced cutaneous signaling, and future experiments will explore the potential of TLR4-directed strategies for prevention of NMSC.

Figure 1. TLR4 is expressed by basal epidermal keratinocytes in normal human skin and is up-regulated in actinic keratosis and SCC

(A) Matched biopsies from sun-protected normal skin, sun-damaged (SD) skin and actinic keratoses (AK) were stained for TLR4 expression. TLR4 strongly stains in the basal layer of the epidermis in normal samples and increases in cell layer involvement during the progression to AK. (B) The thickness of TLR4 stained cell layers in (A) was quantified using Image J and statistically compared using student’s t test. (C) A tissue microarray (NBP-2-30229, Novus) containing 9 normal specimens and 31 cutaneous SCCs was stained for TLR4 (left and middle). H-scores for the samples were statistically compared using the Mann-Whitney test (right). (D) Reverse phase protein microarray (RPPA) was used to quantify expression of TLR4 and key related inflammatory proteins in SD epidermis, AKs and SCCs. The horizontal line in the box is the median of the dataset. Expression of each analyte is significantly increased during progression according to the Kruskal-Wallis test (p < 0.05).

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

(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.

Figure 3. Resatorvid inhibits UV-induced stress signaling in human primary keratinocytes

(A) Primary human keratinocytes were treated with SSL (40 kJ/m² UVA/ 2.7kJ/m² UVB), post-treated with resatorvid and harvested for Western blot 1 hr later. (B, C) Primary human keratinocytes were treated as in (A) and harvested at 18 hr for qRT-PCR analysis of IL-6 and IL-8 mRNA, respectively (p < 0.05).

Figure 4. Resatorvid inhibits UV-induced acute inflammatory signaling and potentiates cell death in mouse epidermis

SKH-1 mice were treated with an acute dose of SSL (105 kJ/m² UVA/6.4 kJ/m² UVB) and post-treated topically with 0.5% resatorvid or acetone (vehicle). 24 hr later, mice were sacrificed and back skins were harvested. (A) Epidermal protein lysates were analyzed by immunoblot analysis for UV-induced phosphorylation of p38 and p65/NF-κB. (B) qRT-PCR of epidermal RNA shows induction of IL-10 mRNA which is significantly inhibited by resatorvid. (C) Skins were sectioned for IHC and stained for cleaved caspase 3, 600× magnification is shown. (D) Quantification of stained cells per field in 400× images from skins in C (p < 0.05). (E) HaCaT keratinocytes were pretreated with 10 µM resatorvid for 1 hr prior to SSL treatment (40 kJ/m² UVA/ 2.7kJ/m² UVB) and post-treated with the same dose of the compound. Cells were harvested 18 hr later for analysis of cell death using annexin V/PI staining via flow cytometry.

Figure 5

TLR4 signaling and TLR4-directed modulatory strategies in keratinocytes. Apart from ligand-dependent activation (exogenous: LPS; endogenous: HMGB1), UV-induced TLR4 activation in keratinocytes plays a role in the control of inflammation, proliferation, cell survival and differentiation, partly though modulation of stress signaling pathways (NF-κB, AP-1). Small molecule antagonism of TLR4-dependent signaling employing resatorvid or other pharmacological modulators may represent a novel approach for the suppression of UV-induced inflammatory signaling and tumorigenesis.