Tapinarof, an Aryl Hydrocarbon Receptor Ligand, Mitigates Fibroblast Activation in Thyroid Eye Disease: Implications for Novel Therapy

Abstract

Purpose: In thyroid eye disease (TED), activation and proliferation of orbital fibroblasts (OFs) promotes remodeling and causes an increase in the volume of orbital tissue. Platelet-derived growth factors (PDGFs) are elevated in TED and promote OF activation. The aryl hydrocarbon receptor (AHR), a ligand activated nuclear receptor, is important in regulating OF activation. AHR ligands have been evaluated as therapeutic agents for inflammatory diseases. Here, we hypothesize that AHR ligands will block PDGF-induced signaling in TED OFs.

Methods: OFs from both non-TED and TED patients were treated with PDGFβ, with or without the AHR ligands 6-Formylindolo[3,2-b]carbazole (FICZ) or tapinarof. Cell viability was measured by the Alamar Blue assay. Cell proliferation was quantified using the BrdU assay. Cell lysates were collected and analyzed by Western blotting and real-time quantitative PCR (RT-qPCR) to measure PDGF and AHR signaling. Scratch assays were used to measure OF migration.

Results: PDGFβ induced proliferation in TED OFs significantly more than in non-TED OFs. Additionally, PDGFβ increased phosphorylation of AKT and expression of thymidylate synthase (TYMS). PDGFβ dependent proliferation and downstream signaling were attenuated by FICZ or tapinarof. TYMS and other PDGF target genes were upregulated by PDGFβ and reduced by AHR activation. PDGFβ induced TED OF migration while both FICZ and tapinarof diminished this effect.

Conclusions: PDGF signaling led to increased proliferation and activation of TED OFs. Treatment of TED OFs with the AHR ligands, FICZ and tapinarof, mitigated PDGF induced effects. These studies support the concept that AHR and PDGF signaling could form the basis for new TED therapeutics.

Figure 1.

PDGFβ increases orbital fibroblast (OF) proliferation, activation, PI3K/AKT signaling, and TYMS expression. (A) Non-TED and TED OFs were treated with vehicle, 1, 10, 25, 50, or 100 ng/mL PDGFβ for 72 hours. The nucleotide analog BrdU was added to measure DNA synthesis. After culture, cells were fixed and the BrdU label was detected by ELISA. PDGFβ treatment resulted in a dose-dependent increase in OF proliferation. TED OFs accumulated significantly more BrdU than non-TED OFs. Results are presented as means ± SEM from triplicate wells. The experiment was performed in four patients with non-TED and four patients with TED OFs. (B) Equal numbers of non-TED or TED OFs were treated with PDGFβ as indicated for 48 to 72 hours. Cells were then treated with Alamar Blue assay reagent for 1 hour. Fluorescent signal showing Alamar Blue reagent reduction was measured. Vehicle (DMSO) treated cells served as control. The experiment was performed in four non-TED and four TED OFs. (C) Non-TED and TED OFs were grown to confluence and treated with PDGFβ for 48 hours. Cells were lysed and analyzed by Western blot for phospho-AKT, total AKT, TYMS, and β-Tubulin (loading control). Phospho-AKT levels were normalized to total AKT levels. TYMS was normalized to β-tubulin levels. (D) Quantification of Western blots for phospho-AKT and TYMS. The experiments were repeated in at least three non-TED and three TED OF strains with representative results shown. One-way ANOVA with Dunnett's multiple comparisons was used to analyze the data. * P ≤ 0.05, ** P = 0.005, *** P < 0.0008, and non-TED versus TED: † P ≤ 0.05.

Figure 2.

PDGFβ signaling is blocked by imatinib. TED OFs were pretreated with imatinib (5 µM) for 1 hour followed by stimulation with PDGFβ (25 ng/mL) for 48 hours. (A) Cells were lysed and analyzed by Western blot for phospho-AKT and total AKT. Phospho-AKT was normalized to total AKT. (B) Quantification of Western blots for phospho-AKT from three TED OF strains. (C) TYMS, AHR, and CYP1B1 were analyzed by Western blot with levels normalized to β-Tubulin. (D) Quantification of Western blots for TYMS, AHR, and CYP1B1. Uncropped blots are presented in the Supplementary Data File. Vehicle (DMSO) treated cells served as control The experiment was repeated in three different TED OF strains with representative results shown. One-way ANOVA with Dunnett's multiple comparisons was used to analyze the data. * P ≤ 0.05, ** P = 0.005, *** P < 0.0008, and PDGF versus treatments: # P ≤ 0.05, ## P ≤ 0.005.

Figure 3.

AHR activation by FICZ or tapinarof attenuates PDGF signaling. (A) The 2D structures of FICZ and tapinarof. (B) TED OFs were treated with vehicle (DMSO) or tapinarof as indicated for 48 hours. Cells were analyzed by Western blot for AHR, CYP1B1, and β-tubulin (loading control). Tapinarof led to a dose dependent reduction in AHR levels and a dose dependent induction of CYP1B1 expression. (C) Quantification of Western blots for AHR and CYP1B1. (D) Non-TED and TED OFs were pretreated for 1 hour with FICZ (1 µM) or tapinarof (1 µM) followed by 48-hour stimulation with PDGFβ (25 ng/mL). Cells were harvested and analyzed by Western blot for phospho-AKT, total AKT, TYMS, CYP1B1, and β-tubulin (loading control). Phospho-AKT was normalized to total AKT, whereas other protein levels were normalized to β-tubulin. FICZ and tapinarof induced CYP1B1 levels. PDGFβ treatment induced phospho-AKT and TYMS, whereas FICZ and tapinarof mitigated these effects. Uncropped blots are presented in the Supplementary Data File. (E) Quantification of Western blots for phospho-AKT, TYMS, and CYP1B1. Vehicle (DMSO) treated cells served as control. The experiment was repeated in three non-TED and three TED OF strains with representative results shown. One-way ANOVA with Dunnett's multiple comparisons was used to analyze the data. * P ≤ 0.05, ** P = 0.005, *** P < 0.0008, and PDGF versus treatments: # P ≤ 0.05, ## P ≤ 0.005, ### P ≤ 0.0005.

Figure 4.

AHR activation by FICZ and tapinarof attenuates PDGFβ dependent gene expression in TED OFs. TED OFs were pretreated for 1 hour with FICZ (1 µM) or tapinarof (1 µM) followed by 48-hour stimulation with PDGFβ (25 ng/mL). Total RNA was isolated and analyzed by RT-qPCR. (A) CYP1B1, (B) thymidylate synthase (TYMS), (C) dihydrofolate reductase (DHFR), and (D) JUN mRNAs were analyzed. Levels were normalized using the ΔΔCT method to the average CT value of the housekeeping genes, 18S rRNA, TATA binding protein (TBP), and β-actin (ACTB). FICZ and tapinarof increase expression of the AHR dependent gene, CYP1B1. Further, PDGFβ induces TYMS, DHFR, and JUN, whereas FICZ and tapinarof block their expression. There were three replicates per treatment. The experiment was repeated in three different TED OF strains with representative results shown. One-way ANOVA with Dunnett's multiple comparisons of vehicle versus treatments: * P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.0001 and PDGF versus treatments: ### P ≤ 0.0005.

Figure 5.

AHR ligands inhibit PDGF-induced cell migration in TED OFs. Non-TED and TED OFs were treated with vehicle or 25 ng/mL PDGFβ with or without 1 uM FICZ or tapinarof for 72 hours. The nucleotide analog BrdU was added to measure DNA synthesis. After culture, cells were fixed and the BrdU label was detected by ELISA as described in the Methods section. (A) PDGFβ treatment increased proliferation, whereas FICZ and tapinarof blocked PDGF induced proliferation in both TED and non-TED OFs. (B) Next, confluent TED OFs were subjected to a wound assay and pretreated with FICZ (1 µM) or tapinarof (1 µM) for 1 hour. PDGFβ (25 ng/mL) was then added to stimulate cell migration. Images were captured at 0, 24, 48, and 72 hours. (C) The area of the scratch was quantified using ImageJ, with the initial wound area normalized to 1.0. Scale bar = 200 µm. Data represent mean ± standard error of the mean from four technical replicates. The experiment was repeated in three different OF strains with representative results shown. One-way ANOVA with Dunnett's multiple comparisons test was used to assess statistical significance vehicle versus treatments: * P ≤ 0.05, ** P ≤ 0.005, *** P ≤ 0.0008 and PDGF versus treatments: ### P ≤ 0.0005, and non-TED versus TED: † P ≤ 0.05.