Kaurenoic acid activates TGF-β signaling

Background: Kaurenoic acid (ent-kaur-16-en-19-oic acid: KA) is a key constituent found in the roots of Aralia continentalis Kitagawa (Araliaceae) that has been used for treating rheumatism in traditional Asian medicine. Hypothesis: Although KA was reported to suppress inflammation by activating Nrf2, the anti-inflammatory function of KA is less characterized. Given the complex nature of the inflammatory response and the critical role of TGF-beta in resolving inflammation, we hypothesized that KA suppresses inflammatory response by activating TGF-beta signaling. Methods: Murine macrophage RAW 264.7, human lung epithelial cell MRC-5, and a TGF beta RII defective cell HCT116 were treated with various amounts of KA. KA was also administered to mouse lung via intratracheal (i.t.) route. Phosphorylated Smad2 and Smad3 were analyzed by western blot. TGF beta-dependent gene expression was determined by immunoblotting of alpha-SMA and luciferase assay. Results: KA induced the phosphorylation of Smad2 and Smad3, key activator molecules in TGF-beta signaling. EW7197, an inhibitor for activin receptor-like kinase 5/TGF-beta receptor I (TGF beta R1) suppressed KA-mediated phosphorylation of Smad2. Similarly, KA failed to phosphorylate Smad2 in HCT116, suggesting that KA acts through the prototypic TGF beta R. KA treatment increased the transcriptional activity driven by a Smad-binding element in a luciferase reporter assay and induced the alpha-smooth muscle actin (alpha-SMA). Similarly, i.t. KA induced the phosphorylation of Smad2 and increased the expression of alpha-SMA in mouse lungs. Conclusion: KA activated TGF-beta signaling, suggesting that TGF beta signaling is associated with KA suppressing inflammation.