The activation of PPARγ by curcumin results in the interruption of canonical Wnt signaling in activated HSCs in vitro
Additional experiments were conducted to further test our above assumption. To evaluate the role of PPARγ activation in the curcumin-caused interruption of canonical Wnt signaling, HSCs were transfected with the plasmid TOPflash or FOPflash. The cells were pre-treated with the PPARγ antagonist PD68235 (20 μM) for 30 min prior to the addition of curcumin (20 μM) for additional 24 hr. Luciferase activity assays in Fig. 10A indicated that compared to the untreated control (the 1st black column on the left), curcumin reduced, as expected, luciferase activities in cells transfected with TOPflash (the 2nd black column). The pretreatment with PD68235 significantly attenuated the inhibitory effect of curcumin (the 4th black column). Curcumin with or without PD68235 caused no significant changes in luciferase activities in cells transfected with the control plasmid FOPflash (white columns). These results suggested that the activation of PPARγ played a critical role in the curcumin-caused interruption of canonical Wnt signaling in HSCs.
To further confirm the inhibitory role of PPARγ activation in the interruption of canonical Wnt signaling, HSCs were co-transfected with the plasmid TOPflash or FOPflash, plus the cDNA expression plasmid pPPARγ. Transfected cells were cultured for 24 hr in media with 10% FBS, which contains enough agonists to activate PPARγ in HSCs 19, 23, 24. Luciferase activity assays in Fig. 10B revealed that forced expression of PPARγ dose-dependently reduced luciferase activities in cells transfected with TOPflash, but not in cells transfected with FOPflash, confirming that the activation of PPARγ interrupted canonical Wnt signaling in HSCs. Additional experiments verified these observations and demonstrated that the activation of PPARγ by PGJ2 dose-dependently reduced luciferase activities in HSCs transfected with TOPflash (Fig. 10C). Taken together, these results indicated that the activation of PPARγ by curcumin resulted in the interruption of canonical Wnt signaling in activated HSCs in vitro.