In order to screen small molecule modulators of Frizzled receptor internalization and develop an assay compatible with high throughput screening, we generated a U2OS cell line stably expressing Frizzled1-GFP (Fzd1GFP-U2OS). The cellular distributions of Frizzled1-GFP chimeras were initially assessed by confocal microscopy. Frizzled1-GFP localized predominantly to the plasma membrane with almost no internalized vesicles present when the cells were not stimulated with Wnt ligands (). When treated with Wnt3A conditioned medium, the cells showed a minimal internalization of receptor fluorescence (), whereas cells exposed to Wnt5A conditioned medium demonstrated a moderate amount of intracellular fluorescence (). These observations indicated that Frizzled1 internalization could provide a readout for agonist/ligand activity.
Wnt-mediated Frizzled1-GFP Internalization
Over 1200 FDA-approved drug and drug-like compounds from the Prestwick Chemical Library were screened at 12.5 μM concentration in a 384-well format. This primary screen revealed the hit niclosamide (Prestwick 01D11), which produced much more robust internalization () than even Wnt3A or Wnt5A stimulation (). To verify the result, the Fzd1GFP-U2OS cells were also treated with niclosamide obtained from an alternate supplier (Sigma), and similarly strong internalization of Frizzled1-GFP was observed (). As a control, human EGF Receptor 2-GFP (Her2-GFP) expressed on the plasma membrane of U2OS cells (X.-R. Ren, unpublished data) did not internalize when treated with an equivalent concentration of niclosamide ().
Niclosamide-induced Frizzled1-GFP Internalization
During our primary screening, we identified an additional 25 small molecule compounds () that had some effect on Frizzled1-GFP internalization, but little or no effect on Wnt signaling as assessed by the TOPFlash luciferase reporter assay (data not shown). These 25 compounds were therefore not studied further, and the small molecule hit niclosamide, which demonstrated a potential for modulating Wnt signaling (see sections below), was investigated in detail.
Summary of Frizzled1-GFP Internalization Screening
To assess the effect of niclosamide on Frizzled1 internalization, we employed a method independent of and complementary to our primary screening methodology using biotin labeling of the Frizzled1-GFP plasma membrane receptors. First, Fzd1GFP-U2OS cells were surface biotinylated at 4 °C to label only the cell surface receptor population. Next the labeled cells were incubated at 37 °C to allow receptor internalization in the presence of niclosamide. Receptors that internalize in this assay will have their biotin label protected from glutathione cleavage and can be visualized using anti-GFP immunoblots. In , Lane 1 shows the total biotin labeled cell surface Frizzled1-GFP, and the nearly complete removal of Frizzled1 bands in Lane 2 demonstrates that receptors on the cell surface (4 °C, not internalized) are susceptible to glutathione cleavage of their biotin label. In contrast, biotinylated Frizzled1-GFP receptors exposed to niclosamide at the permissible internalization temperature of 37 °C (, Lane 4) produce a strong immunoblot signal compared to cells not treated with niclosamide (, Lane 3), indicating that the niclosamide-internalized Frizzled1 receptors originate on the plasma membrane.
Measurement of Frizzled1 Receptor Internalization by Biotinylation
To determine the time-course of internalization for Frizzled1-GFP receptors we measured the accumulation of cytosolic puncta/vesicles over 6 hrs (). is a graphical representation of the internalization time course showing a t1/2 of 2.4 ± 0.5 hr for the niclosamide-induced Frizzled1 internalization. shows the dose-dependence at the 6 hr time point of Frizzled1-GFP internalization in the presence of increasing concentrations of niclosamide. Between 1 to 2 μM niclosamide concentration we observe a significant increase in the number of internalized receptors suggesting that the potency for niclosamide induced internalization is in the low micromolar range ().
Time Course of Niclosamide-stimulated Frizzled1-GFP Internalization
Dose-dependence of Niclosamide Stimulated Frizzled1-GFP Internalization
Many classes of membrane receptors internalize in clathrin coated pits. In particular, β2
-adrenergic receptors are prototypical for clathrin-dependent internalization of G protein-coupled receptors (12
), and Transferrin is a well-documented standard for clathrin-mediated internalization in general (17
). The demonstration that internalized Frizzled1-GFP co-localizes with either β2
-adrenergic receptor-RFP or Transferrin in intracellular vesicles would indicate Frizzled1 also internalizes in a similar manner. show cells expressing both β2
AR and Frizzled1 receptors prior to activation, and under these conditions the two receptors are not intracellularly co-localized. Exposure to isoproterenol and niclosamide for 2 or 6 hrs () results in multiple overlapping intracellular distributions of each receptor. Moreover, internalized Transferrin at 2 hrs has significant co-localization with internalized Frizzled1 (). These data suggest that niclosamide-induced Frizzled1 internalization occurs through clathrin-coated pits.
Colocalization of niclosamide-stimulated Frizzled1-GFP with the β2-adrenergic Receptor or Transferrin
Dishevelled proteins (Dishevelled-1, 2, and 3 in mammalian cells) are intracellular molecules transducing Frizzled signaling. To assess the effect of niclosamide on Wnt signaling mechanism, we examined protein expression of Dishevelled. In U2OS cells stimulated with either control or Wnt3A conditioned medium, treatment of niclosamide for 6 hrs results in dramatic reduction of cytosolic Dishevelled-2 protein (). The half maximal reduction of Dishevelled-2 occurs at a niclosamide concentration of approximately 1 μM (). No endogenous Dishevelled-2 was detected in the membrane fraction, and endogenous Dishevelled-1 and 3 were not detectable using commercial antibodies (Data not shown).
Niclosamide inhibits the cytosolic expression of endogenous Dishevelled-2
LEF/TCF transcription factor reporter (TOPFlash) assay is a general readout for canonical Wnt signaling pathways. To assess the effect of niclosamide on Wnt signaling activity, we generated an HEK293 cell line that stably expressed a LEF/TCF transcription factor reporter plasmid (TOPFlash) that responds to Wnt mediated β-catenin induction and a Renila luciferase plasmid that serves as an internal control. The ability of niclosamide to promote Frizzled1 internalization suggests agonist or partial agonist-like behavior. Niclosamide alone does not produce a statistically significant increase in the TOPFlash (LEF/TCF) reporter signal (). Upon Wnt3A stimulation, a 140 fold induction of the LEF/TCF reporter signal was observed (). Remarkably, the addition of niclosamide to the Wnt3A conditioned medium blocked the increase of the reporter signal observed with Wnt3A alone (), indicating niclosamide inhibits Wnt/Frizzled signaling induced by a full agonist (Wnt in this case). The inhibitory effect is dose-dependent with an IC50 of 0.5 ± 0.05 μM ().
Niclosamide is an Inhibitor of Wnt3A Signaling
The accumulation of cytosolic β-catenin is a measure of canonical Wnt signaling (15
). The reporter assay indicates that Wnt inducible cytosolic β-catenin accumulation should be reduced in the presence of niclosamide. demonstrates that niclosamide prevents Wnt3A-stimulated cytosolic β-catenin stabilization (Compare Lane 3 and 4, upper panel, cytosol). However, the membrane-bound β-catenin levels are relatively unchanged (, bottom panel, membrane), indicating the reduction in the signaling β-catenin pool is not due to a loss of β-catenin expression. The potency of niclosamide inhibition in Wnt-mediated β-catenin stabilization can be determined from the dose dependence presented in the immunoblots of . The half maximal inhibition of Wnt3A signaling occurs at a niclosamide concentration of approximately 1 μM.
In summary, our data indicate that niclosamide promotes Frizzled1 internalization, down regulates the expression of Dishevelled-2 protein, and inhibits Wnt3A-stimulated LEF/TCF (TOPflash) reporter activity and β-catenin stabilization. Therefore niclosamide functions as an inhibitor for Wnt signaling.