Inhibition of Wnt/beta-Catenin Signaling by p38 MAP Kinase Inhibitors Is Explained by Cross-Reactivity with Casein Kinase I delta/epsilon

SummaryWnt/β-catenin signaling plays essential roles in embryonic development, adult stem cell maintenance, and disease. Screening of a small molecule compound library with a β-galactosidase fragment complementation assay measuring β-catenin nuclear entry revealed TAK-715 and AMG-548 as inhibitors of Wnt-3a-stimulated β-catenin signaling. TAK-715 and AMG-548 are inhibitors of p38 mitogen-activated protein kinase, which has been suggested to regulate activation of Wnt/β-catenin signaling. However, two highly selective and equally potent p38 inhibitors, VX-745 and Scio-469, did not inhibit Wnt-3a-stimulated β-catenin signaling. Profiling of TAK-715 and AMG-548 against a panel of over 200 kinases revealed cross-reactivity with casein kinase Iδ and ɛ, which are known activators of Wnt/β-catenin signaling. Our data demonstrate that this cross-reactivity accounts for the inhibition of β-catenin signaling by TAK-715 and AMG-548 and argue against a role of p38 in Wnt/β-catenin signaling

Discovery of Novel Small Molecule Activators of β-Catenin Signaling

Wnt/β-catenin signaling plays a major role in embryonic development and adult stem cell maintenance. Reduced activation of the Wnt/β-catenin pathway underlies neurodegenerative disorders and aberrations in bone formation. Screening of a small molecule compound library with a β-galactosidase fragment complementation assay measuring β-catenin nuclear entry revealed bona fide activators of β-catenin signaling. The compounds stabilized cytoplasmic β-catenin and activated β–catenin-dependent reporter gene activity. Although the mechanism through which the compounds activate β-catenin signaling has yet to be determined, several key regulators of Wnt/β-catenin signaling, including glycogen synthase kinase 3 and Frizzled receptors, were excluded as the molecular target. The compounds displayed remarkable selectivity, as they only induced β-catenin signaling in a human osteosarcoma U2OS cell line and not in a variety of other cell lines examined. Our data indicate that differences in cellular Wnt/β-catenin signaling machinery can be exploited to identify cell type-specific activators of Wnt/β-catenin signaling

A model for signaling specificity of Wnt/Frizzled combinations through co-receptor recruitment

AbstractWnts control mammalian developmental morphogenesis and are critical for adult stem cell maintenance. Wnts initiate several intracellular signaling cascades, such as Wnt/β-catenin-, Wnt/Ca2+- and Wnt/ROR2-signaling. Signaling preference of Wnts for these various pathways is thought to depend on the repertoire of receptors present on recipient cells. Here, we propose a further refinement of this receptor model and hypothesize that Wnt signaling specificity depends on co-receptor recruitment upon binding of Wnt to Frizzled receptor molecules. In this model, recruitment of LRP5/6 leads to activation of Wnt/β-catenin signaling, whereas signaling through other pathways is mediated by recruiting ROR2

beta-Galactosidase enzyme fragment complementation for the measurement of Wnt/beta-catenin signaling

Wnt/beta-catenin signaling is an important regulator of cell polarity, proliferation, and stem cell maintenance during development and adulthood. Wnt proteins induce the nuclear accumulation of beta-catenin, which regulates the expression of Wnt-responsive genes through association with TCF/LEF transcription factors. Aberrant Wnt/beta-catenin signaling has been implicated in a plethora of pathologies and, most notably, underlies initiation and expansion of several cancers. Here, we apply enzyme fragment complementation to measure the nuclear accumulation of beta-catenin. beta-catenin was tagged with a peptide fragment of beta-galactosidase and transfected into cells expressing a corresponding deletion mutant of the enzyme exclusively in the nucleus. Stimulation of the cells with recombinant Wnt-3a restored beta-galactosidase activity in a dose-dependent manner with nanomolar potency. Using the assay, we confirmed that Wnt-5a represses beta-catenin-driven reporter gene activity downstream of nuclear entry of beta-catenin. In addition, we tested a library of >2000 synthetic chemical compounds for their ability to induce beta-catenin nuclear accumulation. The immunosuppressive protein kinase C inhibitor sotrastaurin (AEB-071) was identified as an activator of Wnt/beta-catenin signaling at micromolar concentrations. It was confirmed that the compound stabilizes endogenous beta-catenin protein and can induce TCF/LEF-dependent gene transcription. Subsequent biochemical profiling of >200 kinases revealed both isoforms of glycogen synthase kinase 3, as previously unappreciated targets of sotrastaurin. We show that the beta-catenin nuclear accumulation assay contributes to our knowledge of molecular interactions within the Wnt/beta-catenin pathway and can be used to find new therapeutics targeting Wnt/beta-catenin signaling.-Verkaar, F., Blankesteijn, W. M., Smits, J. F. M., Zaman, G. J. R. beta-Galactosidase enzyme fragment complementation for the measurement of Wnt/beta-catenin signaling. FASEB J. 24, 1205-1217 (2010). www.fasebj.or

Model prediction of Wnt-pathway activation upon WNT addition with AXIN2 feedback.

<p>β-catenin (referred to by its official gene name CTNNB1 in the figure) token levels predicted by our model with arc weight from t11 to AXIN varied from 0 (no feedback; solid lines) to 0.15 (high feedback, dashed lines), and initial WNT token levels at 3, 4 and 5 (top, middle and bottom panels, respectively). We observed three spectra of β-catenin stabilizations depending on initial WNT levels. The highest β-catenin stabilizations correspond to simulations without AXIN2 feedback (solid lines), whereas with high AXIN2 feedback the β-catenin stabilization was attenuated (dashed lines).</p

Illustration of Wnt/β-catenin signaling.

<p>(A) In the absence of an external WNT stimulus β-catenin (referred to by its official gene name CTNNB1 in the figure) is continuously degraded by a ‘destruction complex’ consisting of AXIN1, adenomatous polyposis coli (APC), casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3). (B) Extracellular WNT interacts with the membrane-bound receptors frizzled (FZD) and lipoprotein receptor-related protein (LRP). Dishevelled (DVL) interacts with the intracellular tail of FZD and sequesters AXIN1 to the plasma membrane forming a so-called ‘signalosome’. The ensuing depletion of the cytoplasmic pool of AXIN1 inhibits the formation of the destruction complex. β-catenin thereby stabilizes and translocates to the nucleus, where it interacts with TCF/LEF transcription factors activating transcription of specific target genes, including the negative feedback regulator <i>AXIN2</i>.</p

Petri net model of Wnt/β-catenin signaling.

<p>The model consists of 18 places (circles, representing gene or protein states), 11 transitions (boxes, representing protein complex formation, dissociation, translocation or gene expression) and 41 arcs (arrows, representing the direction of flow of the tokens). WNT initiates signaling by binding to FZD and LRP (t1), forming the WNT/FZD/LRP complex. DVL and AXIN1 then interact with this complex intracellularly (t2 and t3, respectively) forming a so-called ‘signalosome’. The signalosome dissociates (with a rate of once every 10 steps) into WNT/FZD/LRP/DVL and AXIN1 (t4). Note that β-catenin is referred to by its official gene name CTNNB1 in the figure. The β-catenin protein is produced every step (t9) by the <i>β-catenin</i> gene. AXIN1, APC, CK1 and GSK3 interact (t5) and form a ‘destruction complex’. The destruction complex binds β-catenin (t6) to mark it for degradation. The destruction complex is then either reused (t7) for another round of β-catenin degradation or dissociates (t8) into its components AXIN1, APC, CK1 and GSK3. Alternatively, β-catenin may interact with TCF/LEF in the nucleus (t10), leading to transcriptional activation of <i>AXIN2</i> (t11). Initial token levels are 0 (not shown), 1 or 5 (depicted in the places). Most arc weights are 1 (not shown), except for the nuclear translocation and interaction of β-catenin to TCF/LEF transcription factors, which has an incoming arc weight of 3 and an outgoing arc weight of 2 (depicted on the arcs).</p

Model simulation and experimental validation of Wnt-pathway activation upon GSK3 inhibition.

<p>(A) β-catenin (referred to by its official gene name CTNNB1 in the figure) token levels predicted by our model with initial GSK3 token levels ranging from 0 to 5. For GSK3 = 3, 4 or 5, we observed a flat β-catenin response. For GSK3 = 0, 1 or 2 β-catenin increases to low, moderate or high levels, respectively. (B) Graph combining the results from panels C and D to allow easy comparison to the modeling results depicted in panel (A), showing dose- and time-dependent activation of a Wnt/β-catenin responsive TCF/LEF luciferase reporter in HEK293T^WOO cells. For all curves with black data points (corresponding to panel C), luciferase activity was plotted relative to the vehicle control (not shown), which was set at 1 for each of the three time points (3, 8 and 24 hours). For the curve with white data points (corresponding to panel D), luciferase activity was plotted relative to the vehicle control, which was set at 1 for the t = 0 hours condition. (C) Reporter assay in HEK293T^WOO cells, showing dose-dependent activation at 3, 8 and 24 hours after stimulation with CHIR99021 (same concentrations as depicted in B). (D) Reporter assay in HEK293T^WOO cells, showing time-dependent activation upon treatment with 3 μM CHIR99021. Values were plotted relative to the DMSO control, which was set at 1 for t = 0 hours. (E) Western blot from the experiment depicted in (D), showing total and active (non-phosphorylated) β-catenin levels. Tubulin was used as a loading control. (F) Quantification of the Western blot shown in (E). Total and active β-catenin levels were normalized to tubulin. The increase in either total or active β-catenin levels was plotted relative to time point 0, for which the normalized levels were set to 1. Experiments were repeated two (C) or three (D-F) times. A representative experiment is shown.</p

Model simulation and experimental validation of Wnt-pathway activation upon WNT stimulation.

<p>(A) β-catenin (referred to by its official gene name CTNNB1 in the figure) token levels predicted by our model with initial WNT token levels ranging from 0 to 5. For WNT = 0, 1 or 2, we observed a flat β-catenin response. For WNT = 3, 4 and 5 β-catenin increases from low to moderate levels. (B) Graph combining the results from panels C and D to allow easy comparison to the modeling results depicted in panel (A), showing dose- and time-dependent activation of a Wnt/β-catenin responsive TCF/LEF luciferase reporter in HEK293T^WOO cells. For all curves with black data points (corresponding to panel C), luciferase activity was plotted relative to the vehicle control (not shown), which was set at 1 for each of the three time points (3, 8 and 24 hours). For the curve with white data points (corresponding to panel D), luciferase activity was plotted relative to the vehicle control, which was set at 1 for the t = 0 hours condition. (C) Reporter assay in HEK293T^WOO cells, showing dose-dependent activation at 3, 8 and 24 hours after stimulation with purified Wnt3a (same concentrations as depicted in B). (D) Reporter assay in HEK293T^WOO cells, showing time-dependent activation upon treatment with 100 ng/ml of Wnt3a. Values were plotted relative to the vehicle control, which was set at 1 for t = 0 hours. (E) Western blot from the experiment depicted in (D), showing total and active (i.e. non-phosphorylated) β-catenin levels. Since the soluble, signaling pool of β-catenin constitutes only a minor fraction of the total pool of β-catenin, the use of antibody against active β-catenin ensures that only the pool involved in WNT signaling is visualized. Tubulin was used as a loading control. (F) Quantification of the Western blot shown in (E). Total and active β-catenin levels were normalized to tubulin. The increase in either total or active β-catenin levels was plotted relative to time point 0, for which the normalized levels were set to 1. Experiments were repeated two (C) or three (D-F) times. A representative experiment is shown.</p