Convergence of Canonical and Non-Canonical Wnt Signal: Differential Kat3 Coactivator Usage

Abstract

Background: The ancient and highly evolutionarily conserved Wnt signaling pathway is critical in nearly all tissues and organs for an organism to develop normally from embryo through adult. Wnt signaling is generally parsed into "canonical" or Wnt-β-catenin-dependent or "non-canonical" β-catenin-independent signaling. Even though designating Wnt signaling as either canonical or noncanonical allows for easier conceptual discourse about this signaling pathway, in fact canonical and non-canonical Wnt crosstalk regulates complex nonlinear networks.

Objective: In this perspective, we discuss the integration of canonical and non-canonical Wnt signaling via differential Kat3 (CBP and p300) coactivator usage, thereby regulating and coordinating gene expression programs associated with both proliferation and cellular differentiation and morphogenesis.

Methods: Pharmacologic inhibitors, cell culture, real-time PCR, chromatin immunoprecipitation, protein immunoprecipitation, Western blotting, reporter-luciferase, protein purification, site-directed mutagenesis, in vitro phosphorylation and binding assays, and immunofluorescence were utilized.

Conclusion: Coordinated integration between both canonical and non-canonical Wnt pathways appears to be crucial not only in the control of fundamental morphologic processes but also in the regulation of normal as well as pathologic events. Such integration between both canonical and non-canonical Wnt signaling is presumably effected via reversible phosphorylation mechanism (e.g., protein kinase C) to regulate differential β -catenin/Kat3 coactivator usage in order to coordinate proliferation with differentiation and adhesion.

Keywords: CBP; Kat3 coactivator; Wnt; canonical; non-canonical; p300..

Fig. (1)

Integration of canonical and non-canonical Wnt signaling via differential CBP and p300 usage. Non-canonical Wnt signaling (represented by WNT5a) ƒ?oantagonizesƒ?? canonical Wnt signaling (represented by WNT3a) by inducing a change in Iý-catenin coactivator usage and a corresponding switch in gene expression from a proliferative towards a differentiative program. We have demonstrated that WNT5a induces PKC phosphorylation of Ser89 of p300, thereby increasing the affinity of p300 for Iý-catenin, and we propose this as a mechanism to explain the switch in coactivator usage from CBP/Iý-catenin to p300/Iý-catenin. Small molecule ICG-001 specifically blocks the CBP/Iý-catenin interaction, thus biasing towards p300 usage by Iý-catenin. In contrast, small molecules IQ-1 (indirect antagonist) and YH249/250 (direct antagonist) block the p300/Iý-catenin interaction, thus biasing towards CBP usage by Iý-catenin.

Fig. (2)

ICG-001 blocks the association of CBP with the c-myc promoter and concomitantly increases the level of p300 associated with the c-myc promoter. To evaluate coactivator usage at the endogenous c-myc promoter, chromatin immunoprecipitation (ChIP) analysis was performed on SW480 cells treated with either ICG-001 (25 AæM) or control DMSO. The c-myc promoter is occupied by both coactivators CBP and p300 in control DMSO treated cells. However, treatment with ICG-001 completely and selectively blocks the association of CBP with the c-myc promoter and concomitantly increases the level of p300 associated with this promoter. D, DMSO (control) treatment. T, treatment with ICG-001.

Fig. (3)

Endogenous CBP and p300 expression in and the growth of wild type, CBP (+/-), and p300 (+/-) cells. 3T3 cells were generated from MEF cells that are heterozygous deficient for either CBP or p300. (a) Heterozygous CBP (+/-) MEFs express significantly less CBP than either wild type (WT) or p300 heterozygous p300 (+/-) cells as demonstrated by Western blot. Conversely, p300 (+/-) cells express considerably less p300 than the other two cell types. (b) p300 (+/-) 3T3 cells had a significantly steeper growth curve than either the WT or CBP (+/-) cells.

Fig. (4)

p300 (+/-) versus wild type and CBP (+/-) 3T3 cells display heightened response to canonical Wnt3a and negligible response to non-canonical Wnt5a. (a) When stimulated with Wnt3a conditioned media, 3T3 wild type (WT) and CBP (+/-) cells exhibited a small activation of the TOPFlash reporter gene construct; however, p300 (+/-) cells had a significantly larger increase in TOPFlash activity. Wnt5a alone had almost no effect on basal TOPFlash activity in the 3 cell lines. In WT and CBP (+/-) 3T3 cells, co-administration of Wnt5a and Wnt3a versus Wnt3a alone resulted in an ~20% reduction in TOPFlash activity, whereas a dramatic reduction in TOPFlash activity was observed in the p300 (+/-) cells. n ≥ 2, *p < 0.05, **p < 0.01. (b) To gain additional perspective, our studies were extended to include the survivin promoter luciferase construct pGL3-6270Luc whose activity is more CBP-dependent than the TOPFlash construct. Wnt3a caused an ~2.5-fold increase in survivin luciferase activity in 3T3 WT cells, an ~2-fold increase in 3T3 CBP (+/-) cells, and a dramatic ~50-fold increase in the p300 (+/-) cells. Wnt5a elicited a similar minimal response above control media in the WT and CBP (+/-) cells, yet had essentially no effect on the p300 (+/-) cells. Co-administration of Wnt3a and Wnt5a conditioned media led to modest reductions in survivin luciferase activity in the WT and CBP (+/-) cells compared to Wnt3a or 5a alone, whereas co-administration of Wnt3a and 5a dramatically reduced survivin luciferase activity in the p300 (+/-) cells compared to Wnt3a alone. n ≥ 2, *p < 0.05, **p < 0.01.

Fig. (5)

Wnt3a suppresses c-myc expression in p300 (+/-) 3T3 cells, whereas Wnt5a induces c-myc expression in CBP (+/-) cells. In CBP (+/-) cells in which there is a significantly increased ratio of p300 to CBP, treatment with Wnt5a or Wnt3a+Wnt5a conditioned media (CM) led to increased c-myc mRNA expression, which was not observed with Wnt3a alone, as assessed by real-time PCR. In contrast, Wnt3a dramatically reduced c-myc message in p300 (+/-) cells, whereas Wnt5a and Wnt3a+Wnt5a treatment did not greatly change c-myc expression compared to control media-treated cells. n ≥ 2, *p < 0.05, **p < 0.01.

Fig. (6)

Simultaneous stimulation of both canonical and non-canonical Wnt signaling leads to differential coactivator utilization by nuclear Iý-catenin leading to preferential interaction between Iý-catenin and p300. To examine the interaction of nuclear Iý-catenin with the coactivators CBP or p300 after Wnt stimulation, wild type (WT) 3T3 cells were treated with Wnt3a, Wnt5a, or Wnt3a+Wnt5a conditioned media (CM) versus control for 24 h, after which nuclear lysates were prepared and subjected to immunoprecipitation (IP) with CBP, p300, or control antibody, followed by immunoblot for Iý-catenin. Wnt3a-only conditioned media markedly increased the coimmunoprecipitation of Iý-catenin with both CBP and p300 compared to control treated cells (compare lanes 1 and 2). Whereas Wnt5a-only conditioned media caused no increase in the amount of Iý-catenin associated with p300, a marked decrease in the amount of Iý-catenin associated with CBP compared to control (compare lanes 1 and 3) was observed. Co-treatment with Wnt3a and Wnt5a conditioned media caused a decrease in the amount of Iý-catenin associated with CBP compared to treatment with Wnt3a alone, while a conspicuous increase in p300 associated Iý-catenin was observed under these conditions. Ab, antibody.

Fig. (7)

Inhibition of PKC attenuates p300/Iý-catenin interaction while enhancing CBP/Iý-catenin interaction. To examine the role of PKC activation on differential coactivator usage, wild type 3T3 cells were treated with combined Wnt3a and 5a conditioned media (CM) in the presence or absence of selective small molecule PKC inhibitor Go6976 or CaMKII inhibitor KN93 for 24 h. Nuclear lysates were prepared and subjected to immunoprecipitation (IP) with CBP, p300, or control antibody, followed by immunoblot for Iý-catenin. Compared with DMSO-only control, co-administration of Wnt3a and 5a led to a substantial increase in p300-associated nuclear Iý-catenin (compare lanes 1 and 2). Treatment with Go6976 (1 uM) dramatically and selectively decreased the amount of Iý-catenin associated with p300 (compare lanes 2 and 3), while increasing the amount of Iý-catenin associated with CBP. Similar results were obtained utilizing the selective CaMKII inhibitor KN93 (5 uM) (compare lanes 2 and 4). Ab, antibody.

Fig. (8)

Activation of PKC enhances the interaction of Iý-catenin with p300. After respective treatments (as described below), nuclear lysates of wild type 3T3 cells were prepared and subjected to immunoprecipitation with CBP, p300, or control antibody, followed by immunoblot for Iý-catenin. (a) Treatment of 3T3 cells with specific activator of PKC PMA (100 I1/4M) for 1 h led to a modest, yet highly coactivator selective increase in Iý-catenin associated with p300. (b) 3T3 cells were treated with Wnt3a and 5a conditioned media in the presence (20 I1/4M) or absence of N-myristoylated PKC pseudosubstrate peptide specific PKCIñ inhibitor PKCIñ(20-28) for 24 h. PKCIñ(20-28) selectively inhibited the coimmunoprecipitation of Iý-catenin with p300.

Fig. (9)

Non-canonical Wnt activation of PKC induces PKC phosphorylation of p300. 3T3 cells were treated with Wnt3a, Wnt5a, or Wnt3a+Wnt5a in the presence or absence of PKC inhibitor for 24 h. Nuclear lysates were prepared and immunoblotted for total p300 and for phospho-Ser89 p300 (using anti-Ser-89 PO4 p300 antibody). Although the levels of total p300 remained essentially unchanged, the levels of phospho-Ser89 p300 were markedly increased upon treatment with conditioned media containing Wnt5a, and the phosphorylation could be blocked by small molecule PKC inhibitor (either Go6976 or Ro31-8425). VC, vehicle control. p300 antibody (N-15, Santa Cruz Biotechnology) used to assess total p300.

Fig. (10)

Phosphorylation of p300 Ser89 increases binding to Iý-catenin. To further investigate the biochemical effects of PKC phosphorylation of p300 on binding to Iý-catenin, His-tagged versions of the N-terminal regions (amino acid 1-111) of wild type p300 (P1), of a Ser89Ala point-mutated p300 (mt P1(S89A)), and of CBP (C1) were expressed in E. Coli and purified on a Ni-NTA column. Lysates from wild type (WT) 3T3 cells were prepared and incubated with P1, mt P1(S89A), or C1. Wild type p300 immunoprecipitated with Iý-catenin. In vitro phosphorylation of wild type p300 with PKCIñ markedly enhanced binding to Iý-catenin. Mutant p300 bound with somewhat less avidity to Iý-catenin, whereas the in vitro phosphorylated mutant p300 was dramatically less effective in binding to Iý-catenin than the phosphorylated wild type p300.

Fig. (11)

Wnt5a overexpression in UACC 1273 melanoma cells is associated with differential Wnt/Iý-catenin-regulated gene expression. ƒ?"(a) UACC 1273(4-7) melanoma cells which stably express Wnt5a versus UACC 1273(EV) empty vector control cells were evaluated by real-time PCR for mRNA expression of Wnt5a, survivin, cyclin D1, hnkd, and fra-1. In UACC 1273(4-7) cells which demonstrated a ~13-fold increase in Wnt5a expression, mRNA levels for survivin, cyclin D1, and hnkd all decreased significantly while levels for fra-1 message increased significantly. n ≥ 2, *p < 0.05, **p < 0.01. (b) Treatment of UACC 1273(4-7) cells with PKC inhibitor (either 3 uM Go6976 or 5 uM Ro31-8425) demonstrated suppression of mRNA expression of fra-1 and MITF as assessed by real-time PCR. n ≥ 2, *p < 0.05.

Fig. (12)

Non-canonical planar cell polarity is p300-dependent. To investigate the effects of differential p300 versus CBP coactivator usage on planar cell polarity (PCP), we examined cochlear hair cell development during mouse embryonic development ex vivo. E14.5 mouse cochleae were placed into culture and treated for 6 days, with indirect p300/Iý-catenin antagonist IQ-1 or direct CBP/Iý-catenin antagonist ICG-001 versus vehicle control. Whereas inhibition of CBP/Iý-catenin interaction did not have any deleterious effects on cochlear development and stereocilia orientation, inhibition of the p300/Iý-catenin interaction disrupted PCP and cochlear hair cell orientation. ICG-001, in a dose dependent fashion, reversed the deleterious effects of IQ-1 on PCP. OHC, outer hair cell; IHC, inner hair cell.