p300/β-Catenin Interactions Regulate Adult Progenitor Cell Differentiation Downstream of WNT5a/Protein Kinase C (PKC)

Abstract

Maintenance of stem/progenitor cell-progeny relationships is required for tissue homeostasis during normal turnover and repair. Wnt signaling is implicated in both maintenance and differentiation of adult stem/progenitor cells, yet how this pathway serves these dichotomous roles remains enigmatic. We previously proposed a model suggesting that specific interaction of β-catenin with either of the homologous Kat3 co-activators, p300 or CREB-binding protein, differentially regulates maintenance versus differentiation of embryonic stem cells. Limited knowledge of endogenous mechanisms driving differential β-catenin/co-activator interactions and their role in adult somatic stem/progenitor cell maintenance versus differentiation led us to explore this process in defined models of adult progenitor cell differentiation. We focused primarily on alveolar epithelial type II (AT2) cells, progenitors of distal lung epithelium, and identified a novel axis whereby WNT5a/protein kinase C (PKC) signaling regulates specific β-catenin/co-activator interactions to promote adult progenitor cell differentiation. p300/β-catenin but not CBP/β-catenin interaction increases as AT2 cells differentiate to a type I (AT1) cell-like phenotype. Additionally, p300 transcriptionally activates AT1 cell-specific gene Aqp-5. IQ-1, a specific inhibitor of p300/β-catenin interaction, prevents differentiation of not only primary AT2 cells, but also tracheal epithelial cells, and C2C12 myoblasts. p300 phosphorylation at Ser-89 enhances p300/β-catenin interaction, concurrent with alveolar epithelial cell differentiation. WNT5a, a traditionally non-canonical WNT ligand regulates Ser-89 phosphorylation and p300/β-catenin interactions in a PKC-dependent manner, likely involving PKCζ. These studies identify a novel intersection of canonical and non-canonical Wnt signaling in adult progenitor cell differentiation that has important implications for targeting β-catenin to modulate adult progenitor cell behavior in disease.

Keywords: Wnt pathway; beta-catenin (β-catenin); differentiation; p300; protein kinase C (PKC); protein-protein interaction.

FIGURE 1.

Co-activator usage changes dynamically during AEC differentiation and p300 regulates Aqp5-Luc activity. A, representative co-immunoprecipitation of nuclear extracts at day 2 (D2) and day 6 (D6) during AEC differentiation demonstrates increased p300/β-catenin interaction in AT1-like cells (D6) with concurrent reduction in CBP/β-catenin interaction. Immunoblotting (IB) indicates that immunoprecipitated p300 and CBP (∼300 kDa) levels are comparable during differentiation. n ≥ 2. β-Catenin molecular mass is ∼92 kDa. B, schematic of Aqp5 luciferase (Aqp5-Luc) promoter construct. 4.3-kb of the rat Aqp5 promoter is fused upstream of a luciferase reporter in pGL2 basic vector. Dark gray boxes represent conserved putative TCF/LEF binding elements (TBE) predicted by rVista. C, MLE-15 cells were transiently co-transfected with Aqp5-Luc and constitutively active β-catenin (β-catenin S33Y) with or without p300 expression constructs. n = 3. *, p < 0.05; **, p < 0.001. D, MLE-15 cells were co-transfected with Aqp5-Luc and p300 expression plasmids and concurrently treated with IQ-1 (5 μm) or DMSO for 24 h. n = 3. **, p < 0.001. E, MLE-15 cells were transiently co-transfected with Aqp5-Luc and p300 WT or p300 S89A expression vectors. n = 3. *, p < 0.05. Bars represent mean ± S.E. RLU, relative luciferase units.

FIGURE 2.

Inhibition of p300/β-catenin interactions inhibit normal AEC transdifferentiation. A, qRT-PCR analysis of AT1 cell markers Aqp5 and Cav1 (top) and AT2 cell markers pro-Sftpc and pro-Sftpb (bottom) as functions of time in AEC cultivated in vitro with IQ-1 (20 μm (+)) or vehicle (DMSO (−)) from the time of plating and harvested at the indicated times. n = 5. **, p < 0.01; *, p < 0.05, compared with vehicle sample at the same time point. B, transepithelial electrical resistance across primary rAT2 cell monolayers grown on polycarbonate filters exposed to IQ-1 (20 μm (+)) or vehicle (DMSO (−)) from day 0. n = 3. **, p < 0.001. C, representative immunofluorescence image of primary rat AT2 cells treated with IQ-1 (20 μm) or vehicle (DMSO). Nuclei are labeled with DAPI (blue). n = 3. Scale bars = 50 μm. D, representative Western blot of AQP5 (28 kDa) from AEC cultivated in vitro with IQ-1 (20 μm) (+) or vehicle (DMSO (−)) at the indicated times. The AT2 cell marker is pro-SFTPC (24 kDa). Lamin A/C (55 and 62 kDa) is loading control. n = 3. E, representative Western blot and quantitation of phosphorylated p300 at serine 89 (Ser(P)-89, ∼300 kDa) during differentiation of primary AEC on days 0–4. Lamin A/C is loading control. n = 4. *, p < 0.05. F, representative Western blot and quantification of Ser(P)-89 levels in primary rat AEC exposed to IQ-1 (20 μm (+)) or vehicle (DMSO (−)) in culture for 2 days from the time of plating. Lamin A/C is loading control. n = 4. *, p < 0.05. Bars represent mean ± S.E.

FIGURE 3.

Inhibition of p300/β-catenin interactions inhibit hTEC and myoblast differentiation. A, expression of ciliated cell marker Foxj1 and club cell marker Scgb1a1 RNA in primary hTEC exposed to vehicle (DMSO) or IQ-1. Bars are mean ± S.E. of triplicate samples from independent experiments using cells of two donors and cultured at air-liquid interface for 24 days. *, p < 0.05; **, p < 0.001; ***, p < 0.0001. B, representative photomicrographs of ciliated marker acetylated α-tubulin (green) and club cell marker SCGB1A1 (red), with DAPI counterstain (blue) using the conditions as described in A. Scale bar = 100 μm. n = 2. C and D, C2C12 cell differentiation in the presence of IQ-1 and ICG-001. C, i, immunofluorescence of C2C12 cells cultured in growth medium (GM) under subconfluent conditions. ii, after 5 days in DM, cells undergo fusion and become multinucleated as seen by myosin heavy chain II (green)/DAPI (red) staining as they form myotubes. iii, cells treated with 5 μm IQ-1/DM resist fusion and remain distinct entities even though they are in direct contact with one another. iv, cells in GM + 10 μm ICG-001 become multinucleated and extend to form myotubes in the absence of DM. n = 3. D, qRT-PCR for Myf5 mRNA expression from extracts corresponding to the same experimental conditions as in C. Bars represent mean ± S.E. n = 3. *, p < 0.05 relative to GM or DM as indicated by horizontal bars.

FIGURE 4.

Knockdown of p300 but not CBP in primary cells decreases expression of AT1 cell differentiation marker Aqp5. Primary rat AEC were transduced on day 2 in culture with p300 shRNA or pGIPZ non-silencing (NS) control virus or Cbp shRNA or pLKO.1 non-silencing (NS) control virus. Cells were harvested for RNA and protein 72 h posttransduction (on day 5 in culture). A, qRT-PCR of p300 and Aqp5 mRNA levels following transduction with p300 shRNA lentivirus compared with pGIPZ NS control virus. n = 4. *, p < 0.01. B, qRT-PCR of Cbp and Aqp5 mRNA expression following transduction with Cbp shRNA lentivirus compared with pLKO.1 NS control lentivirus. n = 4. *, p < 0.05. C, Western analysis of p300 and AQP5 protein following transduction with p300 shRNA lentivirus compared with pGIPZ NS controls. Top panel shows a representative Western blot. n = 3. *, p < 0.05; **, p < 0.005. D, Western analysis of CBP and AQP5 protein following transduction with Cbp shRNA lentivirus compared with pLKO.1 NS vector controls. Top panel shows a representative Western blot. Bottom panel shows quantitation. n = 3. *, p < 0.01. Bars represent mean ± S.E.

FIGURE 5.

PKC increases during AEC differentiation and inhibition of PKC signaling inhibits AEC differentiation. A, representative Western blot and quantitative analysis of pan phospho-PKC levels (p-PKC, 77–88 kDa) in cultured primary rat AEC during transdifferentiation from day 0 (D0) to D4. n = 4. *, p < 0.05. GAPDH (37 kDa) was used a loading control. B, representative Western blot of primary AEC exposed to Gö6983 (5 μm (+)) versus vehicle (DMSO (−)). Quantification of p-PKC is shown to the right, normalized to D0. n = 4. *, p < 0.05; **, p < 0.005. Bars represent mean ± S.E. C, quantitation of Western blot analysis of Ser(P)-89 in primary rat AT2 cells exposed to Gö6983 (5 μm) for 1 h. n = 3. *, p < 0.01. Bars represent mean ± S.E. D, quantitation of PKCζ levels by Western analysis. n = 4. *, p < 0.05. Bars represent mean ± S.E. E, representative Western blot of AT1 and AT2 cell markers during differentiation of rAT2 cells treated with vehicle (DMSO (−)) or PKCζ pseudosubstrate (50 μm; PS (+)). Molecular weight of T1α is 40 kDa and RAGE is 50 kDa. n = 3.

FIGURE 6.

Wnt5a^−/− mice exhibit reduced expression of AT1 cell differentiation markers and activated PKC. A, qRT-PCR of AT1 cell markers Aqp5, Rage, T1α, and Cav-1 and AT2 cell marker pro-Sftpc at E18.5. WT: n = 3. Wnt5a^−/−: n = 4. *, p < 0.05; ***, p < 0.0001 compared with WT. B, Western blot quantification of AQP5, RAGE, CAV-1, and pro-SFTPC in Wnt5a^−/− E18.5 mouse lung homogenates compared with WT littermates. WT, n = 3. Wnt5a^−/−, n = 4. *, p < 0.05; **, p < 0.001. C, immunofluorescence of AQP5 (green) and T1α (red) in lungs of Wnt5a^−/− mice at E18. Nuclei are stained with DAPI (blue). Scale bars = 50 μm. n = 2. D, Western blot and E, quantitation of p-PKC in Wnt5a^−/− E18.5 lung homogenates. WT, n = 3. Wnt5a^−/−, n = 4. *, p < 0.05. Bars represent mean ± S.E.

FIGURE 7.

WNT5a regulates Ser(P)-89 expression, p300/β-catenin interactions, and AQP5 expression. A, qRT-PCR for Wnt5a performed on rat AEC in primary culture harvested on days 1, 2, and 4 during in vitro transdifferentiation. Aqp5 is shown as control to confirm AT1 cell transdifferentiation. n ≥ 3. **, p < 0.001 relative to day 1. B, representative Western blot and quantification of AQP5 protein expression at 24 h post-plating following exposure to recombinant WNT5a (100 ng/ml) versus H₂O vehicle control from D0. n = 3. *, p < 0.05. C, representative Western blot and quantification of AQP5 protein expression at 24 h post-plating in the presence of recombinant WNT5a (100 ng/ml) and Gö6983 (5 μm (+)) or vehicle (DMSO (−)). Values were normalized to WNT5a vehicle control. n = 3. *, p < 0.05. D, qRT-PCR for Wnt5a and Aqp5 mRNA following lentivirus-mediated shRNA knockdown of Wnt5a in the E10 mouse epithelial cell line. n = 3. ***, p < 0.0001. E, representative co-immunoprecipitation of nuclear extracts harvested 72 h after transduction with pLKO.1 non-silencing (NS) shRNA or Wnt5a shRNA. 135–150 μg of extract was immunoprecipitated (IP) with an antibody recognizing p300 phosphorylated at serine 89 (Ser(P)-89) followed by immunoblotting for Ser(P)-89 and β-catenin. Blots were stripped and re-probed for total p300. n = 3. F, quantification of Ser(P)-89 expression relative to total p300 levels in 5% input samples used in D following knockdown of Wnt5a, normalized to pLKO.1 NS control. n = 4. **, p < 0.005. G, β-catenin pulldown relative to p300 following Wnt5a knockdown calculated as the amount of co-immunoprecipitated β-catenin divided by immunoprecipitated Ser(P)-89 or total p300 as shown in E. n = 3. *, p < 0.01. Bars represent mean ± S.E.

FIGURE 8.

Model for role of p300/β-catenin in adult progenitor cell differentiation. Phosphorylation of p300 at residue serine 89 (Ser(P)-89) enhances p300/β-catenin interactions to promote progenitor cell differentiation. The small molecule inhibitor IQ-1 reduces Ser(P)-89 levels via indirect mechanisms thereby disrupting p300/β-catenin interactions and inhibiting differentiation of AEC, hTEC, and C2C12 cells. Ser(P)-89 expression is regulated by PKC. In turn, WNT5a activates PKC signaling, increases Ser(P)-89 expression and promotes p300/β-catenin interactions to drive differentiation of progenitor cells. These findings support a model in which p300/β-catenin interactions promote differentiation of adult progenitor cells in a PKC- and WNT5a-dependent manner.