Figure 1.
β-catenin forms punctate nuclear lesions in response to PKA activation in primary cultures.
Primary osteoblasts from wild type (WT) bones or from bone tumors arising in Prkar1a+/− mice were studied by immunofluorescence for β-catenin (green). For reference, cell nuclei were stained with DAPI. The left column shows β-catenin, only, whereas the right column shows merger of the β-catenin and DAPI stains. Top) WT osteoblasts. Middle) Tumor osteoblasts. Bottom) WT osteoblasts treated with forskolin (FSK). Note the punctate nuclear localization of β-catenin observed in Tumor cells or WT cells treated with FSK. Magnification: 400x.
Figure 2.
β-catenin (green) localizes to PML bodies in Prkar1a+/− tumor osteoblasts.
A. Triple immunofluorescence for DAPI (blue), β-catenin (green), and PML (red) in WT or Tumor osteoblasts. The bottom right image in each panel represents the merged image. Scale bar: 10 µm. B. Immunofluorescence for H2AX and β-catenin in tumor cells. C. Immunofluorescence for PCNA and β-catenin. Note the lack of co-localization between proteins in B and C. Magnification (B and C): 630x.
Figure 3.
Stimulation of PKA by FSK increases β-catenin phosphorylation and nuclear relocalization in MC3T3-E1 cells.
A. Protein lysates were prepared from MC3T3-E1 cells treated with vehicle for Forskolin (FSK) for the times indicated and blotted with the antibodies shown. Note the increases in pS133-CREB, pS552- and pS675- β-catenin at both timepoints without changes in total protein levels. Actin is shown as a loading control. 20 µg of protein were loaded per lane. B. Immunofluorescence tracking of β-catenin after vehicle (DMSO) or FSK treatment in MC3T3-E1 cells. The bottom row shows a merged images from the panels above. Scale bar: 10 µm.
Figure 4.
PKA activation promotes nuclear relocalization of phospho-β-catenin.
A. Immunofluorescence for pS675-β-catenin (green), PML (red), with DAPI nuclear staining (blue) in MC3T3-E1 cells treated with vehicle (DMSO) or FSK. Note the nuclear accumulation of phospho-β-catenin in response to FSK. B. MC3T3-E1 cells were treated with vehicle or FSK and nuclear and cytosolic protein fractions prepared and blotted for the proteins shown. Specificity of the fractionation is demonstrated by blotting for Lamin A (nuclear marker) and α-tubulin (cytosolic marker). Note the enhanced phospho-β-catenin only in the nuclear fraction in response to FSK. 8 µg of nuclear and 20 µg of cytosolic protein were loaded per lane. C. Control or Prkar1a-knockdown MC3T3-E1 cells were studied by IF as in panel A. Scale bar for all images: 10 µm.
Figure 5.
Mutation of phosphorylation sites affects nuclear localization of β-catenin.
MC3T3-E1 cells were transfected with WT or mutated FLAG-tagged β-catenin constructs, treated with vehicle or forskolin for 6 h and then subjected to confocal microscopy with anti-FLAG antibodies. The bar graph shows the quantification of immunofluorescence data. 30-40 fields were counted for each transfection, yielding 70–100 transfected cells for each condition. Note that very little nuclear localization (<3%) occurs in the absence of FSK with any of the constructs used. Scale bars: 10 µm.
Figure 6.
PKA activation enhances basal and stimulated Wnt/β-catenin -dependent transcriptional activity in MC3T3-E1 cells.
A. Cells were transfected with a Wnt/β-catenin -reporter plasmid (TOPFlash) or the same plasmid with a mutation in the Wnt-responsive elements (FOPFlash). Luciferase assay was performed following treatment with vehicle, FSK, or Wnt3a (100 ng/ml) as indicated (* P<0.05, ** P<0.01). B. Luciferase assay was performed in cells with control or Prkar1a knockdown to measure the Wnt/β-catenin-reporter activity (** P<0.01). C. mRNA expression of Wnt/β-catenin target genes was determined using QPCR analysis. The expression of each was normalized to GAPDH expression (** P<0.01 versus DMSO treated cells). (D) Cells were transfected with TOPFlash or FOPFlash along with FLAG-tagged β-catenin or its PKA phosphorylation mutants, as indicated. The Wnt/β-catenin -reporter activity was measured by luciferase assay following stimulation of cells with forskolin. (** P<0.01 versus WT or single mutants, ∧ P<0.05 versus DMSO treated counterparts).
Figure 7.
CREB and β-catenin cooperation promotes cAMP-dependent transcription.
A. MC3T3-E1 cells were transfected with a cAMP responsive element luciferase construct. Luciferase assay was performed following stimulation of vehicle, FSK, or Wnt3a (100 ng/ml) as indicated (* P<0.05). Error bars represent standard deviation. B. IF co-localization of pS675β-catenin (green) and pS133-CREB (red) in response to FSK stimulation. DAPI (blue) stains the nucleus. The bottom right panel of each group shows the merged image. Scale bar: 10 µm.
Table 1.
Distribution of TCF and CREB binding sites in genes with altered transcription in Prkar1a+/− bone tumors*.
Figure 8.
PKA activation represses Wnt5a/Ror2 pathway.
A. and B. mRNA expression of Wnt5a and Ror2 was determined using QPCR analysis in MC3T3-E1 cells treated with FSK (A) or with Prkar1a knockdown (B) (** P<0.01 versus DMSO or control shRNA treated cells). Error bars represent standard deviation. C. 20 ug of protein lysates from MC3T3-E1 cells were analyzed for Wnt5a/b by Western blotting. Actin was used as the internal control. This experiment was repeated at least twice with similar results, and a representative blot is shown.