Monitoring Expression and Distribution of B-Catenin (Z’ > 0.5)
Introduction:
m-catenin, the mammalian homologue of Drosophila Armadillo is a dual purpose protein of great interest to those biomedical researchers concerned with tumor formation. In normal epithelial cells, β-catenin is found at the plasma membrane where it provides a mechanical linkage between cell-to-cell junctional proteins (e.g., E-cadherin) and cytoskeletal proteins(e.g., α catenin and actinin-4) (Morin 1999; Hayashida et al., 2005). By contrast, in tumor cells, β-catenin is often found in the cytoplasm and nucleus where it associates with TCF family members to form a complex, which activates transcription of pro-mitotic proteins including c-Myc and cyclinD1 (Figure 1). Relocalization of β-catenin also occurs as part of the epithelial-mesenchymal transition (EMT) process, which is essential to organ development in the embryo (Lee et al., 2005). Thus, certain forms of oncogenesis recapitulate the EMT, as tumorgenic cells change from epithelia to the less differentiated mesenchymal phenotype, and β-catenin is a central participant in the process.
Figure 1
Signal transduction pathways modulating β-catenin localization and degradation.
A great deal of current biomedical research is directed towards determining the signal transduction pathways that modulate β-catenin localization, degradation, and function. It has been recently established that phosphorylation of β-catenin by Src is necessary for its dissociation from E-cadherin (Coluccia et al., 2006). β-Catenin is additionally phosphorylated by the glycogen synthase kinase 3-β: adenomatous polyposis coli (GSK:APC) complex leading to its ubiquination and proteosome-mediated degradation. Significantly, GSK3 activity is decreased by the canonical Wnt signaling pathway, which involves the growth factor Wnt, the Wnt receptor Frizzled, and associated regulatory proteins such as Disheveled and Frat (Krishnan et al., 2006). Thus, increased Wnt signaling results in diminished phosphorylation and reduced degradation of β-catenin, and accumulation of β-catenin in the cytoplasm and nucleus.
Figure 2. Redistribution of β-catenin in MDCK cells exposed to GSK-3 inhibitor IX.
A) Control cells stained for β-catenin (green) and nuclei (blue). B) Cells visualized only for β-catenin (same field as A). C) Membrane mask derived from B by Vala's CyteSeerTM software. D Cells exposed to GSK 3 inhibitor IX (10 μM) for 24 hr, stained as in A. E) GSK3 inhibitor-treated cells visualized only for β-catenin (arrow shows example of nuclear β-catenin). F) Membrane mask derived from E by CyteSeer
Assay of β-catenin utilizing high content imaging techniques
Vala Sciences Inc proudly announces the introduction of our new reagent/software kit (catalogue # K006) to quantify expression and cellular location of β-catenin in cultured cells. The kit contains reagents and protocols to optimally visualize β-catenin via fluorescence microscopy, and our CyteSeerTM image processing software for automated quantification of protein distribution within digital cellular images. To demonstrate the functionality of the kit and software, MDCK and HeLa cells were cultured in 96-well plates and exposed to GSK3 or Src inhibitors. The cells were then visualized for β-catenin and nuclei utilizing our optimized reagents and protocols and the cells were photographed utilizing a Beckman IC100 automated fluorescence microscopy work station.
Results for MDCK cells
Figure 2 shows images from a preliminary experiment. Staining for β-catenin in control MDCK cells (exposed to DMSO only) was localized primarily to the plasma membrane (Figure 2 A and B). From these images, Vala Sciences' CyteSeerTM software identified pixels corresponding to the plasma membrane (Figure 2C) with very high fidelity. MDCK cells exposed to 10 μM GSK 3 inhibitor IX (EMD catalogue # 361550) exhibited a larger cell size, and a redistribution of β-catenin to the cytoplasm and nuclei (Figure 2E).
Figure 3. Quantification of β-catenin redistribution in response to GSK inhibitor IX in MDCK cells, as quantified via Vala Sciences™ CyteSeer software.
Each bar is the mean ± SD for n=8 wells/condition.
Figure 4. β-Catenin localization in HeLa cells exposed to GSK 3 and Src inhibitors.
A) Control cells visualized for β-catenin. B) Cells exposed to 10 μM GSK inhibitor IX for 24 hr. Note the prominent nuclear staining exhibited by most cells. C) Cells exposed to 20 μM PP2, a Src inhibitor. D) Cells exposed to 10 μM GSK inhibitor IX plus 20 μM PP2.
To more fully characterize these effects, MDCK cells were exposed to concentrations of the GSK 3 inhibitor IX ranging from 0.6 to 10 μM. A total of 4 fields of view were imaged for each well, with 8 wells used per condition. Each field of view was imaged for nuclei (utilizing DAPI) in addition to β-catenin. Automated analysis of the images was accomplished via CyteSeerTM, which utilizes information from the plasma membrane mask and the nuclear channel to segment each image into the plasma membrane, cytoplasmic, and nuclear regions for each cell within the field of view (approx. 1250 cells were analyzed per well). A variety of data parameters were calculated by CyteSeerTM, including Total Cytoplasmic Area (TCA, the summation of cytoplasmic pixels/cell), Total Cytoplasmic Intensity (TCI, the summation of pixel intensity for cytoplasmic pixels/cell), Total Nuclear Intensity (TNI, summation of pixel intensity for nuclear pixels/cell) and Total Membrane Intensity (TMI, summation of pixel intensity for membrane pixels/cell). Exposure to GSK inhibitor IX lead to a dose-dependent 3.4-fold increase in TCA (Figure 3A, maximal at 10 μM) , a 3.3-fold increase in TCI (Figure 3B), a 1.8-fold increase in TNI (Figure 3C), and a 15% decrease in TMI (not shown). To demonstrate the robustness of the assay, Z' calculations were performed, which compare the data variability to the range of the assay (Z' = 1-((3*SDmin)+(3*SDmax))/(Max-Min)); Z' values above 0.50 are considered excellent and indicate that the assay can be successfully utilized in high throughput screening applications (Zhang et al., 1999). The assay was most robust (Z'= 0.63) for the TCI parameter. Positive Z' values were also obtained for TCA (0.46). Vala Sciences' β-catenin kit and CyteSeerTM software could be used to perform high throughput screens for potential compounds that would modulate GSK 3β activity in live cells, with relatively little chance of false positive readouts for nuclear β-catenin localization.
Figure 5. Effects of GSK inhibitor IX on β-catenin expression and localization in HeLa cells.
Each bar represents the mean +/- SD for n=16 wells.
Results for HeLa cells & our reagent kit
In addition, we tested our β-catenin kit reagents with Hela cells, and explored the relationship between GSK-3 and Src activity in regulating β-catenin distribution. HeLa cells were cultured to confluency, in DMEM+10% FBS, then switched to 2% FBS 48 hr prior to the experiment; test drugs were applied to the cells in media with 2% FBS for 24 hr prior to staining. The overall expression pattern for β-catenin in HeLa cells was similar to that in MDCK cells, with β-catenin primarily localized at the plasma membrane (Figure 4A). Exposure to GSK3 inhibitor IX increased the expression of β-catenin, yielding images which were generally brighter than control, consistent with the hypothesis that β-catenin degradation is increased by GSK3-mediated phosphorylation (Figure 1). Additionally, GSK3 inhibitor IX increased nuclear localization of β-catenin (Figure 4B). Cells exposed to PP2 showed a β-catenin distribution pattern similar to controls (Figure 4C). Interestingly, cells exposed to the combination of GSK3 inhibitor IX and PP2 also showed a β-catenin localization pattern similar to controls (Figure 4D). Thus, PP2 prevented GSK 3 inhibitor IX-mediated β-catenin redistribution.
Results for HeLa cells & CyteSeer
Images from two experiments with HeLa cells utilizing GSK 3 inhibitor IX and the src inhibitor PP2 were then quantified via CyteSeerTM. In the first experiment, a dose-response to GSK 3 inhibitor IX was performed, utilizing concentrations of 1.25 to 10 μM for 24 hrs. Total cellular expression (TCE), of β-catenin, which corresponds to TMI+TCI+TNI, increased a maximum of 2.2-fold at 5 μM. Similarly, TNI and Median Nuclear Intensity (MNI) increased 2.6- and 2.3-fold, with Z' values of 0.45 and 0.50, respectively. Interestingly, the ratio of MNI/MMI (Median Membrane Intensity) varied from a control value of 0.90 to 1.25 at 10 μM GSK inhibitor IX; with relatively little variation in this parameter. The Z' for the MNI/MMI parameter was 0.70, which is an excellent Z' value. In a second experiment, images from HeLa cells treated similarly to the conditions depicted in Figure 4 were quantified. For control cells, MNI averaged 64.0 (since 8-bit images were collected, MNI can vary between 0 and 255). Treatment of the cells with the Src inhibitor PP2, led to a general reduction in nuclear β-catenin staining; the average ANI was 50.5 (-21% vs. control) and 46.9 (-27%), for 5 and 20 μM PP2, respectively. In contrast, GSK 3 inhibitor IX increased nuclear β-catenin; the average ANI was 81.4 (+27% vs. control) and 92.1 (+42%) for 5 and 10 μM, GSK 3 inhibitor IX, respectively. Cells exposed to the combination of 20 μM PP2 plus 10 μM GSK 3 inhibitor IX exhibited an ANI of 56.2, which was reduced by 12% compared to the control value. PP2 substantially blocked the ability of GSK 3 inhibitor IX to stimulate nuclear accumulation of β-catenin.
Figure 6. Analysis of nuclear β-catenin expression in HeLa cells treated with GSK inhibitor IX and/or PP2, an inhibitor of Src.
Each bar represents the mean ± SD for n=16 wells. ##Less than controls; ** greater than control; ^^ greater than controls and also greater than 5 μM GSK inhibitor IX. ANOVA followed by Student Newman Keuls test; p < .001 for all comparisons.
Discussion
In our study, β-catenin was localized to the plasma membrane under control conditions for both MDCK and HeLa cells, and exposure to GSK 3 inhibitor IX led to accumulation of β-catenin in the cytoplasmic and nuclear compartments. Overall, these results are consistent with the hypothesis that GSK activity is a prime determinant of β-catenin distribution. Additionally, exposure to GSK 3 inhibitor IX led to an overall increase in β-catenin expression in HeLa cells, which is consistent with the hypothesis that GSK 3 mediated phosphorylation of β-catenin leads to its' degradation. Finally, the ability of PP2 to block the effect of GSK 3 inhibitor IX is consistent with the emerging hypothesis (Coluccia et al., 2006) that Src activity is essential for dissociation of β-catenin from cadherins localized at the plasma membrane. Thus, inhibitors of Src likely prevent translocation of β-catenin by preventing its dissociation from the junctional complexes involved in cell adhesion. The results from our experiments are consistent with the overall hypothesis that Wnt signaling is a prime determinant of β-catenin localization and degradation and support the hypothesis that Src is "upstream" from GSK in the β-catenin -nuclear translocation pathway.
The results illustrate that Vala's kit reagents visualize β-catenin in both canine (MDCK) and human (HeLa) cell lines. In addition, Vala's CyteSeerTM software, a Windows' compatible stand-alone program, performed a convenient, rapid (within minutes), and quantitative analysis of the results. CyteSeerTM and Vala Sciences' β-catenin kit provides tools that are optimized to describe β-catenin expression and cellular localization and will be useful in chemical genomic studies to further elucidate this pathway, or for high content screening experiments to identify novel modulators of β-catenin, GSK-3 and Src.
References
Coluccia, A. M. L., Benati, D., Dekhil, H., De Filippo, A., Lan, C., Gambacorti-Passerini, C. 2006. SKI-606 decreases growth and motility of colorectal cancer cells by preventing pp60(c-Src)-dependent tyrosine phosphorylation of β-catenin and its nuclear signaling. Cancer Res. 66:2279-2286.
Hayashida, Y., Honda, K., Idogawa, M., Ino, Y., Ono, m., Tsuchida, A., Aoki, T., Hirohashi, S., Yamada, T. 2005. E-cadherin regulates the association between β-catenin and actinin-4. Cancer Res. 65:8836-8845.
Krishnan, V., Bryant, H. U., MacDougald, O. A. 2006. Regulation of bone mass by Wnt signaling. J. Clin. Invest. 116:1202-1209.
Lee, J., M., Dedhar, S., Kalluri, R., Thompson, E. W. 2005. The epithelial-mesenchymal transition: new insights in signaling, development, and disease. J. Cell. Biol. 172:973-981.
Morin, P. J. 1999. β-Catenin signaling and cancer. BioEssays 21:1021-1030.
Zhang et al., 1999. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomolecular Scr. 4:67-73
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