Limits...
MUC1 regulates cyclin D1 gene expression through p120 catenin and β-catenin.

Liu X, Caffrey TC, Steele MM, Mohr A, Singh PK, Radhakrishnan P, Kelly DL, Wen Y, Hollingsworth MA - Oncogenesis (2014)

Bottom Line: We observed a significant enhancement in the activation of cyclin D1 promoter-reporter activity in poorly differentiated Panc1.MUC1F cells that overexpress recombinant MUC1 relative to Panc-1.NEO cells, which express very low levels of endogenous MUC1.Further, MUC1 affected subcellular localization of p120 catenin in association with one of the main effectors of P120 catenin, the transcriptional repressor Kaiso, supporting the hypothesis that p120 catenin relieved transcriptional repression by Kaiso.Our data show MUC1 enhances the activities of both β-catenin and p120 catenin.

View Article: PubMed Central - PubMed

Affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA.

ABSTRACT
MUC1 interacts with β-catenin and p120 catenin to modulate WNT signaling. We investigated the effect of overexpressing MUC1 on the regulation of cyclin D1, a downstream target for the WNT/β-catenin signaling pathway, in two human pancreatic cancer cell lines, Panc-1 and S2-013. We observed a significant enhancement in the activation of cyclin D1 promoter-reporter activity in poorly differentiated Panc1.MUC1F cells that overexpress recombinant MUC1 relative to Panc-1.NEO cells, which express very low levels of endogenous MUC1. In stark contrast, cyclin D1 promoter activity was not affected in moderately differentiated S2-013.MUC1F cells that overexpressed recombinant MUC1 relative to S2-013.NEO cells that expressed low levels of endogenous MUC1. The S2-013 cell line was recently shown to be deficient in p120 catenin. MUC1 is known to interact with P120 catenin. We show here that re-expression of different isoforms of p120 catenin restored cyclin D1 promoter activity. Further, MUC1 affected subcellular localization of p120 catenin in association with one of the main effectors of P120 catenin, the transcriptional repressor Kaiso, supporting the hypothesis that p120 catenin relieved transcriptional repression by Kaiso. Thus, full activation of cyclin D1 promoter activity requires β-catenin activation of TCF-lef and stabilization of specific p120 catenin isoforms to relieve the repression of KAISO. Our data show MUC1 enhances the activities of both β-catenin and p120 catenin.

No MeSH data available.


Related in: MedlinePlus

Re-expression of specific p120 catenin isoform restores cyclin D1 promoter activity and increases its protein expression level. (a) Luciferase reporter assay of cyclin D1 promoter. S2-013 cells with re-expression of different p120 catenin isoforms (with/without MUC1 expression) were transfected with wild-type cyclin D1 reporter construct, which contains wild-type LEF-1/Tcf-4-binding site or reporter plasmids containing mutated LEF-1/Tcf-4-binding site in the cyclin D1 gene promoter (FOPFLASH), together with Renilla luciferase reporter plasmids SV40 as transfection control. Each transfection was carried out in duplicate plates, and the data shown here are relative luminescence units (RLUs) derived from luminescence assays on cell extracts that are representative of three independent experiments. (b) Nuclear extracts from S2-013 cells with re-expression of different p120 catenin isoforms with or without MUC1 expression were subjected to 10% SDS–PAGE and analyzed by immunoblot (IB) with an anti-cyclin D1 catenin mAb or β-tubulin as a loading control. Right panel shows densitometry analysis of the signal for cyclin D1 normalized to the signal for β-tubulin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150213&req=5

fig4: Re-expression of specific p120 catenin isoform restores cyclin D1 promoter activity and increases its protein expression level. (a) Luciferase reporter assay of cyclin D1 promoter. S2-013 cells with re-expression of different p120 catenin isoforms (with/without MUC1 expression) were transfected with wild-type cyclin D1 reporter construct, which contains wild-type LEF-1/Tcf-4-binding site or reporter plasmids containing mutated LEF-1/Tcf-4-binding site in the cyclin D1 gene promoter (FOPFLASH), together with Renilla luciferase reporter plasmids SV40 as transfection control. Each transfection was carried out in duplicate plates, and the data shown here are relative luminescence units (RLUs) derived from luminescence assays on cell extracts that are representative of three independent experiments. (b) Nuclear extracts from S2-013 cells with re-expression of different p120 catenin isoforms with or without MUC1 expression were subjected to 10% SDS–PAGE and analyzed by immunoblot (IB) with an anti-cyclin D1 catenin mAb or β-tubulin as a loading control. Right panel shows densitometry analysis of the signal for cyclin D1 normalized to the signal for β-tubulin.

Mentions: Given the deficiency of p120 catenin expression in S2-013 cells, and the possibility p120 catenin could also influence WNT signaling through β-catenin, we elected to examine cyclin D1 promoter activity in S2-013 cells that expressed different p120 catenin isoforms in the presence of high-level and low-level expression of MUC1 (Figure 3b). Interestingly, different isoforms of p120 catenin expressed in S2-013 cells showed distinct subcellular localizations. P120 catenin 1A was mainly present on the cell membrane and cytoplasm. P120 catenin 3A showed greater localization to the nucleus compared with other isoforms. P120 catenin 4A was mostly distributed at the cell surface with a small amount detected in the cytoplasm (Figure 3c). Upon MUC1 expression, the nuclear localizations of p120 catenin 1A and 3A were slightly increased. P120 catenin 4A was mostly localized on the cell membrane in the presence of MUC1 (Figure 3d). The distinct subcellular localizations of different p120 catenin isoforms is partly associated with distinct functions related to cell adhesion, motility and metastasis.15 The differences in subcellular localization and functional domains in the different p120 catenin isoforms were also predicted to affect WNT signaling and downstream transcriptional regulation, including cyclin D1 promoter activity. Thus, we performed luciferase assays with S2-013 cell lines expressing the different p120 catenin isoforms with and without expression of MUC1. Strikingly, re-expression of p120 catenin 1A or 4A (but not 3A) significantly increased the cyclin D1 promoter activity in S2-013 cells compared with NEO control. Interestingly, superimposing MUC1 expression in this system significantly increased cyclin D1 activity only when co-expressed with p120 catenin isoform 4A (Figure 4a). The effects of MUC1 and p120 catenin on cyclin D1 promoter activity were also reflected in levels of protein expression (Figure 4b).


MUC1 regulates cyclin D1 gene expression through p120 catenin and β-catenin.

Liu X, Caffrey TC, Steele MM, Mohr A, Singh PK, Radhakrishnan P, Kelly DL, Wen Y, Hollingsworth MA - Oncogenesis (2014)

Re-expression of specific p120 catenin isoform restores cyclin D1 promoter activity and increases its protein expression level. (a) Luciferase reporter assay of cyclin D1 promoter. S2-013 cells with re-expression of different p120 catenin isoforms (with/without MUC1 expression) were transfected with wild-type cyclin D1 reporter construct, which contains wild-type LEF-1/Tcf-4-binding site or reporter plasmids containing mutated LEF-1/Tcf-4-binding site in the cyclin D1 gene promoter (FOPFLASH), together with Renilla luciferase reporter plasmids SV40 as transfection control. Each transfection was carried out in duplicate plates, and the data shown here are relative luminescence units (RLUs) derived from luminescence assays on cell extracts that are representative of three independent experiments. (b) Nuclear extracts from S2-013 cells with re-expression of different p120 catenin isoforms with or without MUC1 expression were subjected to 10% SDS–PAGE and analyzed by immunoblot (IB) with an anti-cyclin D1 catenin mAb or β-tubulin as a loading control. Right panel shows densitometry analysis of the signal for cyclin D1 normalized to the signal for β-tubulin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150213&req=5

fig4: Re-expression of specific p120 catenin isoform restores cyclin D1 promoter activity and increases its protein expression level. (a) Luciferase reporter assay of cyclin D1 promoter. S2-013 cells with re-expression of different p120 catenin isoforms (with/without MUC1 expression) were transfected with wild-type cyclin D1 reporter construct, which contains wild-type LEF-1/Tcf-4-binding site or reporter plasmids containing mutated LEF-1/Tcf-4-binding site in the cyclin D1 gene promoter (FOPFLASH), together with Renilla luciferase reporter plasmids SV40 as transfection control. Each transfection was carried out in duplicate plates, and the data shown here are relative luminescence units (RLUs) derived from luminescence assays on cell extracts that are representative of three independent experiments. (b) Nuclear extracts from S2-013 cells with re-expression of different p120 catenin isoforms with or without MUC1 expression were subjected to 10% SDS–PAGE and analyzed by immunoblot (IB) with an anti-cyclin D1 catenin mAb or β-tubulin as a loading control. Right panel shows densitometry analysis of the signal for cyclin D1 normalized to the signal for β-tubulin.
Mentions: Given the deficiency of p120 catenin expression in S2-013 cells, and the possibility p120 catenin could also influence WNT signaling through β-catenin, we elected to examine cyclin D1 promoter activity in S2-013 cells that expressed different p120 catenin isoforms in the presence of high-level and low-level expression of MUC1 (Figure 3b). Interestingly, different isoforms of p120 catenin expressed in S2-013 cells showed distinct subcellular localizations. P120 catenin 1A was mainly present on the cell membrane and cytoplasm. P120 catenin 3A showed greater localization to the nucleus compared with other isoforms. P120 catenin 4A was mostly distributed at the cell surface with a small amount detected in the cytoplasm (Figure 3c). Upon MUC1 expression, the nuclear localizations of p120 catenin 1A and 3A were slightly increased. P120 catenin 4A was mostly localized on the cell membrane in the presence of MUC1 (Figure 3d). The distinct subcellular localizations of different p120 catenin isoforms is partly associated with distinct functions related to cell adhesion, motility and metastasis.15 The differences in subcellular localization and functional domains in the different p120 catenin isoforms were also predicted to affect WNT signaling and downstream transcriptional regulation, including cyclin D1 promoter activity. Thus, we performed luciferase assays with S2-013 cell lines expressing the different p120 catenin isoforms with and without expression of MUC1. Strikingly, re-expression of p120 catenin 1A or 4A (but not 3A) significantly increased the cyclin D1 promoter activity in S2-013 cells compared with NEO control. Interestingly, superimposing MUC1 expression in this system significantly increased cyclin D1 activity only when co-expressed with p120 catenin isoform 4A (Figure 4a). The effects of MUC1 and p120 catenin on cyclin D1 promoter activity were also reflected in levels of protein expression (Figure 4b).

Bottom Line: We observed a significant enhancement in the activation of cyclin D1 promoter-reporter activity in poorly differentiated Panc1.MUC1F cells that overexpress recombinant MUC1 relative to Panc-1.NEO cells, which express very low levels of endogenous MUC1.Further, MUC1 affected subcellular localization of p120 catenin in association with one of the main effectors of P120 catenin, the transcriptional repressor Kaiso, supporting the hypothesis that p120 catenin relieved transcriptional repression by Kaiso.Our data show MUC1 enhances the activities of both β-catenin and p120 catenin.

View Article: PubMed Central - PubMed

Affiliation: Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE, USA.

ABSTRACT
MUC1 interacts with β-catenin and p120 catenin to modulate WNT signaling. We investigated the effect of overexpressing MUC1 on the regulation of cyclin D1, a downstream target for the WNT/β-catenin signaling pathway, in two human pancreatic cancer cell lines, Panc-1 and S2-013. We observed a significant enhancement in the activation of cyclin D1 promoter-reporter activity in poorly differentiated Panc1.MUC1F cells that overexpress recombinant MUC1 relative to Panc-1.NEO cells, which express very low levels of endogenous MUC1. In stark contrast, cyclin D1 promoter activity was not affected in moderately differentiated S2-013.MUC1F cells that overexpressed recombinant MUC1 relative to S2-013.NEO cells that expressed low levels of endogenous MUC1. The S2-013 cell line was recently shown to be deficient in p120 catenin. MUC1 is known to interact with P120 catenin. We show here that re-expression of different isoforms of p120 catenin restored cyclin D1 promoter activity. Further, MUC1 affected subcellular localization of p120 catenin in association with one of the main effectors of P120 catenin, the transcriptional repressor Kaiso, supporting the hypothesis that p120 catenin relieved transcriptional repression by Kaiso. Thus, full activation of cyclin D1 promoter activity requires β-catenin activation of TCF-lef and stabilization of specific p120 catenin isoforms to relieve the repression of KAISO. Our data show MUC1 enhances the activities of both β-catenin and p120 catenin.

No MeSH data available.


Related in: MedlinePlus