Limits...
Downregulation of E-cadherin is an essential event in activating beta-catenin/Tcf-dependent transcription and expression of its target genes in Pdcd4 knockdown cells.

Wang Q, Sun ZX, Allgayer H, Yang HS - Oncogene (2009)

Bottom Line: In addition, Pdcd4 knockdown stimulates urokinase-type plasminogen activator receptor (u-PAR) and c-Myc expression, whereas u-PAR and c-Myc expression can be reversed by over-expressing E-cadherin in Pdcd4 knockdown cells.Using chromatin immunoprecipitation, we showed that beta-catenin/Tcf4 directly binds to the promoters of u-PAR and c-myc in Pdcd4 knockdown cells.Futhermore, knockdown of u-PAR or c-Myc inhibits invasion in Pdcd4 knockdown cells, suggesting that both u-PAR and c-Myc contribute to invasion induced by Pdcd4 knockdown.

View Article: PubMed Central - PubMed

Affiliation: Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, KY 40536, USA.

ABSTRACT
We reported earlier that knockdown of tumor suppressor Pdcd4 (programed cell death 4) downregulates E-cadherin expression and activates beta-catenin/Tcf (T-cell factor)-dependent transcription in colon tumor cells. However, the underlying mechanism of these observations remains unknown. In this study, we showed that knockdown of Pdcd4 downregulates E-cadherin expression through elevated protein level of Snail. Over-expression of Pdcd4 upregulates E-cadherin expression and inhibits beta-catenin/Tcf-dependent transcription. We then showed that knockdown of E-cadherin activates beta-catenin/Tcf-dependent transcription. Conversely, over-expression of E-cadherin in Pdcd4 knockdown cells inhibits beta-catenin/Tcf-dependent transcription. In addition, Pdcd4 knockdown stimulates urokinase-type plasminogen activator receptor (u-PAR) and c-Myc expression, whereas u-PAR and c-Myc expression can be reversed by over-expressing E-cadherin in Pdcd4 knockdown cells. Using chromatin immunoprecipitation, we showed that beta-catenin/Tcf4 directly binds to the promoters of u-PAR and c-myc in Pdcd4 knockdown cells. Futhermore, knockdown of u-PAR or c-Myc inhibits invasion in Pdcd4 knockdown cells, suggesting that both u-PAR and c-Myc contribute to invasion induced by Pdcd4 knockdown. Taken together, our data showed that elevated Snail expression by Pdcd4 knockdown leads to downregulation of E-cadherin resulting in activating beta-catenin/Tcf-dependent transcription and stimulating the expression of c-Myc and u-PAR, thus providing molecular explanation of how Pdcd4 suppresses tumor invasion.

Show MeSH

Related in: MedlinePlus

Over-expression of Pdcd4 up-regulates E-cadherin and inhibits β-catenin/Tcf dependent transcription. (a) Over-expression of Pdcd4 in LoVo cells up-regulates E-cadherin expression. Western blot was performed using antibodies against E-cadherin, Pdcd4, and GAPDH. The ratios of E-cadherin/GAPDH and Pdcd4/GAPDH in LoVo-control cells are designated as 1. The number indicates the relative level of either E-cadherin or Pdcd4 protein. (b) Pdcd4 inhibits β-catenin/Tcf dependent transcription. TOPflash (0.2 μg, filled bar) and FOPflash (0.2 μg, open bar) along with 10 ng of pRL-SV40 were transfected into LoVo-control and LoVo-Pdcd4 cells. The luciferase activity was normalized against Renilla activity. The activity of LoVo-control cells transfected with TOPflash and FOPflash is designated as 100%. The asterisk denotes a significant difference compared with cells transfected with TOPflash as determined by one-way ANOVA (P < 0.005). (c) Pdcd4 inhibits β-catenin/Tcf dependent transcription in a dose-dependent manner. Increasing amount (0-1.5 μg) of pcDNA-Pdcd4 plasmid was transfected along with 0.2 μg of TOPflash (filled bar) or FOPflash (open bar) into LoVo cells. The luciferase activity was normalized against Renilla activity. The total DNA was maintained at 1.7 μg by adding empty vector pcDNA3.1 DNA. The activity of cells transfected with TOPflash or FOPflash plasmid but without pcDNA-Pdcd4 plasmid is designated as 100%. The asterisk denotes a significant difference compared with cells transfected with TOPflash but without pcDNA-Pdcd4 plasmid, as determined by one-way ANOVA (P < 0.005). The experiments in (b) and (c) were repeated three times each with five independent transfections and representative data are shown. Results are expressed as mean ± SD.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2920641&req=5

Figure 3: Over-expression of Pdcd4 up-regulates E-cadherin and inhibits β-catenin/Tcf dependent transcription. (a) Over-expression of Pdcd4 in LoVo cells up-regulates E-cadherin expression. Western blot was performed using antibodies against E-cadherin, Pdcd4, and GAPDH. The ratios of E-cadherin/GAPDH and Pdcd4/GAPDH in LoVo-control cells are designated as 1. The number indicates the relative level of either E-cadherin or Pdcd4 protein. (b) Pdcd4 inhibits β-catenin/Tcf dependent transcription. TOPflash (0.2 μg, filled bar) and FOPflash (0.2 μg, open bar) along with 10 ng of pRL-SV40 were transfected into LoVo-control and LoVo-Pdcd4 cells. The luciferase activity was normalized against Renilla activity. The activity of LoVo-control cells transfected with TOPflash and FOPflash is designated as 100%. The asterisk denotes a significant difference compared with cells transfected with TOPflash as determined by one-way ANOVA (P < 0.005). (c) Pdcd4 inhibits β-catenin/Tcf dependent transcription in a dose-dependent manner. Increasing amount (0-1.5 μg) of pcDNA-Pdcd4 plasmid was transfected along with 0.2 μg of TOPflash (filled bar) or FOPflash (open bar) into LoVo cells. The luciferase activity was normalized against Renilla activity. The total DNA was maintained at 1.7 μg by adding empty vector pcDNA3.1 DNA. The activity of cells transfected with TOPflash or FOPflash plasmid but without pcDNA-Pdcd4 plasmid is designated as 100%. The asterisk denotes a significant difference compared with cells transfected with TOPflash but without pcDNA-Pdcd4 plasmid, as determined by one-way ANOVA (P < 0.005). The experiments in (b) and (c) were repeated three times each with five independent transfections and representative data are shown. Results are expressed as mean ± SD.

Mentions: If E-cadherin expression is regulated by Pdcd4, over-expression of Pdcd4 should increase E-cadherin expression. To test this, we stably expressed Pdcd4 in human colon tumor LoVo cells (LoVo-Pdcd4) using the lentivirus-mediated expression system. As shown in Figure 3a, the protein levels of Pdcd4 and E-cadherin in LoVo-Pdcd4 cells were approximately 8-fold and 2-fold higher than those of cells transduced with lentivirus-empty vector (LoVo-control), respectively. These results suggest that over-expression of Pdcd4 induces E-cadherin expression. To test whether over-expression of Pdcd4 inhibits β-catenin/Tcf dependent transcription, LoVo-control and LoVo-Pdcd4 cells were transfected with TOPflash and FOPflash plasmids. As shown in Figure 3b, the β-catenin/Tcf dependent transcription reduced approximately 50% in LoVo-Pdcd4 cells relative to LoVo-control cells (filled bars). In contrast, a similar luciferase activity was observed when FOPflash was transfected into LoVo-control and LoVo-Pdcd4 cells (open bars). In addition, we found that Pdcd4 inhibited β-catenin/Tcf dependent transcription in a concentration dependent manner when Pdcd4 expression plasmid (pcDNA-Pdcd4) and TOPflash were transiently transfected into LoVo cells (Figure 3c, filled bars). The β-catenin/Tcf dependent transcription was inhibited approximately 60% when 1.5 μg of pcDNA-Pdcd4 was transfected while Pdcd4 does not affect the luciferase activity when FOPflash was co-transfected with either pcDNA-Pdcd4 or control plasmid (Figure 3c). Our findings indicate that not only knock-down of Pdcd4 activates β-catenin/Tcf dependent transcription (Figure 1), but also over-expression of Pdcd4 inhibits β-catenin/Tcf dependent transcription (Figure 2). Thus, Pdcd4 regulates the expression of E-cadherin and activation of β-catenin/Tcf dependent transcription.


Downregulation of E-cadherin is an essential event in activating beta-catenin/Tcf-dependent transcription and expression of its target genes in Pdcd4 knockdown cells.

Wang Q, Sun ZX, Allgayer H, Yang HS - Oncogene (2009)

Over-expression of Pdcd4 up-regulates E-cadherin and inhibits β-catenin/Tcf dependent transcription. (a) Over-expression of Pdcd4 in LoVo cells up-regulates E-cadherin expression. Western blot was performed using antibodies against E-cadherin, Pdcd4, and GAPDH. The ratios of E-cadherin/GAPDH and Pdcd4/GAPDH in LoVo-control cells are designated as 1. The number indicates the relative level of either E-cadherin or Pdcd4 protein. (b) Pdcd4 inhibits β-catenin/Tcf dependent transcription. TOPflash (0.2 μg, filled bar) and FOPflash (0.2 μg, open bar) along with 10 ng of pRL-SV40 were transfected into LoVo-control and LoVo-Pdcd4 cells. The luciferase activity was normalized against Renilla activity. The activity of LoVo-control cells transfected with TOPflash and FOPflash is designated as 100%. The asterisk denotes a significant difference compared with cells transfected with TOPflash as determined by one-way ANOVA (P < 0.005). (c) Pdcd4 inhibits β-catenin/Tcf dependent transcription in a dose-dependent manner. Increasing amount (0-1.5 μg) of pcDNA-Pdcd4 plasmid was transfected along with 0.2 μg of TOPflash (filled bar) or FOPflash (open bar) into LoVo cells. The luciferase activity was normalized against Renilla activity. The total DNA was maintained at 1.7 μg by adding empty vector pcDNA3.1 DNA. The activity of cells transfected with TOPflash or FOPflash plasmid but without pcDNA-Pdcd4 plasmid is designated as 100%. The asterisk denotes a significant difference compared with cells transfected with TOPflash but without pcDNA-Pdcd4 plasmid, as determined by one-way ANOVA (P < 0.005). The experiments in (b) and (c) were repeated three times each with five independent transfections and representative data are shown. Results are expressed as mean ± SD.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2920641&req=5

Figure 3: Over-expression of Pdcd4 up-regulates E-cadherin and inhibits β-catenin/Tcf dependent transcription. (a) Over-expression of Pdcd4 in LoVo cells up-regulates E-cadherin expression. Western blot was performed using antibodies against E-cadherin, Pdcd4, and GAPDH. The ratios of E-cadherin/GAPDH and Pdcd4/GAPDH in LoVo-control cells are designated as 1. The number indicates the relative level of either E-cadherin or Pdcd4 protein. (b) Pdcd4 inhibits β-catenin/Tcf dependent transcription. TOPflash (0.2 μg, filled bar) and FOPflash (0.2 μg, open bar) along with 10 ng of pRL-SV40 were transfected into LoVo-control and LoVo-Pdcd4 cells. The luciferase activity was normalized against Renilla activity. The activity of LoVo-control cells transfected with TOPflash and FOPflash is designated as 100%. The asterisk denotes a significant difference compared with cells transfected with TOPflash as determined by one-way ANOVA (P < 0.005). (c) Pdcd4 inhibits β-catenin/Tcf dependent transcription in a dose-dependent manner. Increasing amount (0-1.5 μg) of pcDNA-Pdcd4 plasmid was transfected along with 0.2 μg of TOPflash (filled bar) or FOPflash (open bar) into LoVo cells. The luciferase activity was normalized against Renilla activity. The total DNA was maintained at 1.7 μg by adding empty vector pcDNA3.1 DNA. The activity of cells transfected with TOPflash or FOPflash plasmid but without pcDNA-Pdcd4 plasmid is designated as 100%. The asterisk denotes a significant difference compared with cells transfected with TOPflash but without pcDNA-Pdcd4 plasmid, as determined by one-way ANOVA (P < 0.005). The experiments in (b) and (c) were repeated three times each with five independent transfections and representative data are shown. Results are expressed as mean ± SD.
Mentions: If E-cadherin expression is regulated by Pdcd4, over-expression of Pdcd4 should increase E-cadherin expression. To test this, we stably expressed Pdcd4 in human colon tumor LoVo cells (LoVo-Pdcd4) using the lentivirus-mediated expression system. As shown in Figure 3a, the protein levels of Pdcd4 and E-cadherin in LoVo-Pdcd4 cells were approximately 8-fold and 2-fold higher than those of cells transduced with lentivirus-empty vector (LoVo-control), respectively. These results suggest that over-expression of Pdcd4 induces E-cadherin expression. To test whether over-expression of Pdcd4 inhibits β-catenin/Tcf dependent transcription, LoVo-control and LoVo-Pdcd4 cells were transfected with TOPflash and FOPflash plasmids. As shown in Figure 3b, the β-catenin/Tcf dependent transcription reduced approximately 50% in LoVo-Pdcd4 cells relative to LoVo-control cells (filled bars). In contrast, a similar luciferase activity was observed when FOPflash was transfected into LoVo-control and LoVo-Pdcd4 cells (open bars). In addition, we found that Pdcd4 inhibited β-catenin/Tcf dependent transcription in a concentration dependent manner when Pdcd4 expression plasmid (pcDNA-Pdcd4) and TOPflash were transiently transfected into LoVo cells (Figure 3c, filled bars). The β-catenin/Tcf dependent transcription was inhibited approximately 60% when 1.5 μg of pcDNA-Pdcd4 was transfected while Pdcd4 does not affect the luciferase activity when FOPflash was co-transfected with either pcDNA-Pdcd4 or control plasmid (Figure 3c). Our findings indicate that not only knock-down of Pdcd4 activates β-catenin/Tcf dependent transcription (Figure 1), but also over-expression of Pdcd4 inhibits β-catenin/Tcf dependent transcription (Figure 2). Thus, Pdcd4 regulates the expression of E-cadherin and activation of β-catenin/Tcf dependent transcription.

Bottom Line: In addition, Pdcd4 knockdown stimulates urokinase-type plasminogen activator receptor (u-PAR) and c-Myc expression, whereas u-PAR and c-Myc expression can be reversed by over-expressing E-cadherin in Pdcd4 knockdown cells.Using chromatin immunoprecipitation, we showed that beta-catenin/Tcf4 directly binds to the promoters of u-PAR and c-myc in Pdcd4 knockdown cells.Futhermore, knockdown of u-PAR or c-Myc inhibits invasion in Pdcd4 knockdown cells, suggesting that both u-PAR and c-Myc contribute to invasion induced by Pdcd4 knockdown.

View Article: PubMed Central - PubMed

Affiliation: Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, KY 40536, USA.

ABSTRACT
We reported earlier that knockdown of tumor suppressor Pdcd4 (programed cell death 4) downregulates E-cadherin expression and activates beta-catenin/Tcf (T-cell factor)-dependent transcription in colon tumor cells. However, the underlying mechanism of these observations remains unknown. In this study, we showed that knockdown of Pdcd4 downregulates E-cadherin expression through elevated protein level of Snail. Over-expression of Pdcd4 upregulates E-cadherin expression and inhibits beta-catenin/Tcf-dependent transcription. We then showed that knockdown of E-cadherin activates beta-catenin/Tcf-dependent transcription. Conversely, over-expression of E-cadherin in Pdcd4 knockdown cells inhibits beta-catenin/Tcf-dependent transcription. In addition, Pdcd4 knockdown stimulates urokinase-type plasminogen activator receptor (u-PAR) and c-Myc expression, whereas u-PAR and c-Myc expression can be reversed by over-expressing E-cadherin in Pdcd4 knockdown cells. Using chromatin immunoprecipitation, we showed that beta-catenin/Tcf4 directly binds to the promoters of u-PAR and c-myc in Pdcd4 knockdown cells. Futhermore, knockdown of u-PAR or c-Myc inhibits invasion in Pdcd4 knockdown cells, suggesting that both u-PAR and c-Myc contribute to invasion induced by Pdcd4 knockdown. Taken together, our data showed that elevated Snail expression by Pdcd4 knockdown leads to downregulation of E-cadherin resulting in activating beta-catenin/Tcf-dependent transcription and stimulating the expression of c-Myc and u-PAR, thus providing molecular explanation of how Pdcd4 suppresses tumor invasion.

Show MeSH
Related in: MedlinePlus