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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.

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Pdcd4 knock-down stimulates the expression of u-PAR and c-Myc. (a) The mRNA levels of u-PAR, c-myc, and GAPDH were determined by qPCR using total RNA isolated from GEO-shLacZ and GEO-shPdcd4 cells. The ratio of u-PAR/GAPDH or c-myc/GAPDH in GEO-shLacZ cells is designated as 1.0. The asterisk indicates a significant difference, as determined by one-way ANOVA (p < 0.01). (b) The protein levels of u-PAR, c-myc, and GAPDH were determined by Western blotting analysis using cell lysates from GEO-shLacZ and GEO-shPdcd4 cells. The ratio of u-PAR/GAPDH and c-Myc/GAPDH in GEO-shLacZ cells is designated as 1. The number indicates the relative level of u-PAR or c-Myc protein. (c) and (d) Over-expression of E-cadherin inhibits u-PAR and c-Myc expression. Either pCMV6-Ecad (E-cad) or pcDNA3.1 (control) was transfected into GEO-shPdcd4 cells. After 72h, the total RNA was isolated and the mRNA levels of E-cadherin, u-PAR and c-myc were determined by qPCR. The ratio of E-cadherin/GAPDH in cells transfected with pcDNA3.1 is designated as 1 (c). The ratio of u-PAR/GAPDH or c-myc/GAPDH in cells transfected with pcDNA3.1 is designated as 100% (d). The asterisk denotes a significant difference, as determined by one-way ANOVA (p < 0.01). (e) and (f) The β-catenin/Tcf4 complex directly binds to the promoters of u-PAR and c-myc in Pdcd4 knock-down cells. ChIP assays were performed with cell lysates from either GEO-shLacZ or GEO-shPdcd4 cells using anti-Tcf4 antibody (Tcf4) or control IgG (Pre). ChIP enriched DNA and input samples (Input) analyzed with PCR using primers for amplifying β-catenin/Tcf4 binding site on promoters of c-myc (e) and u-PAR (f). PCR products were resolved onto 2% agarose gels. M indicates the DNA markers. (g) β-catenin/Tcf4 binding site mediates Pdcd4-regulated u-PAR promoter activity. Wild-type and mutated u-PAR promoter reporter constructs were transiently transfected into GEO-shPdcd4 cells. Luciferase activity was assayed 48 h post-transfection. The luciferase activity was normalized against Renilla activity, and the activity of cells transfected with pu-PAR-1469 plasmid is designated as 100%. These experiments were repeated three times each with five independent transfections, and representative data are shown. Results are expressed as mean ± SD. The asterisk denotes a significant difference compared with cells transfected with pRS-shGFP, as determined by one-way ANOVA (p < 0.001).
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Figure 5: Pdcd4 knock-down stimulates the expression of u-PAR and c-Myc. (a) The mRNA levels of u-PAR, c-myc, and GAPDH were determined by qPCR using total RNA isolated from GEO-shLacZ and GEO-shPdcd4 cells. The ratio of u-PAR/GAPDH or c-myc/GAPDH in GEO-shLacZ cells is designated as 1.0. The asterisk indicates a significant difference, as determined by one-way ANOVA (p < 0.01). (b) The protein levels of u-PAR, c-myc, and GAPDH were determined by Western blotting analysis using cell lysates from GEO-shLacZ and GEO-shPdcd4 cells. The ratio of u-PAR/GAPDH and c-Myc/GAPDH in GEO-shLacZ cells is designated as 1. The number indicates the relative level of u-PAR or c-Myc protein. (c) and (d) Over-expression of E-cadherin inhibits u-PAR and c-Myc expression. Either pCMV6-Ecad (E-cad) or pcDNA3.1 (control) was transfected into GEO-shPdcd4 cells. After 72h, the total RNA was isolated and the mRNA levels of E-cadherin, u-PAR and c-myc were determined by qPCR. The ratio of E-cadherin/GAPDH in cells transfected with pcDNA3.1 is designated as 1 (c). The ratio of u-PAR/GAPDH or c-myc/GAPDH in cells transfected with pcDNA3.1 is designated as 100% (d). The asterisk denotes a significant difference, as determined by one-way ANOVA (p < 0.01). (e) and (f) The β-catenin/Tcf4 complex directly binds to the promoters of u-PAR and c-myc in Pdcd4 knock-down cells. ChIP assays were performed with cell lysates from either GEO-shLacZ or GEO-shPdcd4 cells using anti-Tcf4 antibody (Tcf4) or control IgG (Pre). ChIP enriched DNA and input samples (Input) analyzed with PCR using primers for amplifying β-catenin/Tcf4 binding site on promoters of c-myc (e) and u-PAR (f). PCR products were resolved onto 2% agarose gels. M indicates the DNA markers. (g) β-catenin/Tcf4 binding site mediates Pdcd4-regulated u-PAR promoter activity. Wild-type and mutated u-PAR promoter reporter constructs were transiently transfected into GEO-shPdcd4 cells. Luciferase activity was assayed 48 h post-transfection. The luciferase activity was normalized against Renilla activity, and the activity of cells transfected with pu-PAR-1469 plasmid is designated as 100%. These experiments were repeated three times each with five independent transfections, and representative data are shown. Results are expressed as mean ± SD. The asterisk denotes a significant difference compared with cells transfected with pRS-shGFP, as determined by one-way ANOVA (p < 0.001).

Mentions: In order to understand how Pdcd4 knock-down promotes invasion, we evaluated some target genes of β-catenin/Tcf dependent transcription that are involved in colon tumor cell invasion. It has been reported that u-PAR and c-Myc are the target genes of β-catenin/Tcf dependent transcription (He et al., 1998; Mann et al., 1999). u-PAR is frequently over-expressed in the invasion front of colon cancer (Laufs et al., 2006), whereas c-Myc expression is elevated at both early and late stages of colon carcinogenesis (Smith et al., 1993). To test whether Pdcd4 knock-down elevates u-PAR and c-myc mRNAs, we performed quantitative real-time PCR (qPCR) using total RNAs isolated from GEO-shLacZ and GEO-shPdcd4 cells. As shown in Figure 5a, the u-PAR mRNA level in GEO-shPdcd4 cells was approximately 2.3-fold higher comparing to that in the GEO-shLacZ cells; this is in agreement with a previous report wherein Pdcd4 was transiently knocked-down (Leupold et al., 2007). In addition, the level of c-myc mRNA in GEO-shPdcd4 cells was about 1.7-fold higher comparing to that in GEO-shLacZ cells (Figure 5a). The protein levels of u-PAR and c-Myc in GEO-shPdcd4 cells were approximately 5- and 2.5-fold higher than that in GEO-shLacZ cells, respectively (Figure 5b). These results indicate that Pdcd4 knock-down stimulates the expression of u-PAR and c-Myc.


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)

Pdcd4 knock-down stimulates the expression of u-PAR and c-Myc. (a) The mRNA levels of u-PAR, c-myc, and GAPDH were determined by qPCR using total RNA isolated from GEO-shLacZ and GEO-shPdcd4 cells. The ratio of u-PAR/GAPDH or c-myc/GAPDH in GEO-shLacZ cells is designated as 1.0. The asterisk indicates a significant difference, as determined by one-way ANOVA (p < 0.01). (b) The protein levels of u-PAR, c-myc, and GAPDH were determined by Western blotting analysis using cell lysates from GEO-shLacZ and GEO-shPdcd4 cells. The ratio of u-PAR/GAPDH and c-Myc/GAPDH in GEO-shLacZ cells is designated as 1. The number indicates the relative level of u-PAR or c-Myc protein. (c) and (d) Over-expression of E-cadherin inhibits u-PAR and c-Myc expression. Either pCMV6-Ecad (E-cad) or pcDNA3.1 (control) was transfected into GEO-shPdcd4 cells. After 72h, the total RNA was isolated and the mRNA levels of E-cadherin, u-PAR and c-myc were determined by qPCR. The ratio of E-cadherin/GAPDH in cells transfected with pcDNA3.1 is designated as 1 (c). The ratio of u-PAR/GAPDH or c-myc/GAPDH in cells transfected with pcDNA3.1 is designated as 100% (d). The asterisk denotes a significant difference, as determined by one-way ANOVA (p < 0.01). (e) and (f) The β-catenin/Tcf4 complex directly binds to the promoters of u-PAR and c-myc in Pdcd4 knock-down cells. ChIP assays were performed with cell lysates from either GEO-shLacZ or GEO-shPdcd4 cells using anti-Tcf4 antibody (Tcf4) or control IgG (Pre). ChIP enriched DNA and input samples (Input) analyzed with PCR using primers for amplifying β-catenin/Tcf4 binding site on promoters of c-myc (e) and u-PAR (f). PCR products were resolved onto 2% agarose gels. M indicates the DNA markers. (g) β-catenin/Tcf4 binding site mediates Pdcd4-regulated u-PAR promoter activity. Wild-type and mutated u-PAR promoter reporter constructs were transiently transfected into GEO-shPdcd4 cells. Luciferase activity was assayed 48 h post-transfection. The luciferase activity was normalized against Renilla activity, and the activity of cells transfected with pu-PAR-1469 plasmid is designated as 100%. These experiments were repeated three times each with five independent transfections, and representative data are shown. Results are expressed as mean ± SD. The asterisk denotes a significant difference compared with cells transfected with pRS-shGFP, as determined by one-way ANOVA (p < 0.001).
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Figure 5: Pdcd4 knock-down stimulates the expression of u-PAR and c-Myc. (a) The mRNA levels of u-PAR, c-myc, and GAPDH were determined by qPCR using total RNA isolated from GEO-shLacZ and GEO-shPdcd4 cells. The ratio of u-PAR/GAPDH or c-myc/GAPDH in GEO-shLacZ cells is designated as 1.0. The asterisk indicates a significant difference, as determined by one-way ANOVA (p < 0.01). (b) The protein levels of u-PAR, c-myc, and GAPDH were determined by Western blotting analysis using cell lysates from GEO-shLacZ and GEO-shPdcd4 cells. The ratio of u-PAR/GAPDH and c-Myc/GAPDH in GEO-shLacZ cells is designated as 1. The number indicates the relative level of u-PAR or c-Myc protein. (c) and (d) Over-expression of E-cadherin inhibits u-PAR and c-Myc expression. Either pCMV6-Ecad (E-cad) or pcDNA3.1 (control) was transfected into GEO-shPdcd4 cells. After 72h, the total RNA was isolated and the mRNA levels of E-cadherin, u-PAR and c-myc were determined by qPCR. The ratio of E-cadherin/GAPDH in cells transfected with pcDNA3.1 is designated as 1 (c). The ratio of u-PAR/GAPDH or c-myc/GAPDH in cells transfected with pcDNA3.1 is designated as 100% (d). The asterisk denotes a significant difference, as determined by one-way ANOVA (p < 0.01). (e) and (f) The β-catenin/Tcf4 complex directly binds to the promoters of u-PAR and c-myc in Pdcd4 knock-down cells. ChIP assays were performed with cell lysates from either GEO-shLacZ or GEO-shPdcd4 cells using anti-Tcf4 antibody (Tcf4) or control IgG (Pre). ChIP enriched DNA and input samples (Input) analyzed with PCR using primers for amplifying β-catenin/Tcf4 binding site on promoters of c-myc (e) and u-PAR (f). PCR products were resolved onto 2% agarose gels. M indicates the DNA markers. (g) β-catenin/Tcf4 binding site mediates Pdcd4-regulated u-PAR promoter activity. Wild-type and mutated u-PAR promoter reporter constructs were transiently transfected into GEO-shPdcd4 cells. Luciferase activity was assayed 48 h post-transfection. The luciferase activity was normalized against Renilla activity, and the activity of cells transfected with pu-PAR-1469 plasmid is designated as 100%. These experiments were repeated three times each with five independent transfections, and representative data are shown. Results are expressed as mean ± SD. The asterisk denotes a significant difference compared with cells transfected with pRS-shGFP, as determined by one-way ANOVA (p < 0.001).
Mentions: In order to understand how Pdcd4 knock-down promotes invasion, we evaluated some target genes of β-catenin/Tcf dependent transcription that are involved in colon tumor cell invasion. It has been reported that u-PAR and c-Myc are the target genes of β-catenin/Tcf dependent transcription (He et al., 1998; Mann et al., 1999). u-PAR is frequently over-expressed in the invasion front of colon cancer (Laufs et al., 2006), whereas c-Myc expression is elevated at both early and late stages of colon carcinogenesis (Smith et al., 1993). To test whether Pdcd4 knock-down elevates u-PAR and c-myc mRNAs, we performed quantitative real-time PCR (qPCR) using total RNAs isolated from GEO-shLacZ and GEO-shPdcd4 cells. As shown in Figure 5a, the u-PAR mRNA level in GEO-shPdcd4 cells was approximately 2.3-fold higher comparing to that in the GEO-shLacZ cells; this is in agreement with a previous report wherein Pdcd4 was transiently knocked-down (Leupold et al., 2007). In addition, the level of c-myc mRNA in GEO-shPdcd4 cells was about 1.7-fold higher comparing to that in GEO-shLacZ cells (Figure 5a). The protein levels of u-PAR and c-Myc in GEO-shPdcd4 cells were approximately 5- and 2.5-fold higher than that in GEO-shLacZ cells, respectively (Figure 5b). These results indicate that Pdcd4 knock-down stimulates the expression of u-PAR and c-Myc.

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