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The cAMP responsive element binding protein 1 transactivates epithelial membrane protein 2, a potential tumor suppressor in the urinary bladder urothelial carcinoma.

Li CF, Wu WJ, Wu WR, Liao YJ, Chen LR, Huang CN, Li CC, Li WM, Huang HY, Chen YL, Liang SS, Chow NH, Shiue YL - Oncotarget (2015)

Bottom Line: Multivariate analysis further demonstrated that low EMP2 immunoexpression is an independent prognostic factor for poor disease-specific survival.Genistein treatments, knockdown of EMP2 gene and double knockdown of CREB1 and EMP2 genes significantly inhibited tumor growth and notably downregulated CREB1 and EMP2 protein levels in the mice xenograft models.Therefore, genistein induced CREB1 transcription, translation and upregulated pCREB1(S133) protein level.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Chi Mei Medical Center, Tainan, Taiwan.

ABSTRACT
In this study, we report that EMP2 plays a tumor suppressor role by inducing G2/M cell cycle arrest, suppressing cell viability, proliferation, colony formation/anchorage-independent cell growth via regulation of G2/M checkpoints in distinct urinary bladder urothelial carcinoma (UBUC)-derived cell lines. Genistein treatment or exogenous expression of the cAMP responsive element binding protein 1 (CREB1) gene in different UBUC-derived cell lines induced EMP2 transcription and subsequent translation. Mutagenesis on either or both cAMP-responsive element(s) dramatically decreased the EMP2 promoter activity with, without genistein treatment or exogenous CREB1 expression, respectively. Significantly correlation between the EMP2 immunointensity and primary tumor, nodal status, histological grade, vascular invasion and mitotic activity was identified. Multivariate analysis further demonstrated that low EMP2 immunoexpression is an independent prognostic factor for poor disease-specific survival. Genistein treatments, knockdown of EMP2 gene and double knockdown of CREB1 and EMP2 genes significantly inhibited tumor growth and notably downregulated CREB1 and EMP2 protein levels in the mice xenograft models. Therefore, genistein induced CREB1 transcription, translation and upregulated pCREB1(S133) protein level. Afterward, pCREB1(S133) transactivated the tumor suppressor gene, EMP2, in vitro and in vivo. Our study identified a novel transcriptional target, which plays a tumor suppressor role, of CREB1.

No MeSH data available.


Related in: MedlinePlus

Genistein upregulated CREB1 and pCREB1(S133) protein levels, and pCREB1(Ser133) transactivates EMP2 gene in UBUC-derived cells(A) Phylogenetic footprinting identified two conserved CREB1-responsive elements (CRE1 & CRE2) in the proximal promoter region of human EMP2 and mouse Emp2 orthologs, the first nucleotide of exon 1 was defined as +1. In J82 cells, transfection of the pCMV-CREB1 plasmid notably induced (B) CREB1, pCREB1(S133) and EMP2 protein, and (C)EMP2 mRNA levels. (D, E) Both stably transfection pCMV-CREB1 plasmid and genistein treatments induced G2/M cell cycle arrest in J82 cells. (F, G) Conversely, stable transfection of shRNAi plasmids targeting CREB1 gene suppressed CREB1 and EMP2 mRNA, and CREB1, pCREB1(S133) and EMP2 protein levels in RT4 cells. (H) In J82 cells, genistein treatments (10 μg/mL in DMSO) for 24 and 48 h notably induced CREB1, pCREB1(S133) and EMP2 protein levels. CDKN1A is a well-known target for genistein, was applied as a positive control. (I) Chromatin immunoprecipitation (IP) assay further confirmed that pCREB1(S133) protein interacts with both potential CREs; IgG was served as a negative control. (J) One DNA fragment (–220 to +268) containing two CREs of the EMP2 proximal promoter region was cloned into pGL3 reporter vector, designated as pGL3-C. Site-directed mutagenesis (underlined) at CRE1 (pGL3-C/mCRE1), CRE2 (pGL3-C/mCRE2) and double mutagenesis at both CREs (pGL3-C/dmCREs) were also cloned into the pCL3 reporter vector. (K) In TGSH8301 and J82 cell lines with lower endogenous EMP2 levels, dual luciferase assays demonstrated that transfection of pGL3-C increased promoter activities, compared to those transfections with the pGL3 control. However, the promoter activities were decreased after transfection of pGL3-C/mCRE1, pGL3-C/mCRE2 or pGL3-C/dmCREs plasmid for 24 h, compared to those transfections with the pGL3-C control. The promoter activity was further diminished after transfection of the pGL3-C/dmCREs plasmid, compared to those of transfection with either plasmid with single mutation, pGL3-C/mCRE1 or pGL3-C/mCRE2. (L) Transfection of pCMV-CREB1 for 24 h increased the pGL3-C promoter activity, compared to those of transfection with pCMV-Entry plasmid in both TSGH8301 and J82 cell lines. (M) In J82 cells, treatment with genistein (10 μg/mL in DMSO) increased the activity of pGL3-C, compared to the control (pGL3). However, genistein did not alter the promoter activity with double mutations in CREs (pGL3-C/dmCREs). All experiments were triplicated and results are expressed as mean ± SEM. For immunoblotting analysis, one representative image is shown (B, G, H). Statistical significance: *, p < 0.05; **, p < 0.01 and ***, p < 0.001.
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Figure 2: Genistein upregulated CREB1 and pCREB1(S133) protein levels, and pCREB1(Ser133) transactivates EMP2 gene in UBUC-derived cells(A) Phylogenetic footprinting identified two conserved CREB1-responsive elements (CRE1 & CRE2) in the proximal promoter region of human EMP2 and mouse Emp2 orthologs, the first nucleotide of exon 1 was defined as +1. In J82 cells, transfection of the pCMV-CREB1 plasmid notably induced (B) CREB1, pCREB1(S133) and EMP2 protein, and (C)EMP2 mRNA levels. (D, E) Both stably transfection pCMV-CREB1 plasmid and genistein treatments induced G2/M cell cycle arrest in J82 cells. (F, G) Conversely, stable transfection of shRNAi plasmids targeting CREB1 gene suppressed CREB1 and EMP2 mRNA, and CREB1, pCREB1(S133) and EMP2 protein levels in RT4 cells. (H) In J82 cells, genistein treatments (10 μg/mL in DMSO) for 24 and 48 h notably induced CREB1, pCREB1(S133) and EMP2 protein levels. CDKN1A is a well-known target for genistein, was applied as a positive control. (I) Chromatin immunoprecipitation (IP) assay further confirmed that pCREB1(S133) protein interacts with both potential CREs; IgG was served as a negative control. (J) One DNA fragment (–220 to +268) containing two CREs of the EMP2 proximal promoter region was cloned into pGL3 reporter vector, designated as pGL3-C. Site-directed mutagenesis (underlined) at CRE1 (pGL3-C/mCRE1), CRE2 (pGL3-C/mCRE2) and double mutagenesis at both CREs (pGL3-C/dmCREs) were also cloned into the pCL3 reporter vector. (K) In TGSH8301 and J82 cell lines with lower endogenous EMP2 levels, dual luciferase assays demonstrated that transfection of pGL3-C increased promoter activities, compared to those transfections with the pGL3 control. However, the promoter activities were decreased after transfection of pGL3-C/mCRE1, pGL3-C/mCRE2 or pGL3-C/dmCREs plasmid for 24 h, compared to those transfections with the pGL3-C control. The promoter activity was further diminished after transfection of the pGL3-C/dmCREs plasmid, compared to those of transfection with either plasmid with single mutation, pGL3-C/mCRE1 or pGL3-C/mCRE2. (L) Transfection of pCMV-CREB1 for 24 h increased the pGL3-C promoter activity, compared to those of transfection with pCMV-Entry plasmid in both TSGH8301 and J82 cell lines. (M) In J82 cells, treatment with genistein (10 μg/mL in DMSO) increased the activity of pGL3-C, compared to the control (pGL3). However, genistein did not alter the promoter activity with double mutations in CREs (pGL3-C/dmCREs). All experiments were triplicated and results are expressed as mean ± SEM. For immunoblotting analysis, one representative image is shown (B, G, H). Statistical significance: *, p < 0.05; **, p < 0.01 and ***, p < 0.001.

Mentions: To further identify any transcription factor that might regulate EMP2 expression, phylogenetic footprinting was performed. Two putative cAMP responsive elements (CREs) in the EMP2 proximal promoter region were identified, denoted as CRE1 and CRE2 (Figure 2A). Exogenous expression of cAMP responsive element binding protein 1 (CREB1) in J82 cells notably upregulated CREB1, pCREB1(S133), EMP2 protein and EMP2 mRNA (p < 0.001) levels (Figure 2B, 2C). Stable overexpression of CREB1 gene (p < 0.001) or genistein treatments (10 μg/mL) for 24 h (p < 0.001) and 48 h (p < 0.001) in J82 cells, significantly induced G2/M cell cycle arrest (Figure 2D, 2E). In contrast, stable knockdown of CREB1 gene in RT4 cells downregulated CREB1 (p < 0.001) and EMP2 (p < 0.001) mRNA (Figure 2F); CREB1, pCREB1(S133) and EMP2 protein (Figure 2G) levels. Further, genistein treatments for 24 and 48 h notably induced CREB1, pCREB1(S133) and EMP2 protein abundance in J82 cells (Figure 2H). ChIP assay confirmed that pCREB1(S133) protein interacts with both CRE1 and CRE2 in the EMP2 proximal promoter region, while IgG did not (Figure 2I). Single, double mutations at CRE1 and/or CRE2 were next created (Figure 2J), and a dual luciferase assay additionally demonstrated that the EMP2 promoter activity decreased when either single mutation (pGL3-C/mCRE1 or pGL3-C/mCRE2) was introduced (p < 0.001), compared to those with pGL3-C plasmid (wild type). The promoter activity of EMP2 gene was further diminished when double mutations (pGL3-C/dmCREs) were incorporated, compared to either single mutant (p < 0.05) (Figure 2K). Exogenous expression of the CREB1 gene in both TSGH8301 and J82 cell lines, with low endogenous EMP2 levels, elevated pGL3-C activity (Figure 2L). Genistein increased pGL3-C activity (p < 0.05); however, it did not stimulate the promoter activity when double mutations were introduced (pGL3-C/dmCREs) in J82 cells (Figure 2M). Therefore, genistein induced EMP2 transcription via upregulation of CREB1 mRNA, CREB1 and pCREB1(S133) protein levels, as well as enhancement of the interaction between pCREB1(S133) and CREs on the EMP2 proximal promoter region.


The cAMP responsive element binding protein 1 transactivates epithelial membrane protein 2, a potential tumor suppressor in the urinary bladder urothelial carcinoma.

Li CF, Wu WJ, Wu WR, Liao YJ, Chen LR, Huang CN, Li CC, Li WM, Huang HY, Chen YL, Liang SS, Chow NH, Shiue YL - Oncotarget (2015)

Genistein upregulated CREB1 and pCREB1(S133) protein levels, and pCREB1(Ser133) transactivates EMP2 gene in UBUC-derived cells(A) Phylogenetic footprinting identified two conserved CREB1-responsive elements (CRE1 & CRE2) in the proximal promoter region of human EMP2 and mouse Emp2 orthologs, the first nucleotide of exon 1 was defined as +1. In J82 cells, transfection of the pCMV-CREB1 plasmid notably induced (B) CREB1, pCREB1(S133) and EMP2 protein, and (C)EMP2 mRNA levels. (D, E) Both stably transfection pCMV-CREB1 plasmid and genistein treatments induced G2/M cell cycle arrest in J82 cells. (F, G) Conversely, stable transfection of shRNAi plasmids targeting CREB1 gene suppressed CREB1 and EMP2 mRNA, and CREB1, pCREB1(S133) and EMP2 protein levels in RT4 cells. (H) In J82 cells, genistein treatments (10 μg/mL in DMSO) for 24 and 48 h notably induced CREB1, pCREB1(S133) and EMP2 protein levels. CDKN1A is a well-known target for genistein, was applied as a positive control. (I) Chromatin immunoprecipitation (IP) assay further confirmed that pCREB1(S133) protein interacts with both potential CREs; IgG was served as a negative control. (J) One DNA fragment (–220 to +268) containing two CREs of the EMP2 proximal promoter region was cloned into pGL3 reporter vector, designated as pGL3-C. Site-directed mutagenesis (underlined) at CRE1 (pGL3-C/mCRE1), CRE2 (pGL3-C/mCRE2) and double mutagenesis at both CREs (pGL3-C/dmCREs) were also cloned into the pCL3 reporter vector. (K) In TGSH8301 and J82 cell lines with lower endogenous EMP2 levels, dual luciferase assays demonstrated that transfection of pGL3-C increased promoter activities, compared to those transfections with the pGL3 control. However, the promoter activities were decreased after transfection of pGL3-C/mCRE1, pGL3-C/mCRE2 or pGL3-C/dmCREs plasmid for 24 h, compared to those transfections with the pGL3-C control. The promoter activity was further diminished after transfection of the pGL3-C/dmCREs plasmid, compared to those of transfection with either plasmid with single mutation, pGL3-C/mCRE1 or pGL3-C/mCRE2. (L) Transfection of pCMV-CREB1 for 24 h increased the pGL3-C promoter activity, compared to those of transfection with pCMV-Entry plasmid in both TSGH8301 and J82 cell lines. (M) In J82 cells, treatment with genistein (10 μg/mL in DMSO) increased the activity of pGL3-C, compared to the control (pGL3). However, genistein did not alter the promoter activity with double mutations in CREs (pGL3-C/dmCREs). All experiments were triplicated and results are expressed as mean ± SEM. For immunoblotting analysis, one representative image is shown (B, G, H). Statistical significance: *, p < 0.05; **, p < 0.01 and ***, p < 0.001.
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Figure 2: Genistein upregulated CREB1 and pCREB1(S133) protein levels, and pCREB1(Ser133) transactivates EMP2 gene in UBUC-derived cells(A) Phylogenetic footprinting identified two conserved CREB1-responsive elements (CRE1 & CRE2) in the proximal promoter region of human EMP2 and mouse Emp2 orthologs, the first nucleotide of exon 1 was defined as +1. In J82 cells, transfection of the pCMV-CREB1 plasmid notably induced (B) CREB1, pCREB1(S133) and EMP2 protein, and (C)EMP2 mRNA levels. (D, E) Both stably transfection pCMV-CREB1 plasmid and genistein treatments induced G2/M cell cycle arrest in J82 cells. (F, G) Conversely, stable transfection of shRNAi plasmids targeting CREB1 gene suppressed CREB1 and EMP2 mRNA, and CREB1, pCREB1(S133) and EMP2 protein levels in RT4 cells. (H) In J82 cells, genistein treatments (10 μg/mL in DMSO) for 24 and 48 h notably induced CREB1, pCREB1(S133) and EMP2 protein levels. CDKN1A is a well-known target for genistein, was applied as a positive control. (I) Chromatin immunoprecipitation (IP) assay further confirmed that pCREB1(S133) protein interacts with both potential CREs; IgG was served as a negative control. (J) One DNA fragment (–220 to +268) containing two CREs of the EMP2 proximal promoter region was cloned into pGL3 reporter vector, designated as pGL3-C. Site-directed mutagenesis (underlined) at CRE1 (pGL3-C/mCRE1), CRE2 (pGL3-C/mCRE2) and double mutagenesis at both CREs (pGL3-C/dmCREs) were also cloned into the pCL3 reporter vector. (K) In TGSH8301 and J82 cell lines with lower endogenous EMP2 levels, dual luciferase assays demonstrated that transfection of pGL3-C increased promoter activities, compared to those transfections with the pGL3 control. However, the promoter activities were decreased after transfection of pGL3-C/mCRE1, pGL3-C/mCRE2 or pGL3-C/dmCREs plasmid for 24 h, compared to those transfections with the pGL3-C control. The promoter activity was further diminished after transfection of the pGL3-C/dmCREs plasmid, compared to those of transfection with either plasmid with single mutation, pGL3-C/mCRE1 or pGL3-C/mCRE2. (L) Transfection of pCMV-CREB1 for 24 h increased the pGL3-C promoter activity, compared to those of transfection with pCMV-Entry plasmid in both TSGH8301 and J82 cell lines. (M) In J82 cells, treatment with genistein (10 μg/mL in DMSO) increased the activity of pGL3-C, compared to the control (pGL3). However, genistein did not alter the promoter activity with double mutations in CREs (pGL3-C/dmCREs). All experiments were triplicated and results are expressed as mean ± SEM. For immunoblotting analysis, one representative image is shown (B, G, H). Statistical significance: *, p < 0.05; **, p < 0.01 and ***, p < 0.001.
Mentions: To further identify any transcription factor that might regulate EMP2 expression, phylogenetic footprinting was performed. Two putative cAMP responsive elements (CREs) in the EMP2 proximal promoter region were identified, denoted as CRE1 and CRE2 (Figure 2A). Exogenous expression of cAMP responsive element binding protein 1 (CREB1) in J82 cells notably upregulated CREB1, pCREB1(S133), EMP2 protein and EMP2 mRNA (p < 0.001) levels (Figure 2B, 2C). Stable overexpression of CREB1 gene (p < 0.001) or genistein treatments (10 μg/mL) for 24 h (p < 0.001) and 48 h (p < 0.001) in J82 cells, significantly induced G2/M cell cycle arrest (Figure 2D, 2E). In contrast, stable knockdown of CREB1 gene in RT4 cells downregulated CREB1 (p < 0.001) and EMP2 (p < 0.001) mRNA (Figure 2F); CREB1, pCREB1(S133) and EMP2 protein (Figure 2G) levels. Further, genistein treatments for 24 and 48 h notably induced CREB1, pCREB1(S133) and EMP2 protein abundance in J82 cells (Figure 2H). ChIP assay confirmed that pCREB1(S133) protein interacts with both CRE1 and CRE2 in the EMP2 proximal promoter region, while IgG did not (Figure 2I). Single, double mutations at CRE1 and/or CRE2 were next created (Figure 2J), and a dual luciferase assay additionally demonstrated that the EMP2 promoter activity decreased when either single mutation (pGL3-C/mCRE1 or pGL3-C/mCRE2) was introduced (p < 0.001), compared to those with pGL3-C plasmid (wild type). The promoter activity of EMP2 gene was further diminished when double mutations (pGL3-C/dmCREs) were incorporated, compared to either single mutant (p < 0.05) (Figure 2K). Exogenous expression of the CREB1 gene in both TSGH8301 and J82 cell lines, with low endogenous EMP2 levels, elevated pGL3-C activity (Figure 2L). Genistein increased pGL3-C activity (p < 0.05); however, it did not stimulate the promoter activity when double mutations were introduced (pGL3-C/dmCREs) in J82 cells (Figure 2M). Therefore, genistein induced EMP2 transcription via upregulation of CREB1 mRNA, CREB1 and pCREB1(S133) protein levels, as well as enhancement of the interaction between pCREB1(S133) and CREs on the EMP2 proximal promoter region.

Bottom Line: Multivariate analysis further demonstrated that low EMP2 immunoexpression is an independent prognostic factor for poor disease-specific survival.Genistein treatments, knockdown of EMP2 gene and double knockdown of CREB1 and EMP2 genes significantly inhibited tumor growth and notably downregulated CREB1 and EMP2 protein levels in the mice xenograft models.Therefore, genistein induced CREB1 transcription, translation and upregulated pCREB1(S133) protein level.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Chi Mei Medical Center, Tainan, Taiwan.

ABSTRACT
In this study, we report that EMP2 plays a tumor suppressor role by inducing G2/M cell cycle arrest, suppressing cell viability, proliferation, colony formation/anchorage-independent cell growth via regulation of G2/M checkpoints in distinct urinary bladder urothelial carcinoma (UBUC)-derived cell lines. Genistein treatment or exogenous expression of the cAMP responsive element binding protein 1 (CREB1) gene in different UBUC-derived cell lines induced EMP2 transcription and subsequent translation. Mutagenesis on either or both cAMP-responsive element(s) dramatically decreased the EMP2 promoter activity with, without genistein treatment or exogenous CREB1 expression, respectively. Significantly correlation between the EMP2 immunointensity and primary tumor, nodal status, histological grade, vascular invasion and mitotic activity was identified. Multivariate analysis further demonstrated that low EMP2 immunoexpression is an independent prognostic factor for poor disease-specific survival. Genistein treatments, knockdown of EMP2 gene and double knockdown of CREB1 and EMP2 genes significantly inhibited tumor growth and notably downregulated CREB1 and EMP2 protein levels in the mice xenograft models. Therefore, genistein induced CREB1 transcription, translation and upregulated pCREB1(S133) protein level. Afterward, pCREB1(S133) transactivated the tumor suppressor gene, EMP2, in vitro and in vivo. Our study identified a novel transcriptional target, which plays a tumor suppressor role, of CREB1.

No MeSH data available.


Related in: MedlinePlus