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Estrogen induces Vav1 expression in human breast cancer cells.

Du MJ, Chen XD, Zhou XL, Wan YJ, Lan B, Zhang CZ, Cao Y - PLoS ONE (2014)

Bottom Line: Nevertheless, two regions at the vav1 gene promoter were defined to be responsible for E2-induced activation of vav1 promoter.Consequently, the enhanced expression of Vav1 led to the elevation of Cyclin D1 and the progression of cell cycle.The present study implies that estrogen-ER modulates the transcription and expression of Vav1, which may contribute to the proliferation of cancerous cells.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Microbial Functional Genomics of Ministry of Education, College of Life Sciences, Nankai University, Tianjin, P. R. China.

ABSTRACT
Vav1, a guanine nucleotide exchange factor (GEF) for Rho family GTPases, is a hematopoietic protein involved in a variety of cellular events. In recent years, aberrant expression of Vav1 has been reported in non-hematopoietic cancers including human breast cancer. It remains to be answered how Vav1 is expressed and what Vav1 does in its non-resident tissues. In this study, we aimed to explore the mechanism for Vav1 expression in breast cancer cells in correlation with estrogen-ER pathway. We not only verified the ectopic expression of Vav1 in human breast cancer cell lines, but also observed that Vav1 expression was induced by 17β-estradiol (E2), a typical estrogen receptor (ER) ligand, in ER-positive cell lines. On the other hand, Tamoxifen, a selective estrogen receptor modulator (SERM), and ICI 182,780, an ER antagonist, suppressed the expression of Vav1. The estrogen receptor modulating Vav1 expression was identified to be α form, not β. Furthermore, treatment of E2 increased the transcription of vav1 gene by enhancing the promoter activity, though there was no recognizable estrogen response element (ERE). Nevertheless, two regions at the vav1 gene promoter were defined to be responsible for E2-induced activation of vav1 promoter. Chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP) analyses suggested that ERα might access to the vav1 promoter via interacting with transcription factors, c-Myb and ELF-1. Consequently, the enhanced expression of Vav1 led to the elevation of Cyclin D1 and the progression of cell cycle. The present study implies that estrogen-ER modulates the transcription and expression of Vav1, which may contribute to the proliferation of cancerous cells.

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Analysis of transcription factors involved in ERα-activated vav1 promoter.(A) Depicted deletion mutations in vav1 promoter reporter gene. The deleted regions were indicated by break lines. (B) MCF7 cells were transfected with plasmids containing luciferase under WT or mutated vav1 promoters (D1, D2, and D3) and then treated with E2 (10−7 mol/L) or DMSO for 48 h. The relative fold induction of each group was calculated as the ratio of the luciferase activity induced by E2 to that induced by DMSO, respectively, and plotted as y-axis, and the deletion mutants were presented as in x-axis. All the data represented the mean±S.D. of three independent experiments. “N.S.” and “**” indicates P>0.05 and P<0.01, respectively, versus D1 group by unpaired student T test. (C) ChIP assay. T47D cells were treated with E2 (10−7 mol/L) for 4 h or pre-treated with Tamoxifen (10−6 mol/L) for 30 min before treating with E2 (10−7 mol/L) for 4 h. The immunoprecipitation was performed using antibodies against c-Myb (left panels) or ELF-1 (right panels), with preimmune IgG as control. The precipitated DNAs were analyzed by PCR with the primers corresponding to position −232 to +71 of vav1 promoter. The bar chart below the example blot represents the normalized DNA level of −232 to +71 to Input of three independent experiments. “N.S.” indicates P>0.05 versus DMSO treatment by unpaired student T test. (D) The association of ERα with c-Myb and ELF-1. T47D cells were treated with E2 (10−7 mol/L) or DMSO for 4 h or pretreated with Tamoxifen (10−6 mol/L) for 30 min in prior to E2 treatment and lysed. Antibodies against c-Myb (upper panels) and ELF-1 (lower panels) or control IgG (Lane 1) were used to immunoprecipitate protein complex, which were then resolved by Western Blot with indicated antibodies. (E) The proposed model for ERα modulating the vav1 promoter activity. E2-activated ERα interacts with vav1 promoter via interacting with transcription factors such as c-Myb, ELF-1, or perhaps other unknown coregulators (labeled “?”) to promote the vav1 transcription.
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pone-0099052-g005: Analysis of transcription factors involved in ERα-activated vav1 promoter.(A) Depicted deletion mutations in vav1 promoter reporter gene. The deleted regions were indicated by break lines. (B) MCF7 cells were transfected with plasmids containing luciferase under WT or mutated vav1 promoters (D1, D2, and D3) and then treated with E2 (10−7 mol/L) or DMSO for 48 h. The relative fold induction of each group was calculated as the ratio of the luciferase activity induced by E2 to that induced by DMSO, respectively, and plotted as y-axis, and the deletion mutants were presented as in x-axis. All the data represented the mean±S.D. of three independent experiments. “N.S.” and “**” indicates P>0.05 and P<0.01, respectively, versus D1 group by unpaired student T test. (C) ChIP assay. T47D cells were treated with E2 (10−7 mol/L) for 4 h or pre-treated with Tamoxifen (10−6 mol/L) for 30 min before treating with E2 (10−7 mol/L) for 4 h. The immunoprecipitation was performed using antibodies against c-Myb (left panels) or ELF-1 (right panels), with preimmune IgG as control. The precipitated DNAs were analyzed by PCR with the primers corresponding to position −232 to +71 of vav1 promoter. The bar chart below the example blot represents the normalized DNA level of −232 to +71 to Input of three independent experiments. “N.S.” indicates P>0.05 versus DMSO treatment by unpaired student T test. (D) The association of ERα with c-Myb and ELF-1. T47D cells were treated with E2 (10−7 mol/L) or DMSO for 4 h or pretreated with Tamoxifen (10−6 mol/L) for 30 min in prior to E2 treatment and lysed. Antibodies against c-Myb (upper panels) and ELF-1 (lower panels) or control IgG (Lane 1) were used to immunoprecipitate protein complex, which were then resolved by Western Blot with indicated antibodies. (E) The proposed model for ERα modulating the vav1 promoter activity. E2-activated ERα interacts with vav1 promoter via interacting with transcription factors such as c-Myb, ELF-1, or perhaps other unknown coregulators (labeled “?”) to promote the vav1 transcription.

Mentions: The above results indicated that ERα was in complex with the 5′ region of vav1 gene promoter. Several transcription factors were predicted to bind at the 5′ minimal regulatory region of the human vav1 gene, including ETF, Sp1, E2F, NF-e, c-Myb, TCFα, PU.1, and ELF-1 [30]. We therefore attempted to locate the regions that respond to estrogen. The wild type vav1 promoter (WT) and the truncated mutants (D1, D2, D3) that lack the predicted transcription factor binding sites were depicted in Figure 5A, and the reporter plasmids were constructed [30]. As shown in Figure 5B, the wild type promoter activity was elevated to 3 fold by E2. The deletion mutant D1 that lacks region -(143∼152) exhibited similar extent of induction (2.6 fold), implying that the region −(143∼152) was dispensable in E2-induced vav1 expression. In contrast, the E2 induction of truncated promoters D2 and D3 was severely suppressed to less than 1.5 fold (P<0.01), indicating that these two regions, −(25∼38) and −(5∼22), were required for E2-induced vav1 transcription. As these regions were reported to possess putative binding sites for transcription factors E2F/NF-e/c-Myb at -(25∼38) and TCFα/PU.1/ELF-1 at −(5∼22), respectively, ERα may associate with certain transcription factors within these regions. As reported previously, c-Myb affects vav1 transcription in lung cancer cells [30] and is also involved in the E2-ER regulated gene expression in breast cancer cells [39]. Meanwhile, another breast cancer related transcription factor, ELF-1, is identified to interact with the promoter of vav1 (Genome browser, http://genome.ucsc.edu/) [40]. Firstly, to confirm the binding of these two transcription factors to vav1 promoter, the ChIP analysis was performed. As shown in Figure 5C, both c-Myb and ELF-1 presented positively in complex with the vav1 promoter (Fig. 5C, upper two panels), and the presence of E2 or Tamoxifen had no effects on the complex (Fig. 5C, bottom panel).


Estrogen induces Vav1 expression in human breast cancer cells.

Du MJ, Chen XD, Zhou XL, Wan YJ, Lan B, Zhang CZ, Cao Y - PLoS ONE (2014)

Analysis of transcription factors involved in ERα-activated vav1 promoter.(A) Depicted deletion mutations in vav1 promoter reporter gene. The deleted regions were indicated by break lines. (B) MCF7 cells were transfected with plasmids containing luciferase under WT or mutated vav1 promoters (D1, D2, and D3) and then treated with E2 (10−7 mol/L) or DMSO for 48 h. The relative fold induction of each group was calculated as the ratio of the luciferase activity induced by E2 to that induced by DMSO, respectively, and plotted as y-axis, and the deletion mutants were presented as in x-axis. All the data represented the mean±S.D. of three independent experiments. “N.S.” and “**” indicates P>0.05 and P<0.01, respectively, versus D1 group by unpaired student T test. (C) ChIP assay. T47D cells were treated with E2 (10−7 mol/L) for 4 h or pre-treated with Tamoxifen (10−6 mol/L) for 30 min before treating with E2 (10−7 mol/L) for 4 h. The immunoprecipitation was performed using antibodies against c-Myb (left panels) or ELF-1 (right panels), with preimmune IgG as control. The precipitated DNAs were analyzed by PCR with the primers corresponding to position −232 to +71 of vav1 promoter. The bar chart below the example blot represents the normalized DNA level of −232 to +71 to Input of three independent experiments. “N.S.” indicates P>0.05 versus DMSO treatment by unpaired student T test. (D) The association of ERα with c-Myb and ELF-1. T47D cells were treated with E2 (10−7 mol/L) or DMSO for 4 h or pretreated with Tamoxifen (10−6 mol/L) for 30 min in prior to E2 treatment and lysed. Antibodies against c-Myb (upper panels) and ELF-1 (lower panels) or control IgG (Lane 1) were used to immunoprecipitate protein complex, which were then resolved by Western Blot with indicated antibodies. (E) The proposed model for ERα modulating the vav1 promoter activity. E2-activated ERα interacts with vav1 promoter via interacting with transcription factors such as c-Myb, ELF-1, or perhaps other unknown coregulators (labeled “?”) to promote the vav1 transcription.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048212&req=5

pone-0099052-g005: Analysis of transcription factors involved in ERα-activated vav1 promoter.(A) Depicted deletion mutations in vav1 promoter reporter gene. The deleted regions were indicated by break lines. (B) MCF7 cells were transfected with plasmids containing luciferase under WT or mutated vav1 promoters (D1, D2, and D3) and then treated with E2 (10−7 mol/L) or DMSO for 48 h. The relative fold induction of each group was calculated as the ratio of the luciferase activity induced by E2 to that induced by DMSO, respectively, and plotted as y-axis, and the deletion mutants were presented as in x-axis. All the data represented the mean±S.D. of three independent experiments. “N.S.” and “**” indicates P>0.05 and P<0.01, respectively, versus D1 group by unpaired student T test. (C) ChIP assay. T47D cells were treated with E2 (10−7 mol/L) for 4 h or pre-treated with Tamoxifen (10−6 mol/L) for 30 min before treating with E2 (10−7 mol/L) for 4 h. The immunoprecipitation was performed using antibodies against c-Myb (left panels) or ELF-1 (right panels), with preimmune IgG as control. The precipitated DNAs were analyzed by PCR with the primers corresponding to position −232 to +71 of vav1 promoter. The bar chart below the example blot represents the normalized DNA level of −232 to +71 to Input of three independent experiments. “N.S.” indicates P>0.05 versus DMSO treatment by unpaired student T test. (D) The association of ERα with c-Myb and ELF-1. T47D cells were treated with E2 (10−7 mol/L) or DMSO for 4 h or pretreated with Tamoxifen (10−6 mol/L) for 30 min in prior to E2 treatment and lysed. Antibodies against c-Myb (upper panels) and ELF-1 (lower panels) or control IgG (Lane 1) were used to immunoprecipitate protein complex, which were then resolved by Western Blot with indicated antibodies. (E) The proposed model for ERα modulating the vav1 promoter activity. E2-activated ERα interacts with vav1 promoter via interacting with transcription factors such as c-Myb, ELF-1, or perhaps other unknown coregulators (labeled “?”) to promote the vav1 transcription.
Mentions: The above results indicated that ERα was in complex with the 5′ region of vav1 gene promoter. Several transcription factors were predicted to bind at the 5′ minimal regulatory region of the human vav1 gene, including ETF, Sp1, E2F, NF-e, c-Myb, TCFα, PU.1, and ELF-1 [30]. We therefore attempted to locate the regions that respond to estrogen. The wild type vav1 promoter (WT) and the truncated mutants (D1, D2, D3) that lack the predicted transcription factor binding sites were depicted in Figure 5A, and the reporter plasmids were constructed [30]. As shown in Figure 5B, the wild type promoter activity was elevated to 3 fold by E2. The deletion mutant D1 that lacks region -(143∼152) exhibited similar extent of induction (2.6 fold), implying that the region −(143∼152) was dispensable in E2-induced vav1 expression. In contrast, the E2 induction of truncated promoters D2 and D3 was severely suppressed to less than 1.5 fold (P<0.01), indicating that these two regions, −(25∼38) and −(5∼22), were required for E2-induced vav1 transcription. As these regions were reported to possess putative binding sites for transcription factors E2F/NF-e/c-Myb at -(25∼38) and TCFα/PU.1/ELF-1 at −(5∼22), respectively, ERα may associate with certain transcription factors within these regions. As reported previously, c-Myb affects vav1 transcription in lung cancer cells [30] and is also involved in the E2-ER regulated gene expression in breast cancer cells [39]. Meanwhile, another breast cancer related transcription factor, ELF-1, is identified to interact with the promoter of vav1 (Genome browser, http://genome.ucsc.edu/) [40]. Firstly, to confirm the binding of these two transcription factors to vav1 promoter, the ChIP analysis was performed. As shown in Figure 5C, both c-Myb and ELF-1 presented positively in complex with the vav1 promoter (Fig. 5C, upper two panels), and the presence of E2 or Tamoxifen had no effects on the complex (Fig. 5C, bottom panel).

Bottom Line: Nevertheless, two regions at the vav1 gene promoter were defined to be responsible for E2-induced activation of vav1 promoter.Consequently, the enhanced expression of Vav1 led to the elevation of Cyclin D1 and the progression of cell cycle.The present study implies that estrogen-ER modulates the transcription and expression of Vav1, which may contribute to the proliferation of cancerous cells.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Microbial Functional Genomics of Ministry of Education, College of Life Sciences, Nankai University, Tianjin, P. R. China.

ABSTRACT
Vav1, a guanine nucleotide exchange factor (GEF) for Rho family GTPases, is a hematopoietic protein involved in a variety of cellular events. In recent years, aberrant expression of Vav1 has been reported in non-hematopoietic cancers including human breast cancer. It remains to be answered how Vav1 is expressed and what Vav1 does in its non-resident tissues. In this study, we aimed to explore the mechanism for Vav1 expression in breast cancer cells in correlation with estrogen-ER pathway. We not only verified the ectopic expression of Vav1 in human breast cancer cell lines, but also observed that Vav1 expression was induced by 17β-estradiol (E2), a typical estrogen receptor (ER) ligand, in ER-positive cell lines. On the other hand, Tamoxifen, a selective estrogen receptor modulator (SERM), and ICI 182,780, an ER antagonist, suppressed the expression of Vav1. The estrogen receptor modulating Vav1 expression was identified to be α form, not β. Furthermore, treatment of E2 increased the transcription of vav1 gene by enhancing the promoter activity, though there was no recognizable estrogen response element (ERE). Nevertheless, two regions at the vav1 gene promoter were defined to be responsible for E2-induced activation of vav1 promoter. Chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP) analyses suggested that ERα might access to the vav1 promoter via interacting with transcription factors, c-Myb and ELF-1. Consequently, the enhanced expression of Vav1 led to the elevation of Cyclin D1 and the progression of cell cycle. The present study implies that estrogen-ER modulates the transcription and expression of Vav1, which may contribute to the proliferation of cancerous cells.

Show MeSH
Related in: MedlinePlus