12-O-Tetradecanoylphorbol-13-Acetate Induces Up-Regulated Transcription of Variant 1 but Not Variant 2 of <i>VIL2</i> in Esophageal Squamous Cell Carcinoma Cells via ERK1/2/AP-1/Sp1 Signaling

<div><p>The membrane-cytoskeleton link organizer ezrin may be the most “dramatic” tumor marker, being strongly over-expressed in nearly one-third of human malignancies. However, the molecular mechanisms of aberrant ezrin expression still need to be clarified. Ezrin, encoded by the <i>VIL2 </i>gene, has two transcript variants that differ in the transcriptional start site (TSS): V1 and V2. Both V1 and V2 encode the same protein. Here, we found that 12-O-tetradecanoylphorbol-13-acetate (TPA) induced over-expression of human <i>VIL2 </i>in esophageal squamous cell carcinoma (ESCC) cells. Furthermore, <i>VIL2 </i>V1 but not V2 was up-regulated after TPA stimulation in a time-dependent manner. AP-1 and Sp1 binding sites within the promoter region of <i>VIL2 </i>V1 acted not only as basal transcriptional elements but also as a composite TPA-responsive element (TRE) for the transcription of <i>VIL2 </i>V1. TPA stimulation enhanced c-Jun and Sp1 binding to the TRE via activation of the ERK1/2 pathway and increased protein levels of c-Jun, c-Fos, and Sp1, resulting in over-expression of <i>VIL2 </i>V1, whereas the MEK1/2 inhibitor U0126 blocked these events. Finally, we showed that TPA promoted the migration of ESCC cells whereas MEK1/2 inhibitor or ezrin silencing could partially inverse this alteration. Taken together, these results suggest that TPA is able to induce <i>VIL2 </i>V1 over-expression in ESCC cells by activating MEK/ERK1/2 signaling and increasing binding of Sp1 and c-Jun to the TRE of the <i>VIL2 </i>V1 promoter, and that <i>VIL2 </i>is an important TPA-induced effector.</p></div

Identification of AP-1/Sp1 composite was TPA response element.

<p>(A) EMSA assay of the nuclear extract prepared from EC109 cells bound to the sequence of -87/-46 fragment within the V1 promoter region of <i>VIL2</i> after stimulation with DMSO or TPA for indicated time. Probe WT (-87/-46) was labeled with biotin. (B) The specific DNA-protein complex was confirmed by using biotin-labeled Sm, Am, or SAm oligonucleotides covering the same segment with probe WT but containing a substituted sequence (lanes 3 through 5), which are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124680#pone.0124680.t002" target="_blank">Table 2</a>. The probes were incubated with nuclear extract prepared from EC109 cells treated with DMSO or with TPA for 24 h. (C) Sonicated chromatin isolated from EC109 cells was incubated with antibodies against IgG, Sp1 and c-Jun, respectively. ChIP assay showed that TPA treatment enhanced Sp1 and c-Jun bound to the <i>VIL2</i> promoter region. (D) The effect of site-directed mutagenesis of the AP-1/Sp1 binding sites on the <i>VIL2</i> V1 promoter activity. The mutagenesis constructs were co-transfected with pRL-TK into EC109 or KYSE180 cells. After transfection for 24 h, the cells were treated with DMSO (control; open bars) or TPA (10 ng/ml; filled bars) for another 24 h before analysis. The <i>firefly</i> luciferase activities of mutant constructs were normalized to <i>Renilla</i> luciferase activity and shown as a ratio compared to that of the pGLB-hE (-87/+134) construct treated with DMSO, which was set as 1.Each value represents the mean ± SD, n≥3. The data are representative of at least two independent experiments. *<i>p</i> < 0.05 or **<i>p</i> < 0.01, compared to the pGLB-hE (-87/+134) construct treated with DMSO; ^Δ<i>p</i> < 0.05 or ^ΔΔ<i>p</i> < 0.01, compared to the pGLB-hE (-87/+134) construct treated with TPA.</p

TPA up-regulated <i>VIL2</i> V1 transcription via ERK1/2/AP-1/Sp1 signaling.

<p><b>(A)</b> The expression of Sp1, c-Jun, c-Fos, phospho-ERK1/2 (Thr202/Tyr204) or total ERK was detected after treated with DMSO or TPA (10 ng/ml) for the indicated time in EC109 and KYSE180 cells. β-actin or β-tubulin was served as a loading control. (B) EC109 and KYSE180 cells were treated with DMSO, TPA (10 ng/ml), or pretreated for 1 h with U0126 (10 μM) before adding TPA, respectively, and alteration of ERK1/2/AP-1/Sp1 signaling was determined. (C) Wound healing assay for the cells treated with TPA (10 ng/ml) or TPA and U0126. (D) Wound healing assay was employed to detect the effect of ezrin knockdown on the TPA-mediated cell migration in KYSE180 cells. Left, western blotting analysis for the ezrin silencing in the TPA-treated cells; Right, cell migration assay.</p

Probes for EMSA.

<p>Substituted sites are underlined.</p><p>Probes for EMSA.</p

Expression of the <i>VIL2</i> transcription variants in cell lines.

<p>(A) The genomic structure of <i>VIL2</i> gene. TSS, transcriptional start site; CDS, coding sequencing. The gene information was adapted from NCBI database (Accession number: NC_000006) and Genbank database (Accession number: EF184645). (B) qRT-PCR was used to address the expression of the <i>VIL2</i> transcription variants in a panel of cells lines. Each bar represents the mean ± SD. (C) Alteration of <i>VIL2</i> V1 or V2 mRNA level in response to inducers treatment. The total RNA extract (1 μg) from ESCC cells treated with or without inducers for 24 h, and subjected to qRT-PCR analysis using gene-specific primers. The relative value from the control group was considered equal to one arbitrary unit. Each point represents the mean ± SD. The data are representative of at least two independent experiments.</p

Primers used in this study.

<p>F: forward primer; R: reverse primer.</p><p>Cutting sites are underlined.</p><p>Primers used in this study.</p

TPA increased transcriptional expression of human <i>VIL2</i> transcript variant V1 but not V2.

<p>(A) Alteration of <i>VIL2</i> V1 or V2 mRNA level in response to TPA treatment. The total RNA extract (1 μg) from ESCC cells treated with DMSO or TPA (10 ng/ml) for various time were reversely transcribed, and subsequently use qRT-PCR assay to detect the change. The relative value from the DMSO-treated control group was considered equal to one arbitrary unit. Each point represents the mean ± SD, n≥3. (B) EC109 and KYSE180 cells were stimulated with DMSO or TPA (10 ng/ml) for the indicated time after starvation for 12 h. Western blot was used to determine the protein level of ezrin upon TPA treatment. Signal intensity for the expression of ezrin was quantified by densitometric scanning and normalized by internal control (β-actin). The data represent the means ± SD of triplicate experiments. (C) Relative RT-PCR analysis. The total RNA extract was prepared from EC109 and KYSE180 cells after stimulation with DMSO or TPA (10 ng/ml) for 6 h, or pretreated with actinomycin D (1 μg/ml) for 1 h before TPA was added (10 ng/ml). The target genes were examined using the specific primers. The densitometry values for <i>VIL2</i> V1 levels were normalized to the values for β-actin and then presented relative to that of the DMSO-treated control, which was set as 1. The results of a representative experiment were presented as mean ± SD of the two independent samples. *<i>p</i>< 0.05 or **<i>p</i>< 0.01.</p

Identification of TPA responsive region.

<p>(A) TPA-induced activity of <i>VIL2</i> promoter region. Constructs of pGLB-hE(-87/+134) containing <i>VIL2</i> V1 promoter region, pGLB-hE(-1759/+134) containing both <i>VIL2</i> V1 and V 2 promoter region, pGLB-hE(-1695/-1148) containing <i>VIL2</i> V2 promoter region or empty vector pGL3-basic (pGLB) were co-transfected with pRL-TK into EC109 cells simulated with TPA for 24 h before assay the luciferase activity. The <i>firefly</i> luciferase activity was normalized to <i>Renilla</i> luciferase activity and the relative value from the DMSO-treated control group was considered equal to one arbitrary unit. Each value represents the mean ± SD, n≥3. (B) The schematic of the <i>VIL2</i> promoter 5′-deletion constructs used for transient transfections (<i>Left</i>) and the relative luciferase activity of whole cell extracts from EC109 and KYSE180 cells transiently transfected with the constructs listed in the left for 24 h and then treated with DMSO or TPA for another 24 h (<i>Right</i>). The <i>firefly</i> luciferase activity was normalized to <i>Renilla</i> luciferase activity and the relative value from the DMSO-treated cells transfected with pGLB-hE (-1759/+134) was set to 100%. Each value represents the mean ± SD, n≥3. (C) TPA-induced activity of <i>VIL2</i> promoter region in different cells. pGLB-hE(-87/+134) and pGLB-hE(-1695/-1148) Constructs were used and the relative value from the DMSO-induced control group was considered equal to one arbitrary unit. Each value represents the mean ± SD, n≥3. The data are representative of at least two independent experiments. *<i>p</i>< 0.05 or **<i>p</i>< 0.01.</p

Kaplan-Meier analyses of OS considering a molecular prognostic model and N-stage in generation and validation datasets.

<p>Kaplan-Meier analyses of OS considering a molecular prognostic model and N-stage in generation and validation datasets.</p

The clinicopathological characteristics of two datasets of patients with ESCC.

<p>*, chemo, chemotherapy; radio, radiotherapy.</p