J Breast Cancer.  2011 Mar;14(1):20-27. 10.4048/jbc.2011.14.1.20.

Role of Estrogen Receptor-alpha in the Regulation of Claudin-6 Expression in Breast Cancer Cells

• Yafang L
• Qiong W
• Yue R
• Xiaoming X
• Lina Y
• Mingzi Z
• Ting Z
• Yulin L
• Chengshi Q

• KMID: 2175638
• DOI: http://doi.org/10.4048/jbc.2011.14.1.20

Abstract

PURPOSE
In our previous studies we showed that upregulating claudin-6 (CLDN6) expression may contribute to preventing breast cancer, and that 17beta-estradiol induces a concentration- and time-related effect on CLDN6 mRNA and protein expression in MCF-7 cells. However, the mechanisms of 17beta-estradiol regulation of CLDN6 are still unclear. We determined the role of estrogen receptors in the regulation of CLDN6 expression in human breast cancer tissues and a cell line.
METHODS
CLDN6, estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) expression in breast cancer tissues were examined using immunohistochemistry. The human breast cancer cell line, MCF-7, which expresses ERalpha but not ERbeta was used. CLDN6 and ERalpha expression were measured by reverse transcriptase-PCR, Western blotting and immunofluorescent staining. Treatments with propyl pyrazole triol (PPT) and ICI 182, 780 (ICI) were performed.
RESULTS
The results revealed that CLDN6 expression was related to ERalpha in breast cancer tissues (p=0.033). PPT, an ERalpha-selective ligand, upregulated CLDN6 expression at 10-5 mol/L after 24 hours. The effect of PPT on regulating CLDN6 expression in MCF-7 cells was blocked by ICI.
CONCLUSION
These findings suggest that Eralpha reulates CLDN6 expression in breast cancer tissues and that 17beta-estradiol induces CLDN6 expression through an ERalpha pathway in MCF-7 cells.

Keyword

Breast carcinoma; Claudins; Estrogen; Estrogen receptor alpha; Tight junctions

MeSH Terms

Blotting, Western
Breast
Breast Neoplasms
Cell Line
Claudins
Estrogen Receptor alpha
Estrogen Receptor beta
Estrogens
Humans
Immunohistochemistry
MCF-7 Cells
Pyrazoles
Receptors, Estrogen
RNA, Messenger
Tight Junctions
Claudins
Estrogen Receptor alpha
Estrogen Receptor beta
Estrogens
Pyrazoles
RNA, Messenger
Receptors, Estrogen

Figure

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  Figure 1 Estrogen receptor-α (ERα) expression in breast carcinoma, as detected by immunohistochemistry. Staining was specific for tumor tissue, and nuclear staining was observed. Bar=20 µm. (A) Negative expression of ERα in a breast carcinoma. (B) Positive expression of ERα in a breast carcinoma.

• 

  Figure 2 Claudin-6 (CLDN6) expression in a breast carcinoma, as detected by immunohistochemistry. Staining was specific for tumor tissue, and membrane staining was observed. Bar=20 µm. (A) Negative CLDN6 expression in a breast carcinoma. (B) Positive CLDN6 expression in a breast carcinoma.

• 

  Figure 3 Estrogen receptor-α (ERα) and estrogen receptor-β (ERβ) expression in MCF-7 cells, determined by RT-PCR (A) and Western blot assay (B). MCF-cells, different with MDA MB 231 cells, only expressed ERα, but not ERβ.

• 

  Figure 4 MCF-7 cells were treated with different concentrations of propyl pyrazole triol (PPT) for 1 to 24 hr. PPT induced estrogen receptor-α (ERα) and claudin-6 (CLDN6) expression in a concentration- and time-dependent manner. (A) RT-PCR was used to determine ERα and CLDN6 expression in MCF-7 cells treated with different concentrations of PPT for 24 hr. The results showed that 10-6 mol/L and 10-5 mol/L PPT had the greatest effect on inducing ERα expression but only 10-5 mol/L PPT induced CLDN6 expression. (B) The Western blot assay was performed to detect ERα and CLDN6 protein expression in MCF-7 cells treated with different concentrations of PPT for 24 hr. The changes in protein levels were coincident with mRNA levels. (C) CLDN6 expression in MCF-7 cells treated with 10-5 mol/L PPT for 1 to 24 hr. The results showed that 10-5 mol/L PPT for 24 hr had the greatest effect on inducing ERα and CLDN6 mRNA expression levels. (D) The Western blot assay confirmed that protein levels were associated with mRNA levels. *p<0.05, vs. MCF-7 cells (statistically significant).

• 

  Figure 5 Effect of ICI182780 (ICI) on propyl pyrazole triol (PPT)-induced claudin-6 (CLDN6) expression. (A) The effect of ICI on PPT-induced CLDN6 mRNA expression was determined by RT-PCR. Lane 1 and Lane 2 showed the expression of CLDN6 in control cells (C) and vacant vehicle control cells (VE) respectively. Lanes 3-5 showed that 5×10-9 mol/L 17β-estradiol (E2) and 10-5 mol/L PPT for 24 hr induced CLDN6 expression respectively or collectively. Lane 6 shows that ICI significantly blocked the effect of PPT. (B) The Western blot assay revealed CLDN6 protein expression in the six groups. Lanes 3-5 showed that CLDN6 protein expression was significantly upregulated in MCF-7 cells treated with 17-β-estradiol or (and) PPT for 24 hr. Lane 6 shows that the effect of PPT could be blocked by ICI. (C) Immunofluoresce staining showed CLDN6 expression on the membranes of MCF-7 cells treated with 17β-estradiol or (and) PPT for 24 hr. *p<0.05, vs. MCF-7 cells (statistically significant).

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