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Suppression of estrogen receptor transcriptional activity by connective tissue growth factor.

Cheng L, Yang Z, Wang X, Jiao Y, Xie X, Lin J, Zhang H, Han J, Jiang K, Ye Q - PLoS ONE (2011)

Bottom Line: Reduction of endogenous CTGF with CTGF small interfering RNA enhanced ER transcriptional activity.The interaction between CTGF and ER is required for the repression of estrogen-responsive transcription by CTGF.Moreover, CTGF reduced ER protein expression, whereas the CTGF mutant that did not repress ER transcriptional activity also did not alter ER protein levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Molecular Biology, Beijing Institute of Biotechnology, Beijing, People's Republic of China.

ABSTRACT
Secreted growth factors have been shown to stimulate the transcriptional activity of estrogen receptors (ER) that are responsible for many biological processes. However, whether these growth factors physically interact with ER remains unclear. Here, we show for the first time that connective tissue growth factor (CTGF) physically and functionally associates with ER. CTGF interacted with ER both in vitro and in vivo. CTGF interacted with ER DNA-binding domain. ER interaction region in CTGF was mapped to the thrombospondin type I repeat, a cell attachment motif. Overexpression of CTGF inhibited ER transcriptional activity as well as the expression of estrogen-responsive genes, including pS2 and cathepsin D. Reduction of endogenous CTGF with CTGF small interfering RNA enhanced ER transcriptional activity. The interaction between CTGF and ER is required for the repression of estrogen-responsive transcription by CTGF. Moreover, CTGF reduced ER protein expression, whereas the CTGF mutant that did not repress ER transcriptional activity also did not alter ER protein levels. The results suggested the transcriptional regulation of estrogen signaling through interaction between CTGF and ER, and thus may provide a novel mechanism by which cross-talk between secreted growth factor and ER signaling pathways occurs.

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CTGF inhibits ERα expression.(A) MCF7 cells stably transfected with FLAG-tagged CTGF, which constitutively expressed FLAG-CTGF, or MCF7 cells stably transfected with empty vector were treated with 10 nm E2 for 24 h. Whole cell lysates were blotted with the indicated antibodies. (B) MCF7 cells were treated with the indicated amounts of recombinant human CTGF (rhCTGF) and analyzed as in (A). (C) MCF7 cells transfected with CTGF siRNA2 or control siRNA were treated and analyzed as in (A). Knockdown effect of CTGF siRNA2 on the endogenous CTGF mRNA level was determined by real-time PCR with CTGF and β-actin primers (right panel). (D) ZR75-1 cells transiently transfected with FLAG-tagged CTGF or empty vector were treated and analyzed as in (A). (E) ZR75-1 cells transfected with CTGF siRNA1 or CTGF siRNA2 were treated and analyzed as in (A). Knockdown effect of CTGF siRNA1 or CTGF siRNA2 on the endogenous CTGF mRNA levels was determined as in (C) (lower panel). (F) MCF7 cells were transfected with FLAG-tagged CTGF or CTGF(1–187) and treated with 10 nm E2. Cell lysates were analyzed by immunoblot with the indicated antibodies. (G) MCF7 cells stably transfected with FLAG-tagged CTGF or empty vector were pretreated with 10 µM MG132 for 1 h to block proteasome activity. Cells were then treated with 10 nM E2 for 24 h. Cell lysates were analyzed as in (D). (H) MCF7 cells transfected with FLAG-tagged CTGF or CTGF(1–187) were used for real-time RT-PCR with ERα and GAPDH primers (upper panel). Cell lysates were examined by immunoblot with the indicated antibodies (lower panel). Data shown are means ± SD of triplicates of one representative experiment and have been repeated three times with similar results. *P<0.01 versus empty vector without E2. #P<0.01 versus empty vector with E2.
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pone-0020028-g008: CTGF inhibits ERα expression.(A) MCF7 cells stably transfected with FLAG-tagged CTGF, which constitutively expressed FLAG-CTGF, or MCF7 cells stably transfected with empty vector were treated with 10 nm E2 for 24 h. Whole cell lysates were blotted with the indicated antibodies. (B) MCF7 cells were treated with the indicated amounts of recombinant human CTGF (rhCTGF) and analyzed as in (A). (C) MCF7 cells transfected with CTGF siRNA2 or control siRNA were treated and analyzed as in (A). Knockdown effect of CTGF siRNA2 on the endogenous CTGF mRNA level was determined by real-time PCR with CTGF and β-actin primers (right panel). (D) ZR75-1 cells transiently transfected with FLAG-tagged CTGF or empty vector were treated and analyzed as in (A). (E) ZR75-1 cells transfected with CTGF siRNA1 or CTGF siRNA2 were treated and analyzed as in (A). Knockdown effect of CTGF siRNA1 or CTGF siRNA2 on the endogenous CTGF mRNA levels was determined as in (C) (lower panel). (F) MCF7 cells were transfected with FLAG-tagged CTGF or CTGF(1–187) and treated with 10 nm E2. Cell lysates were analyzed by immunoblot with the indicated antibodies. (G) MCF7 cells stably transfected with FLAG-tagged CTGF or empty vector were pretreated with 10 µM MG132 for 1 h to block proteasome activity. Cells were then treated with 10 nM E2 for 24 h. Cell lysates were analyzed as in (D). (H) MCF7 cells transfected with FLAG-tagged CTGF or CTGF(1–187) were used for real-time RT-PCR with ERα and GAPDH primers (upper panel). Cell lysates were examined by immunoblot with the indicated antibodies (lower panel). Data shown are means ± SD of triplicates of one representative experiment and have been repeated three times with similar results. *P<0.01 versus empty vector without E2. #P<0.01 versus empty vector with E2.

Mentions: To further investigate the mechanisms by which CTGF represses ER transcriptional activity, we determined the effect of CTGF on ERα expression by immunoblotting. As expected [18], E2 decreased ERα protein levels in MCF7 or ZR75-1 cells (Fig. 8 A–E). Importantly, Both FLAG-tagged CTGF and recombinant human CTGF inhibited ERα protein expression both in the presence and in the absence of estrogen, and recombinant human CTGF inhibited ERα protein expression in a dose-dependent manner (Fig. 8 A, B and D). In contrast, knockdown of endogenous CTGF in MCF7 or ZR75-1 cells increased ERα protein levels (Fig. 8 C and E). Although FLAG-tagged full-length CTGF repressed the expression of ERα protein, the CTGF(1–187) mutant that did not decrease ERα transcriptional activity also did not change ERα protein levels in MCF7 cells (Fig. 8F). Reduction of ERα protein levels by CTGF is not mediated through proteosome-dependent protein degradation because MG132, a proteosome inhibitor, had no effect on CTGF-mediated repression of ERα protein expression (Fig. 8G). As a control, MG132 blocked E2-induced dowregulation of ERα.


Suppression of estrogen receptor transcriptional activity by connective tissue growth factor.

Cheng L, Yang Z, Wang X, Jiao Y, Xie X, Lin J, Zhang H, Han J, Jiang K, Ye Q - PLoS ONE (2011)

CTGF inhibits ERα expression.(A) MCF7 cells stably transfected with FLAG-tagged CTGF, which constitutively expressed FLAG-CTGF, or MCF7 cells stably transfected with empty vector were treated with 10 nm E2 for 24 h. Whole cell lysates were blotted with the indicated antibodies. (B) MCF7 cells were treated with the indicated amounts of recombinant human CTGF (rhCTGF) and analyzed as in (A). (C) MCF7 cells transfected with CTGF siRNA2 or control siRNA were treated and analyzed as in (A). Knockdown effect of CTGF siRNA2 on the endogenous CTGF mRNA level was determined by real-time PCR with CTGF and β-actin primers (right panel). (D) ZR75-1 cells transiently transfected with FLAG-tagged CTGF or empty vector were treated and analyzed as in (A). (E) ZR75-1 cells transfected with CTGF siRNA1 or CTGF siRNA2 were treated and analyzed as in (A). Knockdown effect of CTGF siRNA1 or CTGF siRNA2 on the endogenous CTGF mRNA levels was determined as in (C) (lower panel). (F) MCF7 cells were transfected with FLAG-tagged CTGF or CTGF(1–187) and treated with 10 nm E2. Cell lysates were analyzed by immunoblot with the indicated antibodies. (G) MCF7 cells stably transfected with FLAG-tagged CTGF or empty vector were pretreated with 10 µM MG132 for 1 h to block proteasome activity. Cells were then treated with 10 nM E2 for 24 h. Cell lysates were analyzed as in (D). (H) MCF7 cells transfected with FLAG-tagged CTGF or CTGF(1–187) were used for real-time RT-PCR with ERα and GAPDH primers (upper panel). Cell lysates were examined by immunoblot with the indicated antibodies (lower panel). Data shown are means ± SD of triplicates of one representative experiment and have been repeated three times with similar results. *P<0.01 versus empty vector without E2. #P<0.01 versus empty vector with E2.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3101213&req=5

pone-0020028-g008: CTGF inhibits ERα expression.(A) MCF7 cells stably transfected with FLAG-tagged CTGF, which constitutively expressed FLAG-CTGF, or MCF7 cells stably transfected with empty vector were treated with 10 nm E2 for 24 h. Whole cell lysates were blotted with the indicated antibodies. (B) MCF7 cells were treated with the indicated amounts of recombinant human CTGF (rhCTGF) and analyzed as in (A). (C) MCF7 cells transfected with CTGF siRNA2 or control siRNA were treated and analyzed as in (A). Knockdown effect of CTGF siRNA2 on the endogenous CTGF mRNA level was determined by real-time PCR with CTGF and β-actin primers (right panel). (D) ZR75-1 cells transiently transfected with FLAG-tagged CTGF or empty vector were treated and analyzed as in (A). (E) ZR75-1 cells transfected with CTGF siRNA1 or CTGF siRNA2 were treated and analyzed as in (A). Knockdown effect of CTGF siRNA1 or CTGF siRNA2 on the endogenous CTGF mRNA levels was determined as in (C) (lower panel). (F) MCF7 cells were transfected with FLAG-tagged CTGF or CTGF(1–187) and treated with 10 nm E2. Cell lysates were analyzed by immunoblot with the indicated antibodies. (G) MCF7 cells stably transfected with FLAG-tagged CTGF or empty vector were pretreated with 10 µM MG132 for 1 h to block proteasome activity. Cells were then treated with 10 nM E2 for 24 h. Cell lysates were analyzed as in (D). (H) MCF7 cells transfected with FLAG-tagged CTGF or CTGF(1–187) were used for real-time RT-PCR with ERα and GAPDH primers (upper panel). Cell lysates were examined by immunoblot with the indicated antibodies (lower panel). Data shown are means ± SD of triplicates of one representative experiment and have been repeated three times with similar results. *P<0.01 versus empty vector without E2. #P<0.01 versus empty vector with E2.
Mentions: To further investigate the mechanisms by which CTGF represses ER transcriptional activity, we determined the effect of CTGF on ERα expression by immunoblotting. As expected [18], E2 decreased ERα protein levels in MCF7 or ZR75-1 cells (Fig. 8 A–E). Importantly, Both FLAG-tagged CTGF and recombinant human CTGF inhibited ERα protein expression both in the presence and in the absence of estrogen, and recombinant human CTGF inhibited ERα protein expression in a dose-dependent manner (Fig. 8 A, B and D). In contrast, knockdown of endogenous CTGF in MCF7 or ZR75-1 cells increased ERα protein levels (Fig. 8 C and E). Although FLAG-tagged full-length CTGF repressed the expression of ERα protein, the CTGF(1–187) mutant that did not decrease ERα transcriptional activity also did not change ERα protein levels in MCF7 cells (Fig. 8F). Reduction of ERα protein levels by CTGF is not mediated through proteosome-dependent protein degradation because MG132, a proteosome inhibitor, had no effect on CTGF-mediated repression of ERα protein expression (Fig. 8G). As a control, MG132 blocked E2-induced dowregulation of ERα.

Bottom Line: Reduction of endogenous CTGF with CTGF small interfering RNA enhanced ER transcriptional activity.The interaction between CTGF and ER is required for the repression of estrogen-responsive transcription by CTGF.Moreover, CTGF reduced ER protein expression, whereas the CTGF mutant that did not repress ER transcriptional activity also did not alter ER protein levels.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Molecular Biology, Beijing Institute of Biotechnology, Beijing, People's Republic of China.

ABSTRACT
Secreted growth factors have been shown to stimulate the transcriptional activity of estrogen receptors (ER) that are responsible for many biological processes. However, whether these growth factors physically interact with ER remains unclear. Here, we show for the first time that connective tissue growth factor (CTGF) physically and functionally associates with ER. CTGF interacted with ER both in vitro and in vivo. CTGF interacted with ER DNA-binding domain. ER interaction region in CTGF was mapped to the thrombospondin type I repeat, a cell attachment motif. Overexpression of CTGF inhibited ER transcriptional activity as well as the expression of estrogen-responsive genes, including pS2 and cathepsin D. Reduction of endogenous CTGF with CTGF small interfering RNA enhanced ER transcriptional activity. The interaction between CTGF and ER is required for the repression of estrogen-responsive transcription by CTGF. Moreover, CTGF reduced ER protein expression, whereas the CTGF mutant that did not repress ER transcriptional activity also did not alter ER protein levels. The results suggested the transcriptional regulation of estrogen signaling through interaction between CTGF and ER, and thus may provide a novel mechanism by which cross-talk between secreted growth factor and ER signaling pathways occurs.

Show MeSH
Related in: MedlinePlus