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The DNA mismatch repair gene hMSH2 is a potent coactivator of oestrogen receptor alpha.

Wada-Hiraike O, Yano T, Nei T, Matsumoto Y, Nagasaka K, Takizawa S, Oishi H, Arimoto T, Nakagawa S, Yasugi T, Kato S, Taketani Y - Br. J. Cancer (2005)

Bottom Line: Oestrogen receptor alpha/beta bound to hMSH2 through the hMSH3/hMSH6 interaction domain of hMSH2.In a transient expression assay, hMSH2 potentiated the transactivation function of liganded ER alpha, but not that of ER beta.These results suggest that hMSH2 may play an important role as a putative coactivator in ER alpha dependent gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1 Bunkyo-ku, Tokyo 113-8655, Japan. owada-tky@umin.ac.jp

ABSTRACT
The DNA mismatch repair gene is a key regulator in the elimination of base-base mismatches and insertion/deletion loops (IDLs). Human MutS homologue 2 (hMSH2), originally identified as a human homologue of the bacterial MutS, is a tumour suppressor gene frequently mutated in hereditary non-polyposis colorectal cancer. Hereditary non-polyposis colorectal cancer is characterised by the early onset of colorectal cancer and the development of extracolonic cancers such as endometrial, ovarian, and urological cancers. Oestrogen receptor (ER) alpha and beta are members of a nuclear receptor (NR) superfamily. Ligand-dependent transcription of ER is regulated by the p160 steroid receptor coactivator family, the thyroid hormone receptor-associated proteins/the vitamin D receptor-interacting proteins (TRAP/DRIP) mediator complex, and the TATA box-binding protein (TBP)-free TBP associated factor complex (TFTC) type histone acetyltransferase complex. Here, we report the interaction between ER alpha/beta and hMSH2. Immunoprecipitation and glutathione-S-transferase pull-down assay revealed that ER alpha and hMSH2 interacted in a ligand-dependent manner, whereas ER beta and hMSH2 interacted in a ligand-independent manner. Oestrogen receptor alpha/beta bound to hMSH2 through the hMSH3/hMSH6 interaction domain of hMSH2. In a transient expression assay, hMSH2 potentiated the transactivation function of liganded ER alpha, but not that of ER beta. These results suggest that hMSH2 may play an important role as a putative coactivator in ER alpha dependent gene expression.

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In vivo association between hMSH2 and ER α/β and in vitro association between hMSH2 and ER α/β. (A) (i) Ishikawa cells were lysed and subjected to immunoprecipitation (IP) with the antbodies to ER α and IgG. MDA-MB-231 cell lysate was immunoprecipitated with the antbodies to ERβ BRCA1 and IgG. The immunoprecipitates were separated by SDS–PAGE and analysed by immunoblotting (IB) with the anti-hMSH2 antibody. (ii) Reciprocal IP was performed to detect endogenous hMSH2-ER α and hMSH2-ER β interactions by IB. (B) In vitro translated 35S-labelled hMSH2 was pulled down by GST-ER α/β AF-1 or GST-ER α/β AF-2. At the same time, in vitro translated TRAP220 was incubated with GST-ER α AF-2. The mixtures were washed and subjected to SDS–PAGE and analysed.
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fig1: In vivo association between hMSH2 and ER α/β and in vitro association between hMSH2 and ER α/β. (A) (i) Ishikawa cells were lysed and subjected to immunoprecipitation (IP) with the antbodies to ER α and IgG. MDA-MB-231 cell lysate was immunoprecipitated with the antbodies to ERβ BRCA1 and IgG. The immunoprecipitates were separated by SDS–PAGE and analysed by immunoblotting (IB) with the anti-hMSH2 antibody. (ii) Reciprocal IP was performed to detect endogenous hMSH2-ER α and hMSH2-ER β interactions by IB. (B) In vitro translated 35S-labelled hMSH2 was pulled down by GST-ER α/β AF-1 or GST-ER α/β AF-2. At the same time, in vitro translated TRAP220 was incubated with GST-ER α AF-2. The mixtures were washed and subjected to SDS–PAGE and analysed.

Mentions: To determine whether endogenous hMSH2 protein interacted with ER α, ER β, and BRCA1 in the cultured human cells, we performed immunoprecipitation assays by using the antibodies raised against ER α, ER β, and BRCA1. Oestrogen receptor α was immunoprecipitated in Ishikawa cell lysate. Oestrogen receptor β and BRCA1 were immunoprecipitated in MDA-MB-231 cell lysate. Complex formation of the precipitated proteins was confirmed by Western blotting. Immunoblotting revealed the exsistence of hMSH2 in the cell lysate immunoprecipitates (Figure 1A(i)), which supports our hypothesis that hMSH2 physically associates with ER α and ER β in living cells. These results were further confirmed by reciprocal immunoprecipitation with the specific antibody raised against hMSH2. Immunoblotting again revealed the exsistence of ER α and ER β (Figure 1A(ii)).


The DNA mismatch repair gene hMSH2 is a potent coactivator of oestrogen receptor alpha.

Wada-Hiraike O, Yano T, Nei T, Matsumoto Y, Nagasaka K, Takizawa S, Oishi H, Arimoto T, Nakagawa S, Yasugi T, Kato S, Taketani Y - Br. J. Cancer (2005)

In vivo association between hMSH2 and ER α/β and in vitro association between hMSH2 and ER α/β. (A) (i) Ishikawa cells were lysed and subjected to immunoprecipitation (IP) with the antbodies to ER α and IgG. MDA-MB-231 cell lysate was immunoprecipitated with the antbodies to ERβ BRCA1 and IgG. The immunoprecipitates were separated by SDS–PAGE and analysed by immunoblotting (IB) with the anti-hMSH2 antibody. (ii) Reciprocal IP was performed to detect endogenous hMSH2-ER α and hMSH2-ER β interactions by IB. (B) In vitro translated 35S-labelled hMSH2 was pulled down by GST-ER α/β AF-1 or GST-ER α/β AF-2. At the same time, in vitro translated TRAP220 was incubated with GST-ER α AF-2. The mixtures were washed and subjected to SDS–PAGE and analysed.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2361802&req=5

fig1: In vivo association between hMSH2 and ER α/β and in vitro association between hMSH2 and ER α/β. (A) (i) Ishikawa cells were lysed and subjected to immunoprecipitation (IP) with the antbodies to ER α and IgG. MDA-MB-231 cell lysate was immunoprecipitated with the antbodies to ERβ BRCA1 and IgG. The immunoprecipitates were separated by SDS–PAGE and analysed by immunoblotting (IB) with the anti-hMSH2 antibody. (ii) Reciprocal IP was performed to detect endogenous hMSH2-ER α and hMSH2-ER β interactions by IB. (B) In vitro translated 35S-labelled hMSH2 was pulled down by GST-ER α/β AF-1 or GST-ER α/β AF-2. At the same time, in vitro translated TRAP220 was incubated with GST-ER α AF-2. The mixtures were washed and subjected to SDS–PAGE and analysed.
Mentions: To determine whether endogenous hMSH2 protein interacted with ER α, ER β, and BRCA1 in the cultured human cells, we performed immunoprecipitation assays by using the antibodies raised against ER α, ER β, and BRCA1. Oestrogen receptor α was immunoprecipitated in Ishikawa cell lysate. Oestrogen receptor β and BRCA1 were immunoprecipitated in MDA-MB-231 cell lysate. Complex formation of the precipitated proteins was confirmed by Western blotting. Immunoblotting revealed the exsistence of hMSH2 in the cell lysate immunoprecipitates (Figure 1A(i)), which supports our hypothesis that hMSH2 physically associates with ER α and ER β in living cells. These results were further confirmed by reciprocal immunoprecipitation with the specific antibody raised against hMSH2. Immunoblotting again revealed the exsistence of ER α and ER β (Figure 1A(ii)).

Bottom Line: Oestrogen receptor alpha/beta bound to hMSH2 through the hMSH3/hMSH6 interaction domain of hMSH2.In a transient expression assay, hMSH2 potentiated the transactivation function of liganded ER alpha, but not that of ER beta.These results suggest that hMSH2 may play an important role as a putative coactivator in ER alpha dependent gene expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Graduate School of Medicine, The University of Tokyo, Hongo 7-3-1 Bunkyo-ku, Tokyo 113-8655, Japan. owada-tky@umin.ac.jp

ABSTRACT
The DNA mismatch repair gene is a key regulator in the elimination of base-base mismatches and insertion/deletion loops (IDLs). Human MutS homologue 2 (hMSH2), originally identified as a human homologue of the bacterial MutS, is a tumour suppressor gene frequently mutated in hereditary non-polyposis colorectal cancer. Hereditary non-polyposis colorectal cancer is characterised by the early onset of colorectal cancer and the development of extracolonic cancers such as endometrial, ovarian, and urological cancers. Oestrogen receptor (ER) alpha and beta are members of a nuclear receptor (NR) superfamily. Ligand-dependent transcription of ER is regulated by the p160 steroid receptor coactivator family, the thyroid hormone receptor-associated proteins/the vitamin D receptor-interacting proteins (TRAP/DRIP) mediator complex, and the TATA box-binding protein (TBP)-free TBP associated factor complex (TFTC) type histone acetyltransferase complex. Here, we report the interaction between ER alpha/beta and hMSH2. Immunoprecipitation and glutathione-S-transferase pull-down assay revealed that ER alpha and hMSH2 interacted in a ligand-dependent manner, whereas ER beta and hMSH2 interacted in a ligand-independent manner. Oestrogen receptor alpha/beta bound to hMSH2 through the hMSH3/hMSH6 interaction domain of hMSH2. In a transient expression assay, hMSH2 potentiated the transactivation function of liganded ER alpha, but not that of ER beta. These results suggest that hMSH2 may play an important role as a putative coactivator in ER alpha dependent gene expression.

Show MeSH
Related in: MedlinePlus