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.2016 Aug 2;11(8):e0160312.
doi: 10.1371/journal.pone.0160312. eCollection 2016.

Systematic Proteomic Identification of the Heat Shock Proteins (Hsp) that Interact with Estrogen Receptor Alpha (ERα) and Biochemical Characterization of the ERα-Hsp70 Interaction

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Systematic Proteomic Identification of the Heat Shock Proteins (Hsp) that Interact with Estrogen Receptor Alpha (ERα) and Biochemical Characterization of the ERα-Hsp70 Interaction

Ahmed E Dhamad et al. PLoS One..

Abstract

Heat shock proteins (Hsps) are known to associate with estrogen receptors (ER) and regulate ER-mediated cell proliferation. Historically, the studies in this area have focused on Hsp90. However, some critical aspects of the Hsp-ERα interactions remain unclear. For example, we do not know which Hsps are the major or minor ERα interactants and whether or not different Hsp isoforms associate equally with ERα. In the present study, through a quantitative proteomic method we found that 21 Hsps and 3 Hsp cochaperones were associated with ERα in human 293T cells that were cultured in a medium containing necessary elements for cell proliferation. Four Hsp70s (Hsp70-1, Hsc70, Grp75, and Grp78) were the most abundant Hsps identified to associate with ERα, followed by two Hsp90s (Hsp90α and Hsp90β) and three Hsp110s (Hsp105, HspA4, and HspA4L). Hsp90α was found to be 2-3 times more abundant than Hsp90β in the ERα-containing complexes. Among the reported Hsp cochaperones, we detected prostaglandin E synthase 3 (p23), peptidyl-prolyl cis-trans isomerase FKBP5 (FKBP51), and E3 ubiquitin-protein ligase CHIP (CHIP). Studies with the two most abundant ERα-associated Hsps, Hsp70-1 and Hsc70, using human breast cancer MCF7 cells demonstrate that the two Hsps interacted with ERα in both the cytoplasm and nucleus when the cells were cultured in a medium supplemented with fetal bovine serum and phenol red. Interestingly, the ERα-Hsp70-1/Hsc70 interactions were detected only in the cytoplasm but not in the nucleus under hormone starvation conditions, and stimulation of the starved cells with 17β-estradiol (E2) did not change this. In addition, E2-treatment weakened the ERα-Hsc70 interaction but had no effect on the ERα-Hsp70-1 interaction. Further studies showed that significant portions of Hsp70-1 and Hsc70 were associated with transcriptionally active chromatin and inactive chromatin, and the two Hsps interacted with ERα in both forms of the chromatins in MCF7 cells.

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Conflict of interest statement

Competing Interests:The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Verification of the interaction between Flag-ERα and endogenous Hsp70-1/Hsc70.
The 293T cells were transfected with a plasmid that expresses Flag alone (control) or Flag-ERα. Forty eight hours after transfection, the cells were harvested, lysed, and the resulting total protein was pulled down by immobilized anti-Flag antibody. The bound proteins were analyzed with Western blotting using anti-Hsp70-1 and anti-Hsc70 antibodies.
Fig 2
Fig 2. Endogenous ERα interacts with endogenous Hsp70-1 and Hsc70.
(A) The cytosolic and nuclear extracts of MCF7 cells were immunoprecipitated by anti-ERα antibody or an isotype-matched, unrelated control IgG, and the immunoprecipitated protein was analyzed by Western blotting with the indicated antibodies. (B) Left panel, quantification of the IP protein bands in Western blots. Signal intensity values were arbitrary numbers obtained by analyzing the protein bands with ImageJ software. Right panel, validation of the cytosolic and nuclear fractionations. Tubulin and histone H3 were used as markers for the cytosolic and nuclear fractions, respectively. (C) The MCF7 cells were treated with the cell-permeable cross-linking reagent DSP and whole cell lysate of the DSP-treated cells was immunoprecipitated by anti-ERα antibody or a control IgG, followed by Western blot analyses with the indicated antibodies. Values in the Western blot quantifications in (B) and (C) were the means ± S.D. of three separate sample preparations. Cyto, cytosolic; Nuc, nuclear. * and *** denotep < 0.05 andp < 0.001, respectively.
Fig 3
Fig 3. Hsp70-1 and Hsc70 are associated with chromatin.
(A) The MCF7 cell extract was fractionated into soluble protein (S), chromatin-binding protein (CB), and the pellet (P), and then analyzed by Western blotting with the indicated antibodies (left panel). Right panel, quantification of Western blots. (B) The MCF7 cell extract was fractionated into cytoplasmic protein (C), nuclear soluble protein (NS), transcriptionally active chromatin (Ch1) and inactive chromatin (Ch2), and analyzed by Western blotting with the indicated antibodies (left panel). Right panel, quantification of Western blots. Histone H3, p300, and NCoR were used as markers of chromatin-binding protein, active chromatin, and inactive chromatins, respectively. Signal intensity values in the Western blot quantifications were arbitrary numbers obtained by analyzing the protein bands with ImageJ software. Values in the Western blot quantifications were the means ± S.D. of three separate sample preparations.
Fig 4
Fig 4. Hsp70-1 and Hsc70 interact with ERα in chromatin.
Anti-ERα antibody (ERα) and an isotype-matched, unrelated control IgG were used to immunoprecipitate proteins from cytoplasmic (C), nuclear soluble (NS), transcriptionally active chromatin (Ch1), and inactive chromatin (Ch2) fractions prepared from MCF7 cells. The subcellular proteins were prepared as for Fig 3 except that the inactive chromatin (Ch2) was obtained through sonication instead of elution with 600 mM NaCl. Signal intensity values in the Western blot quantifications were arbitrary numbers obtained by analyzing the protein bands with ImageJ software. Values in the Western blot quantifications were the means ± S.D. of three separate sample preparations. W, whole cell lysate. *, **, and *** denotep < 0.05,p < 0.01, andp < 0.001, respectively.
Fig 5
Fig 5. ERα interacts with Hsp70-1 and Hsc70 in the cytoplasm under conditions of hormone starvation/stimulation.
(A) The MCF7 cells were cultured under hormone starvation conditions for 3–4 days and then treated with either 100 nM E2 or ethanol (control) for 24 h. The cytosolic and nuclear extracts of the treated cells were then immunoprecipitated by anti-ERα antibody or a control IgG, and the immunoprecipitated protein was analyzed by Western blotting with the indicated antibodies. (B) Left panel, quantification of Western blots. Only the Hsp70-1 and Hsc70 protein bands in the cytosolic fractions were quantified. Signal intensity values in the Western blot quantifications were arbitrary numbers obtained by analyzing the protein bands with ImageJ software. Values in the Western blot quantifications were the means ± S.D. of four separate sample preparations. Right panel, validation of the cytosolic and nuclear fractionations. Tubulin and histone H3 were used as markers for the cytosolic and nuclear fractions, respectively. W, whole cell lysate. Ctr, control. * and ** denotep < 0.05 andp < 0.01, respectively.
Fig 6
Fig 6. Estradiol does not affect the association of Hsp70-1 and Hsc70 with chromatin.
The MCF7 cell extract was fractionated into cytoplasmic protein (C), nuclear soluble protein (NS), transcriptionally active chromatin (Ch1) and inactive chromatin (Ch2), and analyzed by Western blotting with the indicated antibodies (top panel). Signal intensity values in the Western blot quantifications were arbitrary numbers obtained by analyzing the protein bands with ImageJ software. Values in the Western blot quantifications were the means ± S.D. of three separate sample preparations. * denotesp < 0.05.
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