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ERα36, a Variant of Estrogen Receptor α, is Predominantly Localized in Mitochondria of Human Uterine Smooth Muscle and Leiomyoma Cells

Yan Y, Yu L, Castro L, Dixon D.
PLoS One. 2017 DOI: https://doi.org/10.1371/journal.pone.0186078 PMID: 29020039

Publication

Abstract

ERα36 is a naturally occurring, membrane-associated, isoform of estrogen receptor α. The expression of ERα36 is due to alternative splicing and different promoter usage. ERα36 is a dominant-negative effector of ERα66-mediated transactivational activities and has the potential to trigger membrane-initiated mitogenic, nongenomic, estrogen signaling; however, the subcellular localization of ERα36 remains controversial. To determine the cellular localization of ERα36 in estrogen-responsive human uterine smooth muscle (ht-UtSMC) and leiomyoma (fibroid; ht-UtLM) cells, we conducted systematic confocal microscopy and subcellular fractionation analysis using ERα36 antibodies. With Image J colocalizaton analysis plugin, confocal images were analyzed to obtain a Pearson's Correlation Coefficient (PCC) to quantify signal colocalization of ERα36 with mitochondrial, endoplasmic reticulum, and cytoskeletal components in both cell lines. When cells were double-stained with an ERα36 antibody and a mitochondrial-specific dye, MitoTracker, the PCC for the two channel signals were both greater than 0.75, indicating strong correlation between ERα36 and mitochondrial signals in the two cell lines. A blocking peptide competition assay confirmed that the mitochondria-associated ERα36 signal detected by confocal analysis was specific for ERα36. In contrast, confocal images double-stained with an ERα36 antibody and endoplasmic reticulum or cytoskeletal markers, had PCCs that were all less than 0.4, indicating no or very weak signal correlation. Fractionation studies showed that ERα36 existed predominantly in membrane fractions, with minimal or undetected amounts in the cytosol, nuclear, chromatin, and cytoskeletal fractions. With isolated mitochondrial preparations, we confirmed that a known mitochondrial protein, prohibitin, was present in mitochondria, and by co-immunoprecipitation analysis that ERα36 was associated with prohibitin in ht-UtLM cells. The distinctive colocalization pattern of ERα36 with mitochondria in ht-UtSMC and ht-UtLM cells, and the association of ERα36 with a mitochondrial-specific protein suggest that ERα36 is localized primarily in mitochondria and may play a pivotal role in non-genomic signaling and mitochondrial functions.

Figures

Figure 1. Comparison of ERα36 and ERα66 domain structures and subcellular ERα36 staining patterns.

Comparison of ERα36 and ERα66 domain structures and subcellular ERα36 staining patterns in human uterine smooth muscle (ht-UtSMC) and leiomyoma cells (ht-UtLM). (A) The top structure shows six conserved domains of ERα66 (labeled A-F), the amino acid sequence numbers, and the transactivation function domains (AF-1 and AF-2). The function of each domain is indicated. In the domain structure of ERα36, the last unique 27 amino acids of ERα36 are indicated as a filled box. The diagram is drawn proportionally to domain size. The drawing is based on the publication by Wang et. al., 2005 and GenBank entry CAE45969.1. (B) In ht-UtSMC cells (1), distinctive ERα36 signals were detected along the plasma membrane as shown by the arrow, but mostly localized within a robust intracytoplasmic network. In ht-UtLM cells (2), the ERα36 signals were detected mostly in a robust network structure within the cytoplasm. The cell samples were stained with ERα36 antibody and DAPI.

Figure 2. Colocalization ERα36 with mitochondria, F-actin or endoplasmic reticulum (ht-UtSMC).

Colocalization analysis of ERα36 with mitochondria, F-actin or endoplasmic reticulum in ht-UtSMC cells. Top row: A. ERα36 signal channel, B. MitoTracker signal, C. Merged image, D. Scatter plot; Middle row: E. ERα36 signal channel, F. F-actin dye signal, G. Merged image, H. Scatter plot; Bottom row: I. PDI signal channel, J. ERα36 signal, K. Merged image, L. Scatter plot. In the scatter plot, the symbol r represents the Pearson’s correlation coefficient. Scale bar = 20 μm for all images.

Figure 3. Colocalization ERα36 with mitochondria, F-actin or endoplasmic reticulum (ht-UtLM).

Colocalization analysis of ERα36 with mitochondria, F-actin or endoplasmic reticulum in ht-UtLM cells. Top row: A. ERα36 signal channel, B. MitoTracker signal, C. Merged image, D. Scatter plot; Middle row: E. ERα36 signal channel, F. F-actin dye signal, G. Merged image, H. Scatter plot; Bottom row: I. PDI signal channel, J. ERα36 signal channel, K. Merged image, L. Scatter plot. In the scatter plot, the symbol r represents the Pearson’s correlation coefficient. Scale bar = 20 μm for all images.

Figure 4. ERα36 blocking peptide competition assay in ht-UtLM cells.

Ht-UtLM cells were incubated with MitoTracker and then stained with either neutralized ERα36 antibody (top row) or non-neutralized antibody (bottom row). Abbreviations: A. ERα36 signal; B. MitoTracker; C. Merged images. Scale bar = 10 μm for all images.

Figure 5. ERα36 Expression in subcellular fractions.

Cellular Fraction Abbreviations: CE (cytoplasmic extract); ME (membrane extract); CB (chromatin-bound extract); NE (nuclear extract, nuclear soluble); PE (pellet extract, cytoskeleton); T (total cellular protein extract).

Figure 6. Interactions of ERα36 and prohibitin in human uterine leiomyoma cells.

The interactions between ERα36 and prohibitin were determined with a co-immunoprecipitation (Co-IP). Ht-UtLM cells were treated with vehicle control, 10−8 M E2, and 10−6 M E2 for 24 hours. The blots presented are representative results that were repeated at least three times. PHB: prohibitin; IB: Immunobotting; IP: Immunoprecipitation.

Supplemental Materials

Supplemental Data

S1 Fig. Cross correlation function (CCF) analysis of the confocal images stained by ERα36 antibody and MitoTracker.
A. ht-UtSMC cell culture. B. ht-UtLM cell culture. The CCF graphs were generated by Image J plugin JACoP.

S2 Fig. ERα36 (green) and MitoTracker (red) colocalization analysis in BG-1, MCF-7, Ishikawa and SK-LMS-1 cell lines.
A. DAPI, B. ERα36 signal, C. MitoTracker signal, D. Merged image.

S3 Fig. Subcellular protein fractionations from human uterine cells analyzed with subcellular-specific markers.
The subcellular fractionation procedure was effective in separating the subcellular components, as shown by western blot analyses with respective subcellular markers. Abbreviations: HSP90 (HSP90 Rabbit mAb, Cell Signaling #4877), EGFR (EGFR Rabbit Polyclonal Antibody, Santa Cruz Biotechnology Cat# sc-03), SP1 (SP1 Rabbit mAb, Cell Signaling Cat# 9389), H3 (Histone H3 Rabbit Polyclonal Antibody, Cell Signaling Cat#9715), Vimentin (Vimentin Rabbit polyclonal Antibody, Cell Signaling Cat# 3932). CE (cytoplasmic extract), ME (membrane extract), NE (nuclear extract, nuclear soluble), CB (chromatin-bound extract), PE (pellet extract, cytoskeleton).

S4 Fig. Western blot analysis of mitochondrial fractions with specific mitochondrial markers.
(A) Blot is probed for Mortalin expression. (B) Blot was probed for Src expression. (C). Blot was probed for Prohibitin expression. Abbreviations: C. Cytosol fraction; M. Mitochondrial fraction.