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. 2013 Sep 10;51(2):233-46.
doi: 10.1530/JME-13-0051. Print 2013 Oct.

Estradiol and tamoxifen regulate NRF-1 and mitochondrial function in mouse mammary gland and uterus

Margarita M Ivanova  1 , Brandie N Radde, Jieun Son, Fabiola F Mehta, Sang-Hyuk Chung, Carolyn M Klinge
Affiliations

Estradiol and tamoxifen regulate NRF-1 and mitochondrial function in mouse mammary gland and uterus

Margarita M Ivanova et al. J Mol Endocrinol. .

Abstract

Nuclear respiratory factor-1 (NRF-1) stimulates the transcription of nuclear-encoded genes that regulate mitochondrial (mt) genome transcription and biogenesis. We reported that estradiol (E2) and 4-hydroxytamoxifen (4-OHT) stimulate NRF-1 transcription in an estrogen receptor α (ERα)- and ERβ-dependent manner in human breast cancer cells. The aim of this study was to determine whether E2 and 4-OHT increase NRF-1 in vivo. Here, we report that E2 and 4-OHT increase NRF-1 expression in mammary gland (MG) and uterus of ovariectomized C57BL/6 mice in a time-dependent manner. E2 increased NRF-1 protein in the uterus and MG; however, in MG, 4-OHT increased Nrf1 mRNA but not protein. Chromatin immunoprecipitation assays revealed increased in vivo recruitment of ERα to the Nrf1 promoter and intron 3 in MG and uterus 6 h after E2 and 4-OHT treatment, commensurate with increased NRF-1 expression. E2- and 4-OHT-induced increases in NRF-1 and its target genes Tfam, Tfb1m, and Tfb2m were coordinated in MG but not in uterus due to uterine-selective inhibition of the expression of the NRF-1 coactivators Ppargc1a and Ppargc1b by E2 and 4-OHT. E2 transiently increased NRF-1 and PGC-1α nuclear staining while reducing PGC-1α in uterus. E2, not 4-OHT, activates mt biogenesis in MG and uterus in a time-dependent manner. E2 increased mt outer membrane Tomm40 protein levels in MG and uterus whereas 4-OHT increased Tomm40 only in uterus. These data support the hypothesis of tissue-selective regulation of NRF-1 and its downstream targets by E2 and 4-OHT in vivo.

Keywords: estrogen receptor; mitochondria; mouse; nuclear respiratory factor-1.

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Figures

Figure 1
Figure 1. E2 and 4-OHT increase Nrf1 mRNA and NRF-1 protein expression in mouse mammary gland and uterus
Ovariectomized C57BL/6 mice were given a single sc injection of sesame oil (vehicle control), 100 ng E2, or 50 μg 4-OHT and were euthanized 6, 24, or 72 h post injection. A and B. Q-RT-PCR analysis of Nrf1 mRNA level in mammary gland (MG) (A) and uterus (B). C and D. NRF-1 protein expression was examined relative to β-actin in MG (C) and uterus (D) after the indicated time of treatment. Values are the mean ± SEM of 5-10 mice/treatment group in which control was set to 1 for each treatment time within each blot for comparison. * p < 0.05 versus vehicle control (Student's t test). Representative western blots are shown in Supplementary Fig. 1.
Figure 2
Figure 2. E2 and 4-OHT increase ERα recruitment to the Nrf1 promoter in vivo in a tissue-dependent manner
A, Diagram of the mouse Nrf1 gene promoter showing locations of ERE, AP-1, ½ ERE, and ERR-binding sites (ERRBS), transcription and protein coding start sites, as indicated. Exons (E1, E2, E3, and E4) are indicated as grey boxes. The location of the 3 primers used for ChIP for ERα on Nrf1 are indicated as light grey boxes above the Nrf1 gene. B, Ovex C57BL/6 mice were treated with vehicle (control), E2, or 4-OHT for 6 h. ChIP with ERα antibody or with rabbit pre-immune serum (IgG) was performed with the 3 pairs of ChIP primers (locations shown in A) for the mouse Nrf1 gene promoter. C, Immunoprecipitated DNA was normalized to input and IgG control and Q-PCR data are expressed relative to IP for control-treated mammary gland (MG, left) and uterus (right). Values are the mean ± SEM of 5 mice/treatment group. * p < 0.05 from control for each primer. D and E, ChIP results are diagrammed for E2-ERα and 4-OHT-ERα recruitment to the Nrf1 promoter in MG (D) and uterus (E). The bent arrows indicate transcriptional upregulation of Nrf1 mRNA as measured by Q-PCR (see Fig. 1A).
Figure 3
Figure 3. E2 and 4-OHT selectively regulate the expression of NRF-1 target genes for mtDNA transcription and replication
A and B, Q-RT-PCR analysis of the mRNA expression of Nrf1-regulated Tfam, Tfb1m, and Tfb2m expression in mammary gland (MG) (A) and uterus (B) from ovex mice treated with vehicle control, E2, or 4-OHT for the indicated time. C and D, Tfam protein expression was examined in mammary gland (MG in panel C) and uterus (D) 72 h after E2 or 4-OHT treatment. The data shown are the mean ± SEM of 5-10 mice/treatment group. * p < 0.05 versus vehicle control (Student's t-test). Representative western blots are shown in Supplementary Fig. 3.
Figure 4
Figure 4. E2 and 4-OHT differently regulate mitochondrial biogenesis in mouse mammary gland and uterus
A-B, Total DNA was purified from mammary gland (MG) and uterus of ovex mice treated with vehicle (control), E2, or 4-OHT for 6, 24 and 72 h. The data are the ratio of the mitochondrial genome encoded gene mt-ND5 normalized to nuclear-encoded gene Ctfr as determined by qPCR. Values are the mean ± SEM of 6-15 mice/treatment group. * p < 0.05 versus vehicle control (Student's t-test). C-D, Tom40 protein was examined in MG (C) and uterus (D) after 72 h treatment. The values are the mean ± SEM of 4-7 mice/treatment groups. *p < 0.05 versus vehicle control (Student's t-test). Representative western blots are shown in Supplementary Fig.4.
Figure 5
Figure 5. E2 and 4-OHT differently regulate the level of NRF-1 target genes involved in mitochondrial respiration
A and B, Q-RT-PCR analysis of the mRNA expression of Nrf1-regulated genes Cox4 and CycS in mammary gland (MG) (A) and uterus (B) from ovex mice treated with vehicle (control), E2, or 4-OHT for the indicated time. Values are the mean ± SEM of 10-15 mice/treatment group. *p < 0.05 versus vehicle control (Student's t-test). C-D, Cox4 (C) and cytochrome c (D) protein expression was examined in MG and uterus 72 h after the indicated treatment. Values are the mean ± SEM of 5-12 mice/treatment group. *p < 0.05 versus vehicle control (Student's t-test). Representative Western blots are shown in Supplementary Fig. 5.
Figure 6
Figure 6. E2 and 4-OHT increase Ppargc1b expression in mammary gland and reduce Ppargc1a and Ppargc1b expression in the uterus
Q-RT-PCR analysis of Ppargc1a and Ppargc1b expression in mammary gland (MG) (A) and uterus (B) from ovex C57BL/6 mice treated with vehicle (control), E2, or 4-OHT for the indicated time. Values are the mean ± SEM of 5-10 mice/treatment group. *p < 0.05 versus vehicle control (Student's t-test).
Figure 7
Figure 7. E2 increases NRF-1 and PGC-1a nuclear colocalization in uterus and decreases PGC-1a protein levels
Ovex mice were treated with vehicle (veh, EtOH) or 1 μg E2 for 24 or 48 h. Uterine tissue sections were stained for NRF-1 (green) and PGC-1α (red). Nuclei were stained with Hoechst 33258 (blue). LE, luminal epithelium; GE, glandular epithelium; S, stroma. White scale bar is 10 μm.
Figure 8
Figure 8. Summary of E2 and 4-OHT regulation of Nrf1, Ppargc1a, Ppargc1b, and Nrf1 target gene expression in mouse mammary gland and uterus
Changes in the expression of the indicated genes (A) and proteins (B) in mammary gland (MG) and uterus are expressed relative to vehicle control after the indicated treatment time (6, 24, or 72 h).
Figure 9
Figure 9. Model of E2-ERα and 4-OHT-ERα regulation of Nrf1 transcription, NRF-1 regulation of its target genes, PGC-1 coactivator family expression, Tom40, and mitochondrial biogenesis in mouse mammary gland and uterus
The E2-ERα-stimulated changes are shown in the top two cell models with mammary gland on the left and uterus on the right. The 4-OHT-ERα-stimulated changes are shown in the bottom two cell models with mammary gland on the left and uterus on the right. Of course since we used whole tissue lysates for the experiments in this study, we cannot attribute gene/protein changes to a specific cell type. The time (h) at which changes in gene or protein expression were detected is indicated. Bold black arrows indicate the direction (up- or down-regulation) of the change of gene expression.
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