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. 2008 Mar;22(3):636-48.
doi: 10.1210/me.2007-0359. Epub 2007 Dec 6.

GPR30 contributes to estrogen-induced thymic atrophy

Chunhe Wang  1 , Babak Dehghani, I Jack Magrisso, Elizabeth A Rick, Edna Bonhomme, David B Cody, Laura A Elenich, Sandhya Subramanian, Stephanie J Murphy, Martin J Kelly, Jan S Rosenbaum, Arthur A Vandenbark, Halina Offner
Affiliations

GPR30 contributes to estrogen-induced thymic atrophy

Chunhe Wang et al. Mol Endocrinol. 2008 Mar.

Abstract

The mechanisms by which prolonged estrogen exposures, such as estrogen therapy and pregnancy, reduce thymus weight, cellularity, and CD4 and CD8 phenotype expression, have not been well defined. In this study, the roles played by the membrane estrogen receptor, G protein-coupled receptor 30 (GPR30), and the intracellular estrogen receptors, estrogen receptor alpha (ERalpha) and beta (ERbeta), in 17beta-estradiol (E2)-induced thymic atrophy were distinguished by construction and the side-by-side comparison of GPR30-deficient mice with ERalpha and ERbeta gene-deficient mice. Our study shows that whereas ERalpha mediated exclusively the early developmental blockage of thymocytes, GPR30 was indispensable for thymocyte apoptosis that preferentially occurs in T cell receptor beta chain(-/low) double-positive thymocytes. Additionally, G1, a specific GPR30 agonist, induces thymic atrophy and thymocyte apoptosis, but not developmental blockage. Finally, E2 treatment attenuates the activation of nuclear factor-kappa B in CD25(-)CD4(-)CD8(-) double-negative thymocytes through an ERalpha-dependent yet ERbeta- and GPR30-independent pathway. Differential inhibition of nuclear factor-kappaB by ERalpha and GPR30 might underlie their disparate physiological effects on thymocytes. Our study distinguishes, for the first time, the respective contributions of nuclear and membrane E2 receptors in negative regulation of thymic development.

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Figures

Figure 1
Figure 1
Schematic Organization of GPR30 Exon Structure and Targeting Strategy Coding and noncoding exons of GPR30 are indicated by green and yellow boxes, respectively. Probes used for Southern blot analyses and their location are labeled and indicated by solid black bars above the schematized genomic locus. The positions of PCR primers for genotyping are labeled with black arrowheads. Neo, neomycin.
Figure 2
Figure 2
Validation of Gene Targeting by Southern Blot and Genotyping A, Southern blot analyses of ES cell genomic DNA. Blotting yields a 6.9-kb band for the WT allele, or 4.7 kb for the targeted allele. A single positive clone, T142, was identified after screening approximately 300 ES cell clones. Both 5′- and 3′-probes were then used with secondary digests to confirm clone T142. B, PCR genotyping. Left panel, Genotyping of an initial litter of pups from a heterozygous cross. WT mice yield a 550-bp band upon genotyping, whereas gene-deficient mice yield a 730-bp band. Right panel, Confirmation of lack of expression for GPR30 in gene-deficient mice in selected tissues. Total RNA was isolated from heart, liver, and kidney, and RT-PCR was performed with (RT+) or without (RT−) reverse transcriptase. Lanes 1–2, 5–6, 9–10: WT RT(+) and RT(−) reactions; lanes 3–4, 7–8, and 11–12: gene-deficient RT(+) and RT(−) reactions.
Figure 3
Figure 3
E2-Induced Thymic Atrophy Involves Both ERα and GPR30, But Not ERβ A, In vivo exposure to pregnancy levels of E2 reduced thymic size and cellularity. Thymi were harvested from female C57BL/6J mice implanted with E2 pellets (2.5 mg/60 d release) for 0–11 days. B, E2-induced thymic atrophy was partially attenuated in both ERα- and GPR30-, but not in ERβ-deficient mice. AERKO, BERKO, GPR30KO, and WT C57BL/6J mice were treated with pellets of E2 (2.5 mg/60 d release) or vehicle for 8 d before thymi were harvested and examined. *, P < 0.05; or **, P < 0.01, compared with vehicle control as indicated by one-way ANOVA followed by Newman-Keuls multiple comparisons test. The experiment was repeated three times with at least four mice in each group, with a representative experiment shown.
Figure 4
Figure 4
E2-Dependent Blockage in the Development of DN Thymocytes at the CD44+CD25− (DN1) Stage Was Abrogated in ERα-Deficient Mice A, Histogram and dot plots show differential distribution of DN thymocyte subsets in vehicle- and E2-treated mice after 8 d of E2 treatment. B, E2 gradually depleted CD44+CD25+ (DN2) and CD44lowCD25+ (DN3) cells, while enriching CD44+CD25− (DN1) thymocytes during 11 d of treatment. C, Genetic disruption of ERα, but not ERβ or GPR30 expression, abrogated E2-induced accumulation of DN1 thymocytes and reduction of DN2 thymocytes. *, P < 0.05; or **, P < 0.01, compared with vehicle control as indicated by one-way ANOVA followed by Newman-Keuls multiple comparisons test. The experiment was repeated two times with at least four mice in each group, with a representative experiment shown.
Figure 5
Figure 5
In Vivo E2 Exposure Promoted Apoptosis in TCRβ−/lowCD4+CD8+ Thymocytes A, Annexin V staining shows that thymocyte apoptosis was enhanced by E2 treatment. Left panel, Dot plots of Annexin V staining in thymocytes prepared from mice implanted with pellets of vehicle or E2 (2.5 mg/60 d release) for 8 d. The apoptotic cells distribute in the Annexin V+7AAD− lower right quadrant. Right panel, Time course of E2-induced elevation of apoptotic thymocytes. One-way ANOVA followed by Newman-Keuls multiple comparisons test: *, P < 0.05; **, P < 0.01, compared with vehicle control. B, In situ TUNEL staining showed that E2 treatment elevated apoptosis in the thymus. The number of apoptotic cells in TUNEL-stained sections was counted automatically by Scion Image, and the density was calculated by dividing the total number of apoptotic cells by the total area of the sections. Student’s t test: *, P < 0.05; **, P < 0.01, compared with vehicle control. The experiment was repeated two times with at least four mice in each group. C, The majority of apoptotic cells were TCRβ−/low DP thymocytes. B, TCRβ−/low thymocytes were more vulnerable to apoptosis than TCRβhigh thymocytes. C, Apoptosis developed faster in TCRβ−/low and CD4+CD8+ (DP) populations. The experiment was repeated four times with at least four mice in each group, with a representative experiment shown.
Figure 6
Figure 6
Genetic Disruption of Expression of GPR30, But Not of ERα or ERβ, Prevented TCRβ−/low DN Thymocytes from Undergoing Apoptosis Thymocytes were prepared from mice implanted with pellets of E2 (2.5 mg/60 d release) or vehicle for 8 d. After staining with Annexin V, 7AAD, and Abs for cellular markers, the cells were analyzed by flow cytometry. One-way ANOVA followed by Newman-Keuls multiple comparisons test: *, P < 0.05; **, P < 0.01, compared with vehicle control or otherwise indicated by brackets. The experiment was repeated two times with at least four mice in each group.
Figure 7
Figure 7
GPR30 Agonist, G1, Induces Thymic Atrophy (A) and Thymocyte Apoptosis (B). But Not Thymocyte Developmental Blockage (C, D, and E) E2 (0.04 mg/kg/d) and G1 (0.1 mg/kg/d) were dissolved in vehicle (10% ethanol and 90% oil) and administered sc daily to mice for 8 d before the mice were euthanized to measure the thymic mass, degree of thymocyte apoptosis, and distribution of different DN populations. One-way ANOVA followed by Newman-Keuls multiple comparisons test: *, P < 0.05; **, P < 0.01, compared with vehicle control or otherwise indicated by brackets.
Figure 8
Figure 8
ERα Mediated E2-Induced Inhibition of NFκB in CD25−DN (CD25−CD4−CD8−) Thymocytes A, The activity of NFκB, but not NFAT, was inhibited by E2, but not G1, in CD25−DN thymocytes. CD25−DN thymocytes were prepared from C57BL/6J mice that were injected for 8 d sc with E2 (0.04 mg/kg/d), G1 (0.1 mg/kg/d), or vehicle alone. The activity of NFκB and NFAT in CD25−DN thymocytes was analyzed with the EMSA. B, The level of IκB phosphorylation was reduced by E2, but not by G1. Cell lysates from CD25−DN thymocytes were evaluated by immunoblotting with polyclonal Abs for phosphorylated and total IκB. The bands were scanned and quantified. C, Genetic disruption of expression of ERα, but not ERβ or GPR30, rescued NFκB activity from E2-induced inhibition in CD25−DN thymocytes. The activity of NFκB and NFAT in CD25−DN thymocytes was analyzed with the EMSA. CD25−DN thymocytes were prepared from AERKO, BERKO, GPR30KO, or WT mice. The experiment was repeated two times with four mice in each group.
Figure 9
Figure 9
The Expression of ERα and GPR30 in the Whole Thymus of WT, AERKO, BERKO, and GPR30KO Mice (A) and in the Different CD4−CD8− (DN) Thymocyte Subsets in WT Mice (B) For A, the mRNA was extracted from the whole thymus from different phenotypes of mice. For B, the thymocytes were harvested fresh from WT C57BL6 mice and depleted of CD3+ cells by AutoMACS. The thymocytes at different DN stages were then sorted out by flow cytometry. The amounts of mRNA of ERα and GPR30 relative to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were quantified by real-time PCR using Taqman primers. The experiments were independently repeated two times with two mice in each group. One-way ANOVA followed by Newman-Keuls multiple comparisons test: *, P < 0.05; **, P < 0.01, compared with vehicle control or otherwise indicated by brackets.
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