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A novel function of R-spondin1 in regulating estrogen receptor expression independent of Wnt/β-catenin signaling

  1. Ajun Geng
  2. Ting Wu
  3. Cheguo Cai
  4. Wenqian Song
  5. Jiqiu Wang
  6. Qing Cissy Yu  Is a corresponding author
  7. Yi Arial Zeng  Is a corresponding author
  1. State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, University of Chinese Academy of Sciences, China
  2. Medical Research Institute, Wuhan University, China
  3. Department of Endocrinology and Metabolism, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (SJTUSM), China
  4. School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Chinese Academy of Sciences, China
Research Article
Cite this article as: eLife 2020;9:e56434 doi: 10.7554/eLife.56434
8 figures, 1 table, 1 data set and 1 additional file

Figures

Figure 1 with 2 supplements
Rspo1 enhances Esr1 transcription and ERα signaling activities.

(a) RNA-seq of 3D cultured luminal cells in the presence of RSPO1 (0.5 μg/ml) or vehicle. Increased expression of ERα target genes (Pgr, Greb1) and Esr1 were enlisted in heatmap of differentially expressed genes (DEGs). (b) GO analysis was conducted on upregulated genes and estrogen receptor activity was enhanced in the presence of RSPO1. (c) Sca1+ luminal cells were FACS-isolated. (d, e) qPCR analysis of cultured cells in day two indicating increased expression of Esr1 (e) and its target genes (d) in the presence of RSPO1 (0.5 μg/ml). (f) E2 (1 μM) treatment was used as positive control indicating the upregulation of Esr1 and its target Pgr. (g) Western analysis of cultured cells in day 2 showing increased ERα protein levels after RSPO1 treatment. (h) A luciferase reporter driven by the ESR1 promoter was constructed and transfected into HEK293T cells. RSPO1 treatment activated the ESR1 promoter-luciferase reporter activities in a dose dependent manner. (d–h) Data are presented as mean ± s.e.m. of three independent experiments. Student’s t test: ***p<0.001, **p<0.01, *p<0.05.

Figure 1—figure supplement 1
Rspo1 promotes ERα signaling activities.

(a) Sorted Lin-, CD24+, CD29lo luminal cells were cultured in Matrigel as illustrated. (b) qPCR validation indicating increased Esr1 expression and upregulation of ERα target genes in response to RSPO1 stimulation. (c) Sorted Lin-, CD24+, CD29lo, Sca1+ (ER+) or Sca1- (ER-) luminal cells were cultured with or without the stimulation by RSPO1. Esr1 transcription increased in ER+ luminal cells after RSPO1 treatment but not in ER- luminal cells. (b, c) Data are presented as mean ± s.e.m. Student’s t test: ***p<0.001, **p<0.01, *p<0.05.

Figure 1—figure supplement 2
RSPO1 induces Esr1 expression in a dose-dependent manner in Eph4 cell line.

(a) RSPO1 treatment induced the expression of Esr1 in a dose-dependent manner. (b,c) ERα signaling target genes Pgr (b) and Greb1 (c) were upregulated dose-dependently in the presence of RSPO1. Data are presented as mean ± s.e.m. Student’s t test: ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

Figure 2 with 1 supplement
Rspo1 inducing Esr1 expression is dependent on Lgr4.

(a) qPCR analysis of Lgrs in FACS-isolated basal and luminal cells. Lgr4, Lgr5 and Lgr6 are all expressed in basal cells, while only Lgr4 is distinctively expressed in luminal cells with even distribution in Sca1+ (ER+) and Sca1- (ER-) luminal subpopulations. (b) Lgr4 in situ hybridization (in pink) confirming its expression in basal (arrow) and luminal cells (arrowhead). Nuclei were counterstained with hematoxylin (in purple). Scale bar, 20 μm. (c) qPCR analysis of Lgr4 expression in cultured luminal cells indicating successful knockdown by shRNA. (d) qPCR analysis of Esr1 expression in cultured luminal cells indicating that knockdown of Lgr4 by shRNA counteracts the upregulation of Esr1 by RSPO1. (e) ESR1 promoter-luciferase reporter assays on T47D cells indicating that knockdown of LGR4 mRNA by shRNA counteracts the upregulation of ESR1 by RSPO1, while scramble shRNA cannot. Data in (c–f) are pooled from three independent experiments and are presented as mean ± s.e.m. Student’s t test: ***p<0.001, **p<0.01, *p<0.05; ns, not significant.

Figure 2—figure supplement 1
Validation of LGR4 shRNAs knockdown efficiency.

(a) qPCR analysis of LGR4 expression in T47D cells indicating successful knockdown by shRNA.

Esr1 expression induced by Rspo1 is independent of Wnt/β-catenin signaling.

(a–b) qPCR analysis of cultured luminal cells indicating that Wnt4 alone or in combination with RSPO1 can activate Wnt target Axin2 expression (a). While RSPO1 alone promoted Esr1 expression, Wnt4 was ineffective for Esr1. Combination of RSPO1 and Wnt4 displayed no difference compared with RSPO1 alone (b). (c–d) qPCR analysis of cultured luminal cells indicating that Wnt signaling activators (Wnt3a and GSK3β inhibitor CHIR) cannot activate Esr1 expression, and that Esr1 expression induced by RSPO1 cannot be suppressed by addition of Wnt signaling inhibitor (FzCRD or β-catenin inhibitor XAV939) (d). In contrast, Wnt-target gene Axin2 expression was activated in the presence of Wnt signaling activators, and was suppressed by adding the signaling inhibitors (c). (e–f) HEK293T cells with transiently expressing ESR1-luciferase reporter were cultured in the presence of RSPO1, or in combination with Wnt inhibitors (XAV939 and IWP2). Wnt inhibitors cannot suppress ESR1-luciferase activities induced by RSPO1. Data in (a–f) are pooled from more than three independent experiments and presented as mean ± s.e.m. Student’s t test. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05; ns, not significant.

Figure 4 with 2 supplements
Loss of Rspo1 in mammary luminal cells results in reduced side branching and decreased ERα expression.

(a) Schematic illustration of Rspo1flox knock-in allele generation (see also Figure 4—figure supplement 1). (b) Krt8-CreER;Rspo1fl/fl inducible model specifically knockdown Rspo1 in luminal cells. (c–d) 8-week-old adult virgin mice were Tamoxifen injected twice, 1 day apart (2 mg/25 g body weight per injection). Mammary glands were obtained 4 weeks later. Whole-mount imaging (c) of mammary epithelium and quantification (d) showing decreased side branches in Rspo1-cKO mice. n = 3. Scale bar, 2 mm. More than six views were used for quantification. (e–f) FACS gating strategy for mammary basal and luminal cell isolation. Luminal ER+ and ER- subpopulations were separated based on Sca1 (e). qPCR analyses of luminal cells showing efficient Rspo1 knockdown in Krt8-CreER;Rspo1fl/fl (f). (g–h) Immunostaining indicated decreased ERα+ cell number after Rspo1 knockdown (g). Scale bar, 40 μm. Quantification of ERα+ cells were performed in (h). (i) qPCR analyses of ER+ luminal cells indicated downregulation of Esr1 transcription after Rspo1 knockdown in ER- cells. (j) Sca1+ (ER+) luminal cells were FACS isolated and Western blot was performed to indicate decreased ERα expression after Rspo1 knockdown. (k) qPCR analyses of ER+ luminal cells indicated downregulation of ERα target genes after Rspo1 knockdown in ER- cells. Data are presented as mean ± s.e.m. of more than three independent experiments. Student’s t test: ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. L.N. Lymph node.

Figure 4—figure supplement 1
Generation of Rspo1flox mouse model.

(a) Targeting strategy to generate the Rspo1flox knock-in (KI) mouse. Design of genotyping primers are as indicated. Exon2 of the Rspo1 gene was replaced by inserting cassette. (b) Pups born from crossing heterozygous male and female mice were genotyped by PCR. Pups with KI allele were identified by the presence of a larger 290 bp band. Genotyping PCR indicated the proper distribution of wild type and heterozygous as Mendel’s law of segregation.

Figure 4—figure supplement 2
Loss of Lgr4 reduces Esr1 expression and ERα signaling activities.

(a) Lgr4-/- mammary glands display reduced side branches. (b) Quantification of side branches was performed in designated area. More than six views were used for quantification. (c) Immunostaining of ERα in 8-week-old virgin control and Lgr4-/- mammary gland. More than six views were used for quantification. Scale bar, 50 μm. (d) Quantification of the proportion of ERα+ luminal cell among all luminal cells. (e) qPCR analysis of ER+ luminal cells (Lin-,CD24+,CD29lo,Sca1+) from mammary gland 1) confirming Lgr4 deficiency in Lgr4-/- mammary gland; 2) indicating that Esr1 expression is significantly decreased in Lgr4-/mammary gland, and 3) reduced ERα targets Pgr, Ctsd1 and Wisp2. n = 3 mice. L.N. Lymph node. (d, e) Data are presented as mean ± s.e.m. Student’s t test: ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05. ns, not significant.

Figure 5 with 1 supplement
Loss of Wnt4 increases Esr1 expression in luminal cells.

(a) Schematic illustration of Wnt4flox knock-in allele generation (see also Figure 5—figure supplement 1). (b) Krt8-CreER;Wnt4flox inducible model specifically knockdown Wnt4 in luminal cells. (c–d) 8-week-old adult virgin mice were Tamoxifen administered for 2 courses at 1 day apart, 2 mg/25 g body weight per injection and harvested 4 weeks later. Whole-mount imaging of the mammary epithelium showing decreased side branches in Wnt4-cKO mice (c). n = 3. Scale bar, 1 mm. More than six views were used for quantification. (e) qPCR of isolated ER+ luminal cells validated efficient Wnt4 knockdown in cKO mice. (f) qPCR analysis of ER+ luminal cells indicated Wnt4 loss increased Esr1 expression levels. (g) Western blot analysis indicated increased ERα protein level in Wnt4-cKO. (h) qPCR analysis of ER+ luminal cells indicated increased ERα signaling pathway activity after Wnt4 knockdown. (i) qPCR analysis of basal cells showed Wnt signaling pathway was decreased after Wnt4 knockdown. Data are presented as mean ± s.e.m. Student’s t test: ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05; ns, not significant.

Figure 5—figure supplement 1
Generation of Wnt4flox mouse model.

(a) Targeting strategy to generate the Wnt4flox knock-in (KI) mouse. Design of genotyping primers are as indicated. Exon2 of the Wnt4 gene was replaced by inserting cassette. (b) Pups born form crossing of heterozygous male and female mice were genotyped by PCR. Pups with KI allele were identified by the presence of a larger 322 bp band. Genotyping PCR indicated the proper distribution of wild type and heterozygous as Mendel’s law of segregation.

Figure 6 with 1 supplement
Rspo1 inducing Esr1 expression relies on cAMP-PKA pathway.

(a) HEK293T cells transfected with ESR1-luciferase reporter were cultured in the presence of RSPO1, and in combination with pharmaceutical compounds from a GPCR inhibitor library (Selleck). Bupivacaine HCl (Bup), a cAMP inhibitor, and H89, a PKA inhibitor, suppressed Esr1-luciferase activities induced by RSPO1 (see Figure 6—figure supplement 1a–b). (b–c) qPCR analysis of cultured luminal cells indicating both Bup and H89 counteracted the upregulation of Esr1 expression induced by RSPO1 (b), while Axin2 expression is not affected (c). (d) HEK293T cells with transiently expressing ESR1-luciferase reporter were cultured in the presence of RSPO1 alone or with ERα inhibitor ICI182, 780. Luciferase activities were measured. ICI did not affect the ESR1 upregulation induced by RSPO1. (e) CRE site on ESR1 promoter-luciferase reporter was mutated, and RSPO1 could not activate the reporter with CRE mutation. (f) RSPO1-FL, RSPO1-R66A/Q71A mutant could, but RSPO1-N137Q and RSPO1-F110A/F106A mutants could not induce Esr1 promoter luciferase activities. (g) ΔLuminescence was read out after Eph4 cells were treated with forskolin (FSR) or RSPO1 for 30 min, ΔLuminescence was calculated as Luminescencetreated—Luminescenceuntreated. Rspo1 treatment induced cAMP production in Eph4 cells in a dose dependent manner. (h) Illustration of Rspo1 regulated Esr1 expression mediated by cAMP-PKA pathway. Data in (a–d) are pooled from three independent experiments and presented as mean ± s.e.m. Student’s t test: ***p<0.001, **p<0.01, *p<0.05; ns, not significant.

Figure 6—source data 1

ESR1-luciferase activities induced by RSPO1 in combination with a GPCR inhibitor library.

https://cdn.elifesciences.org/articles/56434/elife-56434-fig6-data1-v1.xls
Figure 6—figure supplement 1
ESR1-luciferase activities induced by RSPO1 in combination with a GPCR inhibitor library HEK293 cells with transiently expressing ESR1-luciferase reporter were cultured in the presence of RSPO1, and screened for the molecules that could suppress the luciferase activities using a GPCR inhibitor library (Selleck, L2200).

Luciferase activities were measured.

Author response image 1
Author response image 2

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Genetic reagent (M. musculus)Krt8-CreERT2PMID:22350718RRID:MGI:5314229Dr. Li Xin (Department of Molecular and Cellular Biology, Baylor College of Medicine, United States)
Genetic reagent (M. musculus)Rspo1flox/+This paperGenerated in our laboratory Detail refer to Figure 4 and Figure 4—figure supplement 1
Genetic reagent (M. musculus)Wnt4flox/+This paperGenerated in our laboratory Detail refer to Figure 5 and Figure 5—figure supplement 1
Genetic reagent (M. musculus)Lgr4LacZ/+PMID:15192078RRID:MGI:3052121Drs. Minyao Liu and Dali Li
Cell line (M. musculus)Eph4PMID:25260709Mouse mammary epithelial cell line
Cell line (H. sapiens)HEK293T (293T)http://www.cellbank.org.cnCat. #: SCSP-502
Cell line (H. sapiens)T47DDr. Gaoxiang Ge's laboratoryHuman breast cancer cell line
Dr. Gaoxiang Ge (Institute of Biochemistry and Cell Biology)
AntibodyRabbit anti Gapdh GAPDH Polyclonal AntibodyProteintechCat. #: 10494–1-AP
RRID:AB_2263076
WB (1:3000)
AntibodyMouse anti β-Actin Monoclonal AntibodySigmaCat. #: A2228
RRID:AB_476697
WB (1:2000)
AntibodyRabbit anti ERα Polyclonal AntibodyMilliporeCat. #: 06–935
RRID:AB_310305
WB (1:1000), IHC (1:200)
AntibodyRat anti Krt8 Monoclonal AntibodyDSHBCat. #: TROMA-1IHC (1:500)
AntibodyRat Anti-Mouse CD31 Monoclonal Antibody, Biotin ConjugatedBD PharMingenCat. #: 553371
RRID:AB_394817
Flow cytometry (1:200)
AntibodyRat Anti-Mouse CD45 Monoclonal Antibody, Biotin ConjugatedBD PharMingenCat. #: 553078
RRID:AB_394608
Flow cytometry (1:200)
AntibodyRat Anti-Mouse TER-119 Monoclonal Antibody, Biotin ConjugatedBD PharMingenCat. #: 553672Flow cytometry (1:200)
AntibodyRat Anti-Mouse CD31 Monoclonal Antibody, FITC ConjugatedBD PharMingenCat. #: 553372
RRID:AB_394818
Flow cytometry (1:200)
AntibodyRat Anti-Mouse CD45 Monoclonal Antibody, FITC ConjugatedBD PharMingenCat. #: 553080
RRID:AB_394610
Flow cytometry (1:200)
AntibodyRat Anti-Mouse TER119 Monoclonal Antibody, FITC ConjugatedBD PharMingenCat. #: 557915Flow cytometry (1:200)
AntibodyPE/Cy7 Rat Anti-Mouse CD24 Monoclonal AntibodyBiolegendCat. #: 101–822
RRID:AB_756048
Flow cytometry (1:200)
AntibodyAPC Armenian Hamster Anti-Mouse/Rat CD29 Monoclonal AntibodyBiolegendCat. #: 102216
RRID:AB_492833
Flow cytometry (1:200)
AntibodyRat Anti-Ly-6A/E (Sca-1) Monoclonal Antibody, PEeBioscienceCat. #: 12-5981-82
RRID:AB_466086
Flow cytometry (1:200)
AntibodyStreptavidin eFluor 450 ConjugateeBioscienceCat. #: 48-4317-82
RRID:AB_10359737
Flow cytometry (1:200)
Peptide, recombinant proteinEpithelial growth factor (EGF)CorningCat. #: 35400150 ng/mL
Peptide, recombinant proteinFzCRDPMID:25260709Inhibition of the Frizzled receptor using its soluble CRD domain. Purified in our laboratory (1:200)
Peptide, recombinant proteinWnt3APMID:20569694Purified in our laboratory 200 ng/ml
Recombinant DNA reagentplko.1 backboneAddgeneRRID:Addgene_30323
Recombinant DNA reagentpGL4.17 basic vectorPromega, Madison, WI, U.S.A.Cat. #: E6721
Recombinant DNA reagentpRL-TK RenillaPromega, Madison, WI, U.S.A.Cat. #: E2241
Recombinant DNA reagentpcDNA3.1-RSPO1 overexpression (RSPO1-OE) plasmidThis paperConstructed in our laboratoryDetail refer to Materials and methods
Chemical compound, drugtamoxifen (TAM)Sigma-AldrichCat. #: T56482 mg/20 g
Chemical compound, drugHEPESSigmaCat. #: H4034-500G25 mM
Chemical compound, drugCollagenase IIIWorthingtonCat. #: LS004183300 U/ml
Chemical compound, drugred blood cell lysing bufferSigmaCat. #: R7757
Chemical compound, drugDNase ISigmaCat. #: D42630.1 mg/mL
Chemical compound, drugCarmineSigmaCat. #: C10222 mg/ml
Chemical compound, drugHistoclearNational DiagnosticsCat. #: HS-200
Chemical compound, drugMatrigelBD BioscienceCat. #: 354230
Chemical compound, drugInsulin-Transferrin-Selenium (ITS)GibcoCat. #: 41400045Cell culture (1:100)
Chemical compound, drugE2SigmaCat. #: E88751 μM
Chemical compound, drugIWP2SelleckCat. #: s70852.5 μM
Chemical compound, drugCHIRSelleckCat. #: S12633 μM
Chemical compound, drugXAV-939SelleckCat. #: S118010 μM
Chemical compound, drugProtein A AgaroseSanta CruzCat. #: sc-2003
Chemical compound, drugDAPILife TechnologiesCat. #: P36931
Chemical compound, drugGPCR compound librarySelleckchem L2200Chemical Biology Core Facility, Institute of Biochemistry and Cell Biology, SIBS, CAS
Commercial assay or kitIn situ hybridization RNAscope kitAdvanced Cell DiagnosticsFollowing the manufacturer’s instructions
Commercial assay or kitDual-Luciferase Reporter Assay SystemPromegaCat. #: E1910Following the manufacturer’s instructions
Commercial assay or kitcAMP-Glo assay kitPromegaCat. #: V1501Following the manufacturer’s instructions
Commercial assay or kitSuperScript III kitInvitrogenCat. #: RR036AFollowing the manufacturer’s instructions
Software, algorithmGraphPad PrismGraphPad Prism (https://graphpad.com)
Software, algorithmImageJImageJ (http://imagej.nih.gov/ij/)

Data availability

RNA-seq data can be viewed online at http://www.biosino.org/node/index, under accession number OEP000754.

The following data sets were generated
    1. Geng A
    (2020) OEP000754
    ID OEP000754. A Novel Function of R-spondin1.

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