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

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8 figures, 1 table and 1 additional file

Figures

Figure 1 with 2 supplements

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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

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Rspo1 promotes ERα signaling activities.

(a) Sorted Lin^-, CD24⁺, CD29^lo 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⁺, CD29^lo, 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

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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

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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

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Validation of LGR4 shRNAs knockdown efficiency.

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

Figure 3

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*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

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Loss of Rspo1 in mammary luminal cells results in reduced side branching and decreased ERα expression.

(a) Schematic illustration of *Rspo1^flox* knock-in allele generation (see also Figure 4—figure supplement 1). (b) *Krt8-CreER;Rspo1^fl/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;Rspo1^fl/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

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Generation of *Rspo1^flox* mouse model.

(a) Targeting strategy to generate the *Rspo1^flox* 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

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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⁺,CD29^lo,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

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Loss of Wnt4 increases *Esr1* expression in luminal cells.

(a) Schematic illustration of *Wnt4^flox* knock-in allele generation (see also Figure 5—figure supplement 1). (b) *Krt8-CreER;Wnt4^flox* 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

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Generation of *Wnt4^flox* mouse model.

(a) Targeting strategy to generate the *Wnt4^flox* 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

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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
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Figure 6—figure supplement 1

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*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.

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Tables

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Genetic reagent (M. musculus)	Krt8-CreERT2	PMID:22350718	RRID:MGI:5314229	Dr. Li Xin (Department of Molecular and Cellular Biology, Baylor College of Medicine, United States)
Genetic reagent (M. musculus)	Rspo1^flox/+	This paper		Generated in our laboratory Detail refer to Figure 4 and Figure 4—figure supplement 1
Genetic reagent (M. musculus)	Wnt4^flox/+	This paper		Generated in our laboratory Detail refer to Figure 5 and Figure 5—figure supplement 1
Genetic reagent (M. musculus)	Lgr4^LacZ/+	PMID:15192078	RRID:MGI:3052121	Drs. Minyao Liu and Dali Li
Cell line (M. musculus)	Eph4	PMID:25260709		Mouse mammary epithelial cell line
Cell line (H. sapiens)	HEK293T (293T)	http://www.cellbank.org.cn	Cat. #: SCSP-502
Cell line (H. sapiens)	T47D	Dr. Gaoxiang Ge's laboratory		Human breast cancer cell line Dr. Gaoxiang Ge (Institute of Biochemistry and Cell Biology)
Antibody	Rabbit anti Gapdh GAPDH Polyclonal Antibody	Proteintech	Cat. #: 10494–1-AP RRID:AB_2263076	WB (1:3000)
Antibody	Mouse anti β-Actin Monoclonal Antibody	Sigma	Cat. #: A2228 RRID:AB_476697	WB (1:2000)
Antibody	Rabbit anti ERα Polyclonal Antibody	Millipore	Cat. #: 06–935 RRID:AB_310305	WB (1:1000), IHC (1:200)
Antibody	Rat anti Krt8 Monoclonal Antibody	DSHB	Cat. #: TROMA-1	IHC (1:500)
Antibody	Rat Anti-Mouse CD31 Monoclonal Antibody, Biotin Conjugated	BD PharMingen	Cat. #: 553371 RRID:AB_394817	Flow cytometry (1:200)
Antibody	Rat Anti-Mouse CD45 Monoclonal Antibody, Biotin Conjugated	BD PharMingen	Cat. #: 553078 RRID:AB_394608	Flow cytometry (1:200)
Antibody	Rat Anti-Mouse TER-119 Monoclonal Antibody, Biotin Conjugated	BD PharMingen	Cat. #: 553672	Flow cytometry (1:200)
Antibody	Rat Anti-Mouse CD31 Monoclonal Antibody, FITC Conjugated	BD PharMingen	Cat. #: 553372 RRID:AB_394818	Flow cytometry (1:200)
Antibody	Rat Anti-Mouse CD45 Monoclonal Antibody, FITC Conjugated	BD PharMingen	Cat. #: 553080 RRID:AB_394610	Flow cytometry (1:200)
Antibody	Rat Anti-Mouse TER119 Monoclonal Antibody, FITC Conjugated	BD PharMingen	Cat. #: 557915	Flow cytometry (1:200)
Antibody	PE/Cy7 Rat Anti-Mouse CD24 Monoclonal Antibody	Biolegend	Cat. #: 101–822 RRID:AB_756048	Flow cytometry (1:200)
Antibody	APC Armenian Hamster Anti-Mouse/Rat CD29 Monoclonal Antibody	Biolegend	Cat. #: 102216 RRID:AB_492833	Flow cytometry (1:200)
Antibody	Rat Anti-Ly-6A/E (Sca-1) Monoclonal Antibody, PE	eBioscience	Cat. #: 12-5981-82 RRID:AB_466086	Flow cytometry (1:200)
Antibody	Streptavidin eFluor 450 Conjugate	eBioscience	Cat. #: 48-4317-82 RRID:AB_10359737	Flow cytometry (1:200)
Peptide, recombinant protein	Epithelial growth factor (EGF)	Corning	Cat. #: 354001	50 ng/mL
Peptide, recombinant protein	FzCRD	PMID:25260709		Inhibition of the Frizzled receptor using its soluble CRD domain. Purified in our laboratory (1:200)
Peptide, recombinant protein	Wnt3A	PMID:20569694		Purified in our laboratory 200 ng/ml
Recombinant DNA reagent	plko.1 backbone	Addgene	RRID:Addgene_30323
Recombinant DNA reagent	pGL4.17 basic vector	Promega, Madison, WI, U.S.A.	Cat. #: E6721
Recombinant DNA reagent	pRL-TK Renilla	Promega, Madison, WI, U.S.A.	Cat. #: E2241
Recombinant DNA reagent	pcDNA3.1-RSPO1 overexpression (RSPO1-OE) plasmid	This paper		Constructed in our laboratoryDetail refer to Materials and methods
Chemical compound, drug	tamoxifen (TAM)	Sigma-Aldrich	Cat. #: T5648	2 mg/20 g
Chemical compound, drug	HEPES	Sigma	Cat. #: H4034-500G	25 mM
Chemical compound, drug	Collagenase III	Worthington	Cat. #: LS004183	300 U/ml
Chemical compound, drug	red blood cell lysing buffer	Sigma	Cat. #: R7757
Chemical compound, drug	DNase I	Sigma	Cat. #: D4263	0.1 mg/mL
Chemical compound, drug	Carmine	Sigma	Cat. #: C1022	2 mg/ml
Chemical compound, drug	Histoclear	National Diagnostics	Cat. #: HS-200
Chemical compound, drug	Matrigel	BD Bioscience	Cat. #: 354230
Chemical compound, drug	Insulin-Transferrin-Selenium (ITS)	Gibco	Cat. #: 41400045	Cell culture (1:100)
Chemical compound, drug	E2	Sigma	Cat. #: E8875	1 μM
Chemical compound, drug	IWP2	Selleck	Cat. #: s7085	2.5 μM
Chemical compound, drug	CHIR	Selleck	Cat. #: S1263	3 μM
Chemical compound, drug	XAV-939	Selleck	Cat. #: S1180	10 μM
Chemical compound, drug	Protein A Agarose	Santa Cruz	Cat. #: sc-2003
Chemical compound, drug	DAPI	Life Technologies	Cat. #: P36931
Chemical compound, drug	GPCR compound library	Selleckchem L2200		Chemical Biology Core Facility, Institute of Biochemistry and Cell Biology, SIBS, CAS
Commercial assay or kit	In situ hybridization RNAscope kit	Advanced Cell Diagnostics		Following the manufacturer’s instructions
Commercial assay or kit	Dual-Luciferase Reporter Assay System	Promega	Cat. #: E1910	Following the manufacturer’s instructions
Commercial assay or kit	cAMP-Glo assay kit	Promega	Cat. #: V1501	Following the manufacturer’s instructions
Commercial assay or kit	SuperScript III kit	Invitrogen	Cat. #: RR036A	Following the manufacturer’s instructions
Software, algorithm	GraphPad Prism	GraphPad Prism (https://graphpad.com)
Software, algorithm	ImageJ	ImageJ (http://imagej.nih.gov/ij/)