A novel function of R-spondin1 in regulating estrogen receptor expression independent of Wnt/β-catenin signaling
- 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
- Medical Research Institute, Wuhan University, China
- Department of Endocrinology and Metabolism, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine (SJTUSM), China
- School of Life Science, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Chinese Academy of Sciences, China
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 …
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 …
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 …
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 …
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.
Figure 3
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 …
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…
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 …
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 …
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) …
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 …
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). …
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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 | 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) | Rspo1flox/+ | This paper | Generated in our laboratory Detail refer to Figure 4 and Figure 4—figure supplement 1 | |
| Genetic reagent (M. musculus) | Wnt4flox/+ | This paper | Generated in our laboratory Detail refer to Figure 5 and Figure 5—figure supplement 1 | |
| Genetic reagent (M. musculus) | Lgr4LacZ/+ | 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/) |
