Fig. 6: GREM1 increases the transcriptional activity of ERRα and the mRNA levels of ERRα target genes through EGFR activation.

a Transcriptional activity of ERRα in breast cancer cells overexpressing GREM1. Each cell line was transfected with the expression vectors encoding ERRα and the coactivator PGC-1α as well as 3xERRE-luc and Renilla. After 48-h incubation, cell lysates were assayed for firefly luciferase and Renilla luciferase activities. b Relative mRNA levels of ERRα target genes in breast cancer cells overexpressing GREM1. The mRNA levels of the indicated genes were quantitated by qPCR analysis. c Effect of GREM1 treatment on the expression of ERRα target genes in MCF-7 cells. Cells were starved overnight and daily stimulated with recombinant GREM1 (10 or 50 ng/ml) for 3 days. RNA was collected and analysed by qPCR analysis. d Effect of GREM1 overexpression on the ERRα target gene transcription in MCF-7 cells. Cells were transfected with expression vectors of mock or GREM1 for 24 h and then incubated with vehicle or XCT790 for another 24 h. The mRNA levels of the indicated genes were measured by qPCR analysis. e, f Effect of EGFR signalling on the transcriptional activity of ERRα induced by GREM1. SKBR3-mock and SKBR3-GREM1 cells were transfected with the expression vectors encoding ERRα and the coactivator PGC-1α as well as 3xERRE-luc and Renilla for 24 h and treated with the indicated pharmacological inhibitors (XCT790, 5 μM; erlotinib, 5 μM; LY294002, 20 μM; U0126, 20 μM) for another 24 h (e). MCF-7 cells were transfected with the expression vectors encoding ERRα and the coactivator PGC-1α as well as 3xERRE-luc and Renilla for 24 h. The cells were pre-treated with recombinant GREM1 or EGF for 24 h and then incubated with the indicated inhibitors for another 24 h (f). g Effect of EGFR signalling on the mRNA expression of ERRα target genes in SKBR3-GREM1 cells. Cells were treated with each indicated compound for 24 h, and mRNA levels of genes were quantitated by qPCR analysis. h Effect of interaction between EGFR and GREM1 on the ERRα transcriptional activity. HEK293 cells were transfected with the expression vectors of EGFR (EGFR-WT, EGFR-ECD or EGFR-ICD), ERRα, PGC-1α, 3xERRE and Renilla for 48 h. The cells were incubated with recombinant GREM1 protein (50 ng/ml) for additional 1 h and analysed by firefly luciferase activity and normalised to Renilla luciferase activity. WT, wild type; ECD, extracellular domain; ICD, intracellular domain. All values in the graphs represent mean ± SD of three independent experiments. Two-sided t test. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.