The Estrogen Receptor-Related Orphan Receptors Regulate Autophagy through TFEB

Abstract

Autophagy is an essential self-degradative and recycling mechanism that maintains cellular homeostasis. Estrogen receptor-related orphan receptors (ERRs) are fundamental in regulating cardiac metabolism and function. Previously, we showed that ERR agonists improve cardiac function in models of heart failure and induce autophagy. Here, we characterized a mechanism by which ERRs induce the autophagy pathway in cardiomyocytes. Transcription factor EB (TFEB) is a master regulator of the autophagy-lysosome pathway and has been shown to be crucial regulator of genes that control autophagy. We discovered that TFEB is a direct ERR target gene whose expression is induced by ERR agonists. Activation of ERR results in increased TFEB expression in both neonatal rat ventricular myocytes and C₂C₁₂ myoblasts. An ERR-dependent increase in TFEB expression results in increased expression of an array of TFEB target genes, which are critical for the stimulation of autophagy. Pharmacologically targeting ERR is a promising potential method for the treatment of many diseases where stimulation of autophagy may be therapeutic, including heart failure. SIGNIFICANCE STATEMENT: Estrogen receptor-related receptor agonists function as exercise mimetics and also display efficacy in animal models of metabolic disease, obesity, and heart failure.

Fig. 1.

ERR agonists induce the expression of autophagy genes and proteins. (A) Tfeb gene and (B) TFEB protein expression are increased after 72 hours treatment with SLU-PP-915 (2.5 μM) in NRVMs. (C) Representative immunoblotting of TFEB in NRVMs quantified in B). (D) Tfeb gene and (E) TFEB protein expression is increased after treatment with SLU-PP-915 (5 μM) in C₂C₁₂ cells (72 h treatment). TFEB induction by SLU-915 is similar to stimulation by starvation (24 h low glucose media). (F) Representative immunoblotting of TFEB protein in C₂C₁₂ cells quantified in E. (G–I) ERR target genes are increased after 72 hours treatment with SLU-PP-915 (5 μM) in C₂C₁₂ cells. *, P < 0.05; **, P < 0.01 relative to DMSO/vehicle control Student’s t test.

Fig. 2.

ERR regulates the transcription of TFEB through binding to ERRE in the TFEB promoter. Dose-response of full-length ERRα (A), ERRβ (B), and ERRγ (C) cotransfection assay in HEK293 cells using TFEB::luc promoter in the absence (dark gray) or presence (light gray) of SLU-PP-915. ****, P < 0.0001 vs. vehicle with no ERR; ++, P < 0.01 and ++++, P < 0.0001 vs. vehicle with ERR; Two-way ANOVA followed by Tukey’s post hoc analysis. Results of ERRα (D), ERRβ (E), and ERRγ (F) cotransfection assay in HEK293 cells illustrating the activation of TFEB promoter by all three isoforms in a dose-response manner presence of SLU-PP-915. Dose-response curve fitting (and EC50’s determined) was performed using GraphPad Prism (log(agonist) vs. response (three parameters) least squares fit). (G) Treatment with ERRγ inverse agonist 4-hydroxy tamoxifen in ERRγ cotransfection assay decreases TFEB response in a dose-responsive manner. (H) Point mutations within the putative ERREs in TFEB gene blunt ERR dependency in cotransfection assays in HEK293. The top shows a schematic of the reporter gene with the TFEB insert as well as the point mutations while the bottom shows the results of the cotransfection assay with either no ERR overexpressed or ERRα, -β or -γ overexpressed. ****, P < 0.0001 vs. vehicle with no ERR; ++, P < 0.01 and ++++, P < 0.0001 vs. vehicle with ERR; two-way ANOVA followed by Tukey’s post hoc analysis. For all graphs error bars represent S.D. and bars in the bar graphs indicate the mean (points are individual determinations) while the points in the dose responses represent the mean of three individual data points.

Fig. 3.

Pharmacological activation of ERR increases TFEB expression. TFEB expression in C2C12 cells was assessed by immunofluorescence. TFEB (green) and nuclei (DAPI; blue) are illustrated. (A–B) Immunofluorescence staining of TFEB in C2C12 cells treated for 48 hours with DMSO or SLU-PP-915 (915) (5 μM). Low magnification (40X). (C–D) Immunofluorescence staining of TFEB in C₂C₁₂ cells treated for 48 hours with DMSO or SLU-PP-915 (5 μM) High magnification (100X). (E) Mean fluorescence of the whole cell is quantified utilizing ImageJ software. F) The fluorescence of nuclear TFEB is quantified utilizing ImageJ software. ****P < 0.0001, Student’s t test.

Fig. 4.

TFEB target genes are increased upon treatment with ERR agonist SLU-PP-915. (A–C) TFEB target genes are modulated in SLU-PP-915 treated NRVMs. The fold change is shown as SLU-PP-915 CPM/DMSO CPM. (D–G) SLU-PP-915 (5 μM) impact on TFEB target genes was validated with qPCR in C₂C₁₂ cells treated for 48 hours. (H) Protein expression of Lamp1-TFEB target gene- increases after 72 hours treatment with SLU-PP-915 (5 μM) in C₂C₁₂ cells. (I) Representative immunoblotting of LAMP1 in C₂C₁₂ cells. *P < 0.05, **P < 0.01; Student’s t test.