Loss of USF2 promotes proliferation, migration and mitophagy in a redox-dependent manner

Abstract

The upstream stimulatory factor 2 (USF2) is a transcription factor implicated in several cellular processes and among them, tumor development seems to stand out. However, the data with respect to the role of USF2 in tumor development are conflicting suggesting that it acts either as tumor promoter or suppressor. Here we show that absence of USF2 promotes proliferation and migration. Thereby, we reveal a previously unknown function of USF2 in mitochondrial homeostasis. Mechanistically, we demonstrate that deficiency of USF2 promotes survival by inducing mitophagy in a ROS-sensitive manner by activating both ERK1/2 and AKT. Altogether, this study supports USF2's function as tumor suppressor and highlights its novel role for mitochondrial function and energy homeostasis thereby linking USF2 to conditions such as insulin resistance, type-2 diabetes mellitus, and the metabolic syndrome.

Keywords: Compromised mitochondria; Migration; Mitophagy; Proliferation; Upstream stimulatory factor 2 (USF2).

Fig. 1

Lack of USF2 in cells affects morphology, proliferation and migration. (A) Representative Western blot analysis of USF2 protein levels in control (scrambled), USF2-deficient (ΔUSF2) and ΔUSF2 MEFs re-expressing a V5-tagged USF2 (ΔUSF2+USF2). (B) Light microscopy images of control, ΔUSF2 and ΔUSF2+USF2 MEFs stained with crystal violet. (C) Real-time proliferation rate of control, ΔUSF2 and ΔUSF2+USF2-MEFs. *significant difference, p ≤ 0.05. (D) Cell cycle distribution of control and ΔUSF2 cells. Histograms display cells in the G1 (blue fraction), S (yellow), and G2/M (green) phase of the cell cycle. (E) Quantification of cell cycle distribution. *significant difference control vs ΔUSF2 cells, p ≤ 0.05. (F, G) Apoptosis in control and ΔUSF2 MEFs was assessed by Annexin-V/P staining, measured by flow cytometry. (H) Real-time cell wound closure analysis of control, ΔUSF2 and ΔUSF2+USF2 MEFs; *significant difference, p ≤ 0.05. (I) Representative images of wound closure of control, ΔUSF2 and ΔUSF2+USF2 MEFs at 0 h, and 16 h after wound introduction. Scale bar 20 μm. (J) Migration was assessed by crystal violet staining and quantified by measuring the absorbance of crystal violet at 595 nm. The OD of ΔUSF2 cells was set to 100%. *significant difference control vs ΔUSF2 cells, **ΔUSF2 cells vs ΔUSF2+USF2 cells, p ≤ 0.05. (K) Representative images of the transwell migration assay. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Lack of USF2 alters mitochondrial morphology and function. (A) Control, ΔUSF2 and ΔUSF2+USF2 MEFs cells were subjected to electron microscopy. Representative images of a cross-section showing the mitochondria (arrows), are presented. Original magnification, 6300 ×. Scale bar 1 μm. (B) Control, ΔUSF2 and ΔUSF2+USF2 cells were stained with TMRE and analyzed with the Operetta high-content imaging system. *significant difference control vs ΔUSF2 cells, **ΔUSF2 cells vs ΔUSF2+USF2 cells, p ≤ 0.05. (C) Representative fluorescent images of TMRE stained cells. (D) The oxygen consumption rate (OCR) of control and ΔUSF2 cells was measured under basal conditions and after the sequential addition of 1 μM oligomycin, 1 μM FCCP, and 0.5 μM rotenone/antimycin to allow calculation of basal respiration, ATP production, proton leak, and spare respiratory capacity (SRC). *significant difference, p ≤ 0.05. (E) Loss of USF2 increases ROS levels. Control, ΔUSF2 and ΔUSF2+USF2 MEFs were stained with CellROX® and the fluorescence intensity was analyzed and quantified with the Operetta high-content imaging system. *significant difference control vs ΔUSF2 cells, **ΔUSF2 cells vs ΔUSF2+USF2 cells, p ≤ 0.05. (F) Representative images of cells displaying CellROX® fluorescence.

Fig. 3

Lack of USF2 alters lysosomes. (A) Control and ΔUSF2 cells were stained with LysoTracker red and quantified with the Operetta high-content imaging system. *significant difference control vs. ΔUSF2 cells, **ΔUSF2 cells vs ΔUSF2+USF2 cells and # control vs ΔUSF2+USF2 cells, p ≤ 0.05. (B) Representative fluorescent images. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Lack of USF2 promotes autophagy and activates ERK1/2 and AKT (A, B) Western blot analyses and densitometric quantification of the protein levels of autophagy-related LC3, ATG5 as well as SDHB, USF2, and α-tubulin in control, ΔUSF2, and ΔUSF2+USF2 cells. The protein levels of the corresponding proteins in control cells were set to 100%. *significant difference control vs. ΔUSF2 and **ΔUSF2 cells vs ΔUSF2+USF2 cells, p ≤ 0.05. (C, D) Representative fluorescence images from control, ΔUSF2, and ΔUSF2+USF2 cells in the presence and absence of chloroquine (Clq; 50 μM for 6 h) probed with an antibody against LC3, stained with DAPI and quantified with the Operetta high-content imaging system. The LC3 levels in control cells were set to 100%. *significant difference control vs. ΔUSF2, ** Control chloroquine treated vs ΔUSF2 chloroquine treated cells, and # untreated vs chloroquine treated p ≤ 0.05. (E, F) Western blot analyses and densitometric quantification of the LC3II and p62 protein levels from control, ΔUSF2, and ΔUSF2+USF2 cells in the presence and absence of chloroquine (Clq; 50 μM for 6 h). The LC3II and p62 levels in control cells were set to 100%. *significant difference control vs. ΔUSF2, **Control chloroquine treated vs ΔUSF2 chloroquine treated cells, and # untreated vs chloroquine treated p ≤ 0.05. (G, H) Western blot analyses and densitometric quantification of the protein levels of pERK1/2, ERK1/2, pAKT, AKT, AMPKα, AMPKβ, USF2, V5-tag, and α-tubulin in control, ΔUSF2 and ΔUSF2+USF2 cells. The protein levels of the corresponding phospho (p) protein levels in the control cells were quantified and normalized to their total levels and the corresponding levels in control cells were set to 100%. *significant difference control vs. ΔUSF2, **ΔUSF2 cells vs ΔUSF2+USF2 cells, and # control vs ΔUSF2+USF2 cells, p ≤ 0.05.

Fig. 5

Lack of USF2 promotes autophagy and activates ERK1/2 and AKT in a redox-sensitive manner. (A) Mitochondrial transmembrane potential in control and ΔUSF2 cells treated with N-acetylcysteine (NAC). Cells were stained with TMRE and analyzed with the Operetta high-content imaging system. *significant difference control vs. ΔUSF2 and **ΔUSF2 cells vs ΔUSF2 cells + NAC, p ≤ 0.05. (B, C) Western blot analyses and densitometric quantification of the protein levels of pERK1/2, ERK1/2, pAKT, AKT and autophagy-related LC3, ATG5 as well as SDHB in control and ΔUSF2 cells treated with NAC. (C) The protein levels of the corresponding phospho (p) ERK and (p) AKT protein levels were quantified and normalized to their respective total levels and the resulting levels in the control cells were set to 100%. *significant difference control vs. ΔUSF2, **ΔUSF2 cells vs ΔUSF2 + NAC cells and # control vs control + NAC, p ≤ 0.05.

Fig. 6

USF2 is crucial for the expression of genes encoding mitochondrial proteins. (A, B, C) Gene ontology (GO) analyses. 12829 genes from the USF2 ENCODE dataset were queried in the PANTHER classification system for statistical overexpression in the GO cellular component complete test. All results shown are valid for an overall FDR <0.05 adjusted P value calculated via the Benjamini–Hochberg procedure. (D, E) RT-qPCR analyses. Data represent log 2 values of fold changes in ΔUSF2 cells relative to control and normalized to the levels of β-Actin, 18S rRNA and Hprt mRNA. *significant difference, p ≤ 0.05.