Fig. 3

Mitochondrial Fitness Fine-Tunes Wnt Signaling in Human Fibroblasts and in Respiratory Chain Complex III Deficiency Zebrafish Models

(A) Reduction of the protein levels of β-catenin and its target genes TCF7 and CD44 and endoplasmic reticulum stress induction was observed in human immortalized fibroblasts. Protein extracts obtained from healthy donors (WT) or GRACILE patients (bcs1l^R73C/F368I) were used. A representative blot and quantification are shown (left and right, respectively; n = 3; means ± SEMs).

(B) Total protein extracts from human immortalized fibroblasts from healthy donors treated overnight with 0.2% DMSO or 1 μM Anti were analyzed by western blot. The quantification is shown at right (n = 3; means ± SEMs).

(C) Transient knockdown of the respiratory chain complex III was performed by two independent splicing morpholinos against Ttc19 and Bcs1l mRNAs (Ttc19 spMO and Bcs1l spMO); a standard control morpholino was also used. Bright-field images of morphants are reported. Bars, 1 mm.

(D) Respiratory efficiency of morpholino-injected embryos (pool of 15 embryos) was analyzed by the Oxygraph+ instrument. Oxygen concentration in the medium and the consumption rate are plotted.

(E) Knock down of the respiratory complex III was performed in Tg(7xTCFX.lasiam:GFP)^ia4 by Ttc19 spMO and Bcs1l spMO and compared to the control. GFP epifluorescence quantification was reported in the graph below; dots and numbers represent treated embryos (means ± SEMs). Statistical significance (ANOVA) was determined as ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001). Bars, 1 mm.

(F) Mito-Wnt axis is an important pathway in which mitochondrial dysfunction (1), due to mutated genes or to pharmacological treatment, leads to reduced mitochondrial ATP synthesis, which directly affects Ca²⁺ uptake by regulating SERCA in the endoplasmic reticulum (2). This in turn decreases Ca²⁺ levels in the endoplasmic reticulum (3), leading to endoplasmic reticulum stress induction by the phosphorylation of eIF2α and activation of its downstream factors ATF4 and CHOP (4), which are able to modulate Wnt signaling by a still-debated mechanism (5). This modulation could regulate several downstream events such as gene transcription of Wnt target genes, leading to the control of cell proliferation, metabolic adaptation, and cell migration, in particular, modulating cancer development and the spreading of metastasis (6). Finally, a possible feedback mechanism of reduced Wnt signaling can further regulate mitochondrial activity (7). As indicated by experiments shown in Figure S2D, glycolysis also contributes to the regulation of Ca²⁺ uptake into the endoplasmic reticulum.