Figure 5.

PGAM5 induces PGC-1α expression to increase mitochondrial mass.A, cells were transfected with 1 μg Topflash (β-catenin reporter containing the TCF promoter) and 35 ng of pRL/TK (Renilla) as transfection control. Cells were then differentiated for 0, 2, or 4 days. Luciferase activity was determined by the dual luciferase reporter assay system. Transcriptional activity of β-catenin is presented for fold change by normalizing with the value of control iPSCs. Days 2 and 4 show a significant increase of β-catenin activity. Mean ± SEM, n = 3 with ∗∗∗p < 0.001. B, cells were treated with 200 ng/ml doxycycline or DMSO to induce PGAM5 knockdown for 3 days and then transfected with Topflash and Renilla. Cells were then differentiated for 2 or 4 days with or without doxycycline. Luciferase activity was determined with the dual luciferase reagent assay. While there is no significant change on day 2 with PGAM5 knockdown, there is a decrease in β-catenin activity on day 4 indicating that cleaved PGAM5 is regulating β-catenin transcriptional activity. Mean ± SEM with ∗p < 0.05. C, iPSC-ECs were treated with the GSK3 inhibitor and Wnt activator CHIR99021 (or DMSO vehicle) for 24 h. mRNA was extracted and levels of PGC-1α were quantified with qPCR. There is an increase in PGC-1α mRNA levels after Wnt activation. B2M (β2-Microglobulin) was used as housekeeping control. Mean ± SEM, n = 3 with ∗p < 0.05. D, Western blot of PGAM5 in hiPSCs transduced with doxycycline-inducible PGAM5 shRNA lentivirus and treated with 200 ng/ml doxycycline for 3 days. β-actin is used as a loading control. E, cells transduced with PGAM5 shRNA lentivirus were differentiated for 7 days. Control cells were treated with DMSO vehicle while KD cells were given doxycycline. mRNA was extracted and levels of PGC-1α were quantified with RT-qPCR. PGAM5 knockdown cells show a decrease in PGC-1α mRNA levels. B2M was used as housekeeping control. Mean ± SEM, n = 3 with ∗p < 0.05. F, as iPSCs differentiate into iPSC-ECs, there is a burst of mitophagy in mesodermal progenitor cells (day 2) followed by a burst of mitochondrial biogenesis in endothelial progenitor cells (day 4). PINK1/Parkin mitophagy in mesodermal progenitors leads to a cleavage of the phosphatase PGAM5 in endothelial progenitors. PGAM5 dephosphorylates β-catenin, which stabilizes the protein and allows it to translocate to the nucleus, interact with TCF, and transcribe for PGC-1α. This results in an increase in mitochondrial mass that contributes to an alteration of metabolism from glutamine metabolism in iPSCs to fatty acid oxidation in iPSC-ECs.