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CHIR-99021 Regulates Mitochondrial Remodelling Via Β-Catenin Signalling

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CHIR-99021 Regulates Mitochondrial Remodelling Via Β-Catenin Signalling © 2019. Published by The Company of Biologists Ltd | Journal of Cell Science (2019) 132, jcs229948. doi:10.1242/jcs.229948 RESEARCH ARTICLE CHIR-99021 regulates mitochondrial remodelling via β-catenin signalling and miRNA expression during endodermal differentiation Yuejiao Ma, Minghui Ma, Jie Sun, Weihong Li, Yaqiong Li, Xinyue Guo and Haiyan Zhang* ABSTRACT been closely linked to cell fate determination and development, and Mitochondrial remodelling is a central feature of stem cell evidence has demonstrated that mitochondrial biogenesis is differentiation. However, little is known about the regulatory essential for the successful differentiation of stem cells (Varum mechanisms during these processes. Previously, we found that a et al., 2011; Chen et al., 2012; Folmes et al., 2012; Michel et al., pharmacological inhibitor of glycogen synthase kinase-3α and -3β, 2012; Xu et al., 2013; Wanet et al., 2015). An increased CHIR-99021, initiates human adipose stem cell differentiation into mitochondrial mass and mitochondrial DNA (mtDNA) copy human definitive endodermal progenitor cells (hEPCs), which were number, together with the elongation of the mitochondrial directed to differentiate synchronously into hepatocyte-like cells after network and maturation of the cristae ultrastructure, has been in vitro further treatment with combinations of soluble factors. In this study, observed during the differentiation of stem cells (Chen et al., we show that CHIR-99021 promotes mitochondrial biogenesis, the 2008; Varum et al., 2011; Wanet et al., 2014, 2017). The expression of PGC-1α (also known as PPARGC1A), TFAM and morphology, localization, abundance and function of NRF1 (also known as NFE2L1), oxidative phosphorylation mitochondria could potentially be used as key markers for the capacities, and the production of reactive oxygen species in differentiation of stem cells into specialized cell types (Varum et al., hEPCs. Blocking mitochondrial dynamics using siRNA targeting 2011; Wanet et al., 2017). However, little is known about the DRP1 (also known as DNM1L) impaired definitive endodermal regulatory mechanisms connecting mitochondrial structural and differentiation. Downregulation of β-catenin (CTNNB1) expression functional remodelling to the differentiation of stem cells. Thus, a weakened the effect of CHIR-99021 on the induction of mitochondrial better understanding and control of mitochondrial remodelling remodelling and the expression of transcription factors for during the differentiation process in the laboratory should translate mitochondrial biogenesis. Moreover, CHIR-99021 decreased the into enhanced efficiency and increased fidelity in the resulting cells. expression of miR-19b-2-5p, miR-23a-3p, miR-23c, miR-130a-3p Previously, we reported that the pharmacological inhibition of α β α β and miR-130a-5p in hEPCs, which target transcription factors for glycogen synthase kinase-3 and -3 (GSK-3 , GSK-3 , also mitochondrial biogenesis. These data demonstrate that CHIR-99021 known as GSK3A and GSK3B) with specific inhibitors (CHIR- plays a role in mitochondrial structure and function remodelling via 99021 and CHIR-98014) initiates the efficient differentiation of activation of the β-catenin signalling pathway and inhibits the human adipose stem cells (hASCs) to human definitive endodermal expression of miRNAs during definitive endodermal differentiation. progenitor cells (hEPCs) by upregulating the transcription factors GATA4, FOXA2 and SOX17 via activation of the Wnt/β-catenin This article has an associated First Person interview with the first pathway (Huang et al., 2017). CHIR-99021 has also been reported author of the paper. as an important cocktail component of endodermal differentiation inducers for pluripotent stem cells (Blauwkamp et al., 2012; Lian KEY WORDS: Human adipose stem cells, Mitochondrial biogenesis, et al., 2014; Teo et al., 2014; Bao et al., 2015; Morrison et al., 2016). Endodermal differentiation, PGC-1α, Oxidative phosphorylation CHIR-99021-induced GSK-3 inhibition during differentiation further resulted in the inhibition of T cell factor 3 (TCF3, also INTRODUCTION known as transcription factor 7-like 1, Tcf7l1), which in turn Mitochondria are highly specialized and dynamic organelles inside relieved the repression of FoxA2 in mouse embryonic stem cells mammalian cells that play crucial roles in several cellular pathways, (Morrison et al., 2016). The inhibition of TCF4 (also known as such as the production of energy in the form of ATP via oxidative Tcf7l2), another member of the transcription factor 7-like family, phosphorylation, apoptosis and reactive oxygen species (ROS) was sufficient to increase the expression of peroxisome proliferator- signalling (Friedman and Nunnari, 2014; Chandel, 2015). To meet activated receptor gamma (PPARγ) coactivator-1α (PGC-1α, also the different energy demands of distinct cell types, cells modulate known as PPARGC1A), the master regulator of mitochondrial mitochondrial numbers and activity through biogenesis and through biogenesis (Ventura-Clapier et al., 2008) and oxidative dynamic events (Eisner et al., 2018). Recently, mitochondria have phosphorylation activity during the early step of human bone marrow mesenchymal stem cell commitment to hepatic differentiation (Wanet et al., 2017). We therefore anticipated that Department of Cell Biology, School of Basic Medical Science, Capital Medical University, Beijing 100069, China. the effects of CHIR-99021 on endodermal induction may be attributable to the recapitulation of mitochondrial biogenesis and *Author for correspondence ([email protected]) glycolysis-oxidative phosphorylation shifts. Y.M., 0000-0001-7237-0527; H.Z., 0000-0001-9005-5774 Mitochondrial biogenesis and oxidative phosphorylation activity are accomplished by the coordinated expression of genes from the Received 14 January 2019; Accepted 17 June 2019 nuclear and mitochondrial genomes. Several transcription and/or Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2019) 132, jcs229948. doi:10.1242/jcs.229948 replication factors have been reported to directly regulate gene of PGC-1α, TFAM and NRF1 in hEPCs were significantly expression in mitochondrial biogenesis. Nuclear respiratory factor 1 increased compared to hASCs (Fig. S2). Next, two fluorescent (NRF1, also known as NFE2L1) and NRF2 (also known as probes were used to assess the mitochondrial mass (MitoTracker NFE2L2) control all ten nucleus-encoded cytochrome oxidase Green) and mitochondrial membrane potential (TMRM) in hASCs subunits, and the orphan nuclear hormone receptor, ERRα (also and hEPCs. The relative intensities of MitoTracker Green FM and known as ESRRA), controls the medium chain acyl-coenzyme A TMRM were calculated, and the relative intensity of TMRM dehydrogenase (MCAD) promoter (Huss and Kelly, 2004). The fluorescence was normalized to MitoTracker Green FM in each cell. PGC-1 family of regulated coactivators [PGC-1α, PGC-1β (also The results showed that the mitochondrial mass and mitochondrial known as PPARGC1B) and PRC (also known as PPRC1)] targets membrane potential in the hEPCs were significantly higher than multiple transcription factors, including NRF1, NRF2, and ERRα those in the hASCs (Fig. 1A,B). plays a central role in a regulatory network governing the To further demonstrate changes in mitochondrial numbers and transcriptional control of mitochondrial biogenesis (Wu et al., structures, we performed ultrastructural analyses using transmission 1999; Scarpulla, 2011; Wanet et al., 2017). PGC-1 binds to and electron microscopy (TEM). Upon definitive endodermal coactivates the transcriptional function of NRF1 on the promoter for differentiation, the hEPCs showed a higher number and density of mitochondrial transcription factor A (TFAM), a direct regulator of mitochondria than did the hASCs (Fig. 1C,D). Mitochondria in mitochondrial DNA replication and/or transcription (Virbasius and hASCs contained functionally immature mitochondria with a Scarpulla, 1994; Picca and Lezza, 2015). Hence, CHIR-99021 may globular shape, poorly developed cristae and perinuclear regulate mitochondrial biogenesis and activities, at least in part, localization, all indicative of a less-active mitochondrial state, through modulating the amounts and/or functions of these key sets while the hEPCs possessed a complex morphology, with developed of regulators. Here, we investigated whether CHIR-99021 can cristae, a denser matrix, and an elongated appearance (Fig. 1E,F). regulate certain mitochondrial regulatory factors to control To investigate aerobic metabolism activity levels during mitochondrion biogenesis and oxidative phosphorylation activities definitive endodermal differentiation, we quantified ATP during the definitive endodermal differentiation of hASCs. production rates by measuring the two major production pathways in mammalian cells; glycolysis and oxidative phosphorylation were RESULTS analysed using Agilent Seahorse XF technology. Differentiating hEPCs induced by CHIR-99021 possess multilineage cells undergo a metabolic switch from highly glycolytic metabolism differentiation potential to active mitochondrial aerobic metabolism and drive ATP Previously, we reported that the efficient differentiation of hASCs to production though oxidative phosphorylation (Khacho and Slack, hEPCs was initiated by activin A, Wnt3a, or the pharmacological 2017). As shown in Fig. 1G, the ATP production rate in hEPCs was inhibition of GSK-3 (CHIR-99021 and CHIR-98014) separately. higher than that
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