FTH1-silencing mediates impairment of ESCs pluripotency.
Iron homeostasis is involved in many biological processes including the maintenance of pluripotency in human PSCs. For instance, results from previous research found that depletion of intracellular iron associates with impairment of pluripotency and self-renewal due to significant reduction of NANOG expression (25). Cells maintain a balanced iron pool and in this regulation ferritin, as iron storage protein, plays a crucial role. Therefore, to speculate on the role of ferritin in the context of hESCs, we first generated FTH1-silenced cells via lentiviral transfection with short hairpin RNA (shRNA); subsequent analysis at both mRNA and protein levels confirmed that FTH1 was successfully knocked-down in transfected hESC (Supplementary Fig. S1B-S1E). Morphologically, colonies from FTH1-KD hESCs were more flattened than those of shSCR control (Supplementary Fig. S1A), and alkaline phosphatase (AP) activity was reduced in FTH1-silenced cells (Fig. 1A). FTH1-KD hESCs colonies were positive for pluripotency markers Oct4, TRA-1-60, and Sox2, while the expression of Nanog was slightly increased (Fig. 1B and Supplementary Fig. S2A). This last finding was further confirmed by immunoblot analysis demonstrating that Nanog expression is indeed higher in FTH1-silenced hESCs. (Fig. 1C). At the transcriptional level, OCT4, SOX2, and NANOG resulted evenly increased in FTH1-KD hESCs (Fig. 1D). We further asked whether FTH1 knockdown could, either directly or indirectly, promote spontaneous differentiation of hESCs. Analysis of the transcript levels of AFP, Nkx2.5, and PAX6, specific of endoderm, mesoderm, and ectoderm germ layers respectively, revealed an enhanced expression of these genes in FTH1-KD hESCs (Fig. 1E, left panel). Next, we induced both SCR control and FTH1-KD hESCs to differentiate using the Embryoid bodies (EBs) formation method (Supplementary S2B). On day 28 of differentiation, EBs were harvested and analyzed for endodermal- (GATA4, FOXA2), mesodermal- (HAND1, CD31), and ectodermal- (NESTIN, PAX6) associated markers. While endodermal and mesodermal transcript levels resulted upregulated in FTH1-KD-derived EBS compared to control EBs, the ectodermal genes NESTIN and PAX6 were significantly downregulated in EBs derived from silenced cells (Fig. 1E, right panel), suggesting that modulation of FTH1 by repression negatively affects neuroectodermal gene expression. Immunofluorescence analysis for Sox17 (endodermal marker), Brachyury (mesodermal marker), and Nestin further confirmed that the percentage of Nestin+ cells is lower in FTH1-silenced hESCs compared to SCR control (Fig. 1F). A similar pattern of results was observed in a previous study, in which overexpression of NANOG was associated with neuroectodermal differentiation impairment of hESCs (80).
ROS levels are reduced upon FTH1-gene silencing in hESCs
The role of ferritin in preventing iron-mediated oxidative stress was observed in many studies reporting a direct correlation between modulation of FTH1 expression and dysregulation of iron homeostasis (75). Specifically, a reduction in FTH1 expression leads to excess labile iron, in turn promoting the formation of oxygen-derived free radicals (39, 67). To speculate on the effects of FTH1 silencing in hESCs, we measured total and mitochondrial ROS levels together with an analysis of the mitochondrial membrane potential (ΔΨM) on three biological replicates of SCR and FTH1-KD hESCs, using DCF, MitoSox, and TMRM dyes, respectively (Supplementary S3A and S3B). Intriguingly, our data revealed a reduction in total ROS levels in FTH1-silenced hESCs compared to SCR controls (FTH1-KD: CM-H2DCFDA MFI: 29785,7 vs SCR: CM-H2DCFDA MFI: 47341,7) (Fig. 2A). Likewise, MitoSox assay highlighted a significant reduction in mitochondrial superoxide in FTH1-deficient cells (FTH1-KD: MitoSOX Red MFI: 251,8 vs SCR: MitoSOX Red MFI: 1002,3) (Fig. 2B). A similar, asymmetric behavior was observed by flow cytometry analysis, which revealed a decrease in TMRM fluorescence, indicative of mitochondrial membrane depolarization, in FTH1-KD hESCs (MFI: 7722) compared to SCR control (MFI: 11086,7) (Fig. 2C). Together, these results led us to conclude that hESCs had to operate and overactivate well established antioxidant systems in order to protect themselves from shFTH1-mediated iron toxicity.
Nrf2 signaling pathway drives the antioxidant response in hESCs
The nuclear factor (erythroid 2-related) factor 2 (Nrf2) is a transcription factor ubiquitously expressed in most eukaryotic cells. Nrf2-Keap1 (Kelch-like ECH-associated protein 1) signaling pathway was shown to play a central role in protecting cells against oxidative stress (41, 50). Under basal conditions, Nrf2 is bound to its negative regulator Keap1 which directs it to proteasomal degradation. In the presence of intracellular ROS, Keap1 is oxidized and releases Nrf2 which is free to translocate into the nucleus, where it binds to antioxidant response elements (AREs) and induces the expression of antioxidant target genes, such as HMOX1, NQO1, GST, GPX (36). Besides its role as a fine regulator of redox and metabolic homeostasis, Nrf2 also acts as a pluripotency master gene: Nrf2 activation in hESCs was shown to enhance NANOG transcriptional activity by delaying Nanog protein degradation through POMP-mediated proteasome ubiquitination (27, 33). We found that FTH1-KD hESCs express higher levels of Nrf2 transcript (Fig. 3A) and protein (Fig. 3B and 3C; Supplementary Fig. S4A), assessed via qRT-PCR, immunoblot, and immunofluorescence (Supplementary Fig. S4F), respectively. Similarly, we could also observe an increased expression of p62/Sqstm1 (Fig. 3D and Supplementary Fig. S4B) which, in the non-canonical Keap1-Nrf2 pathway, is known to bind Keap1, allowing Nrf2 to migrate into the nucleus (37). Moreover, the p62 protein yields a feedback loop that amplifies the Nrf2 system (32). In addition to increased expression of Nrf2 in FTH1-silenced hESCs, we observed the overexpression of some important antioxidant enzymes, such as glutathione peroxidases Gpx2, Gpx3 (Fig. 3E), Gpx4 (Fig. 3F and Supplementary Fig. S4C), and superoxide dismutases (Sod) 1 and 2 (Fig. 3G). Also, more Nrf2-regulated genes (HMOX1, NQO1, HIF1𝛼, HIF2𝛼) (Fig. 3H) and proteins (Hif1𝛼, Ftl) (Fig. 3I and Fig. 3L, respectively and Supplementary Fig. S4D and S4E) were found highly expressed in FTH1-KD hESCs. Altogether, these findings suggest a crosstalk between FTH1 silencing and overactivation of the Nrf2 signaling pathway and its cognate effectors in hESCs.
FTH1 knock-down triggers Apoptosis and DNA damage
Labile iron accumulation is a well-known cell damage effector and pro-apoptotic factor (13, 35, 74); intracellular iron overload by ferric ammonium citrate (FAC) treatment leads to ferroptosis, an iron-regulated cell death (15). To check the effects of FTH1-silencing on apoptosis, expression analysis of intrinsic pro-apoptotic genes such as BAX and BIM, as well as CASP9 and CASP3 was performed, revealing a significant upregulation of their expression in FTH1-KD with respect to SCR control hESCs (Fig. 4A and 4B). Similarly, immunoblot analysis of cleaved Casp3 protein confirmed its overexpression in silenced hESCs (Fig. 4C and Supplementary Fig. S5A). It is by now generally accepted that Akt and Erk1/2 pathways are both linked to apoptosis, but with opposite effects: Erk promotes apoptosis, both intrinsically and extrinsically and its activity can be driven by the presence of ROS (90). By contrast, Akt is linked to cell survival, therefore its activity mediates suppression of apoptosis (38). Based on these observations, we speculated whether Erk and Akt could be involved in our FTH1-KD hESCs system; therefore, we measured their expression levels by immunoblot analysis and found that the active, phosphorylated Akt protein (pAkt) expression was reduced, while the expression of pErk1/2 resulted significantly increased in FTH1-silenced hESCs (Fig. 4D and 4E, respectively; Supplementary Fig. S5B and S5C). Collectively, these findings clearly suggest that an active cell death program occurs in hESCs when ferritin is stably downregulated. In addition, based on the evidence that high ROS levels and FTH1 modulation also associate with DNA damage (8, 65, 73, 83), we measured the expression of some DNA damage-associated genes. In line with previous studies, here we show that BRCA1, DMC1, PCNA, and POLQ are indeed significantly upregulated in FTH1-KD hESCs (Fig. 4F). Similarly, we could also observe an increased expression of Parp1 protein, a substrate of activated caspase 3, in FTH1-silenced hESCs (Fig. 4G and Supplementary Fig. S5D). Overall, these results provide strong evidence that FTH1 silencing induces apoptosis in hESCs and triggers the activation of DNA-damage response programs.
Effects of FTH1 repression on cellular metabolism
Under oxidative stress conditions, a metabolic shift from OXPHOS to glycolysis was reported to occur (87), suggesting a clear and intimate correlation between oxidative stress and metabolic changes. It is also well known that hPSCs rely on glycolysis while OXPHOS takes place as soon as these cells enter differentiation (77, 79). By using the Agilent Seahorse analyzer, we first quantified the fraction of ATP generated by glycolysis and by OXPHOS. The difference between SCR control and FTH1-silenced hESCs in glycolysis vs. OXPHOS ATP fraction was negligible. However, we noticed an overall reduction of total ATP production from both sources (Fig. 5A and Supplementary Fig. S6A). We further sought to speculate on the total glycolytic activity in the two groups of cells (shFTH1 vs. SCR). A lower basal and compensatory glycolytic rate was detected in FTH1-silenced hESCs, suggesting that mitochondrial respiration is somehow inhibited in these cells which retain the capability to manage energy demand despite the genetic modification in the ferritin gene (Fig. 5B and Supplementary Fig. S6B). For a more comprehensive metabolic investigation, we analyzed the expression levels of glycolysis-specific genes such as ALDOA, PGK1, ENO1, PKM, together with genes encoding for enzymes of gluconeogenesis, G6PC1 and FBP1. In line with the results obtained by the glycolytic rate analysis, we could not observe significant changes in the expression of glycolytic genes between SCR and FTH1-silenced hESCs (Fig. 5C); on the other hand, the expression of G6PC1 (Glucose-6-Phosphatase Catalytic Subunit 1) resulted significantly higher in FTH1-silenced cells (Fig. 5D). G6PC1 encodes one of the key enzymes responsible for glucose production from glucose-6-phosphate, and therefore is critically involved in glucose homeostasis. This finding perfectly matches with the fact that high glucose levels are required for the proper functionality of detox systems, such as pentose phosphate pathway (PPP), glucuronidation pathway, and glutathione biosynthesis pathway, all of which are regulated by the Nrf2 signaling pathway (29, 51). In support of this, we found that G6PD (glucose-6-phosphate dehydrogenase) and PGD (6-Phosphogluconate dehydrogenase), two key enzymes involved in PPP, were significantly increased in FTH1-silenced cells (Fig. 5E), strengthening the concept that the PPP may represent a crucial metabolic target by which the Nrf2 signaling potentiates the antioxidant response in hPSCs. The expression of PDK1 (pyruvate dehydrogenase kinase 1) was significantly enhanced in shFTH1-hESCs as well (Fig. 5F); its expression is positively regulated by HIF1𝛼, previously shown to participate in ROS-induced metabolic reprogramming, where PDK1 prevents the pyruvate from entering the TCA cycle (tricarboxylic acid cycle or Krebs cycle) (40). We finally tested the expression level of UCP2 (Uncoupling protein-2), a protein of the inner mitochondrial membrane, involved in uncoupling OXPHOS from ATP synthesis. Similarly, to PDK1, UCP2 expression was higher in FTH1-silenced hESCs (Fig. 5G). As PDK1, UCP2 blocks pyruvate entry into the Krebs cycle and shunts it towards the PPP (31, 60, 85). Altogether, our findings are indicative of a metabolic reorganization occurring in response to iron-mediated oxidative stress.