Cell fate decisions: emerging roles for metabolic signals and cell morphology
2017; Springer Nature; Volume: 18; Issue: 12 Linguagem: Inglês
10.15252/embr.201744816
ISSN1469-3178
AutoresSumitra Tatapudy, Francesca M. Aloisio, Diane L. Barber, Todd Nystul,
Tópico(s)Mitochondrial Function and Pathology
ResumoReview20 November 2017free access Cell fate decisions: emerging roles for metabolic signals and cell morphology Sumitra Tatapudy Departments of Anatomy and OB-GYN/RS, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Francesca Aloisio Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Diane Barber Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Todd Nystul Corresponding Author [email protected] orcid.org/0000-0002-6250-2394 Departments of Anatomy and OB-GYN/RS, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Sumitra Tatapudy Departments of Anatomy and OB-GYN/RS, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Francesca Aloisio Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Diane Barber Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Todd Nystul Corresponding Author [email protected] orcid.org/0000-0002-6250-2394 Departments of Anatomy and OB-GYN/RS, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Author Information Sumitra Tatapudy1, Francesca Aloisio2, Diane Barber2 and Todd Nystul *,1 1Departments of Anatomy and OB-GYN/RS, University of California, San Francisco, San Francisco, CA, USA 2Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA *Corresponding author. Tel: +1 415 476 6883; E-mail: [email protected] EMBO Rep (2017)18:2105-2118https://doi.org/10.15252/embr.201744816 See the Glossary for abbreviations used in this article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Understanding how cell fate decisions are regulated is a fundamental goal of developmental and stem cell biology. Most studies on the control of cell fate decisions address the contributions of changes in transcriptional programming, epigenetic modifications, and biochemical differentiation cues. However, recent studies have found that other aspects of cell biology also make important contributions to regulating cell fate decisions. These cues can have a permissive or instructive role and are integrated into the larger network of signaling, functioning both upstream and downstream of developmental signaling pathways. Here, we summarize recent insights into how cell fate decisions are influenced by four aspects of cell biology: metabolism, reactive oxygen species (ROS), intracellular pH (pHi), and cell morphology. For each topic, we discuss how these cell biological cues interact with each other and with protein-based mechanisms for changing gene transcription. In addition, we highlight several questions that remain unanswered in these exciting and relatively new areas of the field. Glossary ECM extracellular matrix ESCs embryonic stem cells HSCs hematopoietic stem cells iPSCs induced pluripotent stem cells Metabolic reprogramming Changes in metabolism that accompany and can sometimes be necessary or instructive for changes in cell fate MSCs mesenchymal stem cells Niche A specialized microenvironment in the tissue that maintains cells in the stem cell state NSCs neural stem cells pH sensor Selective proteins with post-translational modification by protonation/deprotonation regulating activity or ligand binding. ROS reactive oxygen species SAM S-adenosyl methionine Satellite cells Stem cells of the skeletal muscle Introduction Cell fate decisions are tightly regulated by many layers of control. A change in cell fate is ultimately defined by the acquisition of new characteristics that come about largely through changes in transcription. Protein-based signal transduction cascades leading to changes in transcription factor activity are the most direct causes of transcriptional changes and are among the most well-studied aspects of the cell fate decision process. In contrast, much less is known about how other aspects of cell biology such as changes in metabolite concentration or mechanical forces contribute to cell fate decisions. This is due in part to the difficulty of studying cues that are not directly encoded in the genome. However, technological advances, including the generation of new biosensors that can be used for live cell imaging, improvements in quantitative fluorescence microscopy, and the development of more sensitive biochemical methods for detecting small molecules are making it easier to identify previously unrecognized control mechanisms. In this review, we discuss recent advances in understanding the role of metabolism, reactive oxygen species (ROS), intracellular pH (pHi), and cell morphology and adhesions to cell fate decisions, particularly during differentiation in adult, embryonic, and induced pluripotent stem cell lineages. Metabolism The metabolic state of a cell is the result of a complex array of inputs, including cell signaling, availability of nutrients and oxygen, energy needs, and biomass demands. These inputs and demands combine to influence the rate of ATP production from glycolysis versus oxidative phosphorylation, as well as the rate of side reactions that produce anabolic intermediates. As cells differentiate, the change in these inputs causes the metabolic state to shift. However, the metabolic state of the cell is not merely a consequence of differentiation. Instead, shifts in metabolism can have permissive and, in some cases, even instructive roles in promoting differentiation 1. This perspective positions metabolism as a key node in the regulation of cell fate transitions. In this section, we summarize the metabolic programs of cells at different stages of differentiation, briefly review some of the major cell signaling regulators of metabolic state, and discuss how changes in metabolic state contribute to cellular differentiation (Fig 1). Figure 1. The connections between metabolism and cell fate decisionsMetabolic inputs regulate epigenetics and cell signaling to promote changes in cell fate. Glycolysis produces metabolic intermediates that feed into the folate and one carbon metabolism cycle to produce S-adenosylmethionine (SAM), which is a cofactor for DNA methyltransferases (DNMTs) and histone methyltransferases (HMTs). The energy released from glycolysis and oxidative phosphorylation also converts AMP to ATP and NAD+ to NADH. AMP stimulates AMPK activity, and NAD+ is a cofactor for sirtuins, so increased energy production decreases the activity of these enzymes. Glucose-derived acetyl-CoA enters the tricarboxylic acid (TCA) cycle to form citrate, which can be converted back to acetyl-CoA by ATP-citrate lyase. This source of acetyl-CoA (but not acetyl-CoA derived from fatty acid oxidation) contributes to the pool of nuclear acetyl-CoA that is essential for histone acetylation by histone acetyltransferases (HATs). α-ketoglutarate (α-KG), which is produced in the TCA cycle and in the cytoplasm, is an essential cofactor for TET and Jumonji C enzymes, which demethylate DNA and histones, respectively. The energy released from oxidative phosphorylation converts FAD to FADH2, and FAD is a cofactor for lysine-specific demethylase 1 (LSD1), so a reduction in FAD levels inhibits LSD1 activity. Increased oxidative phosphorylation also generates reactive oxygen species (ROS), which promote oxidation, carbonylation, and hydroxylation as well as increase the levels of JNK and p38/MAPK pathway activity. Low levels of oxygen (O2), for example in the HSC and satellite cell niches, increase the activity of the hypoxia inducible factor-1 (HIF-1), which promotes glycolysis. Download figure Download PowerPoint Changes in metabolism, often collectively referred to as “metabolic reprogramming”, can shift the amount of energy and biomass produced by glycolysis versus oxidative phosphorylation to regulate changes in cell fate. In adult stem cell lineages, less active long-term progenitors, such as quiescent hematopoietic stem cells (HSCs) or satellite cells (stem cells of skeletal muscle) utilize glycolysis over oxidative phosphorylation, whereas more actively growing and proliferating cells are bivalent and utilize both glycolysis and oxidative phosphorylation 2345. Embryonic stem cells (ESCs) transition through several metabolic states during differentiation. ESCs in the most undifferentiated, or “naive” state, have relatively high levels of oxidative phosphorylation 678, although these cells still consume high amounts of glucose and glutamine 69. As ESCs differentiate toward the “primed” state, ATP production becomes decoupled from oxidative phosphorylation, and the metabolic program is shifted toward the use of glycolysis for energy and biomass production 610 through a process that is regulated by the conserved RNA-binding protein, LIN28 7. Energy production from oxidative phosphorylation then increases again as differentiation proceeds beyond the primed state. Likewise, the reprogramming of differentiated somatic cells into induced pluripotent stem cells (iPSCs) requires a shift from a bivalent metabolic program of glycolysis and oxidative phosphorylation toward a primarily glycolytic state that resembles the metabolism of primed ESCs 1112. Recent evidence indicates that this metabolic shift occurs prior to changes in gene expression, suggesting that it is a prerequisite for reprogramming rather than a consequence of the cell fate change 13. Nonetheless, in most cases, metabolic changes are initiated by cell signaling molecules, including AMPK, HIF1α, AKT, and Myc. AMPK, which is activated by high [AMP]/[ATP] ratios that indicate low nutrient availability and metabolic stress, increases glycolytic energy production, activates FOXO proteins to promote the expression of antioxidants and autophagy genes, and restricts growth by inhibiting mTor 1415. This stress response program is important for maintaining cellular homeostasis in general, and thus functions during both self-renewal and differentiation. HIF1α is an oxygen sensor that is stabilized by low oxygen levels and promotes a steady state level of energy production during periods of relatively low activity in quiescent and slowly dividing adult stem cells, such as HSCs 2, mesenchymal stem cells (MSCs) 16, and satellite cells 17. HIF1α shifts the metabolic program toward glycolysis over oxidative phosphorylation, which is conducive to the hypoxic environments of stem cell niches that maintain quiescent stem cells, and also minimizes the damage caused by ROS produced from mitochondrial respiration. In contrast, Akt and Myc promote an increase in energy production from oxidative phosphorylation and a switch in the utilization of glycolysis from a source of energy production to a source of anabolic intermediates. Akt activates mTor by inhibiting the Tsc complex, and several studies have found that this pathway promotes differentiation of adult stem cells including HSCs, NSCs, and ISCs 181920. Akt signaling also increases ROS levels by inhibiting FOXO proteins, which has the effect of further promoting differentiation in some types of stem cells (see next section). Myc is also required for differentiation in the HSC and epidermal stem cell lineages 2122. In addition, Myc is an important factor for reprogramming into iPSCs, and inhibition of mTor or induced expression of metabolic enzymes can substitute for Myc in iPSC reprogramming 2324. Thus, shifts in metabolic state are a prerequisite for differentiation in cases where the shift is needed in order to meet the energetic and anabolic demands of the new cell state. Metabolic state can also influence cell fate decisions by affecting the availability of metabolites that are important for the epigenetic regulation of gene expression 24. Epigenetic regulation occurs primarily through the modification of histones and DNA, and histone acetylation and deacetylation as well as histone and DNA methylation and demethylation all can be regulated by metabolites. Histone acetyltransferases (HATs) use acetyl-CoA, which is a key metabolic intermediate between glycolysis and the TCA cycle, as a substrate for histone acetylation. In the absence of sufficient acetyl-CoA, global histone acetylation is reduced, and thus, the regulation of gene expression is impaired. This connection was clearly demonstrated in a study of in mouse adipocytes 25. The authors found that knockdown of ATP-citrate lyase, which generates acetyl-CoA from citrate, caused a decrease in histone acetylation and prevented the upregulation of genes such as glucose transporters that are required for differentiation. Likewise, deacetylation is also sensitive to acetyl-CoA concentrations in the cell. For example, the addition of acetate (which increases acetyl-CoA levels) to the culture media of human or mouse ESCs blocked histone deacetylation and delayed differentiation, whereas inhibition of glycolysis (which decreases acetyl-CoA levels) accelerated differentiation 26. The effect of glycolysis inhibition could be reversed with the addition of acetate to the media, and pharmacological inhibition of the enzyme that produces acetyl-CoA for histone acetylation produced a similar phenotype, but the effect on histone deacetylation was not tested directly. Deacetylation by sirtuins is also responsive to metabolic inputs 27. Sirtuins are deacetylases with a broad range of targets including histones and transcription factors. These enzymes are considered metabolic sensors because they use NAD+ as a cofactor and thus become more active when [NAD+]/[NADH] ratios are high. In addition, sirtuins both regulate and are regulated by AMPK 28. Sirtuin 1 (SIRT1) has been well studied during mammalian cell differentiation and may function through different mechanisms to both repress differentiation in some contexts and promote differentiation in others. For example, SIRT1 is highly expressed in ESCs, iPSCs, and early morula stage embryos, where it promotes pluripotency and is downregulated upon differentiation 2930. In contrast, genetic and pharmacological studies indicate that SIRT1 promotes differentiation in hematopoietic and neural lineages 3132. The epigenetic regulators that catalyze the addition of methyl groups, DNA methyltransferases (DNMTs) and histone methyltransferases (HMTs), use S-adenosyl methionine (SAM) as a substrate. The rates of histone methylation are different at active versus inactive promoters, and the concentration of intracellular SAM can directly influence these rates. SAM concentrations are relatively high in human and mouse ESCs and iPSCs, and SAM is required for histone methylation to maintain the pluripotent state in these cell types 333435. In adult tissues, there is a well-established role for SAM in the regulation of DNA and histone methylation during oncogenesis 36, and though less is known about the role of SAM in adult stem cell differentiation, many adult progenitors, including HSCs 37, ISCs 38, and epidermal progenitors 39 require DNMTs and HMTs 40. Thus, changes in the concentration of SAM influence cell fate transitions in many different cell types. Likewise, enzymes that catalyze the removal of methyl groups from histones and DNA are sensitive to the availability of specific metabolites. For example, the Jumanji C family of histone demethylases and the TET-family enzymes, which catalyze the first step of DNA demethylation, require both the TCA cycle intermediate α-ketoglutarate (α-KG) and the reduced (Fe2+) form of iron 4142. Iron is more commonly in the Fe3+ form but can be reduced to Fe2+ by vitamin C, and several recent studies revealed the importance of vitamin C for promoting the activity of Jumonji C or TET-family enzymes in ESCs 43444546, adult stem cells 4748, and during iPSC reprogramming 4349. Another important histone demethylase, lysine-specific demethylase 1 (Lsd1), is also sensitive to metabolic changes as it relies on FAD as a cofactor 50. LSD1 is required in mouse ESCs (mESCs) to silence self-renewal genes during differentiation 51, and the homologous gene, Su(var)3-3, is also required in the somatic cells of the Drosophila ovary to promote germ cell differentiation 5253. Collectively, these findings demonstrate that metabolic processes can influence epigenetic regulation of gene expression at multiple levels. In addition to the permissive roles for metabolism in cellular differentiation described above, metabolic cues can also be instructive, causing changes in cell signaling and gene expression sufficient to drive the change in cell fate. For example, in satellite cells, increased glycolysis during exit from quiescence causes a decrease in NAD+, which reduces SIRT activity and thus increases H4K16 acetylation, ultimately leading to the expression of key differentiation genes, such as MyoD 54. Another interesting example comes from a recent study that found that intestinal stem cells (ISCs) utilize lactate provided by the neighboring Paneth cells to sustain a high level of oxidative phosphorylation 55. Increased oxidative phosphorylation in ISCs causes an increase in reactive oxygen species (ROS), which activates the p38-MAPK pathway (as discussed in the following section). Paneth cells are part of the ISC niche, so this suggests that metabolic cues can function as niche signals. Additional examples in which metabolic changes feed into signaling networks to instruct cell fate decisions involve mTOR, which is a master regulator of cell growth and proliferation. Several studies have demonstrated that mTOR is essential for the maintenance of pluripotency and the repression of differentiation genes in ESCs grown under standard conditions 56. In addition, a more recent study found that partial inhibition of mTOR in mESCs induces the cells to adopt a “paused” state resembling embryonic diapause 57. The mechanism of this effect is not fully understood, but the authors speculate that the paused state is induced by the combined effects of mTOR inhibition on transcription, translation, and metabolism. Lastly, in quiescent HSCs, activation of mTOR induces mitochondrial biogenesis, which activates proliferation and induces differentiation 58. Two recent studies demonstrated that changes in pyruvate metabolism can contribute to the regulation of proliferation and differentiation in epidermal and intestinal cell lineages 5960. Pyruvate is the end product of glycolysis and can either enter be converted to lactate in the cytoplasm, or be transported into the mitochondria, where it is converted to acetyl-CoA and oxidized in the TCA cycle. These studies provide evidence that hair follicle and intestinal stem cells are more glycolytic than their non-stem cell progeny, and suggest that increased conversion of pyruvate to lactate drives stem cell proliferation whereas increased mitochondrial oxidation of pyruvate promotes differentiation. The downstream mechanism was not investigated, but both studies provide evidence suggesting that high levels of Myc in the stem cells may promote the shift toward lactate production. Interestingly, a separate study of intestinal differentiation in zebrafish found that Wnt signaling also regulates pyruvate metabolism 61. Wnt signaling is generally high in epithelial stem cells 62 and promotes Myc expression 6364, suggesting a model in which Wnt signaling, Myc, and pyruvate metabolism function together to promote epithelial stem cell identity. Taken together, these studies demonstrate that changes in metabolism influence cell fate decisions in a variety of ways. In many cases, the link between the metabolic cue and the cell fate decision is reactive oxygen species as described in the next section. Reactive oxygen species Metabolic pathways can influence stem cell fate decisions through the activity of ROS (Fig 1). ROS, such as superoxide anion (O2−), hydrogen peroxide (H2O2), and hydroxyl radicals (OH−), are formed by the reduction of molecular oxygen (O2). The toxic effects of these ROS have been studied extensively in the context of cell proliferation, DNA damage, and apoptosis. Additionally, ROS play a crucial role in regulating cellular processes like oxidative stress responses, aging, and stem cell fate decisions. In this section, we review recent advances in the understanding of the role of ROS in cell differentiation. ROS are commonly generated as by-products of metabolic reactions occurring in the mitochondria, mainly in the electron transport chain. ROS levels are controlled by several proteins, such as NADPH oxidases, which have activity that results in formation of superoxides, superoxide dismutases (SOD), which reduce O2− to H2O2, and other enzymes, including thioredoxins, glutathione peroxidases, and peroxiredoxins 6566. Recent studies identified examples in which specific ROS regulators are necessary for stem cell differentiation. For example, Kim et al 67 found that peroxiredoxins, PrxI and Prx II, promote mouse embryonic stem cell differentiation into neurons by regulating ROS levels. In addition, Hochmuth et al 68 found that Nrf2, which controls transcription of antioxidant enzymes like thioredoxins and peroxidases, and Keap1, a negative regulator of Nrf2, regulate Drosophila intestinal stem cell proliferation by altering intracellular ROS. Other studies have focused on the downstream effects of changes to ROS levels, and in general, these studies find that increased ROS levels are associated with differentiation. During Drosophila testes germline stem cell (GSC) differentiation, GSCs maintain reduced levels of ROS, regulated by Keap1 and Nrf2 69. An increase in ROS in GSCs caused a decrease in the number of GSCs and promoted differentiation. In mammalian HSCs, an elevation in ROS levels occurs during differentiation into common myeloid progenitors 70. Likewise, quiescent multipotent hematopoietic progenitor cells in the Drosophila lymph gland have elevated levels of ROS, which promotes differentiation 71. In these studies, scavenging ROS by expressing antioxidant proteins like catalase in vivo or by the addition of N-acetylcysteine, delayed differentiation, whereas increasing ROS by adding paraquat or mutating mitochondrial complex I proteins like ND75, promoted differentiation 697172. During vascular smooth muscle cell differentiation, inhibition of ROS activity decreased the cellular expression of differentiation proteins, whereas an elevation in ROS activity increased expression of these differentiation markers 73. In contrast to this trend, two studies show that elevated ROS levels promote self-renewing, proliferative neural and mouse spermatogonial stem cell fate 7475. Additionally, elevated ROS levels promote Drosophila intestinal stem cell proliferation 68. Reactive oxygen species instruct stem cell fate decisions by regulating key signal transduction pathways. A mechanism by which ROS control signaling pathways that affect stem cell differentiation is by affecting post-translational modifications of regulatory proteins, such as phosphatases. For example, ROS have been shown to mediate cysteine and methionine oxidation, protein carbonylation, and hydroxylation (reviewed by 66). Another mechanism by which ROS influences differentiation decisions is by directly affecting the activity of transcription factors and essential signaling pathway proteins responsible for activating genetic differentiation programs. The most commonly studied signaling pathways in this regard are the JNK and p38 MAPK pathways and an increase in ROS typically activates these pathways to promote differentiation 68717273. For example, in Drosophila hematopoietic progenitor cells, elevated ROS levels stimulate the JNK pathway to promote differentiation by activating transcription factor FoxO and the derepression of polycomb activity 71. However, FoxO also increases antioxidant activity, which reduces ROS levels and thus creates a negative feedback loop that eventually brings FoxO activity back down. Likewise, in mammalian hematopoietic stem cells (HSCs), low levels of ROS are necessary for HSC self-renewal whereas elevated levels of ROS promote differentiation by stimulating the activity of p38 and mTOR signaling pathways 76. During vascular smooth muscle differentiation and mouse spermatogonial stem cell self-renewal, increased ROS levels activate p38 MAPK signaling pathway, which promotes the transcription of serum response factor (SRF), and ultimately increases the activity of differentiation proteins, such as α-actin and calponin 73. Additionally, high levels of ROS in Drosophila GSCs promote differentiation by increasing the transcription of the epidermal growth factor receptor ligand, Spitz, thereby activating the MAPK signaling pathway 69. Collectively, these studies demonstrate that ROS concentrations are tightly controlled during cellular differentiation and that changes in ROS concentrations play important roles in the cell fate decision process. Intracellular pH A long-held view is that pHi is constitutively maintained between 7.2 and 7.4 in normal mammalian cells and only dysregulated from this narrow range in diseases, including being constitutively increased in cancer 7778 and decreased in neurodegenerative disorders 7980. However, emerging evidence indicates there are transient increases in pHi in normal mammalian cells during cell cycle progression 81, directional migration 8283, and differentiation 84858687. Although the role of pHi dynamics in regulating cell fate decisions remains understudied, we highlight recent findings on this topic and emphasize questions that remain to be addressed (Fig 2). Figure 2. Mechanisms by which pHi could regulate cell fate decisionspHi increases during embryonic and adult stem cell differentiation, epithelial-to-mesenchymal transitions, and carcinoma transformations. Theoretically, pH-sensitive proteins (“pH sensors”) that undergo protonation or deprotonation upon changes in pHi could regulate cell fate decisions by affecting proton transporter activity, cellular metabolism, and epigenetic modifications like histone deacetylation and DNA methylation. However, in most cases, the specific mechanisms by which pHi could regulate cell fate decisions are unknown. Download figure Download PowerPoint Increasing evidence suggests that changes in pHi are necessary for embryonic stem cell differentiation. We recently showed a transient increase in pHi during differentiation of clonal naïve mESCs to primed epiblast-like cells (EpiSC), which when prevented, blocks differentiation as indicated by attenuated expression of epiblast cell markers, including Pax6, Brachyury, and Fgf5, as well as the miRNA cluster mir-302 84. The increased pHi from ~7.40 to ~7.65 occurs during the first 3 days of spontaneous differentiation and then returns to pHi values seen in naïve cells, which suggests that the higher pHi is necessary for the differentiation process but not for maintaining a differentiated state. Consistent with this prediction, an earlier study by Edwards et al 88 found that pHi increases from zygote to the morula stage. In a different embryonic cell model, Li et al 87 showed that inhibiting activity of the plasma membrane Na-H exchanger-1 (NHE1), markedly attenuates differentiation of CGR8 clonal mESCs into cardiomyocytes resulting in a decreased expression of the transcription factors Nkx2-5 and Tbx5 and decreased abundance of α-myosin heavy chain. Although changes in pHi during differentiation were not determined, inhibiting NHE1, which is an acid extruder, is predicted to lower pHi. This group also found that NHE1 activity potentiates differentiation of P19 embryonal carcinoma cells into neurons 89. In contrast, umbilical cord-derived human mesenchymal stem cells (MSCs) have a higher pHi than differentiated cells. Lowering pHi of these cells by pharmacological inhibition of NHE1 promotes differentiation to an osteogenic lineage but has no effect on differentiation to an adipogenic lineage 90. Recent studies, including studies from our laboratories, suggest that differentiation occurring during Drosophila adult epithelial follicle stem cell lineages requires changes in pHi. Using the genetically encoded pHi biosensor pHluorin, we showed a lower pHi in follicle stem cells of the adult Drosophila ovary compared with differentiated daughter cells. Preventing the increased pHi by loss of Dnhe2, the Drosophila ortholog of mammalian NHE1, inhibits differentiation, impairs germarium morphology, and results in infertility 84. Krüger and Bohrmann 91 also found an anteroposterior pHi-gradient in follicle and nurse cells of the Drosophila ovary, although significance in oogenesis was not determined. It remains to be determined whether the lower pHi in self-renewing cells or the higher pHi in differentiating cells is an active process. In endometrial epithelial cells, LeftyA inhibits NHE1 to actively maintain a lower pHi 92. Likewise, in mESCs, our findings that the increased pHi with differentiation is transient and seen only during the first 72 h are consistent with an active regulation of pHi 84. Because increased pHi promotes proliferation, the decrease after 72 h may function to limit proliferation. As described below, a constitutively higher pHi is seen in most cancers and can induce hyperproliferation and dysplasia even in the absence of activated oncogenes 93. C
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