Real-Time Monitoring of Glutathione in Living Cells Reveals that High Glutathione Levels Are Required to Maintain Stem Cell Function

摘要

class="head no_bottom_margin" id="sec1title">IntroductionReactive oxygen species (ROS) are important signaling molecules that regulate cellular metabolism, proliferation, and survival (). An increase of ROS induces the thiol oxidation of cysteine residues on signaling proteins, resulting in alterations of protein activities to regulate cellular functions. In particular, ROS-mediated oxidation plays an important role in regulating a variety of signaling proteins in stem cells (SCs) that influence self-renewal capacity, pluripotency, viability, and genomic stability, including OCT4, NRF2, FoxOs, APE1/Ref-1, ATM, HIF-1, p38, and p53 (). For example, OCT4, a pluripotency-related transcription factor, is inactivated via cysteine oxidation under glutamine-depleted conditions, favoring the differentiation and functional maturation of embryonic SCs (ESCs) (). Disruption of Nrf2, a master regulator of redox homeostasis, impinges upon the functions of embryonic and adult SCs such as the self-renewal and pluripotency in ESCs (), the migration and retention of hematopoietic SCs in the bone marrow niche (), and the proliferation and homeostasis in intestinal () and airway basal SCs (). Thus, the cellular redox regulation is critical for maintaining stemness and functional potency of ESCs and adult SCs.Cellular redox homeostasis depends on the balance between ROS production and their elimination via enzymes and antioxidant molecules such as glutathione (GSH), a thiol-containing tripeptide that plays a major role in maintaining redox homeostasis owing to its high concentration (approximately 1–10 mM). GSH is synthesized in the cytosol and is then transported to cellular compartments such as the mitochondria, nucleus, and ER, where redox buffering is required for organelle-specific functions. GSH eliminates hydrogen peroxide (H2O2) through a glutathione peroxidase-catalyzed reaction, producing water and oxidized GSH (GSSG), which is regenerated to GSH by glutathione reductase at the expense of NADPH (). Therefore, changes in the GSH levels in response to oxidative stress can reflect the redox buffering capacity of a particular cell type or cellular compartment. Moreover, GSH also acts as a regulator for ROS-triggered signal transmission. GSH reduces ROS-induced disulfides in signaling proteins, either by glutaredoxin or by forming S-glutathionylated proteins through a thiol-disulfide exchange reaction, thereby modulating the intensity and duration of redox signaling (). Furthermore, ESCs and inducible pluripotent SCs harbor particularly high GSH levels, which confer protection against unfavorable DNA damage (). Thus, monitoring the dynamic changes of GSH levels in living SCs is required to evaluate the redox buffering capacity and signaling processes that modulate SC functions during several physiological and pathological processes. However, obtaining a detailed understanding of the GSH-based redox system has thus far been limited owing to the lack of direct and reliable tools.Various techniques have been used to estimate GSH levels in living cells to date, including redox-sensitive fluorescent proteins such as rxYFP and roGFP (). Since the thiol-disulfide status of these fluorescent proteins is in equilibrium with that of cellular GSH, this method can provide information on the GSH/GSSG redox potential but cannot directly monitor the changes in GSH levels. In addition, the poor efficiency of transfection and consequent cellular damage hinder the application of this genetic method for a wide range of cell types. Several thiol-reactive fluorescent chemical dyes have also been developed as GSH sensors to overcome these problems (). However, these dyes still have drawbacks such as irreversibility, slow kinetics (), low fluorescence quantum yields (), or limited subcellular localization (). Moreover, previous works on GSH monitoring have not paid enough attention to the possible errors caused by reactive thiols in cellular proteins.To overcome the limitations of current GSH probes, we previously reported the synthesis of a coumarin derivative bearing a conjugated 2-cyanoacrylamide group, designated FreSHtracer (fluorescent real-time thiol tracer; href="/pmc/articles/PMC5830891/figure/fig1/" target="figure" class="fig-table-link figpopup" rid-figpopup="fig1" rid-ob="ob-fig1" co-legend-rid="lgnd_fig1">Figure 1A), which reacts reversibly with thiols in aqueous solutions (href="#bib5" rid="bib5" class=" bibr popnode">Cho and Choi, 2012). The present study showed that FreSHtracer is a powerful tool for real-time monitoring of GSH dynamics and heterogeneity in living SCs, and revealed that SCs cultured under conventional conditions exhibit the downregulation of GSH levels that results in deterioration of self-renewal and migration function. Therefore, our results prove that monitoring glutathione contents and dynamics in living SCs can be used as a marker to evaluate SC function and enhance its therapeutic potency.href="/pmc/articles/PMC5830891/figure/fig1/" target="figure" rid-figpopup="fig1" rid-ob="ob-fig1">class="inline_block ts_canvas" href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=5830891_gr1.jpg" target="tileshopwindow">target="object" href="/pmc/articles/PMC5830891/figure/fig1/?report=objectonly">Open in a separate windowclass="figpopup" href="/pmc/articles/PMC5830891/figure/fig1/" target="figure" rid-figpopup="fig1" rid-ob="ob-fig1">Figure 1FreSHtracer Is a Reversible and Ratiometric Probe for Glutathione(A) Changes in the absorption and fluorescence spectra of FreSHtracer when reacted with increasing concentrations of glutathione (GSH).(B) Fluorescence ratio (F510/F580; FR) of FreSHtracer plotted as a function of the GSH concentration.(C) Reversible reaction of FreSHtracer with GSH following treatment with diamide.(D and E) GSH-specific reaction of FreSHtracer. FreSHtracer equilibrated with GSH (5 mM; 15 min) was treated with H2O2 (0–2 mM) (D), and FreSHtracer equilibrated with oxidized GSH (GSSG, 5 mM) was treated with GSH reductase (5 U/mL) and 0.5 mM NADPH (E).(F and G) Fluorescence properties of FreSHtracer in dialyzed cell lysates. The FR change was monitored in dialyzed HeLa cell lysates (F) and then plotted against the protein concentration (G).(H) Effect of oxidants on the PSH-induced FR. Dialyzed cell lysates (25 mg/mL protein) were incubated with FreSHtracer (2 hr) and treated with diamide or H2O2 for 1 hr.(I and J) Fluorescence properties of FreSHtracer in PSH-GSH mixtures. FreSHtracer was added to dialyzed cell lysates (25 mg/mL protein) spiked with various GSH concentrations. The FR change was monitored for 20 min (I). The final FR value was plotted against the GSH concentration (J).(K) Effect of oxidants on the FR of the PSH-GSH mixture. PSH-GSH mixtures (25 mg/mL protein and 2 mM GSH) were incubated with FreSHtracer (2 hr) and treated with diamide or H2O2 for 1 hr.See also href="#mmc1" rid="mmc1" class=" supplementary-material">Figure S1.