Glutathione (GSH), the most abundant nonprotein thiol functioning as an antioxidant, plays critical roles in maintaining the core functions of mesenchymal stem cells (MSCs), which are used as a cellular immunotherapy for graft-versus-host disease (GVHD). However, the role of GSH dynamics in MSCs remains elusive. Genome-wide gene expression profiling and high-throughput live-cell imaging assays revealed that CREB1 enforced the GSH-recovering capacity (GRC) of MSCs through NRF2 by directly up-regulating NRF2 target genes responsible for GSH synthesis and redox cycling. MSCs with enhanced GSH levels and GRC mediated by CREB1-NRF2 have improved self-renewal, migratory, anti-inflammatory, and T cell suppression capacities. Administration of MSCs overexpressing CREB1-NRF2 target genes alleviated GVHD in a humanized mouse model, resulting in improved survival, decreased weight loss, and reduced histopathologic damages in GVHD target organs. Collectively, these findings demonstrate the molecular and functional importance of the CREB1-NRF2 pathway in maintaining MSC GSH dynamics, determining therapeutic outcomes for GVHD treatment.
Aberrant activation of embryogenesis-related molecular programs in urothelial bladder cancer (BC) is associated with stemness features related to oncogenic dedifferentiation and tumor metastasis. Recently, we reported that overexpression of transcription factor CP2-like protein-1 (TFCP2L1) and its phosphorylation at Thr177 by cyclin-dependent kinase-1 (CDK1) play key roles in regulating bladder carcinogenesis. However, the clinical relevance and therapeutic potential of this novel CDK1-TFCP2L1 molecular network remain elusive. Here, we demonstrated that inhibitor of DNA binding-2 (ID2) functions as a crucial mediator by acting as a direct repressive target of TFCP2L1 to modulate the stemness features and survival of BC cells. Low ID2 and high CDK1 expression were significantly associated with unfavorable clinical characteristics. TFCP2L1 downregulated ID2 by directly binding to its promoter region. Consistent with these findings, ectopic expression of ID2 or treatment with apigenin, a chemical activator of ID2, triggered apoptosis and impaired the proliferation, suppressed the stemness features, and reduced the invasive capacity of BC cells. Combination treatment with the specific CDK1 inhibitor RO-3306 and apigenin significantly suppressed tumor growth in an orthotopic BC xenograft animal model. This study demonstrates the biological role and clinical utility of ID2 as a direct target of the CDK1-TFCP2L1 pathway for modulating the stemness features of BC cells.
Glutathione (GSH), an abundant nonprotein thiol antioxidant, participates in several biological processes and determines the functionality of stem cells. A detailed understanding of the molecular network mediating GSH dynamics is still lacking. Here, we show that activating transcription factor-2 (ATF2), a cAMP-response element binding protein (CREB), plays a crucial role in maintaining the level and activity of GSH in human mesenchymal stem cells (MSCs) by crosstalking with nuclear factor erythroid-2 like-2 (NRF2), a well-known master regulator of cellular redox homeostasis. Priming with ascorbic acid 2-glucoside (AA2G), a stable vitamin C derivative, increased the expression and activity of ATF2 in MSCs derived from human embryonic stem cells and umbilical cord. Subsequently, activated ATF2 crosstalked with the CREB1-NRF2 pathway to preserve the GSH dynamics of MSCs through the induction of genes involved in GSH synthesis (GCLC and GCLM) and redox cycling (GSR and PRDX1). Accordingly, shRNA-mediated silencing of ATF2 significantly impaired the self-renewal, migratory, proangiogenic, and anti-inflammatory capacities of MSCs, and these defects were rescued by supplementation of the cells with GSH. In addition, silencing ATF2 attenuated the ability of MSCs to alleviate airway inflammatory responses in an ovalbumin-induced mouse model of allergic asthma. Consistently, activation of ATF2 by overexpression or the AA2G-based priming procedure enhanced the core functions of MSCs, improving the in vivo therapeutic efficacy of MSCs for treating asthma. Collectively, our findings suggest that ATF2 is a novel modulator of GSH dynamics that determines the core functionality and therapeutic potency of MSCs used to treat allergic asthma.
Background
The therapeutic effects of human embryonic stem cell-derived multipotent mesenchymal stem cells (M-MSCs) were evaluated for detrusor underactivity (DUA) in a rat model with atherosclerosis-induced chronic bladder ischemia (CBI) and associated mechanisms.
Methods
Sixteen-week-old male Sprague–Dawley rats were divided into five groups (n = 10). The DUA groups underwent 30 bilateral repetitions of endothelial injury to the iliac arteries to induce CBI, while the sham control group underwent a sham operation. All rats used in this study received a 1.25% cholesterol diet for 8 weeks. M-MSCs at a density of 2.5, 5.0, or 10.0 × 105 cells (250 K, 500 K, or 1000 K; K = a thousand) were injected directly into the bladder 7 weeks post-injury, while the sham and DUA group were treated only with vehicle (phosphate buffer solution). One week after M-MSC injection, awake cystometry was performed on the rats. Then, the bladders were harvested, studied in an organ bath, and prepared for histological and gene expression analyses.
Results
CBI by iliac artery injury reproduced voiding defects characteristic of DUA with decreased micturition pressure, increased micturition interval, and a larger residual volume. The pathological DUA properties were improved by M-MSC treatment in a dose-dependent manner, with the 1000 K group producing the best efficacy. Histological analysis revealed that M-MSC therapy reduced CBI-induced injuries including bladder fibrosis, muscular loss, and apoptosis. Transplanted M-MSCs mainly engrafted as vimentin and NG2 positive pericytes rather than myocytes, leading to increased angiogenesis in the CBI bladder. Transcriptomes of the CBI-injured bladders were characterized by the complement system, inflammatory, and ion transport-related pathways, which were restored by M-MSC therapy.
Conclusions
Single injection of M-MSCs directly into the bladder of a CBI-induced DUA rat model improved voiding profiles and repaired the bladder muscle atrophy in a dose-dependent manner.
Graphical abstract
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