The FASEB Journal最新文献

Nicotinamide adenine dinucleotide (NAD⁺) is a vital molecule, serving as a redox cofactor and the limiting substrate for numerous enzymes. NAD⁺ decline is a key feature of aging, while supplementation with NAD⁺ precursors can efficiently counteract aging traits and prevent age-associated conditions in preclinical models. However, clinical translation remains challenging, likely due to the limited NAD⁺ boosting capacity of classical precursors, such as nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR). This has brought attention to their reduced forms, reduced NMN (NMNH) and reduced NR (NRH), which are more potent NAD⁺ boosters but remain poorly characterized. Here, we performed a comprehensive comparative analysis using RNA sequencing, proteomics, and metabolomics on cultured murine hepatocytes treated with NMN, NMNH, NR, or NRH. Global metabolic profiling revealed that NRH and NMNH induced substantially broader metabolic alterations than NR and NMN, with NRH uniquely suppressing metabolites involved in energy metabolism. The pronounced metabolic effects were reflected at a transcriptional level, with reduced precursors triggering a significantly higher number of differentially expressed genes than oxidized ones. Shared differentially expressed genes between NMNH and NRH revealed upregulation of stress-related glutathione-S-transferases (Gsts) which furthermore were reflected in our proteomic profiling. However, the upregulation of Gsts did not cause a depletion of glutathione or oxiglutathione, suggesting a pseudo-stress response to reduced NAD⁺ precursors. Together, our data demonstrate that reduced NAD⁺ precursors are unique and distinct from the market-available NAD⁺ precursors NR and NMN, not only as more potent NAD⁺ boosters, but also as compounds influencing a broader range of cellular processes.

In asthma, augmented airway wall smooth muscle (ASM) bulk is a major remodeling feature, promoted by increased transforming growth factor (TGF)-β1 and connective tissue growth factor (CTGF). Runt-related transcription factor-2 (RUNX2) represses TGF-β1-induced CTGF through interactions with SMAD3. This study aimed to investigate the expression and role of RUNX2 in asthmatic and nonasthmatic ASM cells. mRNA and protein were detected by microarray, PCR, and western blot in nonasthmatic and asthmatic ASM cells. Immunohistochemistry identified RUNX2 in lung tissues from asthmatic patients and nonasthmatic subjects. Different RUNX2 isoforms were transfected into immortalized-asthmatic ASM cells, and markers of inflammation and airway remodeling were measured. RUNX2 alternatively spliced forms were examined in bronchial biopsies from asthmatic and healthy subjects. The abundance of RUNX2 was decreased in isolated ASM cells from asthmatic compared with nonasthmatic subjects. The ASM layer around airways in lung tissue sections from asthmatic and nonasthmatic patients had a heterogeneous pattern of RUNX2 protein detection. TGF-β1 stimulation increased RUNX2/RUNX2 variant 1 mRNA in nonasthmatic but not asthmatic ASM cells, facilitating SMAD3 activation and nuclear translocation in asthmatic ASM cells. RUNX2 isoform overexpression in immortalized asthmatic ASM cells failed to alter markers of inflammation (IL-6) but significantly reduced markers of remodeling (CTGF), ASM cell hypertrophy (GSK-3β and desmin), and proliferation (pSer⁷⁹⁵ Rb and α-tubulin). In bronchial biopsies, RUNX2 mRNA splicing was higher in asthmatic patients compared with healthy subjects. These data suggest RUNX2 plays a role in the homeostasis of healthy airways. Restoring RUNX2 may provide a new therapeutic approach for asthma.

General anesthetics can adversely affect the heart, negatively impacting chronotropy, electrical conduction, and myocardial contractility. The intravenous sedative-hypnotic, propofol, for example, impairs ventricular contraction at clinically relevant doses and can cause dysrhythmias and atrioventricular block with acute administration. In addition, high cumulative propofol doses can induce bradyarrhythmias, cardiac conduction abnormalities, and myocardial failure. As with propofol, the recently identified intravenous anesthetic agent, ubiquinone-5 (Ub5), causes bradycardia and complete heart block at supratherapeutic doses. However, the cardiac effects of clinically relevant Ub5 doses are unknown. Thus, we aimed to determine how therapeutic doses of Ub5 impact cardiac rhythm, hypothesizing that Ub5 would interfere with dromotropy. We tested our hypothesis in vivo in the young adult mouse and ex vivo in the isolated-perfused murine heart. We then determined mechanistic contributors of Ub5-induced cardiotoxicity in isolated cardiomyocyte mitochondria. We found that Ub5 caused type 1 s-degree heart block and compromised the mitochondrial membrane potential in isolated cardiomyocyte mitochondria by inhibiting electron transport and inducing excessive proton leak. Pharmacological inhibition of the aspartate–glutamate carrier, Aralar, rescued Ub5-mediated disturbances in cardiac rhythm in the isolated-perfused heart. The findings suggest that Ub5 can impact cardiac conduction in a targetable manner, carrying importance for future drug development efforts.

Triple-negative breast cancer (TNBC) remains an aggressive malignancy with limited therapeutic options and poor prognosis, underscoring the critical need for novel therapeutic targets. This investigation elucidates the functional role of the potassium channel tetramerization domain 15 (KCTD15) in TNBC progression, providing mechanistic insights into its potential as a therapeutic target for this challenging disease. KCTD15 exhibited high expression in TNBC tissues, correlating with advanced grade and unfavorable prognosis. Functionally, KCTD15 knockdown in TNBC cell lines (BT-549/MDA-MB-231) markedly suppressed cellular proliferation, migration, and cancer stem cell properties, while concomitantly enhancing apoptosis. Mechanistically, KCTD15 directly interacted with KLF4, facilitating its nuclear translocation and subsequent activation of the β-catenin signaling cascade. Notably, KLF4 knockdown abrogated KCTD15-mediated stemness maintenance and β-catenin pathway activation. In vivo, KCTD15 silencing reduced xenograft tumor growth and downregulated Ki67, KLF4, and β-catenin protein expression in tumor tissues, confirming its oncogenic role through the KLF4/β-catenin axis. Our findings establish KCTD15 as a pivotal regulator of TNBC stemness through modulation of the KLF4/β-catenin signaling axis. These results provide a robust preclinical rationale for developing therapeutic strategies targeting this molecular axis in TNBC management.

Rett syndrome (RTT) is a rare neurodevelopmental disorder caused by loss-of-function mutations in the gene encoding methyl-CpG-binding protein 2 (MECP2) that is located on the X chromosome. Affected individuals also exhibit a variety of non-neurological symptoms such as kyphoscoliosis and osteoporosis. Thus, MECP2 may play a functional role in bone remodeling and osteoblast differentiation. This study aimed to clarify the molecular mechanisms underlying the deregulation of bone remodeling in RTT. Human deciduous tooth-derived mesenchymal stem cells that exhibit osteoblast plasticity were used as a cellular model of RTT. Using a small interfering RNA-mediated MECP2 (MECP2-siR) knockdown system, we quantitatively analyzed the RUNX2-dependent and canonical Wnt signaling pathways during osteoblast differentiation. Expression of active β-catenin, RUNX2, and their downstream targets (osteocalcin and alkaline phosphatase) and mineralization were decreased in MECP2-siR-treated osteoblasts compared to that in control osteoblasts. In contrast, the MECP2-siR-treated osteoblasts exhibited an increase in the endogenous Wnt antagonist DKK1. Notably, MECP2/DKK1 double-knockdown osteoblasts possessed greater β-catenin and RUNX2 levels than MECP2 single-knockdown osteoblasts. Furthermore, microRNA126-3p was upregulated in MECP2-siR-treated osteoblasts, and an antagomir of microRNA126-3p prevented DKK1 upregulation, thereby improving the levels of active β-catenin and other osteoblastic phenotypes. These results suggest that MECP2 insufficiency enhances DKK1 expression via the upregulation of microRNA126-3p, suppressing the canonical Wnt signaling and subsequent RUNX2-dependent osteoblast differentiation. The present study provides insights into the molecular mechanisms involved in impaired osteoblast differentiation that contribute to the development of osteoporosis in RTT.

Nicotinamide adenine dinucleotide (NAD⁺) is a crucial molecule involved in numerous interconnected metabolic processes. Due to its implication in multiple viral infection responses, maintaining NAD⁺ homeostasis has become a promising target for host-directed therapies. Japanese encephalitis virus (JEV) causes severe, fatal encephalitis with irreversible brain damage and long-lasting neurological deficits in survivors. However, the potential interaction between JEV infection and NAD⁺ metabolism remains largely unclear. In this study, we found that JEV infection dysregulates NAD⁺ metabolism and the expression of its pathway enzyme genes in Type I interferon (IFN-α/β) receptor-deficient (A129) mice and human glioblastoma (T98G) cells. Specifically, JEV infection altered the expression of de novo/kynurenine pathway (IDO, KATII, KMO) and salvage pathway (NAMPT, NMNATs) NAD⁺ biosynthetic enzymes, as well as NAD⁺-consuming enzymes (PARPs, SIRTs), culminating in a substantial decrease in NAD⁺ levels. Furthermore, NAD⁺ depletion and JEV production increased when salvage biosynthesis was restrained through NAMPT knockdown, but these effects were reversed by supplementing nicotinamide riboside (NR) in NAMPT knockdown T98G cells. Importantly, restoring NAD⁺ levels with NR supplementation as an anti-JE strategy in A129 mice reduced JEV production and improved infection outcomes. In conclusion, this study demonstrates that JEV infection disrupts NAD⁺ metabolism, and restoring NAD⁺ levels inhibits JE progression. Therefore, maintaining NAD⁺ homeostasis and regulating its metabolic pathway could be a promising therapeutic approach for JE.