Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in murine HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of ATP levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells.
© 2023, Watanuki, Kobayashi et al.

Pseudouridylation plays a regulatory role in various physiological and pathological processes. A prime example is the mitochondrial myopathy, lactic acidosis, and sideroblastic anemia syndrome (MLASA), characterized by defective pseudouridylation resulting from genetic mutations in pseudouridine synthase 1 (PUS1). However, the roles and mechanisms of pseudouridylation in normal erythropoiesis and MLASA-related anemia remain elusive. We established a mouse model carrying a point mutation (R110W) in the enzymatic domain of PUS1, mimicking the common mutation in human MLASA. Pus1-mutant mice exhibited anemia at 4 weeks old. Impaired mitochondrial oxidative phosphorylation was also observed in mutant erythroblasts. Mechanistically, mutant erythroblasts showed defective pseudouridylation of targeted tRNAs, altered tRNA profiles, decreased translation efficiency of ribosomal protein genes, and reduced globin synthesis, culminating in ineffective erythropoiesis. Our study thus provided direct evidence that pseudouridylation participates in erythropoiesis in vivo. We demonstrated the critical role of pseudouridylation in regulating tRNA homeostasis, cytoplasmic translation, and erythropoiesis.
© 2024 The Authors.

Insufficient bone fracture repair represents a major clinical and societal burden and novel strategies are needed to address it. Our data reveal that the transforming growth factor-β superfamily member Activin A became very abundant during mouse and human bone fracture healing but was minimally detectable in intact bones. Single-cell RNA-sequencing revealed that the Activin A-encoding gene Inhba was highly expressed in a unique, highly proliferative progenitor cell (PPC) population with a myofibroblast character that quickly emerged after fracture and represented the center of a developmental trajectory bifurcation producing cartilage and bone cells within callus. Systemic administration of neutralizing Activin A antibody inhibited bone healing. In contrast, a single recombinant Activin A implantation at fracture site in young and aged mice boosted: PPC numbers; phosphorylated SMAD2 signaling levels; and bone repair and mechanical properties in endochondral and intramembranous healing models. Activin A directly stimulated myofibroblastic differentiation, chondrogenesis and osteogenesis in periosteal mesenchymal progenitor culture. Our data identify a distinct population of Activin A-expressing PPCs central to fracture healing and establish Activin A as a potential new therapeutic tool.
© 2023, Yao et al.

Cells have evolved their communication methods to sense their microenvironments and send biological signals. In addition to communication using ligands and receptors, cells use diverse channels including gap junctions to communicate with their immediate neighbors. Current approaches, however, cannot effectively capture the influence of various microenvironments. Here, we propose a novel approach to investigate cell neighbor-dependent gene expression (CellNeighborEX) in spatial transcriptomics (ST) data. To categorize cells based on their microenvironment, CellNeighborEX uses direct cell location or the mixture of transcriptome from multiple cells depending on ST technologies. For each cell type, CellNeighborEX identifies diverse gene sets associated with partnering cell types, providing further insight. We found that cells express different genes depending on their neighboring cell types in various tissues including mouse embryos, brain, and liver cancer. Those genes are associated with critical biological processes such as development or metastases. We further validated that gene expression is induced by neighboring partners via spatial visualization. The neighbor-dependent gene expression suggests new potential genes involved in cell-cell interactions beyond what ligand-receptor co-expression can discover.
© 2023 The Authors. Published under the terms of the CC BY 4.0 license.

Hypodermis is the predominant site of Staphylococcus aureus infections that cause cellulitis. Given the importance of macrophages in tissue remodeling, we examined the hypodermal macrophages (HDMs) and their impact on host susceptibility to infection. Bulk and single-cell transcriptomics uncovered HDM subsets with CCR2-dichotomy. HDM homeostasis required the fibroblast-derived growth factor CSF1, ablation of which abrogated HDMs from the hypodermal adventitia. Loss of CCR2- HDMs resulted in accumulation of the extracellular matrix component, hyaluronic acid (HA). HDM-mediated HA clearance required sensing by the HA receptor, LYVE-1. Cell-autonomous IGF1 was required for accessibility of AP-1 transcription factor motifs that controlled LYVE-1 expression. Remarkably, loss of HDMs or IGF1 limited Staphylococcus aureus expansion via HA and conferred protection against cellulitis. Our findings reveal a function for macrophages in the regulation of HA with an impact on infection outcomes, which may be harnessed to limit the establishment of infection in the hypodermal niche.
Published by Elsevier Inc.