Biology subjects

Karner, C. M.

Publications and source records attributed to Karner, C. M..

12 recordsLinked to original sources

bioRxiv2026

Glutathione Protects Hematopoietic Stem Cells from Proteotoxic Stress

Glutathione is a redox buffer, best known for preventing oxidative stress and ferroptosis, but rarely studied in stem cells. We studied glutathione function in hematopoietic stem cells (HSCs) by deleting the glutamate-cysteine ligase catalytic subunit (GCLC), which synthesizes glutathione, or glutathione peroxidase 4 (GPX4), which employs glutathione to prevent ferroptosis. GCLC deficiency eliminated glutathione from hematopoietic cells, severely impairing HSC function and hematopoiesis. In contrast, GPX4 deficiency did not affect HSC function and had little effect on hematopoiesis, even after vitamin E depletion, suggesting glutathione has ferroptosis-independent functions in HSCs. Consistent with this, GCLC deficiency increased reactive oxygen species levels in hematopoietic cells. But treatment with antioxidants or ferroptosis inhibitors gave little or no rescue of HSC function despite reducing ROS levels. Rather, GCLC-deficient, but not GPX4-deficient, HSCs exhibited unfolded protein accumulation, proteostatic stress, and cell death marked by elevated p53 levels and Annexin V staining. Deficiency for p53 prolonged the function of GCLC-deficient HSCs after transplantation. Glutathione is thus necessary in HSCs for proteostasis but may be dispensable for ferroptosis inhibition in vivo.

cell biology↗

bioRxiv2025

N-acetylcarnosine attenuates age-associated declines in multi-organ systems to improve survival

Histidine containing dipeptides (HCDs) such as N-acetylcarnosine are endogenous metabolites that are ergogenic and mitigate metabolic dysfunction. We previously demonstrated that short-term N-acetylcarnosine treatment is highly efficacious in protecting muscle atrophy induced by disuse. Here we demonstrate that a 6-months treatment of N-acetylcarnosine attenuates a broad spectrum of age-associated maladies and improved survival by [~]50% in female mice. A comprehensive survey of organ systems revealed that N-acetylcarnosine prevents decline in adiposity, diastolic function, vasodilation, muscle strength, and bone density. Together, N-acetylcarnosine substantially delays the onset of system-wide end-stage pathology to prolong lifespan. As an endogenously present metabolite, treatment with N-acetylcarnosine may be a safe and promising intervention to promote healthy aging in humans.

physiology↗

bioRxiv2023

Proteolytic activation of fatty acid synthase signals pan-stress resolution

Chronic stress and inflammation are not only outcomes of pathological states but rather major drivers of many human diseases 1-4. Ideally, a given stress program is downregulated to basal levels upon restoration of homeostasis. Chronic responsiveness despite stress mitigation suggests a failure to sense the resolution of the initiating stressor. Here we show that a proteolytic cleavage event of fatty acid synthase (FASN) activates a global cue for stress resolution. FASN is well-established as the multifunctional enzyme catalyzing de novo biosynthesis of saturated fatty acid 5, 6. Surprisingly, our results demonstrate FASN functioning as a signaling molecule promoting an anti-inflammatory profile apart from fatty acid synthesis. Redox-dependent proteolytic cleavage of FASN by caspase activates a truncated C-terminal enzymatic fragment (FASN-CTF) that is sufficient to down-regulate multiple aspects of stress-responsiveness including gene expression and metabolic programs. Only a fraction of FASN is cleaved allowing for continued fat synthesis. FASN-CTF can signal stress resolution across tissues in a cell non-autonomous manner. Consistent with these findings, FASN processing is also seen in well-fed but not fasted mouse liver. As down-regulation of stress responsiveness is critical to health, our findings provide a potential pathway to control the magnitude for diverse aspects of stress responses.

molecular biology↗

bioRxiv2022

SLC38A2 provides proline to fulfil unique synthetic demands arising during osteoblast differentiation and bone formation.

Cellular differentiation is associated with the acquisition of a unique protein signature which is essential to attain the ultimate cellular function and activity of the differentiated cell. This is predicted to result in unique biosynthetic demands that arise during differentiation. Using a bioinformatic approach, we discovered osteoblast differentiation is associated with increased demand for the amino acid proline. When compared to other differentiated cells, osteoblast-associated proteins including RUNX2, OSX, OCN and COL1A1 are significantly enriched in proline. Using a genetic and metabolomic approach, we demonstrate that the neutral amino acid transporter SLC38A2 acts cell autonomously to provide proline to facilitate the efficient synthesis of proline-rich osteoblast proteins. Genetic ablation of SLC38A2 in osteoblasts limits both osteoblast differentiation and bone formation in mice. Mechanistically, proline is primarily incorporated into nascent protein with little metabolism observed. Collectively, these data highlight a requirement for proline in fulfilling the unique biosynthetic requirements that arise during osteoblast differentiation and bone formation.

developmental biology↗

bioRxiv2021

Distinct roles of glutamine metabolism in benign and malignant cartilage tumors with IDH mutations

Enchondromas and chondrosarcomas are common cartilage neoplasms that are either benign or malignant respectively. The majority of these tumors harbor mutations in either IDH1 or IDH2. Glutamine metabolism has been implicated as a critical regulator of tumors with IDH mutations. Chondrocytes and chondrosarcomas with mutations in the IDH1 or IDH2 genes showed enhanced glutamine utilization in downstream metabolism. Using genetic and pharmacological approaches, we demonstrated that glutaminase-mediated glutamine metabolism played distinct roles in enchondromas and chondrosarcomas with IDH1 or IDH2 mutations. Deletion of glutaminase in chondrocytes with Idh1 mutation increased the number and size of enchondroma-like lesions. Pharmacological inhibition of glutaminase in chondrosarcoma xenografts reduced overall tumor burden. Glutamine affected cell differentiation and viability in these tumors differently through different downstream metabolites. During murine enchondroma-like lesion development, glutamine-derived -ketoglutarate promoted hypertrophic chondrocyte differentiation and regulated chondrocyte proliferation. In human chondrosarcoma, glutamine-derived non-essential amino acids played an important role in preventing cell apoptosis. This study reveals that glutamine metabolism can play distinct roles in benign and malignant cartilage tumors sharing the same genetic mutations. Inhibiting GLS may provide a therapeutic approach to suppress chondrosarcoma tumor growth.

cancer biology↗

bioRxiv2021

SLC1A5 provides glutamine and asparagine necessary for bone development in mice

Osteoblast differentiation is sequentially characterized by high rates of proliferation followed by increased protein and matrix synthesis, processes that require substantial amino acid acquisition and production. How osteoblasts obtain or maintain intracellular amino acid production is poorly understood. Here we identify Slc1a5 as a critical amino acid transporter during bone development. Using a genetic and metabolomic approach, we show Slc1a5 acts cell autonomously in osteoblasts to import glutamine and asparagine. Deleting Slc1a5 or reducing either glutamine or asparagine availability prevents protein synthesis and osteoblast differentiation. Mechanistically, glutamine and asparagine metabolism support amino acid biosynthesis. Thus, osteoblasts depend on Slc1a5 to provide glutamine and asparagine, which are subsequently used to produce non-essential amino acids and support osteoblast differentiation and bone development.

developmental biology↗

bioRxiv2021

Hypertrophic Chondrocytes Serve as a Reservoir for Unique Marrow Associated Skeletal Stem and Progenitor Cells, Osteoblasts, and Adipocytes During Skeletal Development

Hypertrophic chondrocytes give rise to osteoblasts during skeletal development; however, the process by which these non-mitotic cells make this transition is not well understood. Prior studies have also suggested that skeletal stem and progenitor cells (SSPCs) localize to the surrounding periosteum and serve as a major source of marrow associated SSPCs, osteoblasts, osteocytes, and adipocytes during skeletal development. To further understand the cell transition process by which hypertrophic chondrocytes contribute to osteoblasts or other marrow associated cells, we utilized inducible and constitutive hypertrophic chondrocyte lineage tracing and reporter mouse models (Col10a1CreERT2; R26-tdTomatof/+ and Col10a1Cre; R26-tdTomatof/+) in combination with a PDGFRa-H2B-GFP transgenic line, single cell RNA-sequencing, bulk RNA-sequencing, immunofluorescence staining, and cell transplantation assays. Our data demonstrate that hypertrophic chondrocytes undergo a process of dedifferentiation to generate marrow associated SSPCs that serve as a primary source of osteoblasts during skeletal development. These hypertrophic chondrocyte derived SSPCs commit to a CXCL12-abundant reticular (CAR) cell phenotype during skeletal development and demonstrate unique abilities to recruit vasculature and promote bone marrow establishment, while also contributing to the adipogenic lineage.

developmental biology↗

bioRxiv2021

A G protein-coupled receptor is required in cartilaginous and dense connective tissues to maintain spine alignment

Adolescent idiopathic scoliosis (AIS) is the most common spine disorder affecting children worldwide, yet little is known about the pathogenesis of this disorder. Here, we demonstrate that genetic regulation of structural components of the axial skeleton, the intervertebral discs and dense connective tissues (e.g., ligaments and tendons), are essential for maintenance of spinal alignment. We show that the G-coupled protein receptor Adgrg6, previously implicated in human AIS association studies, is required in these tissues to maintain typical spine morphology. We show that Adgrg6 regulates biomechanical properties of tendon and stimulates CREB signaling governing gene expression in cartilaginous tissues of the spine. Treatment with an cAMP agonist was able to mirror aspects of receptor function in culture defining core pathways for regulation of these axial connective tissues. As ADGRG6 is a key gene involved in human AIS, these findings open up novel therapeutic opportunities for human scoliosis. HighlightsO_LIKnockout mice lacking Adgrg6 function in the tendons and ligaments of the spine develop perinatal-onset thoracic scoliosis. C_LIO_LILoss of Adgrg6 function in cartilaginous tissues of the discs contribute to the incidence and severity of scoliosis. C_LIO_LIThe loss of Adgrg6 function in spine tissues provide a model of construct validity for human adolescent idiopathic scoliosis C_LIO_LIFine tuning of the biomechanical properties of dense connective tissues is essential for maintaining spine alignment. C_LI

developmental biology↗

bioRxiv2020

HES1 is a critical mediator of the SHH-GLI3 axis in regulating digit number

Sonic Hedgehog/GLI3 signaling is critical in regulating digit number, such that Gli3-deficiency results in polydactyly and Shh-deficiency leads to digit number reductions. Anterior-posterior SHH/GLI3 signaling gradients regulate cell cycle factors controlling mesenchymal cell proliferation, while simultaneously regulating Grem1 to coordinate BMP-induced chondrogenesis. SHH/GLI3 also coordinates the expression of additional genes, however their importance in digit formation remain unknown. Utilizing genetic and molecular approaches, we identified HES1 as a key transcriptional regulator downstream of SHH/GLI signaling capable of inducing preaxial polydactyly (PPD), required for Gli3-deficient PPD, and capable of overcoming digit number constraints of Shh-deficiency. Our data indicate that HES1, a direct SHH/GLI signaling target, induces mesenchymal cell proliferation via suppression of Cdkn1b, while inhibiting chondrogenic genes and the anterior autopod boundary regulator, Pax9. These findings fill gaps in knowledge regarding digit number and patterning, while creating a comprehensive framework for our molecular understanding of critical mediators of SHH/GLI3 signaling.

developmental biology↗

bioRxiv2020

Hypoxia Depletes Contaminating CD45+ Hematopoietic Cells from Bone Marrow Stromal Cell (BMSC) Cultures: Methods for BMSC Culture Purification

Culture expanded bone marrow stromal cells (BMSCs) are easily isolated, can be grown rapidly en masse, and contain both skeletal stem cells (SSCs) and multipotent mesenchymal progenitors (MMPs). Despite this functional heterogeneity, BMSC cultures continue to be utilized for many applications due to the lack of definitive and universally accepted markers to prospectively identify and purify SSCs. Isolation of BMSCs is widely based on their adherence to tissue culture plastic; however, this method poses unique challenges when working with murine models. In particular, hematopoietic cell contamination is a significant impediment during the isolation or murine BMSCs. Several strategies to reduce hematopoietic contamination have been developed with varying levels of success. In this study, we found that up to 60% of cells isolated in standard BMSC cultures expressed the hematopoietic marker CD45. Remarkably, when cultured at a physiological oxygen tension of 1% O2, there was a 10-fold reduction in hematopoietic cells associated with a concomitant increase in PDGFR+ expressing stromal cells. This finding was due, in part, to a differential response of the two populations to low oxygen tension, or hypoxia. Specifically, in standard tissue culture conditions of 21% O2, CD45+ hematopoietic cells showed increased proliferation coupled with no observable changes in cell death when compared to their counterparts grown at 1% O2. In contrast, PDGFR+ stromal cells responded to hypoxia by increasing proliferation and exhibiting a 10-fold decrease in cell death. In summary, we describe a simple and reliable method exploiting the divergent biological response of hematopoietic and stromal cells to hypoxia to significantly increase the PDGFR+ stromal cell population in murine BMSC cultures.

cell biology↗

bioRxiv2019

Mitochondrial PE potentiates respiratory enzymes to amplify skeletal muscle aerobic capacity

Exercise capacity is a strong predictor of all-cause mortality. Skeletal muscle mitochondrial respiratory capacity, its biggest contributor, adapts robustly to changes in energy demands induced by contractile activity. While transcriptional regulation of mitochondrial enzymes has been extensively studied, there is limited information on how mitochondrial membrane lipids are regulated. Herein, we show that exercise training or muscle disuse alters mitochondrial membrane phospholipids including phosphatidylethanolamine (PE). Addition of PE promoted, whereas removal of PE diminished, mitochondrial respiratory capacity. Surprisingly, skeletal muscle-specific inhibition of mitochondrial-autonomous synthesis of PE caused a respiratory failure due to metabolic insults in the diaphragm muscle. While mitochondrial PE deficiency coincided with increased oxidative stress, neutralization of the latter did not rescue lethality. These findings highlight the previously underappreciated role of mitochondrial membrane phospholipids in dynamically controlling skeletal muscle energetics and function.

physiology↗

bioRxiv2018

Molecular determinants of WNT9b responsiveness in nephron progenitor cells

Primed nephron progenitor cells (NPCs) appear in metanephric mesenchyme by Ell.5 and differentiate in response to the inductive WNT9b signal from the ureteric bud. However, the NPC WNT-receptor complex is unknown. We obtained M15 cells from E10.5 mesonephric mesenchyme and systematically analyzed components required for canonical WNT9b-responsiveness. When M15 cells were transfected with a ({beta}-catenin luciferase reporter plasmid, exposure to recombinant WNT9b resulted in minimal luciferase activity. We then analyzed mRNA-expression of WNT-pathway components and identified Fzdl-6 and Lrp6 transcripts but not RSPO1. When M15 cells were treated with recombinant RSPO1 the response to transfected WNT9b was augmented 4.8-fold. Co-transfection of M15 cells with Fzd5 (but no other Fzd family member) further increased the WNT9b signal to 16.8-fold and siRNA knockdown of Fzd5 reduced the signal by 52%. Knockdown of Lrp6 resulted in 60% WNT9b signal reduction. We confirmed Fzd5, Lrp6 and RSPO1 rrtRNA expression in CITED1(+) NPCs from E15.5 embryonic mouse kidney. Thus, while many WNT signaling-pathway components are present by E10.5, optimum responsiveness of Ell.5 cap mesenchyme requires that NPCs acquire RSPO1, FZD5 and LRP6.\n\nSummary StatementResponsiveness to the inductive WMT9b signal from ureteric bud is crucial for nephrogenesis. Here we analyze the molecules needed to prime nephron progenitor cells in embryonic mouse kidney.

developmental biology↗