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Organic phosphate but not inorganic phosphate regulates expression through MAPK and TGF-ꞵ signaling.IScience Jun 2024One of the main regulators of phosphate homeostasis is fibroblast growth factor 23 (FGF23), secreted by osteocytes. The effects of organic versus inorganic dietary...
One of the main regulators of phosphate homeostasis is fibroblast growth factor 23 (FGF23), secreted by osteocytes. The effects of organic versus inorganic dietary phosphate on this homeostasis are unclear. This study used MC3T3-E1 FGF23-producing cells to examine the transcriptomic responses to these phosphates. Most importantly, the expression and secretion of FGF23 were only increased in response to organic phosphate. Gene ontology terms related to a response to environmental change were only enriched in cells treated with organic phosphate while cells treated with inorganic phosphate were enriched for terms associated with regulation of cellular phosphate metabolism. Inhibition of MAPK signaling diminished the response of to organic phosphate, suggesting it activates FGF23. TGF-β signaling inhibition increased expression after the addition of organic phosphate, while the negative TGF-β regulator decreased this response. In summary, the observed differential response of FGF23-producing to phosphate types may have consequences for phosphate homeostasis.
PubMed: 38883842
DOI: 10.1016/j.isci.2024.109625 -
Journal of Translational Medicine Jun 2024
Correction: Real‑world data suggest effectiveness of the allogeneic mesenchymal stromal cells preparation MSC‑FFM in ruxolitinib‑refractory acute graft‑versus‑host disease.
PubMed: 38872104
DOI: 10.1186/s12967-024-05348-8 -
Nature Communications Jun 2024Generating 3D bone cell networks in vitro that mimic the dynamic process during early bone formation remains challenging. Here, we report a synthetic biodegradable...
Generating 3D bone cell networks in vitro that mimic the dynamic process during early bone formation remains challenging. Here, we report a synthetic biodegradable microporous hydrogel for efficient formation of 3D networks from human primary cells, analysis of cell-secreted extracellular matrix (ECM) and microfluidic integration. Using polymerization-induced phase separation, we demonstrate dynamic in situ formation of microporosity (5-20 µm) within matrix metalloproteinase-degradable polyethylene glycol hydrogels in the presence of living cells. Pore formation is triggered by thiol-Michael-addition crosslinking of a viscous precursor solution supplemented with hyaluronic acid and dextran. The resulting microporous architecture can be fine-tuned by adjusting the concentration and molecular weight of dextran. After encapsulation in microporous hydrogels, human mesenchymal stromal cells and osteoblasts spread rapidly and form 3D networks within 24 hours. We demonstrate that matrix degradability controls cell-matrix remodeling, osteogenic differentiation, and deposition of ECM proteins such as collagen. Finally, we report microfluidic integration and proof-of-concept osteogenic differentiation of 3D cell networks under perfusion on chip. Altogether, this work introduces a synthetic microporous hydrogel to efficiently differentiate 3D human bone cell networks, facilitating future in vitro studies on early bone development.
Topics: Humans; Hydrogels; Mesenchymal Stem Cells; Osteogenesis; Cell Differentiation; Osteoblasts; Extracellular Matrix; Porosity; Cell Culture Techniques, Three Dimensional; Polyethylene Glycols; Tissue Engineering; Hyaluronic Acid; Cells, Cultured; Tissue Scaffolds; Dextrans
PubMed: 38871693
DOI: 10.1038/s41467-024-49280-3 -
Biophysical Journal Jun 2024Exchange of material across two membranes, as in the case of synaptic neurotransmitter release from a vesicle, involves the formation and poration of a hemifusion...
Exchange of material across two membranes, as in the case of synaptic neurotransmitter release from a vesicle, involves the formation and poration of a hemifusion diaphragm (HD). The nontrivial geometry of the HD leads to environment-dependent control, regarding the stability and dynamics of the pores required for this kind of exocytosis. This work combines particle simulations, field-based calculations, and phenomenological modeling to explore the factors influencing the stability, dynamics, and possible control mechanisms of pores in HDs. We find that pores preferentially form at the HD rim, and that their stability is sensitive to a number of factors, including the three line tensions, membrane tension, HD size, and the ability of lipids to "flip-flop" across leaflets. Along with a detailed analysis of these factors, we discuss ways that vesicles or cells may use them to open and close pores and thereby quickly and efficiently transport material.
PubMed: 38867448
DOI: 10.1016/j.bpj.2024.06.009 -
Cell Death Discovery Jun 2024A key feature of cancer is the disruption of cell cycle regulation, which is characterized by the selective and abnormal activation of cyclin-dependent kinases (CDKs)....
A key feature of cancer is the disruption of cell cycle regulation, which is characterized by the selective and abnormal activation of cyclin-dependent kinases (CDKs). Consequently, targeting CDKs via meriolins represents an attractive therapeutic approach for cancer therapy. Meriolins represent a semisynthetic compound class derived from meridianins and variolins with a known CDK inhibitory potential. Here, we analyzed the two novel derivatives meriolin 16 and meriolin 36 in comparison to other potent CDK inhibitors and could show that they displayed a high cytotoxic potential in different lymphoma and leukemia cell lines as well as in primary patient-derived lymphoma and leukemia cells. In a kinome screen, we showed that meriolin 16 and 36 prevalently inhibited most of the CDKs (such as CDK1, 2, 3, 5, 7, 8, 9, 12, 13, 16, 17, 18, 19, 20). In drug-to-target modeling studies, we predicted a common binding mode of meriolin 16 and 36 to the ATP-pocket of CDK2 and an additional flipped binding for meriolin 36. We could show that cell cycle progression and proliferation were blocked by abolishing phosphorylation of retinoblastoma protein (a major target of CDK2) at Ser612 and Thr82. Moreover, meriolin 16 prevented the CDK9-mediated phosphorylation of RNA polymerase II at Ser2 which is crucial for transcription initiation. This renders both meriolin derivatives as valuable anticancer drugs as they target three different Achilles' heels of the tumor: (1) inhibition of cell cycle progression and proliferation, (2) prevention of transcription, and (3) induction of cell death.
PubMed: 38862521
DOI: 10.1038/s41420-024-02056-6 -
BMC Biology Jun 2024Inherited retinal dystrophies (IRDs) are a group of debilitating visual disorders characterized by the progressive degeneration of photoreceptors, which ultimately lead...
BACKGROUND
Inherited retinal dystrophies (IRDs) are a group of debilitating visual disorders characterized by the progressive degeneration of photoreceptors, which ultimately lead to blindness. Among the causes of this condition, mutations in the PCYT1A gene, which encodes the rate-limiting enzyme responsible for phosphatidylcholine (PC) de novo synthesis via the Kennedy pathway, have been identified. However, the precise mechanisms underlying the association between PCYT1A mutations and IRDs remain unclear. To address this knowledge gap, we focused on elucidating the functions of PCYT1A in the retina.
RESULTS
We found that PCYT1A is highly expressed in Müller glial (MG) cells in the inner nuclear layer (INL) of the retina. Subsequently, we generated a retina-specific knockout mouse model in which the Pcyt1a gene was targeted (Pcyt1a-RKO or RKO mice) to investigate the molecular mechanisms underlying IRDs caused by PCYT1A mutations. Our findings revealed that the deletion of Pcyt1a resulted in retinal degenerative phenotypes, including reduced scotopic electroretinogram (ERG) responses and progressive degeneration of photoreceptor cells, accompanied by loss of cells in the INL. Furthermore, through proteomic and bioinformatic analyses, we identified dysregulated retinal fatty acid metabolism and activation of the ferroptosis signalling pathway in RKO mice. Importantly, we found that PCYT1A deficiency did not lead to an overall reduction in PC synthesis within the retina. Instead, this deficiency appeared to disrupt free fatty acid metabolism and ultimately trigger ferroptosis.
CONCLUSIONS
This study reveals a novel mechanism by which mutations in PCYT1A contribute to the development of IRDs, shedding light on the interplay between fatty acid metabolism and retinal degenerative diseases, and provides new insights into the treatment of IRDs.
Topics: Animals; Mice; Choline-Phosphate Cytidylyltransferase; Fatty Acids; Ferroptosis; Mice, Knockout; Retina; Retinal Dystrophies
PubMed: 38858683
DOI: 10.1186/s12915-024-01932-y -
BioRxiv : the Preprint Server For... May 2024The G2019S mutation in the leucine-rich repeat kinase 2 (LRRK2) gene is a major risk factor for the development of Parkinson's disease (PD). LRRK2, although ubiquitously...
The G2019S mutation in the leucine-rich repeat kinase 2 (LRRK2) gene is a major risk factor for the development of Parkinson's disease (PD). LRRK2, although ubiquitously expressed, is highly abundant in cells of the innate immune system. Given the importance of central and peripheral immune cells in the development of PD, we sought to investigate the consequences of the G2019S mutation on microglial and monocyte transcriptome and function. We have generated large-scale transcriptomic profiles of isogenic human induced microglial cells (iMGLs) and patient derived monocytes carrying the G2019S mutation under baseline culture conditions and following exposure to the proinflammatory factors IFNγ and LPS. We demonstrate that the G2019S mutation exerts a profound impact on the transcriptomic profile of these myeloid cells, and describe corresponding functional differences in iMGLs. The G2019S mutation led to an upregulation in lipid metabolism and phagolysosomal pathway genes in untreated and LPS/IFNγ stimulated iMGLs, which was accompanied by an increased phagocytic capacity of myelin debris. We also identified dysregulation of cell cycle genes, with a downregulation of the E2F4 regulon. Transcriptomic characterization of human-derived monocytes carrying the G2019S mutation confirmed alteration in lipid metabolism associated genes. Altogether, these findings reveal the influence of G2019S on the dysregulation of the myeloid cell transcriptome under proinflammatory conditions.
PubMed: 38854101
DOI: 10.1101/2024.05.27.594821 -
Journal of Hematology & Oncology Jun 2024Neutrophils play a crucial role in inflammation and in the increased thrombotic risk in myeloproliferative neoplasms (MPNs). We have investigated how neutrophil-specific...
BACKGROUND
Neutrophils play a crucial role in inflammation and in the increased thrombotic risk in myeloproliferative neoplasms (MPNs). We have investigated how neutrophil-specific expression of JAK2-V617F or CALRdel re-programs the functions of neutrophils.
METHODS
Ly6G-Cre JAK2-V617F and Ly6G-Cre CALRdel mice were generated. MPN parameters as blood counts, splenomegaly and bone marrow histology were compared to wild-type mice. Megakaryocyte differentiation was investigated using lineage-negative bone marrow cells upon in vitro incubation with TPO/IL-1β. Cytokine concentrations in serum of mice were determined by Mouse Cytokine Array. IL-1α expression in various hematopoietic cell populations was determined by intracellular FACS analysis. RNA-seq to analyse gene expression of inflammatory cytokines was performed in isolated neutrophils from JAK2-V617F and CALR-mutated mice and patients. Bioenergetics of neutrophils were recorded on a Seahorse extracellular flux analyzer. Cell motility of neutrophils was monitored in vitro (time lapse microscopy), and in vivo (two-photon microscopy) upon creating an inflammatory environment. Cell adhesion to integrins, E-selectin and P-selection was investigated in-vitro. Statistical analysis was carried out using GraphPad Prism. Data are shown as mean ± SEM. Unpaired, two-tailed t-tests were applied.
RESULTS
Strikingly, neutrophil-specific expression of JAK2-V617F, but not CALRdel, was sufficient to induce pro-inflammatory cytokines including IL-1 in serum of mice. RNA-seq analysis in neutrophils from JAK2-V617F mice and patients revealed a distinct inflammatory chemokine signature which was not expressed in CALR-mutant neutrophils. In addition, IL-1 response genes were significantly enriched in neutrophils of JAK2-V617F patients as compared to CALR-mutant patients. Thus, JAK2-V617F positive neutrophils, but not CALR-mutant neutrophils, are pathogenic drivers of inflammation in MPN. In line with this, expression of JAK2-V617F or CALRdel elicited a significant difference in the metabolic phenotype of neutrophils, suggesting a stronger inflammatory activity of JAK2-V617F cells. Furthermore, JAK2-V617F, but not CALRdel, induced a VLA4 integrin-mediated adhesive phenotype in neutrophils. This resulted in reduced neutrophil migration in vitro and in an inflamed vessel. This mechanism may contribute to the increased thrombotic risk of JAK2-V617F patients compared to CALR-mutant individuals.
CONCLUSIONS
Taken together, our findings highlight genotype-specific differences in MPN-neutrophils that have implications for the differential pathophysiology of JAK2-V617F versus CALR-mutant disease.
Topics: Animals; Neutrophils; Janus Kinase 2; Mice; Myeloproliferative Disorders; Humans; Inflammation; Calreticulin; Mice, Transgenic; Mice, Inbred C57BL; Cytokines
PubMed: 38853260
DOI: 10.1186/s13045-024-01562-5 -
Frontiers in Cellular Neuroscience 2024Mitochondrial homeostasis includes balancing organelle biogenesis with recycling (mitophagy). The ketogenic diet protects retinal ganglion cells (RGCs) from...
Mitochondrial homeostasis includes balancing organelle biogenesis with recycling (mitophagy). The ketogenic diet protects retinal ganglion cells (RGCs) from glaucoma-associated neurodegeneration, with a concomitant increase in mitochondrial biogenesis. This study aimed to determine if the ketogenic diet also promoted mitophagy. MitoQC mice that carry a pH-sensitive mCherry-GFP tag on the outer mitochondrial membrane were placed on a ketogenic diet or standard rodent chow for 5 weeks; ocular hypertension (OHT) was induced via magnetic microbead injection in a subset of control or ketogenic diet animals 1 week after the diet began. As a measure of mitophagy, mitolysosomes were quantified in sectioned retina immunolabeled with RBPMS for RGCs or vimentin for Müller glia. Mitolysosomes were significantly increased as a result of OHT and the ketogenic diet (KD) in RGCs. Interestingly, the ketogenic diet increased mitolysosome number significantly higher than OHT alone. In contrast, OHT and the ketogenic diet both increased mitolysosome number in Müller glia to a similar degree. To understand if hypoxia could be a stimulus for mitophagy, we quantified mitolysosomes after acute OHT, finding significantly greater mitolysosome number in cells positive for pimonidazole, an adduct formed in cells exposed to hypoxia. Retinal protein analysis for BNIP3 and NIX showed no differences across groups, suggesting that these receptors were equivocal for mitophagy in this model of OHT. Our data indicate that OHT and hypoxia stimulate mitophagy and that the ketogenic diet is an additive for mitophagy in RGCs. The different response across RGCs and Müller glia to the ketogenic diet may reflect the different metabolic needs of these cell types.
PubMed: 38841201
DOI: 10.3389/fncel.2024.1409717 -
BioRxiv : the Preprint Server For... May 2024Genetic interactions are adaptive within a species. Hybridization can disrupt such species-specific genetic interactions and creates novel interactions that alter the...
UNLABELLED
Genetic interactions are adaptive within a species. Hybridization can disrupt such species-specific genetic interactions and creates novel interactions that alter the hybrid progeny overall fitness. Hybrid incompatibility, which refers to degenerative genetic interactions that decrease the overall hybrid survival, is one of the results from combining two diverged genomes in hybrids. The discovery of spontaneous lethal tumorigenesis and underlying genetic interactions in select hybrids between diverged species showed that lethal pathological process can result from degenerative genetic interactions. Such genetic interactions leading to lethal phenotype are thought to shield gene flow between diverged species. However, hybrids between certain species do not develop such tumors. Here we report the identification of a locus residing in the genome of one species that represses an oncogene from a different species. Our finding provides insights into normal and pathological pigment cell development, regulation and molecular mechanism in hybrid incompatibility.
SIGNIFICANCE
The Dobzhansky-Muller model states epistatic interactions occurred between genes in diverged species underlies hybrid incompatibility. There are a few vertebrate interspecies hybrid cases that support the Dobzhansky-Muller model. This study reports a fish hybrid system where incompatible genetic interactions are involved in neuronal regulation of pigment cell biology, and also identified a novel point of regulation for pigment cells.
PubMed: 38826429
DOI: 10.1101/2024.05.20.595041