Cancer stem cells (CSCs) represent a subset of cells within tumours that exhibit self-renewal properties and the capacity to seed tumours. CSCs are typically refractory to conventional treatments and ...have been associated to metastasis and relapse. Salinomycin operates as a selective agent against CSCs through mechanisms that remain elusive. Here, we provide evidence that a synthetic derivative of salinomycin, which we named ironomycin (AM5), exhibits a more potent and selective activity against breast CSCs in vitro and in vivo, by accumulating and sequestering iron in lysosomes. In response to the ensuing cytoplasmic depletion of iron, cells triggered the degradation of ferritin in lysosomes, leading to further iron loading in this organelle. Iron-mediated production of reactive oxygen species promoted lysosomal membrane permeabilization, activating a cell death pathway consistent with ferroptosis. These findings reveal the prevalence of iron homeostasis in breast CSCs, pointing towards iron and iron-mediated processes as potential targets against these cells.
Organs and cells must adapt to shear stress induced by biological fluids, but how fluid flow contributes to the execution of specific cell programs is poorly understood. Here we show that shear ...stress favours mitochondrial biogenesis and metabolic reprogramming to ensure energy production and cellular adaptation in kidney epithelial cells. Shear stress stimulates lipophagy, contributing to the production of fatty acids that provide mitochondrial substrates to generate ATP through β-oxidation. This flow-induced process is dependent on the primary cilia located on the apical side of epithelial cells. The interplay between fluid flow and lipid metabolism was confirmed in vivo using a unilateral ureteral obstruction mouse model. Finally, primary cilium-dependent lipophagy and mitochondrial biogenesis are required to support energy-consuming cellular processes such as glucose reabsorption, gluconeogenesis and cytoskeletal remodelling. Our findings demonstrate how primary cilia and autophagy are involved in the translation of mechanical forces into metabolic adaptation.
PiT1 is a Na+-phosphate (Pi) cotransporter located at the plasma membrane that enables Pi entry into the cell. Its broad tissue expression pattern has led to the idea that together with the closely ...related family member PiT2, PiT1 is the ubiquitous supplier of Pi to the cell. Moreover, the role of Pi in phosphorylation reactions, ATP production, DNA structure, and synthesis has led to the view that Pi availability could be an important determinant of cell growth. However, these issues have not been clearly addressed to date, and the role of either Pi or PiT proteins in cell proliferation is unknown. Using RNA interference in HeLa and HepG2 cells, we show that transient or stable PiT1 depletion markedly reduces cell proliferation, delays cell cycle, and impairs mitosis and cytokinesis. In vivo, PiT1 depletion greatly reduced tumor growth when engineered HeLa cells were injected into nude mice. We provide evidence that this effect on cell proliferation is specific to PiT1 and not shared by PiT2 and is not the consequence of impaired membrane Na+-Pi transport. Moreover, we show that modulation of cell proliferation by PiT1 is independent from its transport function because the proliferation of PiT1-depleted cells can be rescued by non-transporting PiT1 mutants. PiT1 depletion leads to the phosphorylation of p38 mitogen-activated protein (MAP) kinase, whereas other MAP kinases and downstream targets of mammalian target of rapamycin (mTOR) remain unaffected. This study is the first to describe the effects of a Pi transporter in cell proliferation, tumor growth, and cell signaling.
The naked mole-rat (
) is characterized by a more than tenfold higher life expectancy compared to another rodent species of the same size, namely, the laboratory mouse (
). We used mass spectrometric ...metabolomics to analyze circulating plasma metabolites in both species at different ages. Interspecies differences were much more pronounced than age-associated alterations in the metabolome. Such interspecies divergences affected multiple metabolic pathways involving amino, bile and fatty acids as well as monosaccharides and nucleotides. The most intriguing metabolites were those that had previously been linked to pro-health and antiaging effects in mice and that were significantly increased in the long-lived rodent compared to its short-lived counterpart. This pattern applies to α-tocopherol (also known as vitamin E) and polyamines (in particular cadaverine, N8-acetylspermidine and N1,N8-diacetylspermidine), all of which were more abundant in naked mole-rats than in mice. Moreover, the age-associated decline in spermidine and N1-acetylspermidine levels observed in mice did not occur, or is even reversed (in the case of N1-acetylspermidine) in naked mole-rats. In short, the present metabolomics analysis provides a series of testable hypotheses to explain the exceptional longevity of naked mole-rats.
Biallelic pathogenic variants in the SLC34A3 gene, encoding for the NPT2c cotransporter, cause Hereditary Hypophosphatemic Rickets with Hypercalciuria (HHRH). However, the associated phenotype is ...highly variable. In addition, mice deleted for Slc34a3 exhibit a different phenotype compared to humans, without urinary phosphate leakage. The mechanisms by which SLC34A3 variants disrupt phosphate/calcium metabolism are un-completely understood. In this study we explored these mechanisms in vitro using SLC34A3 variants identified in patients with urinary phosphate leakage. We analyzed the consequences of these variants on NPT2c function and the link with the phenotype of the patients. We studied 20 patients with recurrent nephrolithiasis and low serum phosphate concentration harboring variants in the SLC34A3 gene. Half of the patients carried homozygous or composite heterozygous variants. Three patients had in addition variants in SLC34A1 and SLC9A3R1 genes. All these patients benefited from a precise analysis of their phenotype. We generated 13 of these mutants by site-directed mutagenesis. Then we carried out transient transfections of these mutants in HEK cells and measured their phosphate uptake capacity under different conditions. Among the 20 patients included, 3 had not only mutations in NPT2c but also in NPT2a or NHERF1 genes. Phosphate uptake was decreased in 8 NPT2c mutants studied and normal for 5. Four variants were initially categorized as variants of uncertain significance. Expression of the corresponding mutants showed that one did not modify phosphate transport, two reduced it moderately and one abolished it. Co-transfection of the NPT2c mutants with the wild-type plasmid of NPT2c or NPT2a did not reveal dominant negative effect of the mutants on NPT2c-mediated phosphate transport. A detailed analysis of patient phenotypes did not find a link between the severity of the disorder and the level of phosphate transport impairment. NPT2c mutations classified as ACMG3 identified in patients with renal phosphate leak should be characterized by in vitro study to check if they alter NPT2c-mediated phosphate transport since phosphate uptake capacity may not be affected. In addition, research for mutations in NHERF1 and NPT2a genes should always be associated to NPT2c sequencing.
PiT1/SLC20A1 is an inorganic phosphate transporter with additional functions including the regulation of TNFα-induced apoptosis, erythropoiesis, cell proliferation and insulin signaling. Recent data ...suggest a relationship between PiT1 and NF-κB-dependent inflammation: (i) Pit1 mRNA is up-regulated in the context of NF-κB pathway activation; (ii) NF-κB target gene transcription is decreased in PiT1-deficient conditions. This led us to investigate the role of PiT1 in lipopolysaccharide (LPS)-induced inflammation. MCP-1 and IL-6 concentrations were impaired in PiT1-deficient bone marrow derived macrophages (BMDMs) upon LPS stimulation. Lower MCP-1 and IL-6 serum levels were observed in Mx1-Cre; Pit1
mice dosed intraperitoneally with LPS. Lower PiT1 expression correlated with decreased in vitro wound healing and lower reactive oxygen species levels. Reduced IκB degradation and lower p65 nuclear translocation were observed in PiT1-deficient cells stimulated with LPS. Conversely, PiT1 expression was induced in vitro upon LPS stimulation. Addition of an NF-κB inhibitor abolished LPS-induced PiT1 expression. Furthermore, we showed that p65 expression activated Pit1 promoter activity. Finally, ChIP assays demonstrated that p65 directly binds to the mPit1 promoter in response to LPS. These data demonstrate a completely novel function of PiT1 in the response to LPS and provide mechanistic insights into the regulation of PiT1 expression by NF-κB.
PiT1 (or SLC20a1) encodes a widely expressed plasma membrane protein functioning as a high-affinity Na(+)-phosphate (Pi) cotransporter. As such, PiT1 is often considered as a ubiquitous supplier of ...Pi for cellular needs regardless of the lack of experimental data. Although the importance of PiT1 in mineralizing processes have been demonstrated in vitro in osteoblasts, chondrocytes and vascular smooth muscle cells, in vivo evidence is missing.
To determine the in vivo function of PiT1, we generated an allelic series of PiT1 mutations in mice by combination of wild-type, hypomorphic and null PiT1 alleles expressing from 100% to 0% of PiT1. In this report we show that complete deletion of PiT1 results in embryonic lethality at E12.5. PiT1-deficient embryos display severely hypoplastic fetal livers and subsequent reduced hematopoiesis resulting in embryonic death from anemia. We show that the anemia is not due to placental, yolk sac or vascular defects and that hematopoietic progenitors have no cell-autonomous defects in proliferation and differentiation. In contrast, mutant fetal livers display decreased proliferation and massive apoptosis. Animals carrying two copies of hypomorphic PiT1 alleles (resulting in 15% PiT1 expression comparing to wild-type animals) survive at birth but are growth-retarded and anemic. The combination of both hypomorphic and null alleles in heterozygous compounds results in late embryonic lethality (E14.5-E16.5) with phenotypic features intermediate between null and hypomorphic mice. In the three mouse lines generated we could not evidence defects in early skeleton formation.
This work is the first to illustrate a specific in vivo role for PiT1 by uncovering it as being a critical gene for normal developmental liver growth.
Long chain polyunsaturated fatty acids (PUFAs) play an important role in cardioprotection. These effects have been largely attributed to membrane docosahexaenoic acid. Conversely, saturated fatty ...acids trigger apoptosis in cardiomyocytes, with modifications of mitochondrial properties including cardiolipin loss, cytochrome
c release and caspase-3 activation. The purpose of this study was to investigate the chronic effect of eicosapentaenoic acid (EPA) on mitochondrial apoptosis induced by palmitate treatment and the associated signalling pathways. Confluent cultures of rat neonatal cardiomyocytes were treated for 2 days in media enriched with either EPA or arachidonic acid (AA) and then exposed to palmitate (0.5 mM) to induce apoptosis, in the absence of PUFA supplements. The EPA treatment resulted in significant membrane enrichment in
n
−
3 PUFAs, especially in docosapentaenoic acid (DPA), and a large decrease in AA. Both AA and EPA treatments prevented caspase-3 activation, translocation of Bax to the mitochondria and release of cytochrome
c induced by palmitate treatment. Furthermore, EPA, but not AA prevented the loss of mitochondrial cardiolipin due to apoptosis. These results suggest that EPA supplementation is able to protect cardiomyocytes against palmitate-induced apoptosis via an implication of different mitochondrial elements, possibly through its elongation to DPA, which is very efficient in cardiomyocytes.
In recent years, primary familial brain calcification (PFBC), a rare neurological disease characterized by a wide spectrum of cognitive disorders, has been associated to mutations in the sodium ...(Na)-Phosphate (Pi) co-transporter SLC20A2. However, the functional roles of the Na-Pi co-transporters in the brain remain still largely elusive. Here we show that Slc20a1 (PiT-1) and Slc20a2 (PiT-2) are the most abundant Na-Pi co-transporters expressed in the brain and are involved in the control of hippocampal-dependent learning and memory. We reveal that Slc20a1 and Slc20a2 are differentially distributed in the hippocampus and associated with independent gene clusters, suggesting that they influence cognition by different mechanisms. Accordingly, using a combination of molecular, electrophysiological and behavioral analyses, we show that while PiT-2 favors hippocampal neuronal branching and survival, PiT-1 promotes synaptic plasticity. The latter relies on a likely Otoferlin-dependent regulation of synaptic vesicle trafficking, which impacts the GABAergic system. These results provide the first demonstration that Na-Pi co-transporters play key albeit distinct roles in the hippocampus pertaining to the control of neuronal plasticity and cognition. These findings could provide the foundation for the development of novel effective therapies for PFBC and cognitive disorders.
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