The Phosphate Transporter PiT1 (Slc20a1) Revealed As a New Essential Gene for Mouse Liver Development

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSION/SIGNIFICANCE: 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

Caractérisation de la réponse des cellules germinales mâles néonatales à un stress génotoxique

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

EKLF-driven PIT1 expression is critical for mouse erythroid maturation in vivo and in vitro

International audienceThe PIT1/SLC20A1 protein, a well-described sodium/phosphate cotransporter and retrovirus receptor, has been identified recently as a modular of proliferation and apoptosis in vitro. The targeted deletion of the PIT1 gene in mice revealed a lethal phenotype due to severe anemia attributed to defects in liver development. However, the presence of immature erythroid cells associated with impaired maturation of the globin switch led us to investigate the role of PIT1 in hematopoietic development. In the present study, specific deletion of PIT1 in the hematopoietic system and fetal liver transplantation experiments demonstrated that anemia was associated with an erythroid cell- autonomous defect. Moreover, anemia was not due to RBC destruction but rather to maturation defects. Because Erythroid Krüppel-like Factor (EKLF)-knockout mice showed similar maturation defects, we investigated the functional link between PIT1 and EKLF. We demonstrated that EKLF increases PIT1 expression during RBC maturation by binding to its promoter in vivo and that shRNA-driven depletion of either PIT1 or EKLF impairs erythroid maturation of G1E cells in vitro, whereas reexpression of PIT1 in EKLF-depleted G1E cells partially restores erythroid maturation. This is the first demonstration of a physiologic involvement of PIT1 in erythroid maturation in vivo

Combined Treatment with Peptide-Conjugated Phosphorodiamidate Morpholino Oligomer-PPMO and AAV-U7 Rescues the Severe DMD Phenotype in Mice

International audienceDuchenne muscular dystrophy (DMD) is a devastating neuromuscular disease caused by an absence of the dystrophin protein, which is essential for muscle fiber integrity. Among the developed therapeutic strategies for DMD, the exon-skipping approach corrects the frameshift and partially restores dystrophin expression. It could be achieved through the use of antisense sequences, such as peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) or the small nuclear RNA-U7 carried by an adeno-associated virus (AAV) vector. AAV-based gene therapy approaches have potential for use in DMD treatment but are subject to a major limitation: loss of the AAV genome, necessitating readministration of the vector, which is not currently possible, due to the immunogenicity of the capsid. The PPMO approach requires repeated administrations and results in only weak cardiac dystrophin expression. Here, we evaluated a combination of PPMO- and AAV-based therapy in a mouse model of severe DMD. Striking benefits of this combined therapy were observed in striated muscles, with marked improvements in heart and diaphragm structure and function, with unrivalled extent of survival, opening novel therapeutic perspectives for patients

Disruption of the Phosphate Transporter Pit1 in Hepatocytes Improves Glucose Metabolism and Insulin Signaling by Modulating the USP7/IRS1 Interaction

International audienceThe liver plays a central role in whole-body lipid and glucose homeostasis. Increasing dietary fat intake results in increased hepatic fat deposition, which is associated with a risk for development of insulin resistance and type 2 diabetes. In this study, we demonstrate a role for the phosphate inorganic transporter 1 (PiT1/SLC20A1) in regulating metabolism. Specific knockout of Pit1 in hepatocytes significantly improved glucose tolerance and insulin sensitivity, enhanced insulin signaling, and decreased hepatic lipogenesis. We identified USP7 as a PiT1 binding partner and demonstrated that Pit1 deletion inhibited USP7/IRS1 dissociation upon insulin stimulation. This prevented IRS1 ubiquitination and its subsequent proteasomal degradation. As a consequence, delayed insulin negative feedback loop and sustained insulin signaling were observed. Moreover, PiT1-deficient mice were protected against high-fat-diet-induced obesity and diabetes. Our findings indicate that PiT1 has potential as a therapeutic target in the context of metabolic syndrome, obesity, and diabetes

The mutated p.H222P A-type Lamins drive Loxl2-mediated extracellular matrix remodeling in both patient-derived cardiomyocytes and mouse models of dilated cardiomyopathy

LMNA cardiomyopathy, caused by mutations in the LMNA gene, is a severe form of dilated cardiomyopathy characterized by arrhythmias, contractile dysfunction, and increased myocardial fibrosis, which impairs left ventricular function and predisposes to heart failure. While the disease has been well characterized, a lack of insight into the pathogenesis impeded the development of therapies. We here used patient-derived LMNA p.H222P cardiomyocytes (hiPSC-CMs) and their isogenic controls and a Lmna H222P/H222P mouse model to dissect abnormal cardiac mechanisms leading to the development of the disease. We showed that LMNA p.H222P hiPSC-CMs exhibit elevated diastolic calcium levels and hypocontractility. They displayed nuclear shape abnormalities, a hallmark of LMNA cardiomyopathy, associated with altered chromosome spatial organization and gene expression profiles. Using transcriptomic analysis, we further revealed that genes related to cardiac extracellular matrix (ECM) remodeling, deposition, and components are dysregulated in both LMNA p.H222P hiPSC-CMs and mutated mice, suggesting a conserved pathogenic mechanism across species. Conversely, molecular inhibition of Loxl2, a key component of the ECM establishment, preserved the cardiac function in vivo . Taken together, our findings suggest that targeting Loxl2 could be a promising therapeutic strategy to maintain cardiac function in LMNA cardiomyopathy