The Complexities of Organ Crosstalk in Phosphate Homeostasis: Time to Put Phosphate Sensing Back in the Limelight.

Phosphate homeostasis is essential for health and is achieved via interaction between the bone, kidney, small intestine, and parathyroid glands and via intricate processes involving phosphate transporters, phosphate sensors, and circulating hormones. Numerous genetic and acquired disorders are associated with disruption in these processes and can lead to significant morbidity and mortality. The role of the kidney in phosphate homeostasis is well known, although it is recognized that the cellular mechanisms in murine models and humans are different. Intestinal phosphate transport also appears to differ in humans and rodents, with recent studies demonstrating a dominant role for the paracellular pathway. The existence of phosphate sensing has been acknowledged for decades; however, the underlying molecular mechanisms are poorly understood. At least three phosphate sensors have emerged. PiT2 and FGFR1c both act as phosphate sensors controlling Fibroblast Growth Factor 23 secretion in bone, whereas the calcium-sensing receptor controls parathyroid hormone secretion in response to extracellular phosphate. All three of the proposed sensors are expressed in the kidney and intestine but their exact function in these organs is unknown. Understanding organ interactions and the mechanisms involved in phosphate sensing requires significant research to develop novel approaches for the treatment of phosphate homeostasis disorders.

Phosphate (Pi)-regulated heterodimerization of the high-affinity sodium-dependent Pi transporters PiT1/Slc20a1 and PiT2/Slc20a2 underlies extracellular Pi sensing independently of Pi uptake.

Extracellular phosphate (Pi) can act as a signaling molecule that directly alters gene expression and cellular physiology. The ability of cells or organisms to detect changes in extracellular Pi levels implies the existence of a Pi-sensing mechanism that signals to the body or individual cell. However, unlike in prokaryotes, yeasts, and plants, the molecular players involved in Pi sensing in mammals remain unknown. In this study, we investigated the involvement of the high-affinity, sodium-dependent Pi transporters PiT1 and PiT2 in mediating Pi signaling in skeletal cells. We found that deletion of PiT1 or PiT2 blunted the Pi-dependent ERK1/2-mediated phosphorylation and subsequent gene up-regulation of the mineralization inhibitors matrix Gla protein and osteopontin. This result suggested that both PiTs are necessary for Pi signaling. Moreover, the ERK1/2 phosphorylation could be rescued by overexpressing Pi transport-deficient PiT mutants. Using cross-linking and bioluminescence resonance energy transfer approaches, we found that PiT1 and PiT2 form high-abundance homodimers and Pi-regulated low-abundance heterodimers. Interestingly, in the absence of sodium-dependent Pi transport activity, the PiT1-PiT2 heterodimerization was still regulated by extracellular Pi levels. Of note, when two putative Pi-binding residues, Ser-128 (in PiT1) and Ser-113 (in PiT2), were substituted with alanine, the PiT1-PiT2 heterodimerization was no longer regulated by extracellular Pi These observations suggested that Pi binding rather than Pi uptake may be the key factor in mediating Pi signaling through the PiT proteins. Taken together, these results demonstrate that Pi-regulated PiT1-PiT2 heterodimerization mediates Pi sensing independently of Pi uptake.

NOTCH activation interferes with cell fate specification in the gastrulating mouse embryo.

NOTCH signalling is an evolutionarily conserved pathway involved in intercellular communication essential for cell fate choices during development. Although dispensable for early aspects of mouse development, canonical RBPJ-dependent NOTCH signalling has been shown to influence lineage commitment during embryonic stem cell (ESC) differentiation. NOTCH activation in ESCs promotes the acquisition of a neural fate, whereas its suppression favours their differentiation into cardiomyocytes. This suggests that NOTCH signalling is implicated in the acquisition of distinct embryonic fates at early stages of mammalian development. In order to investigate in vivo such a role for NOTCH signalling in shaping cell fate specification, we use genetic approaches to constitutively activate the NOTCH pathway in the mouse embryo. Early embryonic development, including the establishment of anterior-posterior polarity, is not perturbed by forced NOTCH activation. By contrast, widespread NOTCH activity in the epiblast triggers dramatic gastrulation defects. These are fully rescued in a RBPJ-deficient background. Epiblast-specific NOTCH activation induces acquisition of neurectoderm identity and disrupts the formation of specific mesodermal precursors including the derivatives of the anterior primitive streak, the mouse organiser. In addition, we show that forced NOTCH activation results in misregulation of NODAL signalling, a major determinant of early embryonic patterning. Our study reveals a previously unidentified role for canonical NOTCH signalling during mammalian gastrulation. It also exemplifies how in vivo studies can shed light on the mechanisms underlying cell fate specification during in vitro directed differentiation.

Slc20a1 and Slc20a2 regulate neuronal plasticity and cognition independently of their phosphate transport ability.

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.

PiT2 deficiency prevents increase of bone marrow adipose tissue during skeletal maturation but not in OVX-induced osteoporosis.

The common cellular origin between bone marrow adipocytes (BMAds) and osteoblasts contributes to the intimate link between bone marrow adipose tissue (BMAT) and skeletal health. An imbalance between the differentiation ability of BMSCs towards one of the two lineages occurs in conditions like aging or osteoporosis, where bone mass is decreased. Recently, we showed that the sodium-phosphate co-transporter PiT2/SLC20A2 is an important determinant for bone mineralization, strength and quality. Since bone mass is reduced in homozygous mutant mice, we investigated in this study whether the BMAT was also affected in PiT2-/- mice by assessing the effect of the absence of PiT2 on BMAT volume between 3 and 16 weeks, as well as in an ovariectomy-induced bone loss model. Here we show that the absence of PiT2 in juveniles leads to an increase in the BMAT that does not originate from an increased adipogenic differentiation of bone marrow stromal cells. We show that although PiT2-/- mice have higher BMAT volume than control PiT2+/+ mice at 3 weeks of age, BMAT volume do not increase from 3 to 16 weeks of age, leading to a lower BMAT volume in 16-week-old PiT2-/- compared to PiT2+/+ mice. In contrast, the absence of PiT2 does not prevent the increase in BMAT volume in a model of ovariectomy-induced bone loss. Our data identify SLC20a2/PiT2 as a novel gene essential for the maintenance of the BMAd pool in adult mice, involving mechanisms of action that remain to be elucidated, but which appear to be independent of the balance between osteoblastic and adipogenic differentiation of BMSCs.

Extracellular phosphate sensing in mammals: what do we know?

The critical role of phosphate (Pi) in countless biological processes requires the ability to control its concentration both intracellularly and extracellularly. At the body level, this concentration is finely regulated by numerous hormones, primarily parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23). While this control of the body's Pi homeostasis is now well documented, knowledge of the mechanisms that allow the cell and the body to detect extracellular Pi variations is much less known. These systems are well described in bacteria, yeasts and plants, but as will be discussed in this review, knowledge obtained from these organisms is not entirely relevant to the requirements of Pi biology in mammals. In this review, we present the latest findings on extracellular Pi sensing in mammals, and describe the mammalian Pi sensors identified to date, such as SLC20A1 (PIT1)/SLC20A2 (PIT2) heterodimers and the calcium-sensing receptor (CaSR). While there are many questions remaining to be resolved, a clarification of the Pi sensing mechanisms in mammals is critical to understanding the deregulation of Pi balance in certain life-threatening disease states, such as end-stage renal disease and associated vascular calcifications, and to proposing relevant therapeutic approaches.

Reporting Guidelines, Review of Methodological Standards, and Challenges Toward Harmonization in Bone Marrow Adiposity Research. Report of the Methodologies Working Group of the International Bone Marrow Adiposity Society.

The interest in bone marrow adiposity (BMA) has increased over the last decade due to its association with, and potential role, in a range of diseases (osteoporosis, diabetes, anorexia, cancer) as well as treatments (corticosteroid, radiation, chemotherapy, thiazolidinediones). However, to advance the field of BMA research, standardization of methods is desirable to increase comparability of study outcomes and foster collaboration. Therefore, at the 2017 annual BMA meeting, the International Bone Marrow Adiposity Society (BMAS) founded a working group to evaluate methodologies in BMA research. All BMAS members could volunteer to participate. The working group members, who are all active preclinical or clinical BMA researchers, searched the literature for articles investigating BMA and discussed the results during personal and telephone conferences. According to the consensus opinion, both based on the review of the literature and on expert opinion, we describe existing methodologies and discuss the challenges and future directions for (1) histomorphometry of bone marrow adipocytes, (2) ex vivo BMA imaging, (3) in vivo BMA imaging, (4) cell isolation, culture, differentiation and in vitro modulation of primary bone marrow adipocytes and bone marrow stromal cell precursors, (5) lineage tracing and in vivo BMA modulation, and (6) BMA biobanking. We identify as accepted standards in BMA research: manual histomorphometry and osmium tetroxide 3D contrast-enhanced µCT for ex vivo quantification, specific MRI sequences (WFI and H-MRS) for in vivo studies, and RT-qPCR with a minimal four gene panel or lipid-based assays for in vitro quantification of bone marrow adipogenesis. Emerging techniques are described which may soon come to complement or substitute these gold standards. Known confounding factors and minimal reporting standards are presented, and their use is encouraged to facilitate comparison across studies. In conclusion, specific BMA methodologies have been developed. However, important challenges remain. In particular, we advocate for the harmonization of methodologies, the precise reporting of known confounding factors, and the identification of methods to modulate BMA independently from other tissues. Wider use of existing animal models with impaired BMA production (e.g., Pfrt-/-, KitW/W-v) and development of specific BMA deletion models would be highly desirable for this purpose.

PiT1/Slc20a1 Is Required for Endoplasmic Reticulum Homeostasis, Chondrocyte Survival, and Skeletal Development.

During skeletal mineralization, the sodium-phosphate co-transporter PiT1Slc20a1 is assumed to meet the phosphate requirements of bone-forming cells, although evidence is missing. Here, we used a conditional gene deletion approach to determine the role of PiT1 in growth plate chondrocytes. We show that PiT1 ablation shortly after birth generates a rapid and massive cell death in the center of the growth plate, together with an uncompensated endoplasmic reticulum (ER) stress, characterized by morphological changes and increased Chop, Atf4, and Bip expression. PiT1 expression in chondrocytes was not found at the cell membrane but co-localized with the ER marker ERp46, and was upregulated by the unfolded protein response cascade. In addition, we identified the protein disulfide isomerase (Pdi) ER chaperone as a PiT1 binding partner and showed that PiT1 ablation impaired Pdi reductase activity. The ER stress induced by PiT1 deficiency in chondrocytes was associated with intracellular retention of aggrecan and vascular endothelial growth factor A (Vegf-A), which was rescued by overexpressing a phosphate transport-deficient mutant of PiT1. Our data thus reveal a novel, Pi-transport independent function of PiT1, as a critical modulator of ER homeostasis and chondrocyte survival during endochondral ossification. © 2018 American Society for Bone and Mineral Research.

Slc20a2, Encoding the Phosphate Transporter PiT2, Is an Important Genetic Determinant of Bone Quality and Strength.

Osteoporosis is characterized by low bone mineral density (BMD) and fragility fracture and affects over 200 million people worldwide. Bone quality describes the material properties that contribute to strength independently of BMD, and its quantitative analysis is a major priority in osteoporosis research. Tissue mineralization is a fundamental process requiring calcium and phosphate transporters. Here we identify impaired bone quality and strength in Slc20a2-/- mice lacking the phosphate transporter SLC20A2. Juveniles had abnormal endochondral and intramembranous ossification, decreased mineral accrual, and short stature. Adults exhibited only small reductions in bone mass and mineralization but a profound impairment of bone strength. Bone quality was severely impaired in Slc20a2-/- mice: yield load (-2.3 SD), maximum load (-1.7 SD), and stiffness (-2.7 SD) were all below values predicted from their bone mineral content as determined in a cohort of 320 wild-type controls. These studies identify Slc20a2 as a physiological regulator of tissue mineralization and highlight its critical role in the determination of bone quality and strength. © 2019 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.

Phosphate-dependent FGF23 secretion is modulated by PiT2/Slc20a2.

OBJECTIVE: The canonical role of the bone-derived fibroblast growth factor 23 (Fgf23) is to regulate the serum inorganic phosphate (Pi) level. As part of a feedback loop, serum Pi levels control Fgf23 secretion through undefined mechanisms. We recently showed in vitro that the two high-affinity Na+-Pi co-transporters PiT1/Slc20a1 and PiT2/Slc20a2 were required for mediating Pi-dependent signaling. Here, we addressed the contribution of PiT1 and PiT2 to the regulation of Fgf23 secretion. METHODS: To this aim, we used PiT2 KO and DMP1Cre; PiT1lox/lox fed Pi-modified diets, as well as ex vivo isolated long bone shafts. Fgf23 secretion and expression of Pi homeostasis-related genes were assessed. RESULTS: In vivo, PiT2 KO mice responded inappropriately to low-Pi diets, displaying abnormally normal serum levels of intact Fgf23. Despite the high iFgf23 level, serum Pi levels remained unaffected, an effect that may relate to lower αKlotho expression in the kidney. Moreover, consistent with a role of PiT2 as a possible endocrine Pi sensor, the iFGF23/cFGF23 ratios were suppressed in PiT2 KO mice, irrespective of the Pi loads. While deletion of PiT1 in osteocytes using the DMP1-Cre mice was inefficient, adenovirus-mediated deletion of PiT1 in isolated long bone shafts suggested that PiT1 does not contribute to Pi-dependent regulation of Fgf23 secretion. In contrast, using isolated bone shafts from PiT2 KO mice, we showed that PiT2 was necessary for the appropriate Pi-dependent secretion of Fgf23, independently from possible endocrine regulatory loops. CONCLUSIONS: Our data provide initial mechanistic insights underlying the Pi-dependent regulation of Fgf23 secretion in identifying PiT2 as a potential player in this process, at least in high Pi conditions. Targeting PiT2, therefore, could improve excess FGF23 in hyperphosphatemic conditions such as chronic kidney disease.

Dendritic-cell-derived osteoclasts: a new game changer in bone-resorption-associated diseases.

Bone-resorbing cells, osteoclasts (OCs), and antigen-presenting cells, dendritic cells (DCs), share several features. They are derived from a common hematopoietic precursor, exhibit phagocytic activities and their functions are dependent upon receptor activator of nuclear factor κB ligand (RANKL). Upon inflammatory conditions, DCs can transdifferentiate toward functional OCs in the presence of RANKL. It has then been assumed that the increase in proinflammatory cytokines could provide a supportive environment for this transdifferentiation. In this review, we emphasize the molecular mechanisms underlying the potential for DCs to give rise to resorbing OCs in the context of bone-destruction-associated diseases upon inflammatory conditions. Whether these mechanisms reveal new strategies for the discovery of therapeutic targets and drugs is discussed extensively.

Expression of phosphate transporters in optimized cell culture models for dental cells biomineralization.

Phosphate is a key component of dental mineral composition. The physiological role of membrane proteins of dental cells is suspected to be crucial for mineralization mechanisms. Contrary to published data related to calcium, data on regulation of phosphate flux through membrane of mineralizing cells are scarce. To address this lack of data, we studied the expression of six membranous phosphate transporters in two dental cell lines: a rat odontoblastic cell line (M2H4) and a mouse ameloblastic cell line (ALC) for which we optimized the mineralizing culture conditions.

Interleukin-33 and RANK-L Interplay in the Alveolar Bone Loss Associated to Periodontitis.

INTRODUCTION: Chronic Periodontitis (CP) is an inflammatory disease of bacterial origin that results in alveolar bone destruction. Porphyromonas gingivalis (Pg), one of the main periopathogens, initiates an inflammatory cascade by host immune cells thereby increasing recruitment and activity of osteoclasts, the bone resorbing cells, through enhanced production of the crucial osteoclastogenic factor, RANK-L. Antibodies directed against some cytokines (IL-1ß, IL-6 and TNF-α) failed to exhibit convincing therapeutic effect in CP. It has been suggested that IL-33, could be of interest in CP. OBJECTIVE: the present study aims to analyze whether and how IL-33 and RANK-L and/or their interplay are involved in the bone destruction associated to CP. MATERIAL AND METHODS: mRNAs and protein expressions of IL-33 and RANK-L were analyzed in healthy and CP human gingival samples by immunohistochemistry (IHC) and RT-qPCR. Murine experimental periodontitis (EP) was induced using Pg infected ligature and Pg free ligature around the first maxillary molar. Alveolar bone loss was recorded by µCT. Mouse gingival explants were stimulated for 24 hours with IL-33 and RANK-L mRNA expression investigated by RT-qPCR. Human oral epithelial cells were infected by Pg for 6, 12; 24 hours and IL-33 and RANK-L mRNA expressions were analyzed by RT-qPCR. RESULTS: IL-33 is overexpressed in gingival epithelial cells in human affected by CP as in the murine EP. In human as in murine gingival cells, RANK-L was independently induced by Pg and IL-33. We also showed that the Pg-dependent RANK-L expression in gingival epithelial cells occured earlier than that of IL-33. CONCLUSION: Our results evidence that IL-33 overexpression in gingival epithelial cells is associated with CP and may trigger RANK-L expression in addition to a direct effect of Pg. Finally, IL-33 may act as an extracellular alarmin (danger signal) showing proinflammatory properties in CP perpetuating bone resorption induced by Pg infection.

Notchless is required for axial skeleton formation in mice.

Maintenance of cell survival is essential for proper embryonic development. In the mouse, Notchless homolog 1 (Drosophila) (Nle1) is instrumental for survival of cells of the inner cell mass upon implantation. Here, we analyze the function of Nle1 after implantation using the Meox2(tm1(cre)Sor) mouse that expresses the Cre recombinase specifically in the epiblast at E5.5. First, we find that NLE1 function is required in epiblast cells, as Nle1-deficient cells are rapidly eliminated. In this report, we also show that the Meox2(Cre) transgene is active in specific tissues during organogenesis. In particular, we detect high Cre expression in the vertebral column, ribs, limbs and tailbud. We took advantage of this dynamic expression profile to analyze the effects of inducing mosaic deletion of Nle1 in the embryo. We show that Nle1 deletion in this context, results in severe developmental anomalies leading to lethality at birth. Mutant embryos display multiple developmental defects in particular during axial skeletal formation. We also provide evidence that axial defects are due to an increase in apoptotic cell death in the somite at E9.5. These data demonstrate an essential role for Nle1 during organogenesis and in particular during axial development.

Notchless-dependent ribosome synthesis is required for the maintenance of adult hematopoietic stem cells.

Blood cell production relies on the coordinated activities of hematopoietic stem cells (HSCs) and multipotent and lineage-restricted progenitors. Here, we identify Notchless (Nle) as a critical factor for HSC maintenance under both homeostatic and cytopenic conditions. Nle deficiency leads to a rapid and drastic exhaustion of HSCs and immature progenitors and failure to maintain quiescence in HSCs. In contrast, Nle is dispensable for cycling-restricted progenitors and differentiated cells. In yeast, Nle/Rsa4 is essential for ribosome biogenesis, and we show that its role in pre-60S subunit maturation has been conserved in the mouse. Despite its implication in this basal cellular process, Nle deletion affects ribosome biogenesis only in HSCs and immature progenitors. Ribosome biogenesis defects are accompanied by p53 activation, which causes their rapid exhaustion. Collectively, our findings establish an essential role for Nle in HSC and immature progenitor functions and uncover previously unsuspected differences in ribosome biogenesis that distinguish stem cells from restricted progenitor populations.

Mice with hypomorphic expression of the sodium-phosphate cotransporter PiT1/Slc20a1 have an unexpected normal bone mineralization.

The formation of hydroxyapatite crystals and their insertion into collagen fibrils of the matrix are essential steps for bone mineralization. As phosphate is a main structural component of apatite crystals, its uptake by skeletal cells is critical and must be controlled by specialized membrane proteins. In mammals, in vitro studies have suggested that the high-affinity sodium-phosphate cotransporter PiT1 could play this role. In vivo, PiT1 expression was detected in hypertrophic chondrocytes of murine metatarsals, but its implication in bone physiology is not yet deciphered. As the complete deletion of PiT1 results in embryonic lethality at E12.5, we took advantage of a mouse model bearing two copies of PiT1 hypomorphic alleles to study the effect of a low expression of PiT1 on bone mineralization in vivo. In this report, we show that a 85% down-regulation of PiT1 in long bones resulted in a slight (6%) but significant reduction of femur length in young mice (15- and 30-day-old). However, despite a defect in alcian blue / alizarin red S and Von Kossa staining of hypomorphic 1-day-old mice, using X-rays micro-computed tomography, energy dispersive X-ray spectroscopy and histological staining techniques we could not detect differences between hypomorphic and wild-type mice of 15- to 300-days old. Interestingly, the expression of PiT2, the paralog of PiT1, was increased 2-fold in bone of PiT1 hypomorphic mice accounting for a normal phosphate uptake in mutant cells. Whether this may contribute to the absence of bone mineralization defects remains to be further deciphered.

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.