Mice Knocked Out for the Primary Brain Calcification-Associated Gene Slc20a2 Show Unimpaired Prenatal Survival but Retarded Growth and Nodules in the Brain that Grow and Calcify Over Time.

Brain calcification of especially the basal ganglia characterizes primary familial brain calcification (PFBC). PFBC is a rare neurodegenerative disorder with neuropsychiatric and motor symptoms, and only symptomatic treatment is available. Four PFBC-associated genes are known; approximately 40% of patients carry mutations in the gene SLC20A2, which encodes the type III sodium-dependent inorganic phosphate transporter PiT2. To investigate the role of PiT2 in PFBC development, we studied Slc20a2-knockout (KO) mice using histology, microcomputed tomography, electron microscopy, and energy-dispersive X-ray spectroscopy. Slc20a2-KO mice showed histologically detectable nodules in the brain already at 8 weeks of age, which contained organic material and were weakly calcified. In 15-week-old mice, the nodules were increased in size and number and were markedly more calcified. The major minerals in overt calcifications were Ca and P, but Fe, Zn, and Al were also generally present. Electron microscopy suggested that the calcifications initiate intracellularly, mainly in pericytes and astrocytes. As the calcification grew, they incorporated organic material. Furthermore, endogenous IgG was detected around nodules, suggesting local increased blood-brain barrier permeabilities. Nodules were found in all 8-week-old Slc20a2-KO mice, but no prenatal or marked postnatal lethality was observed. Thus, besides allowing for the study of PFBC development, the Slc20a2-KO mouse is a potential solid preclinical model for evaluation of PFBC treatments.

Phosphate uptake-independent signaling functions of the type III sodium-dependent phosphate transporter, PiT-1, in vascular smooth muscle cells.

Vascular calcification (VC) is prevalent in chronic kidney disease and elevated serum inorganic phosphate (Pi) is a recognized risk factor. The type III sodium-dependent phosphate transporter, PiT-1, is required for elevated Pi-induced osteochondrogenic differentiation and matrix mineralization in vascular smooth muscle cells (VSMCs). However, the molecular mechanism(s) by which PiT-1 promotes these processes is unclear. In the present study, we confirmed that the Pi concentration required to induce osteochondrogenic differentiation and matrix mineralization of mouse VSMCs was well above that required for maximal Pi uptake, suggesting a signaling function of PiT-1 that was independent of Pi transport. Elevated Pi-induced signaling via ERK1/2 phosphorylation was abrogated in PiT-1 deficient VSMCs, but could be rescued by wild-type (WT) and a Pi transport-deficient PiT-1 mutant. Furthermore, both WT and transport-deficient PiT-1 mutants promoted osteochondrogenic differentiation as measured by decreased SM22α and increased osteopontin mRNA expression. Finally, compared to vector alone, expression of transport-deficient PiT-1 mutants promoted VSMC matrix mineralization, but not to the extent observed with PiT-1 WT. These data suggest that both Pi uptake-dependent and -independent functions of PiT-1 are important for VSMC processes mediating vascular calcification.

Stimulation of V1a receptor increases renal uric acid clearance via urate transporters: insight into pathogenesis of hypouricemia in SIADH.

BACKGROUND: Hypouricemia is pathognomonic in syndrome of inappropriate secretion of antidiuretic hormone (SIADH) but the underlying mechanism remains unclear. Based on the previous studies, we hypothesized that V1a receptor may play a principal role in inducing hypouricemia in SIADH and examined uric acid metabolism using a rat model. METHODS: Terlipressin (25 ng/h), a selective V1a agonist, was subcutaneously infused to 7-week-old male Wistar rats (n = 9). Control rats were infused with normal saline (n = 9). The rats were sacrificed to obtain kidney tissues 3 days after treatment. In addition to electrolyte metabolism, changes in expressions of the urate transporters including URAT1 (SLC22A12), GLUT9 (SLC2A9), ABCG2 and NPT1 (SLC17A1) were examined by western blotting and immunohistochemistry. RESULTS: In the terlipressin-treated rats, serum uric acid (UA) significantly decreased and the excretion of urinary UA significantly increased, resulting in marked increase in fractional excretion of UA. Although no change in the expression of URAT1, GLUT9 expression significantly decreased whereas the expressions of ABCG2 and NPT1 significantly increased in the terlipressin group. The results of immunohistochemistry corroborated with those of the western blotting. Aquaporin 2 expression did not change in the medulla, suggesting the independence of V2 receptor stimulation. CONCLUSION: Stimulation of V1a receptor induces the downregulation of GLUT9, reabsorption urate transporter, together with the upregulation of ABCG2 and NPT1, secretion urate transporters, all changes of which clearly lead to increase in renal UA clearance. Hypouricemia seen in SIADH is attributable to V1a receptor stimulation.

Slc20a1b is essential for hematopoietic stem/progenitor cell expansion in zebrafish.

Hematopoietic stem and progenitor cells (HSPCs) are able to self-renew and can give rise to all blood lineages throughout their lifetime, yet the mechanisms regulating HSPC development have yet to be discovered. In this study, we characterized a hematopoiesis defective zebrafish mutant line named smu07, which was obtained from our previous forward genetic screening, and found the HSPC expansion deficiency in the mutant. Positional cloning identified that slc20a1b, which encodes a sodium phosphate cotransporter, contributed to the smu07 blood phenotype. Further analysis demonstrated that mutation of slc20a1b affects HSPC expansion through cell cycle arrest at G2/M phases in a cell-autonomous manner. Our study shows that slc20a1b is a vital regulator for HSPC proliferation in zebrafish early hematopoiesis and provides valuable insights into HSPC development.

Effects of transgenic Pit-1 overexpression on calcium phosphate and bone metabolism.

The type III inorganic phosphate (Pi) transporter Pit-1 was previously found to be preferentially expressed in developing long bones. Several studies also described a regulation of its expression in cultured bone cells by osteotropic factors, suggesting a role of this transporter in bone metabolism. In the present study, we investigated the effects of the transgenic overexpression of Pit-1 in Wistar male rats on calcium phosphate and bone metabolism. A threefold increase and doubling of Pi transport activity were recorded in primary cultured osteoblastic cells derived from calvaria of two transgenic (Tg) lines compared with wild-type littermates (WT), respectively. Skeletal development was not affected by the transgene, and bone mass, analyzed by DXA, was slightly decreased in Tg compared with WT. Enhanced Pi uptake in calvaria-derived osteoblasts from Pit-1 Tg was associated with a significantly decreased expression of alkaline phosphatase activity and a normal deposition and calcification of the collagenous matrix. In 4-month-old adult Tg rats, serum Pi and renal Pi transport were increased compared with WT. The decrease of serum Ca concentration was associated with increased serum parathyroid hormone levels. Variations in serum Pi in Pit-1 Tg rats were negatively correlated with serum fibroblast growth factor-23, whereas 1,25-dihydroxyvitamin D(3) was not affected by Pit-1 overexpression. In conclusion, transgenic Pit-1 overexpression in rats affected bone and calcium phosphate metabolism. It also decreased alkaline phosphatase activity in osteoblasts without influencing bone matrix mineralization as well as skeletal development.

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.

Vascular smooth muscle cell calcification and SLC20 inorganic phosphate transporters: effects of PDGF, TNF-alpha, and Pi.

Pi transport by vascular smooth muscle cells (VSMC) has been proposed to play an important role in the pathogenesis of vascular calcification. In this study, we have determined the correlation between calcification induced by Pi, platelet-derived growth factor (PDGF)-BB, and tumor necrosis factor-alpha and Pi transport activity in primary cultures of rat aortic VSMC. These agents induced calcification and increased the expression of Cbfa1, Msx2, and Bmp2 osteogene messenger RNA in rat aortic VSMC, while Pi transport rate was not modified per milligram of protein. Only PDGF increased Pi transport when it was expressed per unit of DNA, as PDGF also increased total cell protein by 100%, while DNA content and number of cells were not modified. PDGF increased the expression of the Pi transporter, Pit-1, but membrane protein biotinylation showed that Pit-1 abundance was not modified in the cell surface. Immunofluorescence revealed that, under basal conditions, Pit-1 is only slightly expressed at the cell membrane, but strongly expressed inside the cell. The intracellular signal colocalizes with endoplasmic reticulum (ER) markers, and PDGF increases Pit-1 expression in the ER but not the cell membrane. In conclusion, Pi transport across the plasma membrane does not correlate directly with calcification, but the expression of Pit-1 in the ER opens new possibilities for the study of the pathogenesis of vascular calcification.

Mechanism of phosphate-induced calcification in rat aortic tissue culture: possible involvement of Pit-1 and apoptosis.

BACKGROUND: Hyperphosphataemia is a known contributing factor in the progression of vascular calcification in dialysis patients. The cellular mechanisms underlying phosphate-induced calcification are still unclear despite intense study, so in this study, we investigated the possible involvement of the type III sodium-dependent phosphate cotransporter, Pit-1, in an aortic tissue culture model. METHODS: Aortic segments from 9-week-old male Sprague-Dawley rats were incubated in serum-supplemented medium for 10 days. The phosphate concentration of the medium was elevated to induce calcification, which was assessed by histology and calcium content. Phosphonoformic acid (PFA) was used to inhibit phosphate uptake. The involvement of apoptosis was examined using the terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate (dUTP) nick-end labelling (TUNEL) assay, caspase 3 activation, and inhibition of apoptosis using a general caspase inhibitor. Phenotypic changes in vascular smooth muscle cells (VSMC) were assessed using expression of osteochondrogenic differentiation markers. RESULTS: Medial vascular calcification was induced in aortas cultured in a high phosphate medium. PFA decreased the rates of calcification and apoptosis of VSMC in the media, concomitant with calcification. Caspase inhibitor reduced calcification. No phenotypic transition of VSMC was seen in this model. CONCLUSIONS: These results indicate that phosphate uptake through the type III sodium-dependent phosphate cotransporter, Pit-1, leads to induction of apoptosis and subsequent calcification of VSMC.

[Roles of hyperphosphatemia in vascular calcification].

Derangemenst of mineral metabolism including hyperphosphatemia occur along with progression of chronic kidney disease (CKD) . Recent clinical studies suggest that hyperphosphatemia is a major risk factor for vascular calcification and cardiovascular mortality in dialysis patients. Two pathophysiological processes are involved in the development of vascular calcification : apoptosis and phenotypic transition to chondrocytes or osteoblasts (chondro-/osteogenic differentiation) . Inorganic phosphate has been demonstrated to induce apoptosis and calcification of vascular smooth muscle cells through inhibiting gas6/Axl/PI3K/Akt pathway (cell survival pathway) . Moreover, inorganic phosphate has been shown to promote in vitro calcification of vascular wall cells by stimulating osteoblastic differentiation through a type III sodium-dependent phosphate co-transporter (PiT-1) . These molecular mechanisms suggest that hyperphosphatemia may play a pivotal role in progression of vascular calcification in CKD.

Clinical outcome and mechanism of soft tissue calcification in Werner syndrome.

Werner syndrome (WS) is an autosomal recessive progeroid disorder caused by mutations in RecQ DNA helicase. Ectopic soft tissue calcification is one of the well known symptoms in WS. However, the prevalence, clinical outcome, and mechanism of such calcification remain to be elucidated. The clinical features and mechanism of ectopic calcification were examined in seven patients with WS whose diagnosis were confirmed by a genomic DNA analysis. X-ray examinations revealed subcutaneous calcification in 35 of 41 major joints (85.3%). The patients complained of dermal pain at 23 joints among 35 joints (65.7%) with calcification. Refractory skin ulcers were found at the area of the skin overlaying the calcification in 16 joints (45.7%). In contrast, no pain or ulcers were observed in the joints without calcification. The presence of ectopic calcification could not be explained by a systemic hormonal abnormality. Cultured fibroblasts from WS patients underwent spontaneous mineralization in vitro in the normal phosphate condition, and overexpressed Pit-1, a transmembrane type III Na-Pi cotransporter both at the mRNA and protein levels. Phosphonophormic acid, a specific inhibitor for Pit-1, inhibited mineralization in the WS fibroblasts. Both calcification and Pit-1 overexpression were detected in the skin of WS in situ. WS showed a high prevalence of ectopic calcification, which was associated with dermal pain and refractory skin ulcers. An overexpression of Pit-1 therefore seems to play a key role in the formation of soft tissue calcification in this syndrome.

SLC20A2 Deficiency in Mice Leads to Elevated Phosphate Levels in Cerbrospinal Fluid and Glymphatic Pathway-Associated Arteriolar Calcification, and Recapitulates Human Idiopathic Basal Ganglia Calcification.

Idiopathic basal ganglia calcification is a brain calcification disorder that has been genetically linked to autosomal dominant mutations in the sodium-dependent phosphate co-transporter, SLC20A2. The mechanisms whereby deficiency of Slc20a2 leads to basal ganglion calcification are unknown. In the mouse brain, we found that Slc20a2 was expressed in tissues that produce and/or regulate cerebrospinal fluid, including choroid plexus, ependyma and arteriolar smooth muscle cells. Haploinsufficient Slc20a2 +/- mice developed age-dependent basal ganglia calcification that formed in glymphatic pathway-associated arterioles. Slc20a2 deficiency uncovered phosphate homeostasis dysregulation characterized by abnormally high cerebrospinal fluid phosphate levels and hydrocephalus, in addition to basal ganglia calcification. Slc20a2 siRNA knockdown in smooth muscle cells revealed increased susceptibility to high phosphate-induced calcification. These data suggested that loss of Slc20a2 led to dysregulated phosphate homeostasis and enhanced susceptibility of arteriolar smooth muscle cells to elevated phosphate-induced calcification. Together, dysregulated cerebrospinal fluid phosphate and enhanced smooth muscle cell susceptibility may predispose to glymphatic pathway-associated arteriolar calcification.

Extracellular phosphate as a signaling molecule.

Phosphorus is involved in various biological processes including membrane integrity, maintenance and inheritance of genetic materials, energy metabolism, intracellular signaling and skeletal mineralization. In addition, accumulating evidences have indicated that alteration in the levels of extracellular inorganic phosphate (Pi) itself triggers signaling to regulate gene expression and cellular functions in some cell types. In bone cells such as osteoblasts and chondrocytes, extracellular Pi modulates cell proliferation, differentiation, mineralization and apoptosis. In extraskeletal tissues, extracellular Pi also exerts various effects. For example, increased extracellular Pi results in the calcification associated with the upregulation of osteoblast marker genes in vascular smooth muscle cells. As to the mechanistic aspects, it is suggested that an increase in extracellular Pi triggers signal transduction via the PiT1 type III sodium/phosphate (Na(+)/Pi) cotransporter and ERK1/2 pathway. Unicellular organisms such as bacteria and yeast sense the environmental Pi with a protein complex located in the plasma membrane, which regulates the expression of multiple genes involved in Pi uptake and metabolism to adapt to its availability. In mammals that are multicellular organisms, Pi availability should be sensed both at a cellular level to regulate the function of each cell and as a whole body to maintain the Pi homeostasis of the extracellular fluid. Although the responsiveness to the increased extracellular Pi suggests the existence of Pi-sensing mechanism in mammalian cells as well, it is unknown whether the sensing of Pi availability at a cellular level and that at a whole-body level share the same pathway or not. This chapter will review the findings regarding the regulation of various cellular functions by extracellular Pi, and also discuss the current concept on the mechanism for Pi-sensing.

Roles of major facilitator superfamily transporters in phosphate response in Drosophila.

The major facilitator superfamily (MFS) transporter Pho84 and the type III transporter Pho89 are responsible for metabolic effects of inorganic phosphate in yeast. While the Pho89 ortholog Pit1 was also shown to be involved in phosphate-activated MAPK in mammalian cells, it is currently unknown, whether orthologs of Pho84 have a role in phosphate-sensing in metazoan species. We show here that the activation of MAPK by phosphate observed in mammals is conserved in Drosophila cells, and used this assay to characterize the roles of putative phosphate transporters. Surprisingly, while we found that RNAi-mediated knockdown of the fly Pho89 ortholog dPit had little effect on the activation of MAPK in Drosophila S2R+ cells by phosphate, two Pho84/SLC17A1-9 MFS orthologs (MFS10 and MFS13) specifically inhibited this response. Further, using a Xenopus oocyte assay, we show that MSF13 mediates uptake of [(33)P]-orthophosphate in a sodium-dependent fashion. Consistent with a role in phosphate physiology, MSF13 is expressed highest in the Drosophila crop, midgut, Malpighian tubule, and hindgut. Altogether, our findings provide the first evidence that Pho84 orthologs mediate cellular effects of phosphate in metazoan cells. Finally, while phosphate is essential for Drosophila larval development, loss of MFS13 activity is compatible with viability indicating redundancy at the levels of the transporters.

Sodium-dependent phosphate cotransporters and vascular calcification.

PURPOSE OF REVIEW: Vascular calcification is associated with cardiovascular events in patients with end-stage renal disease and diabetes. Hyperphosphatemia is a risk factor for vascular calcification in these patients. Sodium-dependent phosphate cotransporters are required for cellular phosphate uptake. This review focuses on the potential role of phosphate transport and type III sodium-dependent phosphate cotransporters in the process of vascular calcification. RECENT FINDINGS: Consistent with clinical and animal studies, elevated phosphate induces mineralization of cultured smooth muscle cells in vitro. Calcification is concomitant with osteochondrogenic phenotype change in smooth muscle cells characterized by induction of osteochondrogenic differentiation marker, Runx2, and inhibition of smooth muscle cell lineage marker, SM22. Inhibition of the type III sodium-dependent phosphate cotransporter, Pit-1, blocks phosphate-induced smooth muscle cell calcification. Moreover, the phosphate-induced osteochondrogenic phenotype modulation is also abrogated by Pit-1 inhibition. Pit-1 is upregulated by several calcification-promoting factors, including tumor necrosis factor-alpha, bone morphogenetic protein 2, platelet-derived growth factor and elevated calcium. SUMMARY: Phosphate uptake via Pit-1 is required for osteochondrogenic phenotypic change and calcification of vascular smooth muscle cells in vitro. Modulation of Pit-1 expression or its transport activity may provide a novel therapeutic target for intervention of vascular calcification.

Expression of Pit2 sodium-phosphate cotransporter during murine odontogenesis is developmentally regulated.

Different sodium-dependent inorganic phosphate (P(i)) uptake mechanisms play a major role in cellular P(i) homeostasis. The function and detailed distribution patterns of the type III Na(+)-phosphate cotransporter, PiT-2, in different organs during development are still largely unknown. We therefore examined the temporospatial expression patterns of Pit2 during murine odontogenesis. Odontoblasts were always devoid of Pit2 expression, whereas a transient, but strong, expression was detected in young secretory ameloblasts. However, the stratum intermedium and, later on, the papillary layer and cells of the subodontoblastic layer, exhibited high levels of Pit2 mRNA, which increased gradually as the tooth matured. Hormonal treatment or P(i) starvation of tooth germs in vitro did not alter Pit2 levels or patterns of expression, indicating mechanisms of regulation different from those of PiT-1 or other cell types. PiT-2 also functions as a retroviral receptor, and functional membrane-localized protein was confirmed throughout the dental papilla/pulp by demonstrating cellular permissiveness to infection by a gammaretrovirus that uses PiT-2 as a receptor. The distinct pattern of Pit2 expression during odontogenesis suggests that its P(i)-transporter function may be important for homeostasis of dental cells and not specifically for mineralization of the dental extracellular matrices. The expression of viral receptors in enamel-forming cells and the dental pulp may be of pathological significance.