FeLIX is a restriction factor for mammalian retrovirus infection.

Endogenous retroviruses (ERVs) are remnants of ancestral viral infections. Feline leukemia virus (FeLV) is an exogenous and endogenous retrovirus in domestic cats. It is classified into several subgroups (A, B, C, D, E, and T) based on viral receptor interference properties or receptor usage. ERV-derived molecules benefit animals, conferring resistance to infectious diseases. However, the soluble protein encoded by the defective envelope (env) gene of endogenous FeLV (enFeLV) functions as a co-factor in FeLV subgroup T infections. Therefore, whether the gene emerged to facilitate viral infection is unclear. Based on the properties of ERV-derived molecules, we hypothesized that the defective env genes possess antiviral activity that would be advantageous to the host because FeLV subgroup B (FeLV-B), a recombinant virus derived from enFeLV env, is restricted to viral transmission among domestic cats. When soluble truncated Env proteins from enFeLV were tested for their inhibitory effects against enFeLV and FeLV-B, they inhibited viral infection. Notably, this antiviral machinery was extended to infection with the Gibbon ape leukemia virus, Koala retrovirus A, and Hervey pteropid gammaretrovirus. Although these viruses used feline phosphate transporter 1 (fePit1) and phosphate transporter 2 as receptors, the inhibitory mechanism involved competitive receptor binding in a fePit1-dependent manner. The shift in receptor usage might have occurred to avoid the inhibitory effect. Overall, these findings highlight the possible emergence of soluble truncated Env proteins from enFeLV as a restriction factor against retroviral infection and will help in developing host immunity and antiviral defense by controlling retroviral spread.IMPORTANCERetroviruses are unique in using reverse transcriptase to convert RNA genomes into DNA, infecting germ cells, and transmitting to offspring. Numerous ancient retroviral sequences are known as endogenous retroviruses (ERVs). The soluble Env protein derived from ERVs functions as a co-factor that assists in FeLV-T infection. However, herein, we show that the soluble Env protein exhibits antiviral activity and provides resistance to mammalian retrovirus infection through competitive receptor binding. In particular, this finding may explain why FeLV-B transmission is not observed among domestic cats. ERV-derived molecules can benefit animals in an evolutionary arms race, highlighting the double-edged-sword nature of ERVs.

CTGF regulates mineralization in human mature chondrocyte by controlling Pit-1 and modulating ANK via the BMP/Smad signalling.

OBJECTIVE: Connective tissue growth factor (CTGF) exhibits potent proliferative, differentiated, and mineralizing effects, and is believed to be contribute to cartilage mineralization in Osteoarthritis (OA). However, the underlying mechanism of chondrocyte mineralization induced by CTGF remains obscure. As a key regulator of mineral responses, type III phosphate transporter 1 (Pit-1) has been associated with the pathogenesis of articular mineralization. Therefore, the primary objective of this study was to investigate whether CTGF influences the development of mature chondrocyte mineralization and the underlying mechanisms governing such mineralization. METHODS: The effect of Connective tissue growth factor (CTGF) on human C-28/I2 chondrocytes were investigated. The chondrocytes were treated with CTGF or related inhibitors, and transfected with Overexpression and siRNA transfection of Type III Phosphate Transporter 1(Pit-1). Subsequently, the cells were subjected to Alizarin red S staining, PiPer Phosphate Assay Kit, Alkaline Phosphatase Diethanolamine Activity Kit, ELISA, RT-PCR or Western blot analysis. RESULTS: Stimulation with Connective tissue growth factor (CTGF) significantly upregulated the expression of the Type III Phosphate Transporter 1(Pit-1) and mineralization levels in chondrocytes through activation of α5ß1 integrin and BMP/Samd1/5/8 signaling pathways. Furthermore, treatment with overexpressed Pit-1 markedly increased the expression of Multipass Transmembrane Ankylosis (ANK) transporter in the cells. The inhibitory effect of CTGF receptor blockade using α5ß1 Integrin blocking antibody was demonstrated by significantly suppressed the expression of Pit-1 and ANK transporter, as well as chondrocyte mineralization. CONCLUSIONS: Our data indicate that Connective tissue growth factor (CTGF) plays a critical role inchondrocyte mineralization, which is dependent on the expression of the Type III Phosphate Transporter 1(Pit-1) and Multipass Transmembrane Ankylosis (ANK) transporter. Consequently, inhibition of CTGF activity may represent a novel therapeutic approach for the management of Osteoarthritis (OA).

cis-Golgi phosphate transporters harboring an EXS domain are essential for plant growth and development.

Cell wall synthesis and protein glycosylation require the import of nucleotide diphosphate-sugar conjugates into the Golgi that must be counterbalanced by phosphate (Pi) export. Numerous Golgi nucleotide-sugar transporters have been characterized, but transporters mediating Golgi Pi export remain poorly understood. We used plant and yeast genetics to characterize the role of 2 Arabidopsis (Arabidopsis thaliana) proteins possessing an EXS domain, namely ERD1A and ERD1B, in Golgi Pi homeostasis. ERD1A and ERD1B localized in cis-Golgi and were broadly expressed in vegetative and reproductive tissues. We identified ERD1 putative orthologs in algae, bryophytes, and vascular plants. Expressing ERD1A and ERD1B in yeast complemented the erd1 mutant phenotype of cellular Pi loss via exocytosis associated with reduced Golgi Pi export. The Arabidopsis erd1a mutant had a similar phenotype of apoplastic Pi loss dependent on exocytosis. ERD1A overexpression in Nicotiana benthamiana and Arabidopsis led to partial mislocalization of ERD1A to the plasma membrane and specific Pi export to the apoplastic space. Arabidopsis erd1a had defects in cell wall biosynthesis, which were associated with reduced shoot development, hypocotyl growth, cell wall extensibility, root elongation, pollen germination, pollen tube elongation, and fertility. We identified ERD1 proteins as Golgi Pi exporters that are essential for optimal plant growth and fertility.

Golgi damage caused by dysfunction of PiT-2 in primary familial brain calcification.

The Golgi apparatus is vital for protein modification and molecular trafficking. It is essential for nerve development and activity, and damage thereof is implicated in many neurological diseases. Primary familial brain calcification (PFBC) is a rare inherited neurodegenerative disease characterized by multiple brain calcifications. SLC20A2, which encodes the inorganic phosphate transporter 2 (PiT-2) protein, is the main pathogenic gene in PFBC. The PiT-2 protein is a sodium-dependent phosphate type III transporter, and dysfunction leads to a deficit in the cellular intake of inorganic phosphate (Pi) and calcium deposits. Whether the impaired Golgi apparatus is involved in the PFBC procession requires elucidation. In this study, we constructed induced pluripotent stem cells (iPSCs) derived from two PFBC patients with different SLC20A2 gene mutations (c.613G > A or del exon10) and two healthy volunteers as dependable cell models for research on pathogenic mechanism. To study the mechanism, we differentiated iPSCs into neurons and astrocytes in vitro. Our study found disruptive Golgi structure and damaged autophagy in PFBC neurons with increased activity of mTOR. We also found damaged mitochondria and increased apoptosis in the PFBC dopaminergic neurons and astrocytes. In this study, we prove that dysfunctional PiT-2 leads to an imbalance of cellular Pi, which may disrupt the Golgi apparatus with impaired autophagy, mitochondria and apoptosis in PFBC. Our study provides a new avenue for understanding nerve damage and pathogenic mechanism in brain calcifications.

A SPX domain vacuolar transporter links phosphate sensing to homeostasis in Arabidopsis.

Excess phosphate (Pi) is stored into the vacuole through Pi transporters so that cytoplasmic Pi levels remain stable in plant cells. We hypothesized that the vacuolar Pi transporters may harbor a Pi-sensing mechanism so that they are activated to deliver Pi into the vacuole only when cytosolic Pi reaches a threshold high level. We tested this hypothesis using Vacuolar Phosphate Transporter 1 (VPT1), a SPX domain-containing vacuolar Pi transporter, as a model. Recent studies have defined SPX as a Pi-sensing module that binds inositol polyphosphate signaling molecules (InsPs) produced at high cellular Pi status. We showed here that Pi-deficient conditions or mutation of the SPX domain severely impaired the transport activity of VPT1. We further identified an auto-inhibitory domain in VPT1 that suppresses its transport activity. Taking together the results from detailed structure-function analyses, our study suggests that VPT1 is in the auto-inhibitory state when Pi status is low, whereas at high cellular Pi status InsPs are produced and bind SPX domain to switch on VPT1 activity to deliver Pi into the vacuole. This thus provides an auto-regulatory mechanism for VPT1-mediated Pi sensing and homeostasis in plant cells.

A Coccidia-Specific Phosphate Transporter Is Essential for the Growth of Toxoplasma gondii Parasites.

Toxoplasma gondii is an obligate intracellular parasite that acquires all necessary nutrients from the hosts, but the exact nutrient acquisition mechanisms are poorly understood. Here, we identified three putative phosphate transporters in T. gondii. TgPiT and TgPT2 are mainly on the plasma membrane, whereas TgmPT is localized to the mitochondrion. TgPiT and TgmPT are widely present and conserved in apicomplexan parasites that include Plasmodium and Eimeria species. Nonetheless, they are dispensable for the growth and virulence of Toxoplasma. TgPT2, on the other hand, is restricted to coccidia parasites and is essential for Toxoplasma survival. TgPT2 depletion led to reduced motility and invasion, as well as growth arrest of the parasites both in vitro and in vivo. Both TgPiT and TgPT2 have phosphate transport activities and contribute to parasites' inorganic phosphate (Pi) absorption. Interestingly, the Pi importing activity of Toxoplasma parasites could be competitively inhibited by ATP and AMP. Furthermore, direct uptake of 32P-ATP was also observed, indicating the parasites' ability to scavenge host ATP. Nonetheless, ATP/AMP import is not mediated by TgPiT or TgPT2, suggesting additional mechanisms. Together, these results show the complex pathways of phosphate transport in Toxoplasma, and TgPT2 is a potential target for antitoxoplasmic intervention design due to its essential role in parasite growth. IMPORTANCE To grow and survive within host cells, Toxoplasma must scavenge necessary nutrients from hosts to support its parasitism. Transporters located in the plasma membrane of the parasites play critical roles in nutrient acquisition. Toxoplasma encodes a large number of transporters, but so far, only a few have been characterized. In this study, we identified two phosphate transporters, TgPiT and TgPT2, to localize to the plasma membrane of Toxoplasma. Although both TgPiT and TgPT2 possess phosphate transport activities, only the novel transporter TgPT2 was essential for parasite growth, both in vitro and in vivo. In addition, TgPT2 and its orthologs are only present in coccidia parasites. As such, TgPT2 represents a potential target for drug design against toxoplasmosis. In addition, our data indicated that Toxoplasma can take up ATP and AMP from the environment, providing new insights into the energy metabolism of Toxoplasma.

Multiple PHT1 family phosphate transporters are recruited for mycorrhizal symbiosis in Eucalyptus grandis and conserved PHT1;4 is a requirement for the arbuscular mycorrhizal symbiosis.

Eucalypts engage in a mutualistic endosymbiosis with arbuscular mycorrhizal (AM) fungi to acquire mineral nutrients from soils, particularly inorganic phosphate (Pi). In return, the host plant provides organic carbons to its fungal partners. However, the mechanism by which the Eucalyptus plants acquire Pi released from the AM fungi has remained elusive. In this study, we investigated the characterization of potential PHOSPHATE TRANSPORTER1 (PHT1) family Pi transporters in AM symbiosis in Eucalyptus grandis W. Hill ex Maiden. We show that multiple PHT1 family Pi transporters were recruited for AM symbiosis in E. grandis. We further report that EgPT4, an E. grandis member of the PHT1 family, is conserved across angiosperms and is exclusively expressed in AM roots with arbuscule-containing cells and localizes to the periarbuscular membrane (PAM). EgPT4 was able to complement a yeast mutant strain defective in all inorganic Pi transporters and mediate Pi uptake. Importantly, EgPT4 is essential for improved E. grandis growth, total phosphorus concentration and arbuscule development during symbiosis. Moreover, silencing of EgPT4 led to the induction of polyphosphate accumulation relevant genes of Rhizophagus irregularis DAOM 197198. Collectively, our results unravel a pivotal role for EgPT4 in symbiotic Pi transport across the PAM required for arbuscule development in E. grandis.

MicroRNA-17-5p Promotes Vascular Calcification by Targeting ANKH.

BACKGROUND: MicroRNAs (miRNAs) may participate in the process of vascular calcification. However, the role of microRNA-17-5p in vascular calcification has not been clarified. In this study, we showed the effects of microRNA-17-5p on vascular calcification. MATERIALS AND METHODS: Vascular smooth muscle cells (VSMCs) were transfected with miR-17-5p mimics, a miR-17-5p inhibitor or negative control (NC) using Lipofectamine 2000. Then the cells were induced by an osteogenic medium. Alkaline phosphatase (ALP) activity and mineralization were determined. Osteocalcin (OC), bone morphogenetic protein 2(BMP-2), Collagen Ia (Colla), Runx2, and ankylosis protein homolog (ANKH) gene expressions were determined by reverse transcription-polymerase chain reaction. Vascular calcification was developed using a renal failure model. RESULTS: The ALP activity was increased when miR-17-5p mimics were transfected, whereas the miR-17-5p inhibitor reduced ALP activity (p < 0.05). The number and average area of mineral nodes in the miR-17-5p mimic group was larger than those in the corresponding control and NC groups (p < 0.05). The number and average area of the mineral nodes in the miR-17-5p inhibitor group were smaller than those in the corresponding control and NC groups (p < 0.05). Bmp2, OC, Col1a and Runx2 were higher in the miR-17-5p mimics group compared to those in the control and NC groups. ANKH expression was decreased in VSMCs with the miR-17-5p mimics and increased in VSMCs with miR-17-5p inhibitor. ANKH siRNA intervention also promoted mineralization. The miR-17-5p expression was upregulated and ANKH was down-regulated in the aortic arteries with calcification. CONCLUSION: Our data showed that miR-17-5p may promote vascular calcification by inhibiting ANKH expression.

Hyperglycemic environment disrupts phosphate transporter function and promotes calcification processes in podocytes and isolated glomeruli.

Soft tissue calcification is a pathological phenomenon that often occurs in end-stage chronic kidney disease (CKD), which is caused by diabetic nephropathy, among other factors. Hyperphosphatemia present during course of CKD contributes to impairments in kidney function, particularly damages in the glomerular filtration barrier (GFB). Essential elements of the GFB include glomerular epithelial cells, called podocytes. In the present study, we found that human immortalized podocytes express messenger RNA and protein of phosphate transporters, including NaPi 2c (SLC34A3), Pit 1 (SLC20A1), and Pit 2 (SLC20A2), which are sodium-dependent and mediate intracellular phosphate (Pi) transport, and XPR1, which is responsible for extracellular Pi transport. We found that cells that were grown in a medium with a high glucose (HG) concentration (30 mM) expressed less Pit 1 and Pit 2 protein than podocytes that were cultured in a standard glucose medium (11 mM). We found that exposure of the analyzed transporters in the cell membrane of the podocyte is altered by HG conditions. We also found that the activity of tissue nonspecific alkaline phosphatase increased in HG, causing a rise in Pi generation. Additionally, HG led to a reduction of the amount of ectonucleotide pyrophosphatase/phosphodiesterase 1 in the cell membrane of podocytes. The extracellular concentration of pyrophosphate also decreased under HG conditions. These data suggest that a hyperglycemic environment enhances the production of Pi in podocytes and its retention in the extracellular space, which may induce glomerular calcification.

m6A-mediated upregulation of AC008 promotes osteoarthritis progression through the miR-328-3p‒AQP1/ANKH axis.

Long noncoding RNAs (lncRNAs) have emerged as important regulators of osteoarthritis (OA), but the biological roles and clinical significance of most lncRNAs in OA are not fully understood. Microarray analysis was performed to identify differentially expressed lncRNAs, mRNAs, and miRNAs between normal and osteoarthritic cartilage. We found that AC008440.5 (abbreviated AC008), as well as AQP1 and ANKH, were highly expressed in osteoarthritic cartilage, whereas miR-328-3p was expressed at a low level in osteoarthritic cartilage. Functional assays showed that ectopic expression of AC008, AQP1, and ANKH significantly decreased chondrocyte viability and promoted chondrocyte apoptosis and extracellular matrix (ECM) degradation, whereas knockdown of AC008, AQP1, and ANKH resulted in the opposite effects. Moreover, miR-328-3p overexpression increased chondrocyte viability and attenuated chondrocyte apoptosis and ECM degradation, whereas inhibition of miR-328-3p resulted in the opposite effects. Bioinformatics analysis, RNA immunoprecipitation (RIP), and luciferase assays revealed that AC008 functioned as a competing endogenous RNA (ceRNA) to regulate miR-328-3p, which specifically targeted the AQP1 and ANKH genes. In addition, miR-328-3p significantly ameliorated MIA-induced OA, whereas AC008 accelerated OA progression in vivo. Furthermore, fat mass and obesity-associated (FTO)-mediated N6-methyladenosine demethylation downregulated AC008 transcription, while lower FTO expression led to upregulation of AC008 transcription in OA. In conclusion, our data reveal that AC008 plays a critical role in OA pathogenesis via the miR-328-3p‒AQP1/ANKH pathway, suggesting that AC008 may be a potential therapeutic target for OA.

Activation of nuclear factor-kappa B by TNF promotes nucleus pulposus mineralization through inhibition of ANKH and ENPP1.

Spontaneous mineralization of the nucleus pulposus (NP) has been observed in cases of intervertebral disc degeneration (IDD). Inflammatory cytokines have been implicated in mineralization of multiple tissues through their modulation of expression of factors that enable or inhibit mineralization, including TNAP, ANKH or ENPP1. This study examines the underlying factors leading to NP mineralization, focusing on the contribution of the inflammatory cytokine, TNF, to this pathologic event. We show that human and bovine primary NP cells express high levels of ANKH and ENPP1, and low or undetectable levels of TNAP. Bovine NPs transduced to express TNAP were capable of matrix mineralization, which was further enhanced by ANKH knockdown. TNF treatment or overexpression promoted a greater increase in mineralization of TNAP-expressing cells by downregulating the expression of ANKH and ENPP1 via NF-κB activation. The increased mineralization was accompanied by phenotypic changes that resemble chondrocyte hypertrophy, including increased RUNX2 and COL10A1 mRNA; mirroring the cellular alterations typical of samples from IDD patients. Disc organ explants injected with TNAP/TNF- or TNAP/shANKH-overexpressing cells showed increased mineral content inside the NP. Together, our results confirm interactions between TNF and downstream regulators of matrix mineralization in NP cells, providing evidence to suggest their participation in NP calcification during IDD.

PHO1 family members transport phosphate from infected nodule cells to bacteroids in Medicago truncatula.

Legumes play an important role in the soil nitrogen availability via symbiotic nitrogen fixation (SNF). Phosphate (Pi) deficiency severely impacts SNF because of the high Pi requirement of symbiosis. Whereas PHT1 transporters are involved in Pi uptake into nodules, it is unknown how Pi is transferred from the plant infected cells to nitrogen-fixing bacteroids. We hypothesized that Medicago truncatula genes homologous to Arabidopsis PHO1, encoding a vascular apoplastic Pi exporter, are involved in Pi transfer to bacteroids. Among the seven MtPHO1 genes present in M. truncatula, we found that two genes, namely MtPHO1.1 and MtPHO1.2, were broadly expressed across the various nodule zones in addition to the root vascular system. Expressions of MtPHO1.1 and MtPHO1.2 in Nicotiana benthamiana mediated specific Pi export. Plants with nodule-specific downregulation of both MtPHO1.1 and MtPHO1.2 were generated by RNA interference (RNAi) to examine their roles in nodule Pi homeostasis. Nodules of RNAi plants had lower Pi content and a three-fold reduction in SNF, resulting in reduced shoot growth. Whereas the rate of 33Pi uptake into nodules of RNAi plants was similar to control, transfer of 33Pi from nodule cells into bacteroids was reduced and bacteroids activated their Pi-deficiency response. Our results implicate plant MtPHO1 genes in bacteroid Pi homeostasis and SNF via the transfer of Pi from nodule infected cells to bacteroids.

Molecular Mechanisms of Phosphate Sensing, Transport and Signalling in Streptomyces and Related Actinobacteria.

: Phosphorous, in the form of phosphate, is a key element in the nutrition of all living beings. In nature, it is present in the form of phosphate salts, organophosphates, and phosphonates. Bacteria transport inorganic phosphate by the high affinity phosphate transport system PstSCAB, and the low affinity PitH transporters. The PstSCAB system consists of four components. PstS is the phosphate binding protein and discriminates between arsenate and phosphate. In the Streptomyces species, the PstS protein, attached to the outer side of the cell membrane, is glycosylated and released as a soluble protein that lacks its phosphate binding ability. Transport of phosphate by the PstSCAB system is drastically regulated by the inorganic phosphate concentration and mediated by binding of phosphorylated PhoP to the promoter of the PstSCAB operon. In Mycobacterium smegmatis, an additional high affinity transport system, PhnCDE, is also under PhoP regulation. Additionally, Streptomyces have a duplicated low affinity phosphate transport system encoded by the pitH1-pitH2 genes. In this system phosphate is transported as a metal-phosphate complex in simport with protons. Expression of pitH2, but not that of pitH1 in Streptomyces coelicolor, is regulated by PhoP. Interestingly, in many Streptomyces species, three gene clusters pitH1-pstSCAB-ppk (for a polyphosphate kinase), are linked in a supercluster formed by nine genes related to phosphate metabolism. Glycerol-3-phosphate may be transported by the actinobacteria Corynebacterium glutamicum that contains a ugp gene cluster for glycerol-3-P uptake, but the ugp cluster is not present in Streptomyces genomes. Sugar phosphates and nucleotides are used as phosphate source by the Streptomyces species, but there is no evidence of the uhp gene involved in the transport of sugar phosphates. Sugar phosphates and nucleotides are dephosphorylated by extracellular phosphatases and nucleotidases. An isolated uhpT gene for a hexose phosphate antiporter is present in several pathogenic corynebacteria, such as Corynebacterium diphtheriae, but not in non-pathogenic ones. Phosphonates are molecules that contains phosphate linked covalently to a carbon atom through a very stable C-P bond. Their utilization requires the phnCDE genes for phosphonates/phosphate transport and genes for degradation, including those for the subunits of the C-P lyase. Strains of the Arthrobacter and Streptomyces genera were reported to degrade simple phosphonates, but bioinformatic analysis reveals that whole sets of genes for putative phosphonate degradation are present only in three Arthrobacter species and a few Streptomyces species. Genes encoding the C-P lyase subunits occur in several Streptomyces species associated with plant roots or with mangroves, but not in the laboratory model Streptomyces species; however, the phnCDE genes that encode phosphonates/phosphate transport systems are frequent in Streptomyces species, suggesting that these genes, in the absence of C-P lyase genes, might be used as surrogate phosphate transporters. In summary, Streptomyces and related actinobacteria seem to be less versatile in phosphate transport systems than Enterobacteria.

Expressing Phosphate Transporter PvPht2;1 Enhances P Transport to the Chloroplasts and Increases Arsenic Tolerance in Arabidopsis thaliana.

Arsenic (As) contamination in soils is of great concerns due to its toxicity to plants. As an analogue, phosphorus plays an important role in protecting plants from As toxicity. In this study, we identified a new phosphate transporter 2 (PHT2), PvPht2;1, from As-hyperaccumulator Pteris vittata and analyzed its functions in As and P transport in a yeast mutant, and model plant Arabidopsis thalian. PvPht2;1 contained 12 transmembrane domains, sharing high identity with PHT2 genes in diverse plants. Further, independent of external P or As levels, PvPht2;1 was mainly expressed in P. vittata fronds with the expression being 3-4 folds higher than that in the roots and rhizomes. Localized to the chloroplasts based on GFP-fused PvPht2;1 in model plant tobacco, PvPht2;1 functioned as a low-affinity P transporter. Under As exposure, PvPht2;1 yeast transformants showed comparable growth with the control while high-affinity P transporter PvPht1;3 transformants showed better growth, suggesting that PvPht2;1 transported P but slower than PvPht1;3 transporter. Expressing PvPht2;1 in A. thaliana increased its shoot P concentration without influencing its As accumulation. Further, the chloroplasts' P content in transgenic A. thaliana increased by 37-59% than wild-type (WT) plants. Under As exposure, the photosynthesis of PvPht2;1-expressing A. thaliana remained stable but that of WT plants decreased. The data indicate that, under As stress, expressing PvPht2;1 in A. thaliana enhanced its P transport to the chloroplasts and protected its photosynthesis. In short, highly expressed in the fronds and not impacted by As exposure, chloroplast-located PvPht2;1 may have protected As-hyperaccumulator P. vittata from As toxicity by efficiently transporting only P to its chloroplasts.

Genome-Wide Analysis of the Five Phosphate Transporter Families in Camelina sativa and Their Expressions in Response to Low-P.

Phosphate transporters (PHTs) play pivotal roles in phosphate (Pi) acquisition from the soil and distribution throughout a plant. However, there is no comprehensive genomic analysis of the PHT families in Camelina sativa, an emerging oilseed crop. In this study, we identified 73 CsPHT members belonging to the five major PHT families. A whole-genome triplication event was the major driving force for CsPHT expansion, with three homoeologs for each Arabidopsis ortholog. In addition, tandem gene duplications on chromosome 11, 18 and 20 further enlarged the CsPHT1 family beyond the ploidy norm. Phylogenetic analysis showed clustering of the CsPHT1 and CsPHT4 family members into four distinct groups, while CsPHT3s and CsPHT5s were clustered into two distinct groups. Promoter analysis revealed widespread cis-elements for low-P response (P1BS) specifically in CsPHT1s, consistent with their function in Pi acquisition and translocation. In silico RNA-seq analysis revealed more ubiquitous expression of several CsPHT1 genes in various tissues, whereas CsPHT2s and CsPHT4s displayed preferential expression in leaves. While several CsPHT3s were expressed in germinating seeds, most CsPHT5s were expressed in floral and seed organs. Suneson, a popular Camelina variety, displayed better tolerance to low-P than another variety, CS-CROO, which could be attributed to the higher expression of several CsPHT1/3/4/5 family genes in shoots and roots. This study represents the first effort in characterizing CsPHT transporters in Camelina, a promising polyploid oilseed crop that is highly tolerant to abiotic stress and low-nutrient status, and may populate marginal soils for biofuel production.

Ankylosis progressive homolog upregulation inhibits cell viability and mineralization during fibroblast ossification by regulating the Wnt/ß­catenin signaling pathway.

Ankylosis progressive homolog (ANKH) is associated with fibroblast ossification in ankylosing spondylitis (AS). As the human ANKH gene is poorly characterized relative to its murine counterpart, the aim of the present study was to examine ANKH expression in ligament tissue isolated from patients with AS and the role played by this gene in AS­associated fibroblast ossification. Fibroblasts were isolated from ligament tissue collected from patients with AS and ligament tissue from individuals with spinal cord fractures, then cultured. Fibroblasts from patients with AS were subsequently transfected with an ANKH overexpression vector, while those collected from individuals with spinal cord fractures were transfected with small interfering RNA specific for ANKH. Cell viability, apoptosis and mineralization were analyzed using MTT assays, flow cytometry and Alizarin Red staining, respectively. Furthermore, ANKH mRNA and protein expression levels were analyzed using reverse transcription­quantitative PCR and western blotting analysis, respectively. The expression levels of osteogenesis markers, including alkaline phosphatase, osteocalcin, Runt­related transcription factor 2, c­Myc, as well as the ß­catenin signaling protein, were also determined using western blotting. The results of the present study revealed that ANKH protein expression levels were downregulated in AS total ligament tissue extract, compared with spinal fracture ligament. Moreover, the fibroblasts derived from patients with AS exhibited an increased viability and reduced apoptosis rates, compared with the fibroblasts from patients with spinal fracture. Notably, ANKH overexpression inhibited viability, mineralization and ossification, increased the phosphorylation of ß­catenin and downregulated ß­catenin and c­Myc protein expression levels in fibroblasts from patients with AS. In addition, ANKH overexpression increased the ratio of p­ß­catenin/ß­catenin in fibroblasts from patients with AS. By contrast, ANKH silencing in fibroblasts from patients with spinal fracture resulted in the opposite effect. In conclusion, the findings of the present study suggested that ANKH may inhibit fibroblast viability, mineralization and ossification, possibly by regulating the Wnt/ß­catenin signaling pathway.

The Transcription Factor NIGT1.2 Modulates Both Phosphate Uptake and Nitrate Influx during Phosphate Starvation in Arabidopsis and Maize.

Phosphorus and nitrogen are essential macronutrients for plant growth and crop production. During phosphate (Pi) starvation, plants enhanced Pi but reduced nitrate (NO3 -) uptake capacity, and the mechanism is unclear. Here, we show that a GARP-type transcription factor NITRATE-INDUCIBLE, GARP-TYPE TRANSCRIPTIOANL REPRESSOR1.2 (NIGT1.2) coordinately modulates Pi and NO3 - uptake in response to Pi starvation. Overexpression of NIGT1.2 increased Pi uptake capacity but decreased NO3 - uptake capacity in Arabidopsis (Arabidopsis thaliana). Furthermore, the nigt1.1 nigt1.2 double mutant displayed reduced Pi uptake but enhanced NO3 - uptake under low-Pi stress. During Pi starvation, NIGT1.2 directly up-regulated the transcription of the Pi transporter genes PHOSPHATE TRANSPORTER1;1 (PHT1;1) and PHOSPHATE TRANSPORTER1;4 (PHT1;4) and down-regulated expression of NO3 - transporter gene NITRATE TRANSPORTER1.1 (NRT1.1) by binding to cis-elements in their promoters. Further genetic assays demonstrated that PHT1;1, PHT1;4, and NRT1.1 were genetically epistatic to NIGT1.2 We also identified similar regulatory pathway in maize (Zea mays). These data demonstrate that the transcription factor NIGT1.2 plays a central role in modulating low-Pi-dependent uptake of Pi and NO3 -, tending toward maintenance of the phosphorus to nitrogen balance in plants during Pi starvation.

Phosphate Transporter Profiles in Murine and Human Thymi Identify Thymocytes at Distinct Stages of Differentiation.

Thymocyte differentiation is dependent on the availability and transport of metabolites in the thymus niche. As expression of metabolite transporters is a rate-limiting step in nutrient utilization, cell surface transporter levels generally reflect the cell's metabolic state. The GLUT1 glucose transporter is upregulated on actively dividing thymocytes, identifying thymocytes with an increased metabolism. However, it is not clear whether transporters of essential elements such as phosphate are modulated during thymocyte differentiation. While PiT1 and PiT2 are both phosphate transporters in the SLC20 family, we show here that they exhibit distinct expression profiles on both murine and human thymocytes. PiT2 expression distinguishes thymocytes with high metabolic activity, identifying immature murine double negative (CD4-CD8-) DN3b and DN4 thymocyte blasts as well as immature single positive (ISP) CD8 thymocytes. Notably, the absence of PiT2 expression on RAG2-deficient thymocytes, blocked at the DN3a stage, strongly suggests that high PiT2 expression is restricted to thymocytes having undergone a productive TCRß rearrangement at the DN3a/DN3b transition. Similarly, in the human thymus, PiT2 was upregulated on early post-ß selection CD4+ISP and TCRαß-CD4hiDP thymocytes co-expressing the CD71 transferrin receptor, a marker of metabolic activity. In marked contrast, expression of the PiT1 phosphate importer was detected on mature CD3+ murine and human thymocytes. Notably, PiT1 expression on CD3+DN thymocytes was identified as a biomarker of an aging thymus, increasing from 8.4 ± 1.5% to 42.4 ± 9.4% by 1 year of age (p < 0.0001). We identified these cells as TCRγδ and, most significantly, NKT, representing 77 ± 9% of PiT1+DN thymocytes by 1 year of age (p < 0.001). Thus, metabolic activity and thymic aging are associated with distinct expression profiles of the PiT1 and PiT2 phosphate transporters.

Characterization of In Vivo Function(s) of Members of the Plant Mitochondrial Carrier Family.

Although structurally related, mitochondrial carrier family (MCF) proteins catalyze the specific transport of a range of diverse substrates including nucleotides, amino acids, dicarboxylates, tricarboxylates, cofactors, vitamins, phosphate and H+. Despite their name, they do not, however, always localize to the mitochondria, with plasma membrane, peroxisomal, chloroplast and thylakoid and endoplasmic reticulum localizations also being reported. The existence of plastid-specific MCF proteins is suggestive that the evolution of these proteins occurred after the separation of the green lineage. That said, plant-specific MCF proteins are not all plastid-localized, with members also situated at the endoplasmic reticulum and plasma membrane. While by no means yet comprehensive, the in vivo function of a wide range of these transporters is carried out here, and we discuss the employment of genetic variants of the MCF as a means to provide insight into their in vivo function complementary to that obtained from studies following their reconstitution into liposomes.

The membrane protein ANKH is crucial for bone mechanical performance by mediating cellular export of citrate and ATP.

The membrane protein ANKH was known to prevent pathological mineralization of joints and was thought to export pyrophosphate (PPi) from cells. This did not explain, however, the presence of ANKH in tissues, such as brain, blood vessels and muscle. We now report that in cultured cells ANKH exports ATP, rather than PPi, and, unexpectedly, also citrate as a prominent metabolite. The extracellular ATP is rapidly converted into PPi, explaining the role of ANKH in preventing ankylosis. Mice lacking functional Ank (Ankank/ank mice) had plasma citrate concentrations that were 65% lower than those detected in wild type control animals. Consequently, citrate excretion via the urine was substantially reduced in Ankank/ank mice. Citrate was even undetectable in the urine of a human patient lacking functional ANKH. The hydroxyapatite of Ankank/ank mice contained dramatically reduced levels of both, citrate and PPi and displayed diminished strength. Our results show that ANKH is a critical contributor to extracellular citrate and PPi homeostasis and profoundly affects bone matrix composition and, consequently, bone quality.