伊藤 美紀子

Primary renal inorganic phosphate (Pi) wasting leads to hypophosphatemia, which is associated with skeletal mineralization defects. In humans, mutations in the gene encoding the type IIc sodium-dependent phosphate transporter lead to hereditary hypophophatemic rickets with hypercalciuria, but whether Pi wasting directly causes the bone disorder is unknown. Here, we generated Npt2c-null mice to define the contribution of Npt2c to Pi homeostasis and to bone abnormalities. Homozygous mutants (Npt2c(-/-)) exhibited hypercalcemia, hypercalciuria, and elevated plasma 1,25-dihydroxyvitamin D(3) levels, but they did not develop hypophosphatemia, hyperphosphaturia, renal calcification, rickets, or osteomalacia. The increased levels of 1,25-dihydroxyvitamin D, in Npt2c(-/-) mice compared with age-matched Npt2c(+/+) mice may be the result of reduced catabolism, because we observed significantly reduced expression of renal 25-hydroxyvitamin D-24-hydroxylase mRNA but no change in 1 alpha-hydroxylase mRNA levels. Enhanced intestinal absorption of calcium (Ca) contributed to the hypercalcemia and increased urinary Ca excretion. Furthermore, plasma levels of the phosphaturic protein fibroblast growth factor 23 were significantly decreased in Npt2c(-/-) mice. Sodium-dependent Pi co-transport at the renal brush border membrane, however, was not different among Npt2c(+/+), Npt2c(+/-) and Npt2c/ mice. In summary, these data suggest that Npt2c maintains normal Ca metabolism, in part by modulating the vitamin D/fibroblast growth factor 23 axis.

Long-term supplementation of branched-chain amino acids (BCAA) improves hypoalbuminemia in patients with cirrhosis. Our previous findings have suggested that the binding of polypyrimidine-tract-binding protein (PTB) to rat albumin mRNA attenuates its translation. The aim of the present study was to investigate the role of PTB in the regulation of albumin synthesis by BCAA in human hepatoma cells. HepG2 cells were cultured in a medium containing no amino acids (AA-free medium), a medium containing only I amino acid (a BCAA: valine, leucine or isoleucine) or a medium containing all 20 amino acids (AA-complete medium). HepG2 cells cultured in AA-complete medium secreted much more albumin than cells cultured in AA-free medium, with no difference in albumin mRNA levels. In cells cultured in AA-free medium, nuclear export of PTB was observed, and the level of the albumin mRNA-PTB complex was greater than in cells cultured in AA-complete medium. Addition of amino acids stimulated nuclear import of PTB. However, addition of amino acids with rapamycin inhibited the nuclear import of PTB. The addition of leucine, but not of valine or isoleucine, to AA-free medium increased albumin secretion and stimulated the nuclear import of PTB. These data indicate that the mammalian target of rapamycin is involved in the regulation of PTB localization and that leucine promotes albumin synthesis by inhibiting the formation of the albumin mRNA-PTB complex. (c) 2008 Elsevier Inc. All rights reserved.

Our recent study demonstrates that polypyrimidine tract-binding protein (PTB), which is a sequence specific RNA-binding protein, attenuates albumin synthesis in a cell-free translation system. In this study, the effects of food intake on regulation of albumin synthesis through binding of PTB to albumin messenger RNA (mRNA) were investigated. Rats were divided into 1 of 3 groups: fed; fasted for 36 h; or fasted for 36 h and then refed for 24 h. No significant differences in albumin mRNA levels were found among fed, fasted and refed rats. However, a decrease in the proportion of albumin mRNA associated with polysomes was identified in fasted rats. Furthermore, UV-cross linking analysis demonstrated that levels of albumin mRNA-PTB complex were increased in liver extracts from fasted rats. No significant differences in PTB levels in liver homogenate were found among the experimental groups. However, PTB level in the cytoplasmic fraction was higher in fasted rats than in fed rats. In refed rats, PTB level in the cytoplasmic fraction returned to a level comparable to that in fed rats, but was inhibited by treatment with rapamycin, a mammalian target of rapamycin (mTOR) inhibitor. These results suggest that localization of PTB is regulated by food intake through mTOR signaling, and alterations in level of albumin mRNA-PTB complex play a role in mediating the effects of food intake on albumin synthesis in the rat liver.

Recent studies have demonstrated that klotho protein plays a role in calcium/phosphate homeostasis. The goal of the present study was to investigate the regulation of Na-Pi cotransporters in klotho mutant (kl/kl) mice. The kl/kl mice displayed hyperphosphatemia, high plasma 1,25(OH) 2D3 levels, increased activity of the renal and intestinal sodium-dependent Pi cotransporters, and increased levels of the type IIa, type IIb, and type IIc transporter proteins compared with wild-type mice. Interestingly, transcript levels of the type IIa/type IIc transporter mRNA abundance, but not transcripts levels of type IIb transporter mRNA, were markedly decreased in kl/kl mice compared with wild-type mice. Furthermore, plasma fibroblast growth factor 23 (FGF23) levels were 150-fold higher in kl/kl mice than in wild-type mice. Feeding of a low-Pi diet induced the expression of klotho protein and decreased plasma FGF23 levels in kl/kl mice, whereas colchicine treatment experiments revealed evidence of abnormal membrane trafficking of the type IIa transporter in kl/kl mice. Finally, feeding of a low-Pi diet resulted in increased type IIa Na-Pi cotransporter protein in the apical membrane in the wild-type mice, but not in kl/kl mice. These results indicate that hyperphosphatemia in klotho mice is due to dysregulation of expression and trafficking of the renal type IIa/IIc transporters rather than to intestinal Pi uptake. Copyright © 2007 the American Physiological Society.

Recent studies have demonstrated that klotho protein plays a role in calcium/ phosphate homeostasis. The goal of the present study was to investigate the regulation of Na-P(i) cotransporters in klotho mutant (kl/kl) mice. The kl/kl mice displayed hyperphosphatemia, high plasma 1,25(OH)(2)D(3) levels, increased activity of the renal and intestinal sodium-dependent Pi cotransporters, and increased levels of the type IIa, type IIb, and type IIc transporter proteins compared with wild- type mice. Interestingly, transcript levels of the type IIa/type IIc transporter mRNA abundance, but not transcripts levels of type IIb transporter mRNA, were markedly decreased in kl/kl mice compared with wild- type mice. Furthermore, plasma fibroblast growth factor 23 ( FGF23) levels were 150-fold higher in kl/kl mice than in wild- type mice. Feeding of a low-P(i) diet induced the expression of klotho protein and decreased plasma FGF23 levels in kl/kl mice, whereas colchicine treatment experiments revealed evidence of abnormal membrane trafficking of the type IIa transporter in kl/kl mice. Finally, feeding of a low-P(i) diet resulted in increased type IIa Na-P(i) cotransporter protein in the apical membrane in the wild- type mice, but not in kl/kl mice. These results indicate that hyperphosphatemia in klotho mice is due to dysregulation of expression and trafficking of the renal type IIa/IIc transporters rather than to intestinal P(i) uptake.

Hereditary hypophosphatemic rickets with hypercalciuria results from mutations of the renal type IIc Na-Pi cotransporter gene, suggesting that the type IIc transporter plays a prominent role in renal phosphate handling. The goal of the present study was to investigate the regulation of the type IIc Na-Pi cotransporter by parathyroid hormone (PTH). Type IIc Na-Pi cotransporter levels were markedly increased in thyroparathyroidectomized (TPTX) rats. Four hours after administration of PTH, type IIc transporter protein levels were markedly decreased in the apical membrane fraction but recovered to baseline levels at 24 h. Immunohistochemical analyses demonstrated the presence of the type IIc transporter in the apical membrane and subapical compartments in the proximal tubular cells in TPTX animals. After administration of PTH, the intensity of immunoreactive signals in apical and subapical type IIc transporter decreased in the renal proximal tubular cells in TPTX rats. Colchicine completely blocked the internalization of the type IIc transporter. In addition, leupeptin prevented the PTH-mediated degradation of the type IIa transporter in lysosomes but had no effect on PTH-mediated degradation of the lysosomal type IIc transporter. In PTH-treated TPTX rats, the internalization of the type IIc transporter occurred after administration of PTH(1-34) (PKA and PKC activator) or PTH(3-34) (PKC activator). Thus the present study demonstrated that PTH is a major hormonal regulator of the type IIc Na-Pi cotransporter in renal proximal tubules. Copyright © 2007 the American Physiological Society.

To investigate the mechanism of hypermsulinaemia in rats with acute liver failure induced by the administration of D-galactosamine (GaIN), we focused on the role of polyprimidine tract-binding protein (PTB) in islet insulin synthesis. Recent reports indicate that PTB binds and stabilizes mRNA encoding insulin and insulin secretory granule proteins, including islet cell autoantigen 512 (ICA512), prohormone convertase 1/3 (PC1/3), and PC2. In the present study, glucose-stimulated insulin secretion was significantly increased in GalN-treated rats compared to controls. Levels of mRNA encoding insulin 1, ICA512, and PC1/3 were increased in the pancreatic islets of GalN-treated rats. This mRNA level elevation was not prevented by pretreatment with actinomycin D. When the PTB-binding site in insulin I mRNA was incubated with the islet cytosolic fraction, the RNA-protein complex level was increased in the cytosolic fraction obtained from GalN-treated rats compared to the level in control rats. The cytosolic fraction obtained from pancreatic islets obtained from GalN-treated rats had an increased PTB level compared to the levels obtained from the pancreatic islets of control rats. These findings suggest that, in rats with acute liver failure, cytosolic PTB binds and stabilizes mRNA encoding insulin and its secretory granule proteins. (c) 2006 Elsevier B.V. All rights reserved.

In the present study, we investigated whether a diet deficient in inorganic phosphate (Pi) stimulates an ingestive behavior to seek sources of Pi. Male Wistar rats were placed in individual cages with unrestricted access to tap water and a low (LPD, 0.02% Pi) or normal (NPD, 0.6% Pi) Pi diet for 6 days. On day 7, LDP rats were given unlimited access to a solution of 25 mM potassium phosphate water (Pi-water) for 9 additional days. Rats fed LPD consumed 70-100% more Pi-water then those fed NPD. The increase in Pi-water intake resulted in a marked rise in the growth rate of rats fed LPD during day 9. A similar intake of Pi was induced after only 2 days of LPD and was associated with significant reductions in both plasma and cerebrospinal fluid (CSF) levels of Pi these levels remained low throughout Pi restriction, despite a significant intake of Pi-water. Replenishment with a high-Pi diet rapidly quenched the appetite for Pi-water and was associated with restoration of both plasma and CSF Pi levels. These findings suggest that an appetite for Pi can be induced in rats, perhaps through lowered plasma and CSF Pi levels.

During bone resorption, a large amount of inorganic phosphate (P-i) is generated within the osteoclast hemivacuole. The mechanisms involved in the disposal of this P-i are not clear. In the present study, we investigated the efflux of P-i from osteoclast-like cells. P-i efflux was activated by acidic conditions in osteoclast-like cells derived by the treatment of RAW264.7 cells with receptor activator of nuclear factor-kappa B ligand. Acid-induced P-i influx was not observed in renal proximal tubule-like opossum kidney cells, osteoblast-like MC3T3-E1 cells, or untreated RAW264.7 cells. Furthermore, P-i efflux was stimulated by extracellular P-i and several P-i analogs [phosphonoformic acid (PFA), phosphonoacetic acid, arsenate, and pyrophosphate]. P-i efflux was time dependent, with 50% released into the medium after 10 min. The efflux of P-i was increased by various inhibitors that block P-i uptake, and extracellular P-i did not affect the transport of [C-14] PFA into the osteoclast-like cells. Preloading of cells with P-i did not stimulate P-i efflux by PFA, indicating that the effect of P-i was not due to transstimulation of P-i transport. P-i uptake was also enhanced under acidic conditions. Agents that prevent increases in cytosolic free Ca2+ concentration, including acetoxymethyl ester of 1,2-bis(2-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid, 2-aminoethoxydiphenyl borate, and bongkrekic acid, significantly inhibited P-i uptake in the osteoclast-like cells, suggesting that P-i uptake is regulated by Ca2+ signaling in the endoplasmic reticulum and mitochondria of osteoclast-like cells. These results suggest that osteoclast-like cells have a unique P-i uptake/efflux system and can prevent P-i accumulation within osteoclast hemivacuoles.

Parathyroid hormone-dependent endocytosis of renal type IIc Na-P(i) cotransporter. Am J Physiol Renal Physiol 292: F395-F403, 2007. First published September 19, 2006; doi:10.1152/ajprenal.00100.2006.-Hereditary hypophosphatemic rickets with hypercalciuria results from mutations of the renal type IIc Na-P(i) cotransporter gene, suggesting that the type IIc transporter plays a prominent role in renal phosphate handling. The goal of the present study was to investigate the regulation of the type IIc Na-P(i) cotransporter by parathyroid hormone (PTH). Type IIc Na-P(i) cotransporter levels were markedly increased in thyroparathyroidectomized (TPTX) rats. Four hours after administration of PTH, type IIc transporter protein levels were markedly decreased in the apical membrane fraction but recovered to baseline levels at 24 h. Immunohistochemical analyses demonstrated the presence of the type IIc transporter in the apical membrane and subapical compartments in the proximal tubular cells in TPTX animals. After administration of PTH, the intensity of immunoreactive signals in apical and subapical type IIc transporter decreased in the renal proximal tubular cells in TPTX rats. Colchicine completely blocked the internalization of the type IIc transporter. In addition, leupeptin prevented the PTH-mediated degradation of the type IIa transporter in lysosomes but had no effect on PTH-mediated degradation of the lysosomal type IIc transporter. In PTH-treated TPTX rats, the internalization of the type IIc transporter occurred after administration of PTH(1-34) (PKA and PKC activator) or PTH(3-34) (PKC activator). Thus the present study demonstrated that PTH is a major hormonal regulator of the type IIc Na-P(i) cotransporter in renal proximal tubules.

Abnormalities of the inorganic phosphate ( Pi) reabsorption in the kidney result in various metabolic disorders. Na+-dependent Pi (Na/Pi) transporters in the brush border membrane of proximal tubular cells mediate the rate-limiting step in the overall Pi-reabsorptive process. Type IIa and type IIc Na/Pi cotransporters are expressed in the apical membrane of proximal tubular cells and mediate Na/Pi cotransport; the extent of Pi reabsorption in the proximal tubules is determined largely by the abundance of the type IIa Na/Pi cotransporter. However, several studies suggest that the type IIc cotransporter in Pi reabsorption may also play a role in this process. For example, mutation of the type IIc Na/Pi cotransporter gene results in hereditary hypophosphatemic rickets with hypercalciuria, suggesting that the type IIc transporter plays an important role in renal Pi reabsorption in humans and may be a key determinant of the plasma Pi concentration. The type IIc Na/Pi transporter is regulated by parathyroid hormone, dietary Pi, and fibroblast growth factor 23, and studies suggest a differential regulation of the IIa and IIc transporters. Indeed, differences in temporal and/or spatial expression of the type IIa and type IIc Na/Pi transporters may be required for normal phosphate homeostasis and bone development. This review will briefly summarize the regulation of renal Pi transporters in various Pi-wasting disorders and highlight the role of a relatively new member of the Na/Pi cotransporter family: the type IIc Na/Pi transporter/ SLC34A3. Copyright (c) 2007 S. Karger AG, Basel.

Serotonin transporters (SERTs) are pre-synaptic proteins specialized for the clearance of serotonin following vesicular release at central nervous system (CNS) and enteric nervous system synapses. SERTs are high affinity targets in vivo for antidepressants such as serotonin selective reuptake inhibitors (SSRIs). These include 'medical' psychopharmacological agents such as analgesics and antihistamines, a plant extract called St John's Wort (Hypericum). Osteoclasts are the primary cells responsible for bone resorption. They arise by the differentiation of osteoclast precursors of the monocyte/macrophage lineage. The expression of SERTs was increased in RANKL-induced osteoclast-like cells. Using RANKL stimulation of RAW264.7 cells as a model system for osteoclast differentiation, we studied the direct effects of food factor on serotonin uptake. The SSRIs (fluoxetine and fluvoxamine) inhibited markedly (∼95%) in serotonin transport in differentiated osteoclast cells. The major components of St. John's Wort, hyperforin and hypericine were significantly decreased in serotonin transport activity. Thus, a new in vitro model using RANKL-induced osteoclast-like cells may be useful to analyze the regulation of SERT by food factors and SSRIs.

The mechanisms by which fibroblast growth factor 23 (FGF23) alters inorganic phosphate (Pi) homeostasis is not entirely clear. In the present study, we examined the effect of FGF23 on intestinal sodium-dependent Pi transport in mice. Injection of FGF23(R179Q) markedly reduced serum Pi and 1,25(OH)(2)D-3 levels in normal mice. Those animals show the reduction of intestinal sodiumdependent Pi transport activity and the amount of type Ilb sodium-dependent Pi cotransporter (type Ilb NaPi) protein in the brush border membrane vesicles. In vitamin D receptor null mice (VDR-/-), FGF23(R179Q) had no effect on intestinal sodium-dependent Pi transport activity and type Ilb NaPi protein levels. The present study suggests that FGF23(R179Q) reduces intestinal sodium-dependent Pi transport activity and type Ilb NaPi protein levels by a mechanism that is dependent on VDR.

FGF23 (fibroblast growth factor 23) is a novel phosphaturic factor that influences vitamin D metabolism and renal re-absorption of P-i. The goal of the present study was to characterize the role of the VDR (vitamin D receptor) in FGF23 action using VDR(-/-) (VDR null) mice. Injection of FGF23M (naked DNA encoding the R179Q mutant of human FGF23) into VDR(-/-) and wildtype VDR(+/+) mice resulted in an elevation in serum FGF23 levels, but had no effect on serum calcium or parathyroid hormone levels. In contrast, injection of FGF23M resulted in significant decreases in serum P-i levels, renal Na/P-i co-transport activity and type II transporter protein levels in both groups when compared with controls injected with mock vector or with FGFWT (naked DNA encoding wild-type human FGF23). Injection of FGF23M resulted in a decrease in 25-hydroxyvitamin D 1 alpha-hydroxylase mRNA levels in VDR(-/-) and VDR(+/+) mice, while 25-hydroxyvitamin D 24-hydroxylase mRNA levels were significantly increased in FGF23M-treated animals compared with mock vector control- or FGF23WT-treated animals. The degree of 24-hydroxylase induction by FGF23M was dependent on the VDR, since FGF23M significantly reduced the levels of serum 1,25(OH)(2)D-3 [1,25-hydroxyvitamin D-3] in VDR(+/+) mice, but not in VDR(-/-) mice. We conclude that FGF23 reduces renal P-i transport and 25-hydroxyvitamin D 1 alpha-hydroxylase levels by a mechanism that is independent of the VDR. In contrast, the induction of 25-hydroxyvitamin D 24-hydroxylase and the reduction of serum 1,25(OH)(2)D-3 levels induced by FGF23 are dependent on the VDR.

Fibroblast growth factor-23 (FGF-23) has been recently identified as playing an important pathophysiological role in phosphate homeostasis and vitamin D metabolism. To elucidate the precise physiological regulation of FGF-23, we characterized the mouse FGF-23 5'-flanking region and analyzed its promoter activity. The 5'-flanking region of the mouse FGF-23 gene contained a TFIID site ( TATA box) and several putative transcription factor binding sites, including MZF1, GATA-1 and c-Ets-1 motifs, but it did not contain the typical sequences of the vitamin D response element. Plasmids encoding 554-bp (pGL/-0.6), 364-bp (pGL/-0.4) and 200-bp (pGL/-0.13) promoter regions containing the TFIID element and +1-bp fragments drove the downstream expression of a luciferase reporter gene in transfection assays. We also found that FGF-23 mRNA was expressed in K-562 erythroleukemia cell lines but not in MC3T3-E1, Raji, or Hep G2 human carcinoma cells. Treatment with 1,25-dihydroxyvitamin D-3 in the presence of high phosphate markedly stimulated pGL/-0.6 activity, but calcium had no effect. In addition, the plasma FGF-23 levels were affected by the dietary and plasma inorganic phosphate concentrations. Finally, the levels of plasma FGF-23 in vitamin D receptor-null mice were significantly lower than in wild-type mice. The presents study demonstrated that vitamin D and the plasma phosphate level are important regulators of the transcription of the mouse FGF-23 gene.

Osteoclasts possess inorganic phosphate ( P-i) transport systems to take up external P-i during bone resorption. In the present study, we characterized P-i transport in mouse osteoclast-like cells that were obtained by differentiation of macrophage RAW264.7 cells with receptor activator of NF-kappa B ligand ( RANKL). In undifferentiated RAW264.7 cells, P-i transport into the cells was Na+ dependent, but after treatment with RANKL, Na+-independent P-i transport was significantly increased. In addition, compared with neutral pH, the activity of the Na+-independent P-i transport system in the osteoclast-like cells was markedly enhanced at pH 5.5. The Na+-independent system consisted of two components with K-m of 0.35 mM and 7.5 mM. The inhibitors of P-i transport, phosphonoformic acid, and arsenate substantially decreased P-i transport. The proton ionophores nigericin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone as well as a K+ ionophore, valinomycin, significantly suppressed P-i transport activity. Analysis of BCECF fluorescence indicated that P-i transport in osteoclast-like cells is coupled to a proton transport system. In addition, elevation of extracellular K+ ion stimulated P-i transport, suggesting that membrane voltage is involved in the regulation of P-i transport activity. Finally, bone particles significantly increased Na+- independent P-i transport activity in osteoclast- like cells. Thus, osteoclast- like cells have a Pi transport system with characteristics that are different from those of other Na+- dependent P-i transporters. We conclude that stimulation of P-i transport at acidic pH is necessary for bone resorption or for production of the large amounts of energy necessary for acidification of the extracellular environment.

Dietary phosphate levels regulate the renal brush-border type IIa Na-P i cotransporter. Another Na-Pi cotransporter, type IIc, colocalizes with type IIa Na-Pi cotransporter in the apical membrane of renal proximal tubular cells. The goal of the present study was to determine whether dietary phosphate levels also rapidly regulate the type IIc Na-P i cotransporter. Type IIa and type IIc transporter protein levels were increased in rats chronically fed a low-Pi diet compared with those fed a normal-Pi diet. Two hours after beginning a high-P i diet, type IIa transporter levels were decreased, whereas type IIc protein levels remained unchanged. Western blot analysis of brush-border membrane prepared 4 h after beginning a high-Pi diet showed a significant reduction in type IIc transporter protein levels, and immunohistochemistry showed translocation of the type IIc-immunoreactive signal from the entire brush border to subapical membrane. Membrane fractionation studies revealed a decrease in apical membrane type IIc protein without changes in total cortical type IIc protein, which is compatible with redistribution of type IIc protein from the apical membrane to the dense membrane fraction. The microtubule-disrupting reagent colchicine prevented this reduction in apical type IIc transporter at the apical membrane but had no effect on type IIa transporter levels. These data suggest that the type IIc Na-Pi cotransporter level is rapidly regulated by rapid adaptation to dietary P i in a microtubule-dependent manner. Furthermore, the mechanisms of the internalization of the type IIc transporter are distinct from those of the type IIa transporter.

Dietary phosphate levels regulate the renal brush-border type IIa Na-P(i) cotransporter. Another Na-P(i) cotransporter, type IIc, colocalizes with type IIa Na-P(i) cotransporter in the apical membrane of renal proximal tubular cells. The goal of the present study was to determine whether dietary phosphate levels also rapidly regulate the type IIc Na-P(i) cotransporter. Type IIa and type IIc transporter protein levels were increased in rats chronically fed a low-P(i) diet compared with those fed a normal-P(i) diet. Two hours after beginning a high-P(i) diet, type IIa transporter levels were decreased, whereas type IIc protein levels remained unchanged. Western blot analysis of brush-border membrane prepared 4 h after beginning a high-P(i) diet showed a significant reduction in type IIc transporter protein levels, and immunohistochemistry showed translocation of the type IIc-immunoreactive signal from the entire brush border to subapical membrane. Membrane fractionation studies revealed a decrease in apical membrane type IIc protein without changes in total cortical type IIc protein, which is compatible with redistribution of type IIc protein from the apical membrane to the dense membrane fraction. The microtubule-disrupting reagent colchicine prevented this reduction in apical type IIc transporter at the apical membrane but had no effect on type IIa transporter levels. These data suggest that the type IIc Na-P(i) cotransporter level is rapidly regulated by rapid adaptation to dietary P(i) in a microtubule-dependent manner. Furthermore, the mechanisms of the internalization of the type IIc transporter are distinct from those of the type IIa transporter.

We previously demonstrated that the integration of albumin mRNA into functional polysomes was regulated by the supply of branched chain amino acids (BCAA) in the liver of galactosamine-treated rats. To study the mechanism of this regulation, we investigated interaction between rat liver proteins and albumin transcripts. When albumin transcript was incubated with ribosome salt wash (RSW) extracts prepare from liver, a specific RNA-protein complex (p65) formed. Competition experiments showed that a pyrimidine-rich sequence in the codin region of albumin mRNA was required for the formation of p65. The level of p65 was increased in the RSW extracts prepared from liver c galactosamine-treated rats infused with a standard amino acid formula, compared with a BCAA-enriched amino acid formula. The protein i p65 appears to be polypyrimidine tract-binding protein (PTB), because the formation of p65 was reduced in the RSW extracts pre-incubate with anti-PTB antibody. In cell-free translation analysis, immunodepletion of PTB from rabbit reticulocyte lysate caused an increase albumin translation. These results suggest that binding of PTB to albumin mRNA suppresses its translation. A supply of BCAA may interfe. with this binding and improve the translation efficiency of albumin mRNA in injured liver. (C) 2004 Elsevier B.V. All rights reserved.

Notch (N) signaling is an evolutionarily conserved mechanism that regulates many cell-fate decisions. deltex (dx) encodes an E3-ubiquitin ligase that binds to the intracellular domain of N and positively regulates N signaling. However, the precise mechanism of Dx action is unknown. Here, we found that Dx was required and sufficient to activate the expression of gene targets of the canonical Su(H)-dependent N signaling pathway. Although Dx required N and a cis-acting element that overlaps with the Su(H)-binding site, Dx activated a target enhancer of N signaling, the dorsoventral compartment boundary enhancer of vestigial (vgBE), in a manner that was independent of the Delta (DI)/Serrate (Ser) ligands- or Su(H). Dx caused N to be moved from the apical cell surface into the late-endosome, where it accumulated stably and co-localized with Dx. Consistent with this, the dx gene was required for the presence of N in the endocytic vesicles. Finally, blocking the N transportation from the plasma membrane to the late-endosome by a dominant-negative form of Rab5 inhibited the Dx-mediated activation of N signaling, suggesting that the accumulation of N in the late-endosome was required for the Dx-mediated Su(H)independent N signaling.

Recent studies suggest that vitamin D may play a role in intestinal Na +-dependent phosphate transport adaptation to variable levels of dietary Pi. Therefore, the goal of the current study was to assess Na+-dependent Pi cotransport activity in transgenic mice to determine whether vitamin D is an essential mediator of this process. Intestinal brush-border membrane (BBM), Na+-dependent Pi cotransport activity was significantly decreased in vitamin D receptor (VDR) null [VDR (-/-)] mice compared with wild-type (VDR+/+) mice. While intestinal Na-Pi cotransporter (type IIb) mRNA levels were similar in VDR (-/-) and VDR (+/+) mice, type IIb Na-Pi cotransporter protein expression was markedly suppressed in VDR (-/-) mice compared with VDR (+/+) mice. Furthermore, Na-Pi cotransport activity in renal BBM was similar in VDR (-/-) and VDR (+/+) mice, but type IIa Na-Pi cotransporter protein expression was decreased in VDR (-/-) mice. After administration of a low-Pi diet, type IIb protein expression was significantly increased in VDR (+/+) and VDR (-/-) mice, and type IIb protein expression was present in the intestinal BBM of VDR (-/-) mice. These data demonstrate that intestinal Na-Pi cotransport adaptation to a low-Pi diet occurs independently of vitamin D.

Recent studies suggest that vitamin D may play a role in intestinal Na(+)-dependent phosphate transport adaptation to variable levels of dietary Pi. Therefore, the goal of the current study was to assess Na(+)-dependent P(i) cotransport activity in transgenic mice to determine whether vitamin D is an essential mediator of this process. Intestinal brush-border membrane (BBM), Na(+)-dependent P(i) cotransport activity was significantly decreased in vitamin D receptor (VDR) null [VDR (-/-)] mice compared with wild-type (VDR+/+) mice. While intestinal Na-P(i) cotransporter (type IIb) mRNA levels were similar in VDR (-/-) and VDR (+/+) mice, type IIb Na-P(i) cotransporter protein expression was markedly suppressed in VDR (-/-) mice compared with VDR (+/+) mice. Furthermore, Na-P(i) cotransport activity in renal BBM was similar in VDR (-/-) and VDR (+/+) mice, but type IIa Na-P(i) cotransporter protein expression was decreased in VDR (-/-) mice. After administration of a low-P(i) diet, type IIb protein expression was significantly increased in VDR (+/+) and VDR (-/-) mice, and type IIb protein expression was present in the intestinal BBM of VDR (-/-) mice. These data demonstrate that intestinal Na-P(i) cotransport adaptation to a low-P(i) diet occurs independently of vitamin D.

The physiological regulation of renal Pi reabsorption is mediated by renal type II Na/Pi cotransporters (type IIa and type IIc). The type IIa transporter is regulated, among other factors, by dietary Pi intake and parathyroid hormone (PTH). The PTH-induced inhibition of Pi reabsorption is mediated by endocytosis of the type IIa transporter from the brush-border membrane and subsequent lysosomal degradation. Type IIa is part of the heteromeric protein complexes organized by PDZ proteins. Furthermore, during Pi depletion the type IIc Na/Pi cotransporter is induced in the apical membrane of proximal tubular cells. The type IIc transporter is also regulated by PTH via internalization, but by a vesicular transport pathway distinct from that used by the type IIc transporter. Studying the mechanisms of type IIa and type IIc transporters has increased the understanding of the control of proximal tubular Pi handling and thus of overall Pi homeostasis.

Treatment with PTH (parathyroid hormone) or a high-Pi diet causes internalization of the type IIa sodium-dependent phosphate (Na/P-i IIa) co-transporter from the apical membrane and its degradation in the lysosome. A dibasic amino acid motif (KR) in the third intracellular loop of the co-transporter is essential for protein's PTH-induced retrieval. To elucidate the mechanism of internalization of Na/Pi IIa, we identified the interacting protein for the endocytic motif by yeast two-hybrid screening. We found a strong interaction of the Na/Pi IIa co-transporter with a small protein known as the PEX19 (human peroxisomal farnesylated protein; PxF, Pex19p). PEX19 can bind to the KR motif, but not to a mutant with this motif replaced with NI residues. PEX19 is highly expressed in mouse and rat kidney. Western blot analysis indicates that PEX19 is located in the cytosolic and brush-border membrane fractions (microvilli and the subapical component). Overexpression of PEX19 stimulated the endocytosis of the Na/p(i) IIa co-transporter in opossum kidney cells in the absence of PTH. In conclusion, the present study indicates that PEX19 may be actively involved in controlling the internalization and trafficking of the Na/Pi IIa co-transporter.

Inactivating mutations and/or deletions of PHEX (Phosphate-regulating gene with Homologies to Endopeptidase on the X chromosome) are responsible for X-linked hypophosphatemic rickets in humans. In the present study, three Drosophila PHEX homologues (dPHEX-1, -2, -3) were isolated by the screening of a Drosophila cDNA library and expressed sequence tag (EST) database. The structural region involving motif II: (456)WMXXXTKXXAXXK(468) (numbered according to human PHEX), motif VI: (WW603)-W-602, and motif VIII: (CXLW749)-C-746 was conserved in the dPHEX family. Zinc-coordinating motifs (HEFTH and GENIADNGG) were also conserved in the dPHEX family. All three dPHEX genes were expressed during all stages of Drosophila development. The expression of dPHEX-1 was suppressed by dietary phosphate deprivation, but the expression of dPHEX-2 and that of dPHEX-3 were not affected. In-situ hybridization showed a ubiquitous distribution of dPHEX-1 and dPHEX-2, while dPHEX-3 was highly expressed in the larval brain. In an analysis of subcellular localization, dPHEX-1 was localized to intracellular organelles and dPHEX-3 was localized predominately in the plasma membrane of Drosophila embryonic S2 cells. Homozygosity of a dPHEX-1 mutation, a transposon insertion in the dPHEX-1 promoter region, was completely lethal at an early stage of embryonic development. The present study indicates that three homologues are likely involved in the phosphate homeostasis of Drosophila.

Notch signalling, which is highly conserved from nematodes to mammals, plays crucial roles in many developmental processes. In the Drosophila embryo, deficiency in Notch signalling results in neural hyperplasia, commonly referred to as the neurogenic phenotype. We identify a novel maternal neurogenic gene, neurotic, and show that it is essential for Notch signalling. neurotic encodes a Drosophila homolog of mammalian GDP-fucose protein O-fucosyltransferase, which adds fucose sugar to epidermal growth factor-like repeats and is known to play a crucial role in Notch signalling. neurotic functions in a cell-autonomous manner, and genetic epistasis tests reveal that Neurotic is required for the activity of the full-length but not an activated form of Notch. Further, we show that neurotic is required for Fringe activity, which encodes a fucose-specific beta1,3 N-acetylglucosaminyltransferase, previously shown to modulate Notch receptor activity. Finally, Neurotic is essential for the physical interaction of Notch with its ligand Delta, and for the ability of Fringe to modulate this interaction in Drosophila cultured cells. We present an unprecedented example of an absolute requirement of a protein glycosylation event for a ligand-receptor interaction. Our results suggest that O-fucosylation catalysed by Neurotic is also involved in the Fringe-independent activities of Notch and may provide a novel on-off mechanism that regulates ligand-receptor interactions.

Fibroblast growth factor 23 (FGF23), a phosphaturic factor, is involved in the regulation of renal inorganic phosphate (Pi) reabsorption. Proteolysis-resistant FGF23 mutants expressed in rodents reduce Pi uptake in both intestine and kidney, independent of parathyroid hormone action. In the present study, we investigated whether FGF23 affects dietary regulation of Na+-dependent Pi (Na/Pi) cotransport in the rat kidney using wild-type FGF23 and an R179Q mutant, which disrupts a consensus proteolytic cleavage motif. Rats injected with naked human FGF23 DNA (wild-type or R179Q mutant) expressed the human FGF23 transcript in the liver. In those animals, plasma calcium and parathyroid hormone levels were not affected by FGF23 (either wild-type or R179Q mutant). FGF23-R179Q did, however, significantly decrease plasma Pi and renal Na/Pi cotransport activity and also the level of type-IIc Na/Pi cotransporter protein in brush-border membrane vesicles (BBMVs) from normal rat kidney. Western blot and immunohistochemical analyses in rats fed a low-Pi diet showed the levels of types-IIa and -IIc Na/Pi cotransporters to be markedly increased. After injection of FGF23-R179Q DNA into the rats fed a low-Pi diet, the levels of the types-IIa and -IIc transporter proteins were decreased. The FGF23 mutant thus blunts the signalling of Pi deprivation to the renal type-II Na/Pi cotransporter, suggesting that the FGF23 pathway could be involved in the signalling of dietary Pi.

Dietary phosphate restriction can prevent or retard the progress of chronic renal failure (CRF) and secondary hyperparathyroidism. The klotho gene is involved in the development of a syndrome resembling human ageing, and klotho mutant mice show abnormal calcium/vitamin D metabolism, developing hyperphosphataemia and vascular calcification. Phosphate retention rescues the phenotype of klotho mice. The level of expression of klotho RNA was greatly reduced in the kidneys of all CRF patients. Dietary P-i restriction induced klotho expression, which enhances the beneficial effect of P-i restriction in patients with CRF and/or on haemodialysis.

The Notch pathway is an evolutionarily conserved signaling mechanism that is essential for cell-cell interactions. The Drosophila deltex gene regulates Notch signaling in a positive manner, and its gene product physically interacts with the intracellular domain of Notch through its N-terminal domain. Deltex has two other domains that are presumably involved in protein-protein interactions: a proline-rich motif that binds to SH3-domains, and a RING-H2 finger motif. Using an overexpression assay, we have analyzed the functional involvement of these Deltex domains in Notch signaling. The N-terminal domain of Deltex that binds to the CDC10/Ankyrin repeats of the Notch intracellular domain was indispensable for the function of Deltex. A mutant form of Deltex that lacked the proline-rich motif behaved as a dominant-negative form. This dominant-negative Deltex inhibited Notch signaling upstream of an activated, nuclear form of Notch and downstream of full-length Notch, suggesting the dominant-negative Deltex might prevent the activation of the Notch receptor. We found that Deltex formed a homo-multimer, and mutations in the RING-H2 finger domain abolished this oligomerization. The same mutations in the RING-H2 finger motif of Deltex disrupted the function of Deltex in vivo. However, when the same mutant was fused to a heterologous dimerization domain (Glutathione-S-Transferase), the chimeric protein had normal Deltex activity. Therefore, oligomerization mediated by the RING-H2 finger motif is an integral step in the signaling function of Deltex.

We investigated the regulation of system-L amino acid transporter (LAT1) during T-cell activation. In quiescent T-cells, L-leucine transport is mediated mainly by the system-L amino acid transport system and is increased significantly during T-cell activation by PMA and ionomycin. In quiescent T-cells, the LAT1 protein was heterocomplexed with 4F2 heavy chain (4F2hc) in the plasma membrane. During T-cell activation, the amounts of 4F2hc and LAT1 heterocomplex were significantly elevated compared with those in quiescent T-cells. In addition, by Northern-blot analysis, these increments were found to be due to elevated levels of LAT1 and 4F2hc mRNA. Transient expression of constructs comprising various LAT1 gene promoter fragments, which contained all three of the GC boxes, was sufficient for promoting luciferase expression in Jurkat T-cells, but the promoter of the LAT1 gene did not respond to PMA and ionomycin. Similar observations were observed in the human 4F2hc gene promoter. In nuclear run-on assay, the LAT1 and 4F2hc genes were actively transcribed even in quiescent T-cells, but the low levels of both transcripts were shown to be the result of a block to transcription elongation within the exon 1 intron 1 regions. These findings indicated that a removal of the block to mRNA elongation stimulates the induction of system-L amino acid transporter gene transcripts (LAT1 and 4F2hc) in activated T-cells.

Dietary phosphate (Pi) is an important regulator for renal Pi reabsorption. The type II sodium-dependent phosphate (Na/Pi) cotransporters (NPT2) are located at the apical membranes of renal proximal tubular cells and major functional transporters associated with renal Pi reabsorption. The yeast one-hybrid system was used to clone a transcription factor that binds to a specific sequence (Pi response element) in the promoter of the NPT2 gene. Two cDNA clones that encoded protein of the: mouse transcription factor mu E3 (TFE3) were isolated. TFE3 may participate in the transcriptional regulation of the NPT2 gene by dietary Pi.

Drosophila Argos (Aos), a secreted protein with an epidermal growth factor (EGF)-like domain, has been shown to inhibit the activation of the Drosophila EGF receptor (DER), However, it has not been determined whether Aos binds directly to DER or whether regulation of the DER activation occurs through some other mechanism, Using DER-expressing cells (DER/S2) and a recombinant DER extracellular domain-Fc fusion protein (DER-Fc), we have shown that Aos binds directly to the extracellular domain of DER with its carboxyl-terminal region, including the EGF-like domain. Furthermore, Aos can block the binding of secreted Spitz (sSpi), a transforming growth factor cr-like ligand of DER, to the extracellular domain of DER, We observed that sSpi stimulates the dimerization of both the soluble DER extracellular domain (sDER) and the intact DER in the DER/S2 cells and that Aos can block the sSpi-induced dimerization of both sDER and intact DER, Moreover, we have shown that, by directly interacting with DER, Aos and SpiAos (a chimeric protein that is composed of the N-terminal region of Spi and the C-terminal region of Aos) inhibit the dimerization and phosphorylation of DER that are induced by DER's overexpression in the absence of sSpi, These results indicate that Aos exerts its inhibitory function through dual molecular mechanisms: by blocking both the receptor dimerization and the binding of activating ligand to the receptor. This is the first description of this novel inhibitory mechanism for receptor tyrosine kinases.

Retention of inorganic phosphate (Pi) and associated hyperphosphatemia are important in the development of hyperparathyroidism secondary to renal failure. The beneficial effect of a low-Pi diet in the prevention of hyperparathyroidism can be attributed to the decrease in PTH secretion. This effect of Pi may be mediated by specific molecules in the parathyroid cell membrane. A complementary DNA encoding a Na+-Pi co-transporter, termed rat PiT-1, has been isolated from rat parathyroid. The amount of PiT-1 mRNA in the parathyroid is controlled by vitamin D and dietary Pi, which are the most important regulators of PTH secretion. The parathyroid Pi transporter may mediate the effects of extracellular Pi and PTH secretion in secondary hyperparathyroidism. In this study, we focus on the function of Na/Pi co-transporters in the parathyroid glands as inorganic Pi sensor.

Neuron-derived orphan receptor-1 (NOR-I) is a novel member of the Nur77/NGFI-B subfamily within the nuclear receptor superfamily. Recently, several proteins closely related to NOR-1 have been described. To elucidate the relationships between NOR-1 and these closely related proteins, we analyzed the human NOR-I gene and its transcripts by molecular cloning. We identified two variant NOR-I transcripts in human skeletal muscles. One variant has a different 5'-untranslated region, and the other lacks C-terminal amino acid sequences corresponding to the putative ligand binding domain. These variant sequences share the common exon-intron boundary with NOR-I mRNA, suggesting that they were generated from a single gene by alternative splicing, with the divergent points conserved between the rat and human. We also examined the promoter activities of the 5'-flanking regions of the two NOR-I transcripts (NOR-1 alpha and NOR-1 beta mRNAs) by luciferase gene transfection. We demonstrated that the 5'-flanking region of the previously described NOR-I gene, which has characteristics of a promoter found in housekeeping genes, showed potent promoter activity although the promoter for NOR-1 beta mRNAs could not be determined. (C) 1998 Elsevier Science B.V. All rights reserved.

We identified a human homologue of NOR-1 (neuron-derived orphan receptor) from the fetal brain. There are two transcripts for human NOR-1, encoding 626 amino acid residues with a calculated molecular mass of 68 kDa. The high homology between hNOR-1, mNur77/rNGFI-B/hTR3, and mNurr1/rRNR-1/hNOT indicated that these three orphan receptors form a distinct subfamily within the steroid/thyroid receptor superfamily. Human NOR-1 mRNA was detected in the adult heart and skeletal muscle as well as in the fetal brain, indicating that its expression is not restricted to events that occur during neural development. The hNOR-1 gene is more than 35 kilobases long and interrupted by seven introns. The exon-intron structure of the gene is generally conserved when compared with the steroid/thyroid receptor superfamily and is remarkably similar to that of the Nur77/NGFI-B genes. This suggests that the Nur77/NGFI-B family has evolved from a common ancestral gene. Fluorescence in situ hybridization (FISH) revealed that the gene is located on chromosome 9q.

We identified a human homologue of NOR-1 (neuron-derived orphan receptor) from the fetal brain. There are two transcripts for human NOR-1, encoding 626 amino acid residues with a calculated molecular mass of 68 kDa. The high homology between hNOR-1, mNur77/rNGFI-B/hTR3, and mNurr1/rRNR-1/hNOT indicated that these three orphan receptors form a distinct subfamily within the steroid/thyroid receptor superfamily. Human NOR-1 mRNA was detected in the adult heart and skeletal muscle as well as in the fetal brain, indicating that its expression is not restricted to events that occur during neural development. The hNOR-1 gene is more than 35 kilobases long and interrupted by seven introns. The exon-intron structure of the gene is generally conserved when compared with the steroid/thyroid receptor superfamily and is remarkably similar to that of the Nur77/NGFI-B genes. This suggests that the Nur77/NGFI-B family has evolved from a common ancestral gene. Fluorescence in situ hybridization (FISH) revealed that the gene is located on chromosome 9q.

The cDNA clone encoding HMG 2a of chick liver was isolated from a λgt11 expression library using polyclonal antibodies. DNA sequence analysis revealed an open reading frame of 201 amino acids. Comparison of the nucleotide sequences of cDNA coding for chick liver HMG 2a with pig thymus HMG 2 and human monocytic leukemia cell HMG 2 showed 70% homology, 2.0 kb and 1.2 kb mRNAs were found in newly hatched chick liver and decreased during postnatal development of chicks. © 1992.