Abstract Background This study examined the effects of animal protein‐ and plant protein‐rich diets on postprandial phosphorus metabolism in healthy male subjects. Methods The study was conducted by randomised parallel‐group comparison of healthy men aged 21–24 years. In Study 1, participants were divided into two groups and consumed either a 70% animal protein diet (AD, n = 6) or a 70% plant protein diet (PD, n = 6). In Study 2, participants were divided into three groups and consumed either AD (n = 10), PD (n = 10) or AD + DF, a 70% animal protein diet loaded with the same amount of fibre as PD (n = 9). The phosphorus contents of the diets used in this study were nearly equivalent (AD, 710.1 mg; PD, 709.7 mg; AD + DF, 708.9 mg). Blood and urine samples were collected before, and 2 and 4 h after the meal to measure phosphorus and calcium levels. Results In Study 1, PD consumption resulted in lower blood and urinary phosphorus concentrations 2 h postprandially compared with AD (p < 0.05). In Study 2, blood phosphorus levels in AD + DF after the diet remained lower, but not significantly so compared with AD, and urinary phosphorus levels were significantly lower 2 h postprandially (p < 0.05). Conclusions A plant protein‐rich diet reduced rapid postprandial increases in blood and urinary phosphorus concentrations compared with the animal protein‐rich diets, suggesting that dietary fibre may play a partial role in the postprandial decreases in blood and urinary phosphorus concentrations.

Purpose The management of dietary phosphorus in chronic kidney disease patients is an important issue. Phosphorus is often found with protein in foods. However, excessive protein restriction worsens the nutritional status of the patient; thus, phosphorus must be selectively restricted. This study aims to assess the effects of various pretreatments readily available in ordinary households on phosphorus loss in foodstuffs. Design/methodology/approach This study evaluated the retention of phosphorus in cooked chicken meat (boiled, baked, steamed and microwaved). In addition, this study incorporated various pretreatments (pounding, stabbing, cutting and enzymatic treatment) to the method exhibiting the lowest phosphorus retention (boiling) and assessed the effects on phosphorus retention. Findings Boiling (65%, vs baking, p < 0.001; vs steaming, p = 0.013; vs microwaving, p = 0.002) of the chicken meat resulted in the lowest phosphorus retention compared to the other cooking methods (baking [89%], steaming [73%] and microwaving [75%]). In addition, stabbing (58%, p = 0.009) or cutting (46%, p < 0.001) further reduced the retention of phosphorus in boiled chicken meat. The retention of phosphorus in enzyme-pretreated boiled chicken was reduced by approximately 10% compared to untreated chicken (p = 0.01). The cooking method that reduced phosphorus retention to the greatest extent was enzyme treatment prior to cutting and boiling. Originality/value This paper investigates the effects of common household cooking methods and combinations of methods on the phosphorus content of meat.

X-linked hypophosphatemia (XLH), the most common form of inheritable rickets, is caused by inactivation of phosphate-regulating gene with homologies to endopeptidases on the X chromosome (PHEX) and leads to fibroblast growth factor (FGF) 23-dependent renal inorganic phosphate (Pi) wasting. In the present study, we investigated whether maintaining Pi homeostasis with a potent vitamin D3 analog, eldecalcitol [1α,25-dihydroxy-2β-(3-hydroxypropyloxy) vitamin D3; ED71], could improve hypophosphatemic rickets in a murine model of XLH, the Hyp mouse. Vehicle, ED71, or 1,25-dihydroxyvitamin D was subcutaneously injected five times weekly in wild-type (WT) and Hyp mice for 4 weeks, from 4 to 8 weeks of age. Injection of ED71 into WT mice suppressed the synthesis of renal 1,25-dihydroxyvitamin D and promoted phosphaturic activity. In contrast, administration of ED71 to Hyp mice completely restored renal Pi transport and NaPi-2a protein levels, although the plasma-intact FGF23 levels were further increased. In addition, ED71 markedly increased the levels of the scaffold proteins, renal sodium-hydrogen exchanger regulatory factor 1, and ezrin in the Hyp mouse kidney. Treatment with ED71 increased the body weight and improved hypophosphatemia, the bone volume/total volume, bone mineral content, and growth plate structure in Hyp mice. Thus, ED71 causes FGF23 resistance for phosphate reabsorption and improves rachitic bone phenotypes in Hyp mice. In conclusion, ED71 has opposite effects on phosphate homeostasis in WT and Hyp mice. Analysis of Hyp mice treated with ED71 could result in an additional model for elucidating PHEX abnormalities.

NaPi-IIc/SLC34A3 is a sodium-dependent inorganic phosphate (Pi) transporter in the renal proximal tubules and its mutations cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH). In the present study, we created a specific antibody for opossum SLC34A3, NaPi-IIc (oNaPi-IIc), and analyzed its localization and regulation in opossum kidney cells (a tissue culture model of proximal tubular cells). Immunoreactive oNaPi-IIc protein levels increased during the proliferative phase and decreased during differentiation. Moreover, stimulating cell growth upregulated oNaPi-IIc protein levels, whereas suppressing cell proliferation downregulated oNaPi-IIc protein levels. Immunocytochemistry revealed that endogenous and exogenous oNaPi-IIc proteins localized at the protrusion of the plasma membrane, which is a phosphatidylinositol 4,5-bisphosphate (PIP2) rich-membrane, and at the intracellular vacuolar membrane. Exogenous NaPi-IIc also induced cellular vacuoles and localized in the plasma membrane. The ability to form vacuoles is specific to electroneutral NaPi-IIc, and not electrogenic NaPi-IIa or NaPi-IIb. In addition, mutations of NaPi-IIc (S138F and R468W) in HHRH did not cause cellular PIP2-rich vacuoles. In conclusion, our data anticipate that NaPi-IIc may regulate PIP2 production at the plasma membrane and cellular vesicle formation.

Itai-itai disease is thought to be the result of chronic cadmium (Cd) intoxication. Renal proximal tubules are a major target of Cd toxicity. The whole mechanism of the adverse effects of Cd remains unresolved, especially how renal damage is related to the development of bone lesions. Fibroblast growth factor 23 (FGF23) is a bone-derived phosphaturic factor that regulates vitamin D and inorganic phosphate metabolism in the kidney. To clarify the role of FGF23 on Cd toxicity, we investigated the mechanisms of Cd-induced FGF23 production in the bone. Cd injection into mice significantly increased plasma FGF23 concentrations, but did not change FGF23 mRNA expression in bone. GalNAc-T3 is involved in secreting intact FGF23. To determine potential roles of GalNAc-T3 in Cd-induced FGF23 production, we examined the effect of Cd on GalNAc-T3 mRNA expression in vivo and in vitro. GalNAc-T3 gene expression was significantly increased in the bones of Cd-injected mice. Cd also enhanced the expression of GalNAc-T3 in cultured osteosarcoma UMR106 cells and primary osteocytes. Cd activated aryl hydrocarbon receptors (AhR) and AhR were required for GalNAc-T3 gene expression induced by Cd. In addition, Cd-dependent FGF23 production was completely inhibited by an AhR antagonist. AhR siRNA markedly suppressed the stimulation of transcriptional activity by Cd. Furthermore, Cd induced AhR activation via phosphorylation of Ser-68 by p38 kinase in the nuclear export signal of AhR. Thus, Cd stimulated GalNAc-T3 gene transcription via enhanced AhR binding to the GalNAc-T3 promoter. These findings suggest that the Cd-induced increase in GalNAc-T3 suppresses proteolytic processing of FGF23 and increases serum FGF23 concentrations.

Marked hypophosphatemia is common after major hepatic resection, but the pathophysiologic mechanism remains unknown. We used a partial hepatectomy (PH) rat model to investigate the molecular basis of hypophosphatemia. PH rats exhibited hypophosphatemia and hyperphosphaturia. In renal and intestinal brush-border membrane vesicles isolated from PH rats, Na+-dependent phosphate (Pi) uptake decreased by 50%-60%. PH rats also exhibited significantly decreased levels of renal and intestinal Na+-dependent Pi transporter proteins (NaPi-IIa [NaPi-4], NaPi-IIb, and NaPi-IIc). Parathyroid hormone was elevated at 6 hours after PH. Hyperphosphaturia persisted, however, even after thyroparathyroidectomy in PH rats. Moreover, DNA microarray data revealed elevated levels of nicotinamide phosphoribosyltransferase (Nampt) mRNA in the kidney after PH, and Nampt protein levels and total NAD concentration increased significantly in the proximal tubules. PH rats also exhibited markedly increased levels of the Nampt substrate, urinary nicotinamide (NAM), and NAM catabolites. In vitro analyses using opossum kidney cells revealed that NAM alone did not affect endogenous NaPi-4 levels. However, in cells overexpressing Nampt, the addition of NAM led to a marked decrease in cell surface expression of NaPi-4 that was blocked by treatment with FK866, a specific Nampt inhibitor. Furthermore, FK866-treated mice showed elevated renal Pi reabsorption and hypophosphaturia. These findings indicate that hepatectomy-induced hypophosphatemia is due to abnormal NAM metabolism, including Nampt activation in renal proximal tubular cells.

Endoplasmic reticulum (ER) stress is induced in matrix-producing osteoblasts and plays an essential role in osteoblastogenesis. Although the bone anabolic activity of proteasome inhibitors has been demonstrated, the roles of ER stress induced by proteasome inhibition in osteoblastogenesis remain largely unknown. Here we show that bortezomib translationally increases protein levels of activating transcription factor 4 (ATF4), a downstream mediator of ER stress, in bone marrow stromal cells and MC3T3-E1 preosteoblastic cells. The suppression of ATF4 expression by siRNA abrogated osteocalcin expression and mineralized nodule formation by MC3T3-E1 cells induced by bortezomib, indicating a critical role for ATF4 in bortezomib-mediated osteoblastogenesis. However, bortezomib at 20 nM or higher abolished the mineralized nodule formation along with reductions in the expression of osteoblastogenesis mediators beta-catenin and Osterix. Furthermore, at 50 nM, bortezomib induced the expression of C/EBP homologous protein (CHOP), suggesting activation of the ATF4-CHOP pro-apoptotic pathway. These results suggest that a low dose of bortezomib induces osteogenic activity, but that, in contrast, excessive ER stress caused by bortezomib at higher doses hampers osteoblastogenesis. Therefore, dosing schedules for proteasome inhibitors warrant further study to maximize anabolic actions without compromising anti-MM activity in patients with multiple myeloma (MM).

Endoplasmic reticulum (ER) stress is induced in matrix-producing osteoblasts and plays an essential role in osteoblastogenesis. Although the bone anabolic activity of proteasome inhibitors has been demonstrated, the roles of ER stress induced by proteasome inhibition in osteoblastogenesis remain largely unknown. Here we show that bortezomib translationally increases protein levels of activating transcription factor 4 (ATF4), a downstream mediator of ER stress, in bone marrow stromal cells and MC3T3-E1 preosteoblastic cells. The suppression of ATF4 expression by siRNA abrogated osteocalcin expression and mineralized nodule formation by MC3T3-E1 cells induced by bortezomib, indicating a critical role for ATF4 in bortezomib-mediated osteoblastogenesis. However, bortezomib at 20 nM or higher abolished the mineralized nodule formation along with reductions in the expression of osteoblastogenesis mediators β-catenin and Osterix. Furthermore, at 50 nM, bortezomib induced the expression of C/EBP homologous protein (CHOP), suggesting activation of the ATF4-CHOP pro-apoptotic pathway. These results suggest that a low dose of bortezomib induces osteogenic activity, but that, in contrast, excessive ER stress caused by bortezomib at higher doses hampers osteoblastogenesis. Therefore, dosing schedules for proteasome inhibitors warrant further study to maximize anabolic actions without compromising anti-MM activity in patients with multiple myeloma (MM). © 2013 The Japanese Society of Hematology.

Glucocorticoid (GC) excess causes a rapid loss of bone with a reduction in bone formation. Intermittent PTH(1-34) administration stimulates bone formation and counteracts the inhibition of bone formation by GC excess. We have previously demonstrated that mechanical strain enhances interleukin (IL)-11 gene transcription by a rapid induction of Delta FosB expression and protein kinase C (PKC)-delta-mediated phosphorylation of phosphorylated mothers against decapentaplegic (Smad)-1. Because IL-11 suppresses the expression of dickkopf-1 and -2 and stimulates Wnt signaling, IL-11 appears to mediate at least a part of the effect of mechanical strain on osteoblast differentiation and bone formation. The present study was undertaken to examine the effect of PTH(1-34) and GCs on IL-11 expression in murine primary osteoblasts (mPOBs). PTH(1-34) treatment of mPOBs enhanced IL-11 expression in a time-and dose-dependent manner. PTH(1-34) also stimulated Delta FosB expression and Smad1 phosphorylation, which cooperatively stimulated IL-11 gene transcription. PTH(1-34)-induced Smad1 phosphorylation was mediated via PKC delta and was abrogated in mPOBs from PKC delta knockout mice. Dexamethasone suppressed IL-11 gene transcription enhanced by PTH(1-34) without affecting Delta FosB expression or Smad1 phosphorylation, and dexamethasone-GC receptor complex was bound to JunD, which forms heterodimers with Delta FosB. High doses of PTH(1-34) counteracted the effect of dexamethasone on apoptosis of mPOBs, which was blunted by neutralizing anti-IL-11 antibody or IL-11 small interfering RNA. These results demonstrate that PTH(1-34) and GCs interact to regulate IL-11 expression in parallel with osteoblast differentiation and apoptosis and suggest that PTH(1-34) and dexamethasone may regulate osteoblast differentiation and apoptosis via their effect on IL-11 expression. (Endocrinology 154: 1156-1167, 2013)

The type II sodium-dependent Pi (NaPi) cotransporters (NaPi-IIa, NaPi-IIb and NaPi-IIc) contribute to renal and intestinal Pi absorption. 1,25-Dihydroxyvitamin D [1,25(OH)(2)D-3] is an important factor for NaPi-II transporters in the small intestine and kidney. In a previous study, low levels of 1,25(OH)(2)D-3 appeared to suppress the expression of renal NaPi cotransporters. We identified a functional vitamin D receptor-responsive element in the human NaPi-IIa and NaPi-lIc genes in renal epithelial cells. In an analysis of vitamin D receptor (VDR)-null mice, we observed early onset of hypophosphatemia. The cause of the hypophosphatemia in VDR-null mice before weaning appeared to be increased plasma parathyroid hormone (PTH) levels during the suckling periods. A rescue diet (high calcium diet) decreased plasma PTH levels in VDR-null mice. The reduced plasma PTH levels normalized the renal Npt2a and Npt2c protein levels in weanling animals. Thus, the dietary intervention completely normalized the expression of the renal Pi transporters (Npt2a/Npt2c) in VDR-null mice, suggesting that the lack of VDR activity was not the cause of the impaired renal Pi reabsorption. In suckling animals, 1,25(OH)(2)D-3 may be essential for the prevention of the phosphaturic action of PTH. In adult animals, 1,25(OH)(2)D-3 is thought to be an important factor for posttranscriptional regulation of the Npt2b gene in the small intestine. Fibroblast growth factor 23 (FGF23) is a novel phosphaturic factor that influences vitamin D metabolism and renal reabsorption of Pi. We characterized the role of the VDR in the action of FGF23 using VDR-null mice. FGF23 reduced renal Pi transport and 25-hydroxyvitamin D 1a-hydroxylase levels by a mechanism that was independent of the VDR. By contrast, the induction of 25-hydroxyvitamin D 24-hydroxylase and the reduction in serum 1,25(OH)(2)D-3 levels induced by FGF23 were dependent on the VDR. Thus, the VDR is not essential for the phosphaturic action of FGF23, but is essential for control of the plasma 1,25(OH)(2)D-3 level. Moreover, FGF23 reduces intestinal NaPi transport activity and Npt2b protein levels by a mechanism that is dependent on the VDR. Klotho functions as a co-receptor for FGF23 and is increased by 1,25(OH)(2)D-3. Klotho induces phosphaturia by inhibiting the renal NaPi-Ila transporter. In this review, we discuss the roles of 1,25(OH)(2)D-3/VDR in the regulation of renal type II NaPi cotransporters in the kidney and small intestine. Copyright (C) 2013 S. Karger AG, Basel

Phosphaturia has been documented following cadmium (Cd) exposure in both humans and experimental animals. Fibroblast growth factor 23 (FGF23) serves as an essential phosphate homeostasis pathway in the bone-kidney axis. In the present study, we investigated the effects of Cd on phosphate (Pi) homeostasis in mice. Following Cd injection into C57BL/6J mice, plasma FGF23 concentration significantly increased. The urinary Pi excretion level was significantly higher in the Cd-injected C57BL/6J mice than in the control group. Plasma Pi concentration decreased only slightly in the Cd-injected mice compared with the control group. No changes were observed in the concentration of the plasma parathyroid hormone and 1,25-dihydroxy vitamin D(3) in both groups of mice. We observed a decrease in phosphate transport activity and also a decrease in the expression level of renal phosphate transporter Npt2c, but not that of Npt2a. Furthermore, we examined the effect of Cd on Npt2c in Npt2a-knockout (KO) mice, which expresses Npt2c as a major NaPi cotransporter. Injecting Cd to Npt2aKO mice induced a significant increase in plasma FGF23 concentration and urinary Pi excretion level. Furthermore, we observed decreases in phosphate transport activity and renal Npt2c expression level in the Cd-injected Npt2a KO mice. The present study suggests that hypophosphatemia induced by Cd may be closely associated with FGF23.

Haito-Sugino S, Ito M, Ohi A, Shiozaki Y, Kangawa N, Nishiyama T, Aranami F, Sasaki S, Mori A, Kido S, Tatsumi S, Segawa H, Miyamoto K. Processing and stability of type IIc sodium-dependent phosphate cotransporter mutations in patients with hereditary hypophosphatemic rickets with hypercalciuria. Am J Physiol Cell Physiol 302: C1316-C1330, 2012. First published December 7, 2012; doi:10.1152/ajpcell.00314.2011.-Mutations in the apically located Na+-dependent phosphate (NaPi) cotransporter, SLC34A3 (NaPi-IIc), are a cause of hereditary hypophosphatemic rickets with hypercalciuria (HHRH). We have characterized the impact of several HHRH mutations on the processing and stability of human NaPi-IIc. Mutations S138F, G196R, R468W, R564C, and c. 228delC in human NaPi-IIc significantly decreased the levels of NaPi cotransport activities in Xenopus oocytes. In S138F and R564C mutant proteins, this reduction is a result of a decrease in the V-max for P-i, but not the K-m. G196R, R468W, and c. 228delC mutants were not localized to oocyte membranes. In opossum kidney (OK) cells, cell surface labeling, microscopic confocal imaging, and pulse-chase experiments showed that G196R and R468W mutations resulted in an absence of cell surface expression owing to endoplasmic reticulum (ER) retention. G196R and R468W mutants could be partially stabilized by low temperature. In blue native-polyacrylamide gel electrophoresis analysis, G196R and R468W mutants were either denatured or present in an aggregation complex. In contrast, S138F and R564C mutants were trafficked to the cell surface, but more rapidly degraded than WT protein. The c. 228delC mutant did not affect endogenous NaPi uptake in OK cells. Thus, G196R and R468W mutations cause ER retention, while S138F and R564C mutations stimulate degradation of human NaPi-IIc in renal epithelial cells. Together, these data suggest that the NaPi-IIc mutants in HHRH show defective processing and stability.

Mechanical stress and parathyroid hormone (PTH) are major stimulators, and aging and glucocorticoids excess are important suppressors of osteoblast differentiation. Mechanical stress and PTH stimulate interleukin (IL)-11 expression in cells of osteoblast lineage by enhancing transcription of IL-11 gene via an increase in intracellular Ca2+. The elevated Ca2+ activates extracellular signal-regulated kinase (ERK) to enhance phosphorylation of cyclic AMP response element-binding protein (CREB), which binds to the fosB gene promoter and enhances Delta FosB expression. Delta FosB dimerizes with JunD on the IL-11 gene promoter to enhance its transcription. Both mechanical stress and PTH also stimulate phosphorylation of Smad1 via an activation of protein kinase C delta (PKC delta). Phosphorylated Smad1 binds to the IL-11 gene promoter and forms complex with Delta FosB/JunD to further enhance IL-11 gene transcription. The increased IL-11 then suppresses expression of Wnt inhibitors, including Dickkopf 1 (Dkk1) and 2, and enhances Wnt signaling to stimulate osteoblast differentiation and inhibit adipocyte differentiation. The suppression of osteoblast differentiation by aging involves a decrease in IL-11 gene transcription by a reduction in JunD binding to the activator protein (AP)-1 site of the IL-11 gene promoter. Glucocorticoids inhibit transcriptional activation of IL-11 gene by an interaction of glucocorticoid-glucocorticoid receptor (GR) complex with Delta FosB/JunD heterodimer. Thus, factors that enhance osteoblast differentiation stimulate, and those which suppress osteoblast differentiation inhibit IL-11 gene transcription, and IL-11 enhances Writ signaling by suppressing expression of its inhibitors. These observations are consistent with the notion that IL-11 mediates stimulatory and inhibitory signals of osteoblast differentiation by affecting Wnt signaling.

Mutations in the SLC34A3 gene, a sodium-dependent inorganic phosphate (Pi) cotransporter, also referred to as NaPi IIc, causes hereditary hypophosphatemic rickets with hypercalciuria (HHRH), an autosomal recessive disorder. In human and rodent, NaPi IIc is mainly localized in the apical membrane of renal proximal tubular cells. In this study, we identified mouse NaPi IIc variant (Npt2c-v1) that lacks the part of the exon 3 sequence that includes the assumed translation initiation site of Npt2c. Microinjection of mouse Npt2c-v1 cRNA into Xenopus oocytes demonstrated that Npt2c-v1 showed sodium-dependent Pi cotransport activity. The characterization of pH dependency showed activation at extracellular alkaline-pH. Furthermore, Npt2c-v1 mediated Pi transport activity was significantly higher at any pH value than those of Npt2c. In an in vitro study, the localization of the Npt2c-v1 protein was detected in the apical membrane in opossum kidney cells. The expression of Npt2c-v1 mRNA was detected in the heart, spleen, testis, uterus, placenta, femur, cerebellum, hippocampus, diencephalon and brain stem of mouse. Using mouse bone primary cultured cells, we showed the expression of Npt2c-v1 mRNA. In addition, the Npt2c protein was detected in the spermatozoa head. Thus, Npt2c-v1 was expressed in extra-renal tissues such as epididymal spermatozoa and may function as a sodium-dependent phosphate transporter.

Ohi A, Hanabusa E, Ueda O, Segawa H, Horiba N, Kaneko I, Kuwahara S, Mukai T, Sasaki S, Tominaga R, Furutani J, Aranami F, Ohtomo S, Oikawa Y, Kawase Y, Wada NA, Tachibe T, Kakefuda M, Tateishi H, Matsumoto K, Tatsumi S, Kido S, Fukushima N, Jishage K, Miyamoto K. Inorganic phosphate homeostasis in sodium-dependent phosphate cotransporter Npt2b(+/-) mice. Am J Physiol Renal Physiol 301: F1105-F1113, 2011. First published August 3, 2011; doi:10.1152/ajprenal.00663.2010.-An inorganic phosphate (P-i)-restricted diet is important for patients with chronic kidney disease and patients on hemodialysis. Phosphate binders are essential for preventing hyperphosphatemia and ectopic calcification. The sodium-dependent P-i (Na/P-i) transport system is involved in intestinal P-i absorption and is regulated by several factors. The type II sodium-dependent P-i transporter Npt2b is expressed in the brush-border membrane in intestinal epithelial cells and transports P-i. In the present study, we analyzed the phenotype of Npt2b(-/-) and hetero(+/-) mice. Npt2b(-/-) mice died in utero soon after implantation, indicating that Npt2b is essential for early embryonic development. At 4 wk of age, Npt2b(+/-) mice showed hypophosphatemia and low urinary P-i excretion. Plasma fibroblast growth factor 23 levels were significantly decreased and 1,25(OH)(2)D-3 levels were significantly increased in Npt2b(+/-) mice compared with Npt2b(+/+) mice. Npt2b mRNA levels were reduced to 50% that in Npt2b(+/+) mice. In contrast, renal Npt2a and Npt2c transporter protein levels were significantly increased in Npt2b(+/-) mice. At 20 wk of age, Npt2b(+/-) mice showed hypophosphaturia and reduced Na/P-i cotransport activity in the distal intestine. Npt2b(+/+) mice with adenine-induced renal failure had hyperphosphatemia and high plasma creatinine levels. Npt2b(+/-) mice treated with adenine had significantly reduced plasma Pi levels compared with Npt2b(+/+) mice. Intestinal Npt2b protein and Na+/P-i transport activity levels were significantly lower in Npt2b(+/-) mice than in the Npt2b(+/+) mice. The findings of the present studies suggest that Npt2b is an important target for the prevention of hyperphosphatemia.

An inorganic phosphate (P i)-restricted diet is important for patients with chronic kidney disease and patients on hemodialysis. Phosphate binders are essential for preventing hyperphosphatemia and ectopic calcification. The sodium-dependent P i (Na/P i) transport system is involved in intestinal Pi absorption and is regulated by several factors. The type II sodium-dependent P i transporter Npt2b is expressed in the brush-border membrane in intestinal epithelial cells and transports Pi. In the present study, we analyzed the phenotype of Npt2b -/-and hetero +/-mice. Npt2b -/-mice died in utero soon after implantation, indicating that Npt2b is essential for early embryonic development. At 4 wk of age, Npt2b +/-mice showed hypophosphatemia and low urinary Pi excretion. Plasma fibroblast growth factor 23 levels were significantly decreased and 1,25(OH)2D3 levels were significantly increased in Npt2b +/-mice compared with Npt2b +/+ mice. Npt2b mRNA levels were reduced to 50% that in Npt2b +/+ mice. In contrast, renal Npt2a and Npt2c transporter protein levels were significantly increased in Npt2b +/-mice. At 20 wk of age, Npt2b +/-mice showed hypophosphaturia and reduced Na/P i cotransport activity in the distal intestine. Npt2b +/+ mice with adenine-induced renal failure had hyperphosphatemia and high plasma creatinine levels. Npt2b +/-mice treated with adenine had significantly reduced plasma Pi levels compared with Npt2b +/-mice. Intestinal Npt2b protein and Na +/P i transport activity levels were significantly lower in Npt2b +/-mice than in the Npt2b +/+ mice. The findings of the present studies suggest that Npt2b is an important target for the prevention of hyperphosphatemia. © 2011 the American Physiological Society.

Inorganic phosphate (Pi) is an essential physiological compound, highlighted by the syndromes caused by hypo or hyperphosphatemic states. Hyperphosphatemia is associated with ectopic calcification, cardiovascular disease, and increased mortality in patients with chronic kidney disease (CKD). As phosphate control is not efficient with diet or dialysis, oral Pi binders are used in over 90% of patients with renal failure. However, achieving tight control of serum Pi is difficult, and lower levels of serum Pi (severe hypophosphatemia) do not lead to better outcomes. The inhibition of sodium-dependent Pi (NaPi) transporter would be a preferable method to control serum Pi levels in patients with CKD or patients undergoing dialysis. Three types of NaPi transporters (types I-III) have been identified: solute carrier series SLC17A1 (NPT1/NaPi-I/OATv1), SLC34 (NaPi-IIa, NaPi-IIb, NaPi-IIc), and SLC20 (PiT1, PiT2), respectively. Knockout mice have been created for types I-III NaPi transporters. In this review, we discuss the roles of the NaPi transporters in Pi homeostasis. (C) 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:3719-3730, 2011

Mechanical loading to bone causes mainly two types of stress to bone forming cells, fluid shear stress (FSS) and tensile stress. FSS appears to be a major stress signal that activates osteoblastic/osteocytic intracellular signaling to enhance bone formation. FSS to osteoblasts causes Ca2+ influx by activating a stress-activated cation channel, which activates extracellular signal-regulated kinase (ERK) to the phosphorylate cyclic AMP response element-binding protein (CREB). Phosphorylated CREB binds to the fosB gene promoter to enhance its transcriptional activity, and upregulates the expression of mainly ΔFosB, a C-terminal truncated splice variant of FosB. The increased ΔFosB binds to the 5′AP-1 site on the IL-11 gene promoter and forms a complex with JunD. FSS to osteoblasts also stimulates tyrosine phosphorylation of protein kinase Cδ (PKCδ), which activates Smad1/5. Activated Smad1/5 is bound to the Smad-binding element of the IL-11 gene promoter, and forms a complex with ΔFosB/JunD heterodimer. Thus, Ca2+-ERK-CREB-ΔFosB and PKCδ-Smad1/5 pathways merge together on the IL-11 gene promoter, and the two pathways cooperatively stimulate IL-11 gene expression in response to mechanical stress. The increase in interleukin (IL)-11 then enhances Wnt/β-catenin signaling by suppressing the expression of its inhibitors, dickkopf 1 and 2, in osteoblasts. Thus, the enhanced IL-11 expression by the cooperative AP-1 and Smad1/5 pathways is the upstream signal for stimulation of the main osteogenic pathway, Wnt/β-catenin signaling.

We have analyzed proteome changes associated with bone-forming osteoblast differentiation by quantitative differential proteomic and transcriptomic analyses using in vitro differentiation model. Sixty nine proteins were found up-regulated (>2-fold) and 18 were down-regulated (<0.5-fold) at protein level. The mRNA levels of these proteins were then analyzed by quantitative real-time PCR combined with clustering analysis. The most prominent cluster with increased protein and mRNA levels contains endosomal and lysosomal proteins, demonstrating the drastic induction of degradative endosomal/lysosomal pathways in osteoblasts. Osteoblasts, therefore, are involved not only in the synthesis but also in the turnover of the extracellular matrix proteins such as collagens. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.

Background: Mechanical stress rapidly induces Delta FosB expression in osteoblasts, which binds to interleukin (IL)-11 gene promoter to enhance IL-11 expression, and IL-11 enhances osteoblast differentiation. Because bone morphogenetic proteins (BMPs) also stimulate IL-11 expression in osteoblasts, there is a possibility that BMP-Smad signaling is involved in the enhancement of osteoblast differentiation by mechanical stress. The present study was undertaken to clarify whether mechanical stress affects BMP-Smad signaling, and if so, to elucidate the role of Smad signaling in mechanical stress-induced enhancement of IL-11 gene transcription. Methodology/Principal Findings: Mechanical loading by fluid shear stress (FSS) induced phosphorylation of BMP-specific receptor-regulated Smads (BR-Smads), Smad1/5, in murine primary osteoblasts (mPOBs). FSS rapidly phosphorylated Y311 of protein kinase C (PKC)delta, and phosphorylated PKC delta interacted with BR-Smads to phosphorylate BR-Smads. Transfection of PKC delta siRNA or Y311F mutant PKC delta abrogated BR-Smads phosphorylation and suppressed IL-11 gene transcription enhanced by FSS. Activated BR-Smads bound to the Smad-binding element (SBE) of IL-11 gene promoter and formed complex with Delta FosB/JunD heterodimer via binding to the C-terminal region of JunD. Site-directed mutagenesis in the SBE and the AP-1 site revealed that both SBE and AP-1 sites were required for full activation of IL-11 gene promoter by FSS. Conclusions/Significance: These results demonstrate that PKC delta-BR-Smads pathway plays an important role in the intracellular signaling in response to mechanical stress, and that a cross-talk between PKC delta-BR-Smads and Delta FosB/JunD pathways synergistically stimulates IL-11 gene transcription in response to mechanical stress.

Background: Multiple myeloma (MM) expands almost exclusively in the bone marrow and generates devastating bone lesions, in which bone formation is impaired and osteoclastic bone resorption is enhanced. TGF-beta, a potent inhibitor of terminal osteoblast (OB) differentiation, is abundantly deposited in the bone matrix, and released and activated by the enhanced bone resorption in MM. The present study was therefore undertaken to clarify the role of TGF-beta and its inhibition in bone formation and tumor growth in MM. Methodology/Principal Findings: TGF-beta suppressed OB differentiation from bone marrow stromal cells and MC3T3-E1 preosteoblastic cells, and also inhibited adipogenesis from C3H10T1/2 immature mesenchymal cells, suggesting differentiation arrest by TGF-beta. Inhibitors for a TGF-beta type I receptor kinase, SB431542 and Ki26894, potently enhanced OB differentiation from bone marrow stromal cells as well as MC3T3-E1 cells. The TGF-beta inhibition was able to restore OB differentiation suppressed by MM cell conditioned medium as well as bone marrow plasma from MM patients. Interestingly, TGF-beta inhibition expedited OB differentiation in parallel with suppression of MM cell growth. The anti-MM activity was elaborated exclusively by terminally differentiated OBs, which potentiated the cytotoxic effects of melphalan and dexamethasone on MM cells. Furthermore, TGF-beta inhibition was able to suppress MM cell growth within the bone marrow while preventing bone destruction in MM-bearing animal models. Conclusions/Significance: The present study demonstrates that TGF-beta inhibition releases stromal cells from their differentiation arrest by MM and facilitates the formation of terminally differentiated OBs, and that terminally differentiated OBs inhibit MM cell growth and survival and enhance the susceptibility of MM cells to anti-MM agents to overcome the drug resistance mediated by stromal cells. Therefore, TGF-beta appears to be an important therapeutic target in MM bone lesions.

Phosphaturia has been documented following cadmium (Cd) exposure in both humans and experimental animals. The fibroblast growth factor 23 (FGF23)/klotho axis serves as an essential phosphate homeostasis pathway in the bone-kidney axis. In the present study, we investigated the effects of Cd on phosphate (Pi) homeostasis in mice. Following Cd injection into WT mice, plasma FGF23 concentration was significantly increased. Urinary Pi excretion levels were significantly higher in Cd-injected WT mice than in control group. Plasma Pi concentration decreased only slightly compared with control group. No change was observed in plasma parathyroid hormone and 1,25-dihydroxy vitamin D3 in both group of mice. We observed a decrease in phosphate transport activity and also decrease in expression of renal phosphate transporter SLC34A3 [NaPi-IIc/NPT2c], but not SLC34A1 [NaPi-IIa/NPT2a]. Furthermore, we examined the effect of Cd on Npt2c in Npt2a-knockout (KO) mice which expresses Npt2c as a major NaPi co-transporter. Injecting Cd to Npt2aKO mice induced significant increase in plasma FGF23 concentration and urinary Pi excretion levels. Furthermore, we observed a decrease in phosphate transport activity and renal Npt2c expression in Cd-injected Npt2a KO mice. The present study suggests that hypophosphatemia induced by Cd may be closely associated with the FGF23/klotho axis.

Molecular mechanism of mechanical stress-induced bone formation remains unclear. We demonstrate that mechanical unloading suppresses and reloading enhances Interleukin (IL)-11 gene expression in the hindlimb of mice in vivo. Mechanical stress to osteoblasts by fluid shear stress (FSS) in vitro rapidly and transiently enhances fosB gene transcription, stimulates binding of Delta FosB/JunD complex to activator protein (AP)-1 site of the IL-11 gene promoter, and enhances IL-11 gene transcription. Anti-IL-11 antibody blocks mechanical stress-induced enhancement of osteoblastogenesis and suppression of adipogenesis, suggesting the requirement of IL-11 for the stimulation of osteoblast differentiation by mechanical stress. Downregulation of Delta FosB/JtmD by small interfering RNA (siRNA) suppresses and overexpression of Delta FosB/JunD enhances IL-11 gene promoter activity. Consistent with our previous observations that up-regulation of Delta FosB depends upon activation of cyclic AMP response element-binding protein (CREB) via Ca(2+)-dependent activation of extracellular signal-regulated kinase (ERK) to phosphorylate CREB, mechanical stress-induced activation of IL-11 gene transcription is dependent upon Ca(2+)-ERK pathway. Present results also demonstrated that FSS to osteoblasts enhances canonical Wnt signaling in vitro, and that mechanical unloading induces and reloading suppresses the expression of a canonical Wnt signal inhibitor, dickkopf2 (Dkk2), in vivo. In addition, IL-11 siRNA enhances Dkk2 expression suppressed by FSS, and osteoblasts from IL-11 transgenic mice show reduced Dkk2 mRNA expression than those from wild-type mice. These observations are consistent with the notion that mechanical stress stimulates IL-11 gene transcription via an enhanced OFosB/JunD binding to the IL-11 gene promoter, and that increased IL-11 enhances canonical Wnt signal at least in part via a reduction in Dkk2 expression to stimulate osteoblast differentiation. (C) 2009 Elsevier Inc. All rights reserved.

Monocytes give rise to macrophages, osteoclasts (OCs), and dendritic cells (DCs). Macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappaB (RANK) ligand induce OC differentiation from monocytes, whereas granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) trigger monocytic differentiation into DCs. However, regulatory mechanisms for the polarization of monocytic differentiation are still unclear. The present study was undertaken to clarify the mechanism of triggering the deflection of OC and DC differentiation from monocytes. GM-CSF and IL-4 abolished monocytic differentiation into OCs while inducing DC differentiation even in the presence of M-CSF and RANK ligand. GM-CSF and IL-4 in combination potently up-regulate tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE) and activity in monocytes, causing ectodomain shedding of M-CSF receptor, resulting in the disruption of its phosphorylation by M-CSF as well as the induction of osteoclastogenesis from monocytes by M-CSF and RANK ligand. Interestingly, TACE inhibition robustly causes the resumption of the surface expression of M-CSF receptor on monocytes, facilitating M-CSF-mediated phosphorylation of M-CSF receptor and macrophage/OC differentiation while impairing GM-CSF- and IL-4-mediated DC differentiation from monocytes. These results reveal a novel proteolytic regulation of M-CSF receptor expression in monocytes to control MCSF signaling and monocytic differentiation into macrophage/OC-lineage cells or DCs. (Blood. 2009;114:4517-4526)

Multiple myeloma (MM) cell adhesion to stromal cells via very late antigen (VLA)-4 and vascular cell adhesion molecule (VCAM)-1 interaction causes enhanced secretion of osteoclastogenic activity by MM cells. We have reported that MM cell-derived macrophage inflammatory protein (MIP)-1 alpha and MIP-1 beta are responsible for most of the osteoclastogenic activity in MM. Thus, adhesion-mediated osteoclastogenesis may be caused by enhanced production of MIP-1 via VLA-4-VCAM-1 interaction. The present study was undertaken to clarify whether MM cell-derived MIP-1 plays a role in VLA-4-VCAM-1 adhesion-mediated osteoclastogenesis. Adhesion of MM cells to VCAM-1 upregulated MIP-1 alpha and MIP-1 beta production from MM cells and enhanced production of osteoclastogenic activity by MM cells. Blockade of MIP-1 alpha and MIP-1 beta actions not only abrogated elaboration of osteoclastogenic activity, but also suppressed spontaneous MM cell adhesion to VCAM-1. These results demonstrate that MM cell adhesion to VCAM-1 upregulates MIP-1 production by MM cells to cause enhancement of osteoclastogenesis. In addition, the results suggest that the increased production of MIP-1 further enhances MM cell binding to stromal cells via stimulation of VLA-4-VCAM-1 adhesion, forming a "vicious cycle" between MM cell adhesion to stromal cells and MIP-1 production via VLA-4-VCAM-1 interaction.

Purpose: Similar to osteoclastogenesis, angiogenesis is enhanced in the bone marrow in myeloma in parallel with tumor progression. We showed previously that myeloma cells and osteoclasts are mutually stimulated to form a vicious cycle to lead to enhance both osteoclastogenesis and tumor growth. The present study was undertaken to clarify whether myeloma cell-osteoclast interaction enhances angiogenesis and whether there is any mutual stimulation between osteoclastogenesis and angiogenesis. Experimental Design: Myeloma cells and monocyte-derived osteoclasts were cocultured, and angiogenic activity produced by the cocultures was assessed with in vitro vascular tubule formation assays and human umbilical vascular endothelial cell (HUVEC) migration and survival. Osteoclastogenic activity was determined with rabbit bone cell cultures on dentine slices. Results: Myeloma cells and osteoclasts constitutively secrete proangiogenic factors, vascular endothelial growth factor (VEGF) and osteopontin, respectively. A cell-to-cell interaction between myeloma cells and osteoclasts potently enhanced vascular tubule formation. Blockade of both VEGF and osteopontin actions almost completely abrogated such vascular tubule formation as well as migration and survival of HUVECs enhanced by conditioned medium from cocultures of myeloma cells and osteoclasts. Furthermore, these factors in combination triggered the production of osteoclastogenic activity by HUVEC. Conclusions: Osteoclast-derived osteopontin and VEGF from myeloma cells cooperatively enhance angiogenesis and also induce osteoclastogenic activity by vascular endothelial cells. These observations suggest the presence of a close link between myeloma cells, osteoclasts, and vascular endothelial cells to form a vicious cycle between bone destruction, angiogenesis, and myeloma expansion.

c-Fos is an immediate early gene type proto-oncogene that belongs to the AP (activator protein)-1 transcription factor family. Gene knockout experiments have demonstrated that, among the Fos family, only c-Fos is indispensable for osteoclast differentiation but that c-Fos can be substituted for by other Fos family members including FosB/Delta FosB, Fra-1 and Fra-2, in most other tissues and cells. To further understand a unique role of c-Fos in osteoclastogenesis, we investigated the temporal profile and regulatory mode of expression of c-Fos during the course of osteoclast differentiation. The results indicated that c-Fos protein gradually increased in preosteoclasts during differentiation to a greater extent than that of mRNA induction. We then determined the proteolytic pathway of c-Fos conferring unstable nature on c-Fos protein in a preosteoclastic cell line, RAW264.7. Proteasome inhibitors including MG 132 and Z-LLF caused a rapid increase in c-Fos protein expression in these cells within several hours, but other inhibitors of cysteine protease (E-64), lysosome (chloroquine) and calpain (ALLM) did not. Moreover, the proteasome inhibitors caused an extensive accumulation of ubiquitinated c-Fos protein and an approximately three-fold extension of the c-Fos protein half-life. We therefore conclude that the ubiquitin-proteasome system is the major proteolytic pathway conferring instability on c-Fos protein in preosteoclasts. Our results further imply that c-Fos stabilization due to dynamic changes in the ubiquitin-proteasome-dependent degradation may be involved in the accumulation of c-Fos protein in differentiating preosteoclasts. (c) 2005 Elsevier Inc. All rights reserved.

Multiple myeloma (MM) develops devastating bone destruction with enhanced bone resorption and suppressed bone formation. In contrast to enhanced osteoclastogenesis, little is known about the mechanism of impaired bone formation in MM. Because a canonical Wingless-type (Wnt) signaling pathway has recently been shown to play an important role in osteoblast differentiation, we examined whether MM cells affect a canonical Wnt pathway to suppress bone formation. Conditioned media from RPM18226 and U266 MM cell lines and primary MM cells suppressed in vitro mineralization as well as alkaline phosphatase activity in osteoblasts induced by bone morphogenetic protein 2 (BMP-2). These cell lines constitutively produced a soluble Wnt inhibitor, secreted Frizzled-related protein 2 (sFRP-2), but not other Wnt inhibitors including sFRP-1, sFRP-3, and dickkopf 1 (DKK-1) at the protein level. Most MM cells from patients with advanced bone destructive lesions also expressed sFRP-2. Furthermore, exogenous sFRP-2 suppressed osteoblast differentiation induced by BMP-2, and immunodepletion of sFRP-2 significantly restored mineralized nodule formation in vitro, suggesting a predominant role for MM cell-derived sFRP-2 in the impairment of bone formation by MM. Thus, in addition to enhanced osteolysis, MM cells also suppress bone formation at least in part through an inhibition of the canonical Wnt pathway by secreting sFRP-2.

Purpose: Receptor activator of nuclear factor-kappa B ligand (RANKL) is a key mediator of osteoclastogenesis. Because certain types of tumor cells aberrantly express RANKL, and because bone destruction also develops in B-cell lymphomas of bone origin, we investigated RANKL expression and the mechanisms of osteoclastogenesis in B-lymphoid neoplasms. Experimental Design and Results: Immunohistochemistry of bone specimens resected from patients with primary B-cell lymphoma of bone with bone destruction revealed that lymphoma cells express RANKL as well as vascular endothelial cell growth factor (VEGF). The tumor cells isolated from the bone specimens enhanced osteoclastogenesis in vitro. In contrast, B-cell lymphoma infiltrating to the bone marrow without bone destruction did not express RANKL. Both RANKL and VEGF were expressed by a portion of B-lymphoid cell lines, including Daudi and IM-9. These RANKL-expressing tumor cells enhanced osteoclastogenesis from RAW264.7 cells and human monocyte- derived preosteoclasts in the absence of stromal cells/osteoblasts in a RANKL-dependent manner. Furthermore, conditioned media from Daudi cells enhanced transmigration of preosteoclasts that was inhibited by anti-VEGF antibody, suggesting that tumor cell - derived VEGF mediates recruitment of osteoclast precursors. Moreover, cocultures of B-lymphoid cell lines with osteoclasts enhanced the growth of B-lymphoid cells. Conclusions: Some malignant B cells aberrantly express functional RANKL as well as VEGF to enhance osteoclastogenesis. The coexpression of RANKL and VEGF may also contribute to the close cellular interactions with osteoclastic cells, thereby forming a vicious cycle between osteoclastic bone destruction and tumor expansion in bone.

Mechanical stress to bone plays a critical role in maintaining bone mass and strength. However, the molecular mechanism of mechanical stress-induced bone formation is not fully understood. In the present study, we demonstrate that FosB and its spliced variant DeltaFosB, which is known to increase bone mass by stimulating bone formation in vivo, is rapidly induced by mechanical loading in mouse hind limb bone in vivo and by fluid shear stress (FSS) in mouse calvarial osteoblasts in vitro both at the mRNA and protein levels. FSS induction of FosB/DeltaFosB gene expression was dependent on gadlinium-sensitive Ca2+ influx and subsequent activation of ERK1/2. Analysis of the mouse FosB/DeltaFosB gene upstream regulatory region with luciferase reporter gene assays revealed that the FosB/DeltaFosB induction by FSS occurred at the transcriptional level and was conferred by a short fragment from -603 to -327. DNA precipitation assays and DNA decoy experiments indicated that ERK-dependent activation of CREB binding to a CRE/AP-1 like element (designated "CRE2") at the position of -413 largely contributed to the transcriptional effects of FSS. These results suggest that DeltaFosB participates in mechanical stress-induced intracellular signaling cascades that activate the osteogenic program in osteoblasts.

Multiple myeloma (MM) expands in the bone marrow and causes devastating bone destruction by enhancing osteoclastic bone resorption in its vicinity, suggesting a close interaction between MM cells and osteoclasts (OCs). Here, we show that peripheral blood mononuclear cell-derived OCs enhanced growth and survival of primary MM cells as well as MM cell lines more potently than stromal cells, and that OCs protected MM cells from apoptosis induced by serum depletion or doxorubicin. OCs produced osteopontin (OPN) and interleukin 6 (IL-6), and adhesion of MM cells to OCs increased IL-6 production from OCs. In addition, IL-6 and OPN in combination enhanced MM cell growth and survival. However, the effects of OCs on MM cell growth and survival were only partially suppressed by a simultaneous addition of anti-IL-6 and anti-OPN antibodies and were completely abrogated by inhibition of cellular contact between MM cells and OCs. These results demonstrate that OCs enhance MM cell growth and survival through a cell-cell contact-mediated mechanism that is partially dependent on IL-6 and OPN. It is suggested that interactions of MM cells with OCs augment MM growth and survival and, thereby, form a vicious cycle, leading to extensive bone destruction and MM cell expansion. (C) 2004 by The American Society of Hematology.

Expression of an osteogenic cytokine, IL-11, is decreased in SAMP6. We show here that IL-11 transcription largely depends on AP-1 transcription factors, activities of which are decreased in SAMP6 as well as aged ICR mice. Therefore, diminished AP-1 activity and the resultant decline in IL-11 expression may play a role in impaired bone formation in the aged. Introduction: Evidence suggests that impaired osteoblastogenesis contributes to aging-associated osteopenia. The P6 strain of senescence-accelerated mice (SAM) is an animal model of senile osteoporosis, which exhibits low bone mass caused by impaired bone formation. Bone marrow stromal cells from SAMP6 show decreased osteoblasto-genesis and increased adipogenesis. We previously demonstrated that these abnormalities of SAMP6 stromal cells may be attributed to decreased expression of interleukin (IL)-11. Methods: In this study, we attempted to determine the molecular mechanism of decreased IL-11 expression by SAMP6 stromal cells by cloning and analyzing the mouse IL-11 gene promoter. Results and Conclusions: We found that two tandem activating protein-1 (AP-1) sites that reside immediately upstream of TATA box play critical roles in IL-11 gene transcription. Gel shift analysis showed that binding activity to the IL-11 AP-1 sites was reduced in SAMP6 stromal cell nuclear extracts. Among multiple components of AP-1 transcription factors, Jun D binding was particularly decreased. Furthermore, decreased Jun D binding and IL-11 expression by stromal cells was also observed in aged mice of the ICR strain. Therefore, decreased AP-1 activity and a resultant decline in IL-11 expression by bone marrow stromal cells may play a role in impaired bone formation in the aged.

Receptor activator of nuclear factor (RANK) is a member of the tumor necrosis factor receptor superfamily indispensable for osteoclast differentiation. However. little is known about the regulatory mechanism of RANK expression. In the present study, RANK expression during macrophage/osteoclast differentiation was investigated using a human myelomonocytic cell line, HL60, capable of differentiating into mature osteoclasts under appropriate conditions. RANK mRNA expression was barely detectable in growing HL60 cells. We found that treatment with lalpha,25-dihydroxyvitamin D-3 and TPA resulted in an apparent induction of RANK mRNA and protein in association with differentiation into the macrophage/osteoclast lineage. Induction of RANK was time and dose dependent and lineage specific. Moreover, RANK induction was blocked by an RNA polymerase II inhibitor, suggesting an involvement of a transcriptional mechanism. The induced RANK was functional as it was able to bind RANK ligand and activate NF-kappaB. In the induced HL60 cells expressing both c-Fms and RANK, RANK mRNA expression was further enhanced by RANKL, but not by macrophage colony-stimulating factor. These results suggest a positive feedback regulation of RANK expression by its own intracellular signaling. The in vitro system described here may be a useful model to elucidate the regulatory mechanism of RANK expression and its role in human osteoclastogenesis. (C) 2003 Elsevier Science (USA). All rights reserved.

Multiple myeloma (MM) cells cause devastating bone destruction by activating osteoclasts in the bone marrow milieu. However, the mechanism of enhanced bone resorption in patients with myeloma is poorly understood. In the present study, we investigated a role of C-C chemokines, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta, in MM cell-induced osteolysis. These chemokines were produced and secreted by a majority of MM cell lines as well as primary MM cells from patients. Secretion of MIP-1alpha and MIP-1beta correlated well with the ability of myeloma cells to enhance osteoclastic bone resorption both in vitro and in vivo as well as in MM patients. In osteoclastogenic cultures of rabbit bone cells, cocultures with myeloma cells as well as addition of myeloma cell-conditioned media enhanced both formation of osteoclastlike cells and resorption pits to an extent comparable to the effect of recombinant MIP-1alpha and MIP-1beta. Importantly, these effects were mostly reversed by neutralizing antibodies against MIP-1alpha and MIP-1beta, or their cognate receptor, CCR5, suggesting critical roles of these chemokines. We also demonstrated that stromal cells express CCR5 and that recombinant MIP-1alpha and MIP-1beta induce expression of receptor activator of nuclear factor-kappaB (RANK) ligand by stromal cells, thereby stimulating osteoclast differentiation of preosteoclastic cells. These results suggest that MIP-1alpha and MIP-1beta may be major osteoclast-activating factors produced by MM cells.

Although cells of the osteoblast lineage express functional ERs, direct effects of estrogen on bone formation remain obscure. In the present study, we have investigated estrogen effects on osteoblastic and adipocytic differentiation from a mouse bone marrow stromal cell line, ST-2, which had been manipulated to overexpress either human ERalpha (ST2ERalpha) or ERP (T2ERbeta). Treatment with bone morphogenetic protein-2 increased alkaline phosphatase activity as well as the number of Oil Red O-positive adipocytes, indicating that bone morphogenetic protein-2 stimulated both osteoblastic and adipocytic differentiation from these bipotential cells. In both ST2ERa and ST2ERbeta cells, cotreatment with E2 caused enhancement of alkaline phosphatase activity and suppression of lipid accumulation. These effects were completely reversed by an ER antagonist, ICI182780. Therefore, the estrogen regulation occurred in an ER-specific manner but without ER subtype specificity. Moreover, dose response curves of the opposing effects of estrogen on osteoblastogenesis and adipogenesis formed an apparent mirror image, consistent with a reciprocal regulation of differentiation into the two cell lineages. These results demonstrate that estrogen directly modulates differentiation of bipotential stromal cells into the osteoblast and adipocyte lineages, causing a lineage shift toward the osteoblast. Such effects would lead to direct stimulation of bone formation and thereby contribute to the protective effects of estrogen on bone.

Although cells of the osteoblast lineage express functional ERs, direct effects of estrogen on bone formation remain obscure. In the present study, we have investigated estrogen effects on osteoblastic and adipocytic differentiation from a mouse bone marrow stromal cell line, ST-2, which had been manipulated to overexpress either human ERalpha (ST2ERalpha) or ERP (T2ERbeta). Treatment with bone morphogenetic protein-2 increased alkaline phosphatase activity as well as the number of Oil Red O-positive adipocytes, indicating that bone morphogenetic protein-2 stimulated both osteoblastic and adipocytic differentiation from these bipotential cells. In both ST2ERa and ST2ERbeta cells, cotreatment with E2 caused enhancement of alkaline phosphatase activity and suppression of lipid accumulation. These effects were completely reversed by an ER antagonist, ICI182780. Therefore, the estrogen regulation occurred in an ER-specific manner but without ER subtype specificity. Moreover, dose response curves of the opposing effects of estrogen on osteoblastogenesis and adipogenesis formed an apparent mirror image, consistent with a reciprocal regulation of differentiation into the two cell lineages. These results demonstrate that estrogen directly modulates differentiation of bipotential stromal cells into the osteoblast and adipocyte lineages, causing a lineage shift toward the osteoblast. Such effects would lead to direct stimulation of bone formation and thereby contribute to the protective effects of estrogen on bone.

Osteoprotegerin (OPG) is a recently identified member of the tumor necrosis factor (TNF) receptor superfamily that regulates bone mass through an inhibitory action on osteoclast differentiation and function. To determine its potential roles of OPG in pathological changes in bone metabolism caused by estrogen deficiency, we investigated effects of estrogen on OPG expression by a mouse stromal cell line, ST-2, in vitro. Treatment of ST-2 cells with 17 beta -E-2 resulted in up-regulation of OPG expression at both the messenger RNA and protein levels. The effect was time and dose dependent and steroid specific. The stimulatory action of 17 beta -E-2 on OPG expression appeared to be mediated by the estrogen receptor-alpha (ER alpha) subtype because stable overexpression of ER alpha, but not of ER beta, enhanced the OPG induction by 17 beta -E-2. Moreover, estrogen withdrawal after 5-day pretreatment, mimicking the event occurring in vivo at menopause, dramatically diminished the expression of OPG. These findings suggest that down-regulation of OPG after estrogen withdrawal contributes to the enhanced osteoclastic bone resorption and bone loss after menopause by enhancing RANK ligand-RANK system that lies downstream of a large number of bone-resorbing cytokines.

Dietary phosphate (P-i) is a most important regulator for renal P-i reabsorption. The type II sodium-dependent phosphate (Na/P-i) cotransporters (NPT2) are located at the apical membranes of renal proximal tubular cells and major functional transporters associated with renal P-i reabsorption. The consumption of a low-P-i diet induces the synthesis of NPT2, whereas a high P-i diet decreases it, The molecular mechanisms of regulation by dietary P-i are not yet known. In this report, in weaning mice fed a low-P-i diet for 4 days, the NPT2 mRNA level was increased 1.8-fold compared with mice fed a normal P-i diet. This increase was due to an elevation of the transcriptional activity. In the NPT2 gene promoter, the DNA footprint analysis showed that six regions were masked by the binding protein, but at the position -1010 to -985 upstream of the transcription start site, the binding clearly responded to the levels of dietary P-i, The phosphate response element (PRE) of the NPT2 gene was found to consist of the motif related to the E box, 5'-CACGTG-3'. A yeast one-hybrid system was used to clone a transcription factor that binds to the PRE sequences in the proximal promoter of the NPT2 gene. Two cDNA clones that encoded protein of the mouse transcription factor mu E3 (TFE3) were isolated. This is a DNA-binding protein that activates transcription through the mu E3 site of the immunoglobulin heavy chain enhancer. TFE3 antibody completely inhibited the binding to the PRE. The coexpression of TFE3 in COS-7 cells transfected with the NPT2 gene promoter markedly stimulated the transcriptional activity. The feeding of a low P-i diet significantly increased the amount of TFE3 mRNA in the kidney. These findings suggest that TFE3 may participate in the transcriptional regulation of the NPT2 gene by dietary P-i.

Dietary phosphate (P-i) is a most important regulator for renal P-i reabsorption. The type II sodium-dependent phosphate (Na/P-i) cotransporters (NPT2) are located at the apical membranes of renal proximal tubular cells and major functional transporters associated with renal P-i reabsorption. The consumption of a low-P-i diet induces the synthesis of NPT2, whereas a high P-i diet decreases it, The molecular mechanisms of regulation by dietary P-i are not yet known. In this report, in weaning mice fed a low-P-i diet for 4 days, the NPT2 mRNA level was increased 1.8-fold compared with mice fed a normal P-i diet. This increase was due to an elevation of the transcriptional activity. In the NPT2 gene promoter, the DNA footprint analysis showed that six regions were masked by the binding protein, but at the position -1010 to -985 upstream of the transcription start site, the binding clearly responded to the levels of dietary P-i, The phosphate response element (PRE) of the NPT2 gene was found to consist of the motif related to the E box, 5'-CACGTG-3'. A yeast one-hybrid system was used to clone a transcription factor that binds to the PRE sequences in the proximal promoter of the NPT2 gene. Two cDNA clones that encoded protein of the mouse transcription factor mu E3 (TFE3) were isolated. This is a DNA-binding protein that activates transcription through the mu E3 site of the immunoglobulin heavy chain enhancer. TFE3 antibody completely inhibited the binding to the PRE. The coexpression of TFE3 in COS-7 cells transfected with the NPT2 gene promoter markedly stimulated the transcriptional activity. The feeding of a low P-i diet significantly increased the amount of TFE3 mRNA in the kidney. These findings suggest that TFE3 may participate in the transcriptional regulation of the NPT2 gene by dietary P-i.

Vitamin D is an important regulator of phosphate homeostasis. The effects of vitamin D on the expression of renal Na+-dependent inorganic phosphate (P-i) transporters (types I and II) were investigated. In vitamin D-deficient rats, the amounts of type II Na+-dependent P-i transporter (NaPi-2) protein and mRNA were decreased in the juxtamedullary kidney cortex, but not in the superficial cortex, compared with control rats. The administration of 1,25-dihydroxyvitamin D-3 (1,25-(OH)(2)D-3) to vitamin D-deficient rats increased the initial rate of P-i uptake as well as the amounts of NaPi-2 mRNA and protein in the juxtamedullary cortex. The transcriptional activity of a luciferase reporter plasmid containing the promoter region of the human type II Na+-dependent P-i transporter NaPi-3 gene was increased markedly by 1,25-(OH)(2)D-3 in COS-7 cells expressing the human vitamin D receptor. A deletion and mutation analysis of the NaPi-3 gene promoter identified the vitamin D-responsive element as the sequence 5'-GGGGCAGCAAGGGCA-3' nucleotides -1977 to -1963 relative to the transcription start site. This element bound a heterodimer of the vitamin D receptor and retinoid X receptor, and it enhanced the basal transcriptional activity of the promoter of the herpes simplex virus thymidine kinase gene in an orientation-independent manner. Thus, one mechanism by which vitamin D regulates P-i homeostasis is through the modulation of the expression of type II Na+-dependent P-i transporter genes in the juxtamedullary kidney cortex.

Vitamin D is an important regulator of phosphate homeostasis. The effects of vitamin D on the expression of renal Na+-dependent inorganic phosphate (P-i) transporters (types I and II) were investigated. In vitamin D-deficient rats, the amounts of type II Na+-dependent P-i transporter (NaPi-2) protein and mRNA were decreased in the juxtamedullary kidney cortex, but not in the superficial cortex, compared with control rats. The administration of 1,25-dihydroxyvitamin D-3 (1,25-(OH)(2)D-3) to vitamin D-deficient rats increased the initial rate of P-i uptake as well as the amounts of NaPi-2 mRNA and protein in the juxtamedullary cortex. The transcriptional activity of a luciferase reporter plasmid containing the promoter region of the human type II Na+-dependent P-i transporter NaPi-3 gene was increased markedly by 1,25-(OH)(2)D-3 in COS-7 cells expressing the human vitamin D receptor. A deletion and mutation analysis of the NaPi-3 gene promoter identified the vitamin D-responsive element as the sequence 5'-GGGGCAGCAAGGGCA-3' nucleotides -1977 to -1963 relative to the transcription start site. This element bound a heterodimer of the vitamin D receptor and retinoid X receptor, and it enhanced the basal transcriptional activity of the promoter of the herpes simplex virus thymidine kinase gene in an orientation-independent manner. Thus, one mechanism by which vitamin D regulates P-i homeostasis is through the modulation of the expression of type II Na+-dependent P-i transporter genes in the juxtamedullary kidney cortex.

【背景】健康は栄養・運動・休養の3つのバランスを高いレベルで維持し続けることが必要であるが、大学生は進学などに伴う生活環境の変化によって体調を崩すことが少なくない。近畿大学農学部に在籍する学生の多くは健常であると考えられるが、BMIが基準を大きく外れるなど、健康を維持する上で看過できない潜在的リスクを抱える学生が一部見受けられる。【目的】生活習慣病一次予防に向けた栄養学的介入をおこなうことで、農学部生の健康維持・増進を目指すことを目的に、以下の検討を試みた。【方法】農学部学生支援課（医務室）との連携により4月検診結果を基にBMIを算出した。基準を大きく外れた学生を対象 (BMI≧30)に、栄養学的介入をおこなった。【結果】栄養学的介入を実施した対象者9名の殆どで体重減少を認め、また介入前後で食習慣の改善が見受けられた例もあった。一方で、食事記録並びに食物摂取頻度調査（FFQg）の結果から、対象者の多くに食事変容バイアスが見られるなど、過少申告は特に肥満者で著しい事が改めて示された。【考察】肥満は生活習慣病のリスクとなるため、一部の学生においては将来を見据え生活習慣を整える必要がある。また栄養学的介入は生活習慣病一次予防に向けた支援として有効であった。しかし食物摂取頻度調査においては分量に関する知識や感覚の違いおよび過少申告による誤差の可能性が考えられた。今後我々はBMIが基準を大きく外れている学生に対し、集団的な介入および食への関心や知識を高めるような環境作りを行う必要がある。

【背景】腎機能が低下した慢性腎臓病（CKD）患者では食事からの過剰なリンを尿として排泄できず、高リン血症を招く恐れがある。高リン血症は心血管疾患などの致命的な合併症を引き起こす可能性があることから、CKD患者にとってリンの制限は重要である。しかし、食品中のリンの多くはタンパク質と結合しているためリンのみを制限することは困難である。一方で過度のタンパク質の制限は患者の栄養状態の悪化を招くことから、適切なタンパク質量を確保し、尚且つリンを選択的に制限することが求められる。我々は昨年度の本大会において、健常者を対象に食事中のリン供給源（タンパク質）を植物性食品中心とすることで、リンを選択的に制限できることを明らかにしている。そこで本研究では、健常者において得られた成績がCKD患者においても有効であるか否かについて、臨床研究を試みた。【方法】対象はA医院に通院のCKDステージ3bの70代男性3名とした。動物性タンパク質比率70%（動物70%食）あるいは植物性タンパク質比率70%（植物70%食）に調整した試験食(慢性腎臓病に対する食事療法基準2014年版に準拠し、双方共に３大栄養素、リン、カルシウム、食塩量は等量)の2種類の食事を提供した際の体内リン代謝動態を比較した。実験は、クロスオーバー試験とし、1か月の休息期間後、同様の試験を行った。【結果】食後の尿中リン排泄は、動物70%食群と植物70%食群で差は認められなかった。一方で血中リン濃度において、動物70％食群に比べ植物70％食群で食後血中リン濃度の上昇が穏やかであった。【考察】本成績は、健常者と同様に、CKD患者においても植物性タンパク質中心の食事をとることで、極端にたんぱく質を制限することなく、効率の良いリン管理が可能であることを示唆するものであり、CKD患者の病態改善の一助となることが期待される。

【背景】健常な人の場合、体内のリン並びにカリウムの濃度は、腎臓による排泄と再吸収の機能によって一定に保たれている。しかしながら、腎機能が低下した慢性腎臓病（CKD）患者では、体内にリンやカリウムが蓄積し、副甲状腺機能亢進症や異所性石灰化の原因となる高リン血症や、不整脈や心停止の原因となる高カリウム血症を招く恐れがある。現在CKDの食事療法では病期に合わせてリンやカリウムの制限がおこなわれているが、調理過程で損失の多いカリウムに対して、リンの多くはタンパク質と共存することから食事管理が困難である。一方で我々はこれまでに、同量のリン含量であっても植物性タンパク質の多い食事を摂取することで、動物性タンパク質の多い食事に比べ、食後の血中リン濃度の上昇を穏やかにすることを明らかにしている。しかしながら、植物性タンパク質の多い食事にはカリウムが多く含まれる問題点がある。但し、先行研究では食事の栄養価は食材の生の成分値で算出したため調理による損失を考慮していない。そこで本研究では、タンパク質源の異なる食事におけるリン、カリウムの調理損失に着目し、植物性タンパク質の多い食事と動物性タンパク質の多い食事に含まれるリン並びにカリウム含量を調理前後で比較検討した。【方法】試料は先行研究において健常者に試験食として提供した、動物性タンパク質が多い食事（動物食）と植物性タンパク質が多い食事（植物食）を用いた（双方共に３大栄養素、リン、カルシウム、食 塩量は等量）。試料は、調理前と調理後にミキサーにかけ均一にしたものを用い、リン、カリウム含量を測定し損失率を算出した。【結果】植物食では調理前に比べ調理後でリンは3％、カリウムは53％の損失を示した。一方、動物食では調理前後でリンは5％、カリウムは11％の損失を示した。【考察】本成績は、植物性タンパク質を多く含む食事がリンのみならずカリウム制限も実施できる可能性を示唆するものであり、CKD患者に向けた食事療法として活用できるものと期待される。

【背景】CKDに伴う骨ミネラル代謝異常 (CKD-MBD)の概念が提唱されて以来、血清リン（P）濃度と生命予後との関連性が注目されている。P排泄不全を呈するCKD患者において食事性P管理は極めて重要であるが、Pは多くの食品に含まれていること、その多くはたんぱく質と共存することなどから、その実践は容易ではない。そこで本研究では、食事性P管理の新たな方策として、Pの体内利用率が食材により異なる点に着目し、食事に含まれるたんぱく質の違いが体内P代謝動態に及ぼす影響について検討した。【方法・結果】対象者は当学科所属の20代健常男子学生とした。動物性たんぱく質比率70％(動70%食)あるいは植物性たんぱく質比率70％（植70％食）に調整した試験食（双方共にカロリー、三大栄養素、P量を同等となるよう調整）を喫食後の体内リン代謝動態を検討した。その結果、食後の尿中P排泄は動70％食群で顕著な増加がみられたが、植70％食群では軽微であった。なお双方の血中P濃度に大きな変動は認められなかった。植70％食は食物繊維を多く含む事から、尿中P排泄の低下の一因として食物繊維の関与が考えられる。そこで次に動70％食・植70％食に加えて、植70％食と同等になるよう食物繊維の量を調整した動70％食（動70％DF食）の3種を喫食した際の体内P代謝動態を比較した。その結果、動70％食群でみられた食後尿中P排泄の増加は、食物繊維量を植70％食群と同等に調整すること（動70％DF食）で改善された。【考察】CKD患者に対するたんぱく制限の有効性については未だ議論の分かれるところであり、その要因の1つとして過度のたんぱく制限による栄養状態の悪化が懸念されている。本成績は過度のたんぱく制限を伴わずにP制限を実施できる新たな可能性を示すものであり、CKD患者の病態進展阻止の一助となることが期待される。

【背景】慢性腎臓病（CKD）患者でみられる高リン血症は、心血管イベントなどの致命的な合併症を引き起こすことから、当該患者における食事性リン管理は必須である。血中リン濃度は、食事中のリン含量に左右されるが、リンはタンパク質と結合していることから、リンのみを制限することは容易ではない。そこで本研究では、選択的にリンを制限する新たな手法として、リンの生体内利用率が食材により異なる点に着目し、食事中のリン供給源の違いが体内リン代謝動態に及ぼす影響について検証を試みた。【方法】対象者は当学科に所属の20代の健常学生とした。なお本研究は本学生命倫理員会の承認を得た上で実施した。（実験Ⅰ）動物性由来リン比率70%あるいは植物性由来リン比率70%に調整した試験食(双方共にタンパク質、リン量は等量)を対象者に昼食として供した際の体内リン代謝動態を比較した。（実験Ⅱ）実験Ⅰと同様の栄養価の食事を5日間継続的に被験者に提供し、体内リン代謝動態への影響を検討した。【結果】（実験Ⅰ）の植物性リン70%食群では、動物性リン70%食群に比して試験食摂取後の血中リン濃度並びに尿中リン濃度の上昇が緩やかであった。（実験Ⅱ）の植物性リン70％食群では、動物性リン70％食群と比べて血中リン濃度に差は認められなかったが、尿中リン濃度は、5日間継続的に低い値を維持した。【考察】植物性由来のリンを多く含む食事では食後の血中リン濃度の上昇が穏やかであることが明らかとなった。本成績は、早期リン制限による腎機能悪化進展阻止に向けた新たな食事・栄養療法として、CKD患者の病態改善の一助となる可能性が示唆された。

【背景】慢性腎臓病（CKD）における食事療法ではたんぱく質制限を中心とする厳しい制限により微量栄養素等の欠乏を招く恐れがあるが、その詳細は未だ不明な点が多く残されている。そこで本研究では、既存のCKD患者向け献立を網羅解析することで、その問題点並びに解決策を模索した。【方法】本検討では、腎臓病食品交換表に準拠したCKD患者向け献立を収載する市販書籍を解析対象とした。当該書籍に収載の献立（朝昼夕毎に51食、計153食）をデジタルデータ化し、得られた全ての1日分の組み合わせ（513＝約13万通り）のうち、CKD食事療法基準（2014年版）ステージ3bを満たす組み合わせ約9万通りを抽出し、これらの献立に含まれる栄養素の過不足について検証した。【結果】抽出したCKD食1日分（9万通り）に含まれる各種栄養成分について、「日本人の食事摂取基準2015年版」を基に充足率を求めたところ、ビタミンではVit.B群やVit.Dで、またミネラルではCa、Mg、Znなど、多数の微量栄養素の不足を確認した。【考察】CaやVit.Dの不足はCKDに伴う骨ミネラル代謝障害のリスクであることから、これらの栄養素に配慮した献立作成の必要性が改めて浮き彫りとなった。またこうした微量栄養素の不足は対象群である常食では認められなかったことから、たんぱく質制限を中心とした腎臓病食特有の問題であることが推測される。一方で、Znのように不足しがちであるものの、必ずしもたんぱく質制限との間に強い相関を認めない栄養素も存在したことから、食材の組み合わせによっては、CKD食においても食事摂取基準を十分満たすことが可能であると考えられる。しかしながら、多数の栄養成分を適切に管理しつつ、「単位」のような指標を用いて献立を作成することは困難である。このため、我々の作成したデータベースをはじめとした情報技術を用いることにより、多数の栄養成分に配慮した食事管理法は、今後益々必要とされるものと考えられる。

Na依存性無機リン酸輸送担体Npt2cは高カルシウム尿を伴う遺伝性低リ ン血性クル病（HHRH）の原因遺伝子であることが報告されている。しかしながら、Np t2c欠損マウスでは、ヒトで観察されるような低リン血症やクル病様所見を示さない ため、未だにその役割は不明である。一方、Npt2aは腎近位尿細管上皮細胞に発現し 、リン再吸収の律速段階を担う。げっ歯類においては、Npt2aがリン再吸収の70%を担 うのに対し、Npt2cは補助的な役割を担っている。Npt2cの生理学的な役割を解明する ためには、Npt2cを内因性に発現している細胞を用いる必要がある。OK細胞（フクロ ネズミ由来腎近位尿細管細胞）は、培養日数を経て分化し微絨毛が発達し、Npt2aの 発現が誘導される。本研究において、OK細胞に発現するNpt2cを同定し、特異抗体を 作製して、培養後の変化を調べた。内因性Npt2c発現は、OK細胞の未分化時に高く、 分化後では著しく発現が低下した。未分化細胞においてNpt2c

栄養素データベースを活用することで微量栄養素にも注意を払いつつ制限食の献立を検討することの重要性を確認し，さらに，そのデータを献立推薦システム内に取り込み，複数日で栄養制約の調整を行うことが患者のＱＯＬ向上に有効であることを確認した．また，このような推薦システムにタブレット型拡張現実感技術を導入することに関して，そのユーザインタフェースとしての有用性を評価するための検討を行い，その評価のガイドラインを作成した．

慢性腎臓病（CKD）患者にみられる高リン血症はCKD-MBDと称される骨・ミネラル代謝障害や併発する心血管イベントの発症に深く関わる。血中リン濃度は食事由来のリン含有量に左右されるが、食事由来のリンを選択的に制限することは容易ではない。そこで本研究では、リン利尿因子FGF23に着目し、CKD患者におけるFGF23産生亢進機序解析を試みた。その結果、骨でのFGF23産生はCKD患者体内で蓄積が見られる尿毒症物質の一種（IS）によりAhR依存的に誘導されることを新たなに見いだした。一方で、この作用はAhR阻害活性作用を有する食品由来機能性成分により減弱することが明らかとなった。

繊維芽細胞増殖因子 (FGF23)はリン利尿因子として同定された新規増殖因子であり、慢性腎臓病 (CKD)患者では腎機能の低下に先行して血中FGF23値の上昇がみられるだけでなく、血中FGF23値はCKD患者の生命予後にも関連することなどから、その重要性が示唆されるものの、その誘導機序並びに病態への関与については不明な点が多い。申請者は腎障害並びに骨軟化症を呈するイタイイタイ病患者並びにそのモデル動物の病態解析から、本症患者では血清FGF23値が高値を呈すること、またその機序としてカドミウムはFGF23の翻訳後修飾（分子内切断の抑制）に関わる糖鎖付加酵素GalNAc-T3の転写を芳香族炭化水素受容体 (AhR)依存的に誘導することでFGF23産生を促すといった新たな調節系の存在を明らかにした。AhRはダイオキシンなどの化学物質やカドミウムなどの重金属だけでなく、尿毒症物質の一種であるインドール誘導体によっても活性化を受けることから、CKD患者体内で蓄積がみられる尿毒症物質が当該経路を活性化する可能性が予測された。そこで本研究では尿毒症物質（インドキシル硫酸）が当該経路の活性化を介して骨でのFGF23産生を誘導され、これが血液を介して全身に運ばれ、CKDの病態（腎障害）並びに合併症としての筋・骨格系障害の発症並びにその進展に関わるものと考え、その検証を試みた。その結果、インドキシル硫酸 (IS)はin vitroおよびin vivoにおいて骨でのGalNAc-T3 mRNAの発現を誘導するとともにFGF23産生を惹起した。またISの作用はAhR依存的にGalNAc-T3遺伝子promoter活性を誘導することを見いだした。以上の成績は尿毒症物質であるISがGalNAc-T3の誘導を介してFGF23の安定化を促すことを示すものであり、CKD患者におけるFGF23異常高値の機序の一端を分子レベルで説明しうるものであると考えられる。

慢性腎臓病の進行や透析患者の生命予後においてリンコントロールは非常に重要な役割を演じている。近年、リン酸(以下リン)代謝調節系を支配する中核的なホルモンとして繊維芽細胞様増殖因子FGF23/klotho系が明らかにされた。本研究では、腎遠位尿細管から近位尿細管へのシグナルを伝えるFGF23/klotho系の解明を中心に、腸管/腎臓/骨を結ぶ未知のリン調節因子の探索を行なった。まず、腎遠位尿細管細胞FGF23/klotho/FGFR複合体のシグナルが、どのようなシステムで近位尿細管に伝わるか検討した結果、様々な条件下で近位尿細管細胞にklothoが誘導され、FGF23のシグナルを伝える機序を明らかにした。FGF23は共受容体Klothoの共存下で特異的受容体FGFR1との結合を介して細胞内の各種シグナル伝達経路を活性化する。FGF23刺激下で活性化される分子についてはERK1/2が最も代表的であるが、同ファミリーに属する他のFGFでの作用を見る限り、その他複数の経路の活性化が示唆された。そこで、膜型Klothoを発現する近位尿細管細胞安定発現株(HEK-MmKL)を用いて、FGF23組換え体で刺激後に活性化、される細胞内シグナル伝達経路を系統的に解析した。次に、得られた結果が受容体特異的であるか否かの検証はFGFR特異的阻害薬(PD173074)による阻害作用を指標に検証した。In vivoにおいてマウスにFGFR阻害剤を投与した実験から、リン輸送の制御とは独立して活性型ビタミンDの調節が行なわれていると考えられた。最後に、腸管から分泌されるリン利尿因子の検索を行い、これらは膜型klothoの切断とリンクしている可能性が得られた。以上、骨細胞、腎臓および小腸を結ぶ臓器相関シグナル因子としてのFGF23/klothoシステムの特性の一部が明らかになった。

慢性腎臓病（CKD）は高リン血症から、副甲状腺機能亢進症、腎性骨症、異所性石灰化などの合併症が惹起され、生命予後を悪化させる。 全身性の代謝、老化制御機構として「NADワールド」と名付けられた新たな概念は、NADが各臓器、組織における様々な代謝のペースメーカーであることを示す。これまでの骨細胞死滅マウスの研究で、腸管，肝臓由来の未同定である因子がニコチンアミド代謝を介してリン代謝を制御する可能性を見いだした。そこで当該年度は、骨細胞死滅マウスを利用し、リン代謝およびニコチンアミド代謝を検討した。結果として、腎臓ナトリウムリン酸トランスポーターであるNaPi-IIa NaPi-IIcの著しい減少により尿からのリン再吸収が抑制され尿中へのリン排泄が増加した。骨細胞より分泌されるリン利尿因子であるFGF23 （fibroblast growth factor 23）は正常マウスの40%以下であり、また副甲状腺ホルモン(PTH)は変化がなかった。そのため、FGF23, PTHとは異なるリン利尿促進因子が腎臓に作用していると考えられた。次に腎臓でのニコチンアミド代謝を検討した結果、腎臓内NAD代謝の亢進が認められた。現在、ニコチンアミド代謝酵素ノックアウトマウスの作成に着手した。またニコチンアミド代謝経路を介したリン酸トランスポーター発現制御機構について培養細胞を使用し検討している。本研究でリン排泄促進機構における、ニコチンアミド代謝の重要性を提示し、未同定である腸管、肝臓由来のニコチンアミド代謝制御因子を同定することは、CKD患者の新たなリン恒常性維持、異所性石灰化の予防、老化促進防御の基盤研究となると考える。

末期腎不全患者である長期透析患者では、カルシウム・リンの貯蔵庫としての骨機能が障害され、「骨不全」とも称される骨のミネラル保持能力低下により、高い頻度で軟部組織に異所性石灰化が起こり、患者の生命予後を左右する重要な問題となっている。骨粗鬆症あるいは血管石灰化は加齢と共に発症する老年病であり、両者はしばしば合併する。これら二つの病態に対する関連性を示唆するエビデンスが蓄積されてきており、その発症並びに病態の進展に老化制御因子が関与する可能性が示唆されている。申請者はこれまでに末期腎不全患者に見られる異所性石灰化、および骨粗鬆症と血管石灰化の発症機能を繋ぐ因子を検索し、「骨不全」と「血管石灰化」を結ぶ新しい老化制御転写因子MBF1を同定した。本研究ではこのMBF1の分子基盤の確立をおこなうことにより、新しいミネラル老化学を解明する。

(1)骨形成障害に基づく病態の解明と治療法の開発力学的負荷の免荷による遺伝子発現変化、免荷後の負荷(強制運動)による遺伝子発現変化を観察するin vivo実験系を構築した。その結果、IL-11の遺伝子発現が免荷により抑制される一方、負荷後早期にΔFosB発現が促進されると共に、Smadlのリン酸化がPKCδ依存性に促進され、IL-11発現が高まることを見出した。更にIL-11遺伝子プロモーター上のAP-1領に結合するΔFosB/JunD複合体とSmad結合領域に結合するSmadlとが転写因子複合体を形成していることも明らかにした。(2)多発性骨髄腫に伴う骨破壊病変の発症機序の解明と治療法の開発骨髄腫細胞が産生するsFRP-2が骨芽細胞のcanonical Wnt経路の阻害を介して骨芽細胞分化を抑制し骨髄腫による骨形成の阻害に重要な役割を果たすこと、骨吸収により骨基質より放出されるTGF-βも骨芽細胞の終末分化を抑制することを示した。更に終末分化した骨芽細胞は骨髄腫細胞の増殖を強力に抑制することを見出し、骨芽細胞分化の抑制が骨髄腫の生育に好適な環境を形成する可能性を示した。また、破骨細胞が骨破壊と共に血管新生を介し腫瘍進展を促進する可能性を示した。(3)骨格系における循環動態の障害に基づく病態の解明と治療法の開発デキサメサゾン投与により作成したグルココルチコイド過剰マウスでは尿中NOx排泄量と大動脈eNOS蛋白発現が著しく減少することを見出し、これにより循環障害が惹起される結果大腿骨頭壊死が発症するとの仮説を提唱した。更に、スタチンの一部がこれらに拮抗する作用を有することを見出した。

(研究結果の概要) 1.力学的負荷によるδfosB及びIL-11の発現誘導機構の解析 (1)力学的負荷によるδfosB及びIL-11の誘導はBAPTA並びにERK阻害薬(UO126)存在下ではほぼ完全に抑制された。従ってfluid shear stress (FSS)による両者の誘導はCa及びERKに依存することが示された。また各々のプロモーター領域を用いたluciferase assayによる解析から、δfosB/IL-11の誘導は転写段階であること、さらにδfosBはAP-1配列を介してIL-11遺伝子の転写活性を促進することを明らかにした。 2.力学的負荷並びにIL-11の生物学的作用(In vitro) (1)新生児マウス頭蓋骨由来の初代培養系骨芽細胞(POB)において、troglitazoneは脂肪細胞分化を誘導したが、この作用は間欠的なFSS刺激(10分/時間)を断続的に与えることでほぼ半分に抑制された。またこの作用はIL-11中和抗体により解除された。 (2)同様の細胞系でFSSあるいはIL-11はdexamethasone (Dex)によるアポトーシスを抑制すること、さらにFSSの有する抗アポトーシス作用はIL-11作用を介することを見いだした。 (3)IL-11はBcl-2の発現をmRNA、タンパクレベルで誘導することを見いだした。 3.In vivoでの解析 IL-11 transgenicマウスに対する尾部懸垂の効果を見たところ、重力免荷による骨量減少がIL-11の過剰発現により抑制される傾向が見られた。またDex投与による骨量減少に対しても同様であった。 (総括) 力学的負荷は骨芽細胞系においてAP-1(δfosB)/IL-11経路の活性化を介して生理的な骨形成並びに骨量を椎持していることが示唆された。本経路の解明は不動、あるいはステロイド過剰により惹起される骨量減少(骨粗鬆症)の病態解明並びにその有効な治療薬の解明につながるものと期待される。