Molecular mechanisms of cadmium-induced fibroblast growth factor 23 upregulation in osteoblast-like cells.

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.

Histological examination on osteoblastic activities in the alveolar bone of transgenic mice with induced ablation of osteocytes.

The purpose of this study was to examine histological alterations on osteoblasts from the alveolar bone of transgenic mice with targeted ablation of osteoctyes. Eighteen weeks-old transgenic mice based on the diphtheria toxin (DT) receptor-mediated cell knockout (TRECK) system were used in these experiments. Mice were injected intraperitoneally with 50 µg/kg of DT in PBS, or only PBS as control. Two weeks after injections, mice were subjected to transcardiac perfusion with 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4), and the available alveolar bone was removed for histochemical analyses. Approximately 75% of osteocytes from alveolar bones became apoptotic after DT administration, and most osteocytic lacunae became empty. Osteoblastic numbers and alkaline phosphatase (ALP) activity were markedly reduced at the endosteum of alveolar bone after DT administration compared with the control. Osteoblastic ALP activity in the periodontal ligament region, on the other hand, hardly showed any differences between the two groups even though numbers were reduced in the experiment group. Silver impregnation showed a difference in the distribution of bone canaliculi between the portions near the endosteum and the periodontal ligament: the former appeared regularly arranged in contrast to the latter's irregular distribution. Under transmission electron microscopy (TEM), the osteoblasts in the periodontal ligament showed direct contact with the Sharpey's fibers. Thus, osteoblastic activity was affected by osteocyte ablation in general, but osteoblasts in contact with the periodontal ligament were less affected than endosteal osteoblasts.

[Fibroblast growth factor 23 mediates the phosphaturic actions of cadmium].

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.

An apical expression signal of the renal type IIc Na+-dependent phosphate cotransporter in renal epithelial cells.

The type IIc Na(+)-dependent phosphate cotransporter (NaPi-IIc) is specifically targeted to, and expressed on, the apical membrane of renal proximal tubular cells and mediates phosphate transport. In the present study, we investigated the signals that determine apical expression of NaPi-IIc with a focus on the role of the N- and the C-terminal tails of mouse NaPi-IIc in renal epithelial cells [opossum kidney (OK) and Madin-Darby canine kidney cells]. Wild-type NaPi-IIc, the cotransporter NaPi-IIa, as well as several IIa-IIc chimeras and deletion mutants, were fused to enhanced green fluorescent protein (EGFP), and their cellular localization was analyzed in polarized renal epithelial cells by confocal microscopy and by cell-surface biotinylation. Fluorescent EGFP-fused NaPi-IIc transporter proteins are correctly expressed in the apical membrane of OK cells. The apical expression of N-terminal deletion mutants (deletion of N-terminal 25, 50, or 69 amino acids) was not affected by truncation. In contrast, C-terminal deletion mutants (deletion of C-terminal 45, 50, or 62 amino acids) did not have correct apical expression. A more detailed mutational analysis indicated that a domain (amino acids WLHSL) in the cytoplasmic C terminus is required for apical expression of NaPi-IIc in renal epithelial cells. We conclude that targeting of NaPi-IIc to the apical cell surface is regulated by a unique amino acid motif in the cytoplasmic C-terminal domain.

Phosphaturic action of fibroblast growth factor 23 in Npt2 null mice.

In the present study, we evaluated the roles of type II and type III sodium-dependent P(i) cotransporters in fibroblast growth factor 23 (FGF23) activity by administering a vector encoding FGF23 with the R179Q mutation (FGF23M) to wild-type (WT) mice, Npt2a knockout (KO) mice, Npt2c KO mice, and Npt2a(-/-)Npt2c(-/-) mice (DKO mice). In Npt2a KO mice, FGF23M induced severe hypophosphatemia and markedly decreased the levels of Npt2c, type III Na-dependent P(i) transporter (PiT2) protein, and renal Na/P(i) transport activity. In contrast, in Npt2c KO mice, FGF23M decreased plasma phosphate levels comparable to those in FGF23M-injected WT mice. In DKO mice with severe hypophosphatemia, FGF23M administration did not induce an additional increase in urinary phosphate excretion. FGF23 administration significantly decreased intestinal Npt2b protein levels in WT mice but had no effect in Npt2a, Npt2c, and DKO mice, despite marked suppression of plasma 1,25(OH)(2)D(3) levels in all the mutant mice. The main findings were as follow: 1) FGF23-dependent phosphaturic activity in Npt2a KO mice is dependent on renal Npt2c and PiT-2 protein; 2) in DKO mice, renal P(i) reabsorption is not further decreased by FGF23M, but renal vitamin D synthesis is suppressed; and 3) downregulation of intestinal Npt2b may be mediated by a factor(s) other than 1,25(OH)(2)D(3). These findings suggest that Npt2a, Npt2c, and PiT-2 are necessary for the phosphaturic activity of FGF23. Thus complementary regulation of Npt2 family proteins may be involved in systemic P(i) homeostasis.

Fibroblast growth factor 23 mediates the phosphaturic actions of cadmium.

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 D(3) 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.

Polypyrimidine tract-binding protein is involved in regulation of albumin synthesis in response to food intake.

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.

Localization of polypyrimidine-tract-binding protein is involved in the regulation of albumin synthesis by branched-chain amino acids in HepG2 cells.

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 1 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.

Targeted ablation of osteocytes induces osteoporosis with defective mechanotransduction.

Bone remodeling is performed by osteoclasts and osteoblasts at the bone surface. Inside of bone is a network of numerous osteocytes, whose specific function has remained an enigma. Here we describe a transgenic mouse model in which inducible and specific ablation of osteocytes is achieved in vivo through targeted expression of diphtheria toxin (DT) receptor. Following a single injection of DT, approximately 70%-80% of the osteocytes, but apparently no osteoblasts, were killed. Osteocyte-ablated mice exhibited fragile bone with intracortical porosity and microfractures, osteoblastic dysfunction, and trabecular bone loss with microstructural deterioration and adipose tissue proliferation in the marrow space, all of which are hallmarks of the aging skeleton. Strikingly, these "osteocyte-less" mice were resistant to unloading-induced bone loss, providing evidence for the role of osteocytes in mechanotransduction. Thus, osteocytes represent an attractive target for the development of diagnostics and therapeutics for bone diseases, such as osteoporosis.

Overexpression of gamma-glutamyltransferase in transgenic mice accelerates bone resorption and causes osteoporosis.

We previously identified gamma-glutamyltransferase (GGT) by expression cloning as a factor inducing osteoclast formation in vitro. To examine its pathogenic role in vivo, we generated transgenic mice that overexpressed GGT in a tissue-specific manner utilizing the Cre-loxP recombination system. Systemic as well as local production of GGT accelerated osteoclast development and bone resorption in vivo by increasing the sensitivity of bone marrow macrophages to receptor activator of nuclear factor-kappaB ligand, an essential cytokine for osteoclastogenesis. Mutated GGT devoid of enzyme activity was as potent as the wild-type molecule in inducing osteoclast formation, suggesting that GGT acts not as an enzyme but as a cytokine. Recombinant GGT protein increased receptor activator of nuclear factor-kappaB ligand expression in marrow stromal cells and also stimulated osteoclastogenesis from bone marrow macrophages at lower concentrations. Thus, GGT is implicated as being involved in diseases characterized by accelerated osteoclast development and bone destruction and provides a new target for therapeutic intervention.

Unique uptake and efflux systems of inorganic phosphate in osteoclast-like cells.

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-kappaB 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 [(14)C]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 Ca(2+) 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 Ca(2+) 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.

Characterization of the molecular mechanisms involved in the increased insulin secretion in rats with acute liver failure.

To investigate the mechanism of hyperinsulinaemia in rats with acute liver failure induced by the administration of d-galactosamine (GalN), 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 1 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.

Growth-related renal type II Na/Pi cotransporter.

Growth is critically dependent on the retention of a variety of nutrients. The kidney contributes to this positive external balance. In the present study, we isolated a cDNA from the human and rat kidney that encodes a growth-related Na(+)-dependent inorganic phosphate (P(i)) cotransporter (type IIc). Microinjection of type IIc cRNA into Xenopus oocytes demonstrated sodium-dependent P(i) cotransport activity. Affinity for P(i) was 0.07 mm in 100 mm Na(+). The transport activity was dependent on extracellular pH. In electrophysiological studies, type IIc Na/P(i) cotransport was electroneutral, whereas type IIa was highly electrogenic. In Northern blotting analysis, the type IIc transcript was only expressed in the kidney and highly in weaning animals. In immunohistochemical analysis, the type IIc protein was shown to be localized at the apical membrane of the proximal tubular cells in superficial and midcortical nephrons of weaning rat kidney. Hybrid depletion experiments suggested that type IIc could function as a Na/P(i) cotransporter in weaning animals, but its role is reduced in adults. The finding of the present study suggest that the type IIc is a growth-related renal Na/P(i) cotransporter, which has a high affinity for P(i) and is electroneutral.