Cystine supplementation sustains plasma mercaptalbumin levels in rats fed low-protein diets more effectively than methionine.

We recently reported that dietary cystine maintained plasma mercaptalbumin levels in rats fed low-protein diets. The present study aimed to compare the influence of low-protein diets supplemented with cystine and methionine, which is another sulfur amino acid, on plasma mercaptalbumin levels in rats. Male Sprague-Dawley rats were fed a 20% soy protein isolate diet (control group), 5% soy protein isolate diet (low-protein group) or 5% soy protein isolate diet supplemented with either methionine (low-protein + Met group) or cystine (low-protein + Cyss group) for 1 week. The percentage of mercaptalbumin within total plasma albumin of the low-protein + Met group was significantly lower than that of the control and low-protein + Cyss groups. No significant differences in the mRNA levels of tumor necrosis factor-α, interleukin-6, interleukin-1ß, and cyclooxygenase 2 in blood cells were observed between the low-protein + Met and low-protein + Cyss groups. Treatment with buthionine-(S,R)-sulfoximine, an inhibitor of glutathione synthesis, did not influence the percentage of mercaptalbumin within total plasma albumin in rats fed the low-protein diet supplemented with cystine. These results suggest that supplementation with cystine may be more effective than that with methionine to maintain plasma mercaptalbumin levels in rats with protein malnutrition. Cystine might regulate plasma mercaptalbumin levels via the glutathione-independent pathway.

[A Clinical Study of Orofacial Defect Reconstruction Using a Modified Submental Island Flap following Oral Cancer Resection].

INTRODUCTION: Pedicled flaps are used in orofacial defect reconstruction in older patients and patients with underlying diseases. The submental island flap(SIF)is one type of cervical pedicled flap; however, the modified submental island flap (MSIF), which includes mylohyoid muscle, is a simpler and safer type. Here, we report a clinical study of orofacial defect reconstruction using the MSIF following oral cancer resection. PATIENTS AND METHODS: From January 2019 to December 2020, we retrospectively examined 10 cases of reconstruction using the MSIF following oral squamous cell carcinoma resection. RESULTS: The study population consisted of 7 men and 3 women with a mean age of 76.0(66-88)years. The primary sites were lower gingiva(n=5), tongue(n=3), and buccal mucosa in(n=2). Surgical procedures included marginal mandibulectomy( n=3), segmental mandibulectomy(n=1), partial glossectomy(n=2), hemiglossectomy(n=1), buccal mucosa resection(n=2), and combined partial glossectomy and segmental mandibulectomy(n=1). The average flap size was 61.4×36.0 mm. The average time of flap elevation was 32.4(23-50)minutes. During orofacial surgery using the MSIF, organs adjacent to the primary site could also be reconstructed. There were no complications in any patients. CONCLUSION: The MSIF is useful for orofacial defect reconstruction in older patients and patients with underlying diseases.

Accuracy and reliability of a smartphone application for measuring the knee joint angle.

[Purpose] Recently, a photo-based smartphone application for angle measurement-"Grid line imaging application Professional"-was developed to evaluate joint disease treatments. The aim of this study was to determine the accuracy and reliability of the application. [Participants and Methods] We measured the knee joint of a mannequin using an application and a universal goniometer. Twelve examiners measured eight knee joints of mannequins at different arbitrary angles using the application and a universal goniometer. Correlations between the application and universal goniometer measurements were examined using scatter plots and correlation coefficients. Systematic errors of the application were visually confirmed using the Bland-Altman method. Intra-class correlation coefficients were used to evaluate the inter-examiner reliability of the application. [Results] The application and universal goniometer measurements showed a good correlation (r=0.99) and no systematic error. The intra-class correlation coefficient for inter-examiner reliability was 0.999. Furthermore, to evaluate intra-examiner reliability, six examiners measured six different knee joints twice using the application on a 2-day interval. The intra-class correlation coefficient for intra-examiner reliability was 0.982. [Conclusion] The accuracy of the application was equivalent to that of a universal goniometer, and both the inter- and intra-examiner reliabilities of the application were almost perfect.

NAD metabolism and the SLC34 family: evidence for a liver-kidney axis regulating inorganic phosphate.

The solute carrier 34 (SLC34) family of membrane transporters is a major contributor to Pi homeostasis. Many factors are involved in regulating the SLC34 family. The roles of the bone mineral metabolism factors parathyroid hormone (PTH) and fibroblast growth factor 23 (FGF23) in Pi homeostasis are well studied. Intracellular Pi is thought to be involved in energy metabolism, such as ATP production. Under certain conditions of altered energy metabolism, plasma Pi concentrations are affected by the regulation of a Pi shift into cells or release from the tissues. We recently investigated the mechanism of hepatectomy-related hypophosphatemia, which is thought to involve an unknown phosphaturic factor. Hepatectomy-related hypophosphatemia is due to impaired nicotinamide adenine dinucleotide (NAD) metabolism through its effects on the SLC34 family in the liver-kidney axis. The oxidized form of NAD, NAD+, is an essential cofactor in various cellular biochemical reactions. Levels of NAD+ and its reduced form NADH vary with the availability of dietary energy and nutrients. Nicotinamide phosphoribosyltransferase (Nampt) generates a key NAD+ intermediate, nicotinamide mononucleotide, from nicotinamide and 5-phosphoribosyl 1-pyrophosphate. The liver, an important organ of NAD metabolism, is thought to release metabolic products such as nicotinamide and may control NAD metabolism in other organs. Moreover, NAD is an important regulator of the circadian rhythm. Liver-specific Nampt-deficient mice and heterozygous Nampt mice have abnormal daily plasma Pi concentration oscillations. These data indicate that NAD metabolism in the intestine, liver, and kidney is closely related to Pi metabolism through the SLC34 family. Here, we review the relationship between the SLC34 family and NAD metabolism based on our recent studies.

Systemic network for dietary inorganic phosphate adaptation among three organs.

Inorganic phosphate (Pi) secretion from the salivary glands and dietary Pi are key Pi sources. The regulatory mechanisms of Pi homeostasis in the salivary glands are unknown. We investigated how salivary Pi concentrations are regulated by dietary Pi in mouse models. Dietary manipulation significantly changed the levels of Npt2b protein in the salivary gland ductal cells. In addition, rapid feeding on a high-Pi diet increased the saliva Pi concentrations and led to rapid endocytosis of Npt2b in the apical membranes of the duct cells. Global Npt2b± mice exhibited increased salivary Pi concentrations and intestine-specific deletion of Npt2b after high Pi loading increased the salivary Pi concentrations. These findings indicate that Npt2b levels in the salivary glands affect the salivary Pi concentration and are regulated by dietary Pi. Intestinal Npt2b levels might also affect salivary Pi concentrations as well as renal Pi excretion. These findings suggest Pi is endogenously recycled by salivary Pi secretion, intestinal Pi absorption, and renal Pi excretion.

Role of the putative PKC phosphorylation sites of the type IIc sodium-dependent phosphate transporter in parathyroid hormone regulation.

BACKGROUND: Injection of parathyroid hormone (PTH) rapidly stimulates renal Pi excretion, in part by downregulating NaPi-IIa (Npt2a/SLC34A1) and NaPi-IIc (Npt2c/SLC34A3) transporters. The mechanisms underlying the effects of PTH on NaPi-IIc are not fully elucidated. METHODS: We analyzed the effect of PTH on inorganic phosphate (Pi) reabsorption in Npt2a-/- mice to eliminate the influence of Npt2a on renal Pi reabsorption. In opossum kidney (OK) cells and Xenopus oocytes, we investigated the effect of NaPi-IIc transporter phosphorylation. Studies of mice with mutations of NaPi-IIc protein in which serine and threonine were replaced with either alanine (A), which prevents phosphorylation, or aspartic acid (D), which mimics the charged state of phosphorylated NaPi-IIc, were also performed to evaluate the involvement of phosphorylation in the regulation of transport function. RESULTS: The Npt2a-/- experiments showed that PTH administration rapidly inactivated NaPi-IIc function in the apical membrane of proximal tubular cells. Analysis of mutant proteins (S71, S138, T151, S174, T583) at putative protein kinase C sites, revealed that S138 markedly suppressed the function and cellular expression of mouse NaPi-IIc in Xenopus oocytes and OK cells. In addition, 138D had a short half-life compared with wild-type protein. CONCLUSIONS: The present study suggests that acute regulation of NaPi-IIc protein by PTH is involved in the inactivation of Na+-dependent Pi cotransporter activity and that phosphorylation of the transporter is involved in the rapid modification.

Analysis of opossum kidney NaPi-IIc sodium-dependent phosphate transporter to understand Pi handling in human kidney.

BACKGROUND: The role of Na+-dependent inorganic phosphate (Pi) transporters in the human kidney is not fully clarified. Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is caused by loss-of-function mutations in the IIc Na+-dependent Pi transporter (NPT2c/Npt2c/NaPi-IIc) gene. Another Na+-dependent type II transporter, (NPT2A/Npt2a/NaPi-IIa), is also important for renal Pi reabsorption in humans. In mice, Npt2c deletion does not lead to hypophosphatemia and rickets because Npt2a compensates for the impaired Pi reabsorption. To clarify the differences between mouse and human, we investigated the relation between NaPi-IIa and NaPi-IIc functions in opossum kidney (OK) cells. METHODS: We cloned NaPi-IIc from OK cells and created opossum NaPi-IIc (oNaPi-IIc) antibodies. We used oNaPi-IIc small interference (si)RNA and investigated the role of NaPi-IIc in Pi transport in OK cells. RESULTS: We cloned opossum kidney NaPi-IIc cDNAs encoding 622 amino acid proteins (variant1) and examined their pH- and sodium-dependency. The antibodies reacted specifically with 75-kDa and 150-kDa protein bands, and the siRNA of NaPi-IIc markedly suppressed endogenous oNaPi-IIc in OK cells. Treatment with siRNA significantly suppressed the expression of NaPi-4 (NaPi-IIa) protein and mRNA. oNaPi-IIc siRNA also suppressed Na+/H+ exchanger regulatory factor 1 expression in OK cells. CONCLUSION: These findings suggest that NaPi-IIc is important for the expression of NaPi-IIa (NaPi-4) protein in OK cells. Suppression of Npt2c may downregulate Npt2a function in HHRH patients.

The sodium phosphate cotransporter family and nicotinamide phosphoribosyltransferase contribute to the daily oscillation of plasma inorganic phosphate concentration.

Circulating inorganic phosphate exhibits a remarkable daily oscillation based on food intake. In humans and rodents, the daily oscillation in response to food intake may be coordinated to control the intestinal absorption, renal excretion, cellular shifts, and extracellular concentration of inorganic phosphate. However, mechanisms regulating the resulting oscillation are unknown. Here we investigated the roles of the sodium phosphate cotransporter SLC34 (Npt2) family and nicotinamide phosphoribosyltransferase (Nampt) in the daily oscillation of plasma inorganic phosphate levels. First, it is roughly linked to urinary inorganic phosphate excretion. Second, expression of renal Npt2a and Npt2c, and intestinal Npt2b proteins also exhibit a dynamic daily oscillation. Analyses of Npt2a, Npt2b, and Npt2c knockout mice revealed the importance of renal inorganic phosphate reabsorption and cellular inorganic phosphate shifts in the daily oscillation. Third, experiments in which nicotinamide and a specific Nampt inhibitor (FK866) were administered in the active and rest phases revealed that the Nampt/NAD+ system is involved in renal inorganic phosphate excretion. Additionally, for cellular shifts, liver-specific Nampt deletion disturbed the daily oscillation of plasma phosphate during the rest but not the active phase. In systemic Nampt+/- mice, NAD levels were significantly reduced in the liver, kidney, and intestine, and the daily oscillation (active and rest phases) of the plasma phosphate concentration was attenuated. Thus, the Nampt/NAD+ system for Npt2 regulation and cellular shifts to tissues such as the liver play an important role in generating daily oscillation of plasma inorganic phosphate levels.

A Role of Intestinal Alkaline Phosphatase 3 (Akp3) in Inorganic Phosphate Homeostasis.

BACKGROUND/AIMS: Hyperphosphatemia is a serious complication of late-stage chronic kidney disease (CKD). Intestinal inorganic phosphate (Pi) handling plays an important role in Pi homeostasis in CKD. We investigated whether intestinal alkaline phosphatase 3 (Akp3), the enzyme that hydrolyzes dietary Pi compounds, is a target for the treatment of hyperphosphatemia in CKD. METHODS: We investigated Pi homeostasis in Akp3 knockout mice (Akp3-/-). We also studied the progression of renal failure in an Akp3-/- mouse adenine treated renal failure model. Plasma, fecal, and urinary Pi and Ca concentration were measured with commercially available kit, and plasma fibroblast growth factor 23, parathyroid hormone, and 1,25(OH)2D3 concentration were measured with ELISA. Brush border membrane vesicles were prepared from mouse intestine using the Ca2+ precipitation method and used for Pi transport activity and alkaline phosphatase activity. In vivo intestinal Pi absorption was measured with oral 32P administration. RESULTS: Akp3-/- mice exhibited reduced intestinal type II sodium-dependent Pi transporter (Npt2b) protein levels and Na-dependent Pi co-transport activity. In addition, plasma active vitamin D levels were significantly increased in Akp3-/- mice compared with wild-type animals. In the adenine-induced renal failure model, Akp3 gene deletion suppressed hyperphosphatemia. CONCLUSION: The present findings indicate that intestinal Akp3 deletion affects Na+-dependent Pi transport in the small intestine. In the adenine-induced renal failure model, Akp3 is predicted to be a factor contributing to suppression of the plasma Pi concentration.

Effect of Npt2b deletion on intestinal and renal inorganic phosphate (Pi) handling.

BACKGROUND: Hyperphosphatemia is common in chronic kidney disease and is associated with morbidity and mortality. The intestinal Na+-dependent phosphate transporter Npt2b is thought to be an important molecular target for the prevention of hyperphosphatemia. The role of Npt2b in the net absorption of inorganic phosphate (Pi), however, is controversial. METHODS: In the present study, we made tamoxifen-inducible Npt2b conditional knockout (CKO) mice to analyze systemic Pi metabolism, including intestinal Pi absorption. RESULTS: Although the Na+-dependent Pi transport in brush-border membrane vesicle uptake levels was significantly decreased in the distal intestine of Npt2b CKO mice compared with control mice, plasma Pi and fecal Pi excretion levels were not significantly different. Data obtained using the intestinal loop technique showed that Pi uptake in Npt2b CKO mice was not affected at a Pi concentration of 4 mM, which is considered the typical luminal Pi concentration after meals in mice. Claudin, which may be involved in paracellular pathways, as well as claudin-2, 12, and 15 protein levels were significantly decreased in the Npt2b CKO mice. Thus, Npt2b deficiency did not affect Pi absorption within the range of Pi concentrations that normally occurs after meals. CONCLUSION: These findings indicate that abnormal Pi metabolism may also be involved in tight junction molecules such as Cldns that are affected by Npt2b deficiency.

Control of phosphate balance by the kidney and intestine.

The prevention and correction of hyperphosphatemia are major goals of the treatment of chronic kidney disease (CKD)-bone mineral disorders, and thus, Pi balance requires special attention. Pi balance is maintained by intestinal absorption, renal excretion, and bone accretion. The kidney is mainly responsible for the plasma Pi concentration. In CKD, reduced glomerular filtration rate leads to various Pi metabolism abnormalities, and Pi absorption in the small intestine also has an important role in Pi metabolism. Disturbances in Pi metabolism are mediated by a series of complex changes in regulatory hormones originating from the skeleton, intestine, parathyroid gland, and kidney. In this review, we describe the regulation of type II sodium-dependent Pi co-transporters by the kidney and intestine, including the regulation of Pi transport, circadian rhythm, and the vicious circle between salivary Pi secretion and intestinal Pi absorption in animals with and without CKD.

[Update on recent progress in vitamin D research. Vitamin D and muscle tissues.]

In elderly people, vitamin D insufficiency may increase the risk of fracture, and may also be involved in the development of sarcopenia or cardiovascular decline. Recent studies using vitamin D receptor knockout mice unveiled multiple functions of vitamin D beyond bone and mineral metabolism. This review describes the actions of vitamin D in skeletal, cardiac, and vascular smooth muscle. Eldecalcitol, an active vitamin D analog developed for the treatment of osteoporosis, also effectively improved skeletal muscle function in vivo, and may have synergistic activities in bone and muscle. Maintaining an optimal vitamin D status protects against fracture, and is expected to have beneficial effects for improving chronic diseases accompanied by muscle function abnormalities.

[Applicability of Pedicled Coronoid Process and Temporal Muscle(Fascial)Combined(PCPTM)Flap for Reconstruction of Orbital Floor Defect Following Hemi-Maxillectomy for Advanced Maxillary Cancer - A Report of Two Cases].

We usually perform surgery for resectable oral and maxillofacial carcinomas. Following complete cancer resection, reconstruction of soft and hard tissues using various types of local flaps and/or vascularized free flaps is usually performed. The maxilla is composed of various anatomical structures. In particular, reconstruction of the orbit is one of the most important and challenging procedures for prevention of functional and esthetic complications. Here we report 2 cases of orbital floor defect reconstruction following advanced maxillary cancer resection using a pedicled coronoid process and temporal muscle (fascial)combined(PCPTM)flap. Case 1: A 69-year-old Japanese man with squamous cell carcinoma of the left maxilla (cT4aN2bM0, Stage IV A). Case 2: An 86-year-old Japanese woman with recurrence of myoepithelial carcinoma of the left maxilla. In both cases, the orbital floor defect was reconstructed following hemi-maxillectomy using a PCPTM flap. Minor infection and/or partial necrosis were observed postoperatively, and a maxillofacial prosthesis was used in one case. A PCPTM flap was feasible for reconstruction of surgical defects of the orbital floor following maxillectomy for cancer.

1,25-Dihydroxyvitamin D regulates expression of the tryptophan hydroxylase 2 and leptin genes: implication for behavioral influences of vitamin D.

To investigate vitamin D-related control of brain-expressed genes, candidate vitamin D responsive elements (VDREs) at -7/-10 kb in human tryptophan hydroxylase (TPH)2 were probed. Both VDREs bound the vitamin D receptor (VDR)-retinoid X receptor (RXR) complex and drove reporter gene transcription in response to 1,25-dihydroxyvitamin D3 (1,25D). Brain TPH2 mRNA, encoding the rate-limiting enzyme in serotonin synthesis, was induced 2.2-fold by 10 nM 1,25D in human U87 glioblastoma cells and 47.8-fold in rat serotonergic RN46A-B14 cells. 1,25D regulation of leptin (Lep), encoding a serotoninlike satiety factor, was also examined. In mouse adipocytes, 1,25D repressed leptin mRNA levels by at least 84%, whereas 1,25D induced leptin mRNA 15.1-fold in human glioblastoma cells. Chromatin immunoprecipitation sequencing analysis of the mouse Lep gene in response to 1,25D revealed a cluster of regulatory sites (cis-regulatory module; CRM) at -28 kb that 1,25D-dependently docked VDR, RXR, C/EBPß, and RUNX2. This CRM harbored 3 VDREs and single C/EBPß and RUNX2 sites. Therefore, the expression of human TPH2 and mouse Lep are governed by 1,25D, potentially via respective VDREs located at -7/-10 kb and -28 kb. These results imply that vitamin D affects brain serotonin concentrations, which may be relevant to psychiatric disorders, such as autism, and may control leptin levels and affect eating behavior.

Regulation of renal phosphate handling: inter-organ communication in health and disease.

In this review, we focus on the interconnection of inorganic phosphate (Pi) homeostasis in the network of the bone-kidney, parathyroid-kidney, intestine-kidney, and liver-kidney axes. Such a network of organ communication is important for body Pi homeostasis. Normalization of serum Pi levels is a clinical target in patients with chronic kidney disease (CKD). Particularly, disorders of the fibroblast growth factor 23/klotho system are observed in early CKD. Identification of phosphaturic factors from the intestine and liver may enhance our understanding of body Pi homeostasis and Pi metabolism disturbances in CKD patients.

Hepatectomy-related hypophosphatemia: a novel phosphaturic factor in the liver-kidney axis.

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.

Short-term dietary phosphate restriction up-regulates ileal fibroblast growth factor 15 gene expression in mice.

Members of the fibroblast growth factor (FGF) 19 subfamily, including FGF23, FGF15/19, and FGF21, have a role as endocrine factors which influence the metabolism of inorganic phosphate (Pi) and vitamin D, bile acid, and energy. It has been reported that dietary Pi regulates circulating FGF23. In this study, the short-term effects of dietary Pi restriction on the expression of FGF19 subfamily members in mice were analyzed. An initial analysis confirmed plasma FGF23 levels positively correlated with the amount of dietary Pi. On the other hand, ileal Fgf15 gene expression, but not hepatic Fgf21 gene expression, was up-regulated by dietary Pi restriction. In addition, we observed the increase of plasma 1,25-dihydroxyvitamin D [1,25(OH)2D] levels by dietary Pi restriction, and the up-regulation of ileal Fgf15 mRNA expression by 1,25(OH)2D3 and vitamin D receptor (VDR). Importantly, dietary Pi restriction-induced Fgf15 gene expression was prevented in VDR-knockout mice. Furthermore, diurnal variations of plasma triglyceride concentrations and hepatic mRNA expression of the bile acid synthesis enzyme Cyp7a1 as one of Fgf15 negative target genes was influenced by dietary Pi restriction. These results suggest that dietary Pi restriction up-regulates ileal Fgf15 gene expression through 1,25(OH)2D3 and VDR, and may affect hepatic bile acid homeostasis.

The Role of Intestinal Cytochrome P450s in Vitamin D Metabolism.

Vitamin D hydroxylation in the liver/kidney results in conversion to its physiologically active form of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. 1,25(OH)2D3 controls gene expression through the nuclear vitamin D receptor (VDR) mainly expressed in intestinal epithelial cells. Cytochrome P450 (CYP) 24A1 is a catabolic enzyme expressed in the kidneys. Interestingly, a recently identified mutation in another CYP enzyme, CYP3A4 (gain-of-function), caused type III vitamin D-dependent rickets. CYP3A are also expressed in the intestine, but their hydroxylation activities towards vitamin D substrates are unknown. We evaluated CYP3A or CYP24A1 activities on vitamin D action in cultured cells. In addition, we examined the expression level and regulation of CYP enzymes in intestines from mice. The expression of CYP3A or CYP24A1 significantly reduced 1,25(OH)2D3-VDRE activity. Moreover, in mice, Cyp24a1 mRNA was significantly induced by 1,25(OH)2D3 in the intestine, but a mature form (approximately 55 kDa protein) was also expressed in mitochondria and induced by 1,25(OH)2D3, and this mitochondrial enzyme appears to hydroxylate 25OHD3 to 24,25(OH)2D3. Thus, CYP3A or CYP24A1 could locally attenuate 25OHD3 or 1,25(OH)2D3 action, and we suggest the small intestine is both a vitamin D target tissue, as well as a newly recognized vitamin D-metabolizing tissue.

1,25-dihydroxyvitamin D(3) regulation of fibroblast growth factor-23 expression in bone cells: evidence for primary and secondary mechanisms modulated by leptin and interleukin-6.

Fibroblast growth factor-23 (FGF23) is a circulating hormone that acts to correct hyperphosphatemic states by inhibiting renal phosphate reabsorption and to prevent hypervitaminosis D by feedback repressing 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) biosynthesis. FGF23 gene expression in the osteoblast/osteocyte is induced by the nuclear vitamin D receptor (VDR) bound to 1,25(OH)2D3, but cycloheximide sensitivity of this induction suggests that it may occur largely via secondary mechanisms requiring cooperating transcription factors. We therefore sought to identify 1,25(OH)2D3-regulated transcription factors that might impact FGF23 expression. Although neither leptin nor interleukin-6 (IL-6) alone affects FGF23 expression, leptin treatment was found to potentiate 1,25(OH)2D3 upregulation of FGF23 in UMR-106 cells, whereas IL-6 treatment blunted this upregulation. Genomic analyses revealed conserved binding sites for STATs (signal transduction mediators of leptin and IL-6 action) along with transcription factor ETS1 in human and other mammalian FGF23 genes. Further, STAT3, STAT1, ETS1, and VDR mRNAs were induced in a dose-dependent manner by 1,25(OH)2D3 in UMR-106 cells. Bioinformatic analysis identified nine potential VDREs in a genomic interval containing human FGF23. Six of the putative VDREs were capable of mediating direct transcriptional activation of a heterologous reporter gene when bound by a 1,25(OH)2D3-liganded VDR complex. A model is proposed wherein 1,25(OH)2D3 upregulates FGF23 production directly via multiple VDREs and indirectly via induction of STAT3, ETS1, and VDR transcription factors that are then activated via cell surface and intracellular signaling to cooperate in the induction of FGF23 through DNA looping and generation of euchromatin architecture.

Molecular mechanisms of vitamin D action.

The hormonal metabolite of vitamin D, 1α,25-dihydroxyvitamin D(3) (1,25D), initiates biological responses via binding to the vitamin D receptor (VDR). When occupied by 1,25D, VDR interacts with the retinoid X receptor (RXR) to form a heterodimer that binds to vitamin D responsive elements in the region of genes directly controlled by 1,25D. By recruiting complexes of either coactivators or corepressors, ligand-activated VDR-RXR modulates the transcription of genes encoding proteins that promulgate the traditional functions of vitamin D, including signaling intestinal calcium and phosphate absorption to effect skeletal and calcium homeostasis. Thus, vitamin D action in a particular cell depends upon the metabolic production or delivery of sufficient concentrations of the 1,25D ligand, expression of adequate VDR and RXR coreceptor proteins, and cell-specific programming of transcriptional responses to regulate select genes that encode proteins that function in mediating the effects of vitamin D. For example, 1,25D induces RANKL, SPP1 (osteopontin), and BGP (osteocalcin) to govern bone mineral remodeling; TRPV6, CaBP(9k), and claudin 2 to promote intestinal calcium absorption; and TRPV5, klotho, and Npt2c to regulate renal calcium and phosphate reabsorption. VDR appears to function unliganded by 1,25D in keratinocytes to drive mammalian hair cycling via regulation of genes such as CASP14, S100A8, SOSTDC1, and others affecting Wnt signaling. Finally, alternative, low-affinity, non-vitamin D VDR ligands, e.g., lithocholic acid, docosahexaenoic acid, and curcumin, have been reported. Combined alternative VDR ligand(s) and 1,25D/VDR control of gene expression may delay chronic disorders of aging such as osteoporosis, type 2 diabetes, cardiovascular disease, and cancer.