Takehisa Yamamoto

INCREASED IL-6 PRODUCTION BY CELLS ISOLATED FROM THE FIBROUS BONE DYSPLASIA TISSUES IN PATIENTS WITH MCCUNE-ALBRIGHT SYNDROME

McCune-Albright syndrome (MAS) is characterized by café-au-lait spot, multiple endocrine hyperfunction, and polyostotic fibrous dysplasia. A somatic point mutation of Gsalpha protein was reported to decrease GTPase activity, leading to increase in the GSalpha-associated hormone actions via cAMP. IL-6 is known to stimulate osteoclast formation and in the IL-6 promoter, a cAMP responsive element has been identified. In this paper, we investigated the role of IL-6 in the bone lesions of MAS, using the isolated fibrous cells from the polyostotic fibrous dysplasia tissues in bones of the two patients with MAS. Bone biopsy specimen revealed the increased osteoclast in number. In both patients, a GSalpha mutation (Arg201 -> His) was identified in the cultured fibrous cells. Intracellular cAMP content and IL-6 secretion by the patient cells were increased. Rp-8Br-cAMP significantly inhibited IL-6 production in the patient cells, while it had no effect on normal control. The addition of dibutyryl cAMP significantly increased the synthesis of IL-6 in normal control cells. In contrast, no effect of dibutyryl cAMP on IL-6 synthesis was observed in the cells from one of the MAS patients. These data suggest that IL-6 is, at least, one of the downstream effectors of cAMP and that the increased IL-6 synthesis has a pathogenic role in the bone lesions of MAS patients via increasing the number of osteoclasts. These results may provide a new strategy for the therapy of MAS patients.

Advanced glycation endproducts stimulate interleukin-6 production by human bone-derived cells

Advanced glycation endproducts (AGEs), which result from nonenzymatic reactions of glucose with tissue proteins, have been shown to accumulate on long‐lived proteins in advanced aging and diabetes mellitus. Thus, AGEs have been implicated in some of the chronic complications associated with these disorders. In this study, we investigated the effects of the glucose‐modified protein on the production of the potent bone resorption factors by cells derived from explants of human bone. AGEs stimulated the release of interleukin‐6 (IL‐6) in the culture supernatants from the bone‐derived cells and increased the levels of IL‐6 mRNA in the cells. By contrast, the levels of IL‐11 in the culture supernatants were not altered by AGEs, and the other bone resorption factors IL‐1α and IL‐1β were undetectable (<1.0 pg/ml) either without or with the treatment of AGEs. Electrophoretic mobility‐shift assays revealed that the transcription nuclear factor‐κB, which is critical for the inducible expression of IL‐6, was activated in the nuclear extracts from mouse osteoblastic MC3T3‐E1 cells treated with AGEs. These results suggest that AGEs are involved in bone remodeling modulation by stimulating IL‐6 production in human bone‐derived cells.

The role of fibroblast growth factor 23 for hypophosphatemia and abnormal regulation of vitamin D metabolism in patients with McCune-Albright syndrome.

McCune–Albright syndrome (MAS) is sometimes complicated by hypophosphatemia and abnormally low levels of 1,25(OH)2D in the presence of hypophosphatemia. Recently, fibroblast growth factor 23 (FGF-23) was reported as a phosphaturic and a causal factor of abnormal vitamin D metabolism. This abnormal phosphate and vitamin D metabolism is well known to be found in oncogenic and X-linked hypophosphatemia. We furthermore reported increased circulating plasma FGF-23 levels in patients with oncogenic and X-linked hypophosphatemia. To determine whether FGF-23 may be involved in the pathogenesis of MAS, we measured plasma FGF-23 levels in six MAS patients. As a control for hypophosphatemia, we also investigated the plasma FGF-23 levels in two patients with hereditary hypophosphatemic rickets with hypercalciuria (HHRH). We also investigated the correlation of plasma FGF-23 levels with serum phosphate and 1,25(OH)2D levels after short-term pamidronate therapy in three MAS patients. Plasma FGF-23 levels were significantly increased in patients with MAS compared to normal controls, whereas they were not increased in HHRH patients. Serum phosphate levels of the MAS patients were significantly lower than those observed in normal controls. Plasma FGF-23 levels showed significant negative correlation with serum phosphate concentrations. In three MAS patients, pamidronate therapy decreased plasma FGF-23 levels, which showed significant negative correlation with serum 1,25(OH)2D concentrations. These data suggested that FGF-23 is a possible causal factor for hypophosphatemia and abnormal vitamin D metabolism in MAS.

Increased Interleukin-6 Production in Mouse Osteoblastic MC3T3-E1 Cells Expressing Activating Mutant of the Stimulatory G Protein

The McCune–Albright syndrome (MAS) is characterized by polyostotic fibrous dysplasia, café‐au‐lait spots, and multiple endocrine hyperfunction. An activating missense mutation of the α subunit of the Gs protein (Gsα) was found in several affected tissues, resulting in prolonged stimulation of adenylate cyclase. Our recent study has indicated that the cells derived from the fibrous bone dysplasia tissues in MAS patients produced increased levels of interleukin‐6 (IL‐6), which may be responsible for the increased bone resorption in this disease. In the present investigation, to analyze the molecular mechanism of the increased IL‐6 production by activating mutant Gsα in bone cells, we established mouse osteoblastic MC3T3‐E1 cells stably transfected with the activating mutant Gsα expression vector. These cells showed a significant increase of intracellular cAMP levels and produced a higher amount of IL‐6 than the cells transfected with control vector or wild‐type Gsα expression vector. Analysis of the IL‐6 promoter revealed that any of the AP‐1, nuclear factor (NF)‐IL6, and NF‐κB binding elements are important for the activating mutant Gsα‐induced gene expression. Electrophoretic mobility‐shift assays using nuclear extracts of the mutant Gsα‐expressing cells showed that phospho(Ser133)‐cAMP‐responsive element binding protein (CREB), AP‐1, NF‐IL6, and NF‐κB were increased, compared with the control cells or the wild‐type Gsα‐expressing cells. These results indicate that activating mutant Gsα increases the transcriptional factors binding to CRE, AP‐1, NF‐IL6, and NF‐κB elements to induce IL‐6 gene expression in the osteoblastic cells.

Hypophosphatemic rickets accompanying McCune-Albright syndrome : evidence that a humoral factor causes hypophosphatemia

Abstract. McCune–Albright syndrome (MAS) is sometimes complicated by hypophosphatemia. However, it remains unclear whether a humoral factor is associated with the cause of hypophosphatemia. We isolated cells with mutations of the Gsα gene from fibrous bone dysplasia tissues of two MAS patients (MAS cells). Severe combined immunodeficiency (SCID) mice were subjected to experiments using from one of these cells patients. Effects of conditioned media (CM) isolated from MAS cells (MAS-CM) on phosphate transport were investigated by using rat renal slices, the renal cell line OK-B, rat intestinal rings and the human intestinal cell line Caco-2. In addition, the effects of MAS-CM on human sodium-dependent phosphate transporter (NPT2) gene promoter activity expression were investigated in the renal cell line OK-B2400 and were compared with the effects of CM isolated from a patient with oncogenic hypophosphatemic osteomalacia (OHO). MAS cells caused significant hypophosphatemia (P < 0.05) and elevated serum alkaline phosphatase activity (P < 0.05) in SCID mice. The MAS-CM significantly inhibited phosphate uptake in everted intestinal rings (P < 0.01), whereas it had no effect on glucose uptake. The MAS-CM had no effect on either phosphate uptake in the kidney or NPT2 gene promoter activity. In contrast, the CM of the OHO patient significantly inhibited phosphate uptake and NPT2 gene promoter activity. These results indicate that the humoral factor derived from fibrous dysplasia cells of the MAS patient is different to that from OHO patients, because the humoral factor from the MAS patient inhibited phosphate transport not in the kidney but in the intestine.

Bone marrow-derived osteoclast-like cells from a patient with craniometaphyseal dysplasia lack expression of osteoclast-reactive vacuolar proton pump.

Craniometaphyseal dysplasia (CMD) is a rare craniotubular bone dysplasia transmitted in autosomal dominant or recessive form. This disease is characterized by cranial bone hyperostosis and deformity of the metaphyses of the long bones. Using osteoclast-like cells formed from patient bone marrow cells, we investigated the pathophysiology of CMD in a 3-yr-old patient. Untreated bone marrow cells from the patient differentiated into osteoclast-like cells in vitro. These cells were shown to have vitronectin beta-receptors using a specific monoclonal antibody, i.e., 23C6 (CD51), which reacts with osteoclasts in human bone biopsy samples. However, the number of these osteoclast-like cells formed from the patients bone marrow was only 40% of the normal controls. 1,25-dihydroxyvitamin-D3, bovine 1-34 parathyroid hormone, recombinant human interleukin-1 beta, recombinant human interleukin-6, or recombinant human macrophage colony-stimulating factor significantly increased, while salmon calcitonin significantly inhibited, the number of osteoclast-like cells. However, these cells could not resorb sperm whale dentin slices and lacked the osteoclast-reactive vacuolar proton pump as evidenced by a monoclonal antibody (E11). Western blot analysis using a monoclonal antibody to pp60c-src (327) revealed that protooncogene c-src expression by the platelets of the CMD patient was comparable to the normal control. These data suggest that: (a) the hyperostosis and the metaphyseal long bone deformity in the present CMD patient might be explained by osteoclast dysfunction due to impaired expression of the osteoclast-reactive vacuolar proton pump; and (b) a protooncogene c-src was not associated with the pathogenesis of the present CMD patient.

Hereditary hypophosphatemic rickets with hypercalciuria: a study for the phosphate transporter gene type IIc and osteoblastic function

Two cases of hereditary hypophosphatemic rickets with hypercalciuria (HHRH) were reported in Japanese female siblings. Both of them manifested short stature and bowed legs, and biochemical examination revealed hypophosphatemia, phosphaturia, and hypercalciuria. The serum concentrations of 1,25-dihydroxyvitamin D (1,25(OH)2D) were elevated. In the oral phosphate loading test, serum phosphate levels were markedly increased in the HHRH patients, and the elevation was much higher than that in patients affected with X-linked hypophosphatemic rickets (XLH), suggesting the increased gastrointestinal absorption of phosphate in HHRH. Bone histology studies showed increased osteoid surface and width in HHRH, which was compatible with osteomalacia. In the HHRH patients, there were no hypomineralized periosteocytic lesions, which was a hallmark of XLH in bone histology. In one of the HHRH patients, phosphate administration alone almost completely cured the osteomalacia within a year, although pharmacological doses of 1,25(OH)2D3 had little effect. In osteoblasts isolated from a HHRH patient, basal alkaline phosphatase (ALP) activities and osteocalcin syntheses by a physiological concentration of 1,25(OH)2D3 were not stimulated by the increased medium phosphate concentrations from 0.5 to 4 mM. In contrast, these two parameters were stimulated by the increased medium phosphate concentrations both in normal and XLH osteoblasts, although the regulatory patterns of increased osteocalcin syntheses were different from normal to XLH osteoblasts; 2 and 4 mM of phosphate concentrations at least were necessary for normal and XLH osteoblasts, respectively. The gene analysis of phosphate transporter revealed a novel heterozygous mutation (R564C) in the exon of phosphate transporter NPT type IIc. These lines of evidence suggested that the pathogenesis of osteomalacia in HHRH was different from XLH in terms of the utility of phosphate in osteoblasts. These abnormalities were speculated to be associated with the abnormal functions of phosphate transporter gene type IIc, although the exact roles of this phosphate transporter in the human osteoblast are still unknown.

Increased Serum IL-10 and Endothelin Levels in Hemolytic Uremic Syndrome Caused by Escherichia coli O157

Background: Shiga toxin, produced by Escherichia coli O157:H7, is important for the pathogenicity of the epidemic form of hemolytic uremic syndrome (HUS). This toxin has recently been found to stimulate endothelin-1 synthesis in cultured endothelial cells in vitro. Methods: We investigated endothelin and cytokine levels in sera during a large outbreak of E. coli O157:H7 infection in Osaka, Japan, in 1996. Eleven patients with HUS and 9 patients with hemorrhagic colitis at the onset of E. coli O157:H7 infection were studied. Results: Serum IL-6 (p < 0.01), IL-8 (p < 0.05), IL-10 (p < 0.001) and endothelin (p < 0.001) levels were significantly increased in patients with HUS compared to those with colitis only. The serum thrombomodulin level, a molecular marker of endothelial damage, also showed a significant positive correlation with serum IL-6 (p < 0.01), IL-8 (p < 0.01), IL-10 (p < 0.01) and endothelin (p < 0.001) levels. In a HUS patient, the increase in serum IL-10 and endothelin levels reached a plateau prior to the peak of serum creatinine levels. Conclusion: Increased serum endothelin synthesis by Shiga toxin in vivo was proven in HUS secondary to E. coli O157:H7 infection. Increased serum endothelin and IL-10 levels were speculated to be associated with the development of HUS through vascular endothelial damage caused by E. coli O157:H7 infection.

Hypophosphatemic osteomalacia and bone sclerosis caused by a novel homozygous mutation of the FAM20C gene in an elderly man with a mild variant of Raine syndrome

BACKGROUND Hypophosphatemia and increased serum fibroblast growth factor 23 (FGF23) levels have been reported in young brothers with compound heterozygous mutations for the FAM20C gene; however, rickets was not observed in these cases. We report an adult case of Raine syndrome accompanying hypophosphatemic osteomalacia with a homozygous FAM20C mutation (R408W) associated with increased periosteal bone formation in the long bones and an increase in bone mineral density in the femoral neck. CASE The patient, a 61-year-old man, was born from a cousin-to-cousin marriage. A short stature and severe dental demineralization were reported at an elementary school age. Hypophosphatemia was noted inadvertently at 27years old, at which time he started to take an active vitamin D metabolite (alphacalcidol) and phosphate. He also manifested ossification of the posterior longitudinal ligament. On bone biopsy performed at the age of 41years, we found severe osteomalacia surrounding osteocytes, which appeared to be an advanced form of periosteocytic hypomineralized lesions compared to those reported in patients with X-linked hypophosphatemic rickets. Laboratory data at 61years of age revealed markedly increased serum intact-FGF23 levels, which were likely to be the cause of hypophosphatemia and the decreased level of 1,25(OH)2D. We recently identified a homozygous FAM20C mutation, which was R408W, in this patient. When expressed in HEK293 cells, the R408W mutant protein exhibited impaired kinase activity and secretion. DISCUSSION Our findings suggest that certain homozygous FAM20C mutations can cause FGF23-related hypophosphatemic osteomalacia and indicate the multiple roles of FAM20C in bone.

TWO CASES OF HYPONATREMIC-HYPERTENSIVE SYNDROME IN CHILDHOOD WITH RENOVASCULAR HYPERTENSION

We report two children with renovascular hypertension and fibromuscular dysplasia. They initially presented with severe hyponatremia, hypokalemia, polyuria, and transient proteinuria. This combination of symptoms is known to occur in patients with renovascular and malignant hypertension, and is known as hyponatremic-hypertensive syndrome (HHS), although it is considered rare in children. Since in both of our patients, the renal arterial stenosis was very severely or almost totally occlusive, we could not perform percutaneous transluminal renal artery angioplasty, and therefore nephrectomy was the only option. A histological study showed partial or complete occlusion with intimal hyperplasia and medial fibroplasia of intrarenal arteries such as the interlobular arteries. Conclusion: Both patients showed rapidly progressive renovascular hypertension and loss of function of the affected kidney. In order to preserve renal function in such cases, early invasive intervention appears to be necessary.