Toshimi Michigami

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.

Consensus Statement: Global Consensus Recommendations on Prevention and Management of Nutritional Rickets

BACKGROUND Vitamin D and calcium deficiencies are common worldwide, causing nutritional rickets and osteomalacia, which have a major impact on health, growth, and development of infants, children, and adolescents; the consequences can be lethal or can last into adulthood. The goals of this evidence-based consensus document are to provide health care professionals with guidance for prevention, diagnosis, and management of nutritional rickets and to provide policy makers with a framework to work toward its eradication. EVIDENCE A systematic literature search examining the definition, diagnosis, treatment, and prevention of nutritional rickets in children was conducted. Evidence-based recommendations were developed using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) system that describe the strength of the recommendation and the quality of supporting evidence. PROCESS Thirty-three nominated experts in pediatric endocrinology, pediatrics, nutrition, epidemiology, public health, and health economics evaluated the evidence on specific questions within five working groups. The consensus group, representing 11 international scientific organizations, participated in a multiday conference in May 2014 to reach a global evidence-based consensus. RESULTS This consensus document defines nutritional rickets and its diagnostic criteria and describes the clinical management of rickets and osteomalacia. Risk factors, particularly in mothers and infants, are ranked, and specific prevention recommendations including food fortification and supplementation are offered for both the clinical and public health contexts. CONCLUSION Rickets, osteomalacia, and vitamin D and calcium deficiencies are preventable global public health problems in infants, children, and adolescents. Implementation of international rickets prevention programs, including supplementation and food fortification, is urgently required.

Global Consensus Recommendations on Prevention and Management of Nutritional Rickets

Background: Vitamin D and calcium deficiencies are common worldwide, causing nutritional rickets and osteomalacia, which have a major impact on health, growth, and development of infants, children, and adolescents; the consequences can be lethal or can last into adulthood. The goals of this evidence-based consensus document are to provide health care professionals with guidance for prevention, diagnosis, and management of nutritional rickets and to provide policy makers with a framework to work toward its eradication. Evidence: A systematic literature search examining the definition, diagnosis, treatment, and prevention of nutritional rickets in children was conducted. Evidence-based recommendations were developed using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) system that describes the strength of the recommendation and the quality of supporting evidence. Process: Thirty-three nominated experts in pediatric endocrinology, pediatrics, nutrition, epidemiology, public health, and health economics evaluated the evidence on specific questions within five working groups. The consensus group, representing 11 international scientific organizations, participated in a multiday conference in May 2014 to reach a global evidence-based consensus. Results: This consensus document defines nutritional rickets and its diagnostic criteria and describes the clinical management of rickets and osteomalacia. Risk factors, particularly in mothers and infants, are ranked, and specific prevention recommendations including food fortification and supplementation are offered for both the clinical and public health contexts. Conclusion: Rickets, osteomalacia, and vitamin D and calcium deficiencies are preventable global public health problems in infants, children, and adolescents. Implementation of international rickets prevention programs, including supplementation and food fortification, is urgently required.

Wnt signaling in bone metabolism.

A variety of in vivo models have increased understanding of the role of Wnt signaling in bone since mutations in the LRP5 gene were found in human bone disorders. Canonical Wnt signaling encourages mesenchymal progenitor cells to differentiate into osteoblasts. In osteoblasts, Wnt pathway also promotes proliferation and mineralization, while blocks apoptosis and osteoclastogenesis by increasing the OPG/RANKL ratio. Lrp6-mediated signaling in osteoblasts may regulate osteoclastogenesis. However, the role of canonical Wnt signaling in osteoclasts remains unknown, and our understanding of the role of non-canonical Wnt signaling in bone biology is also not sufficient. As to pharmacological intervention, many levels may be considered to target in Wnt signaling pathway, although tumorigenicity and toxicity to other tissues are important. Mesd might be one of target molecules to increase the quantity of LRP5/6 in the plasma membrane. Since sclerostin is almost exclusively expressed in osteocytes, abrogating sclerostin is the most promising design.

Clinical usefulness of measurement of fibroblast growth factor 23 (FGF23) in hypophosphatemic patients Proposal of diagnostic criteria using FGF23 measurement

Fibroblast growth factor 23 (FGF23) plays important roles in the development of hypophosphatemic diseases such as tumor-induced osteomalacia (TIO) and X-linked hypophosphatemic rickets/osteomalacia (XLH). However, clinical usefulness of measurement of FGF23 has not been established. The objective of this study is to examine the importance of FGF23 measurement in the diagnosis of hypophosphatemic diseases. Biochemical parameters concerning phosphate metabolism were analyzed in a cross-sectional study. 32 patients with TIO, 28 patients with XLH and 16 hypophosphatemic patients with other causes including vitamin D deficiency, Fanconis syndrome and Cushings syndrome were studied. In patients with TIO and XLH, FGF23 was above the upper limit of the reference range in most patients irrespective of medical treatment. The lowest FGF23 in these patients was 38.0 pg/ml. FGF23 in hypophosphatemic patients with other causes was undetectable (less than 3 pg/ml) in 12 patients and the highest FGF23 in this group was 23.9 pg/ml. Relationship between phosphate and FGF23 indicated that TIO and XLH are diseases with high FGF23 and hypophosphatemia judged by age-dependent reference ranges for serum phosphate. FGF23 measurement is useful for differential diagnosis of hypophosphatemic diseases caused by excess actions of FGF23 and other etiologies. High FGF23 with low phosphate judged by age-dependent reference ranges for phosphate establishes the diagnosis of diseases caused by excess actions of FGF23.

PTH/cAMP/PKA Signaling Facilitates Canonical Wnt Signaling Via Inactivation of Glycogen Synthase Kinase-3β in Osteoblastic Saos-2 Cells

Although the intermittent administration of PTH is known to stimulate the bone formation, the underlying mechanisms are not fully understood. Here we investigated the crosstalk between PTH/cAMP signaling and canonical Wnt signaling using the human osteoblastic cell line Saos‐2. Treatment with PTH or forskolin, an activator of adenylate cyclase, facilitated T‐cell factor (TCF)‐dependent transactivation in a dose‐dependent manner, which was abolished by pre‐treatment with a PKA inhibitor, H89. Wnt3a and forskolin synergistically increased the TCF‐dependent transactivation. Interestingly, intermittent treatment with PTH enhanced the TCF‐dependent transactivation more profoundly than continuous treatment. In addition to the effects on TCF‐dependent reporter activity, treatment with PTH or forskolin resulted in the increased expression of endogenous targets of Wnts, Wnt‐induced secreted protein 2 (WISP2) and naked cuticle 2 (NKD2). We then investigated the convergence point of PTH/cAMP signaling and the canonical Wnt pathway. Western blotting demonstrated that GSK‐3β was rapidly phosphorylated at Ser9 on treatment with PTH or forskolin, leading to its inactivation. Moreover, overexpression of a constitutively active mutant of GSK‐3β abolished the TCF‐dependent transactivation induced by forskolin. On the other hand, overexpression of the Wnt antagonist Dickkopf‐1 (DKK1) failed to cancel the effects of forskolin on the canonical Wnt pathway. Interestingly, treatment with Wnt3a markedly reduced the forskolin‐induced expression of receptor activator of NF‐κB ligand (RANKL), a target gene of PTH/cAMP/PKA. These results suggest that cAMP/PKA signaling activates the canonical Wnt pathway through the inactivation of GSK‐3β, whereas Wnt signaling might inhibit bone resorption through a negative impact on RANKL expression in osteoblasts. J. Cell. Biochem. 104: 304–317, 2008.

A blocking peptide for transforming growth factor-β1 activation prevents hepatic fibrosis in vivo

BACKGROUND/AIMS Thrombospondin-1 is a major activator of transforming growth factor-beta1 (TGF-beta1), and a peptide derived from the latency-associated peptide, Leu-Ser-Lys-Leu (LSKL), inhibits the activation of TGF-beta1. In this study, the effects of LSKL on the hepatocyte damage and fibrogenesis in dimethylnitrosamine (DMN)-induced rat liver fibrosis were examined. METHODS Animals were given an intraperitoneal (i.p.) injection of DMN or saline three times per week for 4 weeks, and treated with LSKL, a control peptide, or saline i.p. daily. RESULTS Liver atrophy caused by DMN-injection was significantly inhibited in the DMN+LSKL group. The degrees of necrosis/degeneration and fibrosis scores were significantly lower in the DMN+LSKL group than in the control groups. The hydroxyproline content was significantly higher in the control groups than in the DMN+LSKL group. The amount of active TGF-beta1 was less in the DMN+LSKL group than in the control groups, and the active/total TGF-beta1 ratio in the DMN+LSKL group was suppressed in the control groups. Phosphorylation of Smad 2 in the liver was significantly decreased in the DMN+LSKL group. CONCLUSIONS The LSKL peptide prevented the progression of hepatic damage and fibrosis through the inhibition of TGF-beta1 activation and its signal transduction in vivo.

Analysis of the Molecular Mechanism for the Antagonistic Action of a Novel 1α,25-Dihydroxyvitamin D3 Analogue toward Vitamin D Receptor Function

We have recently reported that 23(S)-25-dehydro-1α-hydroxyvitamin D3-26,23-lactone (TEI-9647) efficiently blocks the differentiation of HL-60 cells induced by 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) (Miura, D., Manabe, K., Ozono, K., Saito, M., Gao, Q., Norman, A. W., and Ishizuka, S. (1999) J. Biol. Chem. 274, 16392–16399). To clarify the molecular mechanisms of this antagonism, we examined whether TEI-9647 antagonizes the genomic effects of 1α,25(OH)2D3. 10−7 to 10−9 m TEI-9647 inhibited the transactivation effect of 10−8 m1α,25(OH)2D3 in a dose-dependent manner, while TEI-9647 alone did not activate the reporter activity driven by SV40 promoter containing two vitamin D response elements in Saos-2 cells. The antagonistic effect of TEI-9647 was also observed using the rat 24-hydroxylase gene promoter, but the effect was weaker in HeLa and COS-7 cells than in Saos-2 cells. TEI-9647 also exhibited antagonism in an assay system where the VDR fused to the GAL4 DNA-binding domain and the reporter plasmid containing the GAL4 binding site were used in Saos-2 cells, but did not in HeLa cells. TEI-9647 reduced the interaction between VDR and RXRα according to the results obtained from the mammalian two-hybrid system in Saos-2 cells, but did not in HeLa cells. The two-hybrid system also revealed that the interaction between VDR and SRC-1 was reduced by TEI-9647 in Saos-2 cells. These results demonstrate that the novel 1α,25(OH)2D3 analogue, TEI-9647, is the first synthetic ligand for the VDR that efficiently antagonizes the action of 1α,25(OH)2D3, although the extent of its antagonism depends on cell type.

Lrp6 Hypomorphic Mutation Affects Bone Mass Through Bone Resorption in Mice and Impairs Interaction With Mesd

Low‐density lipoprotein receptor‐related protein 5 (LRP5) regulates bone acquisition by controlling bone formation. Because roles of LRP6, another co‐receptor for Wnts, in postnatal bone metabolism have not been fully elucidated, we studied bone phenotype in mice harboring an Lrp6 hypomorphic mutation, ringelschwanz (rs), and characterized the mutant protein. First, we performed pQCT, bone histomorphometry, and immunohistochemistry on tibias of Lrp6rs/rs and Lrp6+/+ mice and determined biochemical parameters for bone turnover. Lrp6rs/rs mice exhibited reduced trabecular BMD in pQCT. Bone histomorphometry showed low bone volume and decreased trabecular number, which were associated with increased eroded surface. Urinary deoxypyridinoline excretion was increased in Lrp6rs/rs mice, whereas levels of serum osteocalcin were comparable between Lrp6rs/rs mice and wildtype littermates. Increase in cell number and mineralization of calvariae‐derived osteoblasts were not impaired in Lrp6rs/rs osteoblasts. Rankl expression was increased in Lrp6rs/rs osteoblasts both in vivo and in vitro, and osteoclastogenesis and bone‐resorbing activity in vitro were accelerated in Lrp6rs/rs cells. Treatment with canonical Wnt suppressed Rankl expression in both in primary osteoblasts and ST2 cells. Overexpression of Lrp6 also suppressed Rankl expression, whereas the Lrp6 rs mutant protein did not. Functional analyses of the Lrp6 rs mutant showed decreased targeting to plasma membrane because of reduced interaction with Mesoderm development (Mesd), a chaperone for Lrp6, leading to impaired Wnt/β‐catenin signaling. These results indicate that Lrp6‐mediated signaling controls postnatal bone mass, at least partly through the regulation of bone resorption. It is also suggested that the interaction with Mesd is critical for Lrp6 to function.

Identification of Amino Acid Sequence in the Hinge Region of Human Vitamin D Receptor That Transfers a Cytosolic Protein to the Nucleus

The localization of human vitamin D receptor (VDR) in the absence of its ligand 1,25-dihydroxyvitamin D3 was investigated using chimera proteins fused to green fluorescent protein (GFP) at either the N or C terminus, and the nuclear localization signal (NLS) was identified. Plasmids carrying the fusion proteins were transiently or stably introduced into COS7 cells, and the subcellular distribution of the fusion proteins was examined. GFP-tagged wild-type VDRs were located predominantly in nuclei but with a significant cytoplasmic presence, while GFP alone was equally distributed throughout the cells. 10−8 m1,25-dihydroxyvitamin D3 promoted the nuclear import of VDR in a few hours. To identify the NLS, we constructed several mutated VDRs fused to GFP. Mutant VDRs that did not bind to DNA were also localized predominantly in nuclei, while the deletion of the hinge region resulted in the loss of preference for nucleus. A short segment of 20 amino acids in the hinge region enabled cytoplasmic GFP-tagged alkaline phosphatase to translocate to nuclei. These results indicate that 1) VDR is located predominantly in nuclei with a significant presence in cytoplasm without the ligand and 2) an NLS consisting of 20 amino acids in the hinge region facilitates the transfer of VDR to the nucleus.