Yasuhiro Takeuchi

FGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis.

We analyzed the effects of an FGF‐23 injection in vivo. FGF‐23 caused a reduction in serum 1,25‐dihydroxyvitamin D by altering the expressions of key enzymes for the vitamin D metabolism followed by hypophosphatemia. This study indicates that FGF‐23 is a potent regulator of the vitamin D and phosphate metabolism.

Cloning and characterization of FGF23 as a causative factor of tumor-induced osteomalacia

Tumor-induced osteomalacia (TIO) is one of the paraneoplastic diseases characterized by hypophosphatemia caused by renal phosphate wasting. Because removal of responsible tumors normalizes phosphate metabolism, an unidentified humoral phosphaturic factor is believed to be responsible for this syndrome. To identify the causative factor of TIO, we obtained cDNA clones that were abundantly expressed only in a tumor causing TIO and constructed tumor-specific cDNA contigs. Based on the sequence of one major contig, we cloned 2,270-bp cDNA, which turned out to encode fibroblast growth factor 23 (FGF23). Administration of recombinant FGF23 decreased serum phosphate in mice within 12 h. When Chinese hamster ovary cells stably expressing FGF23 were s.c. implanted into nude mice, hypophosphatemia with increased renal phosphate clearance was observed. In addition, a high level of serum alkaline phosphatase, low 1,25-dihydroxyvitamin D, deformity of bone, and impairment of body weight gain became evident. Histological examination showed marked increase of osteoid and widening of growth plate. Thus, continuous production of FGF23 reproduced clinical, biochemical, and histological features of TIO in vivo. Analyses for recombinant FGF23 products produced by Chinese hamster ovary cells indicated proteolytic cleavage of FGF23 at the RXXR motif. Recent genetic study indicates that missense mutations in this RXXR motif of FGF23 are responsible for autosomal dominant hypophosphatemic rickets, another hypophosphatemic disease with similar features to TIO. We conclude that overproduction of FGF23 causes TIO, whereas mutations in the FGF23 gene result in autosomal dominant hypophosphatemic rickets possibly by preventing proteolytic cleavage and enhancing biological activity of FGF23.

Targeted ablation of Fgf23 demonstrates an essential physiological role of FGF23 in phosphate and vitamin D metabolism

Inorganic phosphate is essential for ECM mineralization and also as a constituent of important molecules in cellular metabolism. Investigations of several hypophosphatemic diseases indicated that a hormone-like molecule probably regulates serum phosphate concentration. FGF23 has recently been recognized as playing important pathophysiological roles in several hypophosphatemic diseases. We present here the evidence that FGF23 is a physiological regulator of serum phosphate and 1,25-dihydroxyvitamin D (1,25[OH]2D) by generating FGF23-null mice. Disruption of the Fgf23 gene did not result in embryonic lethality, although homozygous mice showed severe growth retardation with abnormal bone phenotype and markedly short life span. The Fgf23(-/-) mice displayed significantly high serum phosphate with increased renal phosphate reabsorption. They also showed an elevation in serum 1,25(OH)2D that was due to the enhanced expression of renal 25-hydroxyvitamin D-1alpha-hydroxylase (1alpha-OHase) from 10 days of age. These phenotypes could not be explained by currently known regulators of mineral homeostasis, indicating that FGF23 is essential for normal phosphate and vitamin D metabolism.

Association between activating mutations of calcium-sensing receptor and Bartter's syndrome

Bartters syndrome is a heterogeneous disorder characterised by deficient renal reabsorption of sodium and chloride, and hypokalaemic metabolic alkalosis with hyper-reninaemia and hyperaldosteronaemia. Mutations in several ion transporters and channels have been associated with the pathogenesis of Bartters syndrome. We describe two hypocalcaemic patients with deficient parathyroid hormone secretion who also showed characteristics of Bartters syndrome. We found activating mutations of the gene for the calcium-sensing receptor (CASR) in both patients. Activation of this calcium-sensing receptor inhibits the activity of a renal outer-medullary potassium channel that is mutated in type 2 Bartters syndrome. We therefore suggest that some activating mutations of CASR could provide new mechanisms for the development of Bartters syndrome.

Interleukin-11 as a stimulatory factor for bone formation prevents bone loss with advancing age in mice.

Cytokines in interleukin (IL)-11 subfamily participate in the regulation of bone cell proliferation and differentiation. We report here positive effects of IL-11 on osteoblasts and bone formation. Overexpression of human IL-11 gene in transgenic mice resulted in the stimulation of bone formation to increase cortical thickness and strength of long bones, and in the prevention of cortical bone loss with advancing age. Bone resorption and osteoclastogenesis were not affected in IL-11 transgenic mice. In experiments in vitro, IL-11 stimulated transcription of the target gene for bone morphogenetic protein (BMP) via STAT3, leading to osteoblastic differentiation in the presence of BMP-2, but inhibited adipogenesis in bone marrow stromal cells. These results indicate that IL-11 is a stimulatory factor for osteoblastogenesis and bone formation to conserve cortical bone, possibly by enhancing BMP actions in bone. IL-11 may be a new therapeutic target for senile osteoporosis.

Focal Adhesion Kinase Activity Is Required for Bone Morphogenetic Protein—Smad1 Signaling and Osteoblastic Differentiation in Murine MC3T3-E1 Cells

Cell‐matrix interactions via integrins are essential for osteoblastic differentiation. We have shown that signals activated by aggregation of α2β1‐integrin with type I collagen are involved in the differentiation of osteoblastic MC3T3‐E1 cells. Focal adhesion kinase (FAK) is an immediate downstream signal of the β1‐integrin, and inactivation of FAK has been shown to disrupt osteoblastic differentiation. To elucidate roles of FAK in osteoblastic cells, we examined MC3T3‐E1 cells stably expressing antisense FAK (asFAK) messenger RNA (mRNA). Alkaline phosphatase (ALP) activity, an osteoblastic marker, did not increase in asFAK cells with a long‐term culture until 21 days or in response to bone morphogenetic protein 2 (BMP‐2). Treatment with BMP‐2 also failed to stimulate the expression of osteocalcin in asFAK cells. In control MC3T3‐E1 cells, BMP‐2 induced translocation of Smad1 into nuclei to stimulate transcriptional activity of the Smad6 promoter gene that contains a Smad1 response element. In contrast, BMP‐2 failed to increase transcriptional activity of Smad6 promoter gene in asFAK cells, although it induced nuclear translocation of Smad1. These results indicate that FAK was involved in Smad1‐dependent transcriptional activity but not in nuclear translocation of Smad1 in osteoblastic cells. Hence, FAK activation by integrins might converge transcriptional activation by BMP of its target genes in osteoblastic cells. These observations suggest that FAK activity is essential for BMP‐Smad signaling to stimulate osteoblastic differentiation.

Stimulation of Smad1 Transcriptional Activity by Ras‐Extracellular Signal‐Regulated Kinase Pathway: A Possible Mechanism for Collagen‐Dependent Osteoblastic Differentiation

Signals from bone morphogenetic protein receptors (BMPRs) and cell adhesion to type I collagen are both important for osteoblastic differentiation and functions. BMP signals are mediated mostly by Smad and collagen signals are transduced by integrins to activate focal adhesion kinase (FAK) and its downstream molecules. This study was undertaken to clarify how extracellular matrix collagen signals converge with BMP actions. We show that integrin activation by collagen was involved in BMP signals because disruption of either collagen synthesis or collagen‐α2β1‐integrin binding inhibited the stimulatory effect of BMP‐2 on osteoblastic MC3T3‐E1 cells. Downstream signals of collagen‐integrin might be FAK‐Ras‐extracellular signal‐regulated kinase (ERK) in osteoblastic cells. We further show that Ras‐ERK signals enhanced the transcriptional activity of Smad1 in response to BMP in these cells transiently transfected with expression plasmids for a constitutively active mutant RasV12, a dominant negative mutant RasN17, and an ERK phosphatase CL100. Ras‐ERK signals did not augment the transcriptional activity of Smad3 in response to transforming growth factor β (TGF‐β) receptor activation but that of Smad1 in response to BMPR activation as examined in COS‐1 cells. These observations suggest that the Ras‐ERK pathway downstream of integrin‐FAK is involved in Smad1 signals activated by BMP and provide a possible mechanism for cooperation between intracellular signals activated by integrin and BMPRs in osteoblastic cells.

Receptor Tyrosine Kinases Inhibit Bone Morphogenetic Protein-Smad Responsive Promoter Activity and Differentiation of Murine MC3T3-E1 Osteoblast-like Cells

Growth factors such as fibroblast growth factor‐2 (FGF‐2) and epidermal growth factor (EGF) that activate extracellular signal‐regulated kinases (ERKs) through receptor tyrosine kinases (RTKs) stimulate proliferation but suppress differentiation of osteoblasts. To study the mechanism of this inhibitory action of these growth factors on osteoblastic differentiation, we evaluated Smad1 transactivity in MC3T3‐E1 osteoblast‐like cells by reporters of promoter activity of mouse Smad6, an early response gene to bone morphogenetic proteins (BMPs). FGF‐2 and EGF inhibited alkaline phosphatase activity and Smad6 promoter activity stimulated by BMP‐2. Overexpression of constitutively active MEK by adenovirus mimicked, but that of dominant negative Ras or treatment with a MEK1 inhibitor, PD098059, reversed, the inhibitory effects of these growth factors on both activities. These effects are mediated by BMP‐responsive elements (BMPREs) on Smad6 promoter, because an artificial reporter driven by three tandem BMPREs gave similar results, and these effects were all abolished when the BMPREs were mutated. RTK‐ERK activation inhibited the promoter activity even when BMP signal was mediated by a mutant Smad1, which lacks phosphorylation sites by ERKs, or by a Smad1 fused to Gal4 DNA binding domain, which constitutively localizes in the nucleus. These results show that the RTK‐Ras‐ERK pathway suppresses BMP signal by interfering with Smad1 transactivity. Because direct phosphorylation of Smad1 by ERKs is not required for the inhibition, other transcriptional factors that are phosphorylated by ERKs might be involved in the regulation of osteoblastic differentiation by ERKs.

Decrease in serum leptin by troglitazone is associated with preventing bone loss in type 2 diabetic patients.

Abstract.u2003The thiazolidinedione (TZD) class of antidiabetic drugs has been shown to inhibit the formation of bone-resorbing osteoclasts in vitro and to decrease bone resorption markers in vivo. These drugs also inhibit the expression of leptin in adipocytes. Less leptin can be associated with higher bone mass, based on analyses of mice deficient in leptin action. Effects of 1-year treatment with troglitazone, a member of the TZDs, on bone mineral density (BMD) and bone metabolism were examined in 25 Japanese type 2 diabetic patients. Glucose metabolism was improved, whereas body mass index and percent body fat did not change throughout the study. The percent change of BMD was negatively correlated with that of serum leptin, whereas it was not associated with changes of bone metabolic markers, type I collagen N-telopeptide (NTx), bone alkaline phosphatase (ALP), body mass index, or HbA1c. Serum leptin decreased in 68% of subjects (responders) after 1-month treatment and was consistently lower than the basal level throughout the treatment. Percent changes of BMD were significantly higher in the responders than in the nonresponders and in nondiabetic subjects at 6 and 12 months. NTx and bone ALP decreased at 1 month but increased thereafter in either group of patients. Thus, it is suggested that the decrease in serum leptin with no reduction in body fat mass by troglitazone is associated with preventing bone loss in type 2 diabetic patients. Hence, TZDs may have an advantage for diabetic patients who have risk factors for osteoporosis.

Decreased AP-1 Activity and Interleukin-11 Expression by Bone Marrow Stromal Cells May Be Associated With Impaired Bone Formation in Aged Mice†

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.