Hideo Orimo

The role of tissue-nonspecific alkaline phosphatase in the phosphate-induced activation of alkaline phosphatase and mineralization in SaOS-2 human osteoblast-like cells.

Tissue-nonspecific alkaline phosphatase (TNAP) plays a key role in mineralization by degrading inorganic pyrophosphate and providing free inorganic phosphate. We have previously reported that TNAP is induced by β-glycerophosphate and NaH2PO4 in short-term cultures of SaOS-2 human osteoblast-like cells and that PHEX (phosphate-regulating gene with homologies to endopeptidase on the X chromosome) mRNA is also induced after TNAP induction. In the present study, we have investigated the effects of levamisole, a TNAP inhibitor, and phosphonoformic acid (PFA), a type III sodium-phosphate cotransporter inhibitor, on the phosphate-induced expression of TNAP and mineralization. Levamisole inhibited β-glycerophosphate-induced mineralization, TNAP and PHEX expression, and the increase in enzymatic activity of NPP1 (5′-nucleotide pyrophosphatase phosphodiesterase 1), but did not inhibit NaH2PO4-induced mineralization. PFA completely inhibited NaH2PO4-induced mineralization and NPP1 enzymatic activation, and partly inhibited β-glycerophosphate-induced mineralization, but did not affect the increase in TNAP activity. These results suggest that phosphate derived from TNAP-induced hydrolysis of β-glycerophosphate yields signals that induce TNAP expression and mineralization, and that PHEX expression may be linked to TNAP expression. However, luciferase assays failed to detect any transcriptional activation of the promoter region of the human TNAP gene by β-glycerophosphate or NaH2PO4, suggesting that the effects of these phosphates may be indirect.

Expression of bone morphogenetic proteins and rat distal-less homolog genes following rat femoral fracture.

Abstract: Expression of the genes encoding bone morphogenetic proteins (BMPs), BMP type IA receptor (BMPR-1A), and rat distal-less homolog (rDlx) was studied in bone, callus, and the surrounding soft tissue following rat femoral closed fracture, using RT-PCR-based techniques. Before fracture, the genes encoding BMP-5, BMP-6, and BMPR-1A were found to be expressed in both bone and the surrounding soft tissue, whereas the BMP-2 gene was expressed only in bone and BMP-7 was not expressed in either tissue. Expression of these genes was unaffected by fracture. The gene encoding BMP-4 was also expressed in both bone and the surrounding soft tissue before fracture. Moreover, although unchanged in bone, 6 h after fracture BMP-4 expression was increased tenfold in the surrounding soft tissue. The increased BMP-4 expression was transient and returned to prefracture levels within 72 h. Expression of rDlx was also increased in bone after fracture, but at later times than were observed with BMP-4: elevated rDlx expression was detected after 48 h and persisted for 30 days or more. No expression of rDlx was observed in the surrounding soft tissue before or after fracture. These findings indicate that BMP-4 and rDlx are selectively expressed following femoral fracture in the rat, and also suggest that they are involved in the formation of the callus at an early point during the postfracture healing of bone.

Functional Analysis of the Single Nucleotide Polymorphism (787T>C) in the Tissue-Nonspecific Alkaline Phosphatase Gene Associated With BMD†

Polymorphisms of the TNSALP gene have not previously been studied as a possible determinant for variations in BMD or as a predisposing genetic factor for osteoporosis. This study showed a significantly higher association between the 787T>C (Tyr246His) TNSALP gene and BMD among 501 postmenopausal women. Furthermore, the effects of amino acid substitution on the catalytic property of the protein translated from the 787T>C gene were examined.

Mutational Analysis and Functional Correlation With Phenotype in German Patients With Childhood‐Type Hypophosphatasia

The tissue‐nonspecific alkaline phosphatase (TNSALP) gene from five German family members with childhood‐type hypophosphatasia (HOPS) was analyzed using the polymerase chain reaction‐single strand conformation polymorphism (PCR‐SSCP)‐direct sequencing method. Four novel missense mutations (T51M, R54S, L258P, and R374H) and two that had been described previously (A160T and R206W) were detected in the respective patients. Mutation A160T was detected in 3 distinct patients, and a polymorphism V505A that had been described previously was detected in the same allele as L258P mutation in 1 patient and in 2 fathers whose V505A alleles were not transmitted to the probands. No other mutations were found in 2 patients. Transient expression of the mutant proteins in COS‐1 cells showed that the four novel mutations and R206W were severe alleles, whereas A160T was a moderate allele. Analysis of its enzymatic activity and genetic transmission patterns confirmed that V505A was a polymorphism. Immunoprecipitation of the transiently expressed proteins showed that levels of the 80‐kDa mature form of the enzyme were diminished or absent with the severe alleles; instead, levels of high‐molecular mass disulfide‐linked aggregates were increased. These results suggest that in compound heterozygotes, the combination of severe and moderate alleles may combine to cause the mild phenotype seen in childhood‐type HOPS.

Prolonged Survival and Phenotypic Correction of Akp2 / Hypophosphatasia Mice by Lentiviral Gene Therapy

Hypophosphatasia (HPP) is an inherited systemic skeletal disease caused by mutations in the gene encoding the tissue‐nonspecific alkaline phosphatase (TNALP) isozyme. The clinical severity of HPP varies widely, with symptoms including rickets and osteomalacia. TNALP knockout (Akp2−/−) mice phenotypically mimic the severe infantile form of HPP; that is, TNALP‐deficient mice are born with a normal appearance but die by 20 days of age owing to growth failure, hypomineralization, and epileptic seizures. In this study, a lentiviral vector expressing a bone‐targeted form of TNALP was injected into the jugular vein of newborn Akp2−/− mice. We found that alkaline phosphatase activity in the plasma of treated Akp2−/− mice increased and remained at high levels throughout the life of the animals. The treated Akp2−/− mice survived for more than 10 months and demonstrated normal physical activity and a healthy appearance. Epileptic seizures were completely inhibited in the treated Akp2−/− mice, and X‐ray examination of the skeleton showed that mineralization was significantly improved by the gene therapy. These results show that severe infantile HPP in TNALP knockout mice can be treated with a single injection of lentiviral vector during the neonatal period.

Novel aggregate formation of a frame‐shift mutant protein of tissue‐nonspecific alkaline phosphatase is ascribed to three cysteine residues in the C‐terminal extension

In the majority of hypophosphatasia patients, reductions in the serum levels of alkaline phosphatase activity are caused by various missense mutations in the tissue‐nonspecific alkaline phosphatase (TNSALP) gene. A unique frame‐shift mutation due to a deletion of T at cDNA number 1559 [TNSALP (1559delT)] has been reported only in Japanese patients with high allele frequency. In this study, we examined the molecular phenotype of TNSALP (1559delT) using in vitro translation/translocation system and COS‐1 cells transiently expressing this mutant protein. We showed that the mutant protein not only has a larger molecular size than the wild type enzyme by ≈ 12 kDa, reflecting an 80 amino acid‐long extension at its C‐terminus, but that it also lacks a glycosylphosphatidylinositol anchor. In support of this, alkaline phosphatase activity of the cells expressing TNSALP (1559delT) was localized at the juxtanucleus position, but not on the cell surface. However, only a limited amount of the newly synthesized protein was released into the medium and the rest was polyubiquitinated, followed by degradation in the proteasome. SDS/PAGE and analysis by sucrose‐density‐gradient analysis indicated that TNSALP (1559delT) forms a disulfide‐bonded high‐molecular‐mass aggregate. Interestingly, the aggregate form of TNSALP (1559delT) exhibited a significant enzyme activity. When all three cysteines at positions of 506, 521 and 577 of TNSALP (1559delT) were replaced with serines, the aggregation disappeared and instead this modified mutant protein formed a noncovalently associated dimer, strongly indicating that these cysteine residues in the C‐terminal region are solely responsible for aggregate formation by cross‐linking the catalytically active dimers. Thus, complete absence of TNSALP on cell surfaces provides a plausible explanation for a severe lethal phenotype of a homozygote hypophosphatasia patient carrying TNSALP (1559delT).

Bone formation following transplantation of genetically modified primary bone marrow stromal cells.

Bone marrow stromal cells contain mesenchymal stem cells that can differentiate into a variety of mesenchymal tissues; in the presence of BMP‐2, for example, they differentiate into osteoblasts. We constructed replication‐deficient adenoviral vectors encoding human BMP‐2 (BMP‐2/Ad) or BMP‐4 (BMP‐4/Ad) and used them to transduce primary bone marrow stromal cells from the femurs of four‐week‐old female C3H mice, which then expressed and processed functional BMP‐2 or BMP‐4 protein. Enzyme assays and histochemical staining showed both groups of cells to possess alkaline phosphatase activity, a marker of differentiation into osteoblasts, though the activity was higher in cells transduced with BMP‐2/Ad. When BMP‐2/Ad‐transduced cells were injected into the thigh muscles of immunocompetent C3H mice, ossicle development was detected on radiographs within four weeks after injection. Moreover, histological analysis indicated that newly developed ossicles contain mature osseous components, including cortical bone and bone marrow, within eight weeks. Thus, syngeneic transplantation of genetically modified primary bone marrow stromal cells induced bone formation in immunocompetent mice, perhaps indicating its potential for use in the development of therapeutic protocols aimed at enhancing bone formation.

FIRST‐TRIMESTER PRENATAL MOLECULAR DIAGNOSIS OF INFANTILE HYPOPHOSPHATASIA IN A JAPANESE FAMILY

We obtained a prenatal molecular diagnosis during the first trimester in a Japanese woman whose first child (the proband) had been a compound heterozygote for infantile hypophosphatasia. We examined chorionic villus DNA samples obtained at 10 weeks of gestation for the base substitutions detected in the proband DNA using polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) and PCR–allele‐specific oligonucleotide (ASO) analysis. The genotype of the fetus was the same as that of the proband. The same mobility shift patterns of single strand conformation polymorphism (SSCP) bands were observed in the fetus and the proband. This molecular approach to prenatal diagnosis appears to be more accurate than the enzymatic method and also more accurate and more rapid than the conventional RFLP method.

Retinoic acid regulates commitment of undifferentiated mesenchymal stem cells into osteoblasts and adipocytes

Evidence indicates that the balance between osteoblastogenesis and adipogenesis of mesenchymal stem cells (MSCs) is regulated by several hormones, growth factors, and their downstream signaling cascades. Previous studies suggest that retinoic acid (RA) plays a role in osteoblastogenesis and adipogenesis. However, it is unknown whether RA regulates commitment of MSCs into osteoblasts and adipocytes. In this study, we investigated the role of RA in differentiation of MSCs using the C3H10T1/2 cell line. RA stimulated activity and expression of alkaline phosphatase (ALP) and upregulated activity of the ALP gene promoter. The effects of RA were further enhanced by bone morphogenetic protein 2 (BMP2) and resultant Smad signaling. Furthermore, overexpression of Runx2 and Msx2, critical transcription factors for bone formation and BMP2-dependent osteoblastogenesis, enhanced RA-dependent ALP activity. In view of these findings, RA likely stimulates osteoblast differentiation through the BMP2–Smad–Runx2/Msx2 pathway. In contrast, RA markedly inhibited BMP2-induced adipocyte differentiation, suppressing expression of peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer-binding protein (C/EBP)α and C/EBPδ, and inhibiting adipogenic function of C/EBPβ, C/EBPδ, and PPARγ. In conclusion, our data suggest that RA regulates commitment of MSCs into osteoblasts and adipocytes by controlling transcriptional regulators.

Importance of deletion of T at nucleotide 1559 in the tissue-nonspecific alkaline phosphatase gene in Japanese patients with hypophosphatasia.

Abstract. The tissue-nonspecific alkaline phosphatase (TNSALP) gene in four unrelated patients with hypophosphatasia was analyzed using polymerase chain reaction–single strand conformation polymorphism and the direct sequencing method. Of the participating patients, one had childhood-type and three had perinatal-type disease. All carried a deletion of T at cDNA number 1559, which causes a frameshift downstream from codon L503, as a heterozygote. In the childhood-type patient, an F310L mutation was detected in the opposite allele. Similarly, a perinatal-type patient carried a V365I mutation in the opposite allele. Mutations in the opposite alleles were not detected in the other two patients with perinatal-type disease. In addition, although both parents carried the deletion as a heterozygote in two families with childhood-type and perinatal-type disease, patients from those families were not homozygous for the deletion. Several single-nucleotide polymorphisms (SNPs) were also detected, which were shown to be useful for haplotype analysis. Allele frequency of the deletion among Japanese patients was 36% (10 of 28 alleles) but none occurred in Caucasian patients. These findings indicate that regardless of clinical type, deletion in the TNSALP gene occurs frequently among Japanese patients. Furthermore, haplotype analysis using SNPs suggested that the deletion might have derived from more than a single founder.