Masae Goseki-Sone

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.

Effects of long-term vitamin K1 (phylloquinone) or vitamin K2 (menaquinone-4) supplementation on body composition and serum parameters in rats

Vitamin K is a cofactor for γ-glutamyl carboxylase, which is an essential enzyme for the γ-carboxylation of vitamin K-dependent proteins such as osteocalcin and matrix Gla protein. Although it has been suggested that vitamin K plays an important role in the improvement of bone metabolism, the relationship between dietary vitamin K intake and bone metabolism has not been thoroughly investigated. Moreover, vitamin K is thought to have other actions beyond influencing the γ-carboxylation status. In the present study, we examined the effects of the long-term addition of phylloquinone (PK) or menaquinone-4 (MK-4) to a control diet on bone mineral density, bone strength, body composition, and serum parameters in rats. A total of 23 female Sprague-Dawley strain rats (6 weeks old) were divided into three groups: basic control diet group, PK diet (PK: 600mg/kg diet) group, and MK diet (MK-4: 600mg/kg diet) group. Three months after starting the experimental diet, the addition of PK to the basic control diet significantly increased the bone mineral density (BMD) of the femur (p<0.05). In the MK group, there was no significant difference in the BMD of the femur. However, two types of bone strength parameter: the minimum cross-sectional moment of inertia and the polar moment of inertia, were significantly higher in the MK group than in the control (p<0.05, respectively). Furthermore, the femoral bone parameters (the width, dry weight and ash weight, and cortical, cancellous, trabecular, and total bone mineral contents) in the MK group were increased significantly compared with the control. Interestingly, the addition of PK or MK-4 significantly decreased the total fat accumulation (p<0.01 and p<0.05, respectively), and serum triglycerides were reduced by 48% in the PK group and 29% in the MK group compared with the control. There were no significant differences in the levels of serum calcium, phosphorus, alkaline phosphatase, growth hormone, insulin-like growth hormone-1, insulin-like growth hormone binding protein-3, and cross-linked N-teleopeptide of type I collagen among the three groups. This is the first study to demonstrate the effect of the long-term addition of PK or MK-4 to the control diet on body composition and serum parameters in an in vivo system using rats. Further studies on the mechanism of vitamin K supplementation in the regulation of bone metabolism would provide valuable data on the prevention of lifestyle-related disorders, including osteoporosis.

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.

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).

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.

Effects of Dietary Lactose on Long‐term High‐fat‐diet‐induced Obesity in Rats

Objective: In this study, we examined the effects of lactose on long‐term high‐fat‐diet‐induced obesity in rats.

Functional assay of the mutant tissue-nonspecific alkaline phosphatase gene using U2OS osteoblast-like cells

Tissue-nonspecific alkaline phosphatase (TNAP) plays a key role in mineralization. A defect in the TNAP gene causes hypophosphatasia, which is characteristic of systemic skeletal hypomineralization. To determine the mineralizing ability of the mutant proteins, we developed a functional assay that uses U2OS osteoblast-like cells. Expression plasmids containing TNAP mutant cDNAs were constructed and introduced into U2OS cells, which are derived from a human osteosarcoma and exhibit very low alkaline phosphatase (ALP) activity and disabled mineralization. U2OS cells, in which active TNAP cDNAs were introduced, expressed high ALP activity and mineralized their circumstance when they were cultured with beta-glycerophosphate. The ALP activity in these U2OS cells corresponded to the activity reported for COS cells in which active TNAP cDNA was introduced. An in vitro mineralization assay of U2OS cells transfected with moderate allele cDNAs showed that approximately 35% of TNAP enzymatic activity may be the threshold value for mineralization. In addition, U2OS cells transfected with wild-type TNAP and polymorphism TNAP cDNA showed PHEX (phosphate-regulating gene with homologies to endopeptidases on the X chromosome) induction as in SaOS-2 cells. In summary, the introduction of active TNAP cDNA into U2OS cells allowed these cells to mineralize, and this technique may be a useful functional assay of TNAP mutant proteins.

Phosphate depletion enhances bone morphogenetic protein-4 gene expression in a cultured mouse marrow stromal cell line ST2.

Alkaline phosphatases (ALPs) are glycosylated, membrane-bound enzymes that hydrolyze various monophosphate esters at an optimum high pH and are present in nearly all living organisms. In Escherichia coli, extracellular phosphate (Pi) limitation induces the ALP gene, indicating a role of extracellular Pi in ALP gene regulation. However, little is known about similar mechanisms in mammalian cells. Previously, we reported that Pi starvation increased the tissue-nonspecific ALP (TNSALP) activity and regulated its expression in the mouse stromal cell line ST2, derived from mouse bone marrow. In the present study, we further examined the effects of Pi starvation on the mechanism of TNSALP induction. The specific activity of TNSALP increased markedly after treatment by Pi starvation for 5 days and RT-PCR analysis revealed that the mRNA of the bone morphogenetic protein-4 (BMP-4) gene was highly stimulated. The combination of Pi depletion and mouse BMP-4 receptor IA/Fc chimera down-regulated the TNSALP activity. These results indicated that Pi depletion stimulates the TNSALP activity for the Pi supplementation, and that this system may involve the signaling pathway of the BMP-4 gene at the transcription level.

EXPRESSION OF MRNA ENCODING TISSUE-NONSPECIFIC ALKALINE PHOSPHATASE IN HUMAN DENTAL TISSUES

Abstract. Tissue-nonspecific-type alkaline phosphatase (TNSALP) is found in the bone, liver, kidney, and other tissues, and its gene consists of 12 exons with the coding sequence beginning in the second exon. Recently, a noncoding first exon was identified in the liver message (liver type) which differed from that of the previously known osteoblast-derived cDNA sequence (bone type). Although these two mRNAs produce an identical protein, they have different promoter regions. It is known that ALPs in dental pulp and periodontal ligament are classified into TNSALP by their enzymatic and immunological properties, but little is known about the expression of ALP mRNAs and the transcriptional mechanisms. In order to examine the expression of their mRNA type, specific oligonucleotide primers corresponding to two types of mRNAs of human TNSALP were designed and amplified by reverse transcription-polymerase chain reaction (RT-PCR). It was found that bone-type mRNA was expressed in the human dental tissues such as dental pulp, periodontal ligament, and dental sac, whereas liver-type mRNA was not expressed. Thus, it was concluded that the human dental tissues express the bone-type isozymes and are regulated by the same transcriptional mechanism as in the bone.

Function of mutant (G1144A) tissue-nonspecific ALP gene from hypophosphatasia.

Hypophosphatasia (HOPS) is a clinically heterogeneous heritable disorder characterized by defective skeletal mineralization, deficiency of tissue‐nonspecific alkaline phosphatase (TNSALP) activity, and premature loss of deciduous teeth. The gene for TNSALP is located on chromosome 1p34–36.1 and consists of 12 exons and 11 introns. In our previous study, we found the novel point mutations (G1144A and T979C) from the genomic TNSALP gene of a patient with childhood HOPS. In this study, we have characterized the protein translated from the mutant G1144A gene. Wild‐type and G1144A mutant‐type TNSALP cDNA expression vector pcDNA3 have been constructed and transfected to COS‐1 cells by lipofectin technique. After 48‐h or 72‐h transfection, cells were collected and homogenized using polytron homogenizer. After centrifugation at 10,000g for 10 minutes, the supernatant was assayed. ALP activity was determined with 10 mM of p‐nitrophenylphosphate as a substrate in 100 mM of 2‐amino‐2‐methyl‐1,3‐propanediol‐HCl buffer containing 5 mM of MgCl2. ALP activity of cells transfected with the mutant cDNA (G1144A) plasmid after 48‐h or 72‐h transfection exhibited 0.063 ± 0.012 U/mg and 0.054 ± 0.012 U/mg, respectively. As the enzymatic activity of the wild type was taken as 100%, the value of the mutant was estimated as 2.7% and 1.7%, respectively. These values were not significantly different from those found with mock‐transfected cells, that is, 2.5% and 1.5%, respectively. This study indicated that the mutation (G1144A) produced the inactive ALP enzyme and would be a disease‐causing mutation of the childhood‐type HOPS.