Histochemical evaluation for the biological effect of menatetrenone on metaphyseal trabeculae of ovariectomized rats.

To evaluate the biological effects of vitamin K2 (menatetrenone, MK-4) on ovariectomy (OVX)-induced bone loss, we have examined histological alterations of femoral metaphyses of sham-operated (sham group), ovariectomized (OVX group), and MK-4 dietary-supplemented OVX (MK-4 group; 50 mg/kg per day) female Fischer rats 1, 2, 5, and 8 weeks after OVX. In the first week, rats of the OVX and MK-4 groups showed discontinuous trabeculae compared with sham-operated rats. At 2 weeks after OVX, the OVX rats revealed many large tartrate resistant acid phosphatase (TRAP)-positive osteoclasts, while osteoclasts in the MK-4-treated rats were similar in size to those of the sham group. At 5 weeks, the OVX and MK-4 groups revealed fragmented trabeculae in femoral metaphyses. The cartilage matrix was partially exposed due to stimulated bone resorption in the OVX group, but not in the MK-4 group. After 8 weeks, the OVX rats had little metaphyseal trabeculae, whereas the MK-4-treated rats had maintained short trabeculae. Despite the presence of intense alkaline phosphatase-positive osteoblasts on trabeculae in the MK-4 group, TRAP-positive osteoclasts were flattened without developing ruffled borders. Therefore, MK-4 appeared to lessen the increase in osteoclastic bone resorption induced by OVX, as well as to maintain the accelerated osteoblastic activity. It is of importance to identify the target cells for MK-4 in bone. Autoradiography localized [3H]-labeled MK-4 mainly in osteoblasts and adjacent bone matrices, but not in osteoclasts, indicating that MK-4 targets osteoblasts. Thus, MK-4 appears to target osteoblasts, consequently inhibiting bone loss induced by ovariectomy.

Histochemical examination of osteoblastic activity in op/op mice with or without injection of recombinant M-CSF.

Osteopetrotic (op/op) mice do not exhibit bone remodeling because of defective osteoclast formation caused by the depletion of macrophage colony-stimulating factor (M-CSF). In the present study, we investigated tibial bones of op/op mice with or without prior injections of M-CSF to determine whether osteoclast formation and subsequent bone resorption could activate osteoblasts, which is known as a "coupling" phenomenon. In op/op mice, no osteoclasts were present, but the metaphyseal osteoblasts adjacent to the growth plate cartilage seemed to be active, revealing an intense alkaline phosphatase (ALPase) immunoreactivity. Consequently, primary trabecular bones were extended continuously to the diaphysis, indicating that bone modeling is well achieved in op/op mice. In contrast with the metaphysis, most of the diaphyseal osteoblasts were flattened and showed weak ALPase activity, and, as a result, they seemed to be less active. Osteopontin (OPN) was localized slightly at the interface between bone and cartilage matrices of the primary trabeculae. In contrast, in op/op mice injected with M-CSF, tartrate-resistant acid phosphatase-positive osteoclasts appeared, resorbing trabecular bones of the diaphyseal region. The diaphyseal osteoblasts in the vicinity of the active osteoclasts were cuboidal and exhibited strong ALPase immunoreactivity. OPN was observed not only at the bone-cartilage interface, but also significantly on the resorption lacunae beneath the bone-resorbing osteoclasts. These observations indicate that the activation of diaphyseal osteoblasts appears to be coupled with osteoclast formation and subsequent osteoclastic bone resorption. Alternatively, the metaphyseal osteoblasts at the chondro-osseous junction seemed to be less affected by osteoclastic activity.

Bisphosphonate acts on osteoclasts independent of ruffled borders in osteosclerotic (oc/oc) mice.

We examined the effects of a third generation bisphosphonate [YM-175; disodium dihydrogen (cycloheptylamino)-methylene-1,1-bisphosphonate] on osteoclasts in osteosclerotic (oc/oc) mice to elucidate the cellular mechanism for incorporation of the bisphosphonate. Osteoclasts of oc/oc mice were in direct contact with bone matrix but devoid of ruffled borders. Tartrate-resistant acid phosphatase (TRAPase) showed spotty localization intercellularly, whereas vacuolar H(+)-ATPase (V-ATPase) immunoreactivity was observed homogeneously in the cytoplasm. Upon injection of bisphosphonate, most osteoclasts lost cell polarity and were detached from bone surfaces. The detached osteoclasts underwent apoptosis as characterized by condensation of chromatin, absence of Golgi apparatus, and formation of many vesicles in the cytoplasm. Both TRAPase and V-ATPase were evenly distributed in the cytoplasm. The pyknotic nuclei of osteoclasts revealed DNA fragments as evidenced by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling (TUNEL) method. The results indicate that osteoclasts lacking ruffled borders in oc/oc mice incorporated the bisphosphonate from a site different from ruffled borders and that bisphosphonate may directly affect osteoclasts without mediating its deposition to the bone matrix.

Histopathological characterization of melorheostosis.

Melorheostotic bone was examined histopathologically. In the severely affected areas, an abundance of osteoid and increased angiogenesis was observed. Increased osteoid without mineralization indicated the overproduction of bone matrix. Bone resorption also appeared to increase because osteoclasts were numerous in melorheostotic bone, thus suggesting a high rate of bone turnover. In addition, transforming growth factor-beta was immunolocalized in the periosteal fibroblasts, mesenchymal cells surrounding vessels, endothelial cells, and osteoblasts, while basic fibroblast growth factor was found in endothelial cells and mast cells near vessels. These cytokines may have some association with the exuberant bone matrix production and angiogenesis in melorheostosis.

Localization of alkaline phosphatase and osteopontin during matrix mineralization in the developing cartilage of coccygeal vertebrae.

We observed the manner in which alkaline phosphatase (ALPase) and osteopontin were localized in the cartilage and intramembranous bone of coccygeal vertebrae during matrix mineralization, shedding considerable light on the manner in which they develop. In the cartilage matrix of coccygeal vertebrae, we observed the localization of ALPase activity in the boundary of the proliferative and the hypertrophic zones. Granular nodules of mineralization were consistently found in the boundary of both zones, and increased in size when close to the hypertrophic zone. While osteopontin was rarely present in the early stages of mineralization, its localization along the margins of mineralized matrices in the hypertrophic zone was prominent. In contrast to cartilage, mineralized nodules in the intramembranous bone in the mid-portion of the vertebra displayed osteopontin-immunoreactivity, indicating its early synthesis and subsequent accumulation to early-stage mineralized nodules. When blood vessels, accompanied by osteoblastic and osteoclastic cell populations, invaded the cartilage, osteopontin was localized in the lower region of the hypertrophic zone, despite its maintaining the localization of ALPase and early-stage mineralization. Thus, our investigation demonstrated ALPase activity consistent with early-stage mineralization in the cartilage matrix. However, the fact that osteopontin-localization could not be pinpointed might account for its multifunctionality as concerns both the regulation of mineralization and the attachment of migrating osteogenic and osteoclastic cells to the mineralized matrix.

Cytochemical and ultrastructural examination of apoptotic odontoclasts induced by bisphosphonate administration.

Since odontoclasts share similar characteristics with osteoclasts, this study has examined whether odontoclasts exhibit cytological alteration after treatment with bisphosphonate, which induces apoptosis of osteoclasts. After the administration of bisphosphonate to 6-day-old rabbits, many odontoclasts detached from the dentine surface of the deciduous teeth, resulting in the reduction of tartrate-resistant acid phosphatase (TRAP-ase) and immunoreactivity for cathepsin K. Transmission electron microscopy revealed a number of odontoclasts showing poorly developed or a lack of ruffled borders, a Golgi apparatus markedly reduced in size, and numerous cytoplasmic vesicles. The bisphosphonate-treated odontoclasts displayed fragmented DNA in the pyknotic nuclei evidenced by terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick-end labeling, indicating that bisphosphonate can induce apoptosis of the odontoclasts. Ultrastructural observations of the apoptotic odontoclasts revealed condensed heterochromatin at the margin of the nuclear envelope, assembled arrays of rough endoplasmic reticulum, and many vacuoles and vesicles. Some apoptotic odontoclasts showed ladder-like structures between the adjacent nuclear envelopes, enlargement of the nuclear envelopes, and the formation of a ribosome-like granular structure in the nuclei. Thus, odontoclasts are able to undergo apoptosis after bisphosphonate treatment; this results in cytological alterations, including reduced resorption activity and the inhibition of protein synthesis/transport as indicated by the diminished TRAPase and cathepsin K and the poorly developed Golgi apparatus, respectively. Nuclear alteration as evidenced by the appearance of ladder-like and ribosome-like structures was characteristic of apoptotic odontoclasts.

Recent studies on the biological action of parathyroid hormone (PTH)-related peptide (PTHrP) and PTH/PTHrP receptor in cartilage and bone.

Mice with a targeted deletion of parathyroid hormone (PTH)-related peptide (PTHrP) develop a form of dyschondroplasia resulting from diminished proliferation and premature maturation of chondrocytes. Abnormal, heterogeneous populations of chondrocytes at different stages of differentiation were seen in the hypertrophic zone of the mutant growth plate. Although the homozygous null animals die within several hours of birth, mice heterozygous for PTHrP gene deletion reach adulthood, at which time they show evidence of osteopenia. Therefore, PTHrP appears to modulate cell proliferation and differentiation in both the pre and post natal period. PTH/PTHrP receptor expression in the mouse is controlled by two promoters. We recently found that, while the downstream promoter controls PTH/PTHrP receptor gene expression in bone and cartilage, it is differentially regulated in the two tissues. 1alpha,25-dihydroxyvitamin D3 downregulated the activity of the downstream promoter in osteoblasts, but not in chondrocytes, both in vivo and in vitro. Most of the biological activity of PTHrP is thought to be mediated by binding of its amino terminus to the PTH/PTHrP receptor. However, recent evidence suggests that amino acids 87-107, outside of the amino terminal binding domain, act as a nucleolar targeting signal. Chondrocytic cell line, CFK2, transfected with wild-type PTHrP cDNA showed PTHrP in the nucleoli as well as in the secretory pathway. Therefore, PTHrP appears to act as a bifunctional modulator of both chondrocyte proliferation and differentiation, through signal transduction linked to the PTH/PTHrP receptor and by its direct action in the nucleolus.

Inefficient function of the signal sequence of PTHrP for targeting into the secretory pathway.

Parathyroid hormone-related peptide (PTHrP) is not only secreted out of cells, but also targeted to the nucleoli due to a nucleolar targeting signal (NTS). We assessed the molecular mechanism underlying the dual targeting of PTHrP by constructing a series of truncated forms of rat PTHrP cDNA and expressing them in CHO cells. Immunostaining was observed in both the Golgi apparatus and nucleoli in the same cell expressing PTHrP with the N-terminal full-length signal sequence. When PTHrP molecules were translated from CUGs downstream of the AUG-initiator codon in the signal sequences, potential alternative initiators of the translation, they were exclusively localized in the nucleoli. In contrast, when a construct containing only the ATG-initiator codon was expressed, PTHrP was found to localize in both the nucleolus and the Golgi apparatus. No nucleolar staining of PTHrP was observed in the CHO cells transfected with PTH/PTHrP receptors even after incubating with a conditioned medium containing PTHrP, ruling out a possibility that PTHrP is, once secreted, internalized via receptor-mediated endocytosis and subsequently conveyed to nucleoli. Compatible with these morphological observations, a preproform of PTHrP was found in the cells expressing PTHrP in addition to proPTHrP, indicative of molecules along the secretory pathway. These results strongly indicate that the signal sequence of PTHrP is not sufficient to direct all the newly synthesized molecules across the endoplasmic reticulum, resulting in part of it being delivered to the nucleoli due to the NTS.

Possible interference between tissue-non-specific alkaline phosphatase with an Arg54-->Cys substitution and acounterpart with an Asp277-->Ala substitution found in a compound heterozygote associated with severe hypophosphatasia.

Tissue-non-specific alkaline phosphatase (TNSALP) with an Arg(54)-->Cys (R54C) or an Asp(277)-->Ala (D277A)substitution was found in a patient with hypophosphatasia [Henthorn,Raducha, Fedde, Lafferty and Whyte (1992) Proc. Natl. Acad. Sci. U.S.A.89, 9924-9928]. To examine effects of these missense mutations onproperties of TNSALP, the TNSALP mutants were expressed ectopically inCOS-1 cells. The wild-type TNSALP was synthesized as a 66-kDa endo-beta-N-acetylglucosaminidase H (Endo H)-sensitive form, and processed to an 80-kDa mature form, which is anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). Although the mutant proteins were found to be modified by GPI, digestion with phosphatidylinositol-specific phospholipase C, cell-surface biotinylation and immunofluorescence observation demonstrated that the cell-surface appearance of TNSALP (R54C) and TNSALP (D277A) was either almost totally or partially retarded respectively. The 66-kDa Endo H-sensitive band was the only form, and was rapidly degraded in the cells expressing TNSALP (R54C). In contrast with cells expressing TNSALP(R54C), where alkaline phosphatase activity was negligible, significant enzyme activity was detected and, furthermore, the 80-kDa mature form appeared on the surface of the cells expressing TNSALP (D277A). Analysis by sedimentation on sucrose gradients showed that a considerable fraction of newly synthesized TNSALP (R54C) and TNSALP(D277A) formed large aggregates, indicating improper folding and incorrect oligomerization of the mutant enzymes. When co-expressed with TNSALP (R54C), the level of the 80-kDa mature form of TNSALP (D277A)was decreased dramatically, with a concomitant reduction in enzyme activity in the co-transfected cell. These findings suggest that TNSALP(R54C) interferes with folding and assembly of TNSALP (D277A) intrans when expressed in the same cell, thus probably explaining why a compound heterozygote for these mutant alleles developed severe hypophosphatasia.

The biological action of parathyroid hormone-related peptide (PTHrP) and fibroblast growth factor receptor 3 (FGFR3) on bone and cartilage.

Parathyroid hormone (PTH)-related peptide (PTHrP) was determined to be a factor inducing malignancy-associated hypercalcemia by activating a common receptor (PTH/PTHrP receptor) with PTH. PTHrP gene "knock out" mice showed a form of dyschondroplasia due to reduced proliferation of chondrocytes. In addition, heterogenous populations of variously-differentiated chondrocytes were present in the hypertrophic zone of the mutant epiphyseal plate. Although the homozygotes die within several hours after birth, the adult mice, heterozygous for PTHrP gene deletion, showed a delayed skeletal abnormality at 3 month old, with a reduced amount of PTHrP transcript, therefore, PTHrP appears to modulate cell proliferation and differentiation at both fetal and adult stages. The co-localization of PTHrP and its receptor in osteoblastic cells and chondrocytes suggested a paracrine/autocine mode of action manner of these molecules. Recently, fibroblast growth factor receptor 3 (FGFR3) deficient mice demonstrated skeletal defects including kyphosis, scoliosis, crooked tails and curvature and overgrowth of long bones and vertebrae, which are caused by an increase in proliferation. Therefore, it seems that PTHrP and FGFR3 serve as positive and negative regulators on the chondrocyte proliferation, respectively. In this paper, we review our recent studies on the histological abnormality of long bone seen in PTHrP gene deficient- and FGFR3 gene deficient-mice.

Ultrastructural and cytochemical studies on cell death of osteoclasts induced by bisphosphonate treatment.

The process of apoptosis and fate of osteoclasts are not well elucidated because dying osteoclasts are rarely seen in normal bone. Histological, cytochemical, and ultrastructural features of osteoclasts undergoing apoptosis were studied in the femur and tibia of rats treated with a third-generation bisphosphonate (disodium dihydrogen (cycloheptylamino)-methylene-1, 1-bisphosphonate). After the bisphosphonate administration, osteoclasts decreased significantly in number. Initially, they became devoid of ruffled borders and detached from the bone surface. In such osteoclasts, the Golgi apparatus was degraded, or dispersed in the cytoplasm. Later, osteoclasts revealed typical features of apoptosis, with pyknotic nuclei showing condensation and margination of heterochromatins and DNA fragmentation. They were often convoluted to give rise to apoptotic bodies. In addition, enlargement and fusion of nuclear envelopes and subsequent disruption leading to leakage of nuclear contents into the cytoplasm were observed in osteoclasts in the late stage of apoptosis. These osteoclasts as well as apoptotic bodies were surrounded by cytoplasmic processes of macrophages, which often contained degenerated cytoplasmic fragments of osteoclasts. Apoptotic osteoclasts migrating into or present in capillaries were also observed in some areas. In conclusion, bisphosphonate induces apoptosis of osteoclasts, which was characterized by ultrastructural changes of the nucleus typical of apoptosis accompanied by degradation of cell organelles. The majority of them are eliminated by macrophages, but there are some that escape into blood vessels.

Identification and characterization of a novel protein, periostin, with restricted expression to periosteum and periodontal ligament and increased expression by transforming growth factor beta.

We had previously identified the cDNA for a novel protein called osteoblast-specific factor 2 (OSF-2) from an MC3T3-E1 cDNA library using subtraction hybridization and differential screening techniques. Here we describe the localization, regulation, and potential function of this protein. Immunohistochemistry using specific antiserum revealed that in adult mice, the protein is preferentially expressed in periosteum and periodontal ligament, indicating its tissue specificity and a potential role in bone and tooth formation and maintenance of structure. Based on this observation and the fact that other proteins have been called OSF-2, the protein was renamed "periostin." Western blot analysis showed that periostin is a disulfide linked 90 kDa protein secreted by osteoblasts and osteoblast-like cell lines. Nucleotide sequence revealed four periostin transcripts that differ in the length of the C-terminal domain, possibly caused by alternative splicing events. Reverse transcription- polymerase chain reaction analysis revealed that these isoforms are not expressed uniformly but are differentially expressed in various cell lines. Both purified periostin protein and the periostin-Fc recombinant protein supported attachment and spreading of MC3T3-E1 cells, and this effect was impaired by antiperiostin antiserum, suggesting that periostin is involved in cell adhesion. The protein is highly homologous to betaig-h3, a molecule induced by transforming growth factor beta (TGF-beta) that promotes the adhesion and spreading of fibroblasts. Because TGF-beta has dramatic effects on periosteal expansion and the recruitment of osteoblast precursors, this factor was tested for its effects on periostin expression. By Western blot analysis, TGF-beta increased periostin expression in primary osteoblast cells. Together, these data suggest that periostin may play a role in the recruitment and attachment of osteoblast precursors in the periosteum.

Maturation ameloblasts of the porcine tooth germ do not express amelogenin.

Amelogenins are the most abundant constituent in the enamel matrix of developing teeth. Recent investigations of rodent incisors and molar tooth germs revealed that amelogenins are expressed not only in secretory ameloblasts but also in maturation ameloblasts, although in relatively low levels. In this study, we investigated expression of amelogenin in the maturation stage of porcine tooth germs by in situ hybridization and immunocytochemistry. Amelogenin mRNA was intensely expressed in ameloblasts from the differentiation to the transition stages, but was not detected in maturation stage ameloblasts. C-terminal specific anti-amelogenin antiserum, which only reacts with nascent amelogenin molecules, stained ameloblasts from the differentiation to the transition stages. This antiserum also stained the surface layer of immature enamel at the same stages. At the maturation stage, no immunoreactivity was found within the ameloblasts or the immature enamel. These results indicate that, in porcine tooth germs, maturation ameloblasts do not express amelogenins, suggesting that newly secreted enamel matrix proteins from the maturation ameloblast are not essential to enamel maturation occurring at the maturation stage.

The primary calcification in bones follows removal of decorin and fusion of collagen fibrils.

To elucidate the mechanisms of primary calcification in bone, ultrastructural changes in collagen fibrils, as well as cytochemical alteration of proteoglycan, especially decorin, were investigated morphologically in 19-day postcoitum embryonic rat calvariae. Below the osteoblast layer, calcification of the osteoid area increased in direct proportion to its distance from the osteoblasts. In the uncalcified osteoid area, collagen fibrils near matrix vesicles possessed sharp contours and were a uniform 50 nm in diameter. Immunoelectron microscopy revealed decorin to be abundantly localized in the vicinity of the collagen fibrils. In the osteoid area undergoing the process of calcification, collagen fibrils tended to fuse side by side. Where calcification was progressed, this fusion was even more so. Some very large fibrils exhibited complicated contours, 400 nm or more in diameter. Although the calcification at this stage affected areas both inside and outside of the collagen fibrils, the interior areas manifested a lower density of calcification. The immunolocalization of decorin was also much decreased around these fibrils. Thus, primary calcification in bone matrix follows the removal of decorin and fusion of collagen fibrils. This phenomenon may aid in the process of calcification and bone formation, because (1) inhibitors of calcification, such as decorin, are removed, (2) the fusion of collagen fibrils provides the room necessary for rapid growth of mineral crystals, and (3) the soft elastic bone matrix containing abundant fused collagen fibrils less subjective to calcification is safe for both maternal and embryonic bodies and is convenient for subsequent bone remodeling.

Vitamin D3 differentially regulates parathyroid hormone/parathyroid hormone-related peptide receptor expression in bone and cartilage.

Transcription of the mouse parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTHR) gene is controlled by promoters P1 and P2. We performed transcript-specific in situ hybridization and found that P2 is the predominant promoter controlling PTHR gene expression in bone and cartilage. Treatment with 1alpha, 25-dihydroxyvitamin D3 (D3) in vivo specifically downregulated P2-specific transcripts in osteoblasts, but not in chondrocytes, under conditions where it enhanced bone resorption. Treatment of the osteoblastic cell line MC3T3-E1 with D3 in vitro reduced expression of both P2-specific transcripts and PTHR protein. This effect was not blocked by cycloheximide, indicating that D3 inhibits PTHR expression by downregulating transcription of the P2 promoter. A similar inhibitory effect of D3 was not observed in the chondrocytic cell line CFK2. Gene-transfer experiments showed that P2, but not P1, is active in both MC3T3-E1 and CFK2 cells, and that D3 specifically inhibited P2 promoter activity in MC3T3-E1, but not in CFK2 cells. Inhibition of P2 activity by D3 required promoter sequences lying more that 1.6 kb upstream of the P2 transcription start site. Thus, the P2 promoter controls PTHR gene expression in both osteoblasts and chondrocytes. D3 downregulates PTHR gene transcription in a cell-specific manner by inhibiting P2 promoter activity in osteoblasts, but not in chondrocytes.

Inactivating mutation in the human parathyroid hormone receptor type 1 gene in Blomstrand chondrodysplasia.

A single homozygous nucleotide exchange in exon E3 of the gene encoding the parathyroid hormone receptor type 1 (PTHR1) was identified in an infant with Blomstrand chondrodysplasia born to consanguineous parents. This alteration changes a strictly conserved proline residue at position 132 in the receptor's amino terminal extracellular domain to leucine. COS-1 cells expressing the mutant receptor did not accumulate cyclic adenosine 3',5'-monophosphate in response to PTH or PTH-related peptide (PTHrP) and did not bind the radiolabeled ligand. Expression of the mutant protein on the cell surface of transiently transfected COS-1 cells and in growth plate chondrocytes derived from the affected infant suggests that proline 132 is critical for the receptor's intrinsic binding activity. These findings suggest that the Blomstrand form of human short-limbed dwarfism arises from defective PTHR1 signaling in the developing cartilaginous skeleton.

Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/osteoprotegerin.

Osteoclasts are multinucleated cells that resorb bone. Osteoclastogenesis inhibitory factor (OCIF), also called osteoprotegerin (OPG), acts as a naturally occurring decoy receptor for osteoclast differentiation factor, which mediates an essential signal to osteoclast progenitors for their differentiation into osteoclasts. Here we show that the OCIF/OPG knockout mice exhibited severe osteoporosis due to enhanced osteoclastogenesis when they grew to be adults. These mice were viable and fertile. They exhibited marked bone loss accompanied by destruction of growth plate and lack of trabecular bone in their femurs. The strength of their bones dramatically decreased. These results demonstrate that OCIF/OPG is a key factor acting as a negative regulator against osteoclastogenesis. The OCIF/OPG knockout mice provide the first animal model for osteoporosis without other obvious abnormalities.

Intracellular retention and degradation of tissue-nonspecific alkaline phosphatase with a Gly317-->Asp substitution associated with lethal hypophosphatasia.

One point mutation which converts glycine-317 to aspartate of tissue-nonspecific alkaline phosphatase (TNSALP) was reported to be associated with lethal hypophosphatasia (Greenberg, C. R., et al. Genomics 17, 215-217, 1993). In order to define the molecular defect of TNSALP underlying the pathogenesis of hypophosphatasia, we have examined the biosynthesis of TNSALP with a Gly317-->Asp substitution. When expressed in COS-1 cells, the mutant did not exhibit alkaline phosphatase activity at all, indicating that the replacement of glycine-317 with aspartate abolishes the catalytic activity of TNSALP. Pulse-chase experiments showed that the newly synthesized mutant failed to acquire Endo H-resistance and to reach the cell surface. Interestingly, this TNSALP mutant was found to form a disulfide-bonded high-molecular-mass aggregate and was rapidly degraded within the cell, though the mutant protein was modified by glycosylphosphatidylinositol (GPI). Lactacystin, an inhibitor of the proteasome, obstructed the degradation of the mutant protein, suggesting the involvement of proteasome as a part of quality control of TNSALP.

Morphological examination of bone synthesis via direct administration of basic fibroblast growth factor into rat bone marrow.

Woven bone induced by direct injection of basic fibroblast growth factor (bFGF) into rat bone marrow was examined. On the first day after injection, fibrous tissues formed in the treated region of the bone marrow. Tissue-nonspecific alkaline phosphatase (TNAPase)-immunopositive osteoblastic cells and osteopontin immunopositive-extracellular matrices were observed in the fibrous tissues, indicating bone induction. On the fifth day, the bFGF-induced bone was found broadly in the bone marrow. In the originally existing bone, osteopontin-immunoreactivity was observed at cement lines, but not in the fully calcified matrix, whereas the woven bone displayed immunoreactivity throughout the matrix. Numerous TRAPase-positive osteoclasts were present on the surfaces of the woven bone, but no obvious cement line was observed. Therefore, both bone formation and resorption appeared highly active, without normal cellular coupling equilibrated between bone formation and resorption performed by osteoblasts and osteoclasts. On the tenth day, the bFGF-induced bone was almost replaced by bone marrow. Thus, bone formation actively occurred in the first half of the experimental period, whereas bone resorption came to be predominant thereafter. This study demonstrated that bFGF stimulates bone formation, which, however, is subject to subsequent resorption, probably due in part to the absence of coordinated cellular coupling between osteoclasts and osteoblasts.

Targeted inactivation of Npt2 in mice leads to severe renal phosphate wasting, hypercalciuria, and skeletal abnormalities.

Npt2 encodes a renal-specific, brush-border membrane Na+-phosphate (Pi) cotransporter that is expressed in the proximal tubule where the bulk of filtered Pi is reabsorbed. Mice deficient in the Npt2 gene were generated by targeted mutagenesis to define the role of Npt2 in the overall maintenance of Pi homeostasis, determine its impact on skeletal development, and clarify its relationship to autosomal disorders of renal Pi reabsorption in humans. Homozygous mutants (Npt2(-/-)) exhibit increased urinary Pi excretion, hypophosphatemia, an appropriate elevation in the serum concentration of 1,25-dihydroxyvitamin D with attendant hypercalcemia, hypercalciuria and decreased serum parathyroid hormone levels, and increased serum alkaline phosphatase activity. These biochemical features are typical of patients with hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a Mendelian disorder of renal Pi reabsorption. However, unlike HHRH patients, Npt2(-/-) mice do not have rickets or osteomalacia. At weaning, Npt2(-/-) mice have poorly developed trabecular bone and retarded secondary ossification, but, with increasing age, there is a dramatic reversal and eventual overcompensation of the skeletal phenotype. Our findings demonstrate that Npt2 is a major regulator of Pi homeostasis and necessary for normal skeletal development.