Contribution of matrix vesicles and alkaline phosphatase to ectopic bone formation.

Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 +/- 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PPi), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.

Anti-idiotypic antibody as an oestrogen mimetic in vivo: stimulation of creatine kinase specific activity in rat animal models.

Previous studies indicated that the anti-idiotypic antibody (clone 1D5) significantly increased the specific activity of creatine kinase (CK) activity in the rat uterus, and in vitro in skeletal cells capable of responding to oestradiol (E2), suggesting that the antibody has oestrogenic-like activity. Moreover, the F(ab')2 dimer of clone 1D5 acted like an antagonist and completely inhibited the increase in CK specific activity by either E2 or clone 1D5 in these skeletal cells. In the present study, we examined the in vivo effects of clone 1D5 and its proteolytic fragment, the F(ab')2 dimer, E2 and dihydrotestosterone (DHT) on CK specific activity in the epiphyseal cartilage, diaphyseal bone, uterus, prostate, thymus and pituitary of immature or gonadectomized female and male rat animal models. In the intact immature animals, clone 1D5 caused an increase in CK in all organs of the female except in the pituitary. In the diaphyseal bone and prostate of male rats there was no stimulation by 1D5. The CK response in the uterus, epiphysis, and diaphysis of immature female rats was dose-dependent and was blocked by either the anti-oestrogen tamoxifen or the F(ab')2 dimer of clone 1D5. E2, DHT, as well as clone 1D5, stimulated CK specific activity in both the diaphysis and epiphysis of ovariectomized female and castrated male rats, whereas sex specificity in the CK response was observed also in the uterus and the prostate of gonadectomized animals. Collectively, these results suggest that, as in cell culture, an intact antibody is necessary for the observed stimulation of CK specific activity and the F(ab')2 dimer can act as an antagonist. Furthermore, the observed biological effects of clone 1D5 which are absolutely parallel to E2, imply that the anti-idiotypic antibody is able to penetrate the cell and reach the nuclear oestrogen receptor and transduces a signal to the nucleus, by as yet uncharacterized mechanisms.

A non-calcemic analog of 1 alpha,25 dihydroxy vitamin D(3) (JKF) upregulates the induction of creatine kinase B by 17 beta estradiol in osteoblast-like ROS 17/2.8 cells and in rat diaphysis.

We have reported that multiple treatments with so-called 'non-hypercalcemic' analogs of 1 alpha,25(OH)(2) vitamin D(3) (1,25(OH)(2)D(3)) stimulate the specific activity of creatine kinase BB (CK) in ROS 17/2.8 osteoblast-like cells, and that pretreatment with these analogs upregulates responsiveness and sensitivity to 17 beta estradiol (E(2)) for the induction of CK. However, since the analogs showed toxicity in vivo, we have now studied the action of a demonstrably non-calcemic hybrid analog of vitamin D in ROS 17/2.8 cells, and prepubertal rats. The analog JKF was designed to separate its calcemic activity from other biological activities by combining a calcemic-lowering 1-hydroxymethyl group with a potentiating C, D-ring side chain modification including 24 difluoronation. Treatment with 1 pM JKF alone significantly stimulated CK specific activity at 4 h by 30+/-10%. However after three daily pretreatments, JKF upregulated the extent of induction by 30 nM E(2) by 33% at 1 pM and by 97% at 1 nM; the E(2) dose needed for a significant stimulation of CK activity was lowered to 30 pM. The action of the SERMS tamoxifen, tamoxifen methiodide and raloxifene, at 3 microM, was also upregulated by three daily pretreatments with 1 nM JKF; unexpectedly, this pretreatment prevented the inhibition of E(2) stimulation by the SERMS. Upregulation of E(2) action by 1 nM JKF was inhibited by 1 nM ZK159222, an inhibitor of the nuclear action of 1,25(OH)(2)D(3). In vivo, three daily injections of 0.05 ng/g body weight of JKF augmented the response of prepubertal female rat diaphysis and epiphysis to E(2). Therefore, demonstrably non-calcemic analogs of 1,25(OH)(2)D(3) may have potential for use in combination with estrogens or SERMS in the prevention and/or treatment of metabolic bone diseases such as postmenopausal osteoporosis.

Immunohistochemical localization of matrix metalloproteinase 13 (MMP-13) in mouse mandibular condylar cartilage.

MMP-13 appears to be one of the most important MMPs in cartilage remodeling and mineralization, because it exhibits a substrate preference for the cartilage-specific type II collagen. The condylar process is constructed by rapid accumulation of hypertrophic chondrocytes during development, but its mechanism is still unclear. To investigate the role of MMP-13 in developing condylar cartilage, we immunohistochemically examined the localization of MMP-13 in the endochondral ossification of the mandibular condyle and tibiae of newborn mice. In the tibiae, the MMP-13 expression was detected only in the deepest layer of the terminal hypertrophic chondrocytes through every examined stage (day 1 to day 10 after birth). On the other hand, in the condylar cartilage at days 1 and 5, MMP-13 was expressed throughout the proliferating and the hypertrophic chondrocytes, and at day 10, MMP-13 was mainly localized in the deepest edge of the hypertrophic layer. A zymographical study showed that the activity of MMP-13 in the condyle was observed at day 1, earlier than in the tibia, and increased until day 7. The time-dependent and cell-specific expression of MMP-13 and its enzymatic property suggest that in the mandibular condylar cartilage, MMP-13 plays a role in making the space for cell enlargement by degradation of the cartilage matrix and in onset of mineralization during the early stage of development.

Structural and cellular features in metaphyseal and diaphyseal periosteum of osteoporotic rats.

Despite the important physiological role of periosteum in the pathogenesis and treatment of osteoporosis, little is known about the structural and cellular characteristics of periosteum in osteoporosis. To study the structural and cellular differences in both diaphyseal and metaphyseal periosteum of osteoporotic rats, samples from the right femur of osteoporotic and normal female Lewis rats were collected and tissue sections were stained with hematoxylin and eosin, antibodies or staining kit against tartrate resistant acid phosphatase (TRAP), alkaline phosphatase (ALP), vascular endothelial growth factor (VEGF), von Willebrand (vWF), tyrosine hydroxylase (TH) and calcitonin gene-related peptide (CGRP). The results showed that the osteoporotic rats had much thicker and more cellular cambial layer of metaphyseal periosteum compared with other periosteal areas and normal rats (P < 0.001). The number of TRAP(+) osteoclasts in bone resorption pits, VEGF(+) cells and the degree of vascularization were found to be greater in the cambial layer of metaphyseal periosteum of osteoporotic rats (P < 0.05), while no significant difference was detected in the number of ALP(+) cells between the two groups. Sympathetic nerve fibers identified by TH staining were predominantly located in the cambial layer of metaphyseal periosteum of osteoporotic rats. No obvious difference in the expression of CGRP between the two groups was found. In conclusion, periosteum may play an important role in the cortical bone resorption in osteoporotic rats and this pathological process may be regulated by the sympathetic nervous system.

Oral administration of beta-cryptoxanthin induces anabolic effects on bone components in the femoral tissues of rats in vivo.

The effect of beta-cryptoxanthin on bone components in the femoral tissues of rats was investigated. Beta-cryptoxanthin was isolated from Satsuma mandarin (Citrus unshiu MARC.). Bone tissues were cultured for 48 h in serum-free Dulbecco's modified Eagle's medium containing either vehicle or beta-cryptoxanthin (10(-7) or 10(-6) M). The presence of beta-cryptoxanthin (10(-7) or 10(-6) M) caused a significant increase in calcium content and alkaline phosphatase activity in the femoral-diaphyseal and femoral-metaphyseal tissues. These increases were completely abolished in the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis. Thus beta-cryptoxanthin had an anabolic effect on bone calcification in vitro. Moreover, beta-cryptoxanthin (10, 25, or 50 microg/100 g body weight) was orally administered once daily for 7 d to young male rats. The administration of beta-cryptoxanthin (10, 25, or 50 microg/100 g body weight) caused a significant increase in calcium content and alkaline phosphatase activity in the femoral-diaphyseal and femoral-metaphyseal tissues. Femoral-diaphyseal and femoral-metaphyseal DNA contents were significantly increased by the dose of 25 or 50 microg/100 g body weight. A significant increase in metaphyseal DNA content was also seen with the dose of 10 microg/100 g body weight of beta-cryptoxanthin. This study demonstrates that beta-cryptoxanthin has an anabolic effect on bone components in rats in vitro and in vivo.

Contribution of matrix vesicles and alkaline phosphatase to ectopic bone formation

Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 ± 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PPi), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.