Showing papers on "Alkaline phosphatase published in 2002"
TL;DR: Gutman et al. as mentioned in this paper showed that the acid phosphatase of serum is reduced in metastatic carcinoma of the prostate by decreasing the activity of androgens through castration or estrogenic injections and that this enzyme is increased by injecting androgens.
861 citations
TL;DR: The results suggest that inhibiting PC-1 function may be a viable therapeutic strategy for hypophosphatasia, and interfere with TNAP activity may correct pathological hyperossification because of PPi insufficiency.
Abstract: Osteoblasts mineralize bone matrix by promoting hydroxyapatite crystal formation and growth in the interior of membrane-limited matrix vesicles (MVs) and by propagating the crystals onto the collagenous extracellular matrix. Two osteoblast proteins, tissue-nonspecific alkaline phosphatase (TNAP) and plasma cell membrane glycoprotein-1 (PC-1) are involved in this process. Mutations in the TNAP gene result in the inborn error of metabolism known as hypophosphatasia, characterized by poorly mineralized bones, spontaneous fractures, and elevated extracellular concentrations of inorganic pyrophosphate (PP(i)). PP(i) suppresses the formation and growth of hydroxyapatite crystals. PP(i) is produced by the nucleoside triphosphate pyrophosphohydrolase activity of a family of isozymes, with PC-1 being the only member present in MVs. Mice with spontaneous mutations in the PC-1 gene have hypermineralization abnormalities that include osteoarthritis and ossification of the posterior longitudinal ligament of the spine. Here, we show the respective correction of bone mineralization abnormalities in knockout mice null for both the TNAP (Akp2) and PC-1 (Enpp1) genes. Each allele of Akp2 and Enpp1 has a measurable influence on mineralization status in vivo. Ex vivo experiments using cultured double-knockout osteoblasts and their MVs demonstrate normalization of PP(i) content and mineral deposition. Our data provide evidence that TNAP and PC-1 are key regulators of the extracellular PP(i) concentrations required for controlled bone mineralization. Our results suggest that inhibiting PC-1 function may be a viable therapeutic strategy for hypophosphatasia. Conversely, interfering with TNAP activity may correct pathological hyperossification because of PP(i) insufficiency.
812 citations
TL;DR: The uremic sera-induced osteopontin expression in BVSMC is partially mediated through alkaline phosphatase activity and a Na/Pi co-transport dependent mechanism, however, other non-Na/Pi dependent mechanisms also contribute to accelerated vascular calcification in patients with ESRD.
324 citations
TL;DR: Hyaff-11 scaffolds may be suitable delivery systems for rhBMP-2 in bone/cartilage repair because of their ability to retain rhBmp-2, release low levels of bioactive rhB MP-2 to the local environment in a sustained manner, and stimulate differentiation of pluripotent stem cells.
Abstract: Bone morphogenetic protein-2 (BMP-2) delivered in a suitable implantable matrix has the potential to repair local skeletal defects by inducing new bone formation from undifferentiated pluripotent stem cells resident in host tissue. In this study, we examined in vitro the potential of a derivatized hyaluronic acid (Hyaff-11) scaffold as a delivery vehicle for recombinant human BMP-2 (rhBMP-2) in bone and cartilage repair therapies. Hyaff-11 scaffolds were fabricated using a phase inversion/particulate leaching method and soak-loaded with rhBMP-2. In vitro release kinetics of rhBMP-2, demonstrated using enzyme-linked immunosorbant assay and alkaline phosphatase (ALP) assay revealed a slow, sustained rhBMP-2 release during 28 days, with a cumulative release of 31.82% of the initial rhBMP-2 loaded. rhBMP-2 was released in bioactive form as demonstrated by ALP induction of pluripotent cell line, C3H10T1/2 (T1/2), down the osteoblast lineage when incubated with the release supernatants. rhBMP-2 retention in Hyaff-11 scaffolds was greater than that from collagen gels, which released most of the initially loaded rhBMP-2 by 14 days. rhBMP-2-loaded Hyaff-11 scaffolds were also seeded with T1/2 cells and evaluated at 3, 7, 14, and 28 days for viability and expression of osteoblast phenotype. Cells remained viable throughout the study and expressed a time- and dose-dependent ALP and osteocalcin expression in the rhBMP-2 groups. Based on these observations, Hyaff-11 scaffolds may be suitable delivery systems for rhBMP-2 in bone/cartilage repair because of their ability to retain rhBMP-2, release low levels of bioactive rhBMP-2 to the local environment in a sustained manner, and stimulate differentiation of pluripotent stem cells.
190 citations
TL;DR: The results strongly suggest that the APs reduced the toxicity of LPS, as a host defense factor against LPS.
164 citations
01 Jan 2002
TL;DR: In this article, the authors show that the production of reactive oxygen species (ROS) by osteoclasts, which assist in bone remodeling under physiological conditions, can lead to hyperoxidant stress.
Abstract: Background: Rampant production in skeletal abnormalities can lead to hyperoxidant stress though the production of ‘‘reactive oxygen species’’ (ROS) by osteoclasts, which assist in bone remodeling under physiological conditions. Methods: Thirty cases each of post-menopausal osteoporosis, renal osteodystrophy and bone fractures constituted the test groups. Thirty healthy subjects made up the control group. Serum total alkaline phosphatase served as an index of osteoblastic activity. Serum calcium and phosphorous indicated bone remodeling status. Serum superoxide dismutase, glutathione peroxidase and glutathione reductase represented the enzymatic antioxidants. Results: Mean values for malondialdehyde were significantly elevated (P<0.001) in test groups, indicating enhanced osteoclastic activity. Significantly depressed (P<0.001) activities of superoxide dismutase and glutathione peroxidase reinforced hyperoxidant stress. Mean values of glutathione reductase remained unaltered. Diminished osteoblastic activity in post-menopausal osteoporosis was indicated by depressed alkaline phosphatase (P<0.001). Increased serum calcium (P<0.001) and decreased serum phosphorous (P<0.001) in renal osteodystrophy indicated compensatory hyperparathyroidism. Conclusions: The findings indicate that ROS have a major role to play in bone metabolism. D 2002 Elsevier Science B.V. All rights reserved.
163 citations
TL;DR: BMP-2 accelerates the differentiation of stromal cells toward an osteoblastic phenotype, whereas glucocorticoids induce their differentiation toward an adipocytic phenotype.
156 citations
TL;DR: The acid and alkaline phosphatase activities of the clam Scrobicularia plana have been partially characterised in different organs and tissues and the 'in vitro' effect of heavy metals on both types of enzymatic activity has been analysed.
Abstract: The acid and alkaline phosphatase activities of the clam Scrobicularia plana have been partially characterised in different organs and tissues (digestive gland, gills, foot, siphon and mantle) and the 'in vitro' effect of heavy metals on both types of enzymatic activity have been analysed. The optimal pH ranged between 4.0 and 5.5 for acid phosphatase activity and 8.5 and 9.5 for alkaline phosphatase activity. The apparent optimum temperature was in the 30-60 degrees range for acid phosphatase activity and in the 30-40 degrees C range for alkaline phosphatase activity. The effect of substrate concentration on enzymatic activities in the tissues showed a good fit to the Michaelis-Menten model. For both types of enzymatic activity, the highest values were found in the digestive gland. The effect of heavy metals was dependent on the tissue analysed. Mercury showed the highest inhibition in the organs/tissues and the parameters Km and Vmax were modified when the inhibitor concentration increased, thus indicating a mixed type of inhibition.
152 citations
TL;DR: To understand better how GPI proteins partition into rafts, the insertion of the GPI‐anchored alkaline phosphatase (AP) was studied in real‐time using atomic force microscopy.
Abstract: Several cell surface eukaryotic proteins have a glycosylphosphatidylinositol (GPI) modification at the C-terminal end that serves as an anchor to the plasma membrane and could be responsible for the presence of GPI proteins in rafts, a type of functionally important membrane microdomain enriched in sphingolipids and cholesterol In order to understand better how GPI proteins partition into rafts, the insertion of the GPI-anchored alkaline phosphatase (AP) was studied in real-time using atomic force microscopy Supported phospholipid bilayers made of a mixture of sphingomyelin-dioleoylphosphatidylcholine containing cholesterol (Chl+) or not (Chl-) were used to mimic the fluid-ordered lipid phase separation in biological membranes Spontaneous insertion of AP through its GPI anchor was observed inside both Chl+ and Chl- lipid ordered domains, but AP insertion was markedly increased by the presence of cholesterol
140 citations
TL;DR: Small shifts in extracellular pH led to significant changes in the ability of BMSCs to express markers of the osteoblast phenotype and suggest that degrading polymer scaffolds may influence the biologic activity of the cells in the immediate environment.
Abstract: The objective of this study was to address the hypothesis that changes in extracellular pH alter collagen gene expression, collagen synthesis, and alkaline phospha- tase activity in bone marrow stromal cells (BMSCs). Poten- tial effects of pH on cell function are of particular impor- tance for tissue engineering because considerable effort is being placed on engineering biodegradable polymers that may generate a local acidic microenvironment on degrada- tion. Human and murine single-cell marrow suspensions were plated at a density o f2×1 0 4 cells/cm 2 . After 7 days in culture, the pH of the culture medium was adjusted to one of six ranges: 7.8, 7.5.-7.7, 7.2-7.4, 6.9-7.1, 6.6-6.8, or 6.5. After 48 h of exposure to an altered pH, alkaline phospha- tase activity and collagen synthesis decreased significantly with decreasing pH. This decrease was two-to threefold as pH decreased from 7.5 to 6.6. In contrast, 1(I) procollagen mRNA levels increased two- to threefold as pH was de- creased. The trend in osteocalcin mRNA expression was op- posite to that of collagen. Small shifts in extracellular pH led to significant changes in the ability of BMSCs to express markers of the osteoblast phenotype. These pH effects po- tentially relate to the microenvironment supplied by a tis- sue-engineering scaffold and suggest that degrading poly- mer scaffolds may influence the biologic activity of the cells in the immediate environment. © 2002 John Wiley & Sons, Inc. J Biomed Mater Res 60: 292-299, 2002; DOI 10.1002/ jbm.10050
139 citations
TL;DR: Collagen-coating alone is not sufficient to accelerate differentiation of rat calvarial osteoblasts on Ti6Al4V, indicating that titanium alloy as an implant material cannot be improved by modification of its surface.
Abstract: Several attempts have been made to improve osseointegration of titanium alloy as an implant material by modification of its surface. In the present study, proliferation, differentiation, and mineralization of osteoblasts on type I collagen-coated Ti6Al4V were investigated. The activity of alkaline phosphatase and the accumulation of calcium by osteoblasts grown on titanium alloy were significantly higher compared to cells grown on polystyrene. Precoating of the implant surface with type I collagen did not extensively affect proliferation, the activity of alkaline phosphatase, collagen synthesis, calcium accumulation, or the mRNA levels for collagen I alpha1, osteopontin, osteocalcin, MMP-2, and TIMP-2. Maximum collagen synthesis by osteoblasts was observed at day 4 of culture independent of the type of implant material. The specific activity of alkaline phosphatase reached its maximum at day 18 of culture. Accumulation of calcium and elevated mRNA levels for osteocalcin were found at day 22. These results indicate that collagen-coating alone is not sufficient to accelerate differentiation of rat calvarial osteoblasts on Ti6Al4V.
TL;DR: Results indicate that cementoblast progenitors are present in bovine dental follicle cells, which can provide a useful model for investigating the molecular mechanisms of cementogenesis.
TL;DR: Cloned and characterized cell lines from the periodontal ligament and demonstrated that a cell line, designated PDL-L2, mimics the gene expression of the PDL in vivo: it expresses genes such as alkaline phosphatase, type I collagen, periostin, runt-related transcription factor-2 (Runx2) and EGF receptor, but does not express genessuch as bone sialoprotein and osteocalcin.
Abstract: The periodontal ligament (PDL) is a connective tissue located between the cementum of teeth and the alveolar bone of the mandibula. It plays an integral role in the maintenance and regeneration of periodontal tissue. The cells responsible for maintaining this tissue are thought to be fibroblasts, which can be either multipotent or composed of heterogenous cell populations. However, as no established cell lines from the PDL are available, it is difficult to assess what type of cell promotes all of these functions. As a first step to circumvent this problem, we have cloned and characterized cell lines from the PDL from mice harboring a temperature-sensitive SV 40 large T-antigen gene. RT-PCR and in situ hybridization studies demonstrated that a cell line, designated PDL-L2, mimics the gene expression of the PDL in vivo: it expresses genes such as alkaline phosphatase, type I collagen, periostin, runt-related transcription factor-2 (Runx2) and EGF receptor, but does not express genes such as bone sialoprotein and osteocalcin. Unlike osteoblastic cells and a mixed cell population from the PDL, PDL-L2 cells do not produce mineralized nodules in the mineralization medium. When PDL-L2 cells were incubated in the presence of recombinant human bone morphogenetic protein-2 alkaline phosphatase activity increased and mineralized nodules were eventually produced, although the extent of mineralization is much less than that in osteoblastic MC3T3-E1 cells. Furthermore, PDL-L2 cells appeared to have a regulatory mechanism by which the function of Runx2 is normally suppressed.
TL;DR: It is indicated that TGF-β-responsive ERK1/2 and JNK cascades negatively regulate Smad3-induced transcriptional activity as well as ALP activity and mineralization in osteoblasts.
TL;DR: Evidence is provided that modification of the particle surface prevents DNA strand breakage in epithelial lung cells from quartz-exposed rats and shows the feasibility of the in vivo model to evaluate the role of inflammation, antioxidant status, and cytotoxicity in particle-induced DNA damage.
Abstract: Respirable quartz has been classified as a human lung carcinogen (IARC, 1997). However, the mechanisms involved in quartz-induced carcinogenesis remain unclear. The aim of the present study was to investigate acute DNA damage in epithelial lung cells from rats exposed to quartz. Since surface reactivity is considered to play a crucial role in the toxicity of quartz, the effect of surface modifying agents polyvinylpyridine-N-oxide (PVNO) and aluminium lactate (AL) was evaluated. Therefore, rats were instilled with quartz (DQ12, 2 mg/rat) or quartz treated with PVNO or AL. After 3 days animals were killed and brochoalveolar lavage (BAL) was performed to evaluate inflammatory cell influx. BAL-fluid levels of lactate dehydrogenase (LDH), alkaline phosphatase (AP) and total protein were used as lung damage markers. Neutrophil activation was assessed by myeloperoxidase (MPO) measurement, and total antioxidant capacity of the BAL-fluid was determined using the TEAC (trolox equivalent antioxidant capacity) assay. Lung epithelial cells were isolated and DNA strand breakage was determined by single cell gel electrophoresis (comet assay). DNA damage was significantly increased in epithelial cells from rats instilled with DQ12, whereas no enhanced DNA strand breakage was observed when quartz was treated with PVNO or AL. Total protein, LDH and TEAC were increased in rats treated with native quartz, and this was inhibited by both coatings. A significant correlation between neutrophil numbers and MPO levels was observed, indicating neutrophil activation. Inhibition of DNA damage by both coatings was paralleled by a reduction of neutrophil influx as well as MPO activity. In this study we provide evidence that modification of the particle surface prevents DNA strand breakage in epithelial lung cells from quartz-exposed rats. Furthermore, the present data show the feasibility of our in vivo model to evaluate the role of inflammation, antioxidant status, and cytotoxicity in particle-induced DNA damage.
TL;DR: A biosensor is constructed to detect heavy metals from inhibition of alkaline phosphatase present on the external membrane of Chlorella vulgaris microalgae, cultivated in the laboratory and used for the determination of those toxic compounds.
TL;DR: Cells from human skeletal muscle, because of their availability to orthopaedic surgeons, their osteocompetence, and their ability to express BMP-2 after genetic engineering, are an attractive cell population for use in B MP-2 gene therapy approaches.
Abstract: We have examined whether primary human muscle-derived cells can be used in ex vivo gene therapy to deliver BMP-2 and to produce bone in vivo. Two in vitro experiments and one in vivo experiment were used to determine the osteocompetence and BMP-2 secretion capacity of cells isolated from human skeletal muscle. We isolated five different populations of primary muscle cells from human skeletal muscle in three patients. In the first in vitro experiment, production of alkaline phosphatase by the cells in response to stimulation by rhBMP-2 was measured and used as an indicator of cellular osteocompetence. In the second, secretion of BMP-2 was measured after the cell populations had been transduced by an adenovirus encoding for BMP-2. In the in vivo experiment, the cells were cotransduced with a retrovirus encoding for a nuclear localised beta-galactosidase gene and an adenovirus encoding for BMP-2. The cotransduced cells were then injected into the hind limbs of severe combined immune-deficient (SCID) mice and analysed radiographically and histologically. The nuclear localised beta-galactosidase gene allowed identification of the injected cells in histological specimens. In the first in vitro experiment, the five different cell populations all responded to in vitro stimulation of rhBMP-2 by producing higher levels of alkaline phosphatase when compared with non-stimulated cells. In the second, the five different cell populations were all successfully transduced by an adenovirus to express and secrete BMP-2. The cells secreted between 444 and 2551 ng of BMP-2 over three days. In the in vivo experiment, injection of the transduced cells into the hind-limb musculature of SCID mice resulted in the formation of ectopic bone at 1, 2, 3 and 4 weeks after injection. Retroviral labelling of the cell nuclei showed labelled human muscle-derived cells occupying locations of osteoblasts in the ectopic bone, further supporting their osteocompetence. Cells from human skeletal muscle, because of their availability to orthopaedic surgeons, their osteocompetence, and their ability to express BMP-2 after genetic engineering, are an attractive cell population for use in BMP-2 gene therapy approaches.
TL;DR: Data indicate that Runx2 expression in osteoblasts is reduced by hypoxia, and may be a mechanism of osteoporosis by decreased vascular supply.
TL;DR: This study has concentrated on the tissue-nonspecific alkaline phosphatase from arctic shrimps, which lacks some features specific for the mammalian enzyme, but their backbones are very similar and may therefore be typical for other higher organisms.
TL;DR: The coincidence of the ALP peak values with downregulation of type I collagen in BMP-4 transfected cells suggested that C3H10T1/2 cells differentiate into chondrocytes via a chondROprogenitor-like cell.
TL;DR: IL-6R deficiency may represent for bone marrow-located mesenchymal progenitors a sort of protective mechanism to escape the osteogenic effect of IL-6, which is produced by the MSC itself as well as by other marrow stromal cells.
TL;DR: In mouse osteoblastic cells, Smad3 inhibited proliferation, but it also enhanced ALP activity, mineralization, and the levels of bone matrix proteins such as type I collagen (COLI), OPN, and MGP.
Abstract: Transforming growth factor (TGF) β is abundantly stored in bone matrix and appears to regulate bone metabolism. Although the Smad family proteins are critical components of the TGF-β signaling pathways, the roles of Smad3 in the expression of osteoblastic phenotypes remain poorly understood. Therefore, this study was performed to clarify the roles of Smad3 in the regulation of proliferation, expression of bone matrix proteins, and mineralization in osteoblasts by using mouse osteoblastic cell line MC3T3-E1 cells stably transfected with Smad3. Smad3 significantly inhibited [3H]thymidine incorporation and fluorescent intensity of the MTT-dye assay, compared with empty vector. Moreover, Smad3 increased the levels of type I procollagen, osteopontin (OPN), and matrix Gla protein (MGP) mRNA in Northern blotting. These effects of Smad3 mimicked the effects of TGF-β on the same cells. On the other hand, Smad3 greatly enhanced ALP activity and mineralization of MC3T3-E1 cells compared with empty vector, although TGF-β inhibited ALP activity and mineralization of wild-type MC3T3-E1 cells. A type I collagen synthesis inhibitor L-azetidine-2-carboxylic acid, as well as osteocalcin (OCN), significantly antagonized Smad3-stimulated ALP activity and mineralization of MC3T3-E1 cells. In conclusion, this study showed that in mouse osteoblastic cells, Smad3 inhibited proliferation, but it also enhanced ALP activity, mineralization, and the levels of bone matrix proteins such as type I collagen (COLI), OPN, and MGP. We propose that Smad3 plays an important role in osteoblastic bone formation and might help to elucidate the transcriptional mechanism of bone formation and possibly lead to the development of bone-forming drugs.
TL;DR: It is shown that enzymatic activity of ALP is necessary for mineralization of MC3T3-E1 cells, but not the presence of ALP protein or anchoring of ALP to the cells, which was consistent with the morphological findings.
TL;DR: Elevated alkaline phosphatase and alpha-fetoprotein are associated with subsequent spontaneous preterm birth in asymptomatic pregnant women at 24 and 28 weeks and Elevated corticotropin-releasing hormone levels at 28 weeks areassociated with spontaneous pre term birth at <35 weeks.
TL;DR: Routine measurements of serum alkaline phosphatase and serum phosphate are of no use in predicting bone mineralisation outcome in premature infants.
Abstract: Background: The bone mineral content of premature infants at term is lower than in mature infants at the same postconceptional age. Serum alkaline phosphatase and serum phosphate are often used as indicators of bone mineralisation.
Objective: To analyse the association between bone mineral content and serum alkaline phosphatase and serum phosphate.
Methods: Serum alkaline phosphatase and phosphate were measured at weekly intervals during admission in 108 premature infants of gestational age below 32 weeks (mean (SD) gestational age 29 (2) weeks; mean (SD) birth weight 1129 (279) g). Bone mineral content was measured at term (mean gestational age 41 weeks) by dual energy x ray absorptiometry and corrected for body size.
Results: Serum alkaline phosphatase was significantly negatively associated with serum phosphate (p < 0.001). Bone mineral content was not associated with mean serum alkaline phosphatase (p = 0.8), peak serum alkaline phosphatase (p = 0.5), or mean serum phosphate (p = 0.2) at term.
Conclusion: Routine measurements of serum alkaline phosphatase and serum phosphate are of no use in predicting bone mineralisation outcome in premature infants.
TL;DR: The effects ofSP on serum IGF-I levels suggest that SP may positively influence bone in men, and longer-duration studies examining the effects of SP or its isoflavones on bone turnover and bone mineral density and content in men are warranted.
Abstract: Recent studies suggest that soy protein (SP) protects bone in women; however, its effects on bone metabolism in men have not been investigated. Healthy men (59.2 ± 17.6 y) were assigned to consume 40 g of either SP or milk-based protein (MP) daily for 3 mo in a double-blind, randomized, controlled, parallel design. Serum insulin-like growth factor-I (IGF-I), which is associated with higher rates of bone formation, was greater (P < 0.01) in men supplemented with SP than in those consuming MP. Serum alkaline phosphatase and bone-specific alkaline phosphatase activities, markers of bone formation, and urinary deoxypyridinoline excretion, a specific marker of bone resorption, were not different between the SP and MP groups. Furthermore, because substantial reductions in bone density occur in men at ∼65 y of age, data were analyzed separately for men ≥65 y and those <65 y of age. The response to protein supplementation was consistent in the two age groups. The effects of SP on serum IGF-I levels suggest that SP may positively influence bone in men. Longer-duration studies examining the effects of SP or its isoflavones on bone turnover and bone mineral density and content in men are warranted.
TL;DR: The authors' mutagenesis data, computer modeling, and docking predictions indicate that the Tyr-367 residue contributes to the formation of the hydrophobic pocket that accommodates and stabilizes the side chain of the inhibitor during uncompetitive inhibition of mammalian APs.
TL;DR: It is concluded that subculture of undifferentiated rat bone marrow cells results in the loss of osteogenic potential of these cells, because expression of alkaline phosphatase and mineralization is higher in cells continuously supplemented with dexamethasone.
Abstract: The aim of our study was to investigate the osteogenic potential of subcultured rat bone marrow cells. Rat bone marrow (RBM) cells were cultured with or without dexamethasone. Subsequently, osteogenic differentiation and expression was studied. When cells were cultured continuously in the presence of dexamethasone, cultures initially showed high alkaline phosphatase expression and abundant mineralization. Expression of differentiation markers decreased with passaging. After cells were passaged three times, no alkaline phosphatase activity and calcification were found. Primary cells cultured without dexamethasone showed low alkaline phosphatase and no calcification, and remained fibroblast-like. When these cells were subcultured in the presence of dexamethasone, the cells did show osteogenic differentiation. Nevertheless, this occurred at a significant lower level than with cells continuously cultured with dexamethasone. In addition, no differentiation was found after second passage. Our results indicate that subcultured undifferentiated RBM cells show osteogenic differentiation after addition of dexamethasone. Expression of alkaline phosphatase and mineralization is higher in cells continuously supplemented with dexamethasone. Still, even when dexamethasone is added continuously, RBM cells loose their osteogenic potential after several passages. Therefore, we conclude that subculture of undifferentiated rat bone marrow cells results in the loss of osteogenic potential of these cells.
TL;DR: The present results indicate that OP‐1 is a potent inducer of C2C12 differentiation into osteoblastic cells.
Abstract: The effects of Osteogenic Protein-1 (OP-1, BMP-7) on the differentiation of the pluripotent mesenchymal cell line, C2C12, were examined. OP-1 at 50 ng/ml partially inhibited myotube formation in C2C12 cells, while OP-1 at 200 ng/ml completely inhibited myotube formation and induced the formation of cells displaying osteoblastic morphology. High concentrations of OP-1 elevated the alkaline phosphatase (AP) activity dramatically, both as a function of time and OP-1 concentration. Osteocalcin (OC) mRNA expression was detected as early as 8 days in OP-1-treated cultures and subsequently increased considerably. Expression of bone sialoprotein (BSP) mRNA was low in control cultures and stimulated by OP-1. Collagen type I mRNA expression was enhanced by OP-1 during the early days in culture, but gradually decreased thereafter. MyoD mRNA expression, high in control cultures, was suppressed by OP-1 in a dose- and time-dependent manner. OP-1 enhanced ActR-I mRNA expression and significantly elevated the mRNA expressions of BMP-1, BMP-4, BMP-5, GDF-6, and GDF-8. The present results indicate that OP-1 is a potent inducer of C2C12 differentiation into osteoblastic cells.
TL;DR: It is suggested that 3MC might have critical effects on the formation of bone both in vivo and in vitro, as well as inhibition of appropriate calcification of bones.
Abstract: 3-Methylcholanthrene (3MC) is a ligand for arylhydrocarbon receptor (AhR), which binds dioxin. We examined the effects of 3MC on the proliferation and differentiation of osteoblasts using cultures of rat calvarial osteoblast-like cells (ROB cells) and mouse calvarial clonal preosteoblastic cells (MC3T3-E1 cells). Analysis by RT-PCR revealed that the mRNAs for AhR and AhR nuclear translocators were expressed in both ROB and MC3T3-E1 cells. Cell proliferation and the synthesis of DNA by ROB cells and MC3T3-E1 cells were markedly inhibited on exposure of cells to 3MC. Furthermore, 3MC reduced the activity of alkaline phosphatase and the rate of deposition of calcium by cells. The level of expression of mRNA for osteocalcin, which is a marker of osteoblastic differentiation, was also depressed by 3MC. Moreover, when 3MC (1 mg/kg body weight) was administered sc to pregnant mice at 10.5, 12.5, and 14.5 d post coitus, fetuses examined subsequently at 15.5 or 17.5 d post coitus revealed evidence of inhibition of appropriate calcification of bones. The treated metacarpals showed no subperiosteal bone matrix histologically. Our findings indicate that 3MC might have critical effects on the formation of bone both in vivo and in vitro.