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PubMed1990

Ultrastructural and immunocytochemical study of bone-derived cells cultured in three-dimensional matrices: influence of chondroitin-4 sulfate on mineralization.

Bone-derived cells were cultured in three-dimensional reconstituted matrices made of type I collagen or type I collagen chondroitin-4-sulfate. As observed by microscope, their characteristics were as follows: The cells deposited a faint extracellular matrix mainly composed of type I collagen. In the collagen-chondroitin-sulfate sponge fibers, a calcification process, which involved the deposition of hydroxyapatite crystals, was demonstrated. Mineralization occurred only in collagen chondroitin sulfate sponge fibers when seeded with bone-derived cells and was not seen with nonosteogenic cells, such as gingival fibroblasts. Gla protein was intracellularly visualized in both types of sponges seeded with bone-derived cells while an extracellular secretion was seen only in the collagen chondroitin sulfate sponge fibers where calcification occurred. These results suggest that collagen chondroitin sulfate promotes in vitro mineralization of three-dimensional collagen matrices when seeded with bone-derived cells.

Animals↗
PubMed1991

In vitro differentiation of bone and hypertrophic cartilage from periosteal-derived cells.

Periosteal cells were enzymatically liberated from the tibiae of young chicks, introduced into cell culture, and allowed to reach confluence. The morphology of the cells gave the impression of a relatively homogeneous population of fibroblast-like cells. These cultured cells did not overtly express osteogenic or chondrogenic properties as judged by their morphology and the lack of reactivity with probes to phenotype-specific antigens of osteoblasts or chondrocytes. The cells were then replated at relatively high density and chronologically evaluated for the differentiation of bone and cartilage. These replated cells formed a multi-layer of fibroblast-like cells, the top portion of which eventually differentiated into bone tissue as evidenced by the presence of mineralization and immunocytochemical reactivity to bone Gla protein- and osteocyte-specific probes. Cells below this distinctive top layer differentiated into chondrocytes, which eventually further developed into hypertrophic chondrocytes as evidenced by their morphology and the presence of immunoreactive type X collagen in the matrix. Mineralization was also observed in the territorial matrix of these hypertrophic chondrocytes, when the culture was augmented with beta-glycerophosphate. Periosteal-derived cells replated at a lower density as controls did not show signs of osteochondrogenic differentiation. These observation suggest that periosteal-derived cells of young chicks contain mesenchymal cells which possess the potential to undergo terminal differentiation into osteogenic or chondrogenic phenotypes depending on local environmental or positional cues.

Animals↗
PubMed2005

Evolution of matrix and bone gamma-carboxyglutamic acid proteins in vertebrates.

The evolution of calcified tissues is a defining feature in vertebrate evolution. Investigating the evolution of proteins involved in tissue calcification should help elucidate how calcified tissues have evolved. The purpose of this study was to collect and compare sequences of matrix and bone gamma-carboxyglutamic acid proteins (MGP and BGP, respectively) to identify common features and determine the evolutionary relationship between MGP and BGP. Thirteen cDNAs and genes were cloned using standard methods or reconstructed through the use of comparative genomics and data mining. These sequences were compared with available annotated sequences (a total of 48 complete or nearly complete sequences, 28 BGPs and 20 MGPs) have been identified across 32 different species (representing most classes of vertebrates), and evolutionarily conserved features in both MGP and BGP were analyzed using bioinformatic tools and the Tree-Puzzle software. We propose that: 1) MGP and BGP genes originated from two genome duplications that occurred around 500 and 400 million years ago before jawless and jawed fish evolved, respectively; 2) MGP appeared first concomitantly with the emergence of cartilaginous structures, and BGP appeared thereafter along with bony structures; and 3) BGP derives from MGP. We also propose a highly specific pattern definition for the Gla domain of BGP and MGP.

Amino Acid Sequence↗
PubMed2003

A gene expression profile for endochondral bone formation: oligonucleotide microarrays establish novel connections between known genes and BMP-2-induced bone formation in mouse quadriceps.

Endochondral bone formation has been fairly well characterized from a morphological perspective and yet this process remains largely undefined at molecular and biochemical levels. In vitro and in vivo studies have shown that human bone morphogenetic protein-2 (hBMP-2) is an important developmental growth and differentiation factor, capable of inducing ectopic bone formation in vivo. This study evaluated several aspects of the osteogenic effect of hBMP-2 protein injected into quadriceps of female C57B1/6J SCID mice. Mice were euthanized 1, 2, 3, 4, 7, and 14 days postinjection and muscles were collected for several methods of analysis. Hematoxylin and eosin-stained sections of muscles injected with formulation buffer showed no evidence of osteogenesis. In contrast, sections of muscles injected with hBMP-2 showed evidence of endochondral bone formation that progressed to mineralized bone by day 14. In addition, radiographs of mice injected with hBMP-2 showed that much of the quadriceps muscle had undergone mineralization by day 14. Labeled mRNA solutions were prepared and hybridized to oligonucleotide arrays designed to monitor approximately 1300 murine, full-length genes. Changes in gene expression associated with hBMP-2 were determined from time-matched comparisons between buffer and hBMP-2 samples. A gene expression profile was created for 215 genes that showed greater than 4-fold changes at one or more of the indicated time points. One hundred twenty-two of these genes have previously been associated with bone or cartilage metabolism and showed significant increases in expression, e.g., aggrecan (Agc1), runt related transcription factor 2 (Runx2), bone Gla protein 1 (Bglap1), and procollagens type II (Col2a1) and X (Col10a1). In addition, there were 93 genes that have not been explicitly associated with bone or cartilage metabolism. Two of these genes, cytokine receptor-like factor-1 (Crlf1) and matrix metalloproteinase 23 (Mmp23), showed peak changes in gene expression of 15- and 40-fold on days 4 and 7, respectively. In situ hybridizations of muscle sections showed that Mmp23 and Crlf1 mRNAs were expressed in chondrocytes and osteoblasts, suggesting a role for both proteins in some aspect of cartilage or bone formation. In conclusion, oligonucleotide arrays enabled a broader view of endochondral bone formation than has been reported to date. An increased understanding of the roles played by these gene products will improve our understanding of skeletogenesis, fracture repair, and pathological conditions such as osteoporosis.

Animals↗
PubMed1997

Gamma linolenic acid regulates expression of maspin and the motility of cancer cells.

Maspin, mammary serine protease inhibitor, is a recently identified tumour suppressor and has a profound effect on cell motility. This study examined the effect of gamma linolenic acid (GLA), an essential fatty acid (EFA) with anticancer properties, on the expression of maspin and motility of cancer cells. Six human cell lines including colon cancer, mammary cancer, and melanoma were used. Expression of maspin protein was determined by immunocytochemistry & Western blotting. Maspin mRNA was detected with reverse transcription-PCR (RT-PCR). Four of the six cell types expressed maspin with MDA MB 231 and ECV304 (endothelial cell) being negative. Treatment of these maspin positive cells with gamma linolenic acid (GLA) resulted in a concentration dependent stimulation of the expression of maspin protein with the effects seen as early as 4 hours. Linoleic acid had an inhibitory effects. Alpha linolenic acid and arachidonic acid had no significant effect. The mRNA levels from cells treated with GLA was seen to increase as shown by RT-PCR. Cell motility, monitored with time-lapse video recording and Hoffmann microscopy, showed a marked reduction in terms of spreading and migration on extracellular matrix coated surface. This reduction was reversed with anti-maspin antibody. It is concluded that GLA, a member of then-6 series of EFAs, up-regulates the expression of maspin which is associated with a reduction in the motility of cancer cells.

Breast Neoplasms↗
PubMed2005

Effects of retinoic acid on the differentiation of chondrogenic progenitor cells, ATDC5.

Chondrocyte differentiation is a fundamental process during endochondral ossification. Retinoic acid (RA) has been shown to regulate this process, however, the mechanisms underlying RA regulation of chondrogenesis are not clearly understood. Chondroprogenitor cells, ATDC5 have been shown to be a useful in vitro model for examining the multiple step differentiation of chondrocytes. The present study investigated the mechanisms underlying RA regulation of chondrogenesis using ATDC5 cell culture. In this study, we show that RA suppresses the cell growth, cartilage nodule formation, accumulation of proteoglycan, alkaline phosphatase (ALPase) activity and mineralization and that RA dose dependently upregulates the levels of type X collagen and matrix metalloproteinase-13 (MMP-13) mRNA which are marker proteins of hypertrophic chondrocytes, in ATDC5 cells. The addition of protein synthesis inhibitor, cycloheximide (CHX), partially inhibits the induction of type X collagen and MMP-13 mRNA by RA. In this system, RA upregulates the mRNA level of Runx2/Cbfa1 (type II), a positive regulator for mineralization, and downregulates the mRNA of Indian hedgehog (Ihh), parathyroid hormone related protein (PTHrP), negative regulators for terminal differentiation. However, RA downregulates ALPase, bone gla protein (BGP) mRNAs and mineralization. These data indicate that RA stimulates cartilage differentiation, however, cell condensation and cartilage nodule formation may be candidates of primary importance in the terminal differentiation of chondrocytes.

Alkaline Phosphatase↗
PubMed1992

Characterization of osteoblast-like cells from normal adult rat femoral trabecular bone.

Osteoblast-like cell cultures have been established from the trabecular surfaces of normal adult rat femoral trabecular bone. The cultured cells responded to stimulation by parathyroid hormone (rPTH), with a rise in intracellular cAMP in excess of 25-fold while failing to respond to incubation with sCT. Furthermore, the osteoblast-like cells exhibited a high level of alkaline phosphatase expression, both histochemically and biochemically. Incubations with 1,25(OH)2 vitamin D3 increased the alkaline phosphatase activity by 50% and stimulated bone Gla-protein (BGP) synthesis. When the cell layers were supplemented with both 50 micrograms/ml ascorbic acid and 10 mM beta-glycerophosphate and allowed to grow past confluency for 3 weeks, they formed calcified ridges and multilayered nodules. Confirmation of the mineralization of an extracellular matrix was made by von Kossa staining. This simple isolation technique now facilitates the availability of normal adult rat osteoblastic cells for investigation of bone and mineral metabolism.

Alkaline Phosphatase↗
PubMed1993

Effect of cadmium on osteogenesis within diffusion chambers by bone marrow cells: biochemical evidence of decreased bone formation capacity.

The biological and biochemical effects of cadmium administration on bone marrow in rats were examined. When young adult rats were administered cadmium (Cd) repeatedly at a dose of 750 micrograms/kg body wt for up to 4 weeks, metallothionein mRNA was detected by a gene expression analysis in their bone marrow at 2 weeks after the first Cd administration, though the amounts were lower than those in liver. To determine the direct effect of cadmium on bone formation, the potential of Cd-treated bone marrow cells and demineralized bone matrix (DBM) to form bone and cartilage was assessed using a diffusion chamber (DC) in vivo, by histological examination, and by biochemical parameters such as alkaline phosphatase (ALP) activity, total calcium and phosphorus content, and the bone-specific vitamin K-dependent Gla-containing protein (BGP) content, relative to mineralization. Diffusion chambers were inoculated with DBM and bone marrow cells from either Cd-treated or nontreated rats (control) and were then implanted subcutaneously into syngeneic nontreated rats. The accumulation of BGP in DCs with Cd-treated bone marrow was significantly lower than that in control DCs. Unlike in control DC, a peak of ALP activity did not occur at 4 weeks postimplantation in DC implants inoculated with Cd-treated bone marrow; the ALP activity and calcium content in these implants were also significantly lower than those of the control bone marrow-containing chambers at the early stage of implantation. Histological examinations of chambers with Cd-treated marrow showed a decreased area of cartilage and bone foci compared with those in control chambers. These findings suggest that Cd administration inhibits the osteoblastic and chondroblastic differentiation pathway in bone marrow through direct effects on these cells.

Animals↗
PubMed1997

Viable bone formation in porous hydroxyapatite: marrow cell-derived in vitro bone on the surface of ceramics.

With the aim of in vitro production of bone fragments more closely resembling autogenous bone, rat cultured bone marrow cells were combined with porous hydroxyapatite (HA) discs and cultured in the presence of dexamethasone (Dex). Bone marrow cells were collected from the femoral diaphyses of a 7-week-old male Wistar rat, and primary culture was performed for six days. Then, cell suspensions were prepared by trypsin treatment, and combined with the porous HA discs. After a 2-hour incubation, the composites were additionally cultured for up to 4 weeks (subculture) in the presence of Dex. In a control group, the subculture was performed without Dex. After 1, 2, 3 and 4 weeks of subculturing, the HA discs were removed, and alkaline phosphatase (ALP) activity, bone Gla protein (BGP), and DNA were quantitated. A portion of the disc was prepared for scanning electron microscopy (SEM), from which bone formation was evaluated morphologically. ALP activity peaked at 2-3 weeks and decreased at 4 weeks. BGP levels began to increase at 3 weeks. In the SEM study, mineralized collagenous extracellular matrix was noted at 3 and 4 weeks. In the control group, neither significant ALP activity nor increased BGP was detected. These biochemical and morphological results suggest that with the culture technique, active bone formation in the pore regions of HA can be fabricated in vitro. It is anticipated that when composites are subcultured in this way they will function as a bone graft with properties similar to those of autogenous bone.

Animals↗
PubMed2003

Human-derived vascular smooth muscle cells produce angiotensin II by changing to the synthetic phenotype.

We investigated whether vascular smooth muscle cells (VSMC)-derived from human produce angiotensin (Ang) II upon change from the contractile phenotype to the synthetic phenotype by incubation with fibronectin (FN). Expression of alpha-smooth muscle (SM) actin, apparent in the contractile phenotype, was decreased by FN. Expressions of matrix Gla and osteopontin, apparent in the synthetic phenotype, were increased by FN. Ang II measured by radioimmunoassay (RIA) was significantly increased in human VSMC by FN. Expression of mRNAs for Ang II-generating proteases cathepsin D, cathepsin G, ACE, and chymase was increased by FN. Expressions of cathepsin D and cathepsin G proteins were also increased by FN. Ang I-generating activity, which was inhibited by an aspartyl protease inhibitor pepstatin A, was readily detected in the conditioned medium from human VSMC. Antisense oligodeoxynucleotides (ODNs) that hybridize with cathepsin D and cathepsin G significantly inhibited FN-increased Ang II in conditioned medium and cell extracts. In VSMC conditioned medium, FN-induced elevation of Ang II was significantly inhibited by temocapril but not by chymostatin. Ang II type 1 receptor antagonist CV11974 completely, and antisense cathepsin D and cathepsin G ODNs partially inhibited the FN-stimulated growth of human VSMC. These results indicate that the change of homogeneous cultures of human VSMC from the contractile to the synthetic phenotype sequentially increases expression of proteases cathepsin D, cathepsin G, and ACE, production of Ang II and productions of growth factors, culminating in VSMC proliferation. These findings implicate a new mechanism for the pathogenesis of human vascular proliferative diseases.

Angiotensin I↗
PubMed1995

Analysis of relationships between sex hormone dynamics and bone metabolism and changes in bone mass in surgically induced menopause.

A 3-year follow-up study was performed of bone metabolism and bone changes induced by surgical menopause as a consequence of hysterectomy and oophorectomy (OVX) is 52 nonmenopausal women. We investigated 22 bone parameters and determined seven bone indices as indicators of bone mineral content by dual energy X-ray absorptiometry (DXA), quantitative computed tomography (QCT), and microdensitometry (MD). The significant correlations between levels of sex hormones and/or bone parameters and bone indices demonstrated that marked sex-steroid deficiency after surgical menopause induced bone uncoupling during high bone turnover and subsequent rapid bone loss on the early period after OVX. Principal component analysis using correlation coefficients suggested a seven-loading-factor matrix composed of bone parameters and a two-loading-factor matrix composed of bone indices. Two groups of parameters--estradiol and estriol, and androstenedione together, and luteinizing hormone and follicle stimulating hormone together--indicated that the rate of bone loss was greater in the trabecular bone than in the cortical bone. Three other groups of parameters--urine calcium, urine hydroxyproline, and serum bone Gla-protein together; serum alkaline phosphatase, serum calcium, and 1,25-dihydroxycholecalciferol [1,25(OH)2D] together; and plasma tartrate-resistant acid phosphatase--indicated that bone uncoupling, with a prevalence of resorption over formation of bone, was greater in trabecular bone than is cortical bone and also that magnitude and rate of bone loss in the axial vertebrae surpassed those in the appendicular metacarpals after OVX. Two other groups of parameters, namely, trabecular bone mineral density (Dd) and bone mineral content (Dc), both measured by DXA, and bone mineral density (L2, L3), measured by QCT, together; and the cortical thickness index (MCI), cortical bone mineral density (sigmaGS/D), and the ratio of GSmin/max, measured by MD, indicated that the relative rates of bone reduction at the 3-year follow-up were greater in the axial vertebrae than in the appendicular metacarpals. Thus, bone change in the trabecular bone was associated with rapid loss during the early phase after OVX, whereas that in the cortical bone was slow during the late phase.

Absorptiometry, Photon↗
PubMed1992

Recent investigations on dentin specific proteins.

Several extracellular matrix (ECM) proteins have been isolated from dentin and shown to be synthesized by odontoblasts. Highly phosphorylated proteins (HP), the phosphophoryns, are specifically found in dentin and are synthesized only by odontoblasts. Phosphophoryns are probably involved in controlling the site and/or the rate of circumpulpal mineralization. Other moderately phosphorylated (MP) and low phosphorylated (LP) proteins have also been detected in dentin, but have not been studied extensively. A 53 kDal dentin sialoprotein (DSP) that resembles bone sialoproteins is expressed by odontoblasts and by pulp cells, but essentially by no other cell type. The function of this protein is unknown. Osteocalcin (bone Gla protein) is synthesized by osteoblasts and by odontoblasts and found in the ECM of bone and dentin. Other tissues or cells (including pulp) do not express osteocalcin. The highly specific nature of these dentin and bone ECM proteins suggests that antibody and molecular probes for the phosphoproteins, DSP and osteocalcin could be valuable in studies on the differentiation and identification of odontoblasts and their precursors. Other bone NCPs shown to be present in dentin may also prove to be important in dentinogenesis.

Dentin↗
PubMed1988

Ectopic bone formation is enhanced in senescent animals implanted with embryonic cells.

Ectopic bone formation induced by the subcutaneous implantation of demineralized bone matrix (DBM) is very significantly reduced in older Fischer 344 rats. Cells originating from calvaria of 20-day-old embryo donors were introduced into cylinders of DBM sealed at the ends with a Millipore filter or collagen sponges prior to subcutaneous implantation. Cells within the chambers had access to vascular channels that could penetrate through the interstices of the DBM. After four weeks of implantation in 26-month-old rats, the cylinders were full of bone. This bone was assessed by histologic techniques, by calcium and bone gamma-carboxyglutamic acid (gla) protein (BGP) concentrations, and by alkaline phosphatase activity. Cylinders to which no cells were added produced no bone. Bone marrow cells enclosed in similar cylinders or injected weekly at the implantation site also enhanced new bone formation but to a much lesser extent. Embryonic muscle cells formed large amounts of cartilage and less bone. Fibroblasts were inactive in this system. Prior treatment of the DBM with trypsin inhibited the myoblast response but not that of calvaria cells.

Aging↗
PubMed1994

High affinity binding sites for activated protein C and protein C on cultured human umbilical vein endothelial cells. Independent of protein S and distinct from known ligands.

Activated protein C (APC) is an antithrombotic serine proteinase having anticoagulant, profibrinolytic and anti-inflammatory activities. Despite its potential clinical utility, relatively little is known about its clearance mechanisms. In the present study we have characterized the interaction of APC and its active site blocked forms with human umbilical vein endothelial cells (HUVEC). At 4 degrees C 125I-APC bound to HUVEC in a specific, time dependent, saturable and reversible manner. Scatchard analysis of the binding isotherm demonstrated a Kd value of 6.8 nM and total number of binding sites per cell of 359,000. Similar binding isotherms were obtained using radiolabeled protein C (PC) zymogen as well as D-phe-pro-arg-chloromethylketone (PPACK) inhibited APC indicating that a functional active site was not required. Competition studies showed that the binding of APC, PPACK-APC and PC were mutually exclusive suggesting that they bound to the same site(s). Proteolytic removal of the N-terminal gamma-carboxyglutamic acid (gla) domain of PC abolished its ability to compete indicating that the gla-domain was essential for cell binding. Surprisingly, APC binding to these cells appeared to be independent of protein S, a cofactor of APC generally thought to be required for its high affinity binding to cell surfaces. The identity of the cell binding site(s), for the most part, appeared to be distinct from other known APC ligands which are associated with cell membranes or extracellular matrix including phospholipid, thrombomodulin, factor V, plasminogen activator inhibitor type 1 (PAI-1) and heparin. Pretreatment of HUVEC with antifactor VIII antibody caused partial inhibition of 125I-APC binding indicating that factor VIII or a homolog accounted for approximately 30% of APC binding. Studies of the properties of surface bound 125I-APC or 125I-PC and their fate at 4 degrees C compared to 37 degrees C were consistent with association of approximately 25% of the initially bound radioligand with an endocytic receptor. However, most of the radioligand appeared not to be bound to an endocytic receptor and dissociated rapidly at 37 degrees C in an intact and functional state. These data indicate the presence of specific, high affinity binding sites for APC and PC on the surface of HUVEC. While a minor proportion of binding sites may be involved in endocytosis, the identity and function of the major proportion is presently unknown. It is speculated that this putative receptor may be a further mechanisms of localizing the PC antithrombitic system to the vascular endothelium.

Amino Acid Chloromethyl Ketones↗
PubMed1989

Osteogenesis in bone defects in rats: the effects of hydroxyapatite and demineralized bone matrix.

Hydroxyapatite (HA) and Demineralized Bone Matrix (DBM) are being investigated as potential osteogenic agents with hopes that these substances can be used to induce bone formation in non-union fractures. This study was done to determine the relative effects of HA and DBM implanted as moldable phospholipid composites in bone defects that result in non-unions. We studied 22 ten-month-old Long-Evans male rats with 5.0 mm unilateral radial defects implanted with HA, DBM, and a combination of both substances. Control defects were left unfilled. Eight weeks after implantation, the histological sections demonstrated a decrease in bone formation with HA relative to controls. The HA crystals were encapsulated by connective tissue stroma made up of collagenous elements, fibroblasts, and blood vessels. There were no indications of bone formation within the fibrous stroma. 45Calcium, alkaline phosphatase, and bone gla protein (BGP) assays demonstrated a 16% increase in bone formation in rats implanted with DBM, an 80% decrease in groups implanted with HA (p = 0.01) and an 80% decrease with DBM plus HA (p = 0.01). Radiologic analysis corresponds well with histological and biochemical results. We conclude that osteogenesis in non-union defects is enhanced by the implantation of DBM, while HA interferes with bone formation in the rat model. In the presence of both substances, HA appears to impede new bone growth, negating any positive effects seen with DBM.

Alkaline Phosphatase↗
PubMed1988

Chemotactic response of embryonic limb bud mesenchymal cells and muscle-derived fibroblasts to transforming growth factor-beta.

Transforming growth factor beta (TGF-beta) was tested for its ability to stimulate a chemotactic response in Stage 24 embryonic chick limb bud mesenchymal cells and muscle-derived fibroblasts. TGF-beta stimulated dose-dependent chemotaxis in both cell populations. Maximal chemotaxis was achieved with a concentration of 5 ng/ml for limb bud cells and as low as 15 pg/ml for muscle-derived fibroblasts. TGF-beta was not chemokinetic at these levels. Several other proteins found in bone, namely fibronectin, type I collagen, and osteonectin, were not chemotactic. However, both Bone Gla-protein and basic-FGF were found to be chemotactic but less effective than TGF-beta. Comparison with extracts of adult bone indicates that while TGF-beta is a potent chemoattractant, it does not account for all the chemotactic activity found in adult bone.

Animals↗
PubMed1986

Stimulation of undermineralized matrix formation by 1,25 dihydroxyvitamin D3 in long bones of rats.

We previously reported that pharmacologic doses of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) given for 2-3 days, inhibited osteoblastic collagen synthesis in young rats. In this study, we tested the effects of 5, 25, and 125 ng of 1,25(OH)2D3 injected subcutaneously into 6-week-old rats for 12 or 18 days. In rats given 125 ng, cortical bone of distal half femurs exhibited decreased calcium (Ca) content but dry weight and hydroxyproline (Hyp) content were no different from control. Trabecular bone Ca was not different from control but dry weight and Hyp were increased. When cortical and trabecular bone were combined, there was a decrease in Ca, an increase in Hyp, and a 50% decrease in Ca:Hyp. Fluorescent labels given after 8 days of treatment were either diffuse or absent in calcified sections from rats given 125 ng, indicating impaired mineralization. The 25 and 125 ng doses produced hypercalcemia with normal serum phosphate. There was a dose-related increase in serum immunoreactive bone gla protein (BGP) and serum 1,25(OH)2D3 and a decrease in serum 25(OH)D3. At the 5 ng dose, no adverse effects were seen on body growth. With 25 ng and 125 ng, growth was inhibited. Increased serum urea nitrogen and histologic evidence of nephrocalcinosis occurred at the 125 ng dose. When 125 ng was given for 12 days and then withdrawn for 6 days, systemic toxicity decreased and bone Hyp and Ca increased so that Ca:Hyp remained low and comparable to that of rats treated with 1,25(OH)2D3 continuously.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Identification of proteins secreted by human osteoblastic cells in culture.

To better understand the biochemistry of matrix-forming cells, we developed a simple and reproducible procedure for the isolation and identification by N-terminal sequencing of proteins secreted by cells into culture medium and applied this procedure to the analysis of the major Coomassie blue-staining proteins under 100 kD that are secreted from three different human osteoblastic cell cultures. The major proteins secreted by normal human osteoblasts from adult trabecular bone were identified by N-terminal sequencing to be gelatinase, osteonectin, the C-terminal propeptides of the alpha 1 and alpha 2 chains of type I collagen, tissue inhibitor of metalloproteinase 1 (TIMP-1), and beta 2-microglobulin. The amounts of each of these proteins secreted into medium over a 24 h interval did not change over the 7 consecutive days of culture under serum-free conditions, which indicates that this pattern of protein secretion is not significantly affected by the serum-free conditions needed for protein identification by this method. In addition, radioimmunoassay for bone gla protein (BGP), a marker for osteoblast phenotype, revealed that BGP secretion remained high over 7 days of culture under serum-free conditions and was comparable to the rate of BGP secretion in control cultures with 10% serum. The major proteins secreted by MG-63 cells were identified by N-terminal sequencing to be gelatinase, a novel 40 kD human bone protein we termed YKL-40, TIMP-1, the recently discovered TIMP-2, and beta 2-microglobulin. Further studies revealed that YKL-40 is the only protein detectable by Coomassie staining of SDS gels of MG-63 media proteins that is induced by extended time at confluence or by treatment with 1,25-(OH)2D3. The apparent absence of detectable Coomassie-stained bands corresponding to the C-terminal propeptides of collagen in the medium of MG-63 cells suggests that these transformed cells may not be a good model for bone matrix formation. The major proteins secreted by normal fetal osteoblastic cells were identified by N-terminal sequencing to be osteonectin and the C-terminal propeptides of the alpha 1 and alpha 2 chains of type I collagen. Gelatinase and TIMP could not be detected among the conditioned medium proteins by these methods. These observations indicate that fetal osteoblasts primarily express proteins that are matrix constituents and adult human osteoblasts secrete, in addition to these, proteins that could function in matrix turnover.

Amino Acid Sequence↗