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PubMed1994

Characterization of the osteogenic stromal cell line MN7: identification of secreted MN7 proteins using two-dimensional polyacrylamide gel electrophoresis, western blotting, and microsequencing.

Proteins secreted by the osteogenic stromal cell line MN7 were analyzed using two-dimensional polyacrylamide gel electrophoresis (PAGE), western blotting, immunodetection, and microsequencing. Trichloroacetic acid-precipitated proteins from the conditioned medium of MN7 cell cultures, harvested at different times of growth, were dissolved in denaturing and reducing sample buffer and separated in the first dimension according to isoelectric point and in the second dimension according to molecular weight. Protein patterns were visualized using silver staining. Among the 350 separated protein spots, we identified type I collagen, bone sialoprotein, osteonectin, and cathepsin B by western blotting and immunodetection using polyclonal antibodies. Osteocalcin could not be detected in the conditioned medium of MN7 cells. Furthermore, 15 MN7-specific protein spots were localized after comparison with two-dimensional PAGE patterns from the conditioned medium of the nonosteogenic stromal cell lines MM1 and MV1. Microsequencing of the internal peptides of five selected spots revealed three known proteins, namely the carboxyl-terminal propeptide of the alpha 2 chain of collagen type I, cathepsin L, and the tissue inhibitor of metalloproteinases-2, an 18 kilodalton peptide fragment from osteopontin that has not previously been described, and a novel glycosylated 85 kD protein with an average isoelectric point of 5.7. All identified proteins did not vary in presence between the different time points analyzed by two-dimensional PAGE. The use of two-dimensional PAGE to investigate the secreted proteins of MN7 cells will enable us to establish a complete protein data base of extracellular osteoblast-specific proteins. Furthermore, two-dimensional PAGE in combination with other techniques is a fast and accurate method for the identification of novel proteins that could function as markers in osteoblast differentiation and/or bone formation.

Amino Acid Sequence↗

PubMed1995

Development and characterization of a conditionally immortalized human fetal osteoblastic cell line.

We report the establishment of a human fetal osteoblast cell line derived from biopsies obtained from a spontaneous miscarriage. Primary cultures isolated from fetal tissue were transfected with a gene coding for a temperature-sensitive mutant (tsA58) of SV40 large T antigen along with a gene coding for neomycin (G418) resistance. Individual neomycin resistant colonies were screened for alkaline phosphatase (AP)-specific staining. The clone with the highest AP level, hFOB 1.19, was examined further for other osteoblast phenotypic markers. Incubation of hFOB cells at the permissive temperature (33.5 degrees C) resulted in rapid cell division, whereas little or no cell division occurred at the restrictive temperature (39.5 degrees C). Both AP activity and osteocalcin (OC) secretion increased in a dose-dependent manner following dihydroxyvitamin D3 (1,25-D3) treatment when cultured at either temperature. However, AP and 1,25-D3-induced OC levels were elevated in confluent hFOB cells cultured at 39.5 degrees C compared with 33.5 degrees C. Treatment of hFOB cells with 1-34 parathyroid hormone (PTH) resulted in an increase in cAMP levels. Upon reaching confluence, hFOB cultures went through programmed differentiation and formed mineralized nodules as observed by von Kossa staining. Further, immunostaining of postconfluent, differentiated hFOB cells showed that high levels of osteopontin, osteonectin, bone sialoprotein, and type I collagen were expressed. Therefore, the clonal cell line hFOB 1.19 provides a homogeneous, rapidly proliferating model system to study certain stages of human osteoblast differentiation.

Alkaline Phosphatase↗

PubMed1996

Long-term extracellular matrix metabolism by cultured human osteogenesis imperfecta osteoblasts.

Osteopenia due to deficient extracellular matrix synthesis is a hallmark of osteogenesis imperfecta (OI), Previous studies carried out within 72 h of osteoblast subculture, at an early stage of matrix synthesis, indicated that for osteoblasts derived from human OI patients the total amounts of collagen, osteonectin, and three proteoglycans were significantly reduced, while total amounts of thrombospondin, fibronectin, and matrix hyaluronan were elevated compared with age-matched controls. The current study was undertaken to follow OI osteoblast matrix metabolism as that matrix is synthesized, deposited, and matured. Steady-state metabolic radiolabeling was used to follow the metabolism of collagen, hyaluronan, and total proteoglycan by OI and normal osteoblasts for up to 5 weeks. Trabecular osteoblasts from non-OI controls showed an increase in total and matrix-associated collagen synthesis during the first and second week, reaching steady-state levels by week 4. In contrast, cultured OI osteoblasts did not increase either the total (medium + matrix-associated) or matrix-associated collagen during the entire 5-week period. Proteoglycan synthesis exhibited a pattern similar to that for collagen. OI-derived proteoglycans differed from controls in that levels in OI cultures did not reflect the normal time-dependent increase in total proteoglycan and proteoglycan matrix deposition. OI osteoblast hyaluronan synthesis was increased when compared with age-matched controls during 4 weeks of culture. In contrast, the ratios of calcium to phosphorus solublized from control and the OI extracellular matrix were not statistically different. Thus, with respect to the synthesis of collagen, proteoglycans, and hyaluronan, OI osteoblasts fail to parallel controls in depositing and elaborating extracellular matrix during 35 days in culture.

Adolescent↗

PubMed2002

Real-time quantitative RT-PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro.

We developed and used real-time RT-PCR assays to investigate how the expression of typical osteoblast-related genes by human bone marrow stromal cells (BMSC) is regulated by (i) the culture time in medium inducing osteogenic differentiation and (ii) the previous expansion in medium enhancing cell osteogenic commitment. BMSC from six healthy donors were expanded in medium without (CTR) or with fibroblast growth factor-2 and dexamethasone (FGF/Dex; these factors are known to increase BMSC osteogenic commitment) and further cultivated for up to 20 days with ascorbic acid, beta-glycerophosphate and dexamethasone (these factors are typically used to induce BMSC osteogenic differentiation). Despite a high variability in the gene expression levels among different individuals, we identified the following statistically significant patterns. The mRNA levels of bone morphogenetic protein-2 (BMP-2), bone sialo protein-II (BSP), osteopontin (OP) and to a lower extent cbfa-1 increased with culture time in osteogenic medium (OM), both in CTR- and FGF/Dex-expanded BMSC, unlike levels of alkaline phosphatase, collagen type I, osteocalcin, and osteonectin. After 20 days culture in OM, BMP-2, BSP, and OP were more expressed in FGF/Dex than in CTR-expanded BMSC (mRNA levels were, respectively, 9.5-, 14.9-, and 5.8-fold higher), unlike all the other investigated genes. Analysis of single-colony-derived strains of BMSC further revealed that after 20 days culture in OM, only a subset of FGF/Dex-expanded clones expressed higher mRNA levels of BMP-2, BSP, and OP than CTR-expanded clones. In conclusion, we provide evidence that mRNA levels of BMP-2, BSP, and OP, quantified using real-time RT-PCR, can be used as markers to monitor the extent of BMSC osteogenic differentiation in vitro; using those markers, we further demonstrated that only a few subpopulations of BMSC display enhanced osteogenic differentiation following FGF/Dex expansion.

Base Sequence↗

PubMed2003

Differential gene expression analysis using paraffin-embedded tissues after laser microdissection.

Recent advances in laser microdissection allow for precise removal of pure cell populations from morphologically preserved tissue sections. However, RNA from paraffin-embedded samples is usually degraded during microdissection. The purpose of this study is to determine the optimal fixative for RNA extractions from laser microdissected paraffin-embedded samples. The integrity of RNA was evaluated with the intactness of 18S and 28S ribosomal RNA by electrophoresis and by the length of individual gene transcripts using RT-PCR. The various fixatives were methacarn (a combination of methanol, chloroform, and acetic acid) and several concentrations of ethanol and isopropanol. Methacarn was the optimal fixative for RNA preservation in paraffin-embedded tissues, which included liver, lung, kidney, muscle, and limb. Based on RT-PCR analysis, methacarn fixed samples exhibited the expected RNA sizes for individual genes such as glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) and bone-related genes (e.g., alkaline phosphatase and osteonectin). The laser microdissection technique with methacarn fixation was then applied to analyze the differential gene expression between hypertrophic and proliferative chondrocytes in the growth plate of long bone. The expression of type X collagen, a specific gene for hypertrophic chondrocytes, was only observed in hypertrophic chondrocytes, while type II collagen was observed more broadly in the growth plate as anticipated. Thus, combining laser microdissection with methacarn fixation facilitates the examination of differentially expressed genes from various tissues.

Acetic Acid↗

PubMed2001

Differential gene expression of cultured human osteoblasts.

Human cells with osteogenic capacity were studied for differential gene expression. In the first part of the study we compared gene expression of marrow stroma cells (MSC) in comparison to matured osteoblasts cultured from trabecular bone (TBC) that were analyzed by RT-PCR for series of messages. High expression was detected for PTH-r, TGFb1 and biglycan in TBC compared to MSC's. The messages for c-MYC, IL-6, IL-11, M-CSF, osteonectin, and osteocalcin were expressed at the same level in the two populations of cells. In the second part of the study, we analyzed gene expression within the MSC derived from 25 donors (2.5-49 years old) with respect to donors' age and gender. Increased message levels for M-CSF and biglycan were measured in correlation with age of the donors. Gender differences did not affect the expression of cytokines studied (IL-6, IL-11, MCSF, TGFb1). We investigated the effect of Dexamethasone treatment on MSC and monitored an increased expression of IL-11, M-CSF, biglycan, and osteocalcin messages. This study employs primary cell systems (MSC and TBC) to illustrate differential gene expression by osteoblastic cells. The expression was correlated with maturation status of the cells with respect to differences between donors.

Adolescent↗

PubMed2004

Analysis of mesenchymal cells derived from an chondrodysplasia punctuate patient and donors.

Conradi-Hunermann syndrome (CDPX2) is X-linked dominant disorder appeared with aberrant punctuate calcification. The skeletal cells derived from the marrow stroma are active in maintaining the skeletal formation. We obtained mesenchymal stem cells from a patient with CDPX2 and studied the formation of colony forming unit-fibroblast (CFU-F) in vitro in comparison cells obtained from normal donors. Cultured cells were studied morphologically and subjected to gene expression analysis. Marrow stromal cells (MSC)-chondrodysplasia punctuate (CDP) cells from CDPX2 were identified by their mosaic morphology formed three phenotypically distinct types of CFU-F colonies. One type consisted of normal fibroblasts with developed cell body and cellular processes; the second type contained pathological small cells without processes; and the third type comprised of mixed cells. We compared gene expression by the MSC-CDP to cells from normal donors. Transcription factors analyzed proliferation potential were similar in both normal and mixed colonies of MSC-CDP and similar to normal MSCs. The message expression for cytokines and extra cellular matrix (ECM) proteins revealed similar expression for biglycan, osteocalcin, and osteonectin, while IL-6, IL-11, and M-CSF mRNA levels were significantly higher in normal cells than in MSC-CDP. Mixed cells had elevated levels for IL-6 and M-CSF mRNA, but expressed IL-11 at the normal range. The studied genes were expressed at lower levels by the pathological (MSC-CDP) cells compared to normal ones. Hence, MSC-CDP was demonstrated to display abnormal morphology and transcription of several investigated genes. This study further illuminates the basis of the mosaic pattern of mesenchymal cells derived from a patient affected with CDPX2, and their gene expression involvement.

Biomarkers↗

PubMed2005

Mechanical modulation of molecular signals which regulate anabolic and catabolic activity in bone tissue.

Identifying the molecular mechanisms that regulate bone's adaptive response to alterations in load bearing may potentiate the discovery of interventions to curb osteoporosis. Adult female mice (BALB/cByJ) were subjected to catabolic (disuse) and anabolic (45 Hz, 0.3g vibration for 10 min/day) signals, and changes in the mRNA levels of thirteen genes were compared to altered indices of bone formation. Age-matched mice served as controls. Following 4 days of disuse, significant (P = 0.05) decreases in mRNA levels were measured for several genes, including collagen type I (-55%), osteonectin (-44%), osterix (-36%), and MMP-2 (-36%) all of which, after 21 days, had normalized to control levels. In contrast, expression of several genes in the vibrated group, which failed to show significant changes at 4 days, demonstrated significant increases after 21 days, including inducible nitric oxide synthase (iNOS) (39%, P = 0.07), MMP-2 (54%), and receptor activator of the nuclear factor kB ligand (RANKL) (32%). Correlations of gene expression patterns across experimental conditions and time points allowed the functional clustering of responsive genes into two distinct groups. Each cluster's specific regulatory role (formation vs. resorption) was reinforced by the 60% suppression of formation rates caused by disuse, and the 55% increase in formation rates stimulated by mechanical signals (P < 0.05). These data confirm the complexity of the bone remodeling process, both in terms of the number of genes involved, their interaction and coordination of resorptive and formative activity, and the temporal sensitivity of the processes. More detailed spatial and temporal correlations between altered mRNA levels and tissue plasticity may further delineate the molecules responsible for the control of bone mass and morphology.

Animals↗

PubMed2005

Molecular profile of catabolic versus anabolic treatment regimens of parathyroid hormone (PTH) in rat bone: an analysis by DNA microarray.

Teriparatide, human PTH (1-34), a new therapy for osteoporosis, elicits markedly different skeletal responses depending on the treatment regimen. In order to understand potential mechanisms for this dichotomy, the present investigation utilized microarrays to delineate the genes and pathways that are regulated by intermittent (subcutaneous injection of 80 microg/kg/day) and continuous (subcutaneous infusion of 40 microg/kg/day by osmotic mini pump) PTH (1-34) for 1 week in 6-month-old female rats. The effect of each PTH regimen was confirmed by histomorphometric analysis of the proximal tibial metaphysis, and mRNA from the distal femoral metaphysis was analyzed using an Affymetrix microarray. Both PTH paradigms co-regulated 22 genes including known bone formation genes (i.e., collagens, osteocalcin, decorin, and osteonectin) and also uniquely modulated additional genes. Intermittent PTH regulated 19 additional genes while continuous treatment regulated 173 additional genes. This investigation details for the first time the broad profiling of the gene and pathway changes that occur in vivo following treatment of intermittent versus continuous PTH (1-34). These results extend previous observations of gene expression changes and reveal the in vivo regulation of BMP3 and multiple neuronal genes by PTH treatment.

Animals↗

PubMed2006

Human endothelial cells inhibit BMSC differentiation into mature osteoblasts in vitro by interfering with osterix expression.

It is well accepted, that there is communication between osteoblasts and endothelial cells. However, the influence of endothelial cells on the differentiation of bone precursors into mature osteoblasts is not yet well understood. We therefore studied the effect of human umbilical vein endothelial cells (HUVEC) on human bone marrow stromal cell (BMSC) differentiation towards an osteoblastic phenotype by culturing them in two different types of HUVEC-BMSC cultures (indirect contact, HUVEC-conditioned medium). Typical bone markers (45Ca incorporation, ALP activity, and gene expression of collagen 1 (COL1), osteonectin (ON), matrix metalloproteinase 13 (MMP-13), bone morphogenetic protein 2 (BMP-2)) and transcription factors (Runx2, osterix (OSX)) were evaluated at different time points. The effect of stimulating HUVEC with vascular endothelial growth factor (VEGF) before co-cultures with BMSC was also evaluated. As expected, BMSC in osteogenic medium (OM) (with dexamethasone) differentiated towards the osteoblastic phenotype, as measured by increased matrix mineralization, high ALP activity, and elevated expression of specific osteoblastic marker genes, when compared to BMSC in non-OM. HUVEC reversibly inhibited osteoblastic differentiation of BMSC in OM, independent of the co-culture type. When HUVEC were removed from the cultures, BMSC differentiated into osteoblasts, albeit at a faster rate than BMSC that were never co-cultured with HUVEC. Stimulation of HUVEC with VEGF before co-culture enhanced the inhibitory effect of HUVEC on BMSC differentiation. This inhibitory effect was connected to a reversible suppression of OSX gene expression and was specific to endothelial cells. We conclude that HUVEC can inhibit dexamethasone-induced BMSC differentiation into osteoblasts in vitro, by interfering with OSX expression, thereby arresting BMSC differentiation at a preosteoblastic stage.

Anti-Inflammatory Agents↗

PubMed1994

Differential effects of retinoic acid and growth factors on osteoblastic markers and CD10/NEP activity in stromal-derived osteoblasts.

The effects of retinoic acid (RA) on the expression of osteoblastic-related cell markers was examined. A marrow stromal osteogenic cell line, MBA-15, was analyzed by Northern blotting for the expression of bone matrix proteins. These cells constitutively express mRNA encoding for procollagen alpha 2 (I), osteonectin, osteopontin, biglycan, and alkaline phosphatase (ALK-P). Gene expression was unchanged in response to RA triggering for 24 hr. Furthermore, cell growth and enzymatic activities of ALK-P and neutral endopeptidase (CD10/NEP) were studied. These parameters were examined in MBA-15 and clonal populations representing different stages of differentiation. The cell's growth rate was unchanged, while ALK-P activity was greatly increased during the culture period under RA treatment in MBA-15 and in the clonal cell lines examined while CD10/NEP activity displayed a different pattern. MBA-15.4, a preosteoblast cell line, exhibited an inhibition in CD10/NEP activity at the beginning of the culture period, reaching basal level with time. This activity was greatly increased over control level in MBA-15.6, a mature stage of osteoblasts. Furthermore, the response of cell lines to various growth factors was tested subsequent to priming the cultures with RA. A synergistic effect was monitored for ALK-P activity in MBA-15.4 and MBA-15.6 cells under rh-bone morphogenic protein (BMP-2) and purified osteogenin (BMP-3), and an antagonist effect was measured when cells were exposed to transforming growth factor beta (TGF beta). Contrarily, BMP-2 and BMP-3 inhibited the CD10/NEP activity that had remained unchanged following priming of the cell with RA. Insulin-like growth factor I (IGF-I) and basic fibroblast growth factors (bFGF) did not affect either ALK-P nor CD10/NEP activities in both cloned cells. Cellular response to bone-seeking hormone, parathyroid hormone (PTH), and prostaglandin E2 (PGE2) was monitored by activation of intracellular cAMP. Treatment with RA caused a dramatic decrease in MBA-15.6 cell responses to PTH and PGE2, but no significant effects could be observed in other clonal lines.

1-Methyl-3-isobutylxanthine↗

PubMed1995

Morphological and biochemical characterization of mineralizing primary cultures of avian growth plate chondrocytes: evidence for cellular processing of Ca2+ and Pi prior to matrix mineralization.

Advances in the culture of mineralizing growth plate chondrocytes provided an opportunity to study endochondral calcification under controlled conditions. Here we report that these cultures synthesize large amounts of proteins characteristically associated with mineralization: type II and X collagens, sulfated proteoglycans, alkaline phosphatase, and the bone-related proteins, osteonectin and osteopontin. Certain chondrocytes appeared to accumulate large amounts of Ca2+ and Pi during the mineralization process: laser confocal imaging revealed high levels of intracellular Ca2+ in their periphery and X-ray microanalytical mapping revealed the presence of many Ca(2+)- and Pi-rich cell surface structures ranging from filamentous processes 0.14 +/- 0.02 microns by 0.5-2.0 microns, to spherical globules 0.70 +/- 0.27 microns in diameter. Removal of organic matter with alkaline sodium hypochlorite revealed numerous deposits of globular (0.77 +/- 0.19 micron) mineral (calcospherites) in the lacunae around these cells. The size and spatial distribution of these mineral deposits closely corresponded to the Ca(2+)-rich cell surface blebs. The globular mineral progressively transformed into clusters of crystallites. Taken with earlier studies, these findings indicate that cellular uptake of Ca2+ and Pi leads to formation of complexes of amorphous calcium phosphate, membrane lipids, and proteins that are released as cell surface blebs analogous to matrix vesicles. These structures initiate development of crystalline mineral. Thus, the current findings support the concept that the peripheral intracellular accumulation of Ca2+ and Pi is directly involved in endochondral calcification.

Animals↗

PubMed1987

Type beta transforming growth factor (TGF beta) regulation of alkaline phosphatase expression and other phenotype-related mRNAs in osteoblastic rat osteosarcoma cells.

TGF beta 1 from porcine platelets increased alkaline phosphatase (AP) activity in the rat osteoblastic cell line ROS 17/2.8 about three-fold. This effect was dose-dependent with an ED50 of about approximately 0.2 ng/ml and was larger during logarithmic growth than at confluence. TGF beta 1 inhibited cell growth by about 30% with similar dose dependence. Thirty min exposure to TGF beta 1 was sufficient to increase AP activity 3 days later by about two-fold but did not affect cell growth, suggesting dissociation between effects on proliferation and differentiation. The rise in AP activity started 6 h after TGF beta 1 addition and was blocked by cycloheximide and actinomycin D. TGF beta 1 also increased AP mRNA by two- to three-fold and this effect was not blocked by cycloheximide. The half-life of AP mRNA, estimated following the addition of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole was about ten h in both control and TGF beta 1-treated cells. The mRNAs for type I procollagen and osteonectin were also increased by TGF beta 1 but fibronectin mRNA was decreased. TGF beta 2 effects on AP and cell growth were similar to those of TGF beta 1, except for lack of activity following transient exposure. At saturating concentrations, TGF beta 2 (2 ng/ml) or dexamethasone (10(-7) M), which has similar effects on these cells, did not further augment the effects of TGF beta 1 (at 2 ng/ml). Above findings suggest that TGF beta promotes osteoblastic differentiation in rat osteosarcoma cells at least in part by acting at the pretranslational level.

Alkaline Phosphatase↗

PubMed1991

Dexamethasone induction of osteoblast mRNAs in rat marrow stromal cell cultures.

We have examined the ability of dexamethasone, retinoic acid, and vitamin D3 to induce osteogenic differentiation in rat marrow stromal cell cultures by measuring the expression of mRNAs associated with the differentiated osteoblast phenotype as well as analyzing collagen secretion and alkaline phosphatase activity. Marrow cells were cultured for 8 days in primary culture and 8 days in secondary culture, with and without 10 nM dexamethasone or 1 microM retinoic acid. Under all conditions, cultures produced high levels of osteonectin mRNA. Cells grown with dexamethasone in both primary and secondary culture contained elevated alkaline phosphatase mRNA and significant amounts of type I collagen and osteopontin mRNA. Addition of 1,25-dihydroxyvitamin D3 to these dexamethasone-treated cultures induced expression of osteocalcin mRNA and increased osteopontin mRNA. The levels of alkaline phosphatase, osteopontin, and osteocalcin mRNAs in Dex/Dex/VitD3 cultures were comparable to those of 1,25-dihydroxyvitamin D3-treated ROS 17/2.8 osteosarcoma cells. Omitting dexamethasone from either primary or secondary culture resulted in significantly less alkaline phosphatase mRNA, little osteopontin mRNA, and no osteocalcin mRNA. Retinoic acid increased alkaline phosphatase activity to a greater extent than did dexamethasone but did not have a parallel effect on the expression of alkaline phosphatase mRNA and induced neither osteopontin or osteocalcin mRNAs. In all conditions, marrow stromal cells synthesized and secreted a mixture of type I and III collagens. However, dexamethasone-treated cells also synthesized an additional collagen type, provisionally identified as type V. The synthesis and secretion of collagens type I and III was decreased by both dexamethasone and retinoic acid. Neither dexamethasone nor retinoic acid induced mRNAs associated with the chondrogenic phenotype. We conclude that dexamethasone, but not retinoic acid, promotes the expression of markers of the osteoblast phenotype in cultures of rat marrow stromal fibroblasts.

Alkaline Phosphatase↗

PubMed2005

Bone and cartilage formation by skeletal muscle derived cells.

In adult individuals when most tissues have progressively lost the ability to regenerate, bone maintains the potential for a continuous self remodeling. The bone marrow has been so far the main recognized source of osteoprogenitor cells that contribute to the turnover of the skeletal scaffold. The possibility though exists that a pool of osteoprogenitor cells resides within other adult tissues and in particular, as reported previously, in other connective tissues such as fat and skeletal muscle. In an attempt to identify an alternative source of osteoprogenitor cells other than bone marrow we looked into the skeletal muscle. A plastic adhering cell population, from now on referred to as skeletal muscle derived cells (SMDCs), was obtained from biopsies of human skeletal muscle. SMDCs were clonogenic and displayed a fibroblast-like morphology. The isolated cell population had a mesenchymal origin as indicated by abundant expression of type I collagen, fibronectin, and vimentin and appeared heterogeneous. SMDCs were positive for alpha smooth actin, and to a lesser extent for desmin and alpha sarcomeric myosin, two specific markers of the myogenic phenotype. Surprisingly though SMDCs expressed early markers of an osteogenic commitment as indicated by positive staining for alkaline phosphatase, osteopontin, and osteonectin. Under the appropriate stimuli, these cells deposited in vitro a mineralized bone matrix and a proteoglycan rich matrix. In addition, SMDCs cultured in the presence of low serum and insulin differentiated towards adipocytes developing abundant lipid droplets in the cytoplasm. Furthermore SMDCs formed three-dimensional bone tissue in vivo when implanted in an immunodeficient mouse, and a mature cartilage rudiment when maintained as a pellet culture. In summary, we report the isolation and characterization of a cell population from the human skeletal muscle not only able to express in vitro specific markers of distinct mesenchymal lineages (adipogenic, chondrogenic, and osteogenic), but most importantly, able to complete the differentiation pathway leading to the formation of bone and cartilage. In this respect SMDCs resemble bone marrow stromal cells (BMSCs).

Adipocytes↗

PubMed2001

Arterial calcification: a review of mechanisms, animal models, and the prospects for therapy.

The causes of arterial calcification are beginning to be elucidated. Macrophages, mast cells, and smooth muscle cells are the primary cells implicated in this process. The roles of a variety of bone-related proteins including bone morphogenetic protein-2 (BMP-2), matrix Gla protein (MGP), osteoprotegerin (OPG), osteopontin, and osteonectin in regulating arterial calcification are reviewed. Animals lacking MGP, OPG, smad6, carbonic anhydrase isoenzyme II, fibrillin-1, and klotho gene product develop varying extents of arterial calcification. Hyperlipidemia, vitamin D, nicotine, and warfarin, alone or in various combinations, produce arterial calcification in animal models. MGP has recently been discovered to be an inhibitor of bone morphogenetic protein-2, the principal osteogenic growth factor. Many of the forces that induce arterial calcification may act by disrupting the essential post-translational modification of MGP, allowing BMP-2 to induce mineralization. MGP requires gamma-carboxylation before it is functional, and this process uses vitamin K as an essential cofactor. Vitamin K deficiency, drugs that act as vitamin K antagonists, and oxidant stress are forces that could prevent the formation of GLA residues on MGP. The potential role of arterial apoptosis in calcification is discussed. Potential therapeutic options to limit the rate of arterial calcification are summarized.

Animals↗

PubMed2006

Expression of genes associated with allantois emergence in ovine and bovine conceptuses.

In the development of ruminant embryos, the emergence and growth of the allantois is critical for the establishment of the chorioallantoic placenta. The allantoic membrane contributes to all the vasculature that perfuses the placental tissues and the fetal membranes. Using suppressive subtractive hybridization to compare mRNA from Day 13 ovine preimplantation conceptuses (prior to allantoic emergence) with Day 17 allantoic membrane, we identified nine genes whose expression was associated with the emergence of the allantoic sac. Collagen alpha 1 type XII, collagen alpha 2 type I, collagen alpha 2 type V, epsilon 4 beta-globin, osteonectin, and uroplakin were expressed at significantly greater levels in ovine Day 17 allantois compared to Day 13 conceptuses. These genes are associated with the extracellular matrix and most likely are involved in establishing and strengthening the structural integrity of the allantoic sac and in the development of the blood vessels. RalB expression increased with development although at significantly greater levels in the allantois only at Day 19. Hoxa-10 and RhoA showed no differential expression during this period. All these genes showed a similar temporal pattern of expression in bovine conceptuses at equivalent stages of development with significantly greater expression of all these genes, except for Hoxa-10, found in Day 24 allantois compared to Day 14 conceptuses. This suggests that the role they play in allantoic emergence, growth and function is conserved in both ruminant species and that their expression is regulated in a similar manner. The interactions and regulation of this process remains to be fully explained.

Allantois↗

PubMed2001

Bone metastatic LNCaP-derivative C4-2B prostate cancer cell line mineralizes in vitro.

BACKGROUND: Prostate cancer frequently metastasizes to bone. However, unlike many other tumors that produce osteolytic lesions, prostate cancer produces osteoblastic lesions through unknown mechanisms. In the current study, we explored the ability and mechanism of an osteotropic prostate cancer cell line (C4-2B) to induce mineralization. METHODS: C4-2B cells were grown in promineralization media. Mineral deposition was characterized using von Kossa staining, calcium retention, alizarin red staining, Raman spectroscopy, and electron microscopy. Expression of osteoblast-related proteins was determined by RT-PCR. The nuclear level of the bone-specific transcription factor Cbfa1 was determined using western analysis and the effect of inhibiting Cbfa1 function, using a "decoy" Cbfa1 response element oligo, on mineralization was determined. RESULTS: The studies demonstrated that C4-2B cells, but not its nonosteotropic parent cell line LNCaP, has an osteoblastlike phenotype including production of alkaline phosphatase, osteocalcin, osteonectin, bone sialoprotein, osteoprotegerin (OPG), and OPG ligand. Most importantly, the C4-2B cells produced hydroxyapatite mineral in vitro. Furthermore, C4-2B cells expressed high nuclear levels of the bone-specific transcription factor Cbfa1, compared to LNCaP cells, which accounts for their ability to produce bone-specific proteins. Inhibition of Cbfa1, using decoy DNA Cbfa1 response elements, abrogated the ability of C4-2B to produce mineral. Finally, we determined that C4-2B cells express bone morphogenic protein-7, a known inducer of Cbfa1 expression. CONCLUSIONS: These data demonstrate a novel mechanism through which prostate cancer cells may directly contribute to the osteoblastic component that characterize their skeletal metastatic lesions. Prostate 47:212-221, 2001.

Anthraquinones↗