Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Proteoglycans (PGs) were extracted from a range of cartilaginous ovine connective tissues using 4 M GuHCl and separated by composite agarose polyacrylamide gel electrophoresis. Individual PG populations resolved by this electrophoretic system were identified in toluidine blue and Stains-All stained gel segments and also by conventional immunoblotting using a range of monoclonal antibodies to defined PG epitopes. These PG species were compared with aggregatable PG populations identified by affinity blotting using a biotinylated hyaluronan, and an avidin alkaline phosphatase/nitro blue tetrazolium 5-bromo-4-chloro indolyl phosphate detection system. Two major chondroitin sulfate- and keratan sulfate-substituted aggrecan populations were readily identified by affinity blotting in all of the connective tissue extracts. An additional slower migrating aggrecan species was also detected by affinity and immunoblotting in fetal disc extracts. This may represent an aggrecan species containing an intact carboxyl terminal G3 domain. Link protein was also detectable by affinity blotting; this was confirmed by immunoblotting using an anti-link protein monoclonal antibody (8-A-4). Fragments of aggrecan which contained a functional G1 domain were also detectable by affinity blotting. The biotinylated hyaluronan affinity blotting technique could detect as little as 100 ng (as hexuronic acid) of aggregatable PG. Affinity blotting therefore represents a useful new detection methodology which complements conventional immunoblotting protocols and yields information regarding the functional status of the G1 domain of individual PG populations.
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PMID:Detection of aggregatable proteoglycan populations by affinity blotting using biotinylated hyaluronan. 947 72

Cartilage-derived morphogenetic proteins-1 and -2 (CDMP-1 and CDMP-2) are members of the bone morphogenetic protein (BMP) family, which play important roles in embryonic skeletal development. We studied the biological activities of recombinant CDMP-1 and CDMP-2 in chondrogenic and osteogenic differentiation and investigated their binding properties to type I and type II serine/threonine kinase receptors. In vivo, CDMP-1 and CDMP-2 were capable of inducing dose-dependently de novo cartilage and bone formation in an ectopic implantation assay. In vitro studies using primary chondrocyte cultures showed that both CDMP-1 and CDMP-2 stimulated equally de novo synthesis of proteoglycan aggrecan in a concentration-dependent manner. This activity was equipotent when compared with osteogenic protein-1 (OP-1). In contrast, CDMPs were less stimulatory than OP-1 in osteogenic differentiation as evaluated by alkaline phosphatase activity and expression levels of bone markers in ATDC5, ROB-C26, and MC3T3-E1 cells. CDMP-2 was the least osteogenic in these assays. Receptor binding studies of CDMP-1 and CDMP-2 revealed that both have affinity for the BMP receptor type IB (BMPR-IB) and BMPR-II, and weakly for BMPR-IA. Moreover, using a promoter/reporter construct, transcriptional activation signal was transduced by BMPR-IB in the presence of BMPR-II upon CDMP-1 and CDMP-2 binding. Our data show that distinct members of the BMP family differentially regulate the progression in the osteogenic lineage, and this may be due to their selective affinity for specific receptor complexes.
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PMID:Cartilage-derived morphogenetic proteins and osteogenic protein-1 differentially regulate osteogenesis. 952 38

The availability of Ca2+ in the extracellular fluid plays an important role in regulating cartilage and bone formation. We hypothesized that chondrocytes detect changes in the extracellular [Ca2+] ([Ca2+]o) and modify their function. The effects of changing [Ca2+]o on the expression of matrix proteins were quantified by staining of cartilage nodules with alcian green and assessing RNA levels of cartilage-specific genes in chondrogenic RCJ3.1C5.18 (C5.18) cells. Alcian green staining in these cells decreased with increasing [Ca2+]o in a dose-dependent and reversible manner (ID50, approximately 2 mM Ca2+). RNA levels for aggrecan and type II collagen decreased with increasing [Ca2+]o (ID50, approximately 2.0 and 4.1 mM Ca2+, respectively). RNA levels for type X collagen and alkaline phosphatase were also reduced by high [Ca2+]o with ID50 values of approximately 2.9 and 1.6 mM Ca2+, respectively. These responses were rapid, in that increasing [Ca2+]o from 1.0 to more than 6 mM suppressed aggrecan RNA levels by about 50%, and lowering [Ca2+]o from 2.9 to 1.0 mM increased aggrecan RNA levels by about 300% within 4 h. As Ca2+ receptors (CaRs) mediate extracellular Ca2+ sensing in parathyroid and kidney, we assessed the expression of CaRs in these cells. C5.18 cells stained positively for CaR protein with an anti-CaR antiserum and for CaR RNA by in situ hybridization. An approximately 150-kDa protein was detected by immunoblotting with anti-CaR antiserum. CaR antisense oligonucleotides suppressed the expression of CaR protein and enhanced RNA levels of aggrecan in C5.18 cells. These data support the idea that CaRs are expressed in this cell system and may be involved in regulating chondrogenic gene expression.
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PMID:Calcium sensing in cultured chondrogenic RCJ3.1C5.18 cells. 1009 31

We have developed a method to form reconstituted mineralized articular cartilagenous tissue in vitro from isolated deep zone chondrocytes. The aim of this study was to characterize further these cultures prior to and during mineralization. Histologic examination of the cells up to 8 days in culture showed that the chondrocytes had formed cartilagenous tissue. Similar to the in vivo cartilage, the chondrocytes expressed aggrecan, types II, I, and X collagens, osteopontin, and alkaline phosphatase (ALP). No osteocalcin mRNA expression was detected in either the in vivo cartilage or in vitro-generated tissue. Addition of beta-glycerophosphate (beta-GP) to the medium on day 5 induced mineralization and changes in gene expression. Expression of type X collagen, type II collagen, aggrecan core protein, and ALP were inhibited significantly between 2 h and 24 h after the addition of beta-GP. At 72 h, expression of these genes were still significantly depressed. These changes correlated with a decrease in collagen and proteoglycan synthesis, and ALP activity. Osteopontin expression increased within 8 h but returned to constitutive levels by 72 h. No change in type I collagen expression was detected. The changes in gene expression were not due to a direct effect of beta-GP itself, because similar gene changes occurred in the presence of phosphoethanolamine, another agent which induces mineralization. No changes in gene expression were seen in nonmineralizing cultures. In summary, articular chondrocytes grown on filter culture show expression of similar genes to the chondrocytes in the deep zone of articular cartilage and that changes in expression of specific genes were observed during tissue mineralization, suggesting that it is a suitable model to use to study the mechanism(s) regulating the localized mineralization of articular cartilage.
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PMID:Deep zone articular chondrocytes in vitro express genes that show specific changes with mineralization. 1057 92

Recently, we cloned a messenger RNA (mRNA) predominantly expressed in chondrocytes from a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8, by differential display PCR and found that its gene, named hcs24, was identical with that of connective tissue growth factor (CTGF). Here we investigated CTGF/Hcs24 function in the chondrocytic cell line HCS-2/8 and rabbit growth cartilage (RGC) cells. HCS-2/8 cells transfected with recombinant adenoviruses that generate CTGF/Hcs24 sense RNA (mRNA) proliferated more rapidly than HCS-2/8 cells transfected with control adenoviruses. HCS-2/8 cells transfected with recombinant adenoviruses that generate CTGF/Hcs24 sense RNA expressed more mRNA of aggrecan and type X collagen than the control cells. To elucidate the direct action of CTGF/Hcs24 on the cells, we transfected HeLa cells with CTGF/Hcs24 expression vectors, obtained stable transfectants, and purified recombinant CTGF/Hcs24 protein from conditioned medium of the transfectants. The recombinant CTGF/Hcs24 effectively promoted the proliferation of HCS-2/8 cells and RGC cells in a dose-dependent manner and also dose dependently increased proteoglycan synthesis in these cells. In addition, these stimulatory effects of CTGF/Hcs24 were neutralized by the addition of anti-CTGF antibodies. Furthermore, the recombinant CTGF/Hcs24 effectively increased alkaline phosphatase activity in RGC cells in culture. Moreover, RT-PCR analysis revealed that the recombinant CTGF/Hcs24 stimulated gene expression of aggrecan and collagen types II and X in RGC cells in culture. These results indicate that CTGF/Hcs24 directly promotes the proliferation and differentiation of chondrocytes.
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PMID:Effects of CTGF/Hcs24, a product of a hypertrophic chondrocyte-specific gene, on the proliferation and differentiation of chondrocytes in culture. 1061 47

Multipotential mesenchymal stem cells capable of chondro-osseous induction contribute to the endochondral callus of healing fractured bone. Microvascular pericytes serving the role of multipotential mesenchymal stem cells are considered osteoprogenitors because they express type I collagen, alkaline phosphatase enzyme activity, osteocalcin immunoreactivity, and bone sialoprotein mRNA. Previous electron microscopic studies indicate that this cell type has a contribution to the fracture callus. Limited data suggest that pericytes may also assume a chondrogenic phenotype. We undertook in vitro studies to understand how the chondro-osseous phenotype of the pericyte might be regulated. Using Northern analysis and semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR), we found that cultured pericytes produce aggrecan and type II collagen mRNA indicating their chondrogenic potential. Aggrecan message is elevated by BMP-2 as analyzed by both Northern hybridization and RT-PCR. This finding suggests a regulatory role for this morphogen on this phenotype in pericytes. RT-PCR amplified versican product was also associated with pericyte cultures but was not affected by BMP-2. Our data strongly support a chondrogenic role for the pericyte and that the phenotype is regulated at least in part by BMP.
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PMID:Microvascular pericytes express aggrecan message which is regulated by BMP-2. 1069 96

Calvarial and facial bones form by intramembranous ossification, in which bone cells arise directly from mesenchyme without an intermediate cartilage anlage. However, a number of studies have reported the emergence of chondrocytes from in vitro calvarial cell or organ cultures and the expression of type II collagen, a cartilage-characteristic marker, in developing calvarial bones. Based on these findings we hypothesized that a covert chondrogenic phase may be an integral part of the normal intramembranous pathway. To test this hypothesis, we analyzed the temporal and spatial expression patterns of cartilage characteristic genes in normal membranous bones from chick embryos at various developmental stages (days 12, 15 and 19). Northern and RNAse protection analyses revealed that embryonic frontal bones expressed not only the type I collagen gene but also a subset of cartilage characteristic genes, types IIA and XI collagen and aggrecan, thus resembling a phenotype of prechondrogenic-condensing mesenchyme. The expression of cartilage-characteristic genes decreased with the progression of bone maturation. Immunohistochemical analyses of developing embryonic chick heads indicated that type II collagen and aggrecan were produced by alkaline phosphatase activity positive cells engaged in early stages of osteogenic differentiation, such as cells in preosteogenic-condensing mesenchyme, the cambium layer of periosteum, the advancing osteogenic front, and osteoid bone. Type IIB and X collagen messenger RNAs (mRNA), markers for mature chondrocytes, were also detected at low levels in calvarial bone but not until late embryonic stages (day 19), indicating that some calvarial cells may undergo overt chondrogenesis. On the basis of our findings, we propose that the normal intramembranous pathway in chicks includes a previously unrecognized transient chondrogenic phase similar to prechondrogenic mesenchyme, and that the cells in this phase retain chondrogenic potential that can be expressed in specific in vitro and in vivo microenvironments.
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PMID:Transient chondrogenic phase in the intramembranous pathway during normal skeletal development. 1075 May 67

Cartilage-derived morphogenetic proteins 1 and 2 (CDMP-1 and CDMP-2) are members of the bone morphogenetic protein (BMP) family which play an important role in embryonic skeletal development. Throughout adult life, bone marrow-derived precursor cells maintain their ability to differentiate into osteoblasts in response to local growth factors. This study examines the osteogenic potential of CDMP-1, CDMP-2, BMP-6 and osteogenic protein 1 (OP-1) in bone marrow stromal cells (BMSC) and investigates the endogenous expression of CDMPs/BMPs and their respective activin receptor-like kinase (ALK) receptors. A 4-day exposure of BMSC to CDMP-1, CDMP-2, BMP-6, and OP-1 under serum-free conditions stimulated the progression of the osteogenic lineage in a dose-dependent manner as evaluated by alkaline phosphatase activity and osteocalcin synthesis. In contrast to the BMPs, CDMP-1 and especially CDMP-2 were significantly less osteogenic, as confirmed by Northern blot analysis. Moreover, BMSC were shown to express endogenously CDMP-2, BMP-2 to -6 and ALK-1, -2, -3, -5 and -6. Phenotypic characterization of BMSC by RT-PCR showed transcripts of the fat marker adipsin and the prechondrocytic marker procollagen type IIA; however, we were unable to detect the mature cartilage markers, procollagen type IIB and aggrecan, even after growth factor treatment. Our data indicate that CDMP-1, CDMP-2, BMP-6 and OP-1 enhance the osteogenic phenotype in BMSC, with CDMPs being clearly less osteogenic than BMPs. The endogenous expression of a variety of CDMPs/BMPs and their respective ALK receptors, suggests a possible involvement of these growth factors in the osteogenic differentiation of bone marrow progenitor cells.
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PMID:Stimulatory effects of cartilage-derived morphogenetic proteins 1 and 2 on osteogenic differentiation of bone marrow stromal cells. 1105 13

The regulation of human bone marrow stromal precursor cell differentiation toward the chondrocyte, osteoblast or adipocyte lineages is not known. In this study, we assessed the lineage-specific differentiation and conversion of immortalized clonal F/STRO-1(+) A human fetal bone marrow stromal cells under the control of dexamethasone (Dex), indomethacin/insulin (Indo/Ins) and linoleic acid (LA). Under basal conditions, F/STRO-1(+) A cells expressed markers mRNAs or proteins of the osteoblast lineage [CBFA1, osteocalcin (OC), alkaline phosphatase (ALP), type 1 collagen], of the chondrocyte lineage (aggrecan, types 2, 9 and 10 collagen), and of the adipocyte lineage (PPARgamma2, C/EBPalpha, aP2, G3PDH, lipoprotein lipase, leptin). Treatment with Dex increased CBFA1, OC and ALP mRNA and protein levels. Exposure to LA enhanced expression of adipocytic genes and cytoplasmic triglycerides accumulation, and suppressed the Dex-induced stimulation of osteoblast marker genes. Indo/Ins stimulated the synthesis of aggrecan and type 2 collagen and increased types 9 and 10 collagen mRNA levels, and suppressed both basal and Dex-promoted expression of osteoblast markers. Conversely, stimulation of osteoblastogenesis by Dex suppressed both basal and Indo/Ins-stimulated chondrocyte genes. Thus, the clonal human fetal bone marrow stromal F/STRO-1(+) A cell line is a lineage-unrestricted common progenitor that expresses tripotential adipocyte, osteoblast or chondrocyte characteristics. Our data also show that differentiation towards one pathway in response to Dex, Indo/Ins and LA restricts expression of other lineage-specific genes, and provide evidence for a controlled reciprocal regulation of osteoblast/chondroblast and osteoblast/adipocyte differentiation of clonal F/STRO-1(+) human bone marrow stromal cells.
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PMID:Reciprocal control of osteoblast/chondroblast and osteoblast/adipocyte differentiation of multipotential clonal human marrow stromal F/STRO-1(+) cells. 1118 Mar 95

Localization studies and genetic evidence have implicated cartilage-derived morphogenetic proteins-1, -2 (CDMP-1 and CDMP-2), and osteogenic protein-1 (OP-1) in the osteochondrogenic differentiation of mesenchymal progenitor cells during embryonic development and in postnatal life. Based on their expression pattern and the evidence that periosteum contains mesenchymal cells in the cambium layer that can undergo bone and cartilage formation, we hypothesized that CDMPs and OP-1 may be involved in long bone development and fracture healing. To test this hypothesis, periosteum-derived cells from young calves were cultured as monolayers under serum-free conditions with and without the addition of recombinant CDMP-1, CDMP-2 and OP-1. Phenotypic analysis indicate that periosteum-derived cell populations prepared, expanded, and cultured under the conditions described below, constitutively express messenger RNAs for the bone markers osteocalcin, osteopontin and collagen type I, and the chondrogenic markers collagen type II and aggrecan as determined by RT-PCR. Moreover, histologic examinations showed positive staining for alcian blue and alkaline phosphatase (AP). Treatment of periosteum-derived cells with CDMPs and OP-1 resulted in a dose-dependent increase of cell proliferation; CDMP-2 was less active in this regard. Furthermore, all growth factors enhanced osteogenic differentiation as assessed by a time- and dose-dependent stimulation of AP activity and OP-1 increased messenger RNA expression for osteocalcin and collagen type I. We further examined the effects of CDMPs and OP-1 on chondrogenic differentiation of periosteum-derived cells. Both CDMPs and OP-1 stimulated (35)S-sulfate incorporation into newly synthesized macromolecules with OP-1 having a more pronounced stimulatory effect when compared with CDMP-1 and CDMP-2. Our results indicate that distinct members of the BMP-family increase the mitotic and metabolic activity of periosteum-derived cells. The enhancement of both the chondrogenic and osteogenic differentiation suggests that these growth factors might contribute to the local regulation of bone formation and fracture repair.
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PMID:Effects of cartilage-derived morphogenetic proteins and osteogenic protein-1 on osteochondrogenic differentiation of periosteum-derived cells. 1131 76


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