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At least 19 recordsLinked to original sources

PubMed1991

Intertissular variations in osteonectin: a monoclonal antibody directed to bone osteonectin shows reduced affinity for platelet osteonectin.

Osteonectin, a major noncollagenous protein of bone, is also synthesized and secreted by various non-mineralized tissues and by platelets. To establish whether there are structural specificities of osteonectin according to its tissular origin, we raised 12 monoclonal antibodies against bovine bone osteonectin and screened them for their ability to recognize bone and platelet osteonectin. When hybridoma culture media were radioimmunoassayed all MAbs showed the same titer for [125I]human platelet osteonectin and for [125I]bovine bone osteonectin, except MAb 2, which poorly bound platelet osteonectin. Immunoprecipitation and immunoblotting experiments were performed on human bone protein extracts and on material secreted by human platelets upon thrombin stimulation; in these experiments MAb 2 recognized human bone osteonectin and only faintly human platelet osteonectin. A "sandwich" immunoradiometric assay was devised in which osteonectin bound to a solid phase by a first MAb was recognized by a 125I-labeled second MAb. In this assay MAb 2, used as a tracer, showed a 100-fold lower affinity for purified human platelet osteonectin than for purified human bone osteonectin. These results suggest the existence of structural variations in osteonectin obtained from bone and platelets. Whether these variations result from differences in sequence, post-translational processing, or postsecretional fate remains to be established.

Amino Acid Sequence↗

PubMed1990

Human platelet osteonectin: release, surface expression, and partial characterization.

Our laboratory has previously shown that osteonectin, an abundant noncollagenous bone protein, is contained in and secreted from human platelets. In this study, the distribution of osteonectin both in the supernatant and on the platelet surface after activation was measured by fluid-phase and solid-phase radioimmunoassay, respectively. Total cellular osteonectin was determined by RIA of guanidinium chloride extracted platelets and ranged from 0.65 to 2.2 micrograms/10(8) platelets or 135,000 to 457,000 molecules/platelet. Platelets treated with varying concentrations of collagen and thrombin released osteonectin in a dose-dependent fashion. Approximately 61% of the total platelet osteonectin was secreted at saturating concentrations of collagen and thrombin. A small fraction of platelet osteonectin is expressed on the surface of platelets in an activation-specific manner as evidenced by the specific and saturable binding of [125I]-anti-osteonectin monoclonal antibody, IIIA3A8, to thrombin-activated platelets. Based on a non-linear least squares regression analysis of the antibody binding, 2,200 IIIA3A8 molecules, or 0.8% of the total platelet osteonectin, is expressed on the platelet surface on activation. Platelet osteonectin was purified from the supernatant of thrombin-activated platelets by immunoaffinity chromatography. Western blotting of proteins secreted by washed, thrombin-stimulated platelets with IIIA3A8 indicated that the osteonectin molecule released from the platelet is a single chain polypeptide. Comparison of immunopurified platelet osteonectin with isolated bovine bone osteonectin and isolated human bone osteonectin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that platelet osteonectin has a greater apparent molecular weight than bone osteonectin. The NH2-terminal sequence of immunopurified platelet osteonectin was obtained by automated Edman degradation and is identical to the sequence of human bone osteonectin derived from the cDNA of SaOS-2 cells. Collectively, these data suggest that platelet osteonectin is structurally distinct from bone osteonectin in a region of the molecule at a distance from the NH2-terminus.

Amino Acid Sequence↗

PubMed1991

The collagen binding specificity of bone and platelet osteonectin is related to differences in glycosylation.

In this study we report that bone and platelet osteonectin are structurally and functionally heterogeneous in terms of glycosylation and collagen binding capacity. The relative sensitivity of bone and platelet osteonectin to specific glycosidases was used to evaluate potential differences in glycosylation. Although native bone and platelet osteonectin are electrophoretically nonidentical, N-glycanase treatment yielded products with the same apparent molecular weight. Bone osteonectin was also susceptible to cleavage by endo H but not to neuraminidase, while platelet osteonectin was susceptible to neuraminidase but not to endo H. In lectin blotting experiments of bone and platelet osteonectin, concanavalin A bound specifically to bone osteonectin but not to platelet osteonectin. However, Lens culinaris agglutinin bound to platelet osteonectin but not to bone osteonectin. These data suggest that bone and platelet osteonectin differ in their oligosaccharide side chain structures, with bone osteonectin possessing a high mannose-type and platelet osteonectin, a complex-type structure. Solid-phase binding techniques were used to functionally evaluate bone and platelet osteonectin in terms of collagen binding. Although bone osteonectin bound specifically to types I, III, and V collagen, platelet osteonectin had no apparent affinity for these collagen types suggesting that the two proteins are also functionally distinct.

Amidohydrolases↗

PubMed1992

Localization of platelet osteonectin at the internal face of the alpha-granule membranes in platelets and megakaryocytes.

Osteonectin is a 32-Kd phosphoglycoprotein originally described in bone but also found in platelets. Platelet and bone osteonectin are different both structurally and immunologically. We have previously shown that platelet osteonectin, by binding to thrombospondin, is involved in the secretion-dependent phase of the platelet aggregation process. In this study, we used antiosteonectin antibodies in combination with immunogold labeling to investigate by electron microscopy the fine localization of osteonectin within normal and gray platelets. Using both a polyclonal and monoclonal antibody ON3, osteonectin was specifically located at the internal face of alpha-granule membranes within normal platelets. Osteonectin was not distributed within all alpha-granules, probably because of its low platelet content. In addition, using immunofluorescence, osteonectin could also be detected in immature and mature megakaryocytes with a granular pattern of staining, suggesting that osteonectin is synthesized by megakaryocytes. Using platelets from two patients with gray platelet syndrome, osteonectin was absent within all abnormal small alpha-granules, but was detected in some rare normal-sized alpha-granules. In separate double-label studies, thrombospondin and von Willebrand factor did not colocalize with osteonectin in resting platelets. However, osteonectin was located at the inner face of the alpha-granules, as it is for alpha-granule membrane protein GMP-140 and glycoprotein IIb-IIIa. These results, taken together with the fact that monoclonal antibodies to osteonectin bind only to the surface of activated platelets, suggest that platelet osteonectin is redistributed to the cell surface during fusion of alpha-granule membranes with the plasma membrane.

Blood Platelets↗

PubMed1991

Demonstration of osteonectin mRNA in megakaryocytes: the use of the polymerase chain reaction.

Platelets have been shown to release osteonectin on thrombin stimulation. The origin of platelet osteonectin was unclear as it may have been synthesized by megakaryocytes or it may have been endocytosed from plasma as other platelet alpha-granule constituents are. Platelet osteonectin has a larger apparent molecular size than the bone species, although the molecular basis for this difference has not been elucidated. These two issues have been addressed here by (1) examining the potential for osteonectin biosynthesis in human megakaryocytes by demonstrating the presence of osteonectin mRNA in purified megakaryocytes, and (2) comparing the coding portion of osteonectin transcript in megakaryocytes to the size of its bone counterpart. Because of the limitations of cell population purity and in obtaining sufficient numbers of megakaryocyte cells for Northern analysis, we have used the polymerase chain reaction (PCR) to detect the presence of human osteonectin mRNA in megakaryocyte and megakaryocyte-depleted bone marrow cells. Isolation of RNA, cDNA synthesis, and PCR were performed on human osteosarcoma SaOS-2 cells, enriched megakaryocytes, and megakaryocyte-depleted cells. Restriction enzyme analysis of PCR DNA products confirmed the identity of the products as those encoding osteonectin for all three cell populations studied. In addition, the sizes of DNA indicate that osteonectin genomic DNA, nuclear RNA, or altered transcript were not amplified, and that the transcript from megakaryocytes is the same size as that from bone cells. These data suggest that the difference in protein size between platelet and bone osteonectin is due to posttranslational modification. To overcome the possibility that megakaryocyte signal originated from contaminating cells (less than 5% by cell count), all three cell populations were diluted to less than one cell per tube and PCR amplification was performed. Limiting dilution analyses demonstrated the presence of osteonectin mRNA in single megakaryocytes as well as in single cells from the cell population depleted of megakaryocytes, suggesting the capacity for osteonectin biosynthesis in all cells studied. The procedure we describe in this report can be used to examine specific characteristics of mRNA molecules in heterogeneous cell populations and in situations where only small quantities of cells can be obtained.

Blotting, Southern↗

PubMed1991

A general method of polymerase-chain-reaction-enabled protein domain mutagenesis: construction of a human protein S-osteonectin gene.

Polymerase chain reaction (PCR) amplification was employed to construct a mosaic gene consisting of the propeptide region of protein S and the glutamic acid-rich domain of osteonectin. The strategy is straightforward, results in large amounts of material, and is universally applicable for the generation of protein domain chimeras. In some cases 10% dimethyl sulfoxide aided the amplification. Four base CCGC "clamp" sequences adjacent to BamHI restriction sites at the ends of the PCR products were used to enhance the ligation of products. A hybrid inverse complement oligonucleotide primer composed of sequences containing 20 nucleotides of protein S and 16 nucleotides of osteonectin was used in the first round of PCR. An additional osteonectin sequence was added to the initial amplified product by performing PCR using a second "boot-strap" primer containing 18 nucleotides of osteonectin. Primers used to amplify osteonectin encompassed the 146-aminoacid NH2-terminal half of osteonectin. The double-stranded first-round fragments of protein S-osteonectin and osteonectin were subsequently mixed together and one elongation cycle of PCR was performed. Annealing occurred as the result of the 34-base-pair overlap region composed of osteonectin sequence. Taq polymerase was used for elongation with subsequent recombinant DNA synthesis. After elongation, external primers were added to amplify the protein S-osteonectin gene construct. The protocol we have developed allows noncoding and coding segments of DNA to be linked, GC-rich areas of DNA to be amplified, hybridization temperatures to be increased, annealing times to be reduced, and PCR of products to be subcloned.

Base Sequence↗

PubMed1992

Characterization of human osteoblast and megakaryocyte-derived osteonectin (SPARC).

Osteonectin is an adhesive, cell, and extracellular matrix-binding glycoprotein found primarily in the matrix of bone and in blood platelets in vivo. Osteonectins isolated from these two sources differ with respect to the complexity of their constituent N-linked oligosaccharide. In this study, osteonectin synthesized by bone-forming cells (osteoblasts) and platelet-producing cells (megakaryocytes) in vitro was analyzed to determine if the proteins produced were analogous in terms of glycosylation to those isolated from bone and platelets, respectively. Immunoblot analyses of osteonectin produced by the osteoblast-like cell lines, SaOS-2 and MG-63, indicated that secreted and intracellular forms of the molecule are structurally distinct. Endoglycosidase treatment and immunoblotting of osteonectin secreted from SaOS-2 and MG-63 cells, under serum-deprived conditions, suggested that the molecule possessed a complex type oligosaccharide unlike the high-mannose moiety found on bone matrix-derived osteonectin. Biosynthetic labeling of SaOS-2 cells and human megakaryocytes indicated that both cell types synthesize osteonectin de novo. Electrophoretic and glycosidase sensitivity analyses of [35S]-osteonectin isolated from lysates of metabolically labeled SaOS-2 cells and megakaryocytes indicated that these two cell types synthesize osteonectin molecules that are identical in oligosaccharide structure to the isolated bone and platelet proteins. These data suggest that the intracellular form of the osteonectin molecule is glycosylated differently in SaOS-2 cells and megakaryocytes but that the extracellular form which is secreted from platelets in vivo and osteoblasts in vitro is characterized by the presence of a complex type N-linked oligosaccharide.

Adult↗

PubMed1991

Osteonectin is an alpha-granule component involved with thrombospondin in platelet aggregation.

We previously showed that thrombospondin, a major alpha-granule glycoprotein of human platelets, forms a specific complex with osteonectin, a phosphoglycoprotein originally described in bone that is also present in human platelets. The storage organelles and the function of osteonectin in platelets are still unknown. In this study, using electron microscopy in combination with immunogold staining, the major storage organelle for platelet-secreted proteins, the alpha-granules. Furthermore, osteonectin was qualitatively and quantitatively assessed by studying normal platelets and the platelets from a patient with gray platelet syndrome. Gray platelet syndrome is a rare congenital bleeding disorder characterized by a selective deficiency in morphologically recognizable platelet alpha-granules and in the alpha-granule secretory proteins. Binding of an iodinated antiosteonectin monoclonal antibody to gray platelet proteins transferred to nitrocellulose from SDS-polyacrylamide gels showed no band corresponding to osteonectin compared to control platelets. Using a polyclonal antiosteonectin antibody-based radioimmunoassay, gray platelets contained 0.2 +/- 0.03 ng osteonectin per 10(6) platelets, which is only 20% of the normal platelet content of osteonectin (0.93 +/- 0.16 ng per 10(6) platelets). Study of the localization of osteonectin to the surface of human platelets demonstrated that a radioiodinated antiosteonectin polyclonal antibody bound specifically to thrombin-stimulated platelets but not to resting platelets. Binding was concentration-dependent, saturable (1710 +/- 453 binding sites per platelet, Kd = 1 microM), and inhibited by an excess of cold antiosteonectin polyclonal antibody. No binding was observed on the surface of thrombin-stimulated gray platelets. To gain further insights into the role of osteonectin released from activated platelets, the effect of an antiosteonectin polyclonal antibody was tested on the aggregation of washed platelets. F(ab')2 fragments from the antiosteonectin polyclonal antibody inhibited in a dose-dependent manner the aggregation of collagen-stimulated, washed human platelets without affecting collagen-induced platelet serotonin release. To characterize the mechanism through which antiosteonectin F(ab')2 fragments inhibit platelet aggregation, the expression of endogenous thrombospondin (TSP) on the surface of thrombin-activated platelets was studied using 125I-labeled anti-TSP monoclonal antibody P10. The endogenous surface expression of TSP to thrombin-stimulated platelets was significantly inhibited in the presence of antiosteonectin F(ab')2 fragments (6286 +/- 2065 molecules of P10 per platelet) compared to 11,230 +/- 766 molecules of P10 per platelet in the presence of nonimmune F(ab')2 fragments.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗

PubMed1991

Histogenesis of clear cell chondrosarcoma. An immunohistochemical study with osteonectin, a non-collagenous structure protein.

The histogenesis of clear cell chondrosarcoma is still unclear: Apart from typical clear cell tumor areas, extensive production of woven bone formation suggests within the clear cell cartilagenous stroma is an intriguing phenomenon. Three cases of clear cell chondrosarcoma documented in the Bone Tumor Registry of Westphalia were examined for their patterns of osteonectin expression, and compared with other bone tumors of either osseous or cartilaginous origin, and with normal cartilage tissue. Found predominantly in osseous structures, the protein osteonectin takes part in the formation of new bone. The three clear cell chondrosarcomas showed a strong immunoexpression of osteonectin in clear cell, chondroid and in osseous tumor areas. Similarly, evidence of osteonectin was also found in osteoblastic and in chondroblastic osteosarcomas as well as in osteoblastomas. In contrast, osteonectin could not be demonstrated in the chondrosarcomas and mesenchymal chondrosarcomas from our registry that were analysed for comparison, and was found only minimally in the fibroblastic areas of dedifferentiated chondrosarcomas. The chondroblastic tumor components were always negative. There was no immunoexpression of osteonectin either in fetal or adult intervertebral disc tissue. The present immunohistochemical study of osteonectin has distinctly separated clear cell chondrosarcoma from the other variants of chondrosarcoma, and aptly verified the specificity of this entity. Moreover, the study would call for further histogenetic evaluation of clear cell chondrosarcoma, since the pattern of osteonectin expression in that tumor seems to indicate an osteogenic rather than a chondrogenic origin.

Adenocarcinoma↗

PubMed1990

Changes in osteonectin distribution and levels are associated with mineralization of the chicken tibial growth cartilage.

Osteonectin is a calcium-binding matrix protein thought to play a role in regulating calcium distribution in a variety of biologic processes. To examine its role in endochondral bone formation, we examined the distribution of the protein during mineralization of the chicken tibial growth cartilage, using immunohistochemistry and immunoelectron microscopy. The synthesis of osteonectin was also determined in chondrocyte populations isolated from premineralizing and mineralizing regions of growth cartilage and assayed in short-term culture. The results show that a very low level of osteonectin is detectable in the resting, proliferating, and early hypertrophic zones of growth cartilage; in these zones, osteonectin is largely cell-associated. In contrast, a large amount of osteonectin is present in the mineralizing zone where it is associated with the matrix. Biosynthetic data from short-term culture experiments indicate, however, that osteonectin is synthesized and secreted by chondrocytes from both premineralizing and mineralizing zones. As indicated by immunoprecipitation, Northern hybridization, in vitro translation of hybrid-selected messenger RNA (mRNA), and electrophoretic analysis, osteonectin synthesized by chondrocytes of the premineralizing zones is not obviously different in structure from that synthesized by chondrocytes of the mineralizing zone. We conclude that osteonectin is a product of chondrocytes in each zone of growth cartilage but accumulates only in the mineralizing zone. The high affinity of the protein for calcium could favor its retention in calcifying matrix.

Animals↗

PubMed1991

Epitope mapping of two monoclonal antibodies to the central portion of human osteonectin.

In this study preliminary characterization of two monoclonal antibodies against osteonectin was undertaken. One monoclonal originally raised against bovine bone osteonectin cross reacts with human bone and platelet osteonectin. The other monoclonal antibody has been reported to react with osteonectin derived from human bone and bovine bone but not to the same extent with that from platelets. Initial mapping of the antigenic determinants for both monoclonals was done by testing their ability to bind to the expressed forms of osteonectin in two overlapping SaOS-2 lambda gt11 osteonectin cDNA clones. One clone contains a 0.54 kb insert and is comprised of 50 nucleotides of 5' noncoding and a coding segment for a 17 amino acid signal peptide and 146 amino acids of the N-terminal region of the mature protein. The other clone has a 1.9 kb insert, and includes amino acid no. 18 to the C-terminus of the molecule (amino acid no. 286), a single termination codon, and 1115 nucleotides of 3' noncoding sequence. Both monoclonals recognized expressed osteonectin from the two lambda gt11 SaOS-2 cDNA clones. These results localize the epitope to a region between amino acids 18-146 of osteonectin.

Amino Acid Sequence↗

PubMed1989

Osteonectin inhibiting de novo formation of apatite in the presence of collagen.

The effect of bone matrix protein of osteonectin on de novo formation of apatite was studied in a wide range of calcium phosphate solutions in the presence of collagen. In every solution, from which amorphous calcium phosphate, octacalcium phosphate, or apatite precipitated as a possible initial phase, osteonectin at concentrations less than 1 microM retarded the precipitation, subsequent transformation to apatite, and ripening crystal growth of apatite. Collagen present as either reconstituted or denatured form had no effect on the osteonectin-associated reactions as well as osteonectin-free reactions, and no structural correlation was observed between collagen fibrils and any of the calcium phosphates that appeared in our system. Direct measurement of free calcium levels in the solutions suggested that the reduction in calcium activity due to complexing with osteonectin hardly explained the inhibitory activity of osteonectin in retarding the formation of apatite. Instead, our transmission electron microscopic (TEM) observation strongly suggested that the primary mechanism for osteonectin to inhibit the formation of apatite is to block growth sites of calcium phosphates nucleated. The apatite thus formed in the presence of osteonectin showed less resolved X-ray diffraction patterns, partly because of smaller crystallites as suggested by TEM.

Animals↗

PubMed1990

Circulating levels of osteonectin in normal subjects and patients with thrombocytopenia.

In order to clarify the relative contributions of activated platelets and other sources to circulating osteonectin, the amounts of osteonectin present in serum and in plasma prepared without significant platelet activation were compared to platelet count in normal subjects and patients with various degrees of thrombocytopenia. Serum osteonectin showed a logarithmic positive correlation with platelet count (r = 0.87, P less than 0.0001, n = 52). Osteonectin concentration was significantly lower in plasma than in matched sera in all subjects sampled, and not significantly different in plasma of thrombocytopenic and normal subjects. These results confirm the major contribution of platelets to serum osteonectin. The positive zero intercept of the plot osteonectin vs. platelet count (19 +/- 2.45 ng/ml, mean +/- SD, P less than 0.0001, n = 52) and the presence of significant amounts of osteonectin in plasma, document the existence of a basic level of circulating osteonectin, independent of platelet activation, and to which the relative contribution of bone remains to be assessed.

Adult↗

PubMed1992

Renal tubular epithelial cells express osteonectin in vivo and in vitro.

Osteonectin (SPARC, culture shock protein, BM-40) is a widely distributed glycoprotein which binds calcium and several extracellular matrix proteins, including interstitial collagens and thrombospondin, but whose physiologic role remains undefined. In the present studies, we have demonstrated that immunoreactive osteonectin is present in the distal cortical tubule and medullary tubules of murine kidney. We surveyed the renal epithelial cell lines LLC-PK1, MDCK, and OK for the expression of mRNA encoding osteonectin. We found that osteonectin mRNA is expressed by LLC-PK1 and OK cells but not by MDCK cells, as well as by adult kidney from several species. Calcitonin and vasopressin, agents which increase cAMP in these cells, were found to decrease steady-state osteonectin mRNA concentrations. We found that LLC-PK1 cells produced osteonectin protein, that the protein was localized to intracellular granules, and that the protein bound hydroxyapatite in vitro. Pulse-chase analysis revealed that osteonectin was secreted from the cell layer to the medium after a lag time of four to six hours and was secreted preferentially from the basolateral domain of the cell. The preferential secretion of the calcium-binding protein osteonectin from the renal epithelial cell is consistent with several possible functions, including a structural extracellular matrix protein, a participant in transepithelial ion transport, and an inhibitor of extracellular calcification.

Animals↗

PubMed1990

Insulinlike growth factor 1 regulates mRNA levels of osteonectin and pro-alpha 1(I)-collagen in clonal preosteoblastic calvarial cells.

A nontransformed rat clonal cell line (UMR-201) with phenotypic characteristics of osteoblastic precursor cells was found to respond to insulinlike growth factor 1 (IGF-1) by increased osteonectin and pro-alpha 1(I)-collagen mRNA expression. Cells were treated for 24 h with insulin, growth hormone, or IGF-1 to study the regulation of messenger RNA for osteonectin and pro-alpha 1(I)-collagen using Northern blot hybridization. UMR-201 cells possess specific high-affinity receptors for growth hormone, although there were no significant effects of growth hormone (10(-9)-10(-7) M) or insulin (10(-9)-10(-6) M) on mRNA species for osteonectin or pro-alpha 1(I)-collagen. However, IGF-1 increased both mRNA species from a concentration of 10(-9) M. The effect on osteonectin mRNA expression was likely due to increased transcription; when 5' flanking osteonectin (ON) genomic fragments were linked to the bacterial reporter gene chloramphenicol acetyltransferase (CAT) and introduced by transfection into UMR-201 cells, the transcriptional activity of the ON-CAT construct was increased 235 and 270% by 10(-8) and 10(-7) M IGF-1, respectively. In contrast, growth hormone did not change the transcriptional activity of the ON-CAT construct. In confirmation of other work, transforming growth factor beta (TGF-beta, 0.1-2.5 ng/ml) increased mRNA for osteonectin and pro-alpha 1(I)-collagen in a dose-dependent manner. Transforming growth factor alpha (TGF-alpha) at 0.1-10 ng/ml had no consistent effects in repeated experiments on osteonectin and pro-alpha 1(I)-collagen mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

The role of osteonectin in human tooth development: an immunohistological study.

We investigated immunohistologically 160 teeth and dental germs in various stages of tooth development taken from human individuals (13th week of pregnancy to the 24th year of life) to study the osteonectin expression in dental hard tissue. In the course of dentinogenesis, the predentin, the odontoblasts, and their cell processes show a positive osteonectin staining reaction. During cementogenesis, osteonectin is synthesized by cement-producing fibroblasts, cementoblasts, and cementocytes. The expression of osteonectin during dentinogenesis and cementogenesis is closely related to the development of the respective calcified tissue. All cells of the inner and outer enamel epithelium, the cells of the stratum reticulare and stratum intermedium, the ameloblasts, and the enamel substance are osteonectin negative, just as dentin and cement are. The results of this study indicate one important physiological role of osteonectin as a protein associated with the formation of collagen containing mineralizing tissues like human bone, as well as human dentin and cement.

Adolescent↗

PubMed1992

Distribution of osteonectin mRNA and protein during human embryonic and fetal development.

We investigated the temporal and spatial distribution of osteonectin during human embryonic and fetal development, using in situ hybridization and immunohistochemistry. Osteonectin gene expression was generally found in cells exhibiting high rates of matrix production/proliferation. In mineralized tissue, a strong signal was obtained in osteoblasts, odontoblasts, and chondrocytes of the upper hypertrophic and proliferative zones. Chondrocytes of the mineralized zone showed no expression throughout the different stages of development. Strong osteonectin expression was found in odontoblasts of developing teeth. In addition, osteonectin mRNA and protein were detected in several non-mineralized tissues: steroid-producing cells of the adrenal gland and the gonads, kidney (glomeruli), lung (bronchi), skin, megacaryocytes, and large vessels. Histochemistry confirmed the results and detected extracellular osteonectin in bone and in the zone of mineralized cartilage only. The localization of osteonectin in bone, cartilage, and teeth is consistent with a role in the initiation of mineralization. However, the organ-specific distribution in non-mineralized tissues suggests an important multifunction role of this protein during human development.

Abortion, Spontaneous↗

PubMed1990

Structure and expression of osteonectin mRNA in human tissue.

A cDNA encoding osteonectin was isolated from a human bone cell cDNA library and used to examine osteonectin protein structure, mRNA structure and expression in human tissue. The deduced protein sequence shows complete identity with a recently isolated placental form and extensive homology to mouse and bovine counterparts. The protein is rich in cysteine residues, which are conserved between species except for cys 194 which is only present in the bovine. In the human, osteonectin mRNA is of two sizes, 2.3 and 3.0 kb, the former being dominant in all tissues studied. Human mRNA was detected in the Ewing sarcoma and in non-bone cell and tissue sources. The potential folded structure of osteonectin mRNA was estimated, based on computer predictions, and indicates the presence of a bulge at the 5' end of the message which includes the start of translation. Southern analysis of human genomic DNA using radiolabeled osteonectin cDNA as probe demonstrates a simple banding pattern confirming earlier studies that the osteonectin gene is present in one copy per haploid human genome.

Amino Acid Sequence↗