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Journal of Bone and Mineral Research :... Apr 1991Osteonectin, a major noncollagenous protein of bone, is also synthesized and secreted by various non-mineralized tissues and by platelets. To establish whether there are... (Comparative Study)
Comparative Study
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.
Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Blood Platelets; Bone and Bones; Epitopes; Hybridomas; Immunoblotting; Immunoglobulin G; Immunoradiometric Assay; Male; Mice; Mice, Inbred BALB C; Osteonectin; Precipitin Tests; Radioimmunoassay
PubMed: 1713400
DOI: 10.1002/jbmr.5650060402 -
Blood Dec 1992Osteonectin is an adhesive, cell, and extracellular matrix-binding glycoprotein found primarily in the matrix of bone and in blood platelets in vivo. Osteonectins... (Comparative Study)
Comparative Study
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.
Topics: Adult; Blood Platelets; Bone Marrow; Bone Marrow Cells; Carbohydrate Sequence; Cell Line; Cells, Cultured; Electrophoresis, Polyacrylamide Gel; Endothelium, Vascular; Glycoside Hydrolases; Humans; Megakaryocytes; Molecular Sequence Data; Molecular Weight; Osteoblasts; Osteonectin; Osteosarcoma; Umbilical Veins
PubMed: 1467517
DOI: No ID Found -
Blood Sep 1991Platelets have been shown to release osteonectin on thrombin stimulation. The origin of platelet osteonectin was unclear as it may have been synthesized by...
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.
Topics: Blotting, Southern; Humans; Megakaryocytes; Nucleic Acid Amplification Techniques; Osteonectin; Osteosarcoma; Poisson Distribution; Polymerase Chain Reaction; RNA, Messenger; Tumor Cells, Cultured
PubMed: 1878589
DOI: No ID Found -
Cancer Letters Oct 2002The clinical significance of osteonectin in human stomach cancer was examined immunohistochemically and molecular biologically in 31 differentiated and eight...
The clinical significance of osteonectin in human stomach cancer was examined immunohistochemically and molecular biologically in 31 differentiated and eight undifferentiated stomach adenocarcinomas and 19 non-cancer stomach tissues. Osteonectin-mAb-stained cells were observed in stroma of 90% differentiated and 63% undifferentiated adenocarcinomas, and of 26% non-cancer stomach tissues. Competitive reverse transcriptase polymerase chain reaction results generally coincided with immunohistochemical data. The present results suggest that osteonectin is highly expressed in reactive stroma associated with invasive differentiated adenocarcinomas and that it may serve as a useful clinical diagnostic marker for stomach cancer.
Topics: Adenocarcinoma; DNA Primers; Humans; Immunoenzyme Techniques; Osteonectin; RNA, Neoplasm; Reverse Transcriptase Polymerase Chain Reaction; Stomach Neoplasms
PubMed: 12104055
DOI: 10.1016/s0304-3835(02)00191-x -
Developmental Biology Nov 2017Collagen IV networks endow basement membranes (BMs) with remarkable tensile strength and function as morphoregulatory substrata for diverse tissue-specific developmental... (Review)
Review
Collagen IV networks endow basement membranes (BMs) with remarkable tensile strength and function as morphoregulatory substrata for diverse tissue-specific developmental events. A complex repertoire of intracellular and extracellular molecular interactions are required for collagen IV secretion and supramolecular assembly into BMs. These include intracellular chaperones such as Heat shock protein 47 (Hsp47) and the chaperone-binding trafficking protein Transport and Golgi organization protein 1 (Tango1). Mutations in these proteins lead to compromised collagen IV protomer stability and secretion, leading to defective BM assembly and function. In addition to intracellular chaperones, a role for extracellular chaperones orchestrating the transport, supramolecular assembly, and architecture of collagen IV in BM is emerging. We present evidence derived from evolutionarily distant model organisms that supports an extracellular collagen IV chaperone-like activity for the matricellular protein SPARC (Secreted Protein, Acidic, Rich in Cysteine). Loss of SPARC disrupts BM homeostasis and compromises tissue biomechanics and physiological function. Thus, the combined contributions of intracellular and extracellular collagen IV-associated chaperones and chaperone-like proteins are critical to ensure proper secretion and stereotypic assembly of collagen IV networks in BMs.
Topics: Animals; Basement Membrane; Collagen Type IV; Evolution, Molecular; Humans; Osteonectin; Protein Folding; Protein Transport
PubMed: 28982537
DOI: 10.1016/j.ydbio.2017.09.037 -
Molecular Pharmaceutics Nov 2017Therapeutics reducing bone turnover, such as denosumab (Dmab), an anti-RANKL antibody, can provide treatments for patients with bone destruction. However, some patients...
Therapeutics reducing bone turnover, such as denosumab (Dmab), an anti-RANKL antibody, can provide treatments for patients with bone destruction. However, some patients with osteoporosis or localized primary bone tumors and many patients with various types of bone-metastatic cancer display unsatisfactory responses to Dmab. For achieving greater efficiency of RANKL neutralization in the bone microenvironment by enhancing the distribution of Dmab to the bone, we reengineered Dmab by fusing with single-chain variable fragments of an antibody specific for osteonectin (On), which is abundantly expressed in osseous tissues. The bispecific antibody, Dmab-FvOn, showed a similar activity as Dmab in inhibiting RANKL as examined in an osteoclast differentiation assay. When administered to mice, Dmab-FvOn was found to localize in increased proportions at the endosteum of the bone where osteonectin is abundant. Our study suggests that by linking anti-RANKL with an osteonectin-targeting moiety, a greater proportion of the therapeutic effector can be distributed in the bone. Future studies are needed to investigate whether the bispecific antibody can achieve higher therapeutic efficacy and lower toxicity.
Topics: Animals; Antibodies, Bispecific; Cell Differentiation; Denosumab; Mice; Osteoclasts; Osteonectin; RANK Ligand
PubMed: 28954509
DOI: 10.1021/acs.molpharmaceut.7b00501 -
European Journal of Cancer (Oxford,... Sep 1997Osteonectin is a secreted glycoprotein which is detected in a number of normal and neoplastic human tissues in vivo. It is an extracellular matrix (ECM)-associated...
Osteonectin is a secreted glycoprotein which is detected in a number of normal and neoplastic human tissues in vivo. It is an extracellular matrix (ECM)-associated protein which is postulated to regulate cell migration, adhesion, proliferation and matrix mineralisation and previous reports suggest that it may be modulated by steroid hormones in target tissues. The aim of this study was to measure osteonectin mRNA and protein expression in breast tumour biopsies and compare these with oestrogen (ER) and progesterone receptor (PR) levels in the same tumours. An inverse correlation was seen between osteonectin mRNA expression and ER level. Samples with low ER protein expression had a mean osteonectin mRNA level which was almost 4-fold greater than the mean level of expression observed in tumours containing high concentrations of ER protein. This inverse correlation was statistically significant. Despite the strong inverse relationship between osteonectin mRNA levels and tumour ER content, no correlation was seen when osteonectin protein concentration was measured in tumour cytosols on immunoblots and compared to ER and PR levels in the same tumours. However, since it is a secreted protein, osteonectin protein expression may not reflect cellular osteonectin levels in breast tumours. In summary, these data suggest that ER-mediated suppression of osteonectin gene expression may contribute to the less aggressive characteristics associated with receptor-positive tumours and that loss of ER expression may lead to over-expression of osteonectin and contribute to a poorer differentiated, more invasive phenotype.
Topics: Blotting, Northern; Breast Neoplasms; Female; Gene Expression; Humans; Neoplasm Proteins; Osteonectin; RNA, Messenger; RNA, Neoplasm; Receptors, Estrogen; Receptors, Progesterone
PubMed: 9389930
DOI: 10.1016/s0959-8049(97)00182-2 -
Molecular Vision Sep 1998To characterize gene expression patterns between epithelia isolated from cataractous and normal human lenses.
PURPOSE
To characterize gene expression patterns between epithelia isolated from cataractous and normal human lenses.
METHODS
Reverse transcriptase differential display was used to identify differential expression between cataractous and normal epithelia. RT-PCR was used to compare pooled and individual RNA samples.
RESULTS
One transcript, up-regulated in cataractous as compared to normal epithelia, was identified as osteonectin which is also known as SPARC (secreted acidic protein rich in cysteines). RT-PCR confirmed over-expression of this RNA. High levels of osteonectin mRNA were also detected in six individual epithelia dissected from cataractous lenses.
CONCLUSIONS
The present study provides evidence for up-regulation of osteonectin in human age-related cataract and suggests that osteonectin, a protein involved in cell-cycle control, extracellular matrix and Ca++ binding, plays an important role in human lens homeostasis and may be involved in processes leading to lens opacity.
Topics: Adult; Aged; Aged, 80 and over; Base Sequence; Cataract; Female; Humans; Lens, Crystalline; Male; Middle Aged; Molecular Sequence Data; Osteonectin; Polymerase Chain Reaction; RNA; Sequence Homology, Nucleic Acid; Up-Regulation
PubMed: 9743541
DOI: No ID Found -
Developmental Dynamics : An Official... May 2015SPARC/osteonectin is an evolutionarily conserved matricellular protein that modulates cell-matrix interaction and cell function. In all vertebrates, SPARC is dynamically...
BACKGROUND
SPARC/osteonectin is an evolutionarily conserved matricellular protein that modulates cell-matrix interaction and cell function. In all vertebrates, SPARC is dynamically expressed during embryogenesis. However, the precise function of SPARC and the regulatory elements required for its expression in particular during early embryogenesis are largely unknown.
RESULTS
The present study was undertaken to explore the molecular mechanisms that regulate sparc gene expression by in vivo functional characterization of the sparc promoter and identification of possible putative regulatory elements that govern basal promoter activity. We report here transient expression analyses of eGFP expression from transgenic zebrafish containing a Sparc-iTol2-eGFP-BAC and/or 7.25 kb-sparc-Tol2-eGFP constructs. eGFP expression was specifically found in the notochord, otic vesicle, fin fold, intermediate cell mass, and olfactory placode of BAC and Tol2 transposon vectors injected embryos. Deletion analysis revealed that promoter activity resides in the unique 5'-untranslated intronic region. Computer-based analysis revealed a putative CpG island immediately proximal to the translation start site within the intron sequence. Global inhibition of methylation with 5-Aza-2-deoxycytidine promoted sparc expression in association with decreasing CpG methylation.
CONCLUSIONS
Taken together, these data identify a contributory role for DNA methylation in regulating sparc expression in zebrafish embryogenesis.
Topics: Animals; Animals, Genetically Modified; DNA Methylation; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Osteonectin; Promoter Regions, Genetic; Zebrafish
PubMed: 25728805
DOI: 10.1002/dvdy.24267 -
Kidney International Jan 1992Osteonectin (SPARC, culture shock protein, BM-40) is a widely distributed glycoprotein which binds calcium and several extracellular matrix proteins, including...
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.
Topics: Animals; Cell Line; Durapatite; Epithelium; Gene Expression; Hormones; Hydroxyapatites; Kidney Tubules; Mice; Mice, Inbred C57BL; Osteonectin; RNA, Messenger
PubMed: 1317480
DOI: 10.1038/ki.1992.8