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PubMed2002

Spatial and temporal localization of transforming growth factor-beta, fibroblast growth factor-2, and osteonectin, and identification of cells expressing alpha-smooth muscle actin in the injured anulus fibrosus: implications for extracellular matrix repair.

STUDY DESIGN: The spatial and temporal localization of fibroblast growth factor-2, transforming growth factor-beta, osteonectin, and alpha-smooth muscle cell actin in the injured anulus fibrosus was investigated. OBJECTIVE: To assess the involvement of fibroblast growth factor-2, transforming growth factor-beta, osteonectin, and alpha-smooth muscle cell actin in anulus fibrosus repair. SUMMARY OF BACKGROUND DATA: Fibroblast growth factor-2 and transforming growth factor-beta have been localized to disc herniation tissue, and alpha-smooth muscle cell actin has been identified in a number of mesenchymal cell types, but their roles have not been evaluated in repair processes in the experimentally injured anulus fibrosus. METHODS: For this study, 32 two adult merinos received a 4-mm deep standard annular incision in their L1L2 and L3L4 discs (lesion group). A similar number of sham-surgery animals served as control subjects. Osteonectin, fibroblast growth factor-2, transforming growth factor-beta, and alpha-smooth muscle cell actin were immunolocalized in sagittal disc sections 3, 6, 12, and 26 months after the operation. Selected specimens also were stained with hematoxylin and eosin, Masson-trichrome, toluidine blue, and picrosirius red. RESULTS: Early focal depletion of proteoglycan was evident in the anulus fibrosus and reorganization of outer annular lamellas 3 to 6 months after the operation. Blood vessel ingrowth and fibroblast infiltration from the outer anulus fibrosus along the plane of the annular defect were maximal 12 months after the operation. Focal upregulation in alpha-smooth muscle cell actin expression was evident with maximal staining in the 12-month lesion samples near infiltrating blood vessels at the lesion site, and also in cells well away from these vessels. Some of the anulus fibrosus cells of the sham sections also stained positively for alpha-smooth muscle cell actin, but this staining was significantly less than in the lesion samples. Staining for fibroblast growth factor-2, transforming growth factor-beta, and osteonectin was strongly localized to blood vessels and cells in the vicinity of the annular lesion. It was maximal 12 months after the operation and diminished by 26 months after the operation. Osteonectin expression also was significantly elevated in outer anulus fibrosus cells distant from the lesion site and its associated blood vessels. In the sham discs, immunoreactivity to fibroblast growth factor-2, transforming growth factor-beta, osteonectin, and alpha-smooth muscle cell actin was confined to sparsely distributed cells in the anulus fibrosus. No matrix staining was observed. CONCLUSIONS: Immunoreactivity for the noted agents was strongly associated with regions of the annular lesions undergoing matrix reorganization consistent with an active repair response. This response extended as far as the middle third of the anulus fibrosus, which also demarcated the extent of blood vessel ingrowth and cellular infiltration in this model. The alpha-smooth muscle cell actin expression suggested an active involvement of myofibroblasts in the anulus fibrosus repair processes.

Actins↗
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↗
PubMed1989

The partial degradation of osteonectin by a bone-derived metalloprotease enhances binding to type I collagen.

Cultured neonatal rat calvaria produce latent metalloproteases capable of degrading collagen, gelatin, and osteonectin. The osteonectin-degrading activity was further characterized and found to be optimally active between pH 6 and 8 and inhibited with EDTA and 1, 10-phenanthroline but not phenylmethylsulfonyl fluoride. Analysis of the degradation products of osteonectin by SDS-PAGE in the presence of dithiothreitol showed the generation of a somewhat stable 32,000 mw cleavage product. Comparison of the binding properties of this cleavage product with intact osteonectin indicated that the fragment retained its ability to bind hydroxyapatite in the presence of high salt (2 M NaCl). Importantly, the binding of osteonectin to type I collagen fibrils was enhanced by limited proteolysis.

Animals↗
PubMed1987

Osteonectin--a differentiation marker of bone cells.

Bone matrix consists of type-I collagen and non-collagenous proteins. The latter represent only 10% of its total protein content. Since type-I collagen is also present in various other connective tissue sites (e.g., skin) it cannot be considered as bone specific. Among the non-collagenous components osteonectin--a 32 kilodalton (KD) glycoprotein linking mineral to collagen fibrils--is thought to be bone specific due to its biochemical properties. In the present study various skeletal and non-skeletal tissues were investigated for the presence of osteonectin by means of immunocytochemical methods. Two polyclonal antibodies against human and bovine osteonectin were applied. Immunocytochemically, osteonectin could be demonstrated in active osteoblasts and osteoprogenitor cells as well as in young osteocytes, while aged, quiescent osteocytes did not contain the protein, suggesting that the protein is a marker of the osteoblastic functional differentiation of bone cells. Osteonectin was absent in all non-skeletal tissues with the exception of chondrocytes in so-called mineralizing chondroid bone.

Antibodies↗
PubMed1995

Conformational changes of bovine bone osteonectin induced by interaction with calcium.

To clarify calcium-induced conformational changes in bovine bone osteonectin, the protein was labeled with fluorescein isothiocyanate (FITC) in the presence and absence of calcium. By calcium titration using fluorescence spectrometry, it was demonstrated that FITC-osteonectin labeled in the presence of 2 mM CaCl2 showed a much higher affinity for calcium ions than did that labeled in the absence of calcium ions. The midpoint for completion of the increase in the intrinsic fluorescence (K0.5) of the two were 1 x 10(-7) M and 5 x 10(-7) M, respectively. By tryptic digestion and isolation of the fluorescent peptide of both FITC-osteonectins, the site of FITC-labeling was determined to be Lys174. Furthermore, it was found that the efficacy of labeling in this specific binding site was three times higher in the FITC-osteonectin labeled in the presence of 2 mM CaCl2 than in that labeled in the absence of calcium. The results indicate that in the presence of 2 mM CaCl2 the microenvironment around Lys174 of osteonectin was more open to modification than in the absence of calcium.

Amino Acid Sequence↗
PubMed1987

Osteonectin content in human osteogenesis imperfecta bone shows a range similar to that of two bovine models of OI.

Samples of human osteogenesis imperfecta (OI) bone were analyzed for osteonectin content by SDS gel electrophoresis and immunodetection on Western blots. The OI bone osteonectin content varied from normal to severely depressed. Previously, we showed that two clinically identical but genetically unrelated bovine models of OI were differentiated biochemically by their bone osteonectin content: one OI model had normal bone osteonectin while the other was severely depressed in this parameter. The data in this pilot study suggest that further investigation of bone osteonectin content may prove useful in the clinical assessment of human OI cases.

Adolescent↗
PubMed1989

Localization of osteonectin expression in human fetal skeletal tissues by in situ hybridization.

The expression of osteonectin gene was studied in developing human fetuses by Northern analysis and in situ hybridization. The highest levels of osteonectin mRNA were detected in RNA extracted from calvarial bones, growth plates, and skin. Low mRNA levels were present in several parenchymal tissues. In situ hybridization of developing long bones revealed three cell types with high osteonectin mRNA levels: osteoblasts, cells of the periosteum, and hypertrophic chondrocytes. Weaker signals were detected in osteocytes, fibroblasts of tendons, ligaments and skin, and in cells of the epidermis. Apart from the hypertrophic chondrocytes, only low osteonectin mRNA levels were seen in cartilage. The localization of osteonectin mRNA in fetal growth plates is consistent with the hypothesis that the protein plays a role in the mineralization of bone and cartilage matrices.

Bone and Bones↗
PubMed1996

Enhanced osteonectin expression in the chondroid matrix of the unloaded mandibular condyle.

Osteonectin provided a spatial and temporal marker for proliferating and differentiating chondrocytes, and during the chondroid matrix formation. The goal of this investigation was to examine early cellular and molecular regulation of mandibular growth. Unloading was induced by anterior functional mastication. The proliferative activity measured by tritiated thymidine incorporation increased 8. 3-fold at 24 hours compared with the corresponding control group. Mandibular unloading for 24 hours increased osteonectin mRNA expression 60% in the condyle over the corresponding control group. Microscopic inspection of the condyle demonstrated osteonectin immunostaining of proliferating, early hypertrophic chondrocytes, and the chondroid matrix across the sagittal section in an anterior-posterior direction. An increasing gradient intensity from a medial-superior to posterior direction was produced with treatment in direct contrast to the control group. The posterior chondroid matrix immunostaining increased 11.7-fold (P = 0.038) after 24 hours treatment over a corresponding control group. Unloading of the mouse mandible caused an increased cellular proliferation, a coincident increase of osteonectin mRNA, and a subsequent increased secretion of the osteonectin protein in the chondroid matrix formation.

Animals↗
PubMed1981

Osteonectin, a bone-specific protein linking mineral to collagen.

Osteonectin is a 32,000 dalton bone-specific protein that binds selectively to both hydroxyapatite and collagen. When osteonectin is bound to insolubilized type I collagen, the resultant complex binds synthetic apatite crystals and free calcium ions. The osteonectin-collagen complexes also nucleate mineral phase deposition from metastable balanced salt solutions, Antibodies to osteonectin cross-react with bone and, to a lesser extent, dentin, but not with other tissues. The protein is localized to mineralized bone trabeculae and occurs at higher levels in the matrix than in the cells of bone. These studies suggest that osteonectin is a tissue-specific protein, linking the bone mineral and collagen phases, perhaps initiating active mineralization in normal skeletal tissue.

Animals↗
PubMed1999

Expression of collagens type I and IV, osteonectin and transforming growth factor beta-1 (TGFbeta1) in biliary atresia and paucity of intrahepatic bile ducts during infancy.

BACKGROUND/AIMS: Biliary atresia and paucity of intrahepatic bile ducts are the main causes of neonatal cholestasis leading to hepatic fibrosis. Fibrotic evolution is slow in paucity of bile ducts as compared to the rapid progression to biliary cirrhosis in biliary atresia when cholestasis persists despite hepatoportoenterostomy. Our aim was to compare the expression of collagens type I and IV, alpha-smooth muscle actin, osteonectin and transforming growth factor beta1 in biliary atresia and paucity of bile ducts. METHODS: Liver biopsies were obtained in 12 children with biliary atresia and in five with paucity of bile ducts. Collagens type I and IV, alpha-smooth muscle actin were detected with immunostaining. Collagens type I and IV, osteonectin and transforming growth factor beta1 mRNAs were detected by in situ hybridization. RESULTS: Expression of mRNA and proteins was roughly parallel. In ductular proliferation areas of biliary atresia: (1) the expression of collagens type I and IV and osteonectin was increased, and was localized to periductular myofibroblasts; (2) transforming growth factor beta1 was expressed around biliary ductules, probably in inflammatory cells, and also in biliary cells. Osteonectin expression was also increased in the lobules. In paucity of bile ducts, there was no overexpression of collagens type I and IV and transforming growth factor beta1, except in the only child with marked fibrosis. However, osteonectin expression was enhanced at the periphery of the lobules, even when fibrosis was mild or absent. CONCLUSIONS: These findings suggest that in biliary atresia ductular proliferation areas are the site of a marked production of extracellular matrix proteins in periductular myofibroblasts, probably secondary to transforming growth factor beta1 production by inflammatory cells and by biliary cells. The weak expression of transforming growth factor beta1 could explain the slow progression of fibrosis in paucity of bile ducts.

Actins↗
PubMed1989

Structure of human osteonectin based upon analysis of cDNA and genomic sequences.

Overlapping human bone osteonectin cDNAs were obtained by screening two independent human SaOS-2 lambda gt11 libraries using antibovine osteonectin monoclonal antibodies. One clone contains a 0.54-kb insert and the other a 1.9-kb insert. Insertion fragments from lambda clones were liberated by restriction digestion and subcloned into pUC19 for sequencing. Digestion of the 1.9-kb insert with EcoRI released 0.4- and 1.5-kb fragments. Sequencing analysis revealed that the 0.54- and 0.4-kb fragments are identical except for 150 nucleotides missing at the 5' region of the 0.4-kb fragment. The composite nucleotide sequence of human osteonectin has a total length of 2091 nucleotides and is comprised of 50 nucleotides of 5'-noncoding sequence, a coding segment for 303 amino acids, a termination codon, and 1114 nucleotides of 3'-noncoding sequence. The primary transcript codes for 286 amino acids of mature protein and a 17-residue amino-terminal hydrophobic signal peptide. Outstanding properties inferred from the primary structure are putative Ca2+ binding domains located in the glutamic acid rich NH2 terminus (residues 1-52) and two "EF"-hand domains in the C-terminal half of the protein (residues 165-176 and 257-286). The mature protein also contains a cysteine-rich, highly hydrophilic region homologous to the ovomucoid serine protease inhibitors (residues 76-132). Overlapping human genomic clones in lambda EMBL3 for osteonectin have been isolated and characterized. Intron/exon junction sequencing of the human osteonectin gene shows the presence of 10 exons and 9 introns. The mature protein is encoded by nine exons separated by eight introns.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Osteonectin cDNA sequence reveals potential binding regions for calcium and hydroxyapatite and shows homologies with both a basement membrane protein (SPARC) and a serine proteinase inhibitor (ovomucoid).

Osteonectin is a prominent noncollagenous protein of developing bone. A 2150-base-pair cDNA coding for osteonectin, isolated from a bovine bone cell lambda gt11 expression library, was sequenced and identified by comparison with protein sequence data. The nucleotide sequence predicts that osteonectin contains 304 amino acids, including a 17-residue signal peptide. Analysis of the deduced protein sequence suggests that the secreted protein contains at least four distinct structural domains. An acidic region at the amino terminus of the protein appears to be a potential hydroxyapatite-binding site. This is followed by a second domain, rich in cysteine, that shows sequence homology with cysteine-rich domains in turkey ovomucoid and other serine proteinase inhibitors. Two sequences homologous with central calcium-binding loops of "EF hands" and thus having potential to be high-affinity calcium-binding sites are located in two other domains within the carboxyl-terminal half of the protein. Finally, the osteonectin sequence shows near identity (greater than 90%) with another protein, SPARC (secreted protein, acidic and rich in cysteine), secreted by mouse parietal endoderm. These data suggest that osteonectin, a protein present in bone and other selected tissues, is a multifunctional protein.

Amino Acid Sequence↗
PubMed1995

Transglutaminase-catalyzed matrix cross-linking in differentiating cartilage: identification of osteonectin as a major glutaminyl substrate.

The expression of tissue transglutaminase in skeletal tissues is strictly regulated and correlates with chondrocyte differentiation and cartilage calcification in endochondral bone formation and in maturation of tracheal cartilage (Aeschlimann, D., A. Wetterwald, H. Fleisch, and M. Paulsson. 1993. J. Cell Biol. 120:1461-1470). We now demonstrate the transglutaminase reaction product, the gamma-glutamyl-epsilon-lysine cross-link, in the matrix of hypertrophic cartilage using a novel cross-link specific antibody. Incorporation of the synthetic transglutaminase substrate monodansylcadaverine (amine donor) in cultured tracheal explants reveals enzyme activity in the pericellular matrix of hypertrophic chondrocytes in the central, calcifying areas of the horseshoe-shaped cartilages. One predominant glutaminyl substrate (amine acceptor) in the chondrocyte matrix is osteonectin as revealed by incorporation of the dansyl label in culture. Indeed, nonreducible osteonectin-containing complexes of approximately 65, 90, and 175 kD can be extracted from mature tracheal cartilage. In vitro cross-linking of osteonectin by tissue transglutaminase gives similar products of approximately 90 and 175 kD, indicating that the complexes in cartilage represent osteonectin oligomers. The demonstration of extracellular transglutaminase activity in differentiating cartilage, i.e., cross-linking of osteonectin in situ, shows that tissue transglutaminase-catalyzed cross-linking is a physiological mechanism for cartilage matrix stabilization.

Antibody Specificity↗
PubMed1992

Osteonectin in osteosarcomas: a marker for differential diagnosis and/or prognosis?

Seventy-five osteosarcomas at various grades of histologic differentiation were investigated for evidence of osteonectin. According to the results of the study, osteonectin was present in all osteosarcomas. An association between the intensity of the osteonectin antibody reaction and prognosis could not be established. Evidence of osteonectin was also found in other bone tumors. Osteonectin is therefore unsuitable for differential diagnosis, cannot be regarded as a bone specific protein and has not prognostic value.

Biomarkers, Tumor↗
PubMed2002

Appearance of osteonectin-expressing fibroblastic cells in early rat stomach carcinogenesis and stomach tumors induced with N-methyl-N'-nitro-N-nitrosoguanidine.

The present study was designed to define molecular alterations in the initiation stage of rat stomach carcinogenesis. Groups of male Lewis rats, 6 weeks old, were given drinking water with or without N-methyl-N'-nitro-N-nitrosoguanidine (MNNG; 100 mg/liter). Total RNA was isolated from the stomach pyloric mucosa, and fluorescent differential display analysis was performed. A cDNA fragment of 125 bp encoding an extracellular matrix-associated matricellular glycoprotein, osteonectin, was identified after 14 days of MNNG exposure. A severalfold increase in expression was observed after 14 and 27 days of MNNG exposure, as determined by northern blot and RT-PCR. Immunohistochemistry revealed that osteonectin-mAb-stained fibroblastic cells appeared in interstitial tissue of pyloric mucosa. Additionally the gene expression of other extracellular matrix proteins, viz., collagen type III, fibronectin, osteopontin, proteoglycan NG2, laminin gamma1 and S-laminin, was also markedly increased, as determined by competitive RT-PCR after 14 days of MNNG exposure. The gene expression of osteonectin and the six other extracellular matrix proteins was elevated in twelve stomach adenocarcinomas and adenomas induced by MNNG in Lewis and WKY rats. Osteonectin-mAb-stained fibroblastic cells were evident in interstitial tissue of stomach tumor. These results suggest that osteonectin-expressing fibroblastic cells appear in the interstitial tissue of pyloric mucosa from the early initiation stage of rat stomach chemical carcinogenesis, and that this phenomenon probably plays a role in cancer development.

Animals↗
PubMed1985

Specific immunohistochemical localization of osteonectin and collagen types I and III in fetal and adult porcine dental tissues.

Affinity-purified antibodies have been used in combination with the peroxidase-antiperoxidase technique to study the distribution of osteonectin and collagen types I and III in porcine dental tissues. Tissue sections (2 mm thick), including unerupted (fetal) or erupted (adult) teeth, were fixed in periodate-lysine-paraformaldehyde, demineralized in 12% w/v ethylenediaminetetraacetic acid, and after embedding, 6 micron sections were prepared for immunolocalization. Strong staining for osteonectin was observed in dentine of unerupted teeth and in the associated alveolar bone. Light to moderate staining was observed in the dental pulp, stratum intermedium, stellate reticulum, and the reticular elements in the endosteal spaces. In erupted teeth, osteonectin staining in dentine was concentrated around dentinal tubules and the associated alveolar bone stained with variable intensity. Cementum was poorly stained. However, the periodontal ligament and reticular material in the endosteal spaces showed moderate to strong staining. Weaker staining was apparent in the pulp and lamina propria of the gingiva. In comparison, type I collagen showed a similar distribution to osteonectin in both fetal and adult tissues, whereas type III collagen was generally restricted to the periodontal ligament, reticular elements of the endosteal spaces, and Sharpey's fibers in bone and cementum. Both odontoblast and ameloblast layers in fetal tissues stained for osteonectin and type III collagen.

Animals↗
PubMed2002

Quantitative analysis of osteonectin mRNA in thyroid carcinomas.

Our recent study of the gene expression profile in thyroid carcinoma showed an overexpression of osteonectin mRNA, an extracellular matrix protein, in an anaplastic carcinoma. To confirm this, we measured the expression levels of osteonectin mRNA in 84 thyroid normal and tumor tissues, including five anaplastic carcinomas by realtime quantitative reverse-transcription PCR. Increased expression of osteonectin mRNA was observed in anaplastic carcinoma tissue. However, in five anaplastic carcinoma cell lines, no increase was observed in the expression levels of osteonectin mRNA. These findings suggest the possibility that increased expression of osteonectin mRNA in anaplastic carcinoma tissue may be due to its overexpression in stromal cells, but not in anaplastic carcinoma cells.

Carcinoma↗