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PubMed1998

BM-40 (osteonectin, SPARC) is expressed both in the epidermal and in the dermal compartment of adult human skin.

BM-40 (Osteonectin, SPARC) is the most abundant glycoprotein secreted by human osteoblasts. In situ hybridization studies on the expression of BM-40 mRNA in murine tissues have demonstrated the highest levels of transcripts in bone, but expression was also observed in several other mesenchymal tissues. In contrast, little is known about the expression of BM-40 in human tissues, especially in skin. Total RNA obtained from normal human skin was analyzed by northern blotting and revealed a marked expression of BM-40. To analyze its expression in vivo, in situ hybridization was performed, demonstrating that BM-40 is expressed in fibroblasts, smooth muscle, and endothelial cells in the dermis. Interestingly, BM-40 mRNA was also detected throughout the basal, spinous, and granular layers in the epidermis of adult human skin. Further analysis by immunohistochemistry revealed a marked deposition in the dermis that was most intense directly below the basement membrane in the papillary dermis and around vascular as well as glandular structures. In the epidermis, BM-40 protein could be detected intercellularly in suprabasal layers. This finding is further supported by the intercellular deposition of BM40 detected by immunofluorescence in cultured keratinocytes. This study demonstrates that BM-40 that has previously been thought to be exclusively expressed in extracellular matrix producing cells may in fact play a role in differentiation and maintenance of the epidermis.

Adult↗
PubMed1984

Osteonectin, bone proteoglycan, and phosphophoryn defects in a form of bovine osteogenesis imperfecta.

Bovine osteogenesis imperfecta is a congenital disease in Holstein cattle having several characteristics in common with human osteogenesis imperfecta syndromes. In particular, affected calves have multiple bone fractures and friable teeth. Bone collagen isolated from the affected animals (Texas variant) showed slightly decreased alpha 1(I) and alpha 2(I) chain electrophoretic mobility and increased hydroxylysine content. Overall collagen was present in the affected bones at 80-90% of normal values. However, osteonectin, a 32,000 Mr bone-specific protein found previously to promote collagen mineralization in vitro and present in abundance (approximately equal to 3% of total protein) in normal calf bone, was severely depleted (less than 2% of normal levels) in the osteogenesis imperfecta bone and dentin. The bone proteoglycan was similarly depleted. In contrast, the bone sialoprotein was not as severely affected. Further, the diseased teeth lacked (less than 10% of normal values) phosphophoryn, a dentin-specific protein normally present as 4-5% of the total calf dentin matrix. The data suggest multiple hard tissue matrix protein deletions, perhaps due to impaired cell development.

Amino Acids↗
PubMed1995

Two adjacent N-terminal glutamines of BM-40 (osteonectin, SPARC) act as amine acceptor sites in transglutaminaseC-catalyzed modification.

The extracellular matrix protein BM-40 (osteonectin, SPARC) has recently been shown to be a major target for transglutaminase-catalyzed cross-linking in differentiating cartilage. In the present study we demonstrate that recombinant human BM-40 can be modified with [3H]putrescine in a 1:1 molar ratio by transglutaminaseC (tissue transglutaminase). Residues Gln3 and Gln4 were identified as major amine acceptor sites. This was confirmed with several mutant proteins, including deletions in the N-terminal domain I of BM-40, site-directed mutagenesis of the reactive glutamines, and fusion of the seven-amino acid-long N-terminal sequence (APQQEAL) to an unrelated protein. The results showed that the N-terminal target site is sufficient for modification by transglutaminase but at a low level. For high efficiency amine incorporation an intact domain I is required. The conservation of at least one of the transglutaminase target glutamines in the known vertebrate BM-40 sequences and their absence in an invertebrate homologue point to an important, but yet unknown, role of this modification in vertebrates.

Amino Acid Sequence↗
PubMed1996

Elements within the first 17 amino acids of human osteonectin are responsible for binding to type V collagen.

The region in human osteonectin (ON) responsible for binding to type V collagen has been identified as the first 17 NH2-terminal residues. This conclusion is based upon binding studies with deletion mutants of ON produced in Escherichia coli, in which parts of the first 17 amino acids have been removed. Wild-type ON from E. coli and mammalian cell-derived nonglycosylated ON bind identically to type V collagen and at least twice as effectively as mammalian cell-derived N-glycosylated ON. In previous studies, it was shown that N-glycosylation at residue 99 significantly reduces the capacity of ON to bind to type V collagen. Results reported in this communication demonstrate that the actual binding site on ON for type V collagen is distal from the site of N-glycosylation in terms of amino acid sequence but may be proximal in the folded, fully glycosylated, three-dimensional structure. Consistent with this conclusion is the ability of a synthetic peptide consisting of amino acids 1-17 to specifically inhibit the binding of ON to type V collagen.

Amino Acid Sequence↗
PubMed1988

Developmental expression of 2ar (osteopontin) and SPARC (osteonectin) RNA as revealed by in situ hybridization.

2ar has been identified as a gene inducible by tumor promoters and growth factors in a variety of cultured mouse cell lines (Smith, J. H., and D. T. Denhardt. 1987. J. Cell. Biochem. 34:13-22). Sequence analysis shows that it codes for mouse osteopontin, an RGDS-containing, phosphorylated, sialic acid-rich Ca++-binding protein originally isolated from bone (Oldberg, A., A. Franzen, and D. Heinegard. 1986. Proc. Natl. Acad. Sci. USA. 83:8819-8823; Prince, C. W., T. Oosawa, W. T. Butler, M. Tomana, A. S. Brown, and R. E. Schrohenloer. 1987. J. Biol. Chem. 262:2900-3907.). In this paper we use Northern blot analysis and in situ hybridization to localize expression of 2ar during mouse embryogenesis. 2ar RNA is first detected in developing limb bones and calvaria at 14.5 d p.c., in a population of cells distinct from those expressing SPARC (osteonectin). High levels of 2ar expression are also seen in the bone marrow-derived granulated metrial gland cells of the deciduum and placenta, and in a number of epithelial tissues, including embryonic and postnatal kidney tubules, uterine epithelium and sensory epithelium of the embryonic ear. The temporal and spatial pattern of 2ar expression seen in vivo suggests that the protein plays a wider role than previously realized, in processes which are not confined to bone development.

Animals↗
PubMed1989

Restriction fragment length polymorphisms for growth hormone, prolactin, osteonectin, alpha crystallin, gamma crystallin, fibronectin and 21-steroid hydroxylase in cattle.

Genomic DNAs from animals representing six breeds of cattle (Angus, Brahman, Hereford, Holstein, Jersey and Texas Longhorn) were screened with cloned gene probes in a search for restriction fragment length polymorphisms (RFLPs). Eleven RFLPs were identified using seven different probes: growth hormone, prolactin, osteonectin, alpha A-crystallin, gamma crystallin, fibronectin and 21-steroid hydroxylase. The frequencies of the alleles identified by each probe were calculated and compared in a limited sampling of the six bovine breeds. These polymorphisms greatly enhance the pool of immunogenetic, biochemical and molecular markers available in cattle for linkage analysis, testing of parentage, and distinction of breeds.

Alleles↗
PubMed1987

Distinct patterns of glycosylation of colligin, a collagen-binding glycoprotein, and SPARC (osteonectin), a secreted Ca2+-binding glycoprotein. Evidence for the localisation of colligin in the endoplasmic reticulum.

Mouse parietal endoderm PYS cells were labelled with [2-3H]mannose for 16-24 h. Colligin, an Mr-47000 collagen-binding protein, and SPARC, a Mr-43000 protein, highly homologous to the Ca2+-binding protein osteonectin, were isolated from labelled cell extracts and culture medium respectively. Glycopeptides obtained by exhaustive digestion with pronase were analysed by lectin-affinity, ion-exchange, and gel-filtration chromatography and by paper chromatography of high-mannose oligosaccharides after endo H release. The results show that the N-linked carbohydrate chains of colligin are exclusively the high-mannose type, of which (Man)8(GlcNAc)2 and (Man)9(GlcNAc)2 make up 77%. This carbohydrate structure provides strong evidence that colligin is a component of the endoplasmic reticulum, and argues against a role in cell-surface interactions. By contrast to colligin, SPARC secreted by PYS cells contains predominantly a diantennary complex type of chain containing a variable number of sialic acid and core-substituted fucose residues. Similar glycosylation patterns to those discussed above were seen in colligin isolated from primary mouse embryonic parietal endoderm cells and the murine 3T3 cell line, and in SPARC secreted by bovine corneal endothelial cells. Unlike the type-IV-collagen-binding glycoprotein studied by Dennis, J., Waller, C. and Schirrmacher, V. [J. Cell Biol. 99, 1416-1423 (1984)], removal of N-linked oligosaccharides from colligin had no effect on its binding to native type IV collagen.

Animals↗
PubMed1992

Heat-shock response in cultured chick embryo chondrocytes. Osteonectin is a secreted heat-shock protein.

We investigated the induction of specific protein expression by heat shock in dedifferentiated and hypertrophic chick embryo chondrocytes in a culture system that allows 'in vitro' differentiation of cartilage cells [Castagnola, P., Moro, G., Descalzi-Cancedda, F. and Cancedda, R. (1986) J. Cell. Biol. 102, 2310-2317]. As control, we used cultures of embryonic fibroblasts from the whole body and from the skin. In the cell lysates of all cultures we identified four major heat-shock proteins (HSP), with a molecular size corresponding to HSP families previously described (HSP 90, HSP 70, HSP 47 and HSP 26). Some of these proteins were constantly induced when the temperature was raised, others were expressed in a more variable manner. Differences also existed in the relative amount of the HSP synthesized by the four cultures. When we specifically investigated HSP species released into the culture medium, we observed a 43-45 kDa protein constantly expressed and secreted in large amount by the cells. On the basis of its biochemical characteristic and its precipitation by specific antibodies, this protein has been identified as osteonectin (SPARC, BM-40).

Animals↗
PubMed2005

Osteonectin is a Schwann cell-secreted factor that promotes retinal ganglion cell survival and process outgrowth.

We have investigated the factors made by Schwann cells (SCs) that stimulate survival and neurite outgrowth from postnatal rat retinal ganglion cells (RGCs). These effects are preserved under K252a blockade of the Trk family of neurotrophin receptors and are not fully mimicked by the action of a number of known trophic factors. To identify novel factors responsible for this regenerative activity, we have used a radiolabelling assay. Proteins made by SCs were labelled radioactively and then fed to purified RGCs. The proteins taken up by the RGCs were then isolated and further characterized. Using this assay we have identified a major 40 kDa factor taken up by RGCs, which was microsequenced and shown to be the matricellular protein osteonectin (ON). Using an in vitro assay of purified RGCs we show that ON promotes both survival and neurite outgrowth. We conclude that ON has a potential new role in promoting CNS repair.

Animals↗
PubMed2004

Osteopontin but not osteonectin messenger RNA expression is a prognostic marker in curatively resected non-small cell lung cancer.

PURPOSE: The purpose of this study was to better define the role of osteopontin (OPN) and osteonectin [also known as secreted protein acidic and rich in cysteine (SPARC)] in lung tumorigenesis by comparing the expressions of these genes in lung tumor tissue and matched normal tissue and by determining the prognostic significance of the gene expressions. EXPERIMENTAL DESIGN: Quantitative real-time reverse transcription-PCR was used to analyze OPN and SPARC mRNA expression in normal lung tissue and matching tumor samples from 82 patients with non-small cell lung cancer. Gene expression data for each patient were matched to survival data. RESULTS: The overall median mRNA expression level of OPN was about 20-fold higher in tumor tissues than in matching normal lung tissues (P < 0.001), whereas SPARC gene expression was not significantly different in both tissue types. Forty of 82 patients had high (>or=4.1) intratumoral OPN expression, and 15 of 82 patients had high (>or15.5) SPARC expression. High OPN expression in the tumor tissue was associated with inferior survival (P = 0.014), whereas high SPARC expression showed a trend toward longer survival (P = 0.095). The impact of high OPN and low SPARC expression on patient survival was additive (P = 0.001). CONCLUSIONS: The large increase in OPN expression in tumors compared with normal tissue and its association with survival suggest a role for OPN in lung tumorigenesis.

Aged↗
PubMed1994

Site-specific expression of mRNAs for osteonectin, osteocalcin, and osteopontin revealed by in situ hybridization in rat periodontal ligament during physiological tooth movement.

We investigated the gene expression for non-collagenous proteins in periodontal ligament (PDL) by in situ hybridization histochemistry with a non-radioisotopic probe with cRNAs for osteocalcin (Osc), osteonectin (Osn), and osteopontin (Opn) in rat maxillary dento-alveolar unit containing molars and intact PDL. A highly intense positive signal for Osn and Osc mRNAs was expressed at all distal surfaces of the interradicular septum of buccal roots of the upper second molar in 7-week-old Sprague-Dawley male rats. Cells showing positive signals for Osn and Osc mRNAs were osteoblasts and osteoprogenitor cells. The distribution of Opn mRNA-positive signal was demonstrable at the mesial surface of the interradicular septum of buccal roots, where physiological bone resorption was specifically restricted during physiological tooth movement. Opn mRNA was expressed in cells on the bone resorption surface, including osteoclasts, and osteocytes. A moderately intense positive signal for Osn mRNA was distributed in fibroblasts throughout the ligament. Odontoblasts and pre-mature odontoblasts exhibited a strong signal for Osn and Osc mRNA. Cementoblasts and cementocytes were positive for Osn, Osc, and Opn mRNAs. These findings suggest physiological roles of Osc, Osn, and Opn in bone remodeling, PDL remodeling, dentinogenesis, and cementogenesis.

Animals↗
PubMed1997

Expression of osteonectin in articular cartilage of osteoarthritic knees.

The expression of osteonectin (ON) in osteoarthritic articular cartilage was investigated by enzyme immunohistochemistry and colloidal gold immunoelectron microscopy. A total of 96 specimens from 9 knees of 8 patients with osteoarthritis (OA) were examined. In OA cartilage, ON-positive cells varied in distribution and were not seen in all the specimens obtained from the same patient. However, in over half of the specimens (56 of 96), especially in the specimens on Mankin's grades from 4 to 9, which corresponds to relatively early stages of OA, ON was expressed in the cartilage above the calcified layer. On the other hand, ON was detected only in the calcified layer below the tidemark in normal articular cartilage. In addition, colloidal gold immunoelectron microscopy revealed ON in chondrocytes and matrix vesicles (MVs). These findings suggest that ON acts through MVs in the early stages of OA as a significant pathogenetic factor involved in intracartilage calcification, which is known to have a close relationship to the progression of OA.

Aged↗
PubMed1998

Expression of osteopontin and osteonectin in breast cancer.

Osteopontin (OP) and osteonectin (ON) are bone matrix proteins produced by mammary and other cancers. These proteins may play a role in tumor invasion and metastasis through integrin-mediated signal transduction. We evaluated expressions of OP and ON in 253 resected infiltrating ductal carcinoma of the breast, using immunohistochemical staining and follow-up data. OP and ON were detected 87.4% and 54.2% of 253 cases, respectively. The OP and ON positive staining were localized in the cytoplasm of carcinoma cells. OP and ON did not correlate with various clinicopathological parameters, such as age, lymph node involvement, tumor size, histologic grade, expression of p53 and estrogen receptor (ER). In the multivariate model, lymph node involvement and histologic grade were statistically significant prognostic factors. Assessed by a log rank test, the 5-year-survival rates of OP and ON positive groups and their negative groups were not statistically different. In conclusion, OP and ON immunopositivity of infiltrating ductal carcinomas of the breast provide no additional prognostic information in this study.

Adult↗
PubMed2000

Synthesis of osteonectin by human retinal pigment epithelial cells is modulated by cell density.

PURPOSE: To determine whether human retinal pigment epithelial (HRPE) cells are able to synthesize the antiadhesive protein osteonectin, also known as secreted protein, acidic and rich in cysteine (SPARC). Additionally, because locally produced SPARC may modulate cellular behavior during tissue repair, to ascertain whether HRPE SPARC production and HRPE proliferation, migration, and/or differentiation are associated, in a simple HRPE wound-healing model. METHODS: Immunohistochemical and Western blot analyses of SPARC protein expression by low- and high-density cultured HRPE cells were undertaken. Total RNA extracted from cultures was studied by reverse transcription-polymerase chain reaction (RT-PCR) and Northern blot analysis. Western and Northern blot analyses were evaluated by densitometry. Experiments were repeated with HRPE cells cultured in the presence of 1, 10, or 100 microM of the differentiating agents butyric acid (BA) and retinoic acid (RA). RESULTS: HRPE cell cultures exhibited SPARC immunoreactivity. Western blot analysis of cell lysates and conditioned media showed a 43-kDa protein. RT-PCR and Northern blot analysis confirmed the presence of SPARC mRNA (with transcripts at 2.2 and 3.0 kb). Protein and mRNA transcript band densitometry revealed a higher proportion of SPARC protein and mRNA in high-density HRPE cell culture than in low-density culture. Neither BA nor RA (at the concentrations assessed) had a significant effect on SPARC production by HRPE cells in high- or low-density culture. CONCLUSIONS: HRPE can synthesize SPARC. Although the findings do not support an invariable association between SPARC production by HRPE and HRPE proliferation, migration, or differentiation, they demonstrate that synthesis of SPARC by HRPE is modulated by cell density.

Blotting, Northern↗
PubMed2005

[Effects of rotation on osteonectin and osteopontin mRNA level of cultured osteoblasts in rats].

Conditions of disuse such as bed rest, space flight, and immobilization result in decreased mechanical loading of bone, which is associated with reduced bone mineral density and increased fracture risk. Mechanisms involved in this process are not well understood except the suppression of osteoblast function. To investigate the effect of simulated weightlessness on mRNA level of extracellular matrix proteins, osteoblasts were rotated in horizontal plane as a model of simulated microgravity. Primordial osteoblasts of rats were grown for 2 d and then rotated for 24, 48 and 72 h, respectively. After isolating total RNA in cells, reverse transcription polymerase chain reaction (PT-PCR) was made to examine the mRNA level of osteopontin (OPN) and osteonectin (ON). Meanwhile, the levels of alkaline phosphatase (ALP) and osteocalcin (BGP) in the cultured medium were measured to evaluate the calcific function of cell. The expression of OPN and ON mRNA fell significantly after rotating for 24, 48 and 72 h, respectively. The contents of BGP descended significantly, meanwhile, the activity of ALP also showed a degressive tendency. Horizontal rotation decreased the expression of ON and OPN as well as diminished the secretion of BGP and ALP, which affected the calcific function of osteoblast. The results obtained suggest that depression of extracellular matrix proteins expression plays a key role in bone loss during weightlessness.

Animals↗
PubMed2006

Estradiol and resveratrol stimulating effect on osteocalcin, but not osteonectin and collagen-1alpha gene expression in primary culture of rat calvarial osteoblast-like cells.

Evidence is available that some endocrine disruptors, acting as selective estrogen receptor modulators (SERMs), interfere with osteoblast differentiation and function. Therefore, we investigated whether 17beta-estradiol, bisphenol-A (BSP), silymarin, genistein, resveratrol, procymidone, linurone and benzophenone-3 (BP3) modulate differentiation of rat calvarial osteoblast-like (ROB) cells in primary in vitro culture. Disruptors were added at day 18 of culture and cells were harvested 48 h later. Real time-PCR revealed that estradiol and resveratrol enhanced osteocalcin mRNA expression in ROB cells, while other disruptors were ineffective. The expression of osteonectin and collagen-1alpha was not affected by any disruptor. Estradiol, resveratrol, genistein and BSP stimulated the proliferative activity of ROB cells. In contrast, procymidone and linurone inhibited the proliferative activity, and silymarin and BP3 were ineffective. The conclusion is drawn that i) only resveratrol is able, like estradiol, to stimulate the specialized functions of ROB cells, and ii) the proliferative activity of ROB cells is more sensitive to endocrine disruptors, some of which could probably act via a mechanism independent of their SERM activity.

Animals↗
PubMed1989

Evidence for positive and negative regulatory elements in the 5'-flanking sequence of the mouse sparc (osteonectin) gene.

We have investigated the role of 5'-flanking DNA sequences in regulating the expression of the murine Sparc (osteonectin) gene in parietal endoderm cells and in F9 embryonal carcinoma cells induced to differentiate into parietal endoderm with retinoic acid and cyclic AMP. Varying lengths of flanking sequences extending up to 3.0 kilobase pairs 5' of the transcription initiation site were linked to the bacterial chloramphenicol transacetylase gene in the Bluescript M13- vector. The constructs were tested in transient assays, using a beta-galactosidase plasmid as a transfection control. Sequences between 78 and 169 base pairs upstream of the cap site are the minimum required for cell-type specific promoter activity; this region is dominated by two oligopurine/oligopyrimidine stretches or "GAGA" boxes and is highly conserved between the mouse and bovine genes. Addition of the sequence between -169 and -449, which includes part or all of a third GAGA box, results in increased parietal endoderm specific transcription, up to a maximum of 6.3-fold higher than in undifferentiated F9 cells. Further addition of sequences between -449 and -638 markedly reduces promoter activity in both cell types but parietal endoderm-specific activity is restored in constructs containing 2.2 and 3.0 kilobase pairs of flanking DNA. In addition, we have identified sequences related to the consensus sequence for steroid response elements, one of which is able to confer progesterone-enhanced transcription when tested with a heterologous promoter in steroid responsive cells. These results suggest that negative and positive elements normally interact to regulate the temporal and tissue-specific patterns of Sparc gene transcription seen in vivo.

Animals↗
PubMed1995

Increased expression of osteonectin and osteopontin, two bone matrix proteins, in human breast cancer.

Microcalcifications are a common phenomenon associated with breast cancer and are often the only mammographic sign of a malignant breast disease. Although microcalcifications are not restricted to breast cancer and can be also associated with benign lesions, it is noteworthy that they are composed exclusively of hydroxyapatite in breast carcinoma. Hydroxyapatite is the bone-associated phosphocalcic crystal the deposition of which in bone tissue requires the coordinated expression of several molecules such as osteonectin (OSN) and osteopontin (OPN), synthesized by cells of the osteoblastic lineage. In this study, we evaluated the expression of these two bone matrix proteins, using an immunoperoxidase technique and specific antibodies, in 79 breast lesions including 28 benign and 51 cancerous specimens. We found that normal mammary tissue associated with the lesions examined expressed generally undetectable or lightly detectable (0 or 1+) amounts of OSN and OPN (92 and 81%, respectively). Benign breast lesions, including fibroadenoma and fibrocystic dysplasia, were generally weakly stained (0 or 1+) with both anti-OSN and anti-OPN antibodies (96.4 and 60.7%, respectively). Interestingly, the majority of both in situ and invasive breast carcinoma lesions showed a strong expression (2+ or 3+) for OSN or OPN (74.5 and 84.3%, respectively). High expression of these two bone matrix proteins was associated with frequent microcalcification deposition in the lesion. This study is the first extensive study of OSN and OPN expression in mammary cancers. Our data suggest that OSN and OPN could play a role in the formation of ectopic microcalcifications often associated with breast cancer. It is also tempting to speculate that the expression of these two glycoproteins by breast cancer cells play a role in the preferred bone homing of breast metastases.

Amino Acid Sequence↗