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PubMed2005

Osteopontin expression in normal and fibrotic liver. altered liver healing in osteopontin-deficient mice.

BACKGROUND/AIMS: Osteopontin has been implicated in numerous physiopathological events. Osteopontin expression in normal and fibrotic liver and liver fibrogenesis in osteopontin-deficient mice were studied. METHODS: Fibrosis was induced in mice and rats by carbon tetrachloride (CCl4) treatment or bile duct ligation. The liver was used for conventional histology, osteopontin immunohistochemistry and in situ hybridization, or protein and RNA extraction. In mice, necrotic areas and fibrosis were evaluated by quantitative image analysis. RESULTS: In normal liver, osteopontin mRNA expression was very low. After CCl4 treatment or bile duct ligation, osteopontin mRNA expression was increased. Osteopontin was expressed by biliary epithelial cells in normal and fibrotic liver. Soon after the beginning of the CCl4 treatment, osteopontin was also present in inflammatory cells of the necrotic areas. In osteopontin-deficient mice, necrotic areas after a single dose of CCl4, and fibrosis after chronic CCl4 treatment were significantly increased as compared with wild-type treated mice. CONCLUSIONS: Our results show that osteopontin expression increases during liver fibrogenesis. Furthermore, osteopontin-deficient mice were more susceptible to CCl4 treatment, displaying more necrosis during the initial steps (probably due to a deficiency in nitric oxide production) and more fibrosis thereafter. The increase in osteopontin expression observed during liver fibrogenesis may play a protective role.

Animals↗

PubMed1995

The adhesive and migratory effects of osteopontin are mediated via distinct cell surface integrins. Role of alpha v beta 3 in smooth muscle cell migration to osteopontin in vitro.

Osteopontin is an arginine-glycine-aspartate containing acidic glycoprotein postulated to mediate adhesion, migration, and biomineralization in diverse tissues. The mechanisms explaining this multifunctionality are not well understood, although it is known that one osteopontin receptor is the alpha v beta 3 integrin. In this work, we studied human smooth muscle cells varying in alpha v beta 3 levels to identify additional osteopontin receptors. We report that, in addition to alpha v beta 3, both alpha v beta 5 and alpha v beta 1 are osteopontin receptors. Moreover, the presence or absence of alpha v beta 3 on the cell surface altered the adhesive and migratory responses of smooth muscle cells to osteopontin. Adhesion of alpha v beta 3-deficient cell populations to osteopontin was only half that of cells containing alpha v beta 3, and migration toward an osteopontin gradient in the Boyden chamber was dependent on cell surface alpha v beta 3. Although alpha v beta 3-deficient smooth muscle cells were unable to migrate to osteopontin, they did migrate significantly in response to vitronectin and fibronectin. These findings represent the first description of alpha v beta 5 and alpha v beta 1 as osteopontin receptors and suggest that, while adhesion to osteopontin is supported by integrins containing beta 1, beta 3, and beta 5, migration in response to osteopontin appears to depend on alpha v beta 3. Thus, interaction with distinct receptors is one mechanism by which osteopontin may initiate multiple functions.

Adult↗

PubMed2006

Papilloma development is delayed in osteopontin-null mice: implicating an antiapoptosis role for osteopontin.

Osteopontin is a secreted, adhesive glycoprotein, whose expression is markedly elevated in several types of cancer and premalignant lesions, implicating its association with carcinogenesis. To test the hypothesis that induced osteopontin is involved in tumor promotion in vivo, osteopontin-null and wild-type (WT) mice were subjected to a two-stage skin chemical carcinogenesis protocol. Mice were initiated with 7,12-dimethylbenz(a)anthracene (DMBA) applied on to the dorsal skin followed by twice weekly application of 12-O-tetradecanoylphorbol-13-acetate (TPA) for 27 weeks. Osteopontin-null mice showed a marked decrease both in tumor/papilloma incidence and multiplicity compared with WT mice. Osteopontin is minimally expressed in normal epidermis, but on treatment with TPA its expression is highly induced. To determine the possible mechanism(s) by which osteopontin regulates tumor development, we examined cell proliferation and cell survival. Epidermis from osteopontin-null and WT mice treated with TPA thrice or with DMBA followed by TPA for 11 weeks showed a similar increase in epidermal hyperplasia, suggesting that osteopontin does not mediate TPA-induced cell proliferation. Bromodeoxyuridine staining of papillomas and adjacent epidermis showed no difference in cell proliferation between groups. However, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analyses indicated a greater number of apoptotic cells in DMBA-treated skin and papillomas from osteopontin-null versus WT mice. These studies are the first to show that induction of the matricellular protein osteopontin facilitates DMBA/TPA-induced cutaneous carcinogenesis most likely through prevention of apoptosis.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed2000

[A study on development of enzyme immunoassay of human osteopontin: molecular fragility of serum osteopontin].

Osteopontin, a secreted acidic phosphoglycoprotein with calcium binding activity, has been reported to have diverse functions, including cell adhesion, calcium remodeling, cell signaling and cellular transformation. This recalls us to a concept that the osteopontin can be a marker for certain clinicopathological states. However, a method for analysis of osteopontin has not successfully been developed to be utilized. We have isolated osteopontin from human milk, prepared antibodies specific to it, and tried to establish an enzyme immunoassay (EIA) system to determine osteopontin in serum. It was found that serum osteopontin but not milk osteopontin showed paradoxical phenomena in reaction with the antibodies, i.e., no detectable reaction in immunoelectrophoresis and in EIA, but showing positive reaction in Western blot and in inhibition tests. Taking these phenomena and other circumstantial evidence into account, it was concluded that the serum osteopontin had high molecular fragility, fragmenting into small peptides with paucity of epitopes during incubation time. These findings indicate the possible presence of osteopontins with different molecular characteristics among tissues, and that these characteristics of osteopontin will interfere with the development of quantitative assay.

Antibodies↗

PubMed2000

Enhanced expression of osteopontin by high glucose. Involvement of osteopontin in diabetic macroangiopathy.

Atherosclerotic vascular disease is a major complication of diabetic patients. Osteopontin has recently been implicated in the development of atherosclerosis. In the present study, we have investigated the effects of high glucose on expression of osteopontin in cultured rat aortic smooth muscle cells. High concentrations of glucose increased osteopontin secretion from the cells, and the increased secretion was completely inhibited by an inhibitor of protein kinase C, GF109203X. Northern blot analysis confirmed the enhanced effect of glucose on expression of osteopontin mRNA. Promoter activity of osteopontin, measured using the osteopontin promoter/luciferase expression vector system, was increased by high glucose, and the enhanced effect was completely inhibited by GF109203X. Glucosamine also increased the promoter activity of osteopontin. Azaserine, an inhibitor of glutamine:fructose-6-phosphate amidotransferase, the key enzyme of the hexosamine pathway, profoundly inhibited high glucose-mediated increase in the promoter activity. Taken together, these data indicate that high glucose enhances the expression of osteopontin at the transcriptional level possibly through the activation of protein kinase C as well as the hexosamine pathway. Our results suggest that osteopontin could play a role in the development of diabetic vascular complications.

Animals↗

PubMed2006

Elevated plasma osteopontin levels were associated with osteopontin expression of CD4+ T cells in patients with unstable angina.

BACKGROUND: Plaque instability in patients with unstable angina (UA) is associated with stimulated CD4+ T cells, so the present study investigated whether there is a relationship among plaque instability, osteopontin and CD4+ T cells. METHODS AND RESULTS: Peripheral blood mononuclear cells were collected from 51 consecutive patients with UA, 60 patients with stable angina (SA), and 39 patients with chest pain syndrome (CPS). Osteopontin-producing CD4+ T cells were quantified by flow cytometry. Plasma osteopontin levels (ng/ml) were measured by ELISA and were higher in patients with UA (792.0 +/- 316.7) than in those with SA (626.0 +/- 195.0, p < 0.005) or CPS (594.7 +/- 239.4, p < 0.005). The frequency (%) of osteopontin-producing CD4+ T cells was higher in patients with UA (26.7 +/- 13.3) than in those with SA (19.5 +/- 11.1, p < 0.05) or CPS (16.6 +/- 9.0, p < 0.005). Furthermore, the plasma osteopontin level correlated with the frequency of osteopontin-producing CD4+ T cells (r = 0.327, p = 0.0004), as did the high-sensitivity C-reactive protein level (r = 0.360, p = 0.0002). CONCLUSIONS: The plasma osteopontin levels are elevated in patients with UA, accompanied by an increase in the number of osteopontin-production of circulating CD4+ T cells. Circulating CD4+ T cells may play a role through osteopontin in the pathophysiology of UA.

Aged↗

PubMed1994

Inhibition of Arg-Gly-Asp (RGD)-mediated cell adhesion to osteopontin by a monoclonal antibody against osteopontin.

Osteopontin (OPN), a secreted phosphoprotein, has been implicated in various biological phenomena (e.g. bone development, sepsis, tumor progression, and metastasis). Its role in any context is poorly understood. OPN contains a conserved Gly-Arg-Gly-Asp-Ser (GRGDS) sequence, and binds to cells via integrin-mediated mechanisms. Using recombinant human osteopontin-glutathione S-transferase fusion protein and our improved hybridoma fusion partner (Sp2/mIL6), we raised murine monoclonal antibodies against osteopontin. We characterized two antibodies that recognize not only recombinant but also native human osteopontin. These antibodies do not cross-react with mouse osteopontin (recombinant protein or that secreted by ras-transformed NIH 3T3 cells), or bovine bone osteopontin, suggesting that they recognize epitopes unique to human OPN. One antibody specifically inhibited adhesion of MDA-MB-435 human breast cancer cells and ras-transformed NIH 3T3 cells to human osteopontin. This antibody failed to recognize osteopontin cleaved by thrombin, which cleaves adjacent to the cell binding domain. We previously showed that thrombin cleavage reduces osteopontin cell binding activity. Thus we postulate that this monoclonal antibody recognizes and interferes with the function of the RGD/thrombin cleavage region of human OPN.

3T3 Cells↗

PubMed2004

In vitro apatite induction by osteopontin: interfacial energy for hydroxyapatite nucleation on osteopontin.

Noncollagenous phosphoproteins that interact with type I collagen are thought to nucleate the mineral phase to the collagen fibril network of mineralized tissues. We previously reported that a specific dentin phosphoprotein, phosphophoryn, crosslinked to an insoluble substrate such as type I collagen fibrils, was an effective nucleator of apatite. In this study, we investigated the capacity of another phosphoprotein in dentin, osteopontin, for apatite nucleation in vitro. Osteopontin purified from bovine milk was either absorbed to agarose beads or crosslinked to agarose beads. Each preparation was incubated at 37 degrees C in metastable solutions. Apatite was induced by osteopontin that was crosslinked to agarose beads, whereas osteopontin adsorbed to agarose beads failed to induce apatite crystal formation. Using classical nucleation theory, the interfacial energy for hydroxyapatite nucleation on osteopontin crosslinked to agarose beads was determined to be 94.7 ergs/cm(2). This value is larger than that for phosphophoryn, though it is similar to that for hydroxyapatite. The present study indicates that osteopontin, like phosphophoryn, has a high potential to induce apatite formation when it is covalently bound to some substrate in vitro, and suggests the possibility that osteopontin bound to type I collagen may induce apatite formation in vivo.

Animals↗

PubMed1992

Isolation and characterization of a cDNA for osteopontin-k: a kidney cell adhesion molecule with high homology to osteopontins.

Screening of a bovine renal cDNA library with MAbs resulted in the isolation of a 1447 bp cDNA. This cDNA (pBk2.1) was sequenced and shown to contain an open reading frame with a putative protein of 261 amino acids, with a molecular weight of 29,573 (minute leader sequence) and a hydrophobic leader sequence of 16 amino acids. pBk2.1 was shown to share a high level of nucleic acid sequence homology over portions of its sequence to human, porcine, mouse, and rat osteopontins (40-60%). The peptide (osteopontin-k) had a potential glycosylation site (Asn-X-Ser/Thr), a GRGDS receptor binding region, a high level of asparagine residues, and a high abundance of acid amino acids characteristic of osteopontin-like cell adhesion molecules. The N-terminal amino acid region of pBk2.1 (the first 82 amino acids) and 42 amino acids at the C terminus had the highest level of homology with the osteopontins at 86%. The middle portion of the peptide had greatly reduced homology, ranging from 50% (amino acids 83-174) to 12% (amino acids 175-219). There were also deletions and additions of sequence in osteopontin-k that were not found in the other osteopontins. The homologies suggest that these proteins are highly related and may be derived from a common gene by alternative splicing. A 678 bp cRNA probe constructed from pBk2.1, containing a region with low homology to the osteopontins (amino acids 183-219 with less than 20% homology, plus amino acids 220-261 and untranslated sequence), was used in northern blots and RNAse protection assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2002

Promotion of malignant astrocytoma cell migration by osteopontin expressed in the normal brain: differences in integrin signaling during cell adhesion to osteopontin versus vitronectin.

The extracellular matrix of the normal adult brain lacks expression of most of the adhesive glycoproteins that are known to promote cell attachment, and it has been thought that the malignant invasion of astrocytoma tumor is mediated primarily by remodeling of the matrix by the tumor cells. It has been reported, however, that normal brain neuropil does contain a protein(s) that promotes cell attachment. Therefore, we explored the possibility that the cell attachment protein, osteopontin, is expressed in the normal human brain. Here, we report that osteopontin is expressed in the cortical gray and white matter of normal adult brain, with the levels of osteopontin expression being equivalent to those in malignant astrocytic tumor biopsies as assessed by Western blot analysis. Immunoblotting identified osteopontin polypeptides with relative molecular weights of 60- and 65-kDa in normal brain white matter and in astrocytic tumors, with an additional 70-kDa polypeptide being identified in normal cortical gray matter and in some astrocytic tumors. Recombinant osteopontin was found to promote attachment of U-251MG human malignant astrocytoma cells in a process that was inhibited by anti-integrin monoclonal antibodies anti-alphavbeta3 (75%), anti-alphavbeta5 (80%), and anti-alpha5 (40%). On attachment, integrins alphavbeta5 and alphavbeta3 localized to focal adhesions, and there was an alteration in cell morphology with the formation of lamellae-like processes. The attachment was associated with activation of Rac in a slow and prolonged fashion and rapid activation of Rho. Similarly, integrins alphavbeta5 and alphavbeta3 localized to focal adhesions on attachment of the U-251MG cells to vitronectin, but on this substrate, the cells assumed a spread and flat morphology, and there was rapid activation of both Rac and Rho. Extracts of normal brain white matter were capable of promoting haptotactic migration, and this response was inhibitable by monoclonal antibodies anti-alphavbeta3 and anti-alpha5. Depletion of the osteopontin in these extracts abrogated the haptotactic response significantly (50%). These data indicate that the cell attachment protein, osteopontin, is expressed in the normal adult brain and that it has the potential to promote malignant astrocytoma cell invasion.

Animals↗

PubMed1991

cDNA cloning and gene expression of chicken osteopontin. Expression of osteopontin mRNA in chondrocytes is enhanced by trypsin treatment of cells.

A cDNA clone, pCP15, specific for the chicken 66-kDa major bone phosphoprotein (osteopontin), was isolated from a subtracted library enriched in DNAs coding for mRNAs expressed in chicken differentiating chondrocytes. Northern blot analysis of RNAs extracted from several chick embryo tissues and organs, confirm and extend the observation that osteopontin mRNA expression is not restricted to tissues involved in phosphate metabolism. Osteopontin mRNA was detected in sternal resting chondrocytes at higher levels than in hypertrophic chondrocytes; therefore osteopontin gene transcription occurs in chondrocytes at many stages of differentiation. The steady state level of osteopontin mRNA was enhanced by trypsin treatment of cultured cells. An increased level of osteopontin mRNA in quail chondrocytes constitutively expressing v-myc oncogene is also shown.

Amino Acid Sequence↗

PubMed1994

Localization of osteopontin in resorption lacunae formed by osteoclast-like cells: a study by a novel monoclonal antibody which recognizes rat osteopontin.

The characteristics of a monoclonal antibody produced against osteoclast-like multinucleated cells (MNCs) formed in rat bone marrow cultures were examined immunohistochemically and biochemically. The in vitro immunization was performed using as immunogen the MNCs from rat bone marrow cell culture, which revealed many characteristics of osteoclasts. After screening and cloning of hybridomas, the monoclonal antibody HOK 1 was obtained. This antibody reacted weakly with stromal cells and intensely with both MNCs and their putative migratory traces on culture dishes. Immunofluorescent examination of paraffin sections revealed intense reactivity on the epithelium of the choroid plexus, the ileum and the proximal-convoluted tubules of the kidney, and also on bone cells such as osteocytes, osteoblasts, and osteoclasts. Western blotting using purified rat osteopontin verified that the antigen recognized by HOK 1 was osteopontin. Positive HOK 1 immunoreactivity was further observed in the resorption lacunae formed by a culture of MNCs on human tooth slices and on the surface of osteoclasts. The present data suggested that osteopontin is preferentially present on the resorption lacunae in resorbing calcified matrices and that osteoclasts under a specific state might trap this protein on their cell surface.

Animals↗

PubMed2000

Expression of osteopontin messenger RNA and protein in rheumatoid arthritis: effects of osteopontin on the release of collagenase 1 from articular chondrocytes and synovial fibroblasts.

OBJECTIVE: Osteopontin (OPN) is an extracellular matrix protein that has been implicated in the interactions between tumor cells and host matrix, including those involved in invasion and spread of tumor cells. Because joint destruction in rheumatoid arthritis (RA) is mediated by the invasive growth of synovial tissue through its attachment to cartilage, we examined the expression of OPN in the synovia of patients with RA and the effect of OPN on the production of collagenase 1 in rheumatoid synovial fibroblasts and articular chondrocytes. METHODS: The expression of OPN messenger RNA (mRNA) and protein in synovia from 10 RA patients was examined by in situ hybridization and immunohistochemistry. Synovial fibroblasts from RA patients and articular chondrocytes from patients without joint disease were cultured in the presence of various concentrations of OPN, and levels of collagenase 1 in the culture supernatants were measured by enzyme-linked immunosorbent assay. RESULTS: The expression of OPN mRNA and protein was observed in 9 of 10 specimens obtained from patients with RA. OPN was expressed in the synovial lining and sublining layer and at the interface of cartilage and invading synovium. Double labeling revealed that the majority of OPN-expressing cells were positive for the fibroblast-specific enzyme prolyl 4-hydroxylase and negative for the macrophage marker CD68, while only a few, single OPN-expressing cells were positive for CD68 at sites of synovial invasion into cartilage. OPN staining was not observed in lymphocytic infiltrates or leukocyte common antigen (CD45)-positive cells. Three of 3 cultures of human articular chondrocytes secreted detectable basal amounts of collagenase, with a dose-dependent increase upon OPN stimulation, while synovial fibroblast cultures produced much lower levels of collagenase, with only 2 of 4 fibroblast cultures responding in a dose-dependent manner. CONCLUSION: These findings suggest that OPN produced by synovial fibroblasts in the synovial lining layer and at sites of cartilage invasion not only mediates attachment of these cells to cartilage, but also contributes to matrix degradation in RA by stimulating the secretion of collagenase 1 in articular chondrocytes.

Aged↗

PubMed2002

Expression of osteopontin at sites of bone erosion in a murine experimental arthritis model of collagen-induced arthritis: possible involvement of osteopontin in bone destruction in arthritis.

OBJECTIVE: To investigate the involvement of osteopontin (OPN) in bone destruction in a murine experimental arthritis model of collagen-induced arthritis (CIA). METHODS: The expression of OPN was examined at both the messenger RNA (mRNA) and protein levels in various arthritic lesions in mice with CIA by in situ hybridization and immunohistochemistry, respectively. In addition, the expression of alpha(v)beta3 integrin, a receptor for OPN, the ligation of which is thought to be essential for bone resorption by osteoclasts, was examined by immunohistochemistry. Plasma concentrations of OPN were measured at different time points in the course of CIA by enzyme-linked immunosorbent assay. RESULTS: OPN mRNA was detected mainly at sites of bone erosion in arthritic lesions, where activated osteoclasts were present; OPN protein was also detected at sites of bone erosion. In the arthritic synovium, OPN was predominantly expressed in the synovial lining layer, but not in lymphoid aggregates. In addition, alpha(v)beta3 integrin was detected coincident with OPN at sites of bone erosion (bone-pannus junction). Plasma OPN levels were markedly elevated at the time points that corresponded to arthritis flares, and higher levels were maintained during the progression of arthritis. CONCLUSION: OPN may mediate bone resorption by osteoclasts in arthritis through ligation with its receptor, alpha(v)beta3 integrin. OPN may be a useful therapeutic target molecule in the prevention of bone destruction in arthritis.

Animals↗

PubMed2004

Osteopontin modulates CD44-dependent chemotaxis of peritoneal macrophages through G-protein-coupled receptors: evidence of a role for an intracellular form of osteopontin.

Expression of osteopontin (OPN) by activated T-cells and macrophages is required for the development of cell-mediated inflammatory responses. Acting through integrin alpha(v)beta(3) and CD44 receptors, OPN can promote chemoattraction and pro-inflammatory cytokine expression by macrophages. In this study, we have used peritoneal macrophages from OPN-/, CD44-/-, and WT mice to study the relationship between OPN and CD44 in macrophage migration. Using confocal microscopy, we show that OPN co-distributes with CD44 inside macrophages at cell edges and in cell processes in a mutually dependent manner. The existence of an intracellular form of OPN is supported by pulse-chase studies in which a thrombin-sensitive, phosphorylated protein immunoprecipitated with OPN antibodies is retained inside macrophages. In OPN-/- and CD44-/- macrophages, the absence of CD44 and OPN, respectively, is associated with the formation of fewer cell processes, reduced cell fusion required to form functional multinucleated osteoclasts in the presence of CSF-1 and RANKL, and impaired chemotaxis. Whereas the chemotaxis of CD44-/- cells to various chemoattractants is almost completely abrogated, a differential effect is seen with the OPN-/- cells. Thus, OPN-/- cells migrate normally towards CSF-1 but not towards fMLP and MCP-1, which signal through G-protein coupled receptors (GPCRs). That the GPCR-mediated migration is dependent upon the level of cell-surface CD44 is indicated by the reduced cell-surface expression of CD44 in OPN-/- cells and a comparable impairment in the chemotaxis of CD44+/- cells. Although chemotaxis of OPN-/- cells could be rescued by an OPN substratum, or by addition of high levels of OPN in solution, no response is evident with physiological levels of OPN, indicating a requirement for the CD44-associated intracellular OPN in CD44 cell-surface expression. These studies indicate, therefore, that the level of cell surface CD44 is critical for GPCR-mediated chemotaxis by peritoneal macrophages and suggest that a novel intracellular form of OPN may modulate CD44 activities involved in these processes.

Actins↗

PubMed2005

Mitigation of ectopic calcification in osteopontin-deficient mice by exogenous osteopontin.

Ectopic calcification is a major cause of bioprosthetic heart valve failure. New therapeutic opportunities are offered by the growing understanding that ectopic calcification is an actively regulated process involving several key gene products. One of these products, osteopontin (OPN), is a glycosylated phosphoprotein previously shown to inhibit apatite crystal formation, induce carbonic anhydrase II, and promote mineral resorption. In this study, OPN-deficient mice (OPN-/-) were utilized as an in vivo model to stimulate the ectopic calcification of glutaraldehyde-fixed bovine pericardium (GFBP) tissue and to examine OPN delivery and structure-function relationships with respect to its anti-calcific activity. Significant calcification of GFBP tissue was obtained within 7 days of subcutaneous implantation in OPN-/- mice. Direct rescue of the calcification phenotype was achieved by the administration of exogenous recombinant rat, histidine-fused OPN (rat His-OPN) to the implant site via soluble injection (up to 72% mitigation achieved) or adsorption onto the implant materials (up to 91% mitigation achieved). Effects were specific, since neither fibronectin nor polyhistidine alone could mitigate calcification of GFBP. The maximum anti-calcific effect was achieved only when rat His-OPN was adequately phosphorylated and contained a functional arginine-glycine-aspartate (RGD) cell adhesive domain. Furthermore, CAII levels in host cells surrounding GFBP were greatest when phosphorylated, RGD-containing rat His-OPN was adsorbed. These data suggest that both physical inhibition, mediated by phosphorylation sites in OPN, as well as the induction of CAII and mineral regression, mediated by the RGD domain, contribute to the unique ability of OPN to mitigate ectopic calcification of bioprosthetic valve tissue.

Animals↗

PubMed2002

Inactivation of the osteopontin gene enhances vascular calcification of matrix Gla protein-deficient mice: evidence for osteopontin as an inducible inhibitor of vascular calcification in vivo.

Osteopontin (OPN) is abundantly expressed in human calcified arteries. To examine the role of OPN in vascular calcification, OPN mutant mice were crossed with matrix Gla protein (MGP) mutant mice. Mice deficient in MGP alone (MGP(-/-) OPN(+/+)) showed calcification of their arteries as early as 2 weeks (wk) after birth (0.33 +/- 0.01 mmol/g dry weight), and the expression of OPN in the calcified arteries was greatly up-regulated compared with MGP wild-types. OPN accumulated adjacent to the mineral and colocalized to surrounding cells in the calcified media. Cells synthesizing OPN lacked smooth muscle (SM) lineage markers, SM alpha-actin and SM22alpha. However, most of them were not macrophages. Importantly, mice deficient in both MGP and OPN had twice as much arterial calcification as MGP(-/-) OPN(+/+) at 2 wk, and over 3 times as much at 4 wk, suggesting an inhibitory effect of OPN in vascular calcification. Moreover, these mice died significantly earlier (4.4 +/- 0.2 wk) than MGP(-/-) OPN(+/+) counterparts (6.6 +/- 1.0 wk). The cause of death in these animals was found to be vascular rupture followed by hemorrhage, most likely due to enhanced calcification. These studies are the first to demonstrate a role for OPN as an inducible inhibitor of ectopic calcification in vivo.

Animals↗

PubMed1999

Ovine osteopontin: II. Osteopontin and alpha(v)beta(3) integrin expression in the uterus and conceptus during the periimplantation period.

Osteopontin (OPN) is an acidic 70-kDa glycoprotein that is cleaved by proteases to yield 45-kDa and 24-kDa fragments. The 70-kDa and 45-kDa proteins contain a Gly-Arg-Gly-Asp-Ser (GRGDS) sequence that binds to cell surface integrins (primarily alpha(v)beta(3) heterodimer) to promote cell-cell attachment and cell spreading. A 70-kDa acidic protein was previously detected by two-dimensional (2D) PAGE in Day 17 pregnant endometrial cytosolic extracts using Stainsall and identified as immunoreactive OPN using Western blotting. Three forms of immunoreactive OPN proteins (70, 45, and 24 kDa) were detected by 1D PAGE and Western blot analysis of endometrial extracts. OPN protein in endometrial extracts did not differ between cyclic and pregnant ewes. However, the amount of 45-kDa OPN increased in uterine flushings from pregnant ewes between Days 11 and 17. Immunoreactive OPN was localized to luminal and glandular epithelia of both cyclic and pregnant ewes, and to trophectoderm of Day 19 conceptuses. The alpha(v) and beta(3) integrins were detected on Day 19 endometrium and conceptuses by immunofluorescence. It was reported that OPN mRNA increases in the uterine glands of pregnant ewes and secretion of OPN protein into the uterine lumen increases during early pregnancy. The present results demonstrate accumulation of OPN protein on endometrial LE and conceptus trophectoderm. Therefore, it is hypothesized that progesterone and/or interferon-tau induce expression, secretion and/or proteolytic cleavage of OPN by uterine epithelium. Secreted OPN is then available as ligand for alpha(v)beta(3) integrin heterodimer on trophectoderm and uterus to 1) stimulate changes in morphology of conceptus trophectoderm and 2) induce adhesion between luminal epithelium and trophectoderm essential for implantation and placentation.

Animals↗