Regulation of osteopontin gene expression in osteoblasts.
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Animals↗
Biomedical subjects
D T Denhardt
Publications and source records attributed to D T Denhardt.
Expression of antisense osteopontin RNA in metastatic mouse fibroblasts is associated with reduced malignancy.
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Animals↗
Activation of a signal transduction pathway by osteopontin.
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3T3 Cells↗
Osteopontin (OPN) may facilitate metastasis by protecting cells from macrophage NO-mediated cytotoxicity: evidence from cell lines down-regulated for OPN expression by a targeted ribozyme.
Osteopontin (OPN) is a GRGDS-containing phosphoglycoprotein that is capable of facilitating cell adhesion and modulating gene expression via integrin receptors. Three hammerhead ribozymes designed to target three different regions of OPN mRNA were shown to cleave the message catalytically in vitro. Plasmid vectors that had been engineered to express the ribozymes in mammalian cells were used to generate stably transfected T24 H-ras-transformed NIH3T3 cells that normally express OPN at high levels. Northern and Western blot analyses showed that OPN mRNA and protein expression were reduced in a subset of these anti-OPN ribozyme-expressing cell lines. Cells whose ability to produce OPN had been impaired exhibited greater sensitivity to the cytotoxic action of activated RAW264.7 macrophage-like cells; they were also less effective at suppressing macrophage NO production. In agreement with previous reports, they were also less tumorigenic and metastatic in an experimental metastasis assay. These results are consistent with the hypothesis that OPN serves as a defense against NO-mediated host cell cytotoxicity and thereby augments the metastatic phenotype.
Animals↗
Osteopontin expression detected in adult cochleae and inner ear fluids.
Localization of protein epitopes and mRNA expression showed that there was a wide-spread distribution of osteopontin (OPN) within the membranous labyrinth of the adult mammalian cochleae. Immunoreaction product and mRNA were found within the stria vascularis, VIIIth cranial nerve, spiral ligament and limbus. Only specific cell types within these regions contained abundant OPN mRNA or protein, the main cell type being fibrocytes that populate the spiral limbus and spiral ligament. Epithelial cells that line the luminal surface of the stria vascularis (marginal cells) and neurons that compose the vestibular and auditory ganglia also showed high opn expression. The pattern of anti-OPN staining within membranous labyrinth was comparable to that observed in tissues such as gall bladder, breast and kidney. In those tissues, luminal epithelial cells, corresponding to the marginal cells of the stria vascularis, may be responsible for manufacturing and secreting OPN into the luminal fluids. consistent with those observations, we detected OPN epitopes in cochlear fluids withdrawn from the scalae media and tympani of the cochlea. We found that the protein species in cochlear fluid differed from those present in cerebrospinal fluid (CSF) suggesting that OPN exists in tissue-specific isoforms that may correspond to particular cellular functions.
Animals↗
Identification of a ras-activated enhancer in the mouse osteopontin promoter and its interaction with a putative ETS-related transcription factor whose activity correlates with the metastatic potential of the cell.
The role of RAS in transducing signals from an activated receptor into altered gene expression is becoming clear, though some links in the chain are still missing. Cells possessing activated RAS express higher levels of osteopontin (OPN), an alpha v beta 3 integrin-binding secreted phosphoprotein implicated in a number of developmental, physiological, and pathological processes. We report that in T24 H-ras-transformed NIH 3T3 cells enhanced transcription contributes to the increased expression of OPN. Transient transfection studies, DNA-protein binding assays, and methylation protection experiments have identified a novel ras-activated enhancer, distinct from known ras response elements, that appears responsible for part of the increase in OPN transcription in cells with an activated RAS. In electrophoretic mobility shift assays, the protein-binding motif GGAGGCAGG was found to be essential for the formation of several complexes, one of which (complex A) was generated at elevated levels by cell lines that are metastatic. Southwestern blotting and UV light cross-linking studies indicated the presence of several proteins able to interact with this sequence. The proteins that form these complexes have molecular masses estimated at approximately 16, 28, 32, 45, 80, and 100 kDa. Because the approximately 16-kDa protein was responsible for complex A formation, we have designated it MATF for metastasis-associated transcription factor. The GGANNNAGG motif is also found in some other promoters, suggesting that they may be similarly controlled by MATF.
3T3 Cells↗
Establishment and characterization of bone marrow stromal cell lines that support osteoclastogenesis.
We established bone marrow stromal cell lines that support tartrate-resistant acid phosphatase-positive multinucleated cell [TRAcP(+)MNC] formation by using transgenic mice harboring simian virus 40 large T antigen gene. The morphology of these TM cell lines (large T-immortalized marrow cells) was spindle-like at sparse cell density, whereas it became smaller and cuboidal at confluence. The TM cell lines showed diverse ranges of activity in supporting TRAcP(+)MNC formation when they were examined in the cocultures with spleen cells in the presence of 1 alpha,25-dihydroxyvitamin D3. Among these cell lines, TM8 supported the TRAcP(+)MNC formation most efficiently (from 400-1500 cells/well) when cocultured with spleen cells. Another bone marrow-derived cell line, TM5, supported TRAcP(+)MNC formation in the coculture assay, whereas the efficiency was approximately one fifth that of TM8. Interestingly, TM8 cells also supported TRAcP(+)MNC formation even in the cocultures at low serum concentration (0.5% fetal bovine serum) with an efficiency yielding over 200 TRAcP(+)MNCs/well. TM8 cells expressed certain levels of macrophage colony-stimulating factor and stem cell factor messenger RNAs (mRNAs), but low levels of c-fms mRNA. Expression of c-kit mRNA in TM5 and TM8 cells was undetectable. 1 alpha,25-Dihydroxyvitamin D3 treatment enhanced the expression of osteopontin mRNA more than 10-fold in these cells, indicating the presence of the receptor for this steroid. These TRAcP(+)MNCs, which developed in the cocultures of the TM8 and spleen cells, formed pits when cultured on bone slices, indicating that they were capable of resorbing bone. The various levels of expression of these genes and the difference in the supporting activities for the TRAcP(+)MNC development in the diverse TM cell lines suggest the heterogeneity in the marrow cell populations in vivo regarding their activity in supporting osteoclastogenesis.
Acid Phosphatase↗
Structure-function studies of mouse tissue inhibitor of metalloproteinases-1.
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3T3 Cells↗
A human promyelocyte mRNA transiently induced by TPA is homologous to yeast IPP isomerase.
A cDNA clone was isolated from HL60 human promyelocyte cells on the basis of the induction of its cognate mRNA by the phorbol ester TPA (12-O-tetradecanoyl phorbol-13-acetate) in the presence of cycloheximide. Northern analysis showed a peak of induction of a 1.9-kb mRNA at 3-6 h after TPA addition. Sequence analysis revealed that this cDNA was likely a human homolog of yeast isopentenyl diphosphate:dimethylallyl diphosphate isomerase, an enzyme important for isoprenoid synthesis and the prenylation of proteins such as ras p21.
Amino Acid Sequence↗
Reduced malignancy of ras-transformed NIH 3T3 cells expressing antisense osteopontin RNA.
Osteopontin (OPN) is a secreted, calcium-binding phosphoprotein that frequently has been associated with the transformed phenotype. To clarify the function of OPN in tumor cells, we designed experiments to: (a) express antisense OPN RNA in murine PAP2 cells (metastatic, ras-transformed NIH 3T3 cells) and (b) examine the effects of antisense OPN expression on the tumorigenic and metastatic properties of the cells. PAP2 cells were transfected with pNMH-asOPN, an inducible, mammalian expression vector that can generate antisense OPN RNA complementary to the OPN mRNA. Two clones have been identified that expressed antisense OPN RNA in vitro. While reduced OPN protein secretion was not detected when the cells were grown in vitro, the in vivo expression of antisense OPN RNA was associated with reduced tumorigenicity. Tumors that did arise, with greatly extended lag time, had lost expression of antisense OPN RNA in vivo, suggesting that antisense OPN RNA expression was associated with reduced tumorigenicity of these cells.
3T3 Cells↗
Osteopontin inhibits induction of nitric oxide synthase gene expression by inflammatory mediators in mouse kidney epithelial cells.
We report that osteopontin (OPN), a secreted, Arg-Gly-Asp-containing phosphoprotein expressed at high levels in the kidney, suppresses nitric oxide (NO) synthesis induced by the inflammatory mediators gamma-interferon and lipopolysaccharide in primary mouse kidney proximal tubule epithelial cells. Northern blot and immunofluorescence analyses of inducible nitric oxide synthase (iNOS) expression revealed that the inflammatory mediators increased iNOS mRNA and protein levels. Recombinant human OPN (purified from both mammalian cells and from Escherichia coli) inhibited this response by a process that was blocked by anti-OPN antiserum and by the peptide GRGDS, but not GRGES. The data suggest that inhibition of NO synthesis by OPN in these kidney cells is mediated by an integrin, possibly the alpha v beta 3 integrin, which is known to be an OPN receptor. NO is believed to control blood flow through the glomerulus, regulating salt and water balance, and to be important as a defense against tumor cells and infecting microorganisms. The ability of OPN to inhibit the induction of iNOS suggests that OPN may be an important regulator of the NO signaling pathway and NO-mediated cytotoxic processes.
Amino Acid Oxidoreductases↗
The mitogen-regulated protein/proliferin transcript is degraded in primary mouse embryo fibroblast but not 3T3 nuclei: altered RNA processing correlates with immortalization.
An understanding of what changes occur in the control of gene expression when mammalian cells "spontaneously" immortalize is important to our knowledge of how cancer develops. We describe here an alteration in regulation that occurs when primary mouse embryo fibroblasts (MEFs) are immortalized according to a 3T3 regimen. Mitogen-regulated protein/proliferin mRNA is undetectable in northern blots of RNA from (mortal) MEFs, whereas it is readily detected in immortal 3T3 cell lines derived from the MEFs. Incompletely processed nuclear transcripts of the mitogen-regulated protein/proliferin gene can be detected in MEF RNA preparations by northern blotting and reverse transcriptase polymerase chain reaction analyses, although at roughly half the abundance observed in 3T3 cells. We hypothesize that some attribute of the primary unprocessed transcript determines its assignment to this unique degradative pathway. These results reveal that during passage of MEFs according to a 3T3 regimen the ability of the primary cells to suppress the expression of certain genes by degrading the nuclear transcript is lost concomitantly with immortalization.
3T3 Cells↗
Overcoming obstacles to metastasis--defenses against host defenses: osteopontin (OPN) as a shield against attack by cytotoxic host cells.
Osteopontin (OPN) serves both a cell attachment function and a cell signalling function via the alpha v beta 3 integrin. In its cell attachment capacity it can promote attachment of both osteoclasts to bone hydroxyapatite and various other cell types to basement membrane/extracellular matrix. In its cell signalling capacity it initiates a signal transduction cascade that includes changes in the intracellular calcium ion levels and the tyrosine phosphorylation status of several proteins including paxillin. Effects on gene expression include suppression of the induction of nitric oxide synthase by inflammatory mediators. OPN can also reduce cell oxidant levels and inhibit the killing of tumor cells by activated macrophages and endothelial cells. We hypothesize that those cancer cells that produce OPN at elevated levels can suppress the oxidative burst, inhibit NO production, and thus protect themselves from killing by specific host cell types.
Amino Acid Oxidoreductases↗
Age and development-related changes in osteopontin and nitric oxide synthase mRNA levels in human kidney proximal tubule epithelial cells: contrasting responses to hypoxia and reoxygenation.
Osteopontin (OPN) encodes a secreted glycosylated phosphoprotein containing a GRGDS motif that can mediate cell attachment through the alpha v beta 3 integrin, and has recently been shown to down-regulate nitric oxide synthase (NOS) expression. We report here that primary cultures of renal proximal tubule epithelial (PTE) cells prepared from human kidneys of different developmental stages and ages show a positive correlation between developmental age and the expression, at the mRNA level, of both OPN and constitutive NOS. However, OPN and NOS responded in different manners, as assessed by mRNA measurements, to hypoxia-reoxygenation injury. The OPN mRNA level, assessed by Northern blotting, increased slightly during 60 min of hypoxia and more substantially during subsequent reoxygenation of primary PTE cells derived from the kidneys of young but not of aged donors. The abundance of NOS mRNA, measured using a cDNA probe to the constitutive form of the enzyme, was enhanced during hypoxia in kidneys derived from humans of all ages, and then decreased during reoxygenation--possibly as the result of increased OPN expression. PTE cells from aged kidneys are more susceptible to cell death under hypoxic conditions that PTE cells from young kidneys. An investigation of the effect of an oxidant on OPN and NOS mRNA levels revealed that within 30 min of exposure to H2O2, NOS mRNA levels decreased simultaneously with an increase in OPN mRNA levels. Nitric oxide (NO), the product of NOS, is at low levels an important signal transduction molecule participating in the regulation of vascular tone and renal reabsorption; at high levels it is cytotoxic. We suggest that the diminished ability of cells from old kidneys to down-regulate NO production and to increase OPN expression after hypoxia-reoxygenation may contribute to their increased susceptibility to oxidant injury.
Aging↗
Production of nitric oxide by differentiated LSTRA cells is associated with expression of macrophage-inducible nitric oxide synthase.
LSTRA is a mouse lymphoma cell line overexpressing the src-related oncogene product and T cell marker p56lck. We have discovered that LSTRA cells, like HL60 and U937 promyelocytic leukemia cells, can be induced to differentiate toward macrophages by the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) or granulocytes by the cyclic nucleotide analogue dibutyryl cAMP (dbcAMP). One property of mature macrophages and granulocytes is the ability to produce nitric oxide, a highly reactive free radical that is important in non-specific host defense. Nitric oxide production by mature macrophages is stimulated by inflammatory mediators via an inducible form of the enzyme, nitric oxide synthase. We report that LSTRA cells acquire the ability to produce nitric oxide following differentiation induced by TPA and, to a lesser extent, by dbcAMP. Nitric oxide production by the cells is dependent on L-arginine and blocked by several inhibitors of nitric oxide synthase, including N-monomethyl-L-arginine, L-canavanine, L-arginine benzyl ester, and L-arginine methyl ester. In macrophage-differentiated LSTRA cells, the inflammatory cytokine interferon-gamma was found to be a potent inducer of nitric oxide production. This was correlated with a marked increase in nitric oxide synthase activity in the cells that was due to interferon-gamma-induced expression of the macrophage-inducible form of nitric oxide synthase protein as well as mRNA. Differentiated LSTRA cells also expressed increased amounts of a constitutive form of nitric oxide synthase that was also present in undifferentiated cells. Taken together with previous findings, these results support the model that LSTRA cells have the capacity to differentiate toward mature macrophages.
Amino Acid Oxidoreductases↗
Expression and secretion of active mouse TIMP-1 using a baculovirus expression vector.
Mouse TIMP-1, one of the tissue inhibitors of metalloproteinases important in regulating turnover of extracellular matrix in both normal and pathological tissues, was previously expressed in E. coli in an inactive, nonglycosylated state that required refolding to become functional. Due to the difficulty of renaturation, an alternative to the prokaryotic expression system was sought. Since we are interested in studying the pharmacodynamics and pharmacokinetics of TIMP locally administered by controlled delivery to mice with experimentally induced arthritis, we also needed an efficient way of producing active TIMP in large quantities. Using the pBlueBacII transfer vector, we generated a recombinant baculovirus that in Sf9 cells could express glycosylated mouse TIMP-1 to about 3 mg of active protein/liter conditioned medium.
Animals↗
Osteopontin expression in angiotensin II-induced tubulointerstitial nephritis.
Osteopontin is an arginine-glycine-aspartate (RGD) containing secreted phosphoprotein recently shown to stimulate a local macrophage influx when injected subcutaneously in mice. We examined the effect of angiotensin II infusion on renal injury and osteopontin expression in the rat kidney by in situ hybridization and immunohistochemistry. Preceding pathologic changes in tubular and interstitial cells, a dramatic increase in renal osteopontin protein and mRNA levels was observed primarily in epithelial cells of the distal tubules, collecting ducts and Bowman's capsule. Although both cortex and medulla showed increased osteopontin levels, the effect was most pronounced in the renal cortex which normally showed very little constitutive osteopontin expression. Interestingly, regions of the kidney expressing high osteopontin levels correlated with sites of monocyte/macrophage accumulation. These observations, coupled with recent findings that osteopontin may be a pro-inflammatory protein, suggests that osteopontin over-expression may facilitate monocyte/macrophage accumulation at the sites of renal tubulointerstitial injury.
Angiotensin II↗
Comparative analysis of two alternative first exons reported for the mouse osteopontin gene.
Two conflicting conclusions regarding the structure of the mouse osteopontin (OPN) gene were tested for their validity. Miyazaki et al. (Miyazaki, Y., Setoguchi, M., Yoshida, S., Higuchi, Y., Akizuki, S., and Yamamoto, S. (1990) J. Biol. Chem. 265, 14432-14438) state that the OPN gene is composed of six exons and spans approximately 4.8 kilobases. Craig and Denhardt (Craig, A. M., and Denhardt, D. T. (1991) Gene (Amst.) 100, 163-171) independently reported an additional exon 5' to the region designated as "exon 1" by Miyazaki and colleagues. To investigate this discrepancy, we generated oligodeoxynucleotide probes to three regions of these reported sequences and used them to hybridize to Northern and Southern blots of RNA and DNA from mouse fibroblasts and macrophages. Two of these regions (probes "A" and "B") represent sequences that are disputed, while one of these regions (probe "C") is predicted to be in the mRNA of both sequences. Our results are consistent only with the OPN gene structure reported by Craig and Denhardt and show that a significant portion of the exon 1 reported by Miyazaki et al. is present in murine genomic DNA but is not found in cytoplasmic message. Our results also show that OPN RNA molecules from mouse fibroblasts and macrophages do not differ significantly in the regions tested.
3T3 Cells↗