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D T Denhardt

Publications and source records attributed to D T Denhardt.

At least 19 recordsLinked to original sources
PubMed1995

Osteopontin-induced modifications of cellular functions.

Osteopontin (OPN) serves both a cell attachment function and a cell signalling function via the alpha v beta 3 integrin. We have investigated the action on mammalian cells of recombinant OPN made both in E. coli and in human cells. 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 pp60src and components of focal adhesion complexes. Effects on gene expression include suppression of the induction of nitric oxide synthase by inflammatory mediators. OPN can also reduce cell peroxide levels, promote the survival of cells exposed to hypoxia, and inhibit the killing of tumor cells by activated macrophages.

Amino Acid Oxidoreductases↗
PubMed1995

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↗
PubMed1995

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↗
PubMed1995

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↗
PubMed1995

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↗
PubMed1994

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↗
PubMed1994

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↗
PubMed1994

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↗
PubMed1994

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↗
PubMed1993

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↗
PubMed1992

Mechanism of action of antisense RNA. Sometime inhibition of transcription, processing, transport, or translation.

Anyone considering the use of AS RNA, generated endogenously, to inhibit gene expression should plan to generate several independent transfectants with nonoverlapping sequences; strategies that maximize both the transcription rate and the stability of the AS RNA are obviously desirable. Reasons why different results are obtained in different systems or with different constructs likely include the specific nucleotide sequence under investigation, the location of the AS gene in the nucleus relative to the endogenous gene, and the rate-limiting step in the expression of the target gene. Splicing may not be necessary, but an efficient polyadenylation signal likely is. Employment of a ribozyme-mediated strategy, discussed by various investigators in this volume, may be beneficial. There is no reason at present to conclude that any gene, however abundant its transcript might be, is inherently recalcitrant to AS-mediated down-regulation of expression.

Biological Transport↗
PubMed1992

Expression and purification of mouse TIMP-1 from E. coli.

Tissue inhibitors of metalloproteinases (TIMPs) constitute a family of secreted glycoproteins involved in regulating extracellular matrix degradation in both normal and malignant tissues. We have expressed a cDNA clone of mouse TIMP-1 as a 22-kDa protein with 12 cysteine residues in E. coli and purified protein that shows inhibitory activity against collagenase following renaturation by chemical means. The low specific activity and circular dichroism measurements suggest, however, that the renaturation of the mouse recombinant (non-glycosylated) protein is not efficient under the conditions we have used, indicative of either thermodynamic instability or the transition to kinetic intermediates which have very low in vitro refolding rates.

Animals↗
PubMed1992

Increased expression of cathepsins L and B and decreased activity of their inhibitors in metastatic, ras-transformed NIH 3T3 cells.

We previously found that the T24 Ha-ras oncogene induces metastatic ability in NIH 3T3 cells and that this change depends on expression of the ras oncogene. As part of our studies on mechanisms by which ras may induce metastasis, we investigated expression and activity of two cysteine proteinases, cathepsin L (major excreted protein) and cathepsin B, as well as cysteine proteinase inhibitor activity, in ras-transformed NIH 3T3 cells. In a series of cel lines that expressed differing amounts of ras, we found a good correlation between levels of ras expression and cathepsin L expression (r = 0.80). There was also a good correlation between secreted procathepsin L protein levels and experimental metastatic ability (r = 0.88). We found a similar but less strong association between cathepsin B levels and metastatic ability in these cells (r = 0.76 and r = 0.72 for 2.2-kb and 4.1-kb transcripts, respectively). Functional cathepsin L plus B activity (both secreted and cell-associated) was found to be higher in ras-transformed cells and was dependent on cell confluency in culture. Coupled with increased expression and activity of cysteine proteinases, ras-transformed NIH 3T3 cells showed reduced cysteine proteinase inhibitor activity. We conclude that the balance between expression of cysteine proteinases and their inhibitors may be coregulated by ras expression. Our results suggest that ras-induced increases in production of degradative enzymes such as cathepsins L and B, along with decreased activities of their inhibitors, may contribute to the increased malignant properties of ras-transformed NIH 3T3 cells.

3T3 Cells↗
PubMed1992

Regulated temporal and spatial expression of the calcium-binding proteins calcyclin and OPN (osteopontin) in mouse tissues during pregnancy.

In situ hybridization and northern/slot blot analyses were used to quantify the expression of calcyclin (2A9, 5B10), osteopontin (opn, secreted phosphoprotein, 2ar) and calmodulin mRNAs in mouse tissues that support pregnancy. High-to-moderate levels of the mRNAs of all three genes were detected at discrete locations in the uterus, decidua and placenta as a function of gestation time. Calmodulin expression was constant in these tissues; calcyclin mRNA was high during early pregnancy and declined after day 8-9 of gestation; and opn mRNA was undetectable before day 7, with maximal levels on days 9-12 in each of these tissues. Calcyclin, but not opn, expression was also observed in the chorioamnion after day 12. Calcyclin was expressed throughout the decidua on day 8 but became restricted to the primary (antimesometrial) decidual zone and decidua lateralis on day 9, and the decidua capsularis after day 9. By contrast, opn mRNA was localized on day 9 to the mesometrial triangle, which contains a large population of granulated metrial gland cells, and to the decidua basalis. These two genes may serve as markers for the two types of decidual tissue. We suggest that one function of OPN, which may be an indicator of cells in the decidua that have a bone marrow genealogy, is to mediate the flux of calcium from the maternal circulation to the developing embryo.

Animals↗