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M Navre

Publications and source records attributed to M Navre.

34 records · Page 2Linked to original sources
PubMed1993

Expression of the metalloproteinase matrilysin in DU-145 cells increases their invasive potential in severe combined immunodeficient mice.

Human prostate cancer displays a high degree of variability in its rate of spread, which could be due largely to differences in the invasive potential of the tumor cells. The degradation of the basal lamina and stromal extracellular matrix is mediated in part by the secretion of matrix metalloproteinases (MMPs). Matrilysin (PUMP-1, MMP-7) and gelatinase A (M(r) 72,000 type IV collagenase, MMP-2) have been shown to be overexpressed in prostate carcinoma. We have expressed the single MMP matrilysin in the tumorigenic but nonmetastatic human prostate tumor cell line DU-145 to determine if matrilysin has a functional role in prostate tumor cell invasion. DU-145 cells expressing matrilysin were significantly more invasive than vector-only transfected cell lines as assayed by a severe combined immunodeficient mouse model of tumor cell invasion. Vector-only transfected DU-145 cells injected i.p. into severe combined immunodeficient mice invaded the diaphragm in only 1 of 9 mice (11%), whereas matrilysin-transfected DU-145 cells invaded the diaphragm in 12 of 18 mice (66%). The difference between the controls and matrilysin-transfected cells was statistically significant (P < 0.006). These results suggest a functional role for matrilysin in the initial invasion of prostate cancer through the epithelial basal lamina and into the surrounding stroma.

Animals↗
PubMed1993

Expression and localization of matrilysin, a matrix metalloproteinase, in human endometrium during the reproductive cycle.

OBJECTIVE: We studied the expression of a matrix metalloproteinase, matrilysin, in the human endometrium to determine whether metalloproteinase genes are expressed during the reproductive cycle. Matrix metalloproteinases are a tightly regulated family of enzymes that degrade components of the extracellular matrix and basement membrane; they play important roles in growth and development and in invasion and metastasis of tumors and thus are likely enzymes participating in the dynamic structural changes occurring in endometrium during the reproductive cycle. STUDY DESIGN: In situ and Northern nucleic acid hybridization and immunohistochemistry were used to detect and localize matrilysin ribonucleic acid and protein in normal endometrial tissue. RESULTS: Matrilysin protein and matrilysin messenger ribonucleic acid are abundant in proliferative, late secretory, and menstrual endometrial epithelium but are not detected in early or mid secretory endometrium. CONCLUSION: The expression of the matrilysin gene is regulated in endometrium during the reproductive cycle, implying an important role for matrilysin in endometrial physiologic characteristics.

Blotting, Northern↗
PubMed1992

Reduced expression of AP27 protein, the product of a growth factor-repressible gene, is associated with diminished adipocyte differentiation.

We have recently characterized an adipocyte cDNA (clone 5) that is enhanced in expression by environmental and hormonal conditions favoring adipogenic differentiation. Moreover, certain agents including fibroblast growth factor and phorbol 12-myristate 13-acetate (but not epidermal growth factor) markedly inhibit clone 5 gene expression and prevent TA1 cell differentiation. These results led us to propose that a threshold level of the clone 5 gene product (AP27 protein) is required for triggering adipocyte differentiation. We have constructed vectors that direct the synthesis of clone 5 antisense RNA to reduce the levels of AP27 in adipogenic cell lines TA1 and 3T3-L1. We show here that when these cells express clone 5 antisense RNA, they fail to undergo morphological differentiation, whereas adipogenesis is unaffected in cells expressing antisense beta-actin or ferritin heavy-chain RNA. We further show that cells expressing clone 5 antisense RNA (but not the other antisense RNAs) are unable to induce the expression of characteristic "adipocyte-specific" mRNAs. The level of inhibition of differentiation by clone 5 antisense RNA correlates with decreased levels of AP27 protein. These results provide strong evidence that expression of AP27 is linked to adipogenic differentiation and that AP27 may be a component of an as-yet-uncharacterized signal-transduction pathway required for the triggering of adipocyte differentiation.

3T3 Cells↗
PubMed1992

Growth and developmental regulation of wnt-2 (irp) gene in mesenchymal cells of fetal lung.

The wnt gene family encodes a group of proteins implicated as intercellular signaling molecules in vertebrate development. Because many wnt genes are also expressed in the lung, we have examined whether the wnt family member wnt-2 (irp) plays a role in lung development. We have cloned rat wnt-2 and found that this cDNA detects multiple mRNAs expressed at high levels in fetal rat lung. Much lower levels were found in adult rat lung and other tissues, including, surprisingly, the mammary gland. The wnt-2 mRNA was also detected in human fetal lung fibroblast cell lines, where the mRNA levels were dramatically regulated by growth state as well as growth factor stimulation. In situ hybridization showed that, in fetal rat lung, wnt-2 mRNA expression is restricted to the mesenchyme; levels in the developing epithelium were indistinguishable from background. Based on the known properties of other wnt proteins, our data lead us to propose that wnt-2 may play a role in lung development by mediating intercellular interaction(s) between mesenchyme and epithelium.

Amino Acid Sequence↗
PubMed1991

Mutational analysis of the transin (rat stromelysin) autoinhibitor region demonstrates a role for residues surrounding the "cysteine switch".

The family of mammalian extracellular matrix metalloproteases (MMPs) are secreted by cells in an inactive (latent) proenzyme form. A highly conserved amino acid sequence, PRCGVPDV, is found near the COOH-terminal end of the pro-domain of these MMPs and believed to act as an "autoinhibitor." Recent studies (Springman, E. B., Angleton, E. L., Birkedal-Hansen, H., and Wart, H. E. V. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 364-368) indicate the Cys of this sequence ligands to the active-site zinc keeping the proenzyme in an inactive state, and mutational analysis (Sanchez-Lopez, R., Nicholson, R., Gesnel, M. C., Matrisian, L. M., and Breathnach, R. (1988) J. Biol. Chem. 263, 11892-11899) suggests that the conserved residues surrounding this Cys are required for latency. We have constructed 16 new site-directed mutations of the PRCGVPDV autoinhibitor region of the MMP transin (rat stromelysin) and tested whether these mutant enzymes are produced in a latent or activated form. We find that the conserved Arg as well as the Cys are essential for maintaining latency. The Cys cannot be replaced by other zinc-liganding amino acids, and the Arg cannot be replaced by Lys. Residues immediately surrounding the Cys are sensitive to even conservative amino acid substitutions. We show that a synthetic peptide PRCGVPDV is capable of acting as a weak inhibitor of transin and that replacement of the Cys with a Ser abolishes inhibition by the peptide. A review of the current knowledge of MMP substrate specificity in combination with these new results suggests that the PRCGVPDV sequence does not inhibit activity by mimicking the known substrates of the protease.

Amino Acid Sequence↗
PubMed1991

Expression and localization of the matrix metalloproteinase pump-1 (MMP-7) in human gastric and colon carcinomas.

The expression of members of the family of matrix-degrading metalloproteinases (MMPs) is believed to contribute to the complex process of invasion and metastasis. In this study, specific cDNA probes for three members of the stromelysin subfamily of MMPs--stromelysin (MMP-3), stromelysin-2 (MMP-10), and pump-1 (MMP-7)--were used to examine the expression of these three different MMPs in human gastric and colonic carcinomas and in adjacent normal mucosa. The expression of pump-1 mRNA in malignant colon and stomach samples was striking. In a total of 10 gastric carcinoma samples examined, eight (80%) expressed pump-1 transcripts; similarly, 6 of 8 (75%) colon carcinoma samples were also positive. Stromelysin and stromelysin-2 mRNAs were not detected in any of these samples. Expression of the MMPs examined was not detected in any of the adjacent, grossly normal tissue samples. Using in situ hybridization and affinity purified anti-pump-1 antibodies, the expression of pump-1 mRNA and protein was localized to tumor cells and was not detected in stromal or lymphocytic cells. This data suggests that the inappropriate expression of pump-1 by malignant cells may contribute to the neoplastic phenotype.

Carcinoma↗
PubMed1991

The role of the matrix metalloproteinase stromelysin in the progression of squamous cell carcinomas.

The expression of the metalloproteinase stromelysin correlates with the progression of chemically induced squamous cell carcinomas. We demonstrate that the expression of activated stromelysin in papilloma-derived cells enhances in vitro cell invasion. We also demonstrate that the Ha-ras oncogene induces the transcription of the stromelysin gene through an AP-1 dependent pathway. The hypothesis is that alterations in oncogenes and suppressor genes influence stromelysin expression and thus influence subsequent steps of tumor invasion and metastasis.

Carcinoma, Squamous Cell↗
PubMed1991

Glucocorticoid induction of the adipocyte clone 5 gene requires high cell density.

We have previously reported the identification of a glucocorticoid and cell-density inducible gene (clone 5) that appears to play a regulatory role in adipocyte differentiation. In the current studies we have more carefully investigated the interplay between cell growth or differentiation and the glucocorticoid responsiveness of clone 5 RNA. We find that inducibility by steroid hormone is independent of the differentiated state but surprisingly, occurs only when cells are at high cell density. In subconfluent TA1 cells clone 5 RNA is refractory to induction whereas well characterized glucocorticoid-inducible promoters such as the those from mouse mammary tumor virus and alpha-1-acid glycoprotein are induced under all conditions. These results point to the necessity of ancillary factors, absent in low density cell culture, that are required for glucocorticoid responsiveness of the clone 5 gene.

Adipose Tissue↗
PubMed1989

Differential effects of fibroblast growth factor and tumor promoters on the initiation and maintenance of adipocyte differentiation.

Fibroblast growth factor (FGF) has been shown to inhibit the differentiation of myogenic and adipogenic cell lines without inducing a proliferative response. We have previously shown that agents capable of activating protein kinase C (PKC), such as FGF and the phorbol ester tetradecanoyl phorbol-13-acetate (TPA), inhibit the differentiation of the adipogenic cell line TA1, as measured by the rapid loss of adipocyte-specific RNAs. We report here that the treatment of fully differentiated TA1 adipocytes with FGF at 10 ng/ml induces the reversal of adipocyte differentiation, even in cells where PKC activity has been down-regulated by TPA pretreatment. In contrast, TPA or lower concentrations of FGF (1 ng/ml), both effective inducers of c-fos RNA in adipocytes, fail to reverse adipocyte differentiation. The adipocytes, however, will extinguish differentiation-specific functions in response to TPA by the addition of a calcium ionophore. Therefore, we propose that there are two FGF-sensitive pathways in TA1 cells: one responsible for the initiation of differentiation (TPA sensitive) and another required for maintenance of the adipose phenotype (TPA insensitive). These results suggest that activation of two distinct signaling pathways--one PKC and calcium dependent, the other FGF activated but PKC independent--are capable of inhibiting the biochemical events responsible for the maintenance of adipocyte differentiation.

Adipose Tissue↗
PubMed1988

A growth factor-repressible gene associated with protein kinase C-mediated inhibition of adipocyte differentiation.

The conversion of determined adipoblasts to fully differentiated adipocytes requires appropriate environmental conditions. A strict dependence on cell confluence and a facilitation by glucocorticoid hormones have previously been described. We have found that agents that are capable of activating protein kinase C, such as basic fibroblast growth factor and phorbol esters, inhibit the differentiation of the adipogenic cell line TA1 without stimulating cell growth. Here we describe the sequence and characterization of a cDNA (clone 5) that detects an RNA, the expression of which is enhanced by glucocorticoids and increasing cell density. In contrast, activators of protein kinase C including basic fibroblast growth factor, phorbol esters, and synthetic diacylglycerols inhibit clone 5 gene expression. It appears that clone 5 expression is closely linked to environmental and hormonal factors that promote the differentiation of adipogenic cells.

Adipose Tissue↗
PubMed1988

Induction of adenosine 3',5'-monophosphate-dependent protein kinase subunits during adipogenesis in vitro.

Fatty acid metabolism in adipocytes is known to be regulated by the intracellular transducer cAMP. This study was undertaken to determine the temporal and hormonal regulation of cAMP-dependent protein kinase during the differentiation of preadipocyte mesenchymal cells to adipocytes. For this we have used a stable cell line (TA1) in which the undifferentiated preadipocyte acquires adipocyte functions and morphology after growth to confluence. We observed that synthesis of type I and II cAMP-dependent protein kinases was induced during the adipogenic conversion of growth-arrested TA1 cells. In preconfluent cells, neither mRNAs encoding regulatory subunits (RI, RII beta) and catalytic subunit (C alpha) nor the peptides themselves were detectable. Within several days of growth arrest at high cell density, mRNAs for RI, RII beta, and C alpha were detectable in total RNA extracted from cell populations. The subunits themselves were detectable in some, but not all, of the cells by indirect immunofluorescence. Immunoblotting of cytosolic extracts indicated that RI and the beta-isoform of RII (mol wt = 52,000) were expressed in these cells. Analysis of subunit presence or absence in single cells by immunofluorescence also indicated that kinase subunit expression preceded the accumulation of lipid droplets within the cells. Further, the subunits were predominantly associated with a reticular cytoplasmic structure (Golgi apparatus?) abutting the nucleus. Conversion of TA1 cells to adipocytes can be accelerated by indomethacin (125 microM) or dexamethasone (1 microM) treatment, compounds that also enhanced the accumulation of RII beta and C alpha mRNAs. Within 2-3 days of addition of indomethacin to confluent cultures, RII beta message content is increased about 20-fold, and protein content is increased about 5-fold relative to those in untreated cultures. C alpha mRNA content is increased about 5-fold relative to that in untreated cells. The response to dexamethasone requires 6-7 days, and changes in RII beta message levels were the most pronounced. We also observed the induction of mRNAs for the functionally relevant mRNA lipoprotein lipase in indomethacin-treated cells. In addition to this apparent transcriptional regulation of kinase subunit expression, we provide evidence for regulation at the posttranscriptional level. Within a differentiated culture, there exist stem cells that can be selected, will repopulate the dish, and will again differentiate into adipocytes upon growth arrest at high cell density. In preconfluent populations of these stems cells, unlike the preconfluent TA1 cells originally plated, both RII beta and C alpha messages were present.(ABSTRACT TRUNCATED AT 400 WORDS)

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1987

Requirements for triggering of adipocyte differentiation by glucocorticoids and indomethacin.

The stable adipogenic cell line TA1, spontaneously differentiates into mature adipocytes after several days at confluence. Glucocorticoids (e.g. dexamethasone) accelerate the onset of differentiation by precociously activating the transcription of genes that are expressed in the mature adipocyte but not in the preadipocyte. Thus the hormone may induce a critical regulatory factor required for activating the entire set of differentiation-dependent genes. We have found that the nonsteroidal antiinflammatory drug indomethacin also stimulates differentiation of TA1 cells but even more rapidly and completely than does dexamethasone. Contrary to previous suggestions we find that this activity of indomethacin's cannot be ascribed to inhibition of cyclo-oxygenase, the critical enzyme in prostaglandin biosynthesis. Finally, indomethacin's ability to stimulate TA1 cell differentiation synchronously and rapidly has allowed us to document that cell confluence is required for efficient differentiation and that the drug needs only to trigger rather than maintain the differentiation process.

Adipose Tissue↗
PubMed1984

Location of amino acid alterations in mutants of aspartate transcarbamoylase: Structural aspects of interallelic complementation.

Recent genetic studies of the pyrB locus of Escherichia coli resulted in the characterization of 29 mutant strains harboring defects in the structural gene that encodes the catalytic chains of aspartate transcarbamoylase (carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2). Three alleles, pyrB554, pyrB730, and pyrB748, have been cloned, and their nucleotide sequences have been determined along with that of the wild-type pyrBI operon in order to locate the sites of the alterations in the catalytic chains. Missense mutation pyrB554 leads to replacement of serine-52 by phenylalanine, and the inactive mutant enzyme has properties similar to those of wild-type aspartate transcarbamoylase. The amber mutation pyrB730 results in unstable truncated polypeptide chains 27 amino acids shorter than wild-type chains. Deletion mutation pyrB748 causes the removal of 181 amino acids. Combining these results with knowledge of the crystallographic structure of the wild-type enzyme provides a basis for tentative structural mechanisms for the observed complementation behavior of the mutant proteins.

Alleles↗
PubMed1983

Synthesis of aspartate transcarbamoylase in Escherichia coli: transcriptional regulation of the pyrB-pyrI operon.

The first committed reaction in pyrimidine biosynthesis in Escherichia coli and Salmonella typhimurium is catalyzed by the allosteric enzyme aspartate transcarbamoylase (aspartate carbamoyltransferase; carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), the product of the pyrB-pyrI operon. Regulation of the pyrimidine pathway is achieved in part by changes in the enzyme's catalytic activity as a function of the concentration of substrates and other metabolites as well as by variations in enzyme synthesis in response to changes in cellular levels of pyrimidine nucleotides. Although there is substantial evidence that UTP concentration has a marked influence on expression of the pyrB-pyrI operon, the mechanism of this control is not known. We have cloned the operon and determined the nucleotide sequence of the region preceding the first structural gene (pyrB). These studies show two regions sharing considerable homology with the consensus sequence of E. coli promoters, a segment that can code for a 44-amino-acid leader peptide, and a sequence very similar to that of the attenuator of the trp operon. RNA transcripts from several bacterial strains were studied by S1 nuclease mapping. Under conditions leading to extensive enzyme synthesis there was a large production of transcript whose 5' end correlated with the putative promoter closer to the structural genes. At low levels of operon expression there was little transcript in the extracts and both promoters appeared to serve as initiation sites. The results are interpreted in terms of transcriptional control of the pyrB-pyrI operon according to an attenuation model that differs in novel ways from the mechanisms proposed for the regulation of amino acid biosynthesis.

Amino Acid Sequence↗
PubMed1982

Genes encoding Escherichia coli aspartate transcarbamoylase: the pyrB-pyrI operon.

In both Escherichia coli and Salmonella typhimurium there is approximately balanced synthesis of the six catalytic and six regulatory polypeptide chains of the regulatory enzyme aspartate transcarbamoylase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2). This control is achieved by the contiguous pyrB and pyrI genes, which encode the catalytic and regulatory chains, respectively. Evidence for this single transcriptional unit was obtained by a study of various deletion mutations and from the DNA sequence of the intercistronic region between pyrB and pyrI. One pyrB deletion, pyrB748, produced a normal level of regulatory chains even though it removed a substantial portion of the pyrB gene. Another deletion, pyrB740, shares a similar terminus at one end within pyrB, but the promoter region is removed. In this deletion mutation, there is no production of the regulatory polypeptide, indicating that a single region adjacent to pyrB controls transcription of pyrI as well. Molecular cloning and subsequent DNA analysis demonstrated that the pyrB and pyrI genes are contiguous with pyrI as the distal gene in the operon. The cistrons are separated by a 15-nucleotide untranslated region containing a sequence capable of interacting with the 16S ribosomal RNA and allowing translation of the pyrI cistron.

Amino Acid Sequence↗