Transcriptional control of the expression of the c-fms gene encoding the receptor for macrophage colony-stimulating factor (CSF-1).
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Animals↗
Biomedical subjects
M C Ostrowski
Publications and source records attributed to M C Ostrowski.
Ras-mediated phosphorylation of a conserved threonine residue enhances the transactivation activities of c-Ets1 and c-Ets2.
The Ras oncogene products regulate the expression of genes in transformed cells, and members of the Ets family of transcription factors have been implicated in this process. To determine which Ets factors are the targets of Ras signaling pathways, the abilities of several Ets factors to activate Ras-responsive enhancer (RRE) reporters in the presence of oncogenic Ras were examined. In transient transfection assay, reporters containing RREs composed of Ets-AP-1 binding sites could be activated 30-fold in NIH 3T3 fibroblasts and 80-fold in the macrophage-like line RAW264 by the combination of Ets1 or Ets2 and Ras but not by several other Ets factors that were tested in the assay. Ets2 and Ras also superactivated an RRE composed of Ets-Ets binding sites, but the Ets-responsive promoter of the c-fms gene was not superactivated. Mutation of a threonine residue to alanine in the conserved amino-terminal regions of Ets1 and Ets2 (threonine 38 and threonine 72, respectively) abrogated the ability of each of these proteins to superactivate reporter gene expression. Phosphoamino acid analysis of radiolabeled Ets2 revealed that Ras induced normally absent threonine-specific phosphorylation of the protein. The Ras-dependent increase in threonine phosphorylation was not observed in Ets2 proteins that had the conserved threonine 72 residue mutated to alanine or serine. These data indicate that Ets1 and Ets2 are specific nuclear targets of Ras signaling events and that phosphorylation of a conserved threonine residue is a necessary molecular component of Ras-mediated activation of these transcription factors.
Amino Acid Sequence↗
Cyclic AMP-dependent activation of Rap1b.
Rap1 proteins belong to the Ras superfamily of small molecular weight GTP-binding proteins. Although Rap1 and Ras share approximately 50% overall amino acid sequence identity, the effector domains of the two proteins are identical, suggesting either similar or antagonistic signaling roles. Several pathways leading to Ras activation have been defined, including those initiated by agonist binding to tyrosine kinase or Gi-coupled receptors. Nothing is known about such events for Rap1 proteins. The cAMP-mediated inhibition of Ras-dependent MAP kinase activation is well documented and resembles that caused by expression of GTPase-deficient Rap1. We have developed a system whereby signals leading to Rap1b activation, i.e. an increase in Rap1b-bound GTP/GDP ratio, can be measured. We report here that treatment of cells with agents that elevate intracellular cAMP levels result in Rap1b activation. These results demonstrate for the first time agonist-dependent activation of Rap1 proteins.
3T3 Cells↗
Alterations in differentiation and behavior of monocytic phagocytes in transgenic mice that express dominant suppressors of ras signaling.
To address the role of ras signaling in monocytic phagocytes in vivo, the expression of two dominant suppressors of in vitro ras signaling pathways, the carboxyl-terminal region of the GTPase-activating protein (GAP-C) and the DNA binding domain of the transcription factor ets-2, were targeted to this cell compartment. A 5-kb portion of the human c-fms proximal promoter was shown to direct expression of the transgenes to the monocytic lineage. As a result of the GAP-C transgene expression, ras-GTP levels were reduced in mature peritoneal macrophages by 70%. The terminal differentiation of monocytes was altered, as evidence by the accumulation of atypical monocytic cells in the blood. Mature peritoneal macrophages exhibited changes in colony-stimulating factor 1-dependent survival and structure. Further, expression of the colony-stimulating factor 1-stimulated gene urokinase plasminogen activator was inhibited in peritoneal macrophages. The results indicate that ras action is critical in monocytic cells after these cells have lost the capacity to traverse the cell cycle.
Animals↗
Regulation of urokinase-type plasminogen activator gene transcription by macrophage colony-stimulating factor.
The mouse urokinase-type plasminogen activator (uPA) gene was used as a model macrophage colony-stimulating factor 1 (CSF-1)-inducible gene to investigate CSF-1 signalling pathways. Nuclear run-on analysis showed that induction of uPA mRNA by CSF-1 and phorbol myristate acetate (PMA) was at the transcriptional level in bone marrow-derived macrophages. CSF-1 and PMA synergized strongly in the induction of uPA mRNA, showing that at least some components of CSF-1 action are mediated independently of protein kinase C. Promoter targets of CSF-1 signalling were investigated with NIH 3T3 cells expressing the human CSF-1 receptor (c-fms). uPA mRNA was induced in these cells by treatment with CSF-1, and a PEA3/AP-1 element at -2.4 kb in the uPA promoter was involved in this response. Ets transcription factors can act through PEA3 sequences, and the involvement of Ets factors in the induction of uPA was confirmed by use of a dominant negative Ets-2 factor. Expression of the DNA binding domain of Ets-2 fused to the lacZ gene product prevented CSF-1-mediated induction of uPA mRNA in NIH 3T3 cells expressing the CSF-1 receptor. Examination of ets-2 mRNA expression in macrophages showed that it was also induced synergistically by CSF-1 and PMA. In the macrophage cell line RAW264, the uPA PEA3/AP-1 element mediated a response to both PMA and cotransfected Ets-2. uPA promoter constructs were induced 60- to 130-fold by Ets-2 expression, and the recombinant Ets-2 DNA binding domain was able to bind to the uPA PEA3/AP-1 element. This work is consistent with a proposed pathway for CSF-1 signalling involving sequential activation of fms, ras, and Ets factors.
3T3 Cells↗
Opposing actions of c-ets/PU.1 and c-myb protooncogene products in regulating the macrophage-specific promoters of the human and mouse colony-stimulating factor-1 receptor (c-fms) genes.
The receptor for macrophage colony stimulating factor (CSF-1), the c-fms gene product, is a key determinant in the differentiation of monocytic phagocytes. Dissection of the human and mouse c-fms proximal promoters revealed opposing roles for nuclear protooncogenes in the transcriptional regulation of this gene. On the one hand, c-ets-1, c-ets-2, and the macrophage-specific factor PU.1, but not the ets-factor PEA3, trans-activated the c-fms proximal promoter. On the other hand c-myb repressed proximal promoter activity in macrophages and blocked the action of c-ets-1 and c-ets-2. Basal c-fms promoter activity was almost undetectable in the M1 leukaemia line, which expressed high levels of c-myb, but was activated as cells differentiated in response to leukemia inhibitory factor and expressed c-fms mRNA. The repressor function of c-myb depended on the COOH-terminal domain of the protein. We propose that ets-factors are necessary for the tissue-restricted expression of c-fms and that c-myb acts to ensure correct temporal expression of c-fms during myeloid differentiation.
3T3 Cells↗
Non-nuclear oncogenes and the regulation of gene expression in transformed cells.
Oncogene products not localized to the nucleus regulate the expression of a diverse group of genes. The identities of genes regulated by non-nuclear oncogenes can supply insights into the changes at the cellular level that accompany the altered expression of such genes during the multi-step process of carcinogenesis. For example, one set of genes whose expression is affected by non-nuclear oncogenes are genes encoding extracellular proteases and components of the extracellular matrix. The expression of these genes during tumorigenesis could have important consequences for tumor invasiveness and metastasis. Genes regulated by non-nuclear oncogenes also define signal transduction pathways that allow communication between the plasma membrane and the nucleus. Oncogene-regulated nuclear targets provide a tool to approach the problem of cellular signal transduction and may contribute a clearer view of intracellular signaling pathways and the interactions between these pathways during cell growth and differentiation. Studying the regulation of these genes has revealed that c-jun and members of the ets family of transcription factors are important nuclear targets for the action of several non-nuclear oncogenes. This approach has also indicated that ras p21 is necessary for selective signal transduction events mediated by receptor and nonreceptor tyrosine kinases, including the colony-stimulating factor 1 (CSF-1) receptor, the product of the c-fms gene.
Animals↗
Mitogenic signaling by colony-stimulating factor 1 and ras is suppressed by the ets-2 DNA-binding domain and restored by myc overexpression.
The activity of p21ras is required for the proliferative response to colony-stimulating factor 1 (CSF-1), and signals transduced by both the CSF-1 receptor (CSF-1R) and p21ras stimulate transcription from promoter elements containing overlapping binding sites for Fos/Jun- and Ets-related proteins. A sequence encoding the DNA-binding domain and nuclear localization signal of human c-ets-2, which lacked portions of the c-ets-2 gene product necessary for trans activation, was fused to the bacterial lacZ gene and expressed from an actin promoter in NIH 3T3 cells expressing either the v-ras oncogene or human CSF-1R. Nuclear expression of the Ets-LacZ protein, confirmed by histochemical staining of beta-galactosidase, inhibited the activity of ras-responsive enhancer elements and suppressed morphologic transformation by v-ras as well as CSF-1R-dependent colony formation in semisolid medium. When CSF-1R-bearing cells expressing the Ets-LacZ protein were stimulated by CSF-1, induction of c-ets-2, c-jun, and c-fos ensued, but the c-myc response was impaired. Enforced expression of the c-myc gene overrode the suppressive effect of ets-lacZ and restored the ability of these cells to form colonies in response to CSF-1. NIH 3T3 cells engineered to express a CSF-1R (Phe-809) mutant similarly cannot form CSF-1-dependent colonies in semisolid medium and exhibit an impaired c-myc response, but expression of an exogenous myc gene resensitizes these cells to CSF-1 [M. F. Roussel, J. L. Cleveland, S. A. Shurtleff, and C. J. Sherr, Nature (London) 353:361-363, 1991]. The ability of these cells to respond to CSF-1 was also rescued by enforced expression of an endogenous c-ets-2 gene. The ets family of transcription factors therefore plays a central role in integrating both CSF-1R and ras-induced mitogenic signals and in modulating the myc response to CSF-1 stimulation.
3T3 Cells↗
An enhancer element responsive to ras and fms signaling pathways is composed of two distinct nuclear factor binding sites.
In order to precisely define the sequences that constitute the ras-responsive enhancers element present in the murine retrotransposon NVL3, point mutations were introduced into the previously defined minimal transcriptional enhancer DNA. Analyses of the effects of these point mutations in transient transfection experiments, in gel retention assays, and by methylation interference footprinting indicated that the enhancer element was composed of two binding sites for distinct nuclear factors. Both binding sites were required for activation of the enhancer by either ras or v-fms oncogenes, and the distinct nuclear factors were found in extracts from cells that contained either oncogene. UV cross-linking analysis revealed that the AP1-related binding site, TGACTCT, was recognized by a nuclear factor of apparent molecular size of 50 kilodaltons, that is probably c-jun. The other binding site, CAGGATAT, is very similar to sites recognized by the ets-family of transcription factors, and was recognized by the 120-kilodalton ras-responsive factor-1. Activation of the NVL3 element was reconstituted in an in vitro transcription assay. The ets-related binding site was necessary for this in vitro reconstitution of activity. Thus, the NVL3 enhancer is related to the previously described oncogene-responsive enhancer element present in polyoma virus and is also related to elements identified in several cellular genes known to be ras-responsive, including the transforming growth factor-beta 1 gene.
Base Sequence↗
Analysis of the v-myb structural components important for transactivation of gene expression.
In order to define the domains of the v-myb protein that are important for transactivation of gene expression, we have studied transactivation by the v-myb gene and a set of v-myb deletion mutants using transient transfection assays in NIH 3T3 cells. Analysis of the set of v-myb deletion products demonstrated that a previously unidentified region in the carboxyl-terminal portion of the protein is required for transactivation. This region lies between amino acids 295-356 with respect to the 5' end of the v-myb gene. Switching the v-myb DNA binding domain with the DNA binding domain of the rat glucocorticoid receptor (rGR) switched the cis-element requirement for v-myb action: only reports containing glucocorticoid response elements were activated by myb-rGR fusion proteins. The carboxyl terminal region essential for transactivation by the intact v-myb gene was also necessary for transactivation by the rGR-fusion gene. Carboxyl-terminal deletion mutations that encompassed the novel transactivation region were able to block wild-type v-myb transactivation when tested in transient co-expression assays. In an unexpected sidelight to our studies, we could demonstrate that the lacZ gene present in the prokaryotic vector sequences contained a DNA element that fortuitously can act as a v-myb-dependent enhancer element, and that v-myb protein can bind to this element in vitro. The lacZ enhancer contains the myb consensus DNA binding site YAAC(G/T)G.
Animals↗
The carboxy-terminal catalytic domain of the GTPase-activating protein inhibits nuclear signal transduction and morphological transformation mediated by the CSF-1 receptor.
To determine whether ras p21 products are necessary for signal transduction mediated by the colony stimulating factor-1 receptor (CSF-1R, the c-fms proto-oncogene product), we determined whether CSF-1R and ras activate a common nuclear target and whether the interruption of ras action affects CSF-1R signal transduction. Expression of the NVL3 retrotransposon was activated to the same extent in NIH-3T3 cells by both ras and v-fms oncogenes, and the ras-responsive element located in the long terminal repeat of NVL3 was demonstrated to be a common target for oncogene action. Human recombinant CSF-1 stimulated expression of the NVL3 element 30-fold in NIH-3T3 cells that contained human CSF-1R. Expression of the carboxy-terminal 374 amino acid residues of the human ras GTPase-activating protein (GAP) in cells containing CSF-1R was able to inhibit CSF-1 induction of NVL3 expression by 90%. Expression of the catalytic domain of GAP was also able to suppress transformation by either v-fms or ligand-activated CSF-1R. Expression of the c-jun proto-oncogene was activated by CSF-1R but was insensitive to the action of the catalytic domain of GAP. These results provide genetic evidence that in NIH-3T3 cells, ras p21 is involved in signal transduction mediated by CSF-1R.
3T3 Cells↗
Protein kinase C gamma expression mimics phorbol ester-induced transcriptional activation of a murine VL30 enhancer element.
Protein kinase C (PKC), a critical component in the regulation of cell growth, is thought to participate in transmitting the signals of certain cell surface receptor activation events to the nucleus. We have previously shown that stable expression of the PKC gamma isoenzyme in NIH 3T3 cells causes altered growth and enhanced tumorigenicity. In this report, we show that transient expression of the PKC gamma isoenzyme can trans-activate a murine VL30 enhancer element in a pattern similar to that of the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate. In contrast, ras activation of this element is distinct both quantitatively and qualitatively from PKC gamma and 12-O-tetradecanoylphorbol-13-acetate activation. These results provide direct evidence that PKC is the cellular mediator in the activation of phorbol ester-responsive genes and suggest a mechanism by which abnormal PKC expression might lead to altered growth control by changing the pattern of cellular gene expression.
Animals↗
Transcriptional activation of a conserved sequence element by ras requires a nuclear factor distinct from c-fos or c-jun.
The expression of transforming growth factor beta type 1 mRNA was increased by conditional expression of ras. A 31-base-pair sequence found approximately 420 base pairs upstream of the gene encoding human transforming growth factor beta 1 acted as a ras-responsive enhancer element in transient transfection assays. The human sequence contains the element TGACTCT that also is found in a murine ras-responsive enhancer. Analysis of nuclear factors present in cells stably transformed by ras indicated that both human and murine sequences were recognized by the same nuclear factor. The role of fos and jun in ras transcriptional activation was analyzed in transfection assays using murine elements that contained either TGACTCT or TGAGTAA. These experiments showed that while both elements are activated by fos/jun expression to nearly the same event, only the former element responded to ras. In addition, activation of reporters containing TGACTCT is 6-fold higher by ras than by fos/jun. Gel retention experiments revealed that the nuclear factor present in cells transformed by ras exhibited the same sequence preference as demonstrated in the transient transfection assays. UV-crosslinking experiments identify a protein of apparent molecular mass 120 kDa that recognizes the ras-responsive element. This work identifies a persistent signal transduction pathway that links ras to nuclear transcription and indicates that a 120-kDa protein is a target of this pathway.
Animals↗
ras oncogene activation of a VL30 transcriptional element is linked to transformation.
The activity of a murine VL30 transcriptional element was increased 20-fold in transient assays by coexpression of mutant ras genes. The cis element did not respond to ras in a revertant cell line that was transformation defective. Therefore, ras-dependent alterations in transcription and ras transformation are linked. Deletion analysis of the VL30 long-terminal-repeat U3 region showed that a minimal 53-base-pair segment is required in cis for oncogene activation of transcription. Gel retention assays using a probe that contained the minimal cis element revealed that a unique complex was formed with nuclear proteins prepared from transformed cells. Exonuclease III footprinting and gel retention experiments that used oligonucleotide probes and competitors indicated that two distinct nuclear factors interact with the minimal cis-responsive element. Site-directed deletion of the 5'-proximal binding site (TGACTCT) resulted in a complete loss of ras responsiveness. However, deletion of this site did not affect stimulation by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). These data are consistent with the hypothesis that ras and TPA signal transduction mechanisms for transcriptional activation are distinct.
Animals↗
A nuclear factor that binds to ras-responsive enhancer elements is present in human tumor cells.
The ras protein can be viewed as molecular switches for undetermined signal transduction pathways. We are interested in defining the downstream effectors and targets associated with ras signal transduction. A nuclear target of ras action is the conserved sequence element TGACTCT, which functions as a ras-responsive transcriptional element (RRE). An Mr 120,000 nuclear factor present in transformed murine cells recognizes the RRE. Mutation of the conserved RRE element to the palindromic element AGACTCT created a binding site that is recognized with 5-fold greater affinity by the Mr 120,000 factor. The palindromic element functions as an RRE as determined by transient transfection assays. UV-crosslinking of nuclear factors in human tumor cell lines to the palindromic element revealed that an Mr 120,000 factor that recognized RREs was present in cells that contain activated Ha- or N-ras genes, but not in human tumor cells that lack activated ras. Expression of exogenous activated ras in a human tumor cell line that lacks the oncogene induced the Mr 120,000 factor. The Mr 120,000 factor, which we have termed ras-responsive factor 1, is an intermediate in the signal transduction pathway that links ras to the nucleus and may play a role in the initiation or progression of human tumors containing an activated ras gene.
Base Sequence↗
Negative regulation of transcription in vitro by a glucocorticoid response element is mediated by a trans-acting factor.
In vitro experiments with cell extracts prepared from a mouse mammary epithelial cell line demonstrated that a cis-acting glucocorticoid response element (GRE) of the mouse mammary tumor virus represses transcription from its homologous promoter. Competition transcription experiments, in which a molar excess of a restriction fragment that contains the GRE is added to the cell-free assay, revealed that a nuclear factor mediates in trans the negative regulation of mammary tumor virus transcription in vitro. Gel retention assays indicated that a factor in the extracts specifically recognizes the GRE. One unusual result of the gel retention studies was that heating the GRE probe to 65 degrees C before addition to a binding assay increases the formation of the specific protein-DNA complex 20-fold. Exonuclease III footprinting demonstrated that the sequences recognized by the factor are identical for either untreated or heat-treated probe. The footprinting also demonstrated that this factor recognizes sequences that are distinct from those recognized by the glucocorticoid receptor. A synthetic oligonucleotide based on the sequences identified by the footprinting experiments repressed the activity of a heterologous enhancer-promoter in vivo, as assayed by transient expression assays. We propose that this negative transcription element may control the basal level of expression of some glucocorticoid-modulated genes and may explain the insensitivity of certain tumor cells to steroid hormone action.
Animals↗
Isolation and characterization of minichromosome particles that contain a glucocorticoid-modulated promoter.
A procedure for the enrichment of minichromosomes, composed of bovine papilloma virus and the long terminal repeat element of the mouse mammary tumor virus (MTV), from isolated nuclei is described. Up to 60% of the minichromosomes were extracted as nucleoprotein particles. These particles sediment in sucrose gradients as 160S complexes. Hormone-labeled glucocorticoid receptor co-purifies with these complexes in a specific fashion. Between four and six molecules of receptor are bound per minichromosome molecule. Analysis of DNase I hypersensitivity demonstrates that hypersensitive sites are preserved through the purification procedure in a manner that reflects the hormone-dependent in vivo pattern of digestion. These purified minichromosomes will allow features of chromatin structure that may be important for steroid hormone modulation of transcription to be studied in vitro without resorting to destructive nuclease digestion procedures.
Animals↗
Rapid and selective alterations in the expression of cellular genes accompany conditional transcription of Ha-v-ras in NIH 3T3 cells.
Hormone treatment of NIH 3T3 cells that contain recombinant fusions between the mouse mammary virus long terminal repeat and the v-ras gene of Harvey murine sarcoma virus results in conditional expression of the ras p21 gene product. Levels of ras mRNA and p21 are maximal after 2 to 4 h of hormone treatment. Analysis of cellular RNA by Northern blotting and nuclease S1 protection assays indicates that the expression of two cellular RNA species increases with kinetics similar to v-ras: v-sis-related RNA and retrovirus-related VL30 RNA. Run-on transcription in isolated nuclei shows that the increase in v-sis-related RNA is not dependent on transcription and therefore must arise by a post-transcriptional mechanism. The increase in VL30 expression is a transcriptional effect. Hormone treatment of normal NIH 3T3 cells has no effect on the expression of these DNA sequences. These results suggest that v-ras stimulation of autocrine factors may play a role in transformation of cells by this gene and also suggest a reverse genetic strategy to determine the nucleic acid sequences and cellular factors involved in the regulation of gene expression that is observed.
Animals↗