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Glutaredoxin is a direct target of oncogenic jun
Abstract
We have analysed differential gene expression in v-jun-transformed chicken embryo fibroblasts (CEF) compared to normal CEF by using the directional tag PCR subtraction method. From a first generation of putative Jun targets four clones were selected for study; they are upregulated in jun-transformed cells. Three of these clones showed homology to known genes: glutaredoxin, growth associated protein (GAP)-43/neuromodulin, and phenobarbital-induced cytochrome P450. The expression of these genes was analysed in fibroblasts transformed by various oncogenes. Expression of the glutaredoxin mRNA could be induced by a Jun-estrogen receptor chimaera in the absence of de novo protein biosynthesis. Based on this observation we conclude that glutaredoxin is a direct target of v-Jun.
... Approaches aimed at the identification of genes specifically dysregulated in jun-or fos-transformed fibroblasts have led to the identification of several new potential jun target genes Bister, 1995, 1998;Hadman et al., 1996Hadman et al., , 1998Goller et al., 1998;Fu et al., 1999). For two of these genes it has been demonstrated that they represent direct transcriptional targets of oncogenic Jun. ...
... The complete structural organization of the BKJ gene was determined by nucleotide sequence analysis and transcriptional mapping, and transcriptional transactivation analysis revealed that BKJ is a direct transcriptional target of the AP-1 components Jun and Fos in transformed avian fibroblasts (Hartl and Bister, 1998). The glutaredoxin gene was found to be induced in v-jun-transformed fibroblasts and, based on its activation by a Jun-estrogen receptor chimera (Kruse et al., 1997) in the presence of the protein synthesis inhibitor cycloheximide, classified as a direct transcriptional target of Jun (Goller et al., 1998). Another putative direct target of oncogenic Jun, a gene encoding heparinbinding epidermal growth factor-like growth factor (HB-EGF), is distinguished by its capability to induce partial cell transformation when expressed in a retroviral context (Fu et al., 1999). ...
... Another putative direct target of oncogenic Jun, a gene encoding heparinbinding epidermal growth factor-like growth factor (HB-EGF), is distinguished by its capability to induce partial cell transformation when expressed in a retroviral context (Fu et al., 1999). All of these genes have been isolated from nonconditional transformation systems using either an established jun-transformed cell line or cells chronically infected by a retroviral vector expressing oncogenic Jun (Hadman et al., 1996;Goller et al., 1998;Fu et al., 1999). ...
To investigate the molecular basis of oncogenesis induced by the v-jun oncogene of avian sarcoma virus 17 (ASV17), we developed a conditional cell transformation system in which transcription of the ASV17 v-jun allele is controlled by a doxycycline-sensitive transactivator (tTA) or a reverse (doxycycline-dependent) transactivator (rtTA), respectively. Permanent cell lines of quail embryo fibroblasts conditionally transformed by a doxycycline-controlled v-jun allele revert to the normal phenotype within 3 days and lose their ability to grow in soft agar, strictly dependent on the addition or removal of the drug, respectively. The reverted cells are rapidly retransformed on conditional activation of v-jun. While full-level synthesis of v-jun mRNA and v-Jun protein in these cells is established within 2 and 14 h, respectively, after switching to the permissive conditions, the first morphological alterations are observed after 24 h, and as early as 2 days later the morphology has changed entirely from flat cells resembling normal fibroblasts to spindle-shaped fusiform cells showing a typical jun-transformed phenotype. Kinetic expression analysis revealed that transcriptional activation of the direct jun target gene BKJ precisely coincides with the establishment of full-level v-Jun protein synthesis. Furthermore, we have analyzed the expression of a novel candidate v-jun target gene, termed JAC, which shows no sequence homology to known genes. Similar to BKJ, JAC is specifically activated in jun-transformed fibroblasts, and induction of JAC is tightly linked to the conditional expression of oncogenic v-Jun. These results demonstrate the high stringency of the doxycycline-controlled v-jun expression system, and they also indicate that expression of v-jun in these cells is indispensable for enhanced proliferation, cell transformation, and the induction of specific expression patterns of downstream target genes.
... Further insights into the mechanism of v-Jun transformation will develop from the identi®cation and functional characterization of aberrantly expressed target genes. Previous eorts have identi®ed several upregulated v-Jun targets including quail bkj, encoding a member of the b-keratin family, chicken jtab-1, encoding a cathepsin-like protein and the genes encoding glutaredoxin, neuromodulin, phenobarbital-induced cytochrome P450 and heparinbinding EGF-like growth factor (HB-EGF) (Fu et al., 1999;Goller et al., 1998;Hadman et al., 1996;Hartl and Bister, 1995). One of these targets, HB-EGF, can induce partial oncogenic transformation. ...
... The directional tag PCR subtraction method was used to generate a library of cDNA clones enriched in genes that are overexpressed in v-Jun transformed CEF (Goller et al., 1998;Usui et al., 1994). Two directional cDNA libraries were produced in dierent vectors. ...
... Selected clones identi®ed from a subtractive library were used as probes to isolate full-length cDNAs by screening two cDNA libraries, which were constructed by cloning the cDNA population derived from v-Jun transformed CEF into the pT7T3D plasmid vector or the Uni-ZAP XR l phage vector (Fu et al., 1999;Goller et al., 1998). For screening, the plasmid cDNA library was transformed into XL-1 blue cells, and bacterial colonies were transferred onto Hybond-N ®lters, while phage plaques from the l cDNA library were transferred onto Hybond-C extra nitrocellulose ®lters (Amersham). ...
The transcription factor Jun (c-Jun) functions as a recipient of extracellular growth signals and converts them into patterns of gene expression. An oncogenic variant of c-Jun was isolated from the acutely transforming retrovirus ASV17. Overexpression of this viral Jun (v-Jun) induces transformation of chicken embryo fibroblasts (CEF) in culture and fibrosarcomas in chickens. v-Jun is a constitutively active form of c-Jun and transforms cells presumably by deregulating the expression of specific target genes. In this report, we describe six genes whose transcripts are upregulated in v-Jun-transformed CEF. Three of these genes show homology to known mammalian genes, to MAP kinase phosphatase 2 (MKP-2), to reversion-induced LIM protein (RIL) and to cytokine-inducible SH2-containing protein (CIS). Northern blot analysis, using CEF infected with various Jun mutants or an estrogen-regulatable Jun chimera, revealed distinct induction patterns of individual targets by v-Jun. The chicken RIL homolog showed an expression pattern tightly correlated with the activity of v-Jun. Its expression is also transformation-dependent, suggesting a role for this gene in v-Jun transformation. The newly identified v-Jun targets can serve as molecular markers in the v-Jun transformation process. Oncogene (2000) 19, 3537 - 3545
... Identification of VJT-6 as a v-Jun-Responsive Gene. We have used the directional tag PCR subtraction method to search for genes that are up-regulated in v-Jun-transformed CEF but not in vector-infected CEF (20,31). Several clones representing genes that are up-regulated in Jun-transformed CEF were isolated; among these is clone VJT-6 (viral Jun target 6). ...
... Because of the altered target specificity of v-Jun and the down-regulation of c-Jun in v-Jun-transformed cells, such cells probably contain differentially regulated transformation-specific genes. In recent years, several genes that are specifically up-regulated in v-Jun-transformed cells have been identified, but little is known about their role in the transformation process (18)(19)(20). None of them has been shown to induce cellular properties characteristic of the transformed phenotype. ...
Jun is a transcription factor belonging to the activator protein 1 family. A mutated version of Jun (v-Jun) transduced by the avian retrovirus ASV17 induces oncogenic transformation in avian cell cultures and sarcomas in young galliform birds. The oncogenicity of Jun probably results from transcriptional deregulation of v-Jun-responsive target genes. Here we describe the identification and characterization of a growth-related v-Jun target, a homolog of heparin-binding epidermal growth factor-like growth factor (HB-EGF). HB-EGF is strongly expressed in chicken embryo fibroblasts (CEF) transformed by v-Jun. HB-EGF expression is not detectable or is marginal in nontransformed CEF. Using a hormone-inducible Jun-estrogen receptor chimera, we found that HB-EGF expression is correlated with v-Jun activity. In this system, induction of v-Jun is followed within 1 hr by elevated levels of HB-EGF. In CEF infected with various Jun mutants, HB-EGF expression is correlated with the oncogenic potency of the mutant. Constitutive expression of HB-EGF conveys to CEF the ability to grow in soft agar and to form multilayered foci of transformed cells on a solid substrate. These observations suggest that HB-EGF is an effector of Jun-induced oncogenic transformation.
... There is increasing evidence that cellular transformation induced by the v-Jun protein involves the aberrant expression of specific genes that are normally regulated by endogenous c-Jun as a component of AP-1. Approaches aimed at the identification of genes specifically deregulated in jun-or fos-transformed fibroblasts have led to the identification of several jun target genes (2,7,15), including the direct transcriptional targets BKJ, encoding a -keratin-related protein (16,17), glutaredoxin (18), the gene encoding heparin-binding epidermal growth factor-like growth factor (HB-EGF) (19), and the TOJ3 gene encoding a protein highly related to microspherule protein 1 (MCRS1) (20). Remarkably, the HB-EGF and TOJ3 genes were shown to induce partial cell transformation when expressed in a retroviral context (19,20). ...
... Several AP-1 target genes have been identified, but the possible role of most of those genes in jun-induced cell transformation has not been assessed yet. However, the recent identification of genes, like BKJ (16,17), glutaredoxin (18), HB-EGF (19), or JAC, which are directly regulated by Jun and whose expression profiles correlate precisely with the jun-transformed cellular phenotype, strongly supports the hypothesis that cell transformation induced by oncogenic transcription factors is a consequence of aberrant transcriptional regulation of distinct target genes. Based on these criteria, and on its capacity to induce partial cell transformation, the JAC gene described here presumably belongs to the class of genes that are directly involved in jun-induced cell transformation and tumorigenesis. ...
Using subtractive hybridization techniques, we have isolated a gene termed JAC that is strongly and specifically activated in avian fibroblasts transformed by the v-jun oncogene of avian sarcoma virus 17 (ASV17), but not in cells transformed by other oncogenic agents. Furthermore, JAC is highly expressed in cell lines derived from jun-induced avian fibrosarcomas. Kinetic analysis using a doxycycline-controlled conditional cell transformation system showed that expression of the 0.8-kb JAC mRNA is induced rapidly upon activation of the oncogenic v-jun allele. Nucleotide sequence analysis and transcriptional mapping revealed that the JAC gene contains two exons, with the longest ORF confined to exon 2. The deduced 68-amino acid chicken JAC protein is rich in cysteine residues and displays 37% sequence identity to mammalian high-sulfur keratin-associated proteins. The promoter region of JAC contains a consensus (5'-TGACTCA-3') and a nonconsensus (5'-TGAGTAA-3') AP-1 binding site in tandem, which are both specifically bound by the Gag-Jun hybrid protein encoded by ASV17. Mutational analysis revealed that the two AP-1 sites confer strong transcriptional activation by Gag-Jun in a synergistic manner. Ectopic expression of JAC in avian fibroblasts leads to anchorage-independent growth, strongly suggesting that deregulation of JAC is an essential event in jun-induced cell transformation and tumorigenesis.
... Approaches aimed at the identi®cation of genes speci®cally deregulated in jun-or fos-transformed primary avian ®broblasts, a cellular system in which these oncogenes autonomously induce cell transformation and tumorigenesis (Maki et al., 1987;Nishizawa et al., 1987), have led to the identi®cation of several jun target genes. The activated genes include BKJ, encoding a hydrophobic protein structurally related to avian epidermal b-keratins Bister, 1995, 1998), JTAP-1, encoding a cathepsin-like proteinase (Hadman et al., 1996), glutaredoxin, encoding a redox cofactor in the biosynthesis of deoxyribonucleotides (Goller et al., 1998), HB-EGF, encoding heparinbinding epidermal growth factor-like growth factor , RIL, encoding reversion-induced LIM protein (Fu et al., 2000), and JAC, encoding a novel cysteine-rich protein with unknown function (Bader et al., 2000). From these genes, BKJ, glutaredoxin, HB-EGF, and JAC represent transcriptional targets directly regulated by v-Jun as demonstrated by promoter analysis of the BKJ gene (Hartl and Bister, 1998) and by usage of conditional cell transformation systems (Kruse et al., 1997;Goller et al., 1998;Bader et al., 2000). ...
... The activated genes include BKJ, encoding a hydrophobic protein structurally related to avian epidermal b-keratins Bister, 1995, 1998), JTAP-1, encoding a cathepsin-like proteinase (Hadman et al., 1996), glutaredoxin, encoding a redox cofactor in the biosynthesis of deoxyribonucleotides (Goller et al., 1998), HB-EGF, encoding heparinbinding epidermal growth factor-like growth factor , RIL, encoding reversion-induced LIM protein (Fu et al., 2000), and JAC, encoding a novel cysteine-rich protein with unknown function (Bader et al., 2000). From these genes, BKJ, glutaredoxin, HB-EGF, and JAC represent transcriptional targets directly regulated by v-Jun as demonstrated by promoter analysis of the BKJ gene (Hartl and Bister, 1998) and by usage of conditional cell transformation systems (Kruse et al., 1997;Goller et al., 1998;Bader et al., 2000). In addition to upregulated targets, several genes are known to be suppressed in v-jun-transformed cells (Hadman et al., 1998;Mettouchi et al., 1994;Vial and Castellazzi, 2000;Cohen et al., 2001). ...
Using the established quail cell line Q/d3 conditionally transformed by the v-jun oncogene, cDNA clones (TOJ2, TOJ3, TOJ5, TOJ6) were isolated by representational difference analysis (RDA) that correspond to genes which were induced immediately upon conditional activation of v-jun. One of these genes, TOJ3, is immediately and specifically activated after doxycycline-mediated v-jun induction, with kinetics similar to the induction of well characterized direct AP-1 target genes. TOJ3 is neither activated upon conditional activation of v-myc, nor in cells or cell lines non-conditionally transformed by oncogenes other than v-jun. Sequence analysis revealed that the TOJ3-specific cDNA encodes a 530-amino acid protein with significant sequence similarities to the murine or human microspherule protein 1 (MCRS1, MSP58), a nucleolar protein that directly interacts with the ICP22 regulatory protein from herpes simplex virus 1 or with p120, a proliferation-related protein expressed at high levels in most human malignant tumor cells. Similar to its mammalian counterparts, the TOJ3 protein contains a bipartite nuclear localization motif and a forkhead associated domain (FHA). Using polyclonal antibodies directed against a recombinant amino-terminal TOJ3 protein segment, the activation of TOJ3 in jun-transformed fibroblasts was also demonstrated at the protein level by specific detection of a polypeptide with an apparent M(r) of 65 000. Retroviral expression of the TOJ3 gene in quail or chicken embryo fibroblasts induces anchorage-independent growth, indicating that the immediate activation of TOJ3 in fibroblasts transformed by the v-jun oncogene contributes to cell transformation.
... The first link of GAP43 to neoplastic cell transformation was obtained by differential gene expression analysis. GAP43 was found to be strongly up-regulated in chicken embryo fibroblasts transformed by several oncogenes (30) suggesting that associated perturbations in PKC-or Ca 2+ /CaM-mediated signal transduction pathways are implicated in oncogenesis. In fact, CaM is involved in the promotion of physiological cell cycle progression but also in tumorigenesis (31)(32)(33). ...
The neuronal proteins GAP43 (neuromodulin), MARCKS, and BASP1 are highly expressed in the growth cones of nerve cells where they are involved in signal transmission and cytoskeleton organization. Although their primary structures are unrelated, these signaling proteins share several structural properties like fatty acid modification, and the presence of cationic effector domains. GAP43, MARCKS, and BASP1 bind to cell membrane phospholipids, a process reversibly regulated by protein kinase C-phosphorylation or by binding to the calcium sensor calmodulin (CaM). GAP43, MARCKS, and BASP1 are also expressed in non-neuronal cells, where they may have important functions to manage cytoskeleton architecture, and in case of MARCKS and BASP1 to act as cofactors in transcriptional regulation. During neoplastic cell transformation, the proteins reveal differential expression in normal vs. tumor cells, and display intrinsic tumor promoting or tumor suppressive activities. Whereas GAP43 and MARCKS are oncogenic, tumor suppressive functions have been ascribed to BASP1 and in part to MARCKS depending on the cell type. Like MARCKS, the myristoylated BASP1 protein is localized both in the cytoplasm and in the cell nucleus. Nuclear BASP1 participates in gene regulation converting the Wilms tumor transcription factor WT1 from an oncoprotein into a tumor suppressor. The BASP1 gene is downregulated in many human tumor cell lines particularly in those derived from leukemias, which display elevated levels of WT1 and of the major cancer driver MYC. BASP1 specifically inhibits MYC-induced cell transformation in cultured cells. The tumor suppressive functions of BASP1 and MARCKS could be exploited to expand the spectrum of future innovative therapeutic approaches to inhibit growth and viability of susceptible human tumors.
... The yeast Saccharomyces cerevisiae contains two TTase genes, which are required for protection against reactive oxygen species (Luikenhuis et al., 1998). In v-jun-transformed chicken embryo fibroblasts, the expression of TTase mRNA could be induced, indicating that TTase is a direct target of v-Jun (Goller et al., 1998). Recently, TTase has been found to play a central role in protection against protein damage caused by menadione and hydrogen peroxide in S. cerevisiae (Rodriguez-Manzaneque et al., 1999). ...
Thioltransferase (TTase), also known as glutaredoxin (Grx), is an enzyme catalyzing the reduction of a variety of disulfide compounds and acting as a cofactor for various enzymes such as ribonucleotide reductase. The Schizosaccharomyces pombe cells, exponentially grown in rich medium at 30 o C, were shifted to 20 o C and 35 o C. The yeast cells, shifted to 35 o C, showed higher TTase activity than the cells continuously grown at 30 o C, whereas the yeast cells, shifted to 20 o C, gave lower TTase activity. The S. pombe cells, exponentially grown in minimal medium and shifted from 30 o C to 35 o C and 40 o C, produced higher TTase activity. When the S. pombe cells were initially incubated in rich and minimal media at three different temperatures (25 o C, 30 o C and 35 o C), they showed higher TTase activity at higher temperature. These results suggest that the TTase activity of S. pombe is regulated by temperature.
... The human Glrx1 gene contains putative activator protein-1 (AP-1) sites in its promoter, which links expression of Glrx1 to signaling pathways that control Fos and Jun family members [27]. Indeed, the chicken Glrx1 gene was demonstrated to be a direct target of oncogenic Jun [38], and similarly, under conditions of oxidative stress, in lens epithelial cells the human Glrx1 gene was induced in an AP-1 dependent manner [39]. Results from the present study demonstrate that both in RAW 264.7 macrophages and C10 lung epithelial cells, Grx1 protein expression was increased following activation of NF-κB through expression of CA-IKKβ. ...
The transcription factor nuclear factor κB (NF-κB) is a critical regulator of inflammation and immunity and is negatively regulated via S-glutathionylation. The inhibitory effect of S-glutathionylation is overcome by glutaredoxin-1 (Grx1), which under physiological conditions catalyzes deglutathionylation and enhances NF-κB activation. The mechanisms whereby expression of the Glrx1 gene is regulated remain unknown. Here we examined the role of NF-κB in regulating activation of Glrx1. Transgenic mice that express a doxycycline-inducible constitutively active version of inhibitory κB kinase-β (CA-IKKβ) demonstrate elevated expression of Grx1. Transient transfection of CA-IKKβ also resulted in significant induction of Grx1. A 2-kb region of the Glrx1 promoter that contains two putative NF-κB binding sites was activated by CA-IKKβ, RelA/p50, and lipopolysaccharide (LPS). Chromatin immunoprecipitation experiments confirmed binding of RelA to the promoter of Glrx1 in response to LPS. Stimulation of C10 lung epithelial cells with LPS caused transient increases in Grx1 mRNA expression and time-dependent increases in S-glutathionylation of IKKβ. Overexpression of Grx1 decreased S-glutathionylation of IKKβ, prolonged NF-κB activation, and increased levels of proinflammatory mediators. Collectively, this study demonstrates that the Glrx1 gene is positively regulated by NF-κB and suggests a feed-forward mechanism to promote NF-κB signaling by decreasing S-glutathionylation.
... These proteins are expressed in neuronal growth cones and are substrates of protein kinase C (Maekawa et al., 1994). Recently, the dysregulation of the GAP-43/neuromodulin gene in chicken embryo fibroblasts transformed by the v-src, v-qin, or v-jun oncogene has been reported (Goller et al., 1998). Possibly, perturbations of protein kinase C or Ca 2ϩ -mediated signal transduction pathways caused by deregulation of genes encoding calmodulin-binding proteins is implicated in cell transformation. ...
To investigate the molecular basis of oncogenesis induced by the v-myc oncogene of avian myelocytomatosis virus MC29, we developed a conditional cell transformation system in which expression of the MC29 v-myc allele is dependent on a doxycycline-sensitive transactivator (tTA). Clonal lines of quail embryo fibroblasts transformed by doxycycline-controlled v-myc revert to the normal phenotype and lose their ability to grow in soft agar after the addition of doxycycline. Repression of v-myc causes the cells to withdraw from the cell cycle, and long-term survival in culture requires reexpression of v-myc. Although complete repression of v-myc mRNA and v-Myc protein in these cells occurs within 14 h after the addition of doxycycline, the first morphological alterations are observed after 24 h, and after 3 days, the morphology changed entirely from small rounded cells showing a typical myc-transformed phenotype to large flat cells resembling normal fibroblasts. Cells exposed to doxycycline for 3 days reexpressed v-myc within 24 h after withdrawal of the drug from the culture medium, partial retransformation occurred after 2 days, and complete morphological transformation was reestablished after 6 days. Analogous results were obtained with a cell line in which expression of the v-myc allele is dependent on a reverse transactivator (rtTA) that is activated by doxycycline. The striking differential expression of known transformation-sensitive genes and of new candidate v-myc target genes revealed the tightness of the doxycycline-controlled v-myc expression system. The data also indicate that expression of v-myc in these cells is indispensable for enhanced proliferation, transformation, and immortalization.
... An important question that remains to be answered concerns the identi®cation of these oncogenically relevant target genes. We and others have identi®ed several targets associated with the v-Jun transformed phenotype (Fu et al., 1999;Goller et al., 1998;Hadman et al., 1996Hadman et al., , 1998Hartl and Bister, 1998;Hussain et al., 1998). As more and more targets are identi®ed, the problem of how to sort out the`important' from the `non important' becomes a tremendous task. ...
Overexpression of v-Jun in chicken embryo fibroblasts (CEF) leads to oncogenic transformation phenotypically characterized by anchorage independent growth and release from contact inhibition (focus formation). The mechanisms involved in this oncogenic conversion however, are not yet clear. Because Jun is a transcription factor, it has been assumed that oncogenic transformation results directly from deregulated AP-1 target gene expression. However, a number of experimental observations in avian cell culture models fail to correlate oncogenesis with AP-1 activity suggesting that transformation induced by v-Jun may occur through an indirect mechanism. To test this possibility, we introduced point mutations into the basic DNA binding domain of v-Jun and created mutants that exhibit altered binding specificity. When expressed in CEF, these mutants fail to deregulate three known v-Jun target genes (JTAP-1, apolipoprotein A1, c-Jun) thus demonstrating in vivo specificity changes. Each of the binding specificity mutants was also tested for its ability to induce oncogenic transformation. Interestingly, expression of these mutants in CEF results in a phenotype indistinguishable from the vector control with respect to growth rate, focus formation and the ability to form colonies in soft agar. These results are consistent with a model requiring direct AP-1 target deregulation as a prerequisite of v-Jun induced cell transformation. With this in mind, we generated a series of additional mutants that retain the ability to bind AP-1 sequence elements, but vary in their oncogenic potential. We demonstrate the use of these mutants to screen v-Jun induced gene targets for a functional role in cell transformation.
... An understanding of transformation requires the identi®cation and characterization of these targets. Previous work has revealed several genes that are up-regulated in v-Jun-transformed avian cells including bkj (Hartl and Bister, 1995), jtab (Hadman et al., 1996), glutaredoxin (Goller et al., 1998), hbegf (Fu et al., 1999) and ril (Fu et al., 2000). These genes were identi®ed by various forms of substractive hybridization which typically cover only a small fraction of the transcriptome. ...
Line 10T1/2 mouse fibroblast overexpressing the v-Jun oncoprotein were morphologically altered, grew into multilayered foci in culture and formed colonies when suspended in agar. The growth rate of the v-Jun-transformed 10T1/2 cells was not changed significantly from that of the untransformed parental cells, but the saturation density of the transformed cultures exceeded that of normal controls by a factor of 2. mRNA extracted from v-Jun-transformed 10T1/2 cells was analysed for differential gene expression with DNA micro-array technology. One of the targets downregulated by v-Jun was identified as SSeCKS (Src-suppressed C kinase substrate). Re-expression of SSeCKS in v-Jun-transformed fibroblasts reversed the transformed phenotype of the cells. Their ability to form foci was reduced to background levels, the number and size of agar colonies was lowered by a factor of 10 and the saturation density was significantly diminished. However, expression of SSeCKS had little effect on the morphology of v-Jun-transformed 10T1/2 cells. These data suggest that the SSeCKS protein has growth-attenuating properties. Down-regulation of SSeCKS may be essential for Jun-induced transformation.
... Potential cJun/AP-1 target genes such as transin, collagenase, metallothionein, proliferin, and nerve growth factor have been identified by analysing functional AP-1-binding sites in their promoters by in vitro mutagenesis and transient cotransfection experiments (Angel and Karin, 1991). More recently, other approaches to screen differentially expressed genes in cells with or without overexpressed AP-1 components identified Fra-1, Fit-1, annexin II and V and dnmt1 as cfos target genes in rat fibroblasts (Braselmann et al., 1992;Bakin and Curran, 1999), and bkj, glutaredoxin, neuromodulin, cytochrome P450, heparin-binding epidermal growth factor, JAC and TOJ3 as v-jun or v-jun/ c-jun/junD hybrid target genes in avian fibroblasts (Hartl and Bister, 1995;Goller et al., 1998;Hadman et al., 1998;Fu et al., 1999;Bader et al., 2001;Hartl et al., 2001). It is noteworthy that dnmt1, identified as a cFos target gene, induces morphological transformation in rat fibroblasts and that the inhibition of its expression can revert c-fos transformation (Bakin and Curran, 1999). ...
cJun is a major component of the transcription factor AP-1 and mediates a diverse set of biologic properties including proliferation, differentiation, and apoptosis. To identify cJun-responsive genes, we inducibly expressed cJun in Rat-1a cells and observed two distinct phenotypes: changes in cellular morphology with adherent growth and anchorage-independent growth. The biologic effects of cJun were entirely reversible demonstrating that they require the continued presence of cJun. To determine the genes, which mediate the biologic effects of cJun, we employed multiple methods including differential gene analysis, suppression subtractive hybridization, and cDNA microarrays. We identified 38 cJun-responsive genes including three uncharacterized genes under adherent and/or nonadherent conditions. Half of the known 36 genes were cytoskeleton- and adhesion-related genes, suggesting a major role of cJun in the regulation of the genes related to cell morphology. As proof of the principle that this approach could identify genes whose upregulation was necessary for nonadherent growth, we investigated one gene, stathmin whose upregulation by cJun was observed only under these conditions. Although overexpression of stathmin did not result in nonadherent growth, inhibition of stathmin protein expression by antisense oligonucleotides in cJun-induced Rat-1a cells prevented nonadherent growth. These results suggest that stathmin plays an essential role in anchorage-independent growth by cJun and may be a potential target for specific inhibitors for AP-1-dependent processes involved in carcinogenesis.
Thioltransferase, also known as glutaredoxin, was previously purified and characterized from Chinese cabbage (Brassica campestris ssp, napus var. pekinensis), However, in the process of gel filtration on Sephadex G-75, there were two activity peaks, In this study, a second thioltransferase (TTase CC-2) in the minor peak of the Sephadex G-75 elution profile was further purified using affinity chromatography on an S-hexylglutathione-agarose column by eluting with buffer solution containing 2.5 mM S-hexylglutathione. It showed a single band on SDS-PAGE indicating that TTase CC-2 is electrophoretically homogeneous, The molecular weight of TTase CC-2 was estimated to be about 22,000 daltons, and its isoelectric point was determined to be 6.73, Its size appears to be atypical and much larger than that of the first thioltransferase (TTase CC-1) from Chinese cabbage, and it can utilize 2-hydroxyethyl disulfide, S-sulfocysteine, and insulin as substrates. S-sulfocysteine was found to be a superior substrate for TTase CC-2, TTase CC-2 also displayed the reducing activity for non-disulfides such as dehydroascorbic acid. Its optimum pH was 8.5, which was consistent with that of TTase CC-1, TTase CC-2 activity was greatly activated by L-cysteine and reduced glutathione, and was found to be less heat-stable compared with TTase CC-1, Molecular and physiological differences between TTase CC-1 and TTase CC-2 remain to be elucidated. Chinese cabbage is the first plant which is known to contain two kinds of thioltransferases.
Two types of the thioltransferase (also called glutaredoxin) have been previously detected in the cytosolic extract of Schizosaccharomyces pombe, a fission yeast. Previously, the one with a smaller molecular mass (14 kDa) was purified and characterized. In the present study, the second thioltransferase was purified. The purification procedure included ammonium sulfate fractionation (40-80%), Sephadex G-200 gel filtration, DEAE-cellulose ion-exchange chromatography, Sephadex G-50 gel filtration, and glutathione-agarose affinity chromatography. The purified enzyme showed a single band on SDS-PAGE, and its molecular mass was determined to be 23 kDa, It utilizes various compounds as substrates, including 2-hydroxyethyl disulfide. Interestingly, we found that the purified thioltransferase also contains significant glutathione S-transferase activity.
Schizosaccharomyces pombe gene encoding redox enzymes, such as thioltransferase (TTase) and thioredoxin (TRX), were previously cloned and induced by oxidative stress. In this investigation, their expressions were examined using P-galactosidase fusion plasmids. The expression of the two cloned genes appeared to be growth-dependent. The synthesis of P-galactosidase from the TTase-lacZ fusion was increased in the medium with the low glucose level, whereas it was significantly decreased in the medium without glucose or with galactose. It was also decreased in the nitrogen-limited medium. The synthesis of beta -galactosidase from the TRX-lacZ fusion was unaffected by galactose or low glucose. However, it was lowered the absence of glucose. The synthesis of beta -galactosidase from the TTase-lacZ fusion was shown to be enhanced in a higher medium pH. Our findings indicate that S. pombe TTase and TRX genes may be regulated by carbon and nitrogen sources, as well as medium pH.
Two types of the thioltransferase (also called glutaredoxin) have been previously detected in the cytosolic extract of Schizosaccharomyces pombe, a fission yeast. Previously, the one with a smaller molecular mass (14kDa) was purified and characterized. In the present study, the second thioltransferase was purified. The purification procedure included ammonium sulfate fractionation (40-80%), Sephadex G-200 gel filtration, DEAE-cellulose ion-exchange chromatography, Sephadex G-50 gel filtration, and glutathione-agarose affinity chromatography. The purified enzyme showed a single band on SDS-PAGE, and its molecular mass was determined to be 23 kDa. It utilizes various compounds as substrates, including 2-hydroxyethyl disulfide. Interestingly, we found that the purified thioltransferase also contains significant glutathione S-transferase activity.
Schizosaccharomyces pombe gene encoding redox enzymes, such as thioltransferase (TTase) and thioredoxin (TRX), were previously cloned and induced by oxidative stress. In this investigation, their expressions were examined using -galactosidase fusion plasmids. The expression of the two cloned genes appeared to be growth-dependent. The synthesis of -galactosidase from the TTase-lacZ fusion was increased in the medium with the low glucose level, whereas it was significantly decreased in the medium without glucose or with galactose. It was also decreased in the nitrogen-limited medium. The synthesis of galactosidase from the TRX-lacZ fusion was unaffected by galactose or low glucose. However, it was lowered the absence of glucose. The synthesis of -galactosidase from the TTase-lacZ fusion was shown to be enhanced in a higher medium pH. Our findings indicate that S. pombe TTase and TRX genes may be regulated by carbon and nitrogen sources, as well as medium pH.
Thioltransferase (TTase), also known as glutaredoxin (Grx), is an enzyme that catalyzes the reduction of a variety of disulfide compounds, including protein disulfides, in the presence of reduced glutathione. TTase acts as a cofactor for various enzymes such as ribonucleotide reductase. We previously purified a TTase from Schizosaccharomyces pombe and its molecular size was determined. In the present study, a cDNA coding TTase was isolated from a cDNA library of Schizosaccharomyces pombe by colony hybridization, which was constructed in a plasmid vector pGAD GH, and its corresponding insert was confirmed by Southern hybridization. The nucleotide sequence of the 375 bp long cDNA clone reveals an open reading frame, which encodes a protein of 101 amino acids. The coding region of the original clone was transferred after the lac promoter of pUC13 vector for expression in E. coli, and simultaneously, a suitable Shine-Dalgarno (SD) sequence was added in front of the coding region by PCR. The two primers used for PCR also separately contained BamHI and HindIII restriction sites. The E. coli strain (A434) harboring the pUC13 derivative pKU10 showed a 17.3-fold increase in TTase activity compared to the strain with only the vector plasmid.
The genomic DNA encoding thioltransferase was isolated from Schizosaccharomyces pombe using the polymerase chain reaction. The amplified DNA fragment was confirmed by Southern hybridization, completely digested with HindIII and BamHI, and then ligated into the yeast-Escherichia coli shuttle vector pRS316, which resulted in plasmid pEH1. The insert of plasmid pEH1 was transferred into the multi-copy vector YEp357 to generate plasmid pYEH1. The determined nucleotide sequence harbors an open reading frame consisting of four exons and three introns, which encodes a polypeptide of 101 amino acids with a molecular mass of 11261 Da. Thioltransferase activity was increased 1.6-fold in Saccharomyces cerevisiae containing plasmid pYEH1, and 1.8- and 2.7-fold in S. pombe containing plasmid pEH1 and pYEH1, respectively. The upstream sequence and the region encoding the N-terminal six amino acids were fused into promoterless beta-galactosidase gene of the shuttle vector YEp357R to generate the fusion plasmid pYEHR1. Synthesis of beta-galactosidase from the fusion plasmid was found to be enhanced by zinc and NO-generating S-nitroso-N-acetylpenicillamine.
Fos-lacZ and Jun-lacZ transgenic mice were used to assess the involvement of immediate-early genes in the axotomy-transcription coupling pathway triggered by sciatic nerve injury in neonates and adults. Nerve transection transiently induced Fos-lacZ in degenerating (neonatal) and regenerating (adult) motor, but not sensory, neurons. In contrast, Jun-lacZ was persistently up-regulated in both axotomized motor and sensory neurons in neonates and adults. Thus, expression of these genes did not predict neuronal death or survival. As Jun-lacZ was induced in some undamaged sensory neurons, this gene can be regulated by direct (axotomy) and indirect (transcellular) mechanisms. Indirect mechanisms also mediate expression of both genes in denervated muscle, Schwann cells in the distal and proximal stumps, and satellite cells in the DRG following axotomy. Thus, either these genes may regulate distinct sets of target genes in different cell types or they may subserve a single mechanism that is common to many cell types.
Jun : Fos and Jun : ATF complexes represent two classes of AP-1 dimers that (1) preferentially bind to either heptameric or octameric AP-1 binding sites, and (2) are differently regulated by cellular signaling pathways and oncogene products. To discriminate between the functions of Jun : Fos, Jun : ATF and Jun : Jun, mutants were developed that restrict the ability of Jun to dimerize either to itself, or to Fos(-like) or ATF(-like) partners. Introduction of these mutants in chicken embryo fibroblasts shows that Jun : Fra2 and Jun : ATF2 dimers play distinct, complementary roles in in vitro oncogenesis by inducing either anchorage independence or growth factor independence, respectively. v-Jun : ATF2 rather than v-Jun : Fra2 triggers the development of primary fibrosarcomas in the chicken wing. Genes encoding extracellular matrix components seem to constitute an important subset of v-Jun : ATF2-target genes. Repression of the matrix component SPARC by Jun is essential for the induction of fibrosarcomas. Avian primary cells transformed by either Jun : Fra2 or Jun : ATF2 thus provide powerful tools for the investigation of the downstream pathways involved in oncogenesis. Further genetic studies with Jun dimerization mutants will be required to be precise and extend the specific roles of the Jun : Fos and Jun : ATF dimers during cancer progression in avian and mammalian systems.
Cellular Jun (c-Jun) and viral Jun (v-Jun) can induce oncogenic transformation. For this activity, c-Jun requires an upstream signal, delivered by the Jun N-terminal kinase (JNK). v-Jun does not interact with JNK; it is autonomous and constitutively active. v-Jun and c-Jun address overlapping but not identical sets of genes. Whether all genes essential for transformation reside within the overlap of the v-Jun and c-Jun target spectra remains to be determined. The search for transformation-relevant targets of Jun is moving into a new stage with the application of DNA microarrays technology. Genetic screens and functional tests remain a necessity for the identification of genes that control the oncogenic phenotype.
The Jun oncoprotein is a major component of the transcription factor complex AP-1, which regulates the expression of multiple genes essential for cell proliferation, differentiation and apoptosis. Constitutive activation of endogenous AP-1 is required for tumor formation in avian and mammalian cell transformation systems, and also occurs in distinct human tumor cells suggesting that AP-1 plays an important role in human oncogenesis. The highly oncogenic v-jun allele capable of inducing neoplastic transformation in avian fibroblasts and fibrosarcomas in chicken as a single oncogenic event, was generated by mutation of the cellular c-jun gene during retroviral transduction. Hence, avian cells represent an excellent model system to investigate molecular mechanisms underlying jun-induced cell transformation. Approaches aimed at the identification of genes specifically deregulated in jun-transformed fibroblasts have led to the identification of several genes targeted by oncogenic Jun. Some of the activated genes represent direct transcriptional targets of Jun encoding proteins, which are presumably involved in cell growth and differentiation. Genes suppressed in v-jun-transformed cells include several extracellular proteins like components of the extracellular matrix or proteins involved in extracellular signalling. Due to aberrant regulation of multiple genes by the Jun oncoprotein, it is assumed that only the combined differential expression of Jun target genes or of a subset thereof contributes to the conversion of a normal fibroblast into a tumor cell displaying a phenotype typical of jun-induced cell transformation. It has already been shown that distinct activated targets exhibit partial transforming activity upon over-expression in avian fibroblasts. Also, distinct target genes silenced by v-Jun inhibit tumor formation when re-expressed in v-jun-transformed cells. The protein products of these transformation-relevant genes may thus represent potential drug targets for interference with jun-induced tumorigenesis.
We have constructed artificial AP-1 proteins containing elements derived from yeast GCN4 and from the herpes simplex virus activator VP16. These proteins can only homodimerize but do not heterodimerize, and lacking significant homology to Jun outside the DNA-binding domain, they are largely unaffected by proteins that modulate Jun. Constructs in which the transactivation domain of GCN4 is replaced by that of VP16 induce oncogenic transformation in cultures of chicken embryo fibroblasts. The availability of transforming VP16-GCN4 fusion proteins permits an evaluation of downstream target genes, based on the hypothesis that transformation-relevant targets should be common between Jun and the artificial AP-1 proteins. In a pilot study, we examined the expression of several Jun target genes in cells transformed by the VP16-GCN4 fusions and found that some of the Jun targets are not upregulated by the GCN4-derived transforming construct, suggesting that their upregulation in Jun-transformed cells is not essential for cell transformation. We have further constructed a regulatable GCN4-VP16 protein that will permit a kinetic characterization of target gene responses and will facilitate discrimination between direct and indirect targets.
The Fos/Jun and ATF/CREB families of transcription factors function in coupling extracellular signals to alterations in expression of specific target genes. Like many eukaryotic transcription factors, these proteins bind to DNA as dimers. Dimerization is mediated by a structure known as the "leucine-zipper" motif. Although Fos/Jun and ATF/CREB were previously thought to interact preferentially with different DNA regulatory elements (the AP-1/TRE and ATF/CRE sites, respectively), we find that members of these two families form selective cross-family heterodimers. The resulting heterodimers display distinguishable DNA binding specificities from each other and from their parental homodimers. These findings indicate that the Fos/Jun and ATF/CREB families of transcription factors are not as distinct as was previously thought. We suggest that they can be grouped into a superfamily of transcription factors.
We have previously described several helper independent vector constructions (S. Hughes and E. Kosik, Virology 136:89-99, 1984; J. Sorge and S. H. Hughes, J. Mol. Appl. Genet. 1:547-599, 1982; J. Sorge, B. Ricci, and S. Hughes, J. Virol. 48:667-675, 1983), all of which derive from Rous sarcoma virus. In this report we describe three improvements in the earlier constructions. First, the vectors have been restructured as proviruses, which considerably improves the efficiency of virus production following acute transfection. Second, a series of miniplasmids have been developed, which we call adaptors, and these miniplasmids can be used to convert virtually any DNA segment into a ClaI fragment suitable for insertion into the retroviral (or other) vectors. Adaptors have been developed that supply regions of functional significance, including a splice acceptor and an initiator ATG. Finally, the region of env defining subgroup specificity, A in the original vectors, has been substituted by the corresponding regions of subgroup B and D viruses, giving vectors with additional subgroup specificities and increased host ranges.
A 30-amino-acid segment of C/EBP, a newly discovered enhancer binding protein, shares notable sequence similarity with a segment
of the cellular Myc transforming protein. Display of these respective amino acid sequences on an idealized alpha helix revealed
a periodic repetition of leucine residues at every seventh position over a distance covering eight helical turns. The periodic
array of at least four leucines was also noted in the sequences of the Fos and Jun transforming proteins, as well as that
of the yeast gene regulatory protein, GCN4. The polypeptide segments containing these periodic arrays of leucine residues
are proposed to exist in an alpha-helical conformation, and the leucine side chains extending from one alpha helix interdigitate
with those displayed from a similar alpha helix of a second polypeptide, facilitating dimerization. This hypothetical structure
is referred to as the "leucine zipper," and it may represent a characteristic property of a new category of DNA binding proteins.
Mutants in the leucine zipper and basic regions of mouse c-jun were tested for transformation in chicken embryo fibroblast cultures. Reduction or elimination of the ability of Jun to dimerize or to bind to DNA severely decreased transformation. A chicken v-jun gene from which the major transactivation domain was deleted also failed to transform. We conclude that an intact leucine zipper, basic region and transactivation domain are required for Jun-induced oncogenic transformation. Coexpression of chicken c-Fos increased formation of transformed foci by Jun proteins of moderate to low oncogenic potency but had no effect on highly transforming Jun. Chicken c-Fos could also transform chicken embryo fibroblasts on its own, albeit after prolonged culture and at a low efficiency.
To assess the transforming capability of the c-Jun protein, we introduced the chicken c-jun proto-oncogene into a replication competent avian retroviral expression vector (RCAS). Viral Jun efficiently transformed chicken embryo fibroblasts (CEFs) when expressed from this vector. Overexpression of c-Jun leads to transformation of CEFs with an efficiency that is 15- to 25-fold less than that seen for v-Jun, suggesting that v-Jun contains structural features that increase its oncogenic potential relative to c-Jun. There are four structural differences between v-Jun and c-Jun. To determine the relative contribution that each of these structural differences between v-Jun and c-Jun has on oncogenic activity, several deletion and substitution mutants were constructed. Each of these mutants was expressed in CEF and assayed for transformation by focus formation. Analysis of the results reveals that deletion of a region of 27 amino acids near the amino terminus of c-Jun and deletion of 3'-untranslated sequences are critical in activating the full oncogenic potential of Jun.
Growth-associated protein (GAP)-43 is a neuron-specific phosphoprotein whose expression is associated with axonal outgrowth during neuronal development and regeneration. In order to investigate the expression of this gene product in the early developing nervous system we have isolated and sequenced a cDNA for chicken GAP-43. The predicted amino acid sequence for chicken GAP-43 displays extensive similarity to that of the mammalian protein, particularly in the amino-terminal region, to which functional domains of the protein have been assigned. The cDNA hybridizes with two RNAs of differing molecular weights on Northern blots; both appear to be regulated similarly. These RNAs first appear in the brain on embryonic day 3 (E3), suggesting that GAP-43 begins to be expressed when neuroblasts become post-mitotic. In situ hybridization analysis reveals that GAP-43 RNA is expressed by several neural structures in the chick embryo, including derivatives of the neural tube, neural crest, and neuroectodermal placodes.
Biologically active molecular clones of avian sarcoma virus 17 (ASV 17) contain a replication-defective proviral genome of 3.5 kilobases (kb). The genome retains partial gag and env sequences, which flank a cell-derived putative oncogene of 0.93 kb, termed jun. The jun gene lacks preserved coding domains of tyrosine-specific protein kinases. It also shows no significant nucleic acid homology with other known oncogenes. The probable transformation-specific protein in ASV 17-transformed cells is a 55-kDa gag-jun fusion product.
Avian sarcoma virus 31 contains an oncogene that we have named qin. qin codes for a nuclear protein, Qin, that is a member of the HNF-3/fork head family of transcriptional regulators. Within this family Qin is particularly closely related to rat brain factor 1 (BF-1), a telencephalon-specific gene presumed to play an important role in the development of the mammalian brain.
We have analyzed differential gene expression in normal versus jun-transformed avian fibroblasts by using subtracted nucleic acid probes and differential nucleic acid hybridization techniques for the isolation of cDNA clones. One clone corresponded to a gene that was strongly expressed in a previously established quail (Coturnix japonica) embryo fibroblast line (VCD) transformed by a chimeric jun oncogene but whose expression was undetectable in normal quail embryo fibroblasts. Furthermore, the gene was expressed in quail or chicken fibroblast cultures that were freshly transformed by retroviral constructs carrying various viral or cellular jun alleles and in chicken fibroblasts transformed by the avian retrovirus ASV17 carrying the original viral v-jun allele. However, its expression was undetectable in a variety of established avian cell lines or freshly prepared avian fibroblast cultures transformed by other oncogenes or a chemical carcinogen. The nucleotide and deduced amino acid sequences of the cDNA clone were not identical to any sequence entries in the data bases but revealed significant similarities to avian beta-keratin genes; the highest degree of amino acid sequence identity was 63%. The gene, which we termed bkj, may represent a direct or indirect target for jun function.
The oncogenic potential of Jun in chicken embryo fibroblasts (CEF) varies depending on its structure. V-Jun, which has a number of structural differences from c-Jun is highly transforming and tumorigenic. C-Jun however, is only weakly transforming and is not tumorigenic. We have used this difference in oncogenic potential between v-Jun and c-Jun to screen for downstream target genes associated with the v-Jun induced transformed phenotype. We describe here the identification, cloning and characterization of one of these genes, JTAP-1. JTAP-1 is consistently overexpressed 7 to 10-fold in CEF transformed by v-Jun compared with c-Jun overexpressing or normal CEF. This pattern of expression suggests that JTAP-1 is associated with the transformed phenotype. DNA and amino acid homology search analysis revealed that JTAP-1 shares a high degree of similarity with over 100 cysteine proteases from a variety of species and is likely the chicken homolog of cathepsin O. Analysis of expression of JTAP-1 in CEF overexpressing other oncogenes including v-Ha-ras, v-Src, c-Fos, and Myc revealed that it's overexpression is unique to v-Jun transformed cells. Thus, JTAP-1 is likely a specific target of v-Jun overexpression and not simply a consequence of cell transformation.
The v-jun oncogene encodes a nuclear DNA binding protein that functions as a transcription factor and is part of the activator protein 1 complex. Oncogenic transformation by v-jun is thought to be mediated by the aberrant expression of specific target genes. To identify such Jun-regulated genes and to explore the mechanisms by which Jun affects their expression, we have fused the full-length v-Jun and an amino-terminally truncated form of v-Jun to the hormone-binding domain of the human estrogen receptor. The two chimeric proteins function as ligand-inducible transactivators. Expression of the fusion proteins in chicken embryo fibroblasts causes estrogen-dependent transformation.
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