Biomedical subjects

M Castellazzi

Publications and source records attributed to M Castellazzi.

At least 19 recordsLinked to original sources

PubMed2001

Opposing functions of ATF2 and Fos-like transcription factors in c-Jun-mediated myogenin expression and terminal differentiation of avian myoblasts.

With the aim to identify the oncoprotein partners implicated in the c-Jun myogenic influence, we carried out stable transfection experiments of c-Jun and/or ATF2, Fra2, c-Fos overexpression in avian myoblasts. Before induction of differentiation, c-Jun repressed myoblast withdrawal from the cell cycle, as did a TPA treatment. However, after serum removal, unlike TPA, c-Jun significantly stimulated myoblast differentiation. In search for specific partners involved in this dual influence, we found that a reduction in the amounts of c-Fos and Fra2 and an increase in c-Jun proteins occurred at cell confluence, a situation likely to favor cooperation between c-Jun and ATF2 during terminal differentiation. Whereas c-Fos and Fra2 cooperated with c-Jun to abrogate myoblast withdrawal from the cell cycle and terminal differentiation, ATF2 co-expression potentiated the positive myogenic c-Jun influence. In addition, myogenin expression was a positive target of this cooperation and this regulation occurred through a stimulation of myogenin promoter activity: (1) whereas c-Fos or Fra2 co-expression abrogated c-Jun stimulatory activity on this promoter, ATF2 co-expression potentiated this influence; (2) using a dominant negative ATF2 mutant, we established that c-Jun transcriptional activity required functionality of endogenous ATF2. These data suggest that through this dual myogenic influence due to cooperations with different partners, c-Jun is involved in the control of duration of myoblast proliferation and thereafter of fusion efficiency.

Activating Transcription Factor 2↗

PubMed2001

JAC, a direct target of oncogenic transcription factor Jun, is involved in 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.

Amino Acid Sequence↗

PubMed2001

The defective transforming phenotype of c-Jun Ala(63/73) is rescued by mutation of the C-terminal phosphorylation site.

Cotransfection of primary rat embryo fibroblasts (REF) with c-Jun and activated Ras leads to oncogenic transformation and this process requires the phosphorylation of the N-terminal domain of c-Jun. Ras augments this phosphorylation and, consequently activates the c-Jun transactivation property of TRE (TPA Responsive Element)-dependent promoters. To analyse the role of the c-Jun C-terminal phosphorylation site in oncogenic cooperation we tested the activities of N-terminal c-Jun Ala(63/73) (named Nt), C-terminal c-Jun Ala(234/242/246/252) (named Ct) and (Nt+Ct)-with both mutations-non-phosphorylatable c-Jun mutants. In cooperation with Ras, the Ct mutant and wt c-Jun display similar oncogenic properties whereas the Nt form was defective in transforming REF cells. Unexpectedly, the Nt+Ct mutant exhibited identical oncogenic properties to wt c-Jun, demonstrating that the Ct mutation rescues in cis the Nt mutation. The transcriptional activity and the capacity to bind the c-Jun coactivator CREB Binding Protein (CBP) were enhanced by Ras for the wt and Ct proteins but not for the Nt mutant. Interestingly, the Nt+Ct mutant presents identical transactivation and CBP binding activities to wt c-Jun. Therefore the rescue in cis of the defective Nt mutation by the Ct mutation seems to be due to the recovery of CBP binding. Our results revealed that the process of oncogenic cooperation can occur between Ras and the Nt+Ct non-phosphorylatable c-Jun protein.

Alanine↗

PubMed2001

Assessment of HIV-intrathecal humoral immune response in AIDS-related neurological disorders.

Intrathecal synthesis of IgG directed to HIV antigens was investigated by antibody specific index (ASI), affinity-mediated immunoblot (AMI) and Western blot (WB) assay in a group of 88 AIDS patients of which 28 with HIV-associated neurological disorders (HAND), 13 without associated neurological disorders (WAND) and 47 with non-HIV-associated neurological disorders (non-HAND). CD4+ count was above 50 cells/mm3 (CD4+>50) in 30 and below 50/mm3 (CD4+<50) in 58 patients, respectively. A significantly higher frequency for CSF complete anti-gag profile (p<0.001), and for HIV-specific oligoclonal patterns ("mixed" pattern=p<0.01) was observed in HAND as compared to patterns from the other clinical groups. A decrease in complete anti-env, anti-pol and anti-gag reactivity was present in CSF of patients with CD4+<50 as compared to those with CD4+>50. Our findings suggest that AIDS appears to be characterized by an anti-HIV intrathecal humoral immune response which is principally directed to env products with a prevalence of oligoclonal patterns and CSF complete anti-gag profile in HIV-associated neurological involvement.

AIDS Dementia Complex↗

PubMed2001

JunD regulates transcription of the tissue inhibitor of metalloproteinases-1 and interleukin-6 genes in activated hepatic stellate cells.

Activation of hepatic stellate cells (HSCs) to a myofibroblast-like phenotype is the pivotal event in hepatic wound healing and fibrosis. Rat HSCs activated in vitro express JunD, Fra2, and FosB as the predominant AP-1 DNA-binding proteins, and all three associate with an AP-1 sequence that is essential for activity of the tissue inhibitor of metalloproteinases-1 (TIMP-1) promoter. In this study, we used expression vectors for wild-type, dominant-negative, and forced homodimeric (Jun/eb1 chimeric factors) forms of JunD and other Fos and Jun proteins to determine the requirement for JunD in the transcriptional regulation of the TIMP-1 and interleukin-6 (IL-6) genes. JunD activity was required for TIMP-1 gene promoter activity, whereas overexpression of Fra2 or FosB caused a repression of promoter activity. The ability of homodimeric JunD/eb1 to elevate TIMP-1 promoter activity supports a role for JunD homodimers as the major AP-1-dependent transactivators of the TIMP-1 gene. IL-6 promoter activity was induced upon activation of HSCs and also required JunD activity; however, expression of JunD/eb1 homodimers resulted in transcriptional repression. Mutagenesis of the IL-6 promoter showed that an AP-1 DNA-binding site previously reported to be an activator of transcription in fibroblasts functions as a suppressor of promoter activity in HSCs. We conclude that JunD activates IL-6 gene transcription as a heterodimer and operates at an alternative DNA-binding site in the promoter. The relevance of these findings to events occurring in the injured liver was addressed by showing that AP-1 DNA-binding complexes are induced during HSC activation and contain JunD as the predominant Jun family protein. JunD is therefore an important transcriptional regulator of genes responsive to Jun homo- and heterodimers in activated HSCs.

Animals↗

PubMed2001

Distinct roles of Jun : Fos and Jun : ATF dimers in oncogenesis.

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.

Activating Transcription Factor 2↗

PubMed2001

Transcription factor ATF3 partially transforms chick embryo fibroblasts by promoting growth factor-independent proliferation.

Activating Transcription Factor 3 (ATF3) is a member of the bZip family of transcription factors. Previous studies in mammalian cells suggested that like other bZip family members e.g. Jun and Fos, ATF3 might play a role in the control of cell proliferation and participate in oncogenic transformation. To investigate this putative ATF3 function directly, the rat ATF3 protein was compared with v-Jun for its ability to transform primary cultures of chick embryo fibroblasts (CEFs). Like CEFs accumulating v-Jun, CEFs accumulating the ATF3 protein displayed a typical, fusiform morphology, associated with an enhanced capacity to grow in medium with reduced amount of serum. However, in contrast to v-Jun-transformed CEFs, the ATF3 overexpressing cells could not promote colony formation from single cells in agar. Partial transformation induced by ATF3 was found to be associated with repression of multiple cellular genes that are also down-regulated by v-Jun, including those coding for the extracellular components fibronectin, decorin, thrombospondin 2, and the pro-apoptotic protein Par-4. These data demonstrate that, at least in primary avian cells, rat ATF3 possesses an intrinsic oncogenic potential. Moreover, the results suggest that ATF3 might induce growth factor independence by down-regulating a subset of the genes repressed by v-Jun.

Activating Transcription Factor 3↗

PubMed2000

Transcriptional control of SPARC by v-Jun and other members of the AP1 family of transcription factors.

Transformation of chick embryo fibroblasts by the v-Jun oncoprotein correlates with a down-regulation of the extracellular matrix protein SPARC and repression of the corresponding mRNA. Alteration in SPARC expression has been repeatedly reported in human cancers of various origin, and is thought to contribute to the remodeling of the extracellular matrix during neoplastic progression. Transcriptional control of SPARC is poorly understood. We show here that (i) v-Jun-mediated repression of the endogenous SPARC gene is enhanced by Fra2 but alleviated by ATF2, Fra2 and ATF2 being the two major partners of v-Jun in the transformed cells; (ii) high basal activity as well as repression by v-Jun and modulation by Fra2 and ATF2 is restricted to a small proximal fragment (-124/+16) of the chicken SPARC promoter; (iii) the activity of this minimal promoter is modulated by all the AP1 family members known in chickens (c-Jun and JunD; c-Fos and Fra2; ATF2; c-Maf, MafA, and MafB). Taken together these data demonstrate that, at least in avian primary cells, SPARC expression is under the control of the AP1 transcription factor. Further studies with the minimal (-124/+16) promoter fragment are needed to understand how this control takes place at the molecular level.

Activating Transcription Factor 2↗

PubMed2000

Evidence of cerebrospinal fluid free kappa light chains in AIDS patients with Toxoplasma gondii encephalitis.

Cerebrospinal fluid (CSF) free light chains of kappa or lambda (FLC kappa/lambda) type were investigated by affinity mediated blotting technique (AMI) and ELISA in 28 patients of which nine with AIDS and Toxoplasma gondii encephalitis (AIDS, TE), 11 with AIDS with or without other CNS AIDS-related opportunistic infections (non-TE AIDS) and eight control patients with or without inflammatory neurological disorders (control group). CSF restricted oligoclonal FLC bands either of k or lambda isotype or both were found by AMI in 18 (90%) out of 20 AIDS patients, while a CSF pattern predominantly characterized by FkappaLC rather than FlambdaLC was observed in eight (88.8%) out of nine TE patients. No FLC components were detected in the matched sera of TE or non-TE AIDS patients or in the CSF and sera from control group. The anti-parasite-specific FkappaLC CSF/serum mean levels and the T. gondii-specific FkappaLC index values were found by ELISA to be significantly more elevated in TE patients when compared to non-TE AIDS or control group. These findings suggest that the increased production of T. gondii-specific FkappaLC could provide insights into pathogenesis of reactivated TE in immunocompromised patients and may have important diagnostic usefulness.

AIDS-Related Opportunistic Infections↗

PubMed2000

Down-regulation of the extracellular matrix protein SPARC in vSrc- and vJun-transformed chick embryo fibroblasts contributes to tumor formation in vivo.

In vitro transformation of primary cultures of chick embryo fibroblasts by the membrane-bound vSrc or the nuclear vJun oncoproteins is correlated with a down-regulation of the secreted glycoprotein SPARC (also called BM-40 or osteonectin). This protein is a nonstructural component of the extracellular matrix that is thought to regulate cell-matrix interaction during development, wound repair, and carcinogenesis. Its precise function remains unclear. To estimate the contribution of SPARC down-regulation to the major aspects of the transformed phenotype, we have reexpressed this protein from a self-replicating retrovirus Rcas, designated R-SPARC, in the transformed cultures. These R-SPARC-infected cultures display the following main properties: (i) they accumulate the SPARC protein to a level identical to or only slightly higher than the level in normal chick embryo fibroblasts; (ii) they retain the main phenotypic properties characteristic of in vitro transformed cells, that is, altered morphology, capacity to grow in a reduced amount of serum, and capacity to develop colonies from single cells in agar; (iii) they display a clearly reduced capacity to develop local fibrosarcomas in vivo. Taken together, these data strongly suggest that down-regulation of SPARC contributes to the transformed phenotype triggered by vSrc and vJun in primary avian fibroblasts, by facilitating in vivo tumorigenesis.

Animals↗

PubMed1998

Autocrine growth and anchorage independence: two complementing Jun-controlled genetic programs of cellular transformation.

Cellular transformation can be achieved by constitutive activation of growth-regulatory signaling pathways, which, in turn, activate nuclear transcription factors thought to execute a transformation-specific program of gene expression. Members of the dimeric transcription factor family AP-1 are at the receiving end of such growth-regulating pathways and the viral form of the AP-1 subunit Jun establishes one important aspect of transformation in chick embryo fibroblasts (CEFs): enhanced growth in agar and in low serum. Enhanced Jun activity is likely to target several different genetic programs as Jun forms heterodimers with one of several members of the Fos and ATF2 subfamilies, resulting in transcription factors with different sequence specificities. To identify the programs relevant for transformation, we have reduced the complexity of AP-1 factors by constructing Jun bZip mutants that can efficiently dimerize and transactivate with only a restricted set of partner subunits. Upon introduction into CEFs, a Jun mutant selective for the Fos family induced anchorage-independent growth but no growth factor-independence. In contrast, a c-Jun mutant with preference for ATF2-like proteins caused growth factor-independence, but no growth in agar. Coexpression of both mutants reestablished the combined transformation program as induced by wild-type Jun. These data show that Jun-dependent cell transformation can be resolved into at least two distinct and independent processes, anchorage and growth factor independence, obviously triggered by two classes of Jun heterodimers likely regulating different sets of target genes.

Activating Transcription Factor 2↗

PubMed1998

Transcription factor ATF2 cooperates with v-Jun to promote growth factor-independent proliferation in vitro and tumor formation in vivo.

ATF2 belongs to the bZIP family of transcription factors and controls gene expression via 8-bp ATF/CREB motifs either as a homodimer or as a heterodimer-for instance, with Jun-but has never been shown to be directly involved in oncogenesis. Experiments were designed to evaluate a possible role of ATF2 in oncogenesis in chick embryo fibroblasts (CEFs) in the presence or absence of v-Jun. We found that (i) forced expression of ATF2 cannot alone cause transformation, (ii) overexpression of ATF2 plus v-Jun specifically stimulates v-Jun-induced growth in medium with a reduced amount of serum, and (iii) the efficiency of low-serum growth correlates with the activity of a Jun-ATF2-dependent model promoter in stably transformed CEFs. Analysis of ATF2 and Jun dimerization mutants showed that the growth-stimulatory effect of ATF2 is likely to be mediated by v-Jun-ATF2 heterodimers since (i) v-Jun-m1, a mutant with enhanced affinity for ATF2, induces growth in low-serum medium much more efficiently than v-Jun, when expressed alone or in combination with ATF2; and (ii) ATF2/fos, a mutant that efficiently binds to v-Jun but is unable to form stable homodimers, shows enhanced oncogenic cooperation with v-Jun. In addition, we examined the role of ATF2 in tumor formation by subcutaneous injection of CEFs into chickens. In contrast to v-Jun, v-Jun-m1 gave rise to numerous fibrosarcomas while coexpression of ATF2 and v-Jun-m1 led to a dramatic development of fibrosarcomas visible within 1 week. Together these data demonstrate that overexpressed ATF2 potentiates the ability of v-Jun-transformed CEFs to grow in low-serum medium in vitro and contributes to the formation of tumors in vivo.

Activating Transcription Factor 2↗

PubMed1996

Stepwise transformation of rat embryo fibroblasts: c-Jun, JunB, or JunD can cooperate with Ras for focus formation, but a c-Jun-containing heterodimer is required for immortalization.

Among the Jun family of transcription factors, only c-Jun displays full transforming potential in cooperation with activated c-Ha-Ras in primary rat embryo fibroblasts. c-Jun in combination with Ras can both induce foci of transformed cells from rat embryo fibroblast monolayers and promote the establishment of these foci as tumoral cell lines. JunB can also cooperate with Ras to induce foci but is unable to promote immortalization. We report here that JunD, in cooperation with Ras, induces foci with an efficiency similar to that of JunB. Artificial Jun/eb1 derivatives from each of the three Jun proteins were also analyzed. These constructs carry a heterologous homodimerization domain from the viral EB1 transcription factor and are thought to form only homodimers in the cell. We show here that these Jun/eb1 chimeras are potent transactivators of AP1 sites and that they can cooperate with c-Ha-Ras to induce foci. However, among all the Ras-Jun and Ras-Jun/eb1 combinations tested, only foci from Ras-c-Jun can be efficiently expanded and maintained as long-term growing cultures. Therefore, we suggest that a heterodimer containing c-Jun might be required for in vitro establishment of these primary mammalian cells.

Animals↗

PubMed1995

Tumor induction by v-Jun homodimers in chickens.

To study the contribution of v-Jun homodimers to oncogenesis, we constructed artificial v-Jun derivatives in which the natural dimerization domain of v-Jun was replaced by an heterologous homodimerization domain from either the viral EB1 or the yeast GCN4 transcription factor. The resulting v-Jun chimeric proteins, called v-Juneb1 and v-Jungcn4, which can no longer dimerize with Jun or Fos, should only form homodimers in the cell. Helper-independent retroviruses expressing v-Jun, v-Juneb1 and v-Jungcn4 were generated. All three viruses transformed primary cultures of chick embryo cells with the same high efficiency and promoted local tumor growth after subcutaneous injection of infected cells in young animals. In contrast, after intravenous injection of viral suspensions into chick embryos, only the chimeric proteins produced internal tumors that were lethal. These tumors were leiomyosarcomas located within the liver and along the digestive tract. Thus, in vivo, v-Juneb1 and v-Jungcn4 are more potent oncoproteins than v-Jun. These data demonstrate that when forced to accumulate, v-Jun homodimers can induce tumors efficiently. They also show that the oncogenic potential of v-Jun can be regulated through the properties of its dimerization domain.

Animals↗

PubMed1995

Increased transforming activity of JunB and JunD by introduction of an heterologous homodimerization domain.

The closely-related proteins c-Jun, JunB and JunD form a family of transcription factors which require dimerization for DNA-binding and transcriptional activity. Dimerization is mediated by a conserved amphipathic alpha-helix located adjacent to a highly charged DNA-binding domain. The Jun proteins can form both homo- and heterodimers within the Jun family and can also cross-dimerize with the Fos proteins. When expressed at high levels in primary chicken cells, each mouse Jun displays distinct transforming capacities: c-Jun transforms efficiently, JunB transforms poorly, and JunD does not transform at all. The composition of the transforming dimers, however, is unknown. To study the activity of Jun-Jun homodimers we constructed artificial derivatives, denoted Juneb1, in which the naturally occurring dimerization domain has been replaced by an heterologous homodimerization domain from the Epstein-Barr virus transcription factor EB1. These derivatives were introduced into chicken cells and assayed for their ability to affect growth. Unexpectedly, all three Juneb1 proteins conferred a transformed phenotype to primary cultures, promoting sustained growth in low-serum medium and colony formation from single cells in agar. These data demonstrate that when forced to accumulate as homodimers, both JunB and JunD can transform cells. They also suggest that the poor transforming activity of JunB and the absence of transforming activity of JunD may be due to their inability to accumulate to high levels as homodimers.

Amino Acid Sequence↗

PubMed1993

Transforming growth factor beta 1-mediated growth inhibition in chick embryo fibroblasts: reversion by virally-expressed nuclear oncogenes.

Transforming growth factor beta 1 (TGF-beta 1) inhibits growth of primary cultures of chick embryo fibroblasts by affecting G1 and strongly increasing the generation time. This inhibition is reversed by the nuclear oncogenes v-jun, v-fos, v-myc, but not v-erbA and v-ets. It is also reversed by v-myb from either avian myeloblastosis virus or avian E26 retrovirus. Taken together, these results strongly suggest that independent, functional interferences may take place between the TGF-beta 1-induced growth inhibitory pathway and the oncogen-driven stimulatory pathway(s) at the level of the AP-1, Myc, and Myb transcription factors.

Animals↗

PubMed1993

[The C-Jun oncoprotein].

Jun and Fos are major components of the transcriptional complex AP-1 (Activator Protein-1), a collection of dimeric transcriptional activators composed of members of the Jun and Fos family of bZIP proteins, that bind to a common site known as TRE (TPA Responsive Element) or the AP-1 site. Transcription of c-jun is rapidly induced by exposure to different extra-cellular signals like growth factors, cytokines, tumor promoters (TPA), UV and other DNA-damaging agents. Transcriptional activation of c-jun is a two step mechanism. First, the pre-existing c-Jun protein is activated by posttranscriptional modifications, and second, modified c-Jun activates its own transcription, and the expression of AP-1-dependent genes. Modifications of c-Jun include dephosphorylations, phosphorylations and oxydo-reduction. The transcriptional activation by c-Jun is modulated by heterodimerization with other members of the bZIP family of proteins, and by transcriptional interference with other transcription factors like some members of the hormone nuclear receptors, or MyoD. AP-1 is tightly associated to both the control of cell proliferation and the oncogenic process. Constitutive activation of AP-1 leads to cell transformation in vitro, probably due to the accumulation of homodimeric c-Jun:c-Jun complexes. This hypothesis has been directly confirmed by constructing c-Jun hybrid proteins capable to form only homodimers. Deregulated expression of such proteins efficiently transforms primary cells in culture. These hybrid proteins constitute a powerful tool in order to identify new cellular functions AP-1-dependent, involved in the control of cell proliferation.

Alpharetrovirus↗

PubMed1993

In vitro transforming capacities of mouse c-jun:junD chimeric genes.

Among the murine Jun family of transcription factors, c-Jun and JunD are closely-related proteins with similar dimerization, DNA binding and transactivating properties. However, when expressed from a self-replicating retroviral RCAS vector, c-jun, but not junD, transforms chick embryo fibroblasts. We attempted to map the regions of c-jun which are important for transformation by constructing hybrids between c-jun and junD. Using common restriction sites, we prepared six different chimeric molecules. All of these c-jun:junD hybrids code for transactivators of AP1-containing promoters. An N-terminal segment of 79 amino acids of c-Jun converts JunD into a strong transforming protein, while other segments of c-Jun contribute to a lesser extent. Contrary to what has been reported with rat embryo fibroblasts, a c-Jun derivative with serines substituted by alanines in positions 63 and 73 still transforms CEFs efficiently.

Amino Acid Sequence↗