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S Gerondakis

Publications and source records attributed to S Gerondakis.

At least 37 records · Page 2Linked to original sources
PubMed1998

B lymphocytes differentially use the Rel and nuclear factor kappaB1 (NF-kappaB1) transcription factors to regulate cell cycle progression and apoptosis in quiescent and mitogen-activated cells.

Rel and nuclear factor (NF)-kappaB1, two members of the Rel/NF-kappaB transcription factor family, are essential for mitogen-induced B cell proliferation. Using mice with inactivated Rel or NF-kappaB1 genes, we show that these transcription factors differentially regulate cell cycle progression and apoptosis in B lymphocytes. Consistent with an increased rate of mature B cell turnover in naive nfkb1-/- mice, the level of apoptosis in cultures of quiescent nfkb1-/-, but not c-rel-/-, B cells is higher. The failure of c-rel-/- or nfkb1-/- B cells to proliferate in response to particular mitogens coincides with a cell cycle block early in G1 and elevated cell death. Expression of a bcl-2 transgene prevents apoptosis in resting and activated c-rel-/- and nfkb1-/- B cells, but does not overcome the block in cell cycle progression, suggesting that the impaired proliferation is not simply a consequence of apoptosis and that Rel/NF-kappaB proteins regulate cell survival and cell cycle control through independent mechanisms. In contrast to certain B lymphoma cell lines in which mitogen-induced cell death can result from Rel/NF-kappaB-dependent downregulation of c-myc, expression of c-myc is normal in resting and stimulated c-rel-/- B cells, indicating that target gene(s) regulated by Rel that are important for preventing apoptosis may differ in normal and immortalized B cells. Collectively, these results are the first to demonstrate that in normal B cells, NF-kappaB1 regulates survival of cells in G0, whereas mitogenic activation induced by distinct stimuli requires different Rel/NF-kappaB factors to control cell cycle progression and prevent apoptosis.

Animals↗
PubMed1998

The regulation and roles of Rel/NF-kappa B transcription factors during lymphocyte activation.

The activation of B and T cells by a wide range of stimuli can rapidly induce specific gene expression via a mechanism that promotes the nuclear translocation of different Rel/nuclear factor-kappa B (NF-kappa B) transcription factors which are normally resident in the cytoplasm. Recent findings highlight the crucial roles of specific Rel/NF-kappa B family members in the processes of cell division, apoptosis and differentiation that accompany lymphocyte activation.

Animals↗
PubMed1996

The Rel subunit of NF-kappaB-like transcription factors is a positive and negative regulator of macrophage gene expression: distinct roles for Rel in different macrophage populations.

The role of Rel in the monocyte/macrophage lineage was examined in mice with an inactivated c-rel gene. Although the frequency of monocytic cells was normal in Rel-/- mice, we show that Rel serves distinct roles in regulating gene expression and immune effector function in different mature macrophage populations. Stimulated Rel-/- resident peritoneal macrophages produced higher than normal levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and interleukin-6 (IL-6), but tumour necrosis factor-alpha (TNF-alpha) production was not induced. Diminished cytotoxic activity exhibited by resident Rel-/- macrophages was consistent with reduced nitric oxide production resulting from impaired up-regulation of inducible nitric oxide synthase expression. While a similar altered pattern of IL-6 and TNF-alpha expression was observed in stimulated Rel-/- peritoneal effusion macrophages, cytotoxic activity, nitric oxide, GM-CSF and G-CSF production by these cells was normal. The alternate regulation of certain genes in the two macrophage populations coincided with different patterns of nuclear Rel/NF-kappaB complexes expressed in normal resident and elicited cells. Collectively, these results establish that Rel is a positive or negative regulator of transcription in macrophages and that Rel has distinct roles in different macrophage populations.

Animals↗
PubMed1996

Rel-deficient T cells exhibit defects in production of interleukin 3 and granulocyte-macrophage colony-stimulating factor.

The c-rel protooncogene encodes a subunit of the NF-kappa B-like family of transcription factors. Mice lacking Rel are defective in mitogenic activation of B and T lymphocytes and display impaired humoral immunity. In an attempt to identify changes in gene expression that accompany the T-cell stimulation defects associated with the loss of Rel, we have examined the expression of cell surface activation markers and cytokine production in mitogen-stimulated Rel-/- T cells. The expression of cell surface markers including the interleukin 2 receptor alpha (IL-2R alpha) chain (CD25), CD69 and L-selectin (CD62) is normal in mitogen-activated Rel-/- T cells, but cytokine production is impaired. In Rel-/- splenic T cell cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin, the levels of IL-3, IL-5, granulocyte- macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) were only 2- to 3-fold lower compared with normal T cells. In contrast, anti-CD3 and anti-CD28 stimulated Rel-/- T cells, which fail to proliferate, make little or no detectable cytokines. Exogenous IL-2, which restitutes the proliferative response of the anti-CD3- and anti-CD28-treated Rel-/- T cells, restores production of IL-5, TNF-alpha, and IFN-gamma, but not IL-3 and GM-CSF expression to approximately normal levels. In contrast to mitogen-activated Rel-/- T cells, lipopolysaccharide-stimulated Rel-/- macrophages produce higher than normal levels of GM-CSF. These findings establish that Rel can function as an activator or repressor of gene expression and is required by T lymphocytes for production of IL-3 and GM-CSF.

Animals↗
PubMed1996

Activation of the mitogen-activated protein kinase pathway induces transcription of the PAC-1 phosphatase gene.

PAC-1, an early-response gene originally identified in activated T cells, encodes a dual-specificity mitogen-activated protein kinase phosphatase. Here we report on the regulation of PAC-1 expression in murine hemopoietic cells. PAC-1 mRNA levels rapidly increase in mitogen-stimulated lymphocytes, with the induced expression being transient in B cells but sustained in activated T cells. Transfection analysis of murine PAC-1 promoter-reporter constructs established that in T cells, sequences necessary for basal and induced transcription reside within a 200-bp region located immediately upstream of the transcription initiation sites. Basal transcription is regulated in part by an E-box element that binds a 53-kDa protein. PAC-1 transcription induced by phorbol myristate acetate stimulation and the expression of the v-ras or v-raf oncogene is mediated via the E-box motif and an AP-2-related site and coincides with increased binding activity of the constitutive 53-kDa E-box-binding protein and induced binding of AP-2. The ability of an interfering ERK-2 mutant to block phorbol myristate acetate and v-ras-dependent PAC-1 transcription indicates that mitogen-activated protein kinase activation is necessary for these stimuli to induce transcription of the PAC-1 gene in T cells.

Amino Acid Sequence↗
PubMed1995

Mice lacking the c-rel proto-oncogene exhibit defects in lymphocyte proliferation, humoral immunity, and interleukin-2 expression.

The c-rel proto-oncogene, which is expressed predominantly in hemopoietic cells encodes a subunit of the NF-kappa B-like family of transcription factors. In mice with an inactivated c-rel gene, whereas development of cells from all hemopoietic lineages appeared normal, humoral immunity was impaired and mature B and T cells were found to be unresponsive to most mitogenic stimuli. Phorbol ester and calcium ionophore costimulation, in contrast to certain membrane receptor-mediated signals, overcame the T cell-proliferative defect, demonstrating that T cell proliferation occurs by Rel-dependent and -independent mechanisms. The ability of exogenous interleukin-2 to restore T Cell, but not B cell, proliferation indicates that Rel regulates the expression of different genes in B and T cells that are crucial for cell division and immune function.

Aging↗
PubMed1994

Structure of the gene encoding the murine dual specificity tyrosine-threonine phosphatase PAC1.

The mitogen-induced early-response gene, PAC-1, encodes a nuclear 32-kDa tyrosine-threonine dual specificity phosphatase, which has been shown to specifically dephosphorylate the mitogen activated protein (MAP) kinases, ERK1 and ERK2. Here, we describe the structure and sequence of the murine PAC-1 gene. Transcription starts at three major sites located between 80 and 90 nucleotides upstream of the murine PAC-1 initiation codon within a highly G/C-rich region. The gene comprises three exons, with exon 1 encoding the unique N-terminal half of the protein, while exons 2 and 3 encode the C-terminus that is homologous to the closely related phosphatases, 3CH134 and VH1. The conserved catalytic domain common to all tyrosine phosphatases is encompassed by exon 3. The organization of the murine PAC-1 gene suggests that the PAC-1 N-terminus, which may serve a regulatory function, has evolved as a separate domain from the C-terminal catalytic domain.

Animals↗
PubMed1994

Alternative splicing of RNA transcripts encoded by the murine p105 NF-kappa B gene generates I kappa B gamma isoforms with different inhibitory activities.

The gene encoding the 105-kDa protein (p105) precursor of the p50 subunit of transcription factor NF-kappa B also encodes a p70 I kappa B protein, I kappa B gamma, which is identical to the C-terminal 607 amino acids of p105. Here we show that alternative RNA splicing generates I kappa B gamma isoforms with properties different from those of p70. One 63-kDa isoform, termed I kappa B gamma-1, which lacks 59 amino acids C-terminal to ankyrin repeat 7, has a novel 35-amino acid C terminus encoded by an alternative reading frame of the p105 gene. A 55-kDa isoform, I kappa B gamma-2, lacks the 190 C-terminal amino acids of p70I kappa B gamma. In contrast to p70I kappa B gamma, which is a cytoplasmic protein, I kappa B gamma-1 is found in both the cytoplasm and nucleus, whereas I kappa B gamma-2 is predominantly nuclear. The I kappa B gamma isoforms also display differences in specificity and affinity for Rel/NF-kappa B proteins. While p70I kappa B gamma inhibits p50-, p65-, and c-Rel-mediated transactivation and/or DNA binding, both I kappa B gamma-1 and I kappa B gamma-2 are specific for p50 and have different affinities for this subunit. The absence in I kappa B gamma-1 and I kappa B gamma-2 of a protein kinase A site whose phosphorylation modulates p70I kappa B gamma inhibitory activity suggests that alternative RNA splicing may be used to generate I kappa B gamma isoforms that respond differently to intracellular signals.

Alternative Splicing↗
PubMed1994

Requirement for nuclear factor (NF)-kappa B p65 and NF-interleukin-6 binding elements in the tumor necrosis factor response region of the granulocyte colony-stimulating factor promoter.

Granulocyte colony-stimulating factor (G-CSF) is a hematopoietic growth factor produced by mesenchymal and myeloid cells following activation by inflammatory stimuli. It has previously been shown that a region of the G-CSF promoter, (-200 to -165) containing the decanucleotide CK-1 element and two repeated sequences that resemble nuclear factor (NF)-interleukin-6 (IL-6) binding sites, is required for activation of the G-CSF gene by tumor necrosis factor-alpha (TNF-alpha) and IL-1 beta. We now show that the NF-kappa B p65 protein can bind to and activate this TNF response region. There are several unusual features of this p65 interaction with the TNF response region. First, NF-kappa B p65 but not the related NF-kappa B p50 binds to the CK-1 element and a p50/65 hybrid protein that relies on the p50 rel homology domain for DNA binding does not transactivate the TNF response region. Second, p65 transactivation of this region is cell specific and requires not only its own binding site but also the NF-IL6 consensus sites. NF-IL6 also binds to the TNF response region of the G-CSF promoter. Electrophoretic mobility shift studies show that p65 and NF-IL6 can bind cooperatively to the TNF response region. The ability of this region to respond to TNF-alpha or p65 is correlated with the ability to form the p65/NF-IL6 ternary complex.

Animals↗
PubMed1994

Alternate RNA splicing of murine nfkb1 generates a nuclear isoform of the p50 precursor NF-kappa B1 that can function as a transactivator of NF-kappa B-regulated transcription.

The NF-kappa B1 subunit of the transcription factor NF-kappa B is derived by proteolytic cleavage from the N terminus of a 105-kDa precursor protein. The C terminus of p105NF-kappa B1, like those of I kappa B proteins, contains ankyrin-related repeats that inhibit DNA binding and nuclear localization of the precursor and confer I kappa B-like properties upon p105NF-kappa B1. Here we report the characterization of two novel NF-kappa B1 precursor isoforms, p84NF-kappa B1 and p98NF-kappa B1, that arise by alternate splicing within the C-terminal coding region of murine nfkb1. p98NF-kappa B1, which lacks the 111 C-terminal amino acids (aa) of p105NF-kappa B1, has a novel 35-aa C terminus encoded by an alternate reading frame of the gene. p84NF-kappa B1 lacks the C-terminal 190 aa of p105NF-kappa B1, including part of ankyrin repeat 7. RNA and protein analyses indicated that the expression of p84NF-kappa B1 and p98NF-kappa B1 is restricted to certain tissues and that the phorbol myristate acetate-mediated induction of p84NF-kappa B1 and p105NF-kappa B1 differs in a cell-type-specific manner. Both p84NF-kappa B1 and p98NF-kappa B1 are found in the nuclei of transfected cells. Transient transfection analysis revealed that p98NF-kappa B1, but not p105NF-kappa B1 or p84NF-kappa B1, acts as a transactivator of NF-kappa B-regulated gene expression and that this is dependent on sequences in the Rel homology domain required for DNA binding and on the novel 35 C-terminal aa of this isoform. In contrast to previous findings, which indicated that p105NF-kappa B1 does not bind DNA, all of the NF-kappa B1 precursors were found to specifically bind with low affinity to a highly restricted set of NF-kappa B sites in vitro, thereby raising the possibility that certain of the NF-kappa B1 precursor isoforms may directly modulate gene expression.

Alternative Splicing↗
PubMed1994

The subunit composition of NF-kappa B complexes changes during B-cell development.

The transcription factor NF-kappa B, shown to be essential for expression of the immunoglobulin C kappa gene, is a key regulatory component in pre-B to B-cell differentiation. While previous studies have used lymphoid cell line models, here we examine the expression and subunit composition of rel/NF-kappa B complexes in normal murine pre-B and B lymphocytes. Two major NF-kappa B complexes are detected in pre-B and B cells. A high mobility complex, found in pre-B (Cb) and B cells (C beta) is a homodimer of the NF-kappa B subunit p50. In pre-B cells, the slower migrating complex (Ca), which is predominantly cytoplasmic, is largely comprised of p50 and p65, whereas in B cells, a nuclear and cytoplasmic complex (C alpha) of identical mobility to Ca mainly consists of p50 and p75c-rel. While p50 and p65 levels do not change during pre-B to B-cell differentiation, p75c-rel is 5- to 6-fold more abundant in B cells compared to pre-B cells, a finding consistent with the switch in NF-kappa B subunit usage. During lipopolysaccharide-induced B-cell proliferation, transient up-regulation of both the nuclear p50 homodimer and p75c-rel containing complex is mirrored by a concurrent increase in c-rel and p105 but not p65 mRNA expression, a finding consistent with rel-NF-kappa B expression in B cells being controlled by an autoregulatory mechanism.

Animals↗
PubMed1993

The IL-4 induced increase in the frequency of resting murine splenic B cells expressing germline Ig heavy chain gamma 1 transcripts correlates with subsequent switching to IgG1.

Cytokine induced germline immunoglobulin heavy chain gene transcription appears to signal commitment to an isotype switch and may be the mechanism by which specific switch regions are targeted as the sites for recombination. In this study, the structure and expression of mouse germline gamma 1 RNAs are described. The 5'-ends of these transcripts are derived from an exon denoted I gamma 1, located upstream of the gamma 1 switch region and initiate at multiple sites over a 200 nucleotide region. Sequence analysis of cDNA and genomic clones reveals that these RNAs, unlike other germline CH transcripts, may encode a novel I gamma 1/C gamma 1 heavy chain protein, of which the N-terminal 27 residues are encoded by I gamma 1. In vitro culture of resting or pre-activated splenic B cells in the presence of lipopolysaccharides and interleukin-4 (IL-4) generates clones that secrete both IgM and IgG1 or either isotype alone. IL-4 increases the frequency of clones secreting both IgM and IgG1 and IgG1 alone, suggesting that commitment to IgG1 secretion may be independent of, or associated with, IgM secretion. PCR analysis of gamma 1 germline transcript expression in clonal B cell cultures or single pre-activated B cells, shows that the IL-4 induced increase in the frequency of cells expressing gamma 1 germline transcripts directly correlates with the increased frequency of cells switching from IgM to IgG1 production. This finding statistically confirms at a clonal level the relationship between cytokine induced germline transcription and isotype switching.

Amino Acid Sequence↗
PubMed1993

rel/NF-kappa B nuclear complexes that bind kB sites in the murine c-rel promoter are required for constitutive c-rel transcription in B-cells.

The c-rel protooncogene, a member of a transcription factor family that includes NF-kappa B, displays a complex pattern of gene expression. To understand the basis of this expression, the regulatory region upstream of the murine c-rel transcription start sites has been cloned and characterized. Transcription of the murine c-rel gene initiates at multiple sites downstream of a GC-rich region conserved in the chicken c-rel promoter. This conserved region contains consensus transcription factor binding sites for SP-1 and NF-kappa B (kB3 site) and is sufficient for basal expression in Jurkat T-cells. In contrast, two additional NF-kappa B-like sites (kB1 and kB2) and an octamer consensus binding site, all located upstream of the conserved region, are required for expression of promoter-reporter gene constructs in the B-cell line I29B. NF-kappa B sites kB1 and kB3 bind p50/65 and p50 homodimers, whereas kB2 binds a distinct complex. The consensus octamer site, although only able to bind Oct1 and Oct2 with low affinity, appears to overlap with a binding site for a novel protein(s) expressed in I29B cells. Cotransfection studies show that p75-c-rel and a carboxyl-terminal truncated c-rel protein that lacks the known trans-activating domain both up-regulate the c-rel promoter in I29B cells via a mechanism independent of the NF-kappa B motifs, whereas a mutant c-rel protein lacking the DNA binding domain has no effect. Together, these findings suggest that, in this B-cell line, trans-activation of the c-rel promoter by rel proteins is via an indirect mechanism.

Animals↗
PubMed1993

The activity of a 70 kilodalton I kappa B molecule identical to the carboxyl terminus of the p105 NF-kappa B precursor is modulated by protein kinase A.

The p50 subunit of NF-kappa B is derived from the amino terminus of a 105 kilodalton precursor. The p105 carboxyl terminus, which contains ankyrin-like repeats, a feature of I kappa B molecules, regulates the cytoplasmic retention of p105 and inhibits DNA binding by the precursor. Here, we describe an I kappa B protein identical to the carboxyl-terminal region of p105. Probes spanning the COOH terminus but not the rel homology domain of p105 hybridize to a distinct 2.6-kilobase mRNA expressed in a wide range of murine tissues. The nucleotide sequence of complementary DNA clones for this transcript, in vitro translation, and immune precipitation of metabolically labeled cell lysates establish that it encodes a 70 kilodalton protein that corresponds to the COOH-terminal 607 amino acids of p105. p70 suppresses p65 and p75c-rel mediated transactivation of reporter genes under the control of NF-kappa B elements and in vitro can prevent DNA binding of p50 and p75c-rel homodimers to NF-kappa B sites. The ability of p70 to stably associate with p49 and p65 in vitro, but not inhibit DNA binding by these proteins, suggests that the specific inhibitory properties of this I kappa B may reflect its relative affinity for different rel targets. p70 phosphorylated by protein kinase A fails to inhibit DNA binding by p50 or the c-rel protein, and sequencing of radiolabeled p70 tryptic phosphopeptides establishes that protein kinase A phosphorylates serine residue 576 of p70. This finding suggests that the inhibitory activity of p70 can be regulated by signaling via the adenylate cyclase pathway.

Amino Acid Sequence↗
PubMed1992

Transcriptional regulation of the germline immunoglobulin C alpha and C epsilon genes: implications for commitment to an isotype switch.

Lymphokine directed isotype switching is preceded by the induced expression of the corresponding germline Ig heavy chain constant region (CH) gene. This association favors a model in which lymphokine induced germline CH gene expression promotes switch recombination by increasing the accessibility of the switch region to a recombinase(s). An important prediction of this model is that the induction of germline CH RNAs represents increased specific de novo transcription. To test if this prediction is fulfilled by the switch commitment factors, IL-4 and transforming growth factor-beta (TGF-beta), we have utilized a B cell line, 1.29, that switches from IgM to IgE and IgA in vitro. In this cell line, IL-4 and TGF-beta increase germline C epsilon and C alpha RNA levels respectively, predominantly by elevating transcription of these genes. Transcription of germline C epsilon and C alpha genes appears to be independently regulated and is not affected by lipopolysaccharide or IL-5. These results are discussed in the context of the molecular events necessary to commit a B cell to an isotype switch.

Animals↗
PubMed1991

Structure and expression of mouse germline immunoglobulin gamma 3 heavy chain transcripts induced by the mitogen lipopolysaccharide.

Germline immunoglobulin heavy chain gene transcription is though to direct isotype switching by modulating the accessibility of specific switch regions to a recombinase. In this study, cloned cDNA copies of mouse germline Igh-8 RNAs have been used to characterize the Igh-8 transcription unit. The 5' end of these transcripts are derived from an exon denoted Ig3, located 1 kilobase 5' of the Igh-8 switch region. Sequence analysis of cDNA and genomic clones reveals that these RNAs are noncoding. In splenic B cell cultures treated with lipopolysaccharide (LPS), germline Igh-8 transcript levels are upregulated after 8 h due to increased transcription. This induction is consistent with the identification of a putative binding site for the LPS inducible transcription factor NF-kappa B approximately 150 nucleotides upstream of the sites of transcript initiation. Furthermore, nucleotide sequence comparisons reveal that the region encompassing the site of germline Igh-8 transcription initiation is highly homologous to part of the Ig2b exon, and is also conserved upstream of the Igh-1 switch region. The implications of these findings for the control of germline Igh-8 transcription is discussed.

Animals↗
PubMed1990

Structure and expression of murine germ-line immunoglobulin epsilon heavy chain transcripts induced by interleukin 4.

The murine lymphokine, interleukin 4 (IL-4) is able to specifically promote isotype switching to IgG1 and IgE in cultures of mitogen-stimulated B cells. Emerging evidence suggests that germ-line immunoglobulin heavy chain gene transcription may direct switching by modulating switch-region accessibility to a recombinase. In this study, cloned cDNA copies of the germ-line epsilon heavy chain transcript have been used to determine the genomic organization of this transcription unit. The 5' end of these transcripts are derived from an exon, denoted I epsilon, located 2 kilobases 5' of the C epsilon switch region [C epsilon = epsilon heavy chain constant (C) region gene]. Nucleotide sequence analysis reveals that this RNA does not encode a protein, as the I epsilon exon contains termination codons in all reading frames. Germ-line epsilon chain transcripts can be detected in cultures of normal splenic B cells treated with IL-4 within 24 hr, and this expression correlates with subsequent switching to C epsilon. Consistent with the IL-4 inducibility of this RNA is the identification of a motif upstream from the site of transcription initiation that closely resembles a transcription element implicated in the IL-4 regulation of the gene encoding the murine class II histocompatibility antigen, A alpha k. These data lend support to the accessibility model of isotype switching and implicate IL-4 in the transcriptional activation of the C epsilon locus.

Animals↗