S Gerondakis

Sequence of the murine and human cellular myc oncogenes and two modes of myc transcription resulting from chromosome translocation in B lymphoid tumours.

The 15;12 chromosome translocation in murine plasmacytomas and the 8;14 in human Burkitt lymphomas often link the cellular myc oncogene to the locus for constant regions of immunoglobulin heavy chains (CH locus). To clarify how and why c‐myc translocation occurs, we have sequenced the mouse and human c‐myc genes and correlated c‐myc transcription with c‐myc rearrangement. Both genes comprise three exons; the second and third encode the myc polypeptide, which is conserved between mammals and birds, particularly in its more basic C‐terminal half. Southern blots showed that four of 12 Burkitt lines have c‐myc linked near CH switch regions and two near the joining region (JH) locus. Hence, immunoglobulin recombination machinery may participate in translocation, although the common myc breakpoint region around exon 1 does not resemble a switch region. Tumours with breakpoints just 5′ to exon 1, or distant from c‐myc, had normal c‐myc mRNAs of 2.25 and 2.4 kb, which differ at their 5′ ends, while tumours with breakpoints within exon 1 or intron 1 had altered c‐myc mRNAs (2.1‐2.7 kb in Burkitt lines), initiated within intron 1. Both types of mRNAs probably yield the same polypeptide. Since the untranslocated c‐myc allele was generally silent, translocation to the CH locus must induce constitutive c‐myc expression. The presence of c‐myc mRNA in immortal but non‐tumorigenic lymphoblastoid cell lines may implicate c‐myc in an immortalization step.

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.

NF-κB control of T cell development.

The NF-κB signal transduction pathway is best known as a major regulator of innate and adaptive immune responses, yet there is a growing appreciation of its importance in immune cell development, particularly of T lineage cells. In this Review, we discuss how the temporal regulation of NF-κB controls the stepwise differentiation and antigen-dependent selection of conventional and specialized subsets of T cells in response to T cell receptor and costimulatory, cytokine and growth factor signals.

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.

Interchromosomal recombination of the cellular oncogene c-myc with the immunoglobulin heavy chain locus in murine plasmacytomas is a reciprocal exchange.

The 15:12 chromosome translocations found in most murine plasmacytomas involve the cellular gene (c‐myc) homologous to the oncogene (v‐myc) of avian retrovirus MC29, Translocation links the c‐myc gene of chromosome 15 to the immunoglobulin heavy (H) chain locus of chromosome 12, often within the switch recombination (S) region 5′ to the alpha constant region (C alpha) gene. We have investigated c‐myc rearrangements in 21 BALB/c plasmacytomas and three B lymphomas by Southern blot analysis. We show that the t(15;12) is a reciprocal chromosome exchange since most tumours contain not only a c‐myc gene linked to the S alpha C alpha region but also a separate structure with S mu or S alpha linked to the c‐myc 5′‐flanking region. Analysis of the two rearrangement products cloned from plasmacytoma J558 suggests that one type of H locus target for translocation is an S alpha region recombined with S mu; two other targets appear to be other switched heavy chain genes and an unrearranged C alpha gene. Nearly all the chromosome 15 breakpoints fall within a 1.1‐kb region spanning a 5′ c‐myc exon; hence scission of the transcriptional unit by translocation can account for the altered c‐myc transcription in plasmacytomas. The c‐myc breakpoint region lacks substantial homology with S mu or S alpha, arguing against homologous recombination as the translocation mechanism.

NF-κB1 and c-Rel cooperate to promote the survival of TLR4-activated B cells by neutralizing Bim via distinct mechanisms

The nuclear factor-kappaB (NF-kappaB) pathway is crucial for the survival of B cells stimulated through Toll-like receptors (TLRs). Here, we show that the heightened death of TLR4-activated nfkb1(-/-) B cells is the result of a failure of the Tpl(2)/MEK/ERK pathway to phosphorylate the proapo-ptotic BH3-only protein Bim and target it for degradation. ERK inactivation of Bim after TLR4 stimulation is accompanied by an increase in A1/Bim and Bcl-x(L)/Bim complexes that we propose represents a c-Rel-dependent mechanism for neutralizing Bim. Together these findings establish that optimal survival of TLR4-activated B cells depends on the NF-kappaB pathway neutralizing Bim through a combination of Bcl-2 prosurvival protein induction and Tpl2/ERK-dependent Bim phosphorylation and degradation.

Fusion of DNA region to murine immunoglobulin heavy chain locus corresponds to plasmacytoma-associated chromosome translocation.

Murine plasmacytomas frequently exhibit a translocation of the distal region of chromosome 15 to the end of chromosome 12, where the immunoglobulin heavy chain locus resides. A candidate for the DNA across the chromosome fusion point is a cloned region of non‐immunoglobulin DNA which in most plasmacytomas has recombined near the alpha heavy chain constant region gene. That the incoming DNA, provisionally designated LyR (lymphoid rearranging) DNA, does derive from chromosome 15 is shown here by blot analysis of DNA from two panels of somatic cell hybrids: hybridomas between an AKR T‐lymphoma (Tikaut) and CBA mouse cells with a cytogenetically distinctive chromosome 15, and between mouse and Chinese hamster cells. LyR DNA segregated with chromosome 15 in all lines and the results assign LyR to the distal two thirds of that chromosome. This assignment, together with the previously reported high frequency of recombination between LyR and C(alpha) in plasmacytomas and associated alteration of LyR transcription suggests that translocation activates a LyR gene involved in plasmacytoma oncogenesis. Moreover, LyR rearrangement in certain T‐lymphomas, such as the Tikaut line examined here, also implicate that gene in oncogenesis of some T‐lymphomas.

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.

The acetyltransferase HAT1 moderates the NF-κB response by regulating the transcription factor PLZF

To date, the activities of protein kinases have formed the core of our understanding of cell signal transduction. Comprehension of the extent of protein acetylation has raised expectations that this alternate post-transcriptional modification will be shown to rival phosphorylation in its importance in mediating cellular responses. However, limited instances have been identified. Here we show that signalling from Toll-like or TNF-α receptors triggers the calcium/calmodulin-dependent protein kinase (CaMK2) to activate histone acetyltransferase-1 (HAT1), which then acetylates the transcriptional regulator PLZF. Acetylation of PLZF promotes the assembly of a repressor complex incorporating HDAC3 and the NF-κB p50 subunit that limits the NF-κB response. Accordingly, diminishing the activity of CaMK2, the expression levels of PLZF or HAT1, or mutating key residues that are covalently modified in PLZF and HAT1, curtails control of the production of inflammatory cytokines. These results identify a central role for acetylation in controlling the inflammatory NF-κB transcriptional programme.

Tumor progression locus 2 (Tpl2) deficiency does not protect against obesity-induced metabolic disease.

Obesity is associated with a state of chronic low grade inflammation that plays an important role in the development of insulin resistance. Tumor progression locus 2 (Tpl2) is a serine/threonine mitogen activated protein kinase kinase kinase (MAP3K) involved in regulating responses to specific inflammatory stimuli. Here we have used mice lacking Tpl2 to examine its role in obesity-associated insulin resistance. Wild type (wt) and tpl2−/− mice accumulated comparable amounts of fat and lean mass when fed either a standard chow diet or two different high fat (HF) diets containing either 42% or 59% of energy content derived from fat. No differences in glucose tolerance were observed between wt and tpl2−/− mice on any of these diets. Insulin tolerance was similar on both standard chow and 42% HF diets, but was slightly impaired in tpl2−/− mice fed the 59% HFD. While gene expression markers of macrophage recruitment and inflammation were increased in the white adipose tissue of HF fed mice compared with standard chow fed mice, no differences were observed between wt and tpl2−/− mice. Finally, a HF diet did not increase Tpl2 expression nor did it activate Extracellular Signal-Regulated Kinase 1/2 (ERK1/2), the MAPK downstream of Tpl2. These findings argue that Tpl2 does not play a non-redundant role in obesity-associated metabolic dysfunction.