Impaired TNFalpha-induced A20 expression in E1A/Ras-transformed cells.

BACKGROUND: Tumour necrosis factor (TNF) is capable of activating the cell death pathway, and has been implicated in killing transformed cells. However, TNF also activates survival signals, including NF-kappaB activation and the subsequent expression of anti-apoptotic genes, leading to protection against TNF toxicity. METHODS: In this study, we show that, although untransformed mouse embryonic fibroblasts (MEFs) were resistant to TNF killing, E1A/Ras-transformed MEFs were susceptible to extensive apoptosis induced by TNF. The key factors for determining TNF sensitivity were explored by comparing wild-type and E1A/Ras-transformed MEFs. RESULTS: TNF signalling to NF-kappaB and to its target genes such as IkappaBalpha seemed to be mostly intact in E1A/Ras-transformed cells. Instead, the induction of A20 was completely abolished in E1A/Ras-transformed MEFs, although A20 is known to be NF-kappaB dependent. Reintroduction of A20 into E1A/Ras-transformed MEFs rescued these cells from TNF-induced death and reduced the formation of the FADD/caspase-8 complex. This impaired A20 induction in E1A/Ras MEFs was not because of the stabilisation of p53 or a defective TNF-induced p38 and Jun N-terminal kinase (JNK) signalling. Consistently, we found a reduced A20 promoter activity but normal NF-kappaB activity in TNF-treated E1A/Ras MEFs. However, Bcl-3 seemed to have a role in the transactivation of the A20 promoter in E1A/Ras cells. CONCLUSIONS: Our results suggest that specific inhibition of certain survival factors, such as A20, may determine the sensitivity to TNF-induced apoptosis in transformed cells such as E1A/Ras MEFs.

Deficiency of T2K leads to apoptotic liver degeneration and impaired NF-kappaB-dependent gene transcription.

Induction of NF-kappaB-dependent transcription requires phosphorylation and subsequent degradation of I-kappaB, an inhibitor of NF-kappaB, followed by nuclear translocation and DNA binding of NF-kappaB. Tumor necrosis factor receptor-associated factor 2 (TRAF2) plays a role in NF-kappaB activation in response to cytokines such as tumor necrosis factor alpha (TNFalpha). In this study, we purified and characterized a novel kinase (T2K, also known as TBK1 or NAK), which associates with TRAF2 and exhibits kinase activity towards I-kappaBalpha in vitro. The physiological function of T2K was investigated using T2K-deficient mice. Heterozygotes appear normal, but t2k(-/-) animals die at approximately E14.5 of massive liver degeneration and apoptosis. Never theless, hematopoietic progenitors from T2K-deficient fetal liver support normal lymphocyte development. Furthermore, t2k(-/-) embryonic fibroblasts and thymocytes do not display increased sensitivity to TNFalpha-induced apoptosis. In response to either TNFalpha or IL-1 induction, t2k(-/-) embryonic fibroblasts exhibit normal degradation of I-kappaB and kappaB-binding activity. However, NF-kappaB-directed transcription is dramatically reduced. These results demonstrate that, like I-kappaB kinase beta and the RelA subunit of NF-kappaB, T2K is critical in protecting embryonic liver from apoptosis. However, T2K has a unique role in the activation of NF-kappaB-directed transcription, apparently independent of I-kappaB degradation and NF-kappaB DNA binding.

Requirement for Casper (c-FLIP) in regulation of death receptor-induced apoptosis and embryonic development.

Casper (c-FLIP) associates with FADD and caspase-8 in signaling complexes downstream of death receptors like Fas. We generated Casper-deficient mice and cells and noted a duality in the physiological functions of this molecule. casper-/- embryos do not survive past day 10.5 of embryogenesis and exhibit impaired heart development. This phenotype is reminiscent of that reported for FADD-/- and caspase-8-/- embryos. However, unlike FADD-/- and caspase-8-/- cells, casper-/- embryonic fibroblasts are highly sensitive to FasL- or TNF-induced apoptosis and show rapid induction of caspase activities. NF-kappaB and JNK/SAPK activation is intact in TNF-stimulated casper-/- cells. These results suggest that Casper has two distinct roles: to cooperate with FADD and caspase-8 during embryonic development and to mediate cytoprotection against death factor-induced apoptosis.

Targeted disruption in murine cells reveals variable requirement for Smad4 in transforming growth factor beta-related signaling.

The tumor suppressor gene Smad4 has been proposed to be a common mediator of transforming growth factor beta (TGFbeta)-related signaling pathways. We investigated the role of Smad4 in TGFbeta-related pathways by targeted disruption of its locus in murine cell lines. TGFbeta responses, including growth arrest, induction of the endogenous PAI-1 gene, and other extracellular matrix components, were normal in Smad4-deficient fibroblasts. Assembly of a TGFbeta-induced DNA-binding complex on one of two regulatory regions in the human plasminogen activator inhibitor (PAI)-1 promoter did not require Smad4 but was, instead, dependent on a TFE-3 binding site. In contrast, Smad4 was required for activation of the Xenopus Mix.2 promoter in response to TGFbeta/activin. Smad4 was also involved in the regulation of the Msx homeobox protein family members in response to bone morphogenetic protein (BMP). Interestingly, the expression of the endogenous Msx-2 was reduced, whereas that of Msx-3 was activated in differentiating Smad4(-/-) ES cells relative to wild-type cells. Moreover, reporter assays of the Msx-2 promoter revealed an absolute requirement for Smad4 in fibroblasts and ES cells for activation. Our results indicate that Smad4 is dispensable for critical TGFbeta-induced responses but is required for others in murine fibroblasts. We have identified transcriptional targets for Smad4 in the BMP signaling pathway, which may contribute to the genetic defect observed in the Smad4-deficient embryos.

TRAF2 deficiency results in hyperactivity of certain TNFR1 signals and impairment of CD40-mediated responses.

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) can interact with various members of the TNF receptor family. Previously, we reported that TRAF2-deficient mice die prematurely and have elevated serum TNF levels. In this study, we demonstrate that TRAF2-deficient macrophages produce increased amounts of nitric oxide (NO) and TNF in response to TNF stimulation. Furthermore, we could enhance the survival of TRAF2-deficient mice by eliminating either TNF or TNFR1. Using these double-knockout mice, we show that in the absence of TRAF2, the T helper-dependent antibody response, CD40-mediated proliferation, and NF-kappaB activation are defective. These data demonstrate two important roles of TRAF2, one as a negative regulator of certain TNFR1 signals and the other as a positive mediator of CD40 signaling.

Interleukin 13 is secreted by and stimulates the growth of Hodgkin and Reed-Sternberg cells.

Gene expression patterns can provide vital clues to the pathogenesis of neoplastic diseases. We investigated the expression of 950 genes in Hodgkin's disease (HD) by analyzing differential mRNA expression using microarrays. In two independent microarray experiments, the HD-derived cell lines L428 and KMH2 were compared with an Epstein-Barr virus (EBV)-immortalized lymphoblastoid B cell line, LCL-GK. Interleukin (IL)-13 and IL-5 were found to be highly expressed in the HD-derived cell lines. Examination of IL-13 and IL-5 expression by Northern blot analysis and enzyme-linked immunosorbent assay confirmed these results and revealed the expression of IL-13 in a third HD-derived cell line, HDLM2. Control LCL and EBV-negative non-Hodgkin lymphoma-derived cell lines did not express IL-13. In situ hybridization of lymph node tissue from HD patients showed that elevated levels of IL-13 were specifically expressed by Hodgkin/Reed-Sternberg (H/RS) tumor cells. Treatment of a HD-derived cell line with a neutralizing antibody to IL-13 resulted in a dose-dependent inhibition of H/RS cell proliferation. These data suggest that H/RS cells produce IL-13 and that IL-13 plays an important role in the stimulation of H/RS cell growth, possibly by an autocrine mechanism. Modulation of the IL-13 signaling pathway may be a logical objective for future therapeutic strategies.

Negative regulation of PKB/Akt-dependent cell survival by the tumor suppressor PTEN.

PTEN is a tumor suppressor with sequence homology to protein tyrosine phosphatases and the cytoskeletal protein tensin. mPTEN-mutant mouse embryos display regions of increased proliferation. In contrast, mPTEN-deficient immortalized mouse embryonic fibroblasts exhibit decreased sensitivity to cell death in response to a number of apoptotic stimuli, accompanied by constitutively elevated activity and phosphorylation of protein kinase B/Akt, a crucial regulator of cell survival. Expression of exogenous PTEN in mutant cells restores both their sensitivity to agonist-induced apoptosis and normal pattern of PKB/Akt phosphorylation. Furthermore, PTEN negatively regulates intracellular levels of phosphatidylinositol (3,4,5) trisphosphate in cells and dephosphorylates it in vitro. Our results show that PTEN may exert its role as a tumor suppressor by negatively regulating the PI3'K/PKB/Akt signaling pathway.

The tumor suppressor gene Smad4/Dpc4 is required for gastrulation and later for anterior development of the mouse embryo.

Mutations in the SMAD4/DPC4 tumor suppressor gene, a key signal transducer in most TGFbeta-related pathways, are involved in 50% of pancreatic cancers. Homozygous Smad4 mutant mice die before day 7.5 of embryogenesis. Mutant embryos have reduced size, fail to gastrulate or express a mesodermal marker, and show abnormal visceral endoderm development. Growth retardation of the Smad4-deficient embryos results from reduced cell proliferation rather than increased apoptosis. Aggregation of mutant Smad4 ES cells with wild-type tetraploid morulae rescues the gastrulation defect. These results indicate that Smad4 is initially required for the differentiation of the visceral endoderm and that the gastrulation defect in the epiblast is secondary and non-cell autonomous. Rescued embryos show severe anterior truncations, indicating a second important role for Smad4 in anterior patterning during embryogenesis.

Proto-oncoprotein Vav interacts with c-Cbl in activated thymocytes and peripheral T cells.

The molecular adapter c-Cbl is rapidly tyrosine phosphorylated following stimulation through the TCR and associates with Src homology domain-2 (SH2)/SH3 domain-containing adapters such as Grb2, Crk, and Crk-L, which interact with guanine nucleotide exchange factors specific for the Ras family. This suggests that c-Cbl may link TCR activation to molecules that regulate GTP binding proteins. The SH2/SH3-containing protein Vav also contains a guanine nucleotide exchange factor domain, and Vav has a crucial role in thymocyte development and activation of peripheral T cells following stimulation through the TCR. Here we show that Vav and c-Cbl form inducible molecular complexes in TCR-activated murine thymocytes and peripheral T cells as well as pervanadate-treated T cells. Vav/c-Cbl interactions are also detectable in freshly isolated T cells from gene-targeted mice that lack the T cell-specific inhibitory receptor CTLA-4, in which c-Cbl is hyperphosphorylated on tyrosine residues. The interaction between Vav and c-Cbl is directly mediated via the SH2 domain of Vav and is dependent on tyrosine phosphorylation of c-Cbl. In addition, we show that the conserved motif Y699 MTP present in c-Cbl is the binding site for the Vav SH2 domain in vitro. These data imply that c-Cbl is a molecular adapter that regulates the function of Vav in thymocytes and peripheral T cells.