Interactive Fly, Drosophila

The proper growth and elaboration of neural processes is essential for the establishment of a functional nervous system during development and is an integral feature of neural plasticity throughout life. Nuclear factor-kappa B (NF-kappaB) is classically known for its ubiquitous roles in inflammation, immune and stress-related responses and regulation of cell survival in all tissues, including the nervous system. NF-kappaB participation in other cellular processes remains poorly understood. A mechanism is reported for controlling the growth of neural processes in developing peripheral and central neurons involving the transcription factor NF-kappaB. Inhibiting NF-kappaB activation with super-repressor IkappaB-alpha, BAY 11 7082 (IkappaB-alpha phosphorylation inhibitor) or N-acetyl-Leu-Leu-norleucinal (proteosomal degradation inhibitor), or inhibiting NF-kappaB transcriptional activity with kappaB decoy DNA substantially reduces the size and complexity of the neurite arbors of sensory neurons cultured with brain-derived neurotrophic factor while having no effect on their survival. NF-kappaB exerts this effect during a restricted period of development following the phase of naturally occurring neuronal death when the processes and connections of the remaining neurons are extensively modified and refined. Inhibiting NF-kappaB activation or NF-kappaB transcriptional activity in layer 2 pyramidal neurons in postnatal somatosensory cortical slices reduces dendritic arbor size and complexity. This function of NF-kappaB has important implications for neural development and may provide an explanation for reported involvement of NF-kappaB in learning and memory (Gutierrez, 2005).

For a given cell type, particular extracellular signals generate characteristic patterns of activity in intracellular signalling networks that lead to distinctive cell-type specific responses. This paper reports the first known occurrence of a developmental switch in the intracellular signalling network required for an identical cellular response to the same extracellular signal in the same cell type. Although NF-kappaB signalling is required for BDNF-promoted neurite growth from both foetal and postnatal mouse sensory neurons, there is a developmental switch between these stages in the NF-kappaB activation mechanism and the phosphorylation status of the p65 NF-kappaB subunit required for neurite growth. Shortly before birth, BDNF activates NF-kappaB by an atypical mechanism that involves tyrosine phosphorylation of IkappaBalpha by Src family kinases, and dephosphorylates p65 at serine 536. Immediately after birth, BDNF-independent constitutive activation of NF-kappaB signalling by serine phosphorylation of IkappaBalpha and constitutive dephosphorylation of p65 at serine 536 are required for BDNF-promoted neurite growth. This abrupt developmental switch in NF-kappaB signalling in a highly differentiated cell type illustrates an unsuspected plasticity in signalling networks in the generation of identical cellular responses to the same extracellular signal (Gavaldà, 2009).

NF kappa B and programmed cell death

Tumor necrosis factor alpha (TNF-alpha) binding to the TNF receptor (TNFR) potentially initiates apoptosis and activates the transcription factor nuclear factor kappa B (NF-kappaB), which suppresses apoptosis by an unknown mechanism. The activation of NF-kappaB blocks the activation of caspase-8. TRAF1 (TNFR-associated factor 1), TRAF2, and the inhibitor-of-apoptosis (IAP) proteins c-IAP1 and c-IAP2 have been identified as gene targets of NF-kappaB transcriptional activity. In cells in which NF-kappaB is inactive, all of these proteins are required to fully suppress TNF-induced apoptosis, whereas c-IAP1 and c-IAP2 are sufficient to suppress etoposide-induced apoptosis. Thus, NF-kappaB activates a group of gene products that function cooperatively at the earliest checkpoint to suppress TNF-alpha-mediated apoptosis and that function more distally to suppress genotoxic agent-mediated apoptosis (Wang, 1998).

Binding of the proinflammatory cytokine tumor necrosis factor (TNFalpha) to its receptor triggers competing signaling pathways that determine whether a cell lives or dies. Whereas one pathway is conducive to cell death, the other leads to activation of Rel/NF-kappaB transcription factors and the coincident inhibition of apoptosis. Accumulating evidence supports a proactive role for NF-kappaB in the inhibition of cell death induced by TNFalpha and other death-causing agents. Whereas the activation of NF-kappaB blocks cell killing, its inhibition enhances the cytotoxicity of TNFalpha and promotes apoptosis in various cell systems, demonstrating the need for NF-kappaB function for cell survival. Bcl-2-family proteins are key regulators of the apoptotic response (see Rob-1. The pro-survival Bcl-2 homolog Bfl-1/A1 is a direct transcriptional target of NF-kappaB. bfl-1 gene expression is dependent on NF-kappaB activity and it can substitute for NF-kappaB to suppress TNFalpha-induced apoptosis. bfl-1 promoter analysis has identified an NF-kappaB site responsible for its Rel/NF-kappaB-dependent induction. The expression of bfl-1 in immune tissues supports the protective role of NF-kappaB in the immune system. The activation of Bfl-1 may be the means by which NF-kappaB functions in oncogenesis and promotes cell resistance to anti-cancer therapy (Zong, 1999).

The transcription factor NFkappaB is an important regulator of gene expression during immune and inflammatory responses, and can also protect against apoptosis. Endothelial cells undergo apoptosis when deprived of growth factors. Surviving viable cells exhibit increased activity of NFkappaB, whereas apoptotic cells show caspase-mediated cleavage of the NFkappaB p65/RelA subunit. This cleavage leads to loss of carboxy-terminal transactivation domains and a transcriptionally inactive p65 molecule. The truncated p65 acts as a dominant-negative inhibitor of NFkappaB, promoting apoptosis, whereas an uncleavable, caspase-resistant p65 protects the cells from apoptosis. The generation of a dominant-negative fragment of p65 during apoptosis may be an efficient pro-apoptotic feedback mechanism between caspase activation and NF-kappaB inactivation (Levkau, 1999).

The maintenance of NF-kappaB activity by endothelial cells is required for cell survival, because overexpression of a transcriptionally active p65 clearly prevents apoptosis in this system. the results presented here are consistent with several reports that show that NF-kappaB protects cells from a number of apoptotic stimuli, such as TNF-alpha, ionizing radiation and chemotherapeutic agents, and mediates anti-apoptotic signals from the matrix through integrin alpha_vbeta₃. Transcriptional activation of survival genes is believed to be the mechanism by which NF-kappaB provides protection from apoptosis. However, an absolute requirement for NF-kappaB as a transcription factor that universally prevents cell death is far from established (Levkau, 1999).

Other results indicate that NF-kappaB's transcriptional activity may be involved in glutamate-induced toxicity in neuronal cells, and can accelerate growth-factor-deprivation-induced apoptosis in certain transformed cell lines. Although classic NF-kappaB activators, such as TNF-alpha, do not induce apoptosis unless NF-kappaB is blocked, other apoptotic stimuli, such as ligation of the cell-surface-located receptor Fas, induce cell death independent of their ability to activate NF-kappaB. Therefore, growth-factor deprivation in endothelial cells may be an apoptotic stimulus that activates NF-kappaB as an initial protective reaction. NF-kappaB's persistence in the viable population is at least responsible for the cells' resistance to apoptosis. In contrast, cells committed to apoptosis appear to repress NF-kappaB. These results indicate that, as long as cells are capable of maintaining transcriptionally active NF-kappaB, they can resist apoptosis induced by growth-factor deprivation (Levkau, 1999 and references therein).

The maintenance of lymphocyte homeostasis by apoptosis is a critical regulatory mechanism in the normal immune system. The transcription factor NF-kappaB, which has been shown to play a role in protecting cells against death mediated by TNF. NF-kappaB, also has a role in regulating Fas/APO-1/CD95-mediated death, a major pathway of peripheral T cell death. Transfection of Jurkat cells with the NF-kappaB subunits p50 and p65 confers resistance against Fas-mediated apoptosis. Reciprocally, inhibition of NF-kappaB activation by a soluble peptide inhibitor or a dominant form of the NF-kappaB inhibitor, IkappaB, makes the cells more susceptible to Fas-mediated apoptosis. Furthermore, inhibition of NF-kappaB activation by a soluble peptide inhibitor renders a T cell hybridoma more susceptible to TCR-mediated apoptosis. Correspondingly, transfection of p50 and p65 provides considerable protection from TCR-mediated apoptosis. These observations are corroborated by studies on Fas-mediated death in primary T cells. Concanavalin A-activated cycling T cell blasts from mice that are transgenic for the dominant IkappaB molecule have increased sensitivity to Fas-mediated apoptosis, associated with a down-regulation of NF-kappaB complexes in the nucleus. In addition, blocking TNF (itself a positive regulator of NF-kappaB) with neutralizing antibodies renders the cells more susceptible to anti-Fas-mediated apoptosis. In summary, these results provide compelling evidence that NF-kappaB protects against Fas-mediated death and is likely to be an important regulator of T cell homeostasis and tolerance (Dudley, 1999).

Activation of CD40 is essential for thymus-dependent humoral immune responses and rescuing B cells from apoptosis. Many of the effects of CD40 are believed to be achieved through altered gene expression. In addition to Bcl-x, a known CD40-regulated antiapoptotic molecule, a related antiapoptotic molecule, A1/Bfl-1, has been identified as a CD40-inducible gene. Inhibition of the NF-kappaB pathway by overexpression of a dominant-active inhibitor of NF-kappaB abolishes CD40-induced up-regulation of both the Bfl-1 and Bcl-x genes and also eliminates the ability of CD40 to rescue Fas-induced cell death. Within the upstream promoter region of Bcl-x, a potential NF-kappaB-binding sequence was found to support NF-kappaB-dependent transcriptional activation. Furthermore, expression of physiological levels of Bcl-x protects B cells from Fas-mediated apoptosis in the absence of NF-kappaB signaling. Thus, these results suggest that CD40-mediated cell survival proceeds through NF-kappaB-dependent up-regulation of Bcl-2 family members (Lee, 1999).

The death domain of tumor necrosis factor (TNF) receptor-1 (TNFR1) triggers distinct signaling pathways leading to apoptosis and NF-kappa B activation through its interaction with the death domain protein TRADD. TRADD interacts strongly with RIP, another death domain protein that was shown previously to associate with Fas antigen. RIP is a serine-threonine kinase that is recruited by TRADD to TNFR1 in a TNF-dependent process. Overexpression of the intact RIP protein induces both NF-kappa B activation and apoptosis. However, expression of the death domain of RIP Induces apoptosis, but potently inhibits NF-kappa B activation by TNF. These results suggest that distinct domains of RIP participate in the TNF signaling cascades leading to apoptosis and NF-kappa B activation (Hsu, 1996a).

The TNFR1 signaling complex leads to activation of at least three distinct effector functions, JNK activation, NF-kappaB activation, and induction of apoptosis. In the induction of apoptosis, TNFR1 recruits TNFR1-associated death domain protein (TRADD) which in turn interacts directly with two other proteins, TNFR-1 associated protein 1 (TRAF1) and Fas-associated protein with death domain (FADD) (Hsu, 1996b). TRADD is required for induction of apoptosis by TNFR1, and expression of both TRADD and RIP, another death domain protein, is sufficient to activate this process. Apoptosis induction by TNFR1 also appears to require FADD, but unlike TRADD and RIP, this activity is mediated by the FADD N-terminal domain rather than its death domain. In fact, the FADD death domain blocks TNF-induced apoptosis. The N-terminal death effector domain of FADD interacts with an ICE-like protease which appears to be a direct activator of the apoptotic protease cascade. As far as the two other effector functions of TNFR1, recruitment of FADD to the TNFR1 does not activate NF-kappaB or JNK. Two other signal transducers, RIP and TRAF2 mediate both JNK and NF-kappaB activation. These two responses diverge downstream to TRAF2. JNK activation is not involved in induction of apoptosis but may be a protective response. Likewise, NF-kappaB activation may also be a protective response. The NF-kappaB activated antiapoptotic genes remain to be identified, but one likely candidate is manganese superoxide dismutase (Liu, 1996 and references).

Engagement of the CD95 (APO-1/Fas) receptor induces apoptosis in a variety of cell types. However, the nature of the cytotoxic signal and the intermediate messenger molecules remain to be elucidated. In an effort to understand CD95-mediated signaling, possible changes in the DNA binding activity of NF-kappaB were assessed as a result of CD95 engagement in various tumor cells. CD95 can stimulate the DNA binding activity of NF-kappaB in a variety of cells, irrespective of their sensitivity or resistance to CD95-mediated cytotoxicity. Moreover, deletion of 37 carboxyl-terminal residues from the cytoplasmic domain of CD95, which abrogates CD95-mediated apoptosis, only marginally affects NF-kappaB activation. Taken together, these observations indicate that CD95 has a function that involves activation of NF-kappaB and that appears to be unrelated to its role as an inducer of apoptotic cell death (Ponton, 1996).

Tumor necrosis factor receptor 1 (TNF-R1) mediates most of the biological properties of TNF including activation of the transcription factor NF-kappaB and programmed cell death. An approximately 80-amino acid region within the intracellular domain of the receptor, termed the death domain, is required for signaling NF-kappaB activation and cytotoxicity. A TNF-R1-associated protein TRADD has been discovered that interacts with the death domain of the receptor. Elevated expression of TRADD in cells triggers both NF-kappaB activation and programmed cell death pathways. The biological activities of TRADD have been mapped to a 111-amino acid region within the carboxyl-terminal half of the protein. This region shows sequence similarity to the death domain of TNF-R1 and can self-associate and bind to the TNF-R1 death domain. An alanine scanning mutagenesis was performed in TRADD's death domain to explore the relationship among its various functional properties. Mutations affecting the different activities of TRADD do not map to discrete regions but rather are spread over the entire death domain, suggesting that the death domain is a multifunctional unit. A mutant that separates cell killing from NF-kappaB activation by the TRADD death domain has been identified indicating that these two signaling pathways diverge with TRADD. Additionally, one of the TRADD mutants that fails to activate NF-kappaB was found to act as dominant negative mutant capable of preventing induction of NF-kappaB by TNFalpha. Such observations provide evidence that TRADD performs an obligate role in TNF-induced NF-kappaB activation (Park, 1996).

Tumor necrosis factor (TNF) can induce apoptosis and activate NF-kappa B through signaling cascades emanating from TNF receptor 1 (TNFR1). TRADD is a TNFR1-associated signal transducer that is involved in activating both pathways. TRADD directly interacts with TRAF2 and FADD, signal transducers that respectively activate NF-kappa B and induce apoptosis. A TRAF2 mutant lacking its N-terminal RING finger domain is a dominant-negative inhibitor of TNF-mediated NF-kappa B activation, but does not affect TNF-induced apoptosis. Conversely, a FADD mutant lacking its N-terminal 79 amino acids is a dominant-negative inhibitor of TNF-induced apoptosis, but does not inhibit NF-kappa B activation. Thus, these two TNFR1-TRADD signaling cascades appear to bifurcate at TRADD (Hsu 1996).

Fas (CD95) and Fas ligand (CD95L) are an interacting receptor-ligand pair required for immune homeostasis. Lymphocyte activation results in the upregulation of Fas expression and the acquisition of sensitivity to FasL-mediated apoptosis. Although Fas upregulation is central to the preservation of immunologic tolerance, little is known about the molecular machinery underlying this process. To investigate the events involved in activation-induced Fas upregulation, three processes, mRNA accumulation, fas promoter activity, and protein expression, have been examined in the Jurkat T-cell line treated with phorbol myristate acetate and ionomycin (P/I), two pharmacological mimics of T-cell receptor activation. Although resting Jurkat cells express Fas, Fas mRNA is induced approximately 10-fold, 2 h after P/I stimulation. Using sequential deletion mutants of the human fas promoter in transient transfection assays, a 47-bp sequence (positions -306 to -260 relative to the ATG) has been identified. This sequence is required for activation-driven fas upregulation. Sequence analysis reveals the presence of a previously unrecognized composite binding site for both the Sp1 and NF-kappaB transcription factors at positions -295 to -286. Electrophoretic mobility shift assay (EMSA) and supershift analyses of this region document constitutive binding of Sp1 in unactivated nuclear extracts and inducible binding of p50-p65 NF-kappaB heterodimers after P/I activation. Sp1 and NF-kappaB transcription factor binding has been shown to be mutually exclusive by EMSA displacement studies with purified recombinant Sp1 and recombinant p50. The functional contribution of the kappaB-Sp1 composite site in P/I-inducible fas promoter activation was verified by using kappaB-Sp1 concatamers (-295 to -286) in a thymidine kinase promoter-driven reporter construct and native promoter constructs in Jurkat cells overexpressing IkappaB-alpha. Site-directed mutagenesis of the critical guanine nucleotides in the kappaB-Sp1 element documents the essential role of this site in activation-dependent fas promoter induction (Chan, 1999).

The Epstein-Barr virus latent membrane protein 1 (LMP1) is essential for the transformation of B lymphocytes into lymphoblastoid cell lines. Previous data are consistent with a model that LMP1 is a constitutively activated receptor that transduces signals for transformation through its carboxyl-terminal cytoplasmic tail. One transformation effector site (TES1), located within the 45 residues of the cytoplasmic tail that are most proximal to the membrane, constitutively engages tumor necrosis factor receptor-associated factors. Signals from TES1 are sufficient to drive initial proliferation of infected resting B lymphocytes, but most lymphoblastoid cells infected with a virus that does not express the 155 residues beyond TES1 fail to grow as long-term cell lines. Mutating two tyrosines to an isoleucine at the carboxyl end of the cytoplasmic tail cripples the ability of EBV to cause lymphoblastoid cell outgrowth, thereby marking a second transformation effector site, TES2. A yeast two-hybrid screen identifies TES2 interacting proteins, including the tumor necrosis factor receptor-associated death domain protein (TRADD). TRADD is the only protein that interacts with wild-type TES2 and not with isoleucine-mutated TES2. TRADD associates with wild-type LMP1 but not with isoleucine-mutated LMP1 in mammalian cells, and TRADD constitutively associates with LMP1 in EBV-transformed cells. In transfection assays, TRADD and TES2 synergistically mediate high-level NF-kappaB activation. These results indicate that LMP1 appropriates TRADD to enable efficient long-term lymphoblastoid cell outgrowth. High-level NF-kappaB activation also appears to be a critical component of long-term outgrowth (Izumi, 1997).

TNF-induced activation of the transcription factor NF-kappaB and the c-jun N-terminal kinase (JNK/SAPK) requires TNF receptor-associated factor 2 (TRAF2). The NF-kappaB-inducing kinase (NIK) associates with TRAF2 and mediates TNF activation of NF-kappaB. NIK interacts with additional members of the TRAF family; this interaction requires the conserved "WKI" motif within the TRAF domain. The role of NIK has been investigated in JNK activation by TNF. Whereas overexpression of NIK potently induces NF-kappaB activation, it fails to stimulate JNK activation. A kinase-inactive mutant of NIK is a dominant negative inhibitor of NF-kappaB activation but does not suppress TNF- or TRAF2-induced JNK activation. Thus, TRAF2 is the bifurcation point of two kinase cascades leading to the respective activation of NF-kappaB and JNK (Song, 1997).

Exposure of mammalian skin to UV light results in induced gene transcription, playing a role in inflammation, immunosuppression, and tumor promotion. One important group of transcription factors induced by UV radiation is composed of members of the Rel/NF-kappaB family, which are known to play a major role in the transcriptional activation of many genes encoding inflammatory cytokines, adhesion molecules, and viral proteins. However, the upstream events in the transduction of the UVB signal to Rel protein activity are, as yet, unknown. Biochemical evidence is provided that exposure of keratinocytes to UVB causes rapid association of tumor necrosis factor (TNF) receptor 1 with its downstream partner TRAF-2. The functional relevance of this association is demonstrated by experiments showing that expression of either a dominant negative TNF receptor 1 or a TRAF-2 protein inhibits UVB-induced Rel-dependent transcription. Inclusion of a neutralizing antibody toward TNF has no effect on UVB activation of a Rel-responsive reporter gene. Therefore, UVB-induced activation of Rel proteins via TNF receptor 1, independent of ligand activation, is a key component in the UV response in keratinocytes (Tobin, 1998).

The CD95 (Fas/APO-1) and tumor necrosis factor (TNF) receptor pathways share many similarities, including a common reliance on proteins containing "death domains" for elements of the membrane-proximal signal relay. Mutant cell lines have been created that are unable to activate NF-kappaB in response to TNF. One of the mutant lines lacks RIP, a 74 kDa Ser/Thr kinase originally identified by its ability to associate with Fas/APO-1 and induce cell death. Reconstitution of the line with RIP restores responsiveness to TNF. The RIP-deficient cell line is susceptible to apoptosis initiated by anti-CD95 antibodies. An analysis of cells reconstituted with mutant forms of RIP reveals similarities between the action of RIP and FADD/MORT-1, a Fas-associated death domain protein. RIP mediates TNF receptor 1 activation of NF-kappaB but not Fas/APO-1-initiated apoptosis (Ting, 1996).

The TRAF3 molecule interacts with the cytoplasmic carboxyl terminus (COOH terminus) of the Epstein-Barr virus-encoded oncogene LMP-1. NF-kappaB activation is a downstream signaling event of tumor necrosis factor receptor-associated factor (TRAF) molecules in other signaling systems (CD40 for example) and is an event caused by LMP-1 expression. One region capable of TRAF3 interaction in LMP-1 is the membrane-proximal 45 amino acids (188-242) of the COOH terminus. This region contains the only site for binding of TRAF3 in the 200-amino acid COOH terminus of LMP-1. The site also binds TRAF2 and TRAF5, but not TRAF6. TRAF3 binds to critical residues localized between amino acids 196 and 212 (HHDDSLPHPQQATDDSG), including the PXQX(T/S) motif, that share limited identity to the CD40 receptor TRAF binding site (TAAPVQETL). Mutation of critical residues in the TRAF3 binding site of LMP-1 that prevents binding of TRAF2, TRAF3, and TRAF5 does not affect NF-kappaB-activating potential. Deletion mapping localizes the major NF-kappaB activating region of LMP-1 to critical residues in the distal 4 amino acids of the COOH terminus (383-386). Therefore, TRAF3 binding and NF-kappaB activation occur through two separate motifs at opposite ends of the LMP-1 COOH-terminal sequence (Brodeur, 1997).

Members of both the NF-kappaB/Rel and inhibitor of apoptosis (IAP) protein families have been implicated in signal transduction programs that prevent cell death elicited by the cytokine tumor necrosis factor alpha (TNF). Although NF-kappaB appears to stimulate the expression of specific protective genes, neither the identities of these genes nor the precise role of IAP proteins in this anti-apoptotic process are known. NF-kappaB is required for TNF-mediated induction of the gene encoding human c-IAP2. When overexpressed in mammalian cells, c-IAP2 activates NF-kappaB and suppresses TNF cytotoxicity. Both of these c-IAP2 activities are blocked in vivo by coexpressing a dominant form of IkappaB that is resistant to TNF-induced degradation. In contrast to wild-type c-IAP2, a mutant lacking the C-terminal RING domain inhibits NF-kappaB induction by TNF and enhances TNF killing. These findings suggest that c-IAP2 is critically involved in TNF signaling and exerts positive feedback control on NF-kappaB via an IkappaB targeting mechanism. Functional coupling of NF-kappaB and c-IAP2 during the TNF response may provide a signal amplification loop that promotes cell survival rather than death (Chu, 1997).

The oncoprotein v-Rel, a member of the Rel/NF-kappaB family of transcription factors, induces neoplasias and inhibits apoptosis. To identify differentially regulated cellular genes and to evaluate their relevance to transformation and apoptosis in v-Rel-transformed cells, mRNA differential display has been used. One of the recovered cDNAs corresponds to a gene that is highly expressed in v-Rel-transformed fibroblasts. Analysis of the isolated full-length cDNA of a chicken inhibitor-of-apoptosis protein (ch-IAP1) reveals that it encodes a 68-kDa protein that is highly homologous to members of the IAP family, such as human c-LAP1. Like other IAPs, ch-IAP1 contains the N-terminal baculovirus IAP repeats and C-terminal RING finger motifs. Northern blot analysis identifies a 3.3-kb ch-IAP1 transcript expressed at relatively high levels in the spleen, thymus, bursa, intestine, and lungs. Expression of v-Rel in fibroblasts, a B-cell line, and spleen cells up-regulates the expression of ch-IAP1. In contrast, ch-IAP1 expression levels are low in chicken cell lines transformed by several other unrelated tumor viruses. ch-IAP1 is expressed predominantly in the cytoplasm of the v-Rel-transformed cells. ch-IAP1 suppresses mammalian cell apoptosis induced by the overexpression of the interleukin-1-converting enzyme. Expression of exogenous ch-IAP1 in temperature-sensitive v-Rel transformed spleen cells inhibits apoptosis of these cells at the nonpermissive temperature. Collectively, these results suggest that ch-IAP1 is induced during the v-Rel-mediated transformation process and functions as a suppressor of apoptosis in v-Rel-transformed cells (You, 1997).

The v-Rel oncoprotein belongs to the Rel/NF-kappaB family of transcription factors. It transforms chicken lymphoid cells in vitro and induces fatal lymphomas in vivo. A tetracycline-regulated system was used to characterize the role of v-Rel in cell transformation. The continued expression of v-Rel is necessary to maintain the viability of transformed lymphoid cells and enables primary spleen cells to escape apoptosis in vitro culture. In agreement with a possible role for v-Rel in the inhibition of programmed cell death, its inducible expression in HeLa cells prevents TNFalpha-induced apoptosis. While the repression of v-Rel is accompanied by the rapid degradation of IkappaBalpha, changes in the steady-state levels of the apoptosis inhibitors Bcl-2 and Bcl-X(L) are observed only following the onset of cell death in transformed lymphoid cells. This suggests that the anti-apoptotic activity of v-Rel may affect other apoptosis inhibitors or other factors in the death pathway. Together, these findings demonstrate that v-Rel blocks apoptosis and suggest that this activity may be an important component of its transforming function (Zong, 1997).

The possibility that bacteria may have evolved strategies to overcome host cell apoptosis was explored by using Rickettsia rickettsii, an obligate intracellular Gram-negative bacteria that is the etiologic agent of Rocky Mountain spotted fever. The vascular endothelial cell, the primary target cell during in vivo infection, exhibits no evidence of apoptosis during natural infection and is maintained for a sufficient time to allow replication and cell-to-cell spread prior to eventual death due to necrotic damage. R. rickettsii infection activates the transcription factor NF-kappaB and alters expression of several genes under its control. However, when R. rickettsii-induced activation of NF-kappaB is inhibited, apoptosis of infected endothelial cells (but not uninfected cells) rapidly ensues. Human embryonic fibroblasts stably transfected with a superrepressor mutant inhibitory subunit IkappaB that renders NF-kappaB inactivatable also undergo apoptosis when infected, whereas infected wild-type human embryonic fibroblasts survive. R. rickettsii, therefore, appears to inhibit host cell apoptosis via a mechanism dependent on NF-kappaB activation. Apoptotic nuclear changes correlate with the presence of intracellular organisms, and thus this previously unrecognized proapoptotic signal (masked by concomitant NF-kappaB activation) likely requires intracellular infection. These studies demonstrate that a bacterial organism can exert an antiapoptotic effect, thus modulating the host cell's apoptotic response to its own advantage by potentially allowing the host cell to remain as a site of infection (Clifton, 1998).

Neurotrophins activate multiple signaling pathways in neurons. However, the precise roles of these signaling molecules in cell survival are not well understood. Nerve growth factor (NGF) activates the transcription factors NF-kappaB and AP-1 in cultured sympathetic neurons. Activated NF-kappaB complexes consist of heterodimers of p50 and Rel proteins (RelA, as well as c-Rel), and NF-kappaB activation occurs independently of de novo protein synthesis but in a manner that requires the action of the proteasome complex. Treatment with the NF-kappaB inhibitory peptide SN50 in the continuous presence of NGF results in dose-dependent induction of cell death. Under the conditions used, SN50 selectively inhibits NF-kappaB activation but not the activation of other cellular transcription factors, such as AP-1 and cAMP response element-binding protein. Cells treated with SN50 exhibit the morphological and biochemical hallmarks of apoptosis, and the kinetics of cell killing are accelerated relative to death induced by NGF withdrawal. Experiments were also conducted to test directly whether NF-kappaB could act as a survival factor for NGF-deprived neurons. Microinjection of cells with an expression plasmid encoding NF-kappaB (c-Rel) results in enhanced neuronal survival after withdrawal of NGF, whereas cells that are transfected with a vector encoding a mutated derivative of c-Rel lacking the transactivation domain undergo cell death to the same extent as control cells. Together, these findings suggest that the activation of NF-kappaB/Rel transcription factors may contribute to the survival of NGF-dependent sympathetic neurons (Maggirwar, 1998).

The developing cerebral cortex undergoes a period of substantial cell death. The present studies examine the role of the suicide receptor Fas/Apo[apoptosis]-1 in cerebral cortical development. Fas mRNA and protein are transiently expressed in subsets of cells within the developing rat cerebral cortex during the peak period of apoptosis. Fas-immunoreactive cells have been localized in close proximity to Fas ligand (FasL)-expressing cells. The Fas-associated signaling protein receptor interacting protein (RIP) is expressed by some Fas-expressing cells, whereas Fas-associated death domain (FADD) is undetectable in the early postnatal cerebral cortex. FLICE-inhibitory protein (FLIP), an inhibitor of Fas activation, is also expressed in the postnatal cerebral cortex. Fas expression is more ubiquitous in embryonic cortical neuroblasts in dissociated culture, as compared to in situ, within the developing brain, suggesting that the environmental milieu partly suppresses Fas expression at this developmental stage. Furthermore, FADD, RIP, and FLIP are also expressed by subsets of dissociated cortical neuroblasts in culture. Fas activation by ligand (FasL) or anti-Fas antibody induces caspase-dependent cell death in primary embryonic cortical neuroblast cultures. The activation of Fas is also accompanied by a rapid downregulation of Fas receptor expression, non-cell cycle-related incorporation of nucleic acids and nuclear translocation of the RelA/p65 subunit of the transcription factor NF-kappaB. Together, these data suggest that adult cortical cell number may be established, in part, by an active process of receptor-mediated cell suicide, initiated in situ by killer (FasL-expressing) cells and that Fas may have functions in addition to suicide in the developing brain (Cheema, 1999).

Nuclear factor kappaB (NF-kappaB) appears to participate in the excitotoxin-induced apoptosis of striatal medium spiny neurons. To elucidate molecular mechanisms by which this transcription factor contributes to NMDA receptor-triggered apoptotic cascades in vivo, rats were given the NMDA receptor agonist quinolinic acid (QA) by intrastriatal infusion, and the role of NF-kappaB in the induction of apoptosis-related genes and gene products was evaluated. QA administration induces time-dependent NF-kappaB nuclear translocation. The nuclear NF-kappaB protein after QA treatment is comprised mainly of p65 and c-Rel subunits as detected by gel supershift assay. Levels of c-Myc and p53 mRNA and protein are markedly increased at the time of QA-induced NF-kappaB nuclear translocation. Immunohistochemical analysis shows that c-Myc and p53 induction occurs in the excitotoxin-sensitive medium-sized striatal neurons. NF-kappaB nuclear translocation is blocked in a dose-dependent manner by the cell-permeable recombinant peptide NF-kappaB SN50, but not by the NF-kappaB SN50 control peptide. NF-kappaB SN50 significantly inhibits the QA-induced elevation in levels of c-Myc and p53 mRNA and protein. Pretreatment or posttreatment with NF-kappaB SN50, but not the control peptide, also substantially reduces the intensity of QA-induced internucleosomal DNA fragmentation. The results suggest that NF-kappaB may promote an apoptotic response in striatal medium-sized neurons to excitotoxic insult through upregulation of c-Myc and p53. This study also provides evidence indicating a unique signaling pathway from the cytoplasm to the nucleus, which regulates p53 and c-Myc levels in these neurons during apoptosis (Qin, 1999).

NF-kappaB/Rel transcription factors and IkappaB kinases (IKK; see Drosophila Ird5) are essential for inflammation and immune responses, but also for bone-morphogenesis, skin proliferation and differentiation. Determining their other functions has been impossible, owing to embryonic lethality of NF-kappaB/Rel or IKK-deficient animals. Using a gene targeting approach, an NF-kappaB super-repressor was ubiquitously expressed to investigate NF-kappaB functions in the adult. Mice with suppressed NF-kappaB reveal defective early morphogenesis of hair follicles, exocrine glands and teeth, identical to Eda (tabby) and Edar (downless) mutant mice. These affected epithelial appendices normally display high NF-kappaB activity, suppression of which results in increased apoptosis, indicating that NF-kappaB acts as a survival factor downstream of the tumor necrosis factor receptor family member EDAR. Furthermore, NF-kappaB is required for peripheral lymph node formation and macrophage function (Schmidt-Ullrich, 2001).

The transcription factor NF-kappaB is essential for survival of many cell types. However, cells can undergo apoptosis despite the concurrent NF-kappaB activation. It is unknown how the protection conveyed by NF-kappaB is overridden during apoptosis. IkappaB kinase (IKK) ß is specifically proteolyzed by Caspase-3-related caspases at aspartic acid residues 78, 242, 373, and 546 during tumor necrosis factor (TNF)-alpha-induced apoptosis. Proteolysis of IKKß eliminates its enzymatic activity, interfers with IKK activation, and promotes TNF-alpha killing. Point mutations that abrogate IKKß proteolysis generate a caspase-resistant IKKß mutant, which suppresses TNF-alpha-induced apoptosis. Thus, this study demonstrates that TNF-alpha-induced apoptosis requires caspase-mediated proteolysis of IKKß (Tang, 2001).

What is the mechanism by which the UC-IKKß mutant suppresses TNF-alpha-induced apoptosis? One of the possibilities is that it functions through prolonged induction of NF-kappaB-controlled antiapoptotic proteins. It is envisioned that apoptotic death of a cell requires amplification of the caspase cascade, an event that may depend on destruction of cellular survival factors. Treatment with TNF-alpha triggers rapid activation and reactivation of IKK and NF-kappaB. This results in production of antiapoptotic proteins, such as c-IAP1, which inhibit caspases. The life balance is maintained and cells survive. The addition of cycloheximide (CHX) reduces the production of antiapoptotic proteins, allowing activation of caspases such as Casp3. Activated Casp3 in turn cleaves IKKß, blunting IKK reactivation and subsequent production of antiapoptotic proteins. Activated Casp3 also cleaves antiapoptotic proteins, including c-IAP1. Therefore, the caspase cascade is amplified, switching the life balance toward death. In contrast, the uncleavable IKKß (IKK UCß) mutant is resistant to caspase-mediated proteolysis and can be reactivated over a prolonged period of time. Since 10 µg/ml CHX reduces, but does not completely block protein synthesis, activation of NF-kappaB by the UC IKKß mutant allows the accumulation of antiapoptotic proteins at a level that is sufficient to inhibit caspases. As a result, amplification of the caspase cascade and cell death is suppressed (Tang, 2001).

The c-Jun NH2-terminal kinase (JNK) can cause cell death by activating the mitochondrial apoptosis pathway. However, JNK is also capable of signaling cell survival. The mechanism that accounts for the dual role of JNK in apoptosis and survival signaling has not been established. JNK-stimulated survival signaling can be mediated by JunD. The JNK/JunD pathway can collaborate with NF-kappaB to increase antiapoptotic gene expression, including cIAP-2. This observation accounts for the ability of JNK to cause either survival or apoptosis in different cellular contexts. Furthermore, these data illustrate the general principal that signal transduction pathway integration is critical for the ability of cells to mount an appropriate biological response to a specific challenge (Lamb, 2003).

The survival signaling role of JNK in the response to TNF contrasts with the effects of JNK to mediate apoptosis in response to the exposure of cells to environmental stress. How can one signal transduction pathway mediate two very different responses? There are two general mechanisms that could account for these different roles of JNK in apoptosis signaling. These mechanisms are not mutually exclusive. One mechanism is represented by the time course of JNK activation. Studies of MAP kinases indicate that the time course of activation can determine the cellular response. This may also apply to the response of cells to JNK activation. Sustained JNK activation is required for apoptotic signaling and is sufficient for apoptosis. In contrast, TNF causes transient JNK activation. These considerations indicate that transient JNK activation may be important for mediating a survival response in TNF-treated cells and that chronic JNK activation may contribute to apoptotic responses. A second mechanism that may account for the different roles of JNK in apoptosis signaling is that the biological consequence of JNK function may depend upon the activation state of other signal transduction pathways. For example, increased AKT activation can suppress the apoptotic effects of activated JNK. A plausible hypothesis is that the JNK signaling pathway may cooperate with other signaling pathways to mediate cell survival (e.g., NF-kappaB and AKT). For example, target genes that are induced by the antiapoptotic NF-kappaB pathway may contain JNK-responsive elements in their promoters (e.g., AP-1 sites). The cIAP-2 gene represents an example of this class of gene. JNK increases the expression of such genes in cells with activated NF-kappaB and thus increases cell survival. In contrast, in the absence of a survival pathway that can cooperate with JNK, sustained JNK activation may lead to apoptosis (Lamb, 2003 and references therein).

This analysis demonstrates that JNK mediates a survival response in cells treated with TNF. The role of JNK is mediated by the transcription factor JunD, which can collaborate with NF-kappaB to increase the expression of prosurvival genes, including cIAP-2. In the absence of activated NF-kappaB, the JNK pathway mediates an apoptotic response. Together, these data provide a mechanism that can account for the dual ability of JNK to cause either survival or apoptosis in different cellular contexts (Lamb, 2003).

During inflammation, NF-kappaB transcription factors antagonize apoptosis induced by tumor necrosis factor TNFalpha. This antiapoptotic activity of NF-kappaB involves suppressing the accumulation of reactive oxygen species (ROS) and controlling the activation of the c-Jun N-terminal kinase (JNK) cascade. However, the mechanism(s) by which NF-kappaB inhibits ROS accumulation is unclear. Ferritin heavy chain (FHC) -- the primary iron storage factor -- is an essential mediator of the antioxidant and protective activities of NF-kappaB. FHC is induced downstream of NF-kappaB and is required to prevent sustained JNK activation and, thereby, apoptosis triggered by TNFalpha. FHC-mediated inhibition of JNK signaling depends on suppressing ROS accumulation and is achieved through iron sequestration. These findings establish a basis for the NF-kappaB-mediated control of ROS induction and identify a mechanism by which NF-kappaB suppresses proapoptotic JNK signaling. These results suggest modulation of FHC or, more broadly, of iron metabolism as a potential approach for anti-inflammatory therapy (Pham, 2004).

NF-kappaB and aging

Aging is characterized by specific alterations in gene expression, but their underlying mechanisms and functional consequences are not well understood. A systematic approach was developed to identify combinatorial cis-regulatory motifs that drive age-dependent gene expression across different tissues and organisms. Integrated analysis of 365 microarrays spanning nine tissue types predicted fourteen motifs as major regulators of age-dependent gene expression in human and mouse. The motif most strongly associated with aging was that of the transcription factor NF-kappaB. Inducible genetic blockade of NF-kappaB for 2 wk in the epidermis of chronologically aged mice reverted the tissue characteristics and global gene expression programs to those of young mice. Age-specific NF-kappaB blockade and orthogonal cell cycle interventions revealed that NF-kappaB controls cell cycle exit and gene expression signature of aging in parallel but not sequential pathways. These results identify a conserved network of regulatory pathways underlying mammalian aging and show that NF-kappaB is continually required to enforce many features of aging in a tissue-specific manner (Adler, 2007).

Cellular senescence acts as a potent barrier to tumorigenesis and contributes to the anti-tumor activity of certain chemotherapeutic agents. Senescent cells undergo a stable cell cycle arrest controlled by RB and p53 and, in addition, display a senescence-associated secretory phenotype (SASP) involving the production of factors that reinforce the senescence arrest, alter the microenvironment, and trigger immune surveillance of the senescent cells. Through a proteomics analysis of senescent chromatin, the nuclear factor-kappaB (NF-kapaB) subunit p65 was identified as a major transcription factor that accumulates on chromatin of senescent cells. NF-kappaB acts as a master regulator of the SASP, influencing the expression of more genes than RB and p53 combined. In cultured fibroblasts, NF-kappaB suppression causes escape from immune recognition by natural killer (NK) cells and cooperates with p53 inactivation to bypass senescence. In a mouse lymphoma model, NF-kappaB inhibition bypasses treatment-induced senescence, producing drug resistance, early relapse, and reduced survival. These results demonstrate that NF-kappaB controls both cell-autonomous and non-cell-autonomous aspects of the senescence program and identify a tumor-suppressive function of NF-kappaB that contributes to the outcome of cancer therapy (Chien, 2011).

NF-kappaB and cell transformation

Inflammation is linked clinically and epidemiologically to cancer, and NF-kappaB appears to play a causative role, but the mechanisms are poorly understood. An experimental model of oncogenesis is described involving a derivative of MCF10A, a spontaneously immortalized cell line derived from normal mammary epithelial cells, that contains ER-Src, a fusion of the Src kinase oncoprotein (v-Src) and the ligand binding domain of the estrogen receptor. Treatment of these cells with estrogen receptor antagonist tamoxifen (TAM) for 36 hr results in phenotypic transformation, formation of multiple foci, the ability to form colonies in soft agar, increased motility and invasive ability, and tumor formation upon injection in nude mice. This model permits the opportunity to kinetically follow the pathway of cellular transformation in a manner similar to that used to study viral infection and other temporally ordered processes. Transient activation of Src oncoprotein can mediate an epigenetic switch from immortalized breast cells to a stably transformed line that forms self-renewing mammospheres that contain cancer stem cells. Src activation triggers an inflammatory response mediated by NF-kappaB that directly activates Lin28 transcription and rapidly reduces let-7 microRNA levels. Let-7 directly inhibits IL6 expression, resulting in higher levels of IL6 than achieved by NF-kappaB activation. IL6-mediated activation of the STAT3 transcription factor is necessary for transformation, and IL6 activates NF-kappaB, thereby completing a positive feedback loop. This regulatory circuit operates in other cancer cells lines, and its transcriptional signature is found in human cancer tissues. Thus, inflammation activates a positive feedback loop that maintains the epigenetic transformed state for many generations in the absence of the inducing signal (Iliopoulos, 2009).

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