Lymphoma cell adhesion-induced expression of B cell-activating factor of the TNF family in bone marrow stromal cells protects non-Hodgkin's B lymphoma cells from apoptosis.

This study explores whether lymphoma cell adhesion-induced B cell-activating factor (BAFF) expression in bone marrow stromal cells (BMSCs) protects B lymphoma cells from apoptosis. We first showed protection of lymphoma cells from apoptosis by conditioned medium of a stromal cell-lymphoma cell coculture, either spontaneous or induced by mitoxantrone, implying a role for soluble factor(s) in lymphoma cell survival. Addition of BAFF counteracted mitoxantrone-induced apoptosis and elicited a reduction in spontaneous apoptosis in primary lymphomas, suggesting a role of BAFF in sustaining B-cell survival. Abundant BAFF was detected in the BMSC cell line (HS-5) and primary BMSCs by flow cytometry, RT-PCR and immunoblotting. BAFF levels were 20- to 200-fold higher in BMSCs than in lymphoma cells, and lymphoma cell adhesion to BMSCs augmented BAFF secretion twofold through upregulation of BAFF gene expression. Finally, neutralization of BAFF by TACI-Ig or depletion of BAFF by small hairpin RNA (shRNA) in BMSCs significantly enhanced lymphoma cell response to chemotherapy and overcame stroma-mediated drug resistance, suggesting that lymphoma cells use BMSC-derived BAFF as a survival factor. These findings support the hypothesis that lymphoma cells interact with BMSCs, resulting in stromal niches with high BAFF concentration, and identify BMSC-derived BAFF as a functional determinant for B lymphoma cell survival in the bone marrow environment.

TTDN1 is a Plk1-interacting protein involved in maintenance of cell cycle integrity.

Polo-like kinase 1 (Plk1) is a highly conserved serine/threonine kinase that plays critical roles in many cell cycle events, especially in mitosis. In the present study, we identified TTDN1 as a potential interacting partner of Plk1 in yeast two-hybrid screens. Sequence analysis indicates that TTDN1 contains a consensus Plk1-binding motif at its C terminus. TTDN1 colocalizes with Plk1 at the centrosome in mitosis and the midbody during cytokinesis. TTDN1 is phosphorylated by Cdk1 in mitosis, and this is required for its interaction with Plk1. Site-directed mutagenesis indicates that TTDN1 is phosphorylated at multiple residues, including Ser93 and Ser104. Mutation of Thr120 of TTDN1 abolishes its interaction with Plk1, suggesting phosphorylation of Thr120 in the consensus Plk1-binding motif is required for its interaction with Plk1. Overexpression of TTDN1 or its knockdown by siRNA causes multi-polar spindles and multiple nuclei, suggesting that TTDN1 plays a role in regulating mitosis and cytokinesis.

The p53-inducible apoptotic protein AMID is not required for normal development and tumor suppression.

AMID is an apoptosis-inducing factor (AIF)-homologous and mitochondria-associated protein that has been implicated in caspase-independent apoptosis. Transcription of human AMID gene is upregulated by p53 and downregulated in tumors in comparison to their matched normal tissues, suggesting the possibility that AMID is involved in the downstream effects of p53. To investigate the physiological functions of AMID, we generated AMID-deficient mice by gene targeting. AMID-deficient mice are viable and fertile, develop normally and lack obvious phenotypic changes compared to wild-type mice up to 1 year old. AMID(-/-) mice up to 1 year old have no spontaneous tumors and show similar fibrosarcoma incidence after MCA inoculation compared to wild-type mice. AMID(-/-) embryonic fibroblasts exhibit normal proliferation but slightly increased resistance to genotoxin-induced growth arrest. These findings suggest that AMID is not required for normal development and p53-mediated tumor suppression.

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.

B cell maturation protein is a receptor for the tumor necrosis factor family member TALL-1.

TALL-1 is a recently identified member of the tumor necrosis factor (TNF) family that costimulates B lymphocyte proliferation. Here we show that B cell maturation protein (BCMA), a member of the TNF receptor family that is expressed only by B lymphocytes, specifically binds to TALL-1. A soluble receptor containing the extracellular domain of BCMA blocks the binding of TALL-1 to its receptor on the plasma membrane and inhibits TALL-1-triggered B lymphocyte costimulation. Overexpression of BCMA activates NF-kappaB, and this activation is potentiated by TALL-1. Moreover, BCMA-mediated NF-kappaB activation is inhibited by dominant negative mutants of TNF receptor-associated factor 5 (TRAF5), TRAF6, NF-kappaB-inducing kinase (NIK), and IkappaB kinase (IKK). These data indicate that BCMA is a receptor for TALL-1 and BCMA activates NF-kappaB through a TRAF5-, TRAF6-, NIK-, and IKK-dependent pathway. The identification of BCMA as a NF-kappaB-activating receptor for TALL-1 suggests molecular targets for drug development against certain immunodeficient or autoimmune diseases.

Activation of NF-kappaB by FADD, Casper, and caspase-8.

Fas-associated death domain protein (FADD), caspase-8-related protein (Casper), and caspase-8 are components of the tumor necrosis factor receptor type 1 (TNF-R1) and Fas signaling complexes that are involved in TNF-R1- and Fas-induced apoptosis. Here we show that overexpression of FADD and Casper potently activates NF-kappaB. In the presence of caspase inhibitors, overexpression of caspase-8 also activates NF-kappaB. A caspase-inactive point mutant, caspase-8(C360S), activates NF-kappaB as potently as wild-type caspase-8, suggesting that caspase-8-induced apoptosis and NF-kappaB activation are uncoupled. NF-kappaB activation by FADD and Casper is inhibited by the caspase-specific inhibitors crmA and BD-fmk, suggesting that FADD- and Casper-induced NF-kappaB activation is mediated by caspase-8. FADD, Casper, and caspase-8-induced NF-kappaB activation are inhibited by dominant negative mutants of TRAF2, NIK, IkappaB kinase alpha, and IkappaB kinase beta. A dominant negative mutant of RIP inhibits FADD- and caspase-8-induced but not Casper-induced NF-kappaB activation. A mutant of Casper and the caspase-specific inhibitors crmA and BD-fmk partially inhibit TNF-R1-, TRADD, and TNF-induced NF-kappaB activation, suggesting that FADD, Casper, and caspase-8 function downstream of TRADD and contribute to TNF-R1-induced NF-kappaB activation. Moreover, activation of caspase-8 results in proteolytic processing of NIK, which is inhibited by crmA. When overexpressed, the processed fragments of NIK do not activate NF-kappaB, and the processed C-terminal fragment inhibits TNF-R1-induced NF-kappaB activation. These data indicate that FADD, Casper, and pro-caspase-8 are parts of the TNF-R1-induced NF-kappaB activation pathways, whereas activated caspase-8 can negatively regulate TNF-R1-induced NF-kappaB activation by proteolytically inactivating NIK.

Tumor necrosis factor-related apoptosis-inducing ligand receptors signal NF-kappaB and JNK activation and apoptosis through distinct pathways.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family that interacts with several receptors, including TRAIL-R1, TRAIL-R2, and TRAIL-R4. TRAIL-R1 and TRAIL-R2 can induce apoptosis of cancer cells and activate the transcription factor NF-kappaB. TRAIL-R4 can activate NF-kappaB and protect cells from TRAIL-induced apoptosis. Here we show that TRAIL-R1-, TRAIL-R2-, and TRAIL-R4-induced NF-kappaB activation are mediated by a TRAF2-NIK-IkappaB kinase alpha/beta signaling cascade but is MEKK1 independent. TRAIL receptors also activate the protein kinase JNK. JNK activation by TRAIL-R1 is mediated by a TRAF2-MEKK1-MKK4 but not the TRAF2-NIK/IkappaB kinase alpha/beta signaling pathway. We also show that activation of NF-kappaB or overexpression of TRAIL-R4 does not protect TRAIL-R1-induced apoptosis. Moreover, inhibition of NF-kappaB by IkappaBalpha sensitizes cells to tumor necrosis factor- but not TRAIL-induced apoptosis. These findings suggest that TRAIL receptors induce apoptosis, NF-kappaB and JNK activation through distinct signaling pathways, and activation of NF-kappaB is not sufficient for protecting cells from TRAIL-induced apoptosis.

TALL-1 is a novel member of the TNF family that is down-regulated by mitogens.

Members of the tumor necrosis factor (TNF) family play important roles in modulation of immune responses. We describe the identification and cloning of a novel TNF family member that has been designated as TALL-1. TALL-1 is a 285-amino acid type II transmembrane protein. Its carboxy terminus shares approximately 35% sequence identity with the recently identified APRIL and approximately 20-25% with TNF, FasL, TRAIL, and lymphotoxin-alpha, suggesting that TALL-1 and APRIL belong to a subfamily of the TNF family of ligands. Northern blot analysis suggests that TALL-1 is expressed abundantly in peripheral blood leukocytes and weakly in spleen but is barely detectable in all other tissues examined. Reverse transcriptase-polymerase chain reaction analysis indicates that TALL-1 is specifically expressed in monocytes and macrophages but is undetectable in T and B lymphocytes. Furthermore, TALL-1 expression is dramatically down-regulated by phorbol myristate acetate/ionomycin.

FADD: essential for embryo development and signaling from some, but not all, inducers of apoptosis.

FADD (also known as Mort-1) is a signal transducer downstream of cell death receptor CD95 (also called Fas). CD95, tumor necrosis factor receptor type 1 (TNFR-1), and death receptor 3 (DR3) did not induce apoptosis in FADD-deficient embryonic fibroblasts, whereas DR4, oncogenes E1A and c-myc, and chemotherapeutic agent adriamycin did. Mice with a deletion in the FADD gene did not survive beyond day 11.5 of embryogenesis; these mice showed signs of cardiac failure and abdominal hemorrhage. Chimeric embryos showing a high contribution of FADD null mutant cells to the heart reproduce the phenotype of FADD-deficient mutants. Thus, not only death receptors, but also receptors that couple to developmental programs, may use FADD for signaling.

Casper is a FADD- and caspase-related inducer of apoptosis.

Caspases are cysteine proteases that play a central role in apoptosis. Caspase-8 may be the first enzyme of the proteolytic cascade activated by the Fas ligand and tumor necrosis factor (TNF). Caspase-8 is recruited to Fas and TNF receptor-1 (TNF-R1) through interaction of its prodomain with the death effector domain (DED) of the receptor-associating FADD. Here we describe a novel 55 kDa protein, Casper, that has sequence similarity to caspase-8 throughout its length. However, Casper is not a caspase since it lacks several conserved amino acids found in all caspases. Casper interacts with FADD, caspase-8, caspase-3, TRAF1, and TRAF2 through distinct domains. When overexpressed in mammalian cells, Casper potently induces apoptosis. A C-terminal deletion mutant of Casper inhibits TNF- and Fas-induced cell death, suggesting that Casper is involved in these apoptotic pathways.

The tumor necrosis factor receptor 2 signal transducers TRAF2 and c-IAP1 are components of the tumor necrosis factor receptor 1 signaling complex.

The two cell surface receptors for tumor necrosis factor (TNF) interact with a number of intracellular signal transducing proteins. The association of TRADD, a 34-kDa cytoplasmic protein containing a C-terminal death domain, with aggregated TNF receptor 1 (TNF-R1) through their respective death domains leads to NF-kappa B activation and programmed cell death. In contrast, TNF receptor 2 (TNF-R2) interacts with the TNF receptor associated factors 2/1 (TRAF2/TRAF1) heterocomplex, which mediates the recruitment of two cellular inhibitor of apoptosis proteins (c-IAP1 and c-IAP2) to TNF-R2. Here we show that the TNF-R2 signal transducers TRAF2 and c-IAP1 are a part of the TNF-R1 signaling complex. The recruitment of TRAF2 and c-IAP1 to TNF-R1 is TNF-dependent, is mediated by TRADD, and is independent of TNF-R2. These data establish the physiological involvement of TRAF2 and c-IAP1 in TNF-R1 signaling and help provide a molecular explanation for both the overlapping and distinct signals generated by the two TNF receptors.

I-TRAF is a novel TRAF-interacting protein that regulates TRAF-mediated signal transduction.

Tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins associate with and transduce signals from TNF receptor 2, CD40, and presumably other members of the TNF receptor superfamily. TRAF2 is required for CD40- and TNF-mediated activation of the transcription factor NF-kappa B. Here we describe the isolation and characterization of a novel TRAF-interacting protein, I-TRAF, that binds to the conserved TRAF-C domain of the three known TRAFs. Overexpression of I-TRAF inhibits TRAF2-mediated NF-kappa B activation signaled by CD40 and both TNF receptors. Thus, I-TRAF appears as a natural regulator of TRAF function that may act by maintaining TRAFs in a latent state.

TNF-dependent recruitment of the protein kinase RIP to the TNF receptor-1 signaling complex.

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. Here, we show that TRADD interacts strongly with RIP, another death domain protein that was shown previously to associate with Fas antigen. We also show that 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.

TRADD-TRAF2 and TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways.

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. Here we show that TRADD directly interacts with TRAF2 and FADD, signal transducers that activate NF-kappa B and induce apoptosis, respectively. 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.

A transient association of gamma-tubulin at the midbody is required for the completion of cytokinesis during the mammalian cell division.

gamma-Tubulin, a relatively new member of the tubulin gene family, is localized primarily at the centrosome throughout the mammalian cell cycle and may play a key role in nucleation of cellular microtubule assembly. A transient association of gamma-tubulin at the cytoplasmic bridge of telophase mammalian cells, the midbody, is recently documented. Using immunogold electron microscopy and serial section reconstruction analysis, we show here that the transiently associated midbody gamma-tubulin is localized at the minus ends of microtubules in the midbody structure. Using antisense RNA methods we also demonstrate that a selective depletion of transiently associated midbody gamma-tubulin causes an abortive cytokinesis due to a failure in the morphogenesis of the midbody structure.

Gamma-tubulin can both nucleate microtubule assembly and self-assemble into novel tubular structures in mammalian cells.

alpha-, beta-, and gamma-tubulins are evolutionarily highly conserved members of the tubulin gene superfamily. While the abundant members, alpha- and beta-tubulins, constitute the building blocks of cellular microtubule polymers, gamma-tubulin is a low abundance protein which localized to the pericentriolar material and may play a role in microtubule assembly. To test whether gamma-tubulin mediates the nucleation of microtubule assembly in vivo, and co-assembles with alpha- and beta-tubulins into microtubules or self-assembles into macro-molecular structures, we experimentally elevated the expression of gamma-tubulin in the cell cytoplasm. In most cells, overexpression of gamma-tubulin causes a dramatic reorganization of the cellular microtubule network. Furthermore, we show that when overexpressed, gamma-tubulin causes ectopic nucleation of microtubules which are not associated with the centrosome. In a fraction of cells, gamma-tubulin self-assembles into novel tubular structures with a diameter of approximately 50 nm (named gamma-tubules). Furthermore, unlike microtubules, gamma-tubules are resistant to cold or drug induced depolymerization. These data provide evidence that gamma-tubulin can cause nucleation of microtubule assembly and can self-assemble into novel tubular structures.

Direct transfer of transforming growth factor beta 1 gene into arteries stimulates fibrocellular hyperplasia.

The arterial wall responds to thrombosis or mechanical injury through the induction of specific gene products that increase cellular proliferation and connective tissue formation. These changes result in intimal hyperplasia that is observed in restenosis and the early phases of atherosclerosis. Transforming growth factor beta 1 (TGF-beta 1) is a secreted multi-functional protein that plays an important role in embryonal development and in repair following tissue injury. However, the function of TGF-beta 1 in vascular cell growth in vivo has not been defined. In this report, we have evaluated the role of TGF-beta 1 in the pathophysiology of intimal and medial hyperplasia by gene transfer of an expression plasmid encoding active TGF-beta 1 into porcine arteries. Expression of TGF-beta 1 in normal arteries resulted in substantial extracellular matrix production accompanied by intimal and medial hyperplasia. Increased procollagen, collagen, and proteoglycan synthesis in the neointima was demonstrated by immunohistochemistry relative to control transfected arteries. Expression of TGF-beta 1 induced a distinctly different program of gene expression and biologic response from the platelet-derived growth factor B (PDGF B) gene: procollagen synthesis induced by TGF-beta 1 was greater, and cellular proliferation was less prominent. These findings show that TGF-beta 1 differentially modulates extracellular matrix production and cellular proliferation in the arterial wall in vivo and could play a reparative role in the response to arterial injury.

Differential regulation of vascular cell adhesion molecule 1 gene expression by specific NF-kappa B subunits in endothelial and epithelial cells.

Vascular cell adhesion molecule 1 (VCAM-1) is expressed in both endothelial and epithelial cell types, where it contributes to lymphocyte migration to sites of inflammation. Its expression is regulated by cytokines, in part through two kappa B-like regulatory elements. Because NF-kappa B can be composed of multiple alternative subunits with differential effects on gene expression, the role of different specific NF-kappa B family members subunits in VCAM-1 regulation is unknown. In this report, we define the contribution of different NF-kappa B family members to VCAM-1 gene regulation. We show that both kappa B sites in the VCAM-1 enhancer are required to optimally stimulate gene expression, but the enhancer is differentially regulated by specific combinations of NF-kappa B subunits. At low concentrations, RelA(p65) acted in concert with the approximately 50-kDa product of p105 NF-kappa B, NF-kappa B1(p50), to stimulate transcription, and at high concentrations, RelA(p65) alone stimulated the VCAM-1 promoter. In contrast, NF-kappa B2 inhibited functional activation of the VCAM reporter by p65. Consistent with this finding, an additional binding complex was detected by using recombinant NF-kappa B2(p49)/RelA(p65) with radiolabeled VCAM kappa B site probes. Interestingly, the human immunodeficiency virus enhancer responded differently to stimulation by NF-kappa B subunits, with optimal response to p49(100)/p65. Analysis of NF-kappa B mRNA in human umbilical vein endothelial cells revealed that nfkb1, nfkb2, and relA NF-kappa B but not c-rel were induced by tumor necrosis factor alpha and lipopolysaccharide, which also induce VCAM-1. These data suggest that specific subunits of NF-kappa B regulate VCAM-1 and differentially activate other genes in these cells.