Targeting TRAF6 for Cancer Therapeutical Development

Tumor necrosis factor (TNF)-Receptor Associated Factors (TRAFs) are a family of signal transducer proteins. TRAF6 is a unique member of this family in that it is involved in not only the TNF superfamily, but the toll-like receptor (TLR)/IL-1R (TIR) superfamily. The formation of the complex consisting of Receptor Activator of Nuclear Factor �� B (RANK), with its ligand (RANKL) results in the recruitment of TRAF6, which activates NF-��B, JNK and MAP kinase pathways. TRAF6 is critical in signaling with leading to release of various growth factors in bone, and promotes osteoclastogenesis. TRAF6 has also been implicated as an oncogene in lung cancer and as a target in multiple myeloma. In the hopes of developing small molecule inhibitors of the TRAF6-RANK interaction, multiple steps were carried out. Computational prediction of hot spot residues on the protein-protein interaction of TRAF6 and RANK were examined. Three methods were used: Robetta, KFC2, and HotPoint, each of which uses a different methodology to determine if a residue is a hot spot. These hot spot predictions were considered the basis for resolving the binding site for in silico high-throughput screening using GOLD and the MyriaScreen database of drug/lead-like compounds. Computationally intensive molecular dynamics simulations highlighted the binding mechanism and TRAF6 structural changes upon hit binding. Compounds identified as hits were verified using a GST-pull down assay, comparing inhibition to a RANK decoy peptide. Since many drugs fail due to lack of efficacy and toxicity, predictive models for the evaluation of the LD50 and bioavailability of our TRAF6 hits, and these models can be used towards other drugs and small molecule therapeutics as well. Datasets of compounds and their corresponding bioavailability and LD50 values were curated based, and QSAR models were built using molecular descriptors of these compounds using the k-nearest neighbor (k-NN) method, and quality of these models were cross-validated.

Regulation of myogenesis and suppression of {\it mck\/} 5$\sp\prime$ enhancer elements by TGF$\beta$ and RAS

The regulation of muscle differentiation, like cell differentiation in general, is only now beginning to be understood. Here are described several key features to myogenesis: a beginning, some intermediary events, and an endpoint. Muscle differentiation proceeds spontaneously when myoblasts are cultured in serum-poor medium. Transforming growth factor type $\beta$ (TGF$\beta$), a component of fetal serum, was found to potently suppress muscle differentiation. Prolonged blockade of differentiation required replenishing TGF$\beta$. When TGF$\beta$ was removed, cells rapidly differentiated. Both TGF$\beta$ and RAS, which also blocks myogenesis, suppress the genes for a series of muscle-specific proteins. Regions that regulate transcription of one such gene, muscle creatine kinase (mck), were located by linking progressively smaller parts of the mck 5$\sp\prime$ region to the marker gene cat and testing the constructs for regulated expression of cat in myoblasts and muscle cells. The mck promoter is not muscle-specific but requires activation. Two enhancers were found: a weak, developmentally regulated enhancer within the first intron, and a strong, compact, and tightly developmentally regulated enhancer about 1.2 Kb upstream of the transcription start site. Activity of this enhancer is eliminated by activated ras. Suppression of activated N-RAS restores potency to the upstream enhancer. Further deletion shows the mck 5$\sp\prime$ enhancer to contain an enhancer core with low but significant muscle-specific activity, and at least one peripheral element that augments core activity. The core and this peripheral element were comprised almost entirely of factor-binding motifs. The peripheral element was inactive as a single copy, but was constitutively active in multiple copies. Regions flanking the peripheral element augmented its activity and conferred partial muscle-specificity. The enhancer core is also modulated by its 5$\sp\prime$ flanking region in a complex manner. Site-specific mutants covering most of the enhancer core and interesting flanking sequences have been made; all mutants tested diminish the activity of the 5$\sp\prime$ enhancer. Alteration of the site to which MyoD1 is reported to bind completely inactivates the enhancer. A theoretical analysis of cooperativity is presented, through which the binding of a constitutively expressed nuclear factor is shown to have weak positive cooperativity. In summary, TGF$\beta$, RAS, and enhancer-binding factors are found to be initial, intermediary, and final regulators, respectively, of muscle differentiation. ^

Dysregulated CD40-NF-kappaB signaling in the pathophysiology of aggressive non -Hodgkin's lymphoma B cells

Non-Hodgkin's Lymphomas (NHL) are a group (>30) of important human lymphoid cancers that unlike other tumors today, are showing a marked increase in incidence. The lack of insight to the pathogenesis of B-cell NHL poses a significant problem in the early detection and effective treatment of these malignancies. This study shows that large B-cell lymphoma (LBCL) cells, the most common type of B-cell NHL (account for more than 30% of cases), have developed a novel mechanism for autonomous neoplastic B cell growth. We have identified that the key transcription factor NF-��B, is constitutively activated in LBCL cell lines and primary biopsy-derived LBCL cells, suggesting that they are autonomously activated, and do not require accessory T-cell signaling for cell growth and survival. Further studies have indicated that LBCL cells ectopically express an important T-cell associated co-mitogenic factor, CD154 (CD40 ligand), that is able to internally activate the CD401NF-��B pathway, through constitutive binding to its cognate receptor, CD40, on the lymphoma cell surface. CD40 activation triggers the formation of a ���Signalosome��� comprising virtually the entire canonical CD40/NF-��B signaling pathway that is anchored by CD40 in plasma membrane lipid rafts. The CD40 Signalosome is vulnerable to interdiction by antibody against CD40 that disrupts the Signalosome and induces cell death in the malignant cells. In addition to constitutive NF-��B activation, we have found that the nuclear factor of activated T cells (NFAT) transcription factor is also constitutively activated in LBCL cells. We have demonstrated that the constitutively active NFATc1 and c-rel members of the NFAT and NF-��B families of transcription factors, respectively, interact with each other, bind to the CD154 promoter, and synergistically activate CD154 gene transcription. Down-regulation of NFATc1 and c-rel with small interfering RNA inhibits CD154 gene transcription and lymphoma cell growth. Our findings suggest that continuous CD40 activation not only provides dysregulated proliferative stimuli for lymphoma cell growth and extended tumor cell survival, but also allows continuous regeneration of the CD40 ligand in the lymphoma cell and thereby recharges the system through a positive feedback mechanism. Targeting the CD40/NF-��B signaling pathway could provide potential therapeutic modalities for LBCL cells in the future. ^

Ubiquitin-dependent degradation of I��B�� is mediated by a ubiquitin ligase Skp1/Cul 1/F-box protein FWD1

Activation of the transcription factor nuclear factor kappa B (NF-��B) is controlled by proteolysis of its inhibitory subunit (I��B) via the ubiquitin-proteasome pathway. Signal-induced phosphorylation of I��B�� by a large multisubunit complex containing I��B kinases is a prerequisite for ubiquitination. Here, we show that FWD1 (a mouse homologue of Slimb/��TrCP), a member of the F-box/WD40-repeat proteins, is associated specifically with I��B�� only when I��B�� is phosphorylated. The introduction of FWD1 into cells significantly promotes ubiquitination and degradation of I��B�� in concert with I��B kinases, resulting in nuclear translocation of NF-��B. In addition, FWD1 strikingly evoked the ubiquitination of I��B�� in the in vitro system. In contrast, a dominant-negative form of FWD1 inhibits the ubiquitination, leading to stabilization of I��B��. These results suggest that the substrate-specific degradation of I��B�� is mediated by a Skp1/Cull 1/F-box protein (SCF) FWD1 ubiquitin-ligase complex and that FWD1 serves as an intracellular receptor for phosphorylated I��B��. Skp1/Cullin/F-box protein FWD1 might play a critical role in transcriptional regulation of NF-��B through control of I��B protein stability.

Derepression of human embryonic ��-globin promoter by a locus-control region sequence

A multiple protein���DNA complex formed at a human ��-globin locus-specific regulatory element, HS-40, confers appropriate developmental expression pattern on human embryonic ��-globin promoter activity in humans and transgenic mice. We show here that introduction of a 1-bp mutation in an NF-E2/AP1 sequence motif converts HS-40 into an erythroid-specific locus-control region. Cis-linkage with this locus-control region, in contrast to the wild-type HS-40, allows erythroid lineage-specific derepression of the silenced human ��-globin promoter in fetal and adult transgenic mice. Furthermore, ��-globin promoter activities in adult mice increase in proportion to the number of integrated DNA fragments even at 19 copies/genome. The mutant HS-40 in conjunction with human ��-globin promoter thus can be used to direct position-independent and copy number-dependent expression of transgenes in adult erythroid cells. The data also supports a model in which competitive DNA binding of different members of the NF-E2/AP1 transcription factor family modulates the developmental stage specificity of an erythroid enhancer. Feasibility to reswitch on embryonic/fetal globin genes through the manipulation of nuclear factor binding at a single regulatory DNA motif is discussed.

NF-��B and AP-1 Activation by Nitric Oxide Attenuated Apoptotic Cell Death in RAW 264.7 Macrophages

A toxic dose of the nitric oxide (NO) donor S-nitrosoglutathione (GSNO; 1 mM) promoted apoptotic cell death of RAW 264.7 macrophages, which was attenuated by cellular preactivation with a nontoxic dose of GSNO (200 ��M) or with lipopolysaccharide, interferon-��, and NG-monomethyl-l-arginine (LPS/IFN-��/NMMA) for 15 h. Protection from apoptosis was achieved by expression of cyclooxygenase-2 (Cox-2). Here we investigated the underlying mechanisms leading to Cox-2 expression. LPS/IFN-��/NMMA prestimulation activated nuclear factor (NF)-��B and promoted Cox-2 expression. Cox-2 induction by low-dose GSNO demanded activation of both NF-��B and activator protein-1 (AP-1). NF-��B supershift analysis implied an active p50/p65 heterodimer, and a luciferase reporter construct, containing four copies of the NF-��B site derived from the murine Cox-2 promoter, confirmed NF-��B activation after NO addition. An NF-��B decoy approach abrogated not only Cox-2 expression after low-dose NO or after LPS/IFN-��/NMMA but also inducible protection. The importance of AP-1 for Cox-2 expression and cell protection by low-level NO was substantiated by using the extracellular signal-regulated kinase inhibitor PD98059, blocking NO-elicited Cox-2 expression, but leaving the cytokine signal unaltered. Transient transfection of a dominant-negative c-Jun mutant further attenuated Cox-2 expression by low-level NO. Whereas cytokine-mediated Cox-2 induction relies on NF-��B activation, a low-level NO���elicited Cox-2 response required activation of both NF-��B and AP-1.

I��B Is a Substrate for a Selective Pathway of Lysosomal Proteolysis

In lysosomes isolated from rat liver and spleen, a percentage of the intracellular inhibitor of the nuclear factor �� B (I��B) can be detected in the lysosomal matrix where it is rapidly degraded. Levels of I��B are significantly higher in a lysosomal subpopulation that is active in the direct uptake of specific cytosolic proteins. I��B is directly transported into isolated lysosomes in a process that requires binding of I��B to the heat shock protein of 73 kDa (hsc73), the cytosolic molecular chaperone involved in this pathway, and to the lysosomal glycoprotein of 96 kDa (lgp96), the receptor protein in the lysosomal membrane. Other substrates for this degradation pathway competitively inhibit I��B uptake by lysosomes. Ubiquitination and phosphorylation of I��B are not required for its targeting to lysosomes. The lysosomal degradation of I��B is activated under conditions of nutrient deprivation. Thus, the half-life of a long-lived pool of I��B is 4.4 d in serum-supplemented Chinese hamster ovary cells but only 0.9 d in serum-deprived Chinese hamster ovary cells. This increase in I��B degradation can be completely blocked by lysosomal inhibitors. In Chinese hamster ovary cells exhibiting an increased activity of the hsc73-mediated lysosomal degradation pathway due to overexpression of lamp2, the human form of lgp96, the degradation of I��B is increased. There are both short- and long-lived pools of I��B, and it is the long-lived pool that is subjected to the selective lysosomal degradation pathway. In the presence of antioxidants, the half-life of the long-lived pool of I��B is significantly increased. Thus, the production of intracellular reactive oxygen species during serum starvation may be one of the mechanisms mediating I��B degradation in lysosomes. This selective pathway of lysosomal degradation of I��B is physiologically important since prolonged serum deprivation results in an increase in the nuclear activity of nuclear factor �� B. In addition, the response of nuclear factor �� B to several stimuli increases when this lysosomal pathway of proteolysis is activated.

Myogenic stem cell function is impaired in mice lacking the forkhead/winged helix protein MNF

Myocyte nuclear factor (MNF) is a winged helix transcription factor that is expressed selectively in myogenic stem cells (satellite cells) of adult animals. Using a gene knockout strategy to generate a functional null allele at the Mnf locus, we observed that mice lacking MNF are viable, but severely runted. Skeletal muscles of Mnf���/��� animals are atrophic, and satellite cell function is impaired. Muscle regeneration after injury is delayed and incomplete, and the normal timing of expression of cell cycle regulators and myogenic determination genes is dysregulated. Mnf mutant mice were intercrossed with mdx mice that lack dystrophin and exhibit only a subtle myopathic phenotype. In contrast, mdx mice that also lack MNF die in the first few weeks of life with a severe myopathy. Haploinsufficiency at the Mnf locus (Mnf+/���) also exacerbates the mdx phenotype to more closely resemble Duchenne's muscular dystrophy in humans. We conclude that MNF acts to regulate genes that coordinate the proliferation and differentiation of myogenic stem cells after muscle injury. Animals deficient in MNF may prove useful for evaluation of potential therapeutic interventions to promote muscle regeneration for patients having Duchenne's muscular dystrophy.

Type 2 diabetes: Evidence for linkage on chromosome 20 in 716 Finnish affected sib pairs

We are conducting a genome scan at an average resolution of 10 centimorgans (cM) for type 2 diabetes susceptibility genes in 716 affected sib pairs from 477 Finnish families. To date, our best evidence for linkage is on chromosome 20 with potentially separable peaks located on both the long and short arms. The unweighted multipoint maximum logarithm of odds score (MLS) was 3.08 on 20p (location, x�� = 19.5 cM) under an additive model, whereas the weighted MLS was 2.06 on 20q (x�� = 57 cM, recurrence risk, ����s = 1.25, P = 0.009). Weighted logarithm of odds scores of 2.00 (x�� = 69.5 cM, P = 0.010) and 1.92 (x�� = 18.5 cM, P = 0.013) were also observed. Ordered subset analyses based on sibships with extreme mean values of diabetes-related quantitative traits yielded sets of families who contributed disproportionately to the peaks. Two-hour glucose levels in offspring of diabetic individuals gave a MLS of 2.12 (P = 0.0018) at 9.5 cM. Evidence from this and other studies suggests at least two diabetes-susceptibility genes on chromosome 20. We have also screened the gene for maturity-onset diabetes of the young 1, hepatic nuclear factor 4-a (HNF-4��) in 64 affected sibships with evidence for high chromosomal sharing at its location on chromosome 20q. We found no evidence that sequence changes in this gene accounted for the linkage results we observed.

Development of a sensitive multi-well colorimetric assay for active NF��B

The transcription factor nuclear factor ��B (NF��B) is a key factor in the immune response triggered by a wide variety of molecules such as inflammatory cytokines, or some bacterial and viral products. This transcription factor represents a new target for the development of anti-inflammatory molecules, but this type of research is currently hampered by the lack of a convenient and rapid screening assay for NF��B activation. Indeed, NF��B DNA-binding capacity is traditionally estimated by radioactive gel shift assay. Here we propose a new DNA-binding assay based on the use of multi-well plates coated with a cold oligonucleotide containing the consensus binding site for NF��B. The presence of the DNA-bound transcription factor is then detected by anti-NF��B antibodies and revealed by colorimetry. This assay is easy to use, non-radioactive, highly reproducible, specific for NF��B, more sensitive than regular radioactive gel shift and very convenient for high throughput screening.

Targeted inhibition of calcineurin attenuates cardiac hypertrophy in vivo

The Ca2+-calmodulin-activated Ser/Thr protein phosphatase calcineurin and the downstream transcriptional effectors of calcineurin, nuclear factor of activated T cells, have been implicated in the hypertrophic response of the myocardium. Recently, the calcineurin inhibitory agents cyclosporine A and FK506 have been extensively used to evaluate the importance of this signaling pathway in rodent models of cardiac hypertrophy. However, pharmacologic approaches have rendered equivocal results necessitating more specific or genetic-based inhibitory strategies. In this regard, we have generated Tg mice expressing the calcineurin inhibitory domains of Cain/Cabin-1 and A-kinase anchoring protein 79 specifically in the heart. ��Cain and ��A-kinase-anchoring protein Tg mice demonstrated reduced cardiac calcineurin activity and reduced hypertrophy in response to catecholamine infusion or pressure overload. In a second approach, adenoviral-mediated gene transfer of ��Cain was performed in the adult rat myocardium to evaluate the effectiveness of an acute intervention and any potential species dependency. ��Cain adenoviral gene transfer inhibited cardiac calcineurin activity and reduced hypertrophy in response to pressure overload without reducing aortic pressure. These results provide genetic evidence implicating calcineurin as an important mediator of the cardiac hypertrophic response in vivo.

Regulation of the p21-activated kinase (PAK) by a human G��-like WD-repeat protein, hPIP1

The family of p21-activated protein kinases (PAKs) is composed of serine���threonine kinases whose activity is regulated by the small guanosine triphosphatases (GTPases) Rac and Cdc42. In mammalian cells, PAKs have been implicated in the regulation of mitogen-activated protein cascades, cellular morphological and cytoskeletal changes, neurite outgrowth, and cell apoptosis. Although the ability of Cdc42 and Rac GTPases to activate PAK is well established, relatively little is known about the negative regulation of PAK or the identity of PAK cellular targets. Here, we describe the identification and characterization of a human PAK-interacting protein, hPIP1. hPIP1 contains G protein ��-like WD repeats and shares sequence homology with the essential fission yeast PAK regulator, Skb15, as well as the essential budding yeast protein, MAK11. Interaction of hPIP1 with PAK1 inhibits the Cdc42/Rac-stimulated kinase activity through the N-terminal regulatory domains of PAK1. Cotransfection of hPIP1 in mammalian cells inhibits PAK-mediated c-Jun N-terminal kinase and nuclear factor �� B signaling pathways. Our results demonstrate that hPIP1 is a negative regulator of PAK and PAK signaling pathways.

Regulation of Myosin Heavy Chain Expression during Rat Skeletal Muscle Development In Vitro

Signals that determine fast- and slow-twitch phenotypes of skeletal muscle fibers are thought to stem from depolarization, with concomitant contraction and activation of calcium-dependent pathways. We examined the roles of contraction and activation of calcineurin (CN) in regulation of slow and fast myosin heavy chain (MHC) protein expression during muscle fiber formation in vitro. Myotubes formed from embryonic day 21 rat myoblasts contracted spontaneously, and ���10% expressed slow MHC after 12 d in culture, as seen by immunofluorescent staining. Transfection with a constitutively active form of calcineurin (CN*) increased slow MHC by 2.5-fold as determined by Western blot. This effect was attenuated 35% by treatment with tetrodotoxin and 90% by administration of the selective inhibitor of CN, cyclosporin A. Conversely, cyclosporin A alone increased fast MHC by twofold. Cotransfection with VIVIT, a peptide that selectively inhibits calcineurin-induced activation of the nuclear factor of activated T-cells, blocked the effect of CN* on slow MHC by 70% but had no effect on fast MHC. The results suggest that contractile activity-dependent expression of slow MHC is mediated largely through the CN���nuclear factor of activated T-cells pathway, whereas suppression of fast MHC expression may be independent of nuclear factor of activated T-cells.

Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF-��B

The human inducible nitric oxide synthase (hiNOS) gene is expressed in several disease states and is also important in the normal immune response. Previously, we described a cytokine-responsive enhancer between ���5.2 and ���6.1 kb in the 5���-flanking hiNOS promoter DNA, which contains multiple nuclear factor ���� (NF-��B) elements. Here, we describe the role of the IFN-Jak kinase-Stat (signal transducer and activator of transcription) 1 pathway for regulation of hiNOS gene transcription. In A549 human lung epithelial cells, a combination of cytokines tumor necrosis factor-��, interleukin-1��, and IFN-�� (TNF-��, IL-1��, and IFN-��) function synergistically for induction of hiNOS transcription. Pharmacological inhibitors of Jak2 kinase inhibit cytokine-induced Stat 1 DNA-binding and hiNOS gene expression. Expression of a dominant-negative mutant Stat 1 inhibits cytokine-induced hiNOS reporter expression. Site-directed mutagenesis of a cis-acting DNA element at ���5.8 kb in the hiNOS promoter identifies a bifunctional NF-��B/Stat 1 motif. In contrast, gel shift assays indicate that only Stat 1 binds to the DNA element at ���5.2 kb in the hiNOS promoter. Interestingly, Stat 1 is repressive to basal and stimulated iNOS mRNA expression in 2fTGH human fibroblasts, which are refractory to iNOS induction. Overexpression of NF-��B activates hiNOS promoter���reporter expression in Stat 1 mutant fibroblasts, but not in the wild type, suggesting that Stat 1 inhibits NF-��B function in these cells. These results indicate that both Stat 1 and NF-��B are important in the regulation of hiNOS transcription by cytokines in a complex and cell type-specific manner.

Cross talk between cell death and cell cycle progression: BCL-2 regulates NFAT-mediated activation.

BCL-2-deficient T cells demonstrate accelerated cell cycle progression and increased apoptosis following activation. Increasing the levels of BCL-2 retarded the G0-->S transition, sustained the levels of cyclin-dependent kinase inhibitor p27Kip1, and repressed postactivation death. Proximal signal transduction events and immediate early gene transcription were unaffected. However, the transcription and synthesis of interleukin 2 and other delayed early cytokines were markedly attenuated by BCL-2. In contrast, a cysteine protease inhibitor that also blocks apoptosis had no substantial affect upon cytokine production. InterleUkin 2 expression requires several transcription factors of which nuclear translocation of NFAT (nuclear factor of activated T cells) and NFAT-mediated transactivation were impaired by BCL-2. Thus, select genetic aberrations in the apoptotic pathway reveal a cell autonomous coregulation of activation.