When activated, NF-B, a ubiquitous transcription factor, binds DNA as a heterodimeric complex composed of members of the Rel/NF-B family of polypeptides. Because of its intimate involvement in host defense against disease, this transcription factor is an important target for therapeutic intervention. In the present report we demonstrate that curcumin (diferuloylmethane), a known anti-inflammatory and anticarcinogenic agent, is a potent inhibitor of NF-B activation. Treatment of human myeloid ML-1a cells with tumor necrosis factor (TNF) rapidly activated NF-B, which consists of p50 and p65 subunits, and this activation was inhibited by curcumin. AP-1 binding factors were also found to be down-modulated by curcumin, whereas the Sp1 binding factor was unaffected.Besides TNF, curcumin also blocked phorbol esterand hydrogen peroxide-mediated activation of NF-B. The TNF-dependent phosphorylation and degradation of IB␣ was not observed in curcumin-treated cells; the translocation of p65 subunit to the nucleus was inhibited at the same time. The mechanism of action of curcumin was found to be different from that of protein tyrosine phosphatase inhibitors. Our results indicate that curcumin inhibits NF-B activation pathway at a step before IB␣ phosphorylation but after the convergence of various stimuli.

Caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives, is known to have antimitogenic, anticarcinogenic, antiinflammatory, and immunomodulatory properties. The molecular basis for these diverse properties is not known. Since the role of the nuclear factor NF-cB in these responses has been documented, we examined the effect of CAPE on this transcription factor. Our results show that the activation of NF-KcB by tumor necrosis factor (TNF) is completely blocked by CAPE in a dose-and time-dependent manner. Besides TNF, CAPE also inhibited NF-cB activation induced by other inflammatory agents including phorbol ester, ceramide, hydrogen peroxide, and okadaic acid. Since the reducing agents reversed the inhibitory effect of CAPE, it suggests the role of critical sulfhydryl groups in NF-KcB activation. CAPE prevented the translocation of the p65 subunit of NF-cB to the nucleus and had no significant effect on TNF-induced IKcBa degradation, but did delay IucBa resynthesis. The effect of CAPE on inhibition of NF-cB binding to the DNA was specific, in as much as binding of other transcription factors including AP-1, Oct-i, and TFIID to their DNA were not affected. When various synthetic structural analogues of CAPE were examined, it was found that a bicyclic, rotationally constrained, 5,6-dihydroxy form was superactive, whereas 6,7-dihydroxy variant was least active. Thus, overall our results demonstrate that CAPE is a potent and a specific inhibitor of NF-KcB activation and this may provide the molecular basis for its multiple immunomodulatory and antiinflammatory activities.

The Notch pathway regulates the development of many tissues and cell types and is involved in a variety of human diseases, making it an attractive potential therapeutic target. This promise has been limited by the absence of potent inhibitors or agonists that are specific for individual human Notch receptors (NOTCH1-4). Using an unbiased functional screening, we identified monoclonal antibodies that specifically inhibit or induce activating proteolytic cleavages in NOTCH3. Remarkably, the most potent inhibitory and activating antibodies bind to overlapping epitopes within a juxtamembrane negative regulatory region that protects NOTCH3 from proteolysis and activation in its resting autoinhibited state. The inhibitory antibodies revert phenotypes conveyed on 293T cells by NOTCH3 signaling, such as increased cellular proliferation, survival, and motility, whereas the activating antibody mimics some of the effects of ligand-induced Notch activation. These findings provide insights into the mechanisms of Notch autoinhibition and activation and pave the way for the further development of specific antibody-based modulators of the Notch receptors, which are likely to be of utility in a wide range of experimental and therapeutic settings.

The transcription factor NF-B is retained in the cytoplasm by its interaction with the inhibitory subunit known as IB. Signal-induced serine phosphorylation and subsequent ubiquitination of IB␣ target it for degradation by the 26 S proteasome. Recently, pervanadate, a protein-tyrosine phosphatase inhibitor, was shown to block the degradation of IB␣, thus inhibiting NF-B activation. We investigated the mechanism by which pervanadate inhibits the degradation of IB␣. Western blot analysis of IB␣ from tumor necrosis factor-treated cells revealed a slower migrating IB␣ species that was subsequently degraded. However, pervanadate-treated cells also revealed a slower migrating species of IB␣ that appeared in a time-and dose-dependent manner and was not degraded by tumor necrosis factor. The slower migrating species of IB␣ from pervanadatetreated cells was tyrosine-phosphorylated as revealed by cross-reactivity with anti-phosphotyrosine antibodies, by the ability of the specific tyrosine phosphatase PTP1B to dephosphorylate it, and by phosphoamino acid analysis of IB␣ immunoprecipitated from 32 P-labeled cells. By site-specific mutagenesis and deletion analysis, we identified Tyr-42 on IB␣ as the phosphoacceptor site. Furthermore, in an in vitro reconstitution system, tyrosine-phosphorylated IB␣ was protected from degradation. Our results demonstrate that inducible phosphorylation and degradation of IB␣ are negatively regulated by phosphorylation at Tyr-42, thus preventing NF-B activation.The transcription factor NF-B regulates the expression of many genes that play essential roles in immune and inflammatory responses including the type I human immunodeficiency virus (1-4). Like all members of the Rel/NF-B transcription factor family, NF-B has the unique property of being sequestered in its inactive state in the cytoplasm by a noncovalent association with inhibitory proteins called IB (4). In mammalian species, at least seven structural homologs of IB have been identified (4), but only the IB␣ form has been extensively studied. Recently, IB␣-deficient mice have been generated; they exhibit constitutive NF-B activation, severe runting, dermatitis, extensive granulopoiesis, and neonatal death (5, 6). However, tumor necrosis factor (TNF), 1 which initiates the degradation of IB␣, causes sustained NF-B activation in embryonic fibroblasts from these knockout mice, suggesting that IB␣ is necessary for the postinduction repression of NF-B activity (5).Indeed, IB␣ controls the activation of NF-B by masking the nuclear localization signal located on the p50-p65 heterodimer of NF-B (7). In response to a wide variety of stimuli besides TNF, IB␣ undergoes degradation, allowing the p50-p65 heterodimer to migrate to the nucleus (8, 9). Since protein synthesis is not required for activation of this transcription factor, induction of target genes can occur within minutes of extracellular stimulus.Site-specific mutagenesis and peptide mapping have revealed that inducible phosphorylation of IB␣ occurs at both serines 32 and 36 (10 -12). ...

Most of the inflammatory and proviral effects of tumor necrosis factor (TNF) are mediated through the activation of the nuclear transcription factor NF-kappa B. How TNF activates NF-kappa B, however, is not well understood. We examined the role of protein phosphatases in the TNF-dependent activation of NF-kappa B. Treatment of human myeloid ML-1a cells with TNF rapidly activated (within 30 min) NF-kappa B; this effect was abolished by treating cells with inhibitors of protein-tyrosine phosphatase (PTPase), including phenylarsine oxide (PAO), pervanadate, and diamide. The inhibition was dependent on the dose and occurred whether added before or at the same time as TNF. PAO also inhibited the activation even when added 15 min after the TNF treatment of cells. In contrast to inhibitors of PTPase, okadaic acid and calyculin A, which block serine-threonine phosphatase, had no effect. The effect of PTPase inhibitors was not due to the modulation of TNF receptors. Since both dithiothreitol and dimercaptopropanol reversed the inhibitory effect of PAO, critical sulfhydryl groups in the PTPase must be involved in NF-kappa B activation by TNF. PTPase inhibitors also blocked NF-kappa B activation induced by phorbol ester, ceramide, and interleukin-1 but not that activated by okadaic acid. The degradation of I kappa B protein, a critical step in NF-kappa B activation, was also abolished by the PTPase inhibitors as revealed by immunoblotting. Thus, overall, we demonstrate that PTPase is involved either directly or indirectly in the pathway leading to the activation of NF-kappa B.

Recent cloning of the cDNA for Fas/Apo-1 and its ligand has revealed that they belong to the tumor necrosis factor (TNF) receptor and TNF family, respectively, and play an important role in apoptosis (programmed cell death). Like TNF, antibodies against the Fas antigen (anti-Fas) have been shown to be cytotoxic to Fas-expressing cells. Whether Fas, like TNF receptor, also mediates proliferation of normal human diploid fibroblasts (HDF), is not known. In this study, we show that HDF expresses Fas antigen and the engagement of this antigen signals proliferation of these cells in a dose-dependent manner. Unlike TNF receptor, however, Fas-mediated proliferation of HDF could not be blocked by orthovanadate, a tyrosine phosphatase inhibitor. The difference in the signaling was further evident from our observation that TNF induced the expression of interleukin-6 but anti-Fas did not. Overall, our results demonstrate for the first time that besides cell killing, Fas also mediates proliferation of HDF and that its mechanism is different from that of TNF receptor.

Financial support: All authors are paid employees of the Janssen Pharmaceuticals, Johnsons & Johnson Group of companies and receiving salary and other compensation.

PAX6 is a transcription activator that regulates eye development in animals ranging from Drosophila to human. The C-terminal region of PAX6 is proline/serine/ threonine-rich (PST) and functions as a potent transactivation domain when attached to a heterologous DNAbinding domain of the yeast transcription factor, GAL4. The PST region comprises 152 amino acids encoded by four exons. The transactivation function of the PST region has not been defined and characterized in detail by in vitro mutagenesis. We dissected the PST domain in two independent systems, a heterologous system using a GAL4 DNA-binding site and the native system of PAX6. Our data consistently showed that in both systems all four constituent exons of the PST domain are responsible for the transactivation function. The four exon fragments act synergistically to stimulate transcription, although none of them can function individually as an independent transactivation domain. Combinations of two or more exon fragments can reconstitute substantial transactivation activity when fused to the DNAbinding domain of GAL4, but they surprisingly do not produce much activity in the context of native PAX6, although the mutant PAX6 proteins are stable and their DNA-binding function remains unaffected. Our data suggest that these mutants may antagonize the wildtype PAX6 activity by competing for target DNA-binding sites. We conclude that the PAX6 protein contains an unusually large transactivation domain that is evolutionarily conserved to a high degree and that its full transactivation activity relies on the synergistic action of the four exon fragments.