06-413 Sigma-AldrichAnti-NFκB p52 Antibody

The molecular interactions between B-cell precursor acute lymphoblastic leukemia (pre-B ALL) cells and stromal cells in the bone marrow that provide microenvironmentally-mediated protection against therapeutic drugs are not well-defined. Galectin-3 (Lgals3) is a multifunctional galactose-binding lectin with reported location in the nucleus, cytoplasm and extracellular space in different cell types. We previously reported that ALL cells co-cultured with stroma contain high levels of Galectin-3. We here establish that, in contrast to more mature B-lineage cancers, Galectin-3 detected in and on the ALL cells originates from stromal cells, which express it on their surface, secrete it as soluble protein and also in exosomes. Soluble and stromal-bound Galectin-3 is internalized by ALL cells, transported to the nucleus and stimulates transcription of endogenous LGALS3 mRNA. When human and mouse ALL cells develop tolerance to different drugs while in contact with protective stromal cells, Galectin-3 protein levels are consistently increased. This correlates with induction of Galectin-3 transcription in the ALL cells. Thus Galectin-3 sourced from stroma becomes supplemented by endogenous Galectin-3 production in the pre-B ALL cells that are under continuous stress from drug treatment. Our data suggest that stromal Galectin-3 may protect ALL cells through auto-induction of Galectin-3 mRNA and tonic NFκB pathway activation. Since endogenously synthesized Galectin-3 protects pre-B ALL cells against drug treatment, we identify Galectin-3 as one possible target to counteract the protective effects of stroma.

Ovarian cancer is one of the leading causes of female death and the development of novel therapeutic approaches is urgently required. Nuclear factor-κB (NF-κB) is constitutively activated in several types of cancer including ovarian cancer and is known to support the survival of cancer cells. However, molecular mechanisms of persistent activation of NF-κB in ovarian cancer remain largely unknown. We report here that, in addition to the previously reported canonical activation, NF-κB is activated through the noncanonical pathway in ovarian cancer cells. RNA interference-mediated silencing of NF-κB inducing kinase (NIK), a central regulator of the noncanonical pathway, reduced the NF-κB2/p52 DNA binding activity and NF-κB-dependent reporter gene expression as well as NF-κB target gene expression. Notably, anchorage-dependent and -independent cell growth was impaired in NIK-depleted cells. Depletion of NIK also suppressed tumor formation in the nude mouse xenograft assay. These results indicate that NIK plays a key role in constitutive NF-κB activation and the progression of ovarian cancer cells and suggest that NIK represents an attractive therapeutic target for ovarian cancer.

Dysregulation of Maspin expression and constitutive activation of NF-κB subunits are important events in tumorigenesis of prostate cancer. Recent finding points that RelB, which contributes to the alternative NF-κB activity, interferes with carcinogenesis in the prostate. We report here, that both the classical and the alternative NF-κB activities are constitutively present in androgen-insensitive human prostate cancer cells. Maspin and RelB expression is correlated negatively in prostate cancer tissues at the later stage. TNF-α signaling triggers the nuclear accumulation of RelB and the concomitant reduction of Maspin expression in a time-dependent manner. In addition, the proteasome inhibitor-induced Maspin expression is accompanied by the reduction of RelB expression. A successful depletion of RelB expression, but not RelA expression, induces Maspin expression. RelB-deficiency abrogates the proteasome inhibitor-induced Maspin expression. Moreover, we demonstrate that the enforced expression of RelB protein in prostate cancer cells inhibits Maspin expression. We propose that RelB is an essential molecule controlling the endogenous and the proteasome inhibitor-induced Maspin expression. Developing a RelB-targeted therapeutic intervention, which might be coupled with the induction of a tumor suppressor Maspin, is valuable in treating advanced, metastatic prostate cancer.Copyright © 2011 Elsevier Ltd. All rights reserved.

Proteolytic processing of the nuclear factor (NF)-kappaB2 precursor protein p100 generates the active NF-kappaB2 subunit p52, which in turn transcriptionally up-regulates p100 expression. p100 also functions as an IkappaB molecule capable of repressing p52 activity. The biological significance of this negative feedback control loop has yet to be demonstrated in vivo. Here we show that mice deficient in p100 but with constitutive expression of p52 in lymphocytes developed fatal lung inflammation characterized by diffuse alveolar damage with marked peribronchial fibrosis. In contrast, their littermates with only p100 deficiency or constitutive expression of p52 in lymphocytes developed mild lung inflammation with perivascular lymphocyte infiltration and had a normal life span. The fatal lung inflammation is associated with high-level induction of interferon-gamma and its inducible inflammatory chemokines, suggesting the involvement of a T-helper-1 immune response. These findings demonstrate the physiological relevance of the NF-kappaB2 p100 precursor protein in limiting the potentially detrimental effects of constitutive NF-kappaB2 signaling in lymphocytes.

The expression of interferon-β (IFN-β) in virus-infected HeLa cells established a paradigm of multifactorial gene regulation, in which cooperative assembly of transcription factors (TFs) at the composite DNA element (enhanceosome), is central for amplification of weak activating signals provided by individual TFs. However, whether the same TFs and the same DNA element are essential for IFN-β induction in response to bacterial stimuli are less well understood. Here we report that rapid and transient transcription of IFN-β in response to TLR4 stimulation with bacterial lipopolysaccharide (LPS) follows nuclear factor-κB (NF-κB) RelA activation and recruitment to the IFN-β genomic locus at multiple spatially separated regulatory regions. We demonstrate that the IFN-β enhanceosome region is not sufficient for maximal gene induction in response to LPS and identify an essential cluster of homotypic κB sites in the 3' downstream of the gene. The cluster is characterized by elevated levels of histone 3 lysine 4 mono-methylation, a chromatin signature of enhancers, and efficiently binds RelA-containing NF-κB complexes in vitro and in vivo. These findings demonstrate that IFN-β gene activation via multifactorial enhanceosome assembly is potentiated in LPS-stimulated cells by NF-κB interactions with all functional κB sites in the locus.

GSK-3 is active in the absence of growth factor stimulation and generally acts to induce apoptosis or inhibit cell proliferation. We previously identified a subset of growth factor-inducible genes that can also be induced in quiescent T98G cells solely by inhibition of GSK-3 in the absence of growth factor stimulation. Computational predictions verified by chromatin immunoprecipitation assays identified NF-kappaB binding sites in the upstream regions of 75% of the genes regulated by GSK-3. p50 bound to most of these sites in quiescent cells, and for one-third of the genes, binding of p65 to the predicted sites increased upon inhibition of GSK-3. The functional role of p65 in gene induction following inhibition of GSK-3 was demonstrated by RNA interference experiments. Furthermore, inhibition of GSK-3 in quiescent cells resulted in activation of IkappaB kinase, leading to phosphorylation and degradation of IkappaB alpha and nuclear translocation of p65 and p50. Taken together, these results indicate that the high levels of GSK-3 activity in quiescent cells repress gene expression by negatively regulating NF-kappaB through inhibition of IkappaB kinase. This inhibition of NF-kappaB is consistent with the role of GSK-3 in the induction of apoptosis or cell cycle arrest in cells deprived of growth factors.

The proteasome inhibitor bortezomib can inhibit activation of the transcription factor NF-kappaB, a mechanism implicated in its anti-neoplastic effects observed in mantle cell lymphoma (MCL). However, NF-kappaB can be activated through many distinct mechanisms, including proteasome independent pathways. While MCL cells have been shown to harbor constitutive NF-kappaB activity, what fraction of this activity in primary MCL samples is sensitive or resistant to inhibition by bortezomib remains unclear.Proteasome activity in the EBV-negative MCL cell lines Jeko-1 and Rec-1 is inhibited by greater than 80% after exposure to 20 nM bortezomib for 4 hours. This treatment decreased NF-kappaB activity in Jeko-1 cells, but failed to do so in Rec-1 cells when assessed by electrophoretic mobility shift assay (EMSA). Concurrently, Rec-1 cells were more resistant to the cytotoxic effects of bortezomib than Jeko-1 cells. Consistent with a proteasome inhibitor resistant pathway of activation described in mouse B-lymphoma cells (WEHI231) and a breast carcinoma cell line (MDA-MB-468), the bortezomib-resistant NF-kappaB activity in Rec-1 cells is inhibited by calcium chelators, calmodulin inhibitors, and perillyl alcohol, a monoterpene capable of blocking L-type calcium channels. Importantly, the combination of perillyl alcohol and bortezomib is synergistic in eliciting Rec-1 cell cytotoxicity. The relevance of these results is illuminated by the additional finding that a considerable fraction of primary MCL samples (8 out of 10) displayed bortezomib-resistant constitutive NF-kappaB activity.Our findings show that bortezomib-resistant NF-kappaB activity is frequently observed in MCL samples and suggest that this activity may be relevant to MCL biology as well as serve as a potential therapeutic target.

Phosphatidylinositol (PI) 3-kinase is activated by a variety of growth factor receptors and the PI 3-kinase/Akt signaling pathway is a key regulator of cell proliferation and survival. The downstream targets of PI 3-kinase/Akt signaling include direct regulators of cell cycle progression and apoptosis as well as a number of transcription factors. Growth factor stimulation of quiescent cells leads to robust activation of PI 3-kinase, induction of immediate-early genes, and re-entry into the cell cycle. A lower level of PI 3-kinase signaling is also required for the proliferation and survival of cells maintained in the presence of growth factors, but the gene expression program controlled by PI 3-kinase signaling in proliferating cells has not been elucidated.We used microarray analyses to characterize the changes in gene expression resulting from inhibition of PI 3-kinase in proliferating cells. The genes regulated by inhibition of PI 3-kinase in proliferating cells were distinct from genes induced by growth factor stimulation of quiescent cells and highly enriched in genes that regulate programmed cell death. Computational analyses followed by chromatin immunoprecipitations demonstrated FOXO binding to both previously known and novel sites in promoter regions of approximately one-third of the up-regulated genes, consistent with activation of FOXO1 and FOXO3a in response to inhibition of PI 3-kinase. NFkappaB binding sites were similarly identified in promoter regions of over one-third of the down-regulated genes. RelB was constitutively bound to promoter regions in cells maintained in serum, however binding decreased following PI 3-kinase inhibition, indicating that PI 3-kinase signaling activates NFkappaB via the non-canonical pathway in proliferating cells. Approximately 70% of the genes targeted by FOXO and NFkappaB regulate cell proliferation and apoptosis, including several regulators of apoptosis that were not previously known to be targeted by these transcription factors.PI 3-kinase signaling in proliferating cells regulates a novel transcriptional program that is highly enriched in genes that regulate apoptosis. At least one-third of these genes are regulated either by FOXO transcription factors, which are activated following PI 3-kinase inhibition, or by RelB, which is activated by PI 3-kinase via the non-canonical pathway in proliferating cells.

Normal development of the immune system requires regulated processing of NF-kappaB2 p100 to p52, which activates NF-kappaB2 signaling. Constitutive production of p52 has been suggested as a major mechanism underlying lymphomagenesis induced by NF-kappaB2 mutations, which occur recurrently in a variety of human lymphoid malignancies. To test the hypothesis, we generated transgenic mice with targeted expression of p52 in lymphocytes. In contrast to their counterparts expressing the tumor-derived NF-kappaB2 mutant p80HT, which develop predominantly B cell tumors, p52 transgenic mice are not prone to lymphomagenesis. However, they are predisposed to inflammatory autoimmune disease characterized by multiorgan infiltration of activated lymphocytes, high levels of autoantibodies in the serum, and immune complex glomerulonephritis. p52, but not p80HT, represses Bim expression, leading to defects in apoptotic processes critical for elimination of autoreactive lymphocytes and control of immune response. These findings reveal distinct signaling pathways for actions of NF-kappaB2 mutants and p52 and suggest a causal role for sustained NF-kappaB2 activation in the pathogenesis of autoimmunity.

Bortezomib (Velcade/PS341), a proteasome inhibitor used in the treatment of multiple myeloma (MM), can inhibit activation of nuclear factor-kappaB (NF-kappaB), a family of transcription factors often deregulated and constitutively activated in primary MM cells. NF-kappaB can be activated via several distinct mechanisms, including the proteasome inhibitor-resistant (PIR) pathway. It remains unknown what fraction of primary MM cells harbor constitutive NF-kappaB activity maintained by proteasome-dependent mechanisms. Here, we report an unexpected finding that constitutive NF-kappaB activity in 10 of 14 primary MM samples analyzed is refractory to inhibition by bortezomib. Moreover, when MM cells were cocultured with MM patient-derived bone marrow stromal cells (BMSC), microenvironment components critical for MM growth and survival, further increases in NF-kappaB activity were observed that were also refractory to bortezomib. Similarly, MM-BMSCs caused PIR NF-kappaB activation in the RPMI8226 MM cell line, leading to increased NF-kappaB-dependent transcription and resistance to bortezomib-induced apoptosis. Our findings show that primary MM cells frequently harbor PIR NF-kappaB activity that is further enhanced by the presence of patient-derived BMSCs. They also suggest that this activity is likely relevant to the drug resistance development in some patients. Further elucidation of the mechanism of PIR NF-kappaB regulation could lead to the identification of novel diagnostic biomarkers and/or therapeutic targets for MM treatment.