Targeted Lymphoma Therapy Using a Gold Nanoframework-Based Drug Delivery System.

Precision nanomedicine can be employed as an alternative to chemo- or radiotherapy to overcome challenges associated with the often narrow therapeutic window of traditional treatment approaches, while safely inducing effective, targeted antitumor responses. Herein, we report the formulation of a therapeutic nanocomposite comprising a hyaluronic acid (HA)-coated gold nanoframework (AuNF) delivery system and encapsulated IT848, a small molecule with potent antilymphoma and -myeloma properties that targets the transcriptional activity of nuclear factor kappa B (NF-κB). The porous AuNFs fabricated via a liposome-templated approach were loaded with IT848 and surface-functionalized with HA to formulate the nanotherapeutics that were able to efficiently deliver the payload with high specificity to myeloma and lymphoma cell lines in vitro. In vivo studies characterized biodistribution, pharmacokinetics, and safety of HA-AuNFs, and we demonstrated superior efficacy of HA-AuNF-formulated IT848 vs free IT848 in lymphoma mouse models. Both in vitro and in vivo results affirm that the AuNF system can be adopted for targeted cancer therapy, improving the drug safety profile, and enhancing its efficacy with minimal dosing. HA-AuNF-formulated IT848 therefore has strong potential for clinical translation.

Inhibition of NF-κB DNA Binding Suppresses Myeloma Growth via Intracellular Redox and Tumor Microenvironment Modulation.

Multiple myeloma is a plasma cell malignancy that is still largely incurable, despite considerable progress in recent years. NF-κB is a well-established therapeutic target in multiple myeloma, but none of the currently available treatment options offer direct, specific pharmacologic targeting of NF-κB transcriptional activity. Thus, we designed a novel direct NF-κB inhibitor (IT848) as a drug candidate with strong potential for clinical translation and conducted comprehensive in vitro and in vivo mechanistic studies in multiple myeloma cell lines, primary multiple myeloma cells, xenograft models, and immunocompetent mouse models of multiple myeloma. Here, we show that IT848 inhibits NF-κB activity through inhibition of DNA binding of all five NF-κB subunits. IT848 treatment of multiple myeloma cell lines and patient samples inhibited proliferation and induced caspase-dependent and independent apoptosis. In addition to direct NF-κB inhibitory effects, IT848 treatment altered the redox homeostasis of multiple myeloma cells through depletion of the reduced glutathione pool, selectively inducing oxidative stress in multiple myeloma but not in healthy cells. Multiple myeloma xenograft studies confirmed the efficacy of IT848 as single agent and in combination with bortezomib. Furthermore, IT848 significantly improved survival when combined with programmed death protein 1 inhibition, and correlative immune studies revealed that this clinical benefit was associated with suppression of regulatory T-cell infiltration of the bone marrow microenvironment. In conclusion, IT848 is a potent direct NF-κB inhibitor and inducer of oxidative stress specifically in tumor cells, displaying significant activity against multiple myeloma cells in vitro and in vivo, both as monotherapy as well as in combination with bortezomib or immune checkpoint blockade.

Targeting metabolism and survival in chronic lymphocytic leukemia and Richter syndrome cells by a novel NF-κB inhibitor.

IT-901 is a novel and selective NF-κB inhibitor with promising activity in pre-clinical models. Here we show that treatment of chronic lymphocytic leukemia cells (CLL) with IT-901 effectively interrupts NF-κB transcriptional activity. CLL cells exposed to the drug display elevated mitochondrial reactive oxygen species, which damage mitochondria, limit oxidative phosphorylation and ATP production, and activate intrinsic apoptosis. Inhibition of NF-κB signaling in stromal and myeloid cells, both tumor-supportive elements, fails to induce apoptosis, but impairs NF-κB-driven expression of molecules involved in cell-cell contacts and immune responses, essential elements in creating a pro-leukemic niche. The consequence is that accessory cells do not protect CLL cells from IT-901-induced apoptosis. In this context, IT-901 shows synergistic activity with ibrutinib, arguing in favor of combination strategies. IT-901 is also effective in primary cells from patients with Richter syndrome (RS). Its anti-tumor properties are confirmed in xenograft models of CLL and in RS patient-derived xenografts, with documented NF-κB inhibition and significant reduction of tumor burden. Together, these results provide pre-clinical proof of principle for IT-901 as a potential new drug in CLL and RS.

Characterization of a c-Rel Inhibitor That Mediates Anticancer Properties in Hematologic Malignancies by Blocking NF-κB-Controlled Oxidative Stress Responses.

NF-κB plays a variety of roles in oncogenesis and immunity that may be beneficial for therapeutic targeting, but strategies to selectively inhibit NF-κB to exert antitumor activity have been elusive. Here, we describe IT-901, a bioactive naphthalenethiobarbiturate derivative that potently inhibits the NF-κB subunit c-Rel. IT-901 suppressed graft-versus-host disease while preserving graft-versus-lymphoma activity during allogeneic transplantation. Further preclinical assessment of IT-901 for the treatment of human B-cell lymphoma revealed antitumor properties in vitro and in vivo without restriction to NF-κB-dependent lymphoma. This nondiscriminatory, antilymphoma effect was attributed to modulation of the redox homeostasis in lymphoma cells resulting in oxidative stress. Moreover, NF-κB inhibition by IT-901 resulted in reduced stimulation of the oxidative stress response gene heme oxygenase-1, and we demonstrated that NF-κB inhibition exacerbated oxidative stress induction to inhibit growth of lymphoma cells. Notably, IT-901 did not elicit increased levels of reactive oxygen species in normal leukocytes, illustrating its cancer selective properties. Taken together, our results provide mechanistic insight and preclinical proof of concept for IT-901 as a novel therapeutic agent to treat human lymphoid tumors and ameliorate graft-versus-host disease.

The NFKB Inducing Kinase Modulates Hematopoiesis During Stress.

The genetic programs that maintain hematopoiesis during steady state in physiologic conditions are different from those activated during stress. Here, we show that hematopoietic stem cells (HSCs) with deficiencies in components of the alternative NFκB pathway (the NFκB inducing kinase, NIK, and the downstream molecule NFκB2) had a defect in response to stressors such as supraphysiological doses of cytokines, chemotherapy, and hematopoietic transplantation. NIK-deficient mice had peripheral blood and bone marrow leukocyte numbers within normal ranges (except for the already reported defects in B-cell maturation); however, HSCs showed significantly slower expansion capacity in in vitro cultures compared to wild-type HSCs. This was due to a delayed cell cycle and increased apoptosis. In vivo experiments showed that NIK-deficient HSCs did not recover at the same pace as controls when challenged with myeloablative chemotherapy. Finally, NIK-deficient HSCs showed a significantly decreased competitive repopulation capacity in vivo. Using HSCs from mice deficient in one of two downstream targets of NIK, that is, either NFκB2 or c-Rel, only NFκB2 deficiency recapitulated the defects detected with NIK-deficient HSCs. Our results underscore the role of NIK and the alternative NFκB pathway for the recovery of normal levels of hematopoiesis after stress.

A small-molecule c-Rel inhibitor reduces alloactivation of T cells without compromising antitumor activity.

Preventing unfavorable GVHD without inducing broad suppression of the immune system presents a major challenge of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We developed a novel strategy to ameliorate GVHD while preserving graft-versus-tumor (GVT) activity by small molecule-based inhibition of the NF-κB family member c-Rel. Underlying mechanisms included reduced alloactivation, defective gut homing, and impaired negative feedback on interleukin (IL)-2 production, resulting in optimal IL-2 levels, which, in the absence of competition by effector T cells, translated into expansion of regulatory T cells. c-Rel activity was dispensable for antigen-specific T-cell receptor (TCR) activation, allowing c-Rel-deficient T cells to display normal GVT activity. In addition, inhibition of c-Rel activity reduced alloactivation without compromising antigen-specific cytotoxicity of human T cells. Finally, we were able to demonstrate the feasibility and efficacy of systemic c-Rel inhibitor administration. Our findings validate c-Rel as a promising target for immunomodulatory therapy and demonstrate the feasibility and efficacy of pharmaceutical inhibition of c-Rel activity.

GATA-6 and NF-κB activate CPI-17 gene transcription and regulate Ca2+ sensitization of smooth muscle contraction.

Protein kinase C (PKC)-potentiated inhibitory protein of 17 kDa (CPI-17) inhibits myosin light chain phosphatase, altering the levels of myosin light chain phosphorylation and Ca(2+) sensitivity in smooth muscle. In this study, we characterized the CPI-17 promoter and identified binding sites for GATA-6 and nuclear factor kappa B (NF-κB). GATA-6 and NF-κB upregulated CPI-17 expression in cultured human and mouse bladder smooth muscle (BSM) cells in an additive manner. CPI-17 expression was decreased upon GATA-6 silencing in cultured BSM cells and in BSM from NF-κB knockout (KO) mice. Moreover, force maintenance by BSM strips from KO mice was decreased compared with the force maintenance of BSM strips from wild-type mice. GATA-6 and NF-κB overexpression was associated with CPI-17 overexpression in BSM from men with benign prostatic hyperplasia (BPH)-induced bladder hypertrophy and in a mouse model of bladder outlet obstruction. Thus, aberrant expression of NF-κB and GATA-6 deregulates CPI-17 expression and the contractile function of smooth muscle. Our data provide insight into how GATA-6 and NF-κB mediate CPI-17 transcription, PKC-mediated signaling, and BSM remodeling associated with lower urinary tract symptoms in patients with BPH.

Late-onset Parkinsonism in NFκB/c-Rel-deficient mice.

Activation of the nuclear factor κB/c-Rel can increase neuronal resilience to pathological noxae by regulating the expression of pro-survival manganese superoxide dismutase (MnSOD, now known as SOD2) and Bcl-xL genes. We show here that c-Rel-deficient (c-rel(-/-)) mice developed a Parkinson's disease-like neuropathology with ageing. At 18 months of age, c-rel(-/-) mice exhibited a significant loss of dopaminergic neurons in the substantia nigra pars compacta, as assessed by tyrosine hydroxylase-immunoreactivity and Nissl staining. Nigral degeneration was accompanied by a significant loss of dopaminergic terminals and a significant reduction of dopamine and homovanillic acid levels in the striatum. Mice deficient of the c-Rel factor exhibited a marked immunoreactivity for fibrillary α-synuclein in the substantia nigra pars compacta as well as increased expression of divalent metal transporter 1 (DMT1) and iron staining in both the substantia nigra pars compacta and striatum. Aged c-rel(-/-) mouse brain were characterized by increased microglial reactivity in the basal ganglia, but no astrocytic reaction. In addition, c-rel(-/-) mice showed age-dependent deficits in locomotor and total activity and various gait-related deficits during a catwalk analysis that were reminiscent of bradykinesia and muscle rigidity. Both locomotor and gait-related deficits recovered in c-rel(-/-) mice treated with l-3,4-dihydroxyphenylalanine. These data suggest that c-Rel may act as a regulator of the substantia nigra pars compacta resilience to ageing and that aged c-rel(-/-) mice may be a suitable model of Parkinson's disease.

The Th17 immune response is controlled by the Rel-RORγ-RORγ T transcriptional axis.

The Th17 cells use the retinoid-related orphan receptor-γ (Rorg or Rorc) to specify their differentiation and lineage-specific function. However, how Rorg is switched on during Th17 differentiation is unknown. We report here that c-Rel and RelA/p65 transcription factors drive Th17 differentiation by binding to and activating two distinct Rorg promoters that control RORγT and RORγ expression, respectively. Similar to RORγT, RORγ is selectively expressed in Th17 cells and is effective in specifying the Th17 phenotype. T cells deficient in c-Rel or RelA are significantly compromised in Th17 differentiation, and c-Rel-deficient mice are defective in Th17 responses. Thus, Th17 immunity is controlled by a Rel-RORγ-RORγT axis, and strategies targeting Rel/NF-κB can be effective for controlling Th17 cell-mediated diseases.

The roles of c-rel and interleukin-2 in tolerance: a molecular explanation of self-nonself discrimination.

The molecular mechanisms responsible for the exquisite discrimination between self and nonself molecules have remained enigmatic despite intense investigation. However, with the availability of adequate amounts of anergic lymphocytes produced by double transgenic mice, large numbers of immature B cells from sublethaly irradiated, hematopoietically-synchronized mice, as well as critical gene-deleted mice, it has been possible for the first time to uncover plausible molecular mechanisms that lead to tolerance versus immunity. The Rel family of transcription factors is expressed at different stages of lymphocyte maturation and differentiation. C-Rel is not activated by immature lymphocytes, which undergo either anergy or apoptosis when triggered by antigen receptors, but c-Rel is activated in mature lymphocytes. Antigen receptor triggering induces c-Rel-dependent survival and proliferative genetic programs. In T cells, a critical c-Rel-dependent gene encodes the T-cell growth factor interleukin-2 (IL-2). Thus, T cells from c-Rel gene-deleted mice produce inadequate quantities of IL-2, which renders them immunocompromised and unable to mount normal T-cell proliferative and differentiative responses. In the face of absolute IL-2 deficiency from birth, severe, multiorgan autoimmunity gradually ensues. Also, with more subtle IL-2 deficiency, organ/tissue-specific autoimmune disease becomes evident. Accordingly, both c-Rel and IL-2 appear to be key molecules for tolerance versus immunity, and doubtless will become foci for continued investigation, as well as future therapeutic targets in autoimmune diseases.

Role of the NF-κB transcription factor c-Rel in the generation of CD8+ T-cell responses to Toxoplasma gondii.

The nuclear factor κB transcription factor c-Rel is exclusively expressed in immune cells and plays a role in numerous cellular functions including proliferation, survival and production of chemokines and cytokines. c-Rel has also been implicated in the regulation of multiple genes involved in innate and adaptive immune responses to the intracellular protozoan parasite Toxoplasma gondii, in particular IL-12. To better understand how this transcription factor controls the CD8(+) T-cell response to this organism, wild-type (WT) and c-Rel(-/-) mice were challenged with a replication-deficient strain of T. gondii that expresses the model antigen ovalbumin (OVA). These studies revealed that c-Rel was required for optimal primary expansion of OVA-specific CD8(+) T cells and that immunized c-Rel-deficient mice were susceptible to challenge with a virulent strain of T. gondii. However, when c-Rel(-/-) cells specific for OVA were adoptively transferred into a WT recipient, or c-Rel(-/-) mice were treated with IL-12 at the time of immunization, there was no apparent proliferative defect. Surprisingly, upon secondary challenge, antigen-specific CD8(+) T cells in c-Rel(-/-) mice expanded to a much greater degree in terms of frequency as well as numbers when compared with WT mice. Despite this, the cytokine responses of c-Rel(-/-) mice remained defective, consistent with their susceptibility to secondary challenge. Together, these results indicate that in this infection model, the major influence of c-Rel in generation of CD8(+) T-cell responses is through its regulation of the inflammatory environment, rather than playing a substantial T-cell-intrinsic role.

The c-Rel subunit of nuclear factor-kappaB regulates murine liver inflammation, wound-healing, and hepatocyte proliferation.

UNLABELLED: In this study, we determined the role of the nuclear factor-kappaB (NF-kappaB) subunit c-Rel in liver injury and regeneration. In response to toxic injury of the liver, c-Rel null (c-rel(-/-)) mice displayed a defect in the neutrophilic inflammatory response, associated with impaired induction of RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted; also known as CCL5). The subsequent fibrogenic/wound-healing response to both chronic carbon tetrachloride and bile duct ligation induced injury was also impaired and this was associated with deficiencies in the expression of fibrogenic genes, collagen I and alpha-smooth muscle actin, by hepatic stellate cells. We additionally report that c-Rel is required for the normal proliferative regeneration of hepatocytes in response to toxic injury and partial hepatectomy. Absence of c-Rel was associated with blunted and delayed induction of forkhead box M1 (FoxM1) and its downstream targets cyclin B1 and Cdc25C. Furthermore, isolated c-rel(-/-) hepatocytes expressed reduced levels of FoxM1 and a reduced rate of basal and epidermal growth factor-induced DNA synthesis. Chromatin immunoprecipitation revealed that c-Rel binding to the FoxM1 promoter is induced in the regenerating liver. CONCLUSION: c-Rel has multiple functions in the control of liver homeostasis and regeneration and is a transcriptional regulator of FoxM1 and compensatory hepatocyte proliferation.

RNAi-mediated c-Rel silencing leads to apoptosis of B cell tumor cells and suppresses antigenic immune response in vivo.

c-Rel is a member of the Rel/NF-kappaB transcription factor family and is predominantly expressed in lymphoid and myeloid cells, playing a critical role in lymphocyte proliferation and survival. Persistent activation of the c-Rel signal transduction pathway is associated with allergies, inflammation, autoimmune diseases, and a variety of human malignancies. To explore the potential of targeting c-Rel as a therapeutic agent for these disorders, we designed a small interfering RNA (siRNA) to silence c-Rel expression in vitro and in vivo. C-Rel-siRNA expression via a retroviral vector in a B cell tumor cell line leads to growth arrest and apoptosis of the tumor cells. Silencing c-Rel in primary B cells in vitro compromises their proliferative and survival response to CD40 activation signals, similar to the impaired response of c-Rel knockout B cells. Most important, in vivo silencing of c-Rel results in significant impairment in T cell-mediated immune responses to antigenic stimulation. Our study thus validates the efficacy of c-Rel-siRNA, and suggests the development of siRNA-based therapy, as well as small molecular inhibitors for the treatment of B cell tumors as well as autoimmune diseases.

Development of Foxp3(+) regulatory t cells is driven by the c-Rel enhanceosome.

Regulatory T (Treg) cells are essential for maintaining immune homeostasis. Although Foxp3 expression marks the commitment of progenitors to Treg cell lineage, how Treg cells are generated during lymphocyte development remains enigmatic. We report here that the c-Rel transcription factor controlled development of Treg cells by promoting the formation of a Foxp3-specific enhanceosome. This enhanceosome contained c-Rel, p65, NFAT, Smad, and CREB. Although Smad and CREB first bound to Foxp3 enhancers, they later moved to the promoter to form the c-Rel enhanceosome. c-Rel-deficient mice had up to 90% reductions of Treg cells compared to wild-type mice, and c-Rel-deficient T cells were compromised in Treg cell differentiation. Thus, Treg cell development is controlled by a c-Rel enhanceosome, and strategies targeting Rel-NF-kappaB can be effective for manipulating Treg cell function.

BCR-mediated apoptosis associated with negative selection of immature B cells is selectively dependent on Pten.

The molecular basis of B cell receptor (BCR)-induced apoptosis during the negative selection of immature B cells is largely unknown. We use transitional immature B cells that are highly susceptible to BCR-induced apoptosis to show that Pten is selectively required for BCR-mediated initiation of the mitochondrial death pathway. Specifically, deleting Pten, but not other pro-apoptotic molecules, abrogates BCR-elicited apoptosis and improves viability in wild-type immature B cells. We further identify a physiologically and significantly higher intracellular Pten level in immature B cells, as compared to mature B cells, which is responsible for low AKT activity and the propensity towards death in immature B cells. Restoration of AKT activity using a constitutive form of AKT or reduction of Pten to a level comparable with that seen in mature B cells rescues immature B cells from BCR-induced apoptosis. Thus, we provide evidence that Pten is an essential mediator of BCR-induced cell death, and that differential regulation of intracellular Pten levels determines whether BCR ligation promotes cell death or survival. Our findings provide a valuable insight into the mechanisms underlying negative selection and clonal deletion of immature B cells.

Leptin is induced in the ischemic cerebral cortex and exerts neuroprotection through NF-kappaB/c-Rel-dependent transcription.

BACKGROUND AND PURPOSE: Leptin is an adipose hormone endowed with angiopoietic, neurotrophic, and neuroprotective properties. We tested the hypothesis that leptin might act as an endogenous mediator of recovery after ischemic stroke and investigated whether nuclear transcription factors kappaB activation is involved in leptin-mediated neuroprotection. METHODS: The antiapoptotic effects of leptin were evaluated in cultured mouse cortical neurons from wild-type or NF-kappaB/c-Rel(-/-) mice exposed to oxygen-glucose deprivation. Wild-type, c-Rel(-/-) and leptin-deficient ob/ob mice were subjected to permanent middle cerebral artery occlusion. Leptin production was measured in brains from wild-type mice with quantitative reverse transcriptase-polymerase chain reaction and immunostaining. Mice received a leptin bolus (20 microg/g) intraperitoneally at the onset of ischemia. RESULTS: Leptin treatment activated the nuclear translocation of nuclear transcription factors kappaB dimers containing the c-Rel subunit, induced the expression of the antiapoptotic c-Rel target gene Bcl-xL in both control and oxygen-glucose deprivation conditions, and counteracted the oxygen-glucose deprivation-mediated apoptotic death of cultured cortical neurons. Leptin-mediated Bcl-xL induction and neuroprotection against oxygen-glucose deprivation were hampered in cortical neurons from c-Rel(-/-) mice. Leptin mRNA was induced and the protein was detectable in microglia/macrophage cells from the ischemic penumbra of wild-type mice subjected to permanent middle cerebral artery occlusion. Ob/ob mice were more susceptible than wild-type mice to the permanent middle cerebral artery occlusion injury. Leptin injection significantly reduced the permanent middle cerebral artery occlusion-mediated cortical damage in wild-type and ob/ob mice, but not in c-Rel(-/-) mice. CONCLUSIONS: Leptin acts as an endogenous mediator of neuroprotection during cerebral ischemia. Exogenous leptin administration protects against ischemic neuronal injury in vitro and in vivo in a c-Rel-dependent manner.

Direct Rel/NF-κB inhibitors: structural basis for mechanism of action.

The Rel/NF-κB transcription factors have emerged as novel therapeutic targets for a variety of human diseases and pathological conditions, including inflammation, autoimmune diseases, cancer, ischemic injury, osteoporosis, transplant rejection and neurodegeneration. Several US FDA-approved drugs may, in part, attribute their therapeutic effects to the inhibition of the Rel/NF-κB pathway. Strategies for blocking the Rel/NF-κB signaling pathway have inspired the pharmaceutical industry to develop inhibitors for I-κB kinase, however, this article focuses instead on identifying natural compounds that directly target and inhibit DNA binding and transcription activity of Rel/NF-κB. These include compounds containing a quinone core, an α,ß unsaturated carbonyl and a benzene diamine. By investigating the mechanisms of action of existing natural inhibitors, novel strategies and synthetic approaches can be devised that will facilitate the development of novel and selective Rel/NF-κB inhibitors with better safety profiles.

c-Rel, an NF-kappaB family transcription factor, is required for hippocampal long-term synaptic plasticity and memory formation.

Transcription is a critical component for consolidation of long-term memory. However, relatively few transcriptional mechanisms have been identified for the regulation of gene expression in memory formation. In the current study, we investigated the activity of one specific member of the NF-kappaB transcription factor family, c-Rel, during memory consolidation. We found that contextual fear conditioning elicited a time-dependent increase in nuclear c-Rel levels in area CA1 and DG of hippocampus. These results suggest that c-rel is active in regulating transcription during memory consolidation. To identify the functional role of c-Rel in memory formation, we characterized c-rel(-/-) mice in several behavioral tasks. c-rel(-/-) mice displayed significant deficits in freezing behavior 24 h after training for contextual fear conditioning but showed normal freezing behavior in cued fear conditioning and in short-term contextual fear conditioning. In a novel object recognition test, wild-type littermate mice exhibited a significant preference for a novel object, but c-rel(-/-) mice did not. These results indicate that c-rel(-/-) mice have impaired hippocampus-dependent memory formation. To investigate the role of c-Rel in long-term synaptic plasticity, baseline synaptic transmission and long-term potentiation (LTP) at Schaffer collateral synapses in c-rel(-/-) mice was assessed. c-rel(-/-) slices had normal baseline synaptic transmission but exhibited significantly less LTP than did wild-type littermate slices. Together, our results demonstrate that c-Rel is necessary for long-term synaptic potentiation in vitro and hippocampus-dependent memory formation in vivo.

Role for IkappaBalpha, but not c-Rel, in skeletal muscle atrophy.

Skeletal muscle atrophy is associated with a marked and sustained activation of nuclear factor-kappaB (NF-kappaB) activity. Previous work showed that p50 is one of the NF-kappaB family members required for this activation and for muscle atrophy. In this work, we tested whether another NF-kappaB family member, c-Rel, is required for atrophy. Because endogenous inhibitory factor kappaBalpha (IkappaBalpha) was activated (i.e., decreased) at 3 and 7 days of muscle disuse (i.e., hindlimb unloading), we also tested if IkappaBalpha, which binds and retains Rel proteins in the cytosol, is required for atrophy and intermediates of the atrophy process. To do this, we electrotransferred a dominant negative IkappaBalpha (IkappaBalphaDeltaN) in soleus muscles, which were either unloaded or weight bearing. IkappaBalphaDeltaN expression abolished the unloading-induced increase in both NF-kappaB activation and total ubiquitinated protein. IkappaBalphaDeltaN inhibited unloading-induced fiber atrophy by 40%. The expression of certain genes known to be upregulated with atrophy were significantly inhibited by IkappaBalphaDeltaN expression during unloading, including MAFbx/atrogin-1, Nedd4, IEX, 4E-BP1, FOXO3a, and cathepsin L, suggesting these genes may be targets of NF-kappaB transcription factors. In contrast, c-Rel was not required for atrophy because the unloading-induced markers of atrophy were the same in c-rel(-/-) and wild-type mice. Thus IkappaBalpha degradation is required for the unloading-induced decrease in fiber size, the increase in protein ubiquitination, activation of NF-kappaB signaling, and the expression of specific atrophy genes, but c-Rel is not. These data represent a significant advance in our understanding of the role of NF-kappaB/IkappaB family members in skeletal muscle atrophy, and they provide new candidate NF-kappaB target genes for further study.

Essential roles of c-Rel in TLR-induced IL-23 p19 gene expression in dendritic cells.

IL-23 plays crucial roles in both immunity against pathogens and autoimmunity against self. Although it is well recognized that IL-23 expression is restricted to the myeloid lineage and is tightly regulated at the transcriptional level, the nature of transcription factors required for IL-23 expression is poorly understood. We report, in this study, that murine dendritic cells deficient in c-Rel, a member of the NF-kappaB family, are severely compromised in their ability to transcribe the p19 gene, one of the two genes that encode the IL-23 protein. The p19 gene promoter contains three putative NF-kappaB binding sites, two of which can effectively bind c-Rel as determined by chromatin immunoprecipitation and EMSA. Unexpectedly, mutation of either of these two c-Rel binding sites completely abolished the p19 promoter activity induced by five TLRs (2, 3, 4, 6, and 9) and four members of the NF-kappaB family (c-Rel, p65, p100, and p105). Based on these observations, we conclude that c-Rel controls IL-23 p19 gene expression through two kappaB sites in the p19 promoter, and propose a c-Rel-dependent enhanceosome model for p19 gene activation.