NF-κB Superrepressor Research Articles

Regulation of Vascular Smooth Muscle Cell Proliferation

The transcription factor NF-kappaB regulates cell cycle progression and proliferation in a number of cell types. An important unresolved issue is the potential role of NF-kappaB in the proliferation of vascular smooth muscle cells (VSMCs) as a basis for the development of vascular disease. To investigate the contribution of NF-kappaB to mitogen-induced proliferation of VSMCs, a knock-in mouse model expressing the NF-kappaB superrepressor IkappaBalphaDeltaN (c(IkappaBalphaDeltaN)) was used. Comparing wild-type and IkappaBalphaDeltaN-expressing VSMCs, we found that proliferation rates did not differ after mitogenic stimulation by platelet-derived growth-factor-BB (PDGF-BB) or serum. In line with this, NF-kappaB activation was not observed in VSMCs derived from transgenic mice expressing an NF-kappaB-dependent lacZ reporter (c((Igk)3conalacZ)). We further show, that classical mitogenic signaling pathways (namely mitogen-activated protein kinase [MAPK] and the phosphatidyl-inositol-3-OH-kinase [PI3K] pathways) control VSMC proliferation, but independently of NF-kappaB activation. In contrast to VSMCs, mouse embryonic fibroblasts (MEFs) derived from IkappaBalphaDeltaN-expressing mice showed significantly impaired proliferation rates after mitogenic stimulation. This was reflected by strongly impaired cyclin D1 expression in serum-stimulated MEFs derived from (c(IkappaBalphaDeltaN)) mice. These results implicate that essential pathogenetic functions of NF-kappaB in the development of atherosclerosis involve apoptotic and inflammatory signaling of VSMCs rather than proliferation. They further provide genetic evidence for a cell-type restricted requirement of NF-kappaB in the control of cellular proliferation.

NF-kappaB inhibits TNF-induced accumulation of ROS that mediate prolonged MAPK activation and necrotic cell death.

NF-kappaB downregulates tumor necrosis factor (TNF)-induced c-Jun N-terminal kinase (JNK) activation that promotes cell death, but the mechanism is not yet fully understood. By using murine embryonic fibroblasts (MEFs) that are deficient in TNF receptor-associated factor (TRAF) 2 and TRAF5 (DKO) or p65 NF-kappaB subunit (p65KO), we demonstrate here that TNF stimulation leads to accumulation of reactive oxygen species (ROS), which is essential for prolonged mitogen-activated protein kinase (MAPK) activation and cell death. Interestingly, dying cells show necrotic as well as apoptotic morphological changes as assessed by electron microscopy and flow cytometry, and necrotic, but not apoptotic, cell death is substantially inhibited by antioxidant. Importantly, TNF does not induce ROS accumulation or prolonged MAPK activation in wild-type MEFs, indicating that TRAF-mediated NF-kappaB activation normally suppresses the TNF-induced ROS accumulation that subsequently induces prolonged MAPK activation and necrotic cell death

Hypoxia-inducible factor induction by tumour necrosis factor in normoxic cells requires receptor-interacting protein-dependent nuclear factor kappa B activation.

Tumour necrosis factor alpha (TNF-alpha) binds to its receptor (TNFR1) and activates both death- and inflammation/survival-related signalling pathways. The inflammation and survival-related signalling cascade results in the activation of the transcription factor, nuclear factor kappa B (NF-kappa B) and requires recruitment of receptor-interacting protein (RIP) to TNFR1. The indispensable role of RIP in TNF-induced NF-kappa B activation has been demonstrated in RIP(-/-) mice and in cell lines derived from such mice. In the present study, we show that the TNF-alpha-induced accumulation of hypoxia-inducible factor 1 alpha (HIF-1 alpha) protein in normoxic cells is RIP-dependent. Exposing fibroblasts derived from RIP(-/-) mice to either cobalt or PMA resulted in an equivalent HIF-1 alpha induction to that seen in RIP(+/+) fibroblasts. In contrast, RIP(-/-) cells were unable to induce HIF-1 alpha in response to TNF-alpha. Further, transient transfection of NIH 3T3 cells with an NF-kappa B super-repressor plasmid (an inhibitor of NF-kappa B activation) also prevented HIF-1 alpha induction by TNF-alpha. Surprisingly, although HIF-1 alpha mRNA levels remained unchanged after induction by TNF, induction of HIF-1 alpha protein by the cytokine was completely blocked by pretreatment with the transcription inhibitors actinomycin D and 5,6-dichlorobenzimidazole riboside. Finally, TNF failed to induce both HIF-1 alpha, made resistant to von Hippel-Lindau (VHL), and wild-type HIF-1 alpha transfected into VHL(-/-) cells. These results indicate that HIF-1 alpha induction by TNF-alpha in normoxic cells is mediated by protein stabilization but is nonetheless uniquely dependent on NF-kappa B-driven transcription. Thus the results describe a novel mechanism of HIF-1 alpha up-regulation and they identify HIF-1 alpha as a unique component of the NF-kappa B-mediated inflammatory/survival response.

Alpha 5 beta 1 integrin activates an NF-kappa B-dependent program of gene expression important for angiogenesis and inflammation.

GeneCalling, a genome-wide method of mRNA profiling, reveals that endothelial cells adhering to fibronectin through the alpha 5 beta 1 integrin, but not to laminin through the alpha 2 beta 1 integrin, undergo a complex program of gene expression. Several of the genes identified are regulated by the NF-kappa B transcription factor, and many are implicated in the regulation of inflammation and angiogenesis. Adhesion of endothelial cells to fibronectin activates NF-kappa B through a signaling pathway requiring Ras, phosphatidylinositol 3-kinase, and Rho family proteins, whereas adhesion to laminin has a limited effect. Retroviral transfer of the superrepressor of NF-kappa B, I kappa B-2A, blocks basic fibroblast growth factor-induced angiogenesis in vivo. These results suggest that engagement of the alpha 5 beta 1 integrin promotes an NF-kappa B-dependent program of gene expression that coordinately regulates angiogenesis and inflammation.

NF-κB Inhibition Enhances Peroxynitrite-Induced Enterocyte Apoptosis

Background. Sustained overproduction of nitric oxide and peroxynitrite (ONOO−) in conditions such as necrotizing enterocolitis and inflammatory bowel disease may promote gut barrier failure by inducing enterocyte apoptosis. NF-κB is upregulated in the gut during inflammation and, in addition to its proinflammatory effects, may upregulate protective or antiapoptotic factors such as inhibitor of apoptosis proteins (IAPs). We have previously demonstrated that NF-κB inhibition increases cytokine-induced enterocyte apoptosis; however, the effect of NF-κB on ONOO−-induced enterocyte apoptosis is unknown.Materials and methods. Rat intestinal epithelial cells (IEC-6) were transfected with the adenoviral vector AdIκB or AdlacZ. AdIκB contains a mutated form of IκB which functions as a superrepressor of NF-κB. Cells were then treated with 50 μM ONOO− or decomposed ONOO−. Apoptosis was then determined by flow cytometry with annexin V–FITC and propidium iodide staining. Caspase activation and IAP, Bcl-2, Bad, and Bax expression were examined using Western blot analysis, and NF-κB activation was determined via electrophoretic mobility shift assay (EMSA).Results. Inhibition of NF-κB with AdIκB significantly enhanced ONOO−-induced apoptosis in IEC-6 cells. ONOO− treatment did not activate NF-κB in IEC-6 cells as determined by EMSA. There was no difference in IAP, Bcl-2, Bad, and Bax expression between nontransfected, AdlacZ-transfected, and AdIκB-transfected cells. Baseline procaspase 3 activation was increased in AdIκB-transfected cells.Conclusions. NF-κB inhibition enhances ONOO−-induced enterocyte apoptosis, suggesting that NF-κB upregulates a protective factor. This protective factor does not appear to be an IAP or Bcl-2 family member and may be expressed constitutively, since ONOO− did not activate NF-κB over baseline levels of activation.

Inhibition of cytokine-induced NF-kappaB activation by adenovirus-mediated expression of a NF-kappaB super-repressor prevents beta-cell apoptosis.

Cytokine-induced beta-cell death is an important event in the pathogenesis of type 1 diabetes. The transcription factor nuclear factor-kappaB (NF-kappaB) is activated by interleukin-1beta (IL-1beta), and its activity promotes the expression of several beta-cell genes, including pro- and anti-apoptotic genes. To elucidate the role of cytokine (IL-1beta + gamma-interferon [IFN-gamma])-induced expression of NF-kappaB in beta-cell apoptosis, rat beta-cells were infected with the recombinant adenovirus AdIkappaB((SA)2), which contained a nondegradable mutant form of inhibitory kappaB (IkappaB((SA)2), with S32A and S36A) that locks NF-kappaB in a cytosolic protein complex, preventing its nuclear action. Expression of IkappaB((SA)2) inhibited cytokine-stimulated nuclear translocation and DNA-binding of NF-kappaB. Cytokine-induced gene expression of several NF-kappaB targets, namely inducible nitric oxide synthase, Fas, and manganese superoxide dismutase, was prevented by AdIkappaB((SA)2), as established by reverse transcriptase-polymerase chain reaction, protein blot, and measurement of nitrite in the medium. Finally, beta-cell survival after IL-1beta + IFN-gamma treatment was significantly improved by IkappaB((SA)2) expression, mostly through inhibition of the apoptotic pathway. Based on these findings, we conclude that NF-kappaB activation, under in vitro conditions, has primarily a pro-apoptotic function in beta-cells.

Requirement of NF-kappaB/Rel for the development of hair follicles and other epidermal appendices.

NF-kappaB/Rel transcription factors and IkappaB kinases (IKK) are essential for inflammation and immune responses, but also for bone-morphogenesis, skin proliferation and differentiation. Determining their other functions has previously been impossible, owing to embryonic lethality of NF-kappaB/Rel or IKK-deficient animals. Using a gene targeting approach we have ubiquitously expressed an NF-kappaB super-repressor to investigate NF-kappaB functions in the adult. Mice with suppressed NF-kappaB revealed defective early morphogenesis of hair follicles, exocrine glands and teeth, identical to Eda (tabby) and Edar (downless) mutant mice. These affected epithelial appendices normally display high NF-kappaB activity, suppression of which resulted in increased apoptosis, indicating that NF-kappaB acts as a survival factor downstream of the tumor necrosis factor receptor family member EDAR. Furthermore, NF-kappaB is required for peripheral lymph node formation and macrophage function.

Adenovirus-Mediated Transfer of Caspase-8 in Combination with Superrepressor of NF-κB Drastically Induced Apoptosis in Gliomas

Inhibition of NF-κB in the presence of tumor necrosis factor-α (TNF) is supposed to be a promising cancer therapeutic approach, since it disrupts the protective mechanism of NF-κB activated by TNF. To test this approach in gliomas, we introduced a superrepressor of NF-κB, an N-terminal deleted form of inhibitor κ B α (IκBdN) gene, to human glioma cells (U251 and U-373MG) via adenoviral vector (Adv) in the presence of TNF. U-373MG cells were refractory to TNF-induced apoptosis even when they were transduced with the IκBdN gene. On the other hand, transduction of IκBdN drastically augmented caspase-8-mediated apoptosis in U-373MG cells. Similar results were obtained in U251 cells. Cotransduction of IκBdN and caspase-8 induced cleavage of PARP. Taken together, Adv-mediated transfer of IκBdN plus caspase-8 may be a promising therapeutic approach to treat gliomas.

NF-kappaB function in growth control: regulation of cyclin D1 expression and G0/G1-to-S-phase transition.

Nuclear factor kappa B (NF-kappaB) has been implicated in the regulation of cell proliferation, transformation, and tumor development. We provide evidence for a direct link between NF-kappaB activity and cell cycle regulation. NF-kappaB was found to stimulate transcription of cyclin D1, a key regulator of G1 checkpoint control. Two NF-kappaB binding sites in the human cyclin D1 promoter conferred activation by NF-kappaB as well as by growth factors. Both levels and kinetics of cyclin D1 expression during G1 phase were controlled by NF-kappaB. Moreover, inhibition of NF-kappaB caused a pronounced reduction of serum-induced cyclin D1-associated kinase activity and resulted in delayed phosphorylation of the retinoblastoma protein. Furthermore, NF-kappaB promotes G1-to-S-phase transition in mouse embryonal fibroblasts and in T47D mammary carcinoma cells. Impaired cell cycle progression of T47D cells expressing an NF-kappaB superrepressor (IkappaBalphaDeltaN) could be rescued by ectopic expression of cyclin D1. Thus, NF-kappaB contributes to cell cycle progression, and one of its targets might be cyclin D1.