A cell cycle regulatory network controlling NF-kappaB subunit activity and function

Aberrantly active NF-kappaB complexes can contribute to tumorigenesis by regulating genes that promote the growth and survival of cancer cells. We have investigated NF-kappaB during the cell cycle and find that its ability to regulate the G1-phase expression of key proto-oncogenes is subject to regulation by the integrated activity of IkappaB kinase (IKK)alpha, IKKbeta, Akt and Chk1. The coordinated binding of NF-kappaB subunits to the Cyclin D1, c-Myc and Skp2 promoters is dynamic with distinct changes in promoter occupancy and RelA(p65) phosphorylation occurring through G1, S and G2 phases, concomitant with a switch from coactivator to corepressor recruitment. Akt activity is required for IKK-dependent phosphorylation of NF-kappaB subunits in G1 and G2 phases, where Chk1 is inactive. However, in S-phase, Akt is inactivated, while Chk1 phosphorylates RelA and associates with IKKalpha, inhibiting the processing of the p100 (NF-kappaB2) subunit, which also plays a critical role in the regulation of these genes. These data reveal a complex regulatory network integrating NF-kappaB with the DNA-replication checkpoint and the expression of critical regulators of cell proliferation.     

The endogenous reactive oxygen species promote NF-kappaB activation by targeting on activation of NF-kappaB-inducing kinase in oral squamous carcinoma cells

Reactive oxygen species (ROS) could stimulate or inhibit NF-κB pathways. However, most results have been obtained on the basis of the exogenous ROS and the molecular target of ROS in NF-κB signalling pathways has remained unclear. Here, the oral squamous carcinoma (OSC) cells, with a mild difference in the endogenous ROS level, were used to investigate how slight fluctuation of the endogenous ROS regulates NF-κB activation. This study demonstrates that NF-κB-inducing kinase (NIK) is a critical target of the endogenous ROS in NF-κB pathways. The results indicate that ROS may function as a physiological signalling modulator on NF-κB signalling cascades through its ability to facilitate the activity of NIK and subsequent NF-κB transactivation. In addition, the data are useful to explain why the altered intracellular microenvironment related to redox state may influence biological behaviours of cancer cells.     

Specific blockade by CD54 and MHC II of CD40-mediated signaling for B cell proliferation and survival

Regulation of B lymphocyte proliferation is critical to maintenance of self-tolerance, and intercellular interactions are likely to signal such regulation. Here, we show that coligation of either the adhesion molecule ICAM-1/CD54 or MHC II with CD40 inhibited cell cycle progression and promoted apoptosis of mouse splenic B cells. This resulted from specific blockade of NF-kappa B induction, which normally inhibits apoptosis. LPS- or B cell receptor (BCR)-induced proliferation was not inhibited by these treatments, and mAb-induced association of CD40 with other B cell surface molecules did not have these effects. Addition of BCR or IL-4 signals did not overcome the effect of ICAM-1 or MHC II on CD40-induced proliferation. FasL expression was not detected in B cell populations. These results show that MHC II and ICAM-1 specifically modulate CD40-mediated signaling, so inhibiting proliferation and preventing inhibition of apoptosis.     

PIASy represses TRIF-induced ISRE and NF-kappaB activation but not apoptosis

The TIR domain-containing adapter protein TRIP is critically involved in TLR3-induced IFN-β production through activation of NF-κB and ISRE. In addition, TRIF also induces apoptosis when overexpressed in 293 cells. In this report, we demonstrate that PIASy, a member of the PIAS SUMO-ligase family, interacts with TRIP, IRF-3 and IRF-7. In reporter gene assays, PIASy dramatically inhibits TRIF-induced NF-κB, ISRE and IFN-β activation but not TRIF-induced apoptosis. Furthermore, PIASy also inhibits IRF-3, IRF-7 and Sendai virus-induced ISRE activation. Our results suggest that PIASy is an inhibitor of TRIF-induced ISRE and NF-κB activation but not apoptosis.     

Ginsenosides promote proliferation of chicken primordial germ cells via PKC-involved activation of NF-kappaB

The effect of ginsenosides on proliferation of chicken primordial germ cells (PGCs) was evaluated and involvement of nuclear factor (NF)-kappaB in the signaling pathway was investigated. PGCs were isolated from the genital ridge of 3.5-4 day embryos and cultured in Medium 199 supplemented with 5% FCS and 10 ng/ml LIF. PGCs subcultured on chicken embryonic fibroblast feeder were challenged with ginsenosides alone or in combination with PKC inhibitor H(7) or activator phorbol 12-myristate 13-acetate (PMA) for 24h. Moreover, the translocation of NF-kappaB and degradation level of IkappaBalpha were investigated by Western blot analysis. Results show that PGCs were identified by periodic acid-Schiff, alkaline phosphatase histochemistry as well as c-kit, SSEA-1 and Oct-4 immunocytochemistry. Treatment with ginsenosides at 1-100 microg/ml significantly increased the number and area of PGC colonies in a dose-dependent manner. However, this proliferating effect was obviously attenuated by combined treatment of H(7) (10(-7)-10(-5)M). Similarly, PKC staining of PGC colonies was more intensive after ginsenosides treatment compared with the control group. In addition, treatment with ginsenosides at 1-10 microg/ml stimulated the translocation of NF-kappaB (p65). However, the NF-kappaB translocation and the degradation of IkappaBalpha were significantly blocked by combined treatment with 10(-6)M H(7). These results indicated that ginsenosides promote proliferation of chicken PGCs through activation of PKC-involved NF-kappaB signaling pathway.     

NF-kappaB dictates the degradation pathway of IkappaBalpha

IkappaB proteins are known as the regulators of NF-kappaB activity. They bind tightly to NF-kappaB dimers, until stimulus-responsive N-terminal phosphorylation by IKK triggers their ubiquitination and proteasomal degradation. It is known that IkappaBalpha is an unstable protein whose rapid degradation is slowed upon binding to NF-kappaB, but it is not known what dynamic mechanisms control the steady-state level of total IkappaBalpha. Here, we show clearly that two degradation pathways control the level of IkappaBalpha. Free IkappaBalpha degradation is not controlled by IKK or ubiquitination but intrinsically, by the C-terminal sequence known as the PEST domain. NF-kappaB binding to IkappaBalpha masks the PEST domain from proteasomal recognition, precluding ubiquitin-independent degradation; bound IkappaBalpha then requires IKK phosphorylation and ubiquitination for slow basal degradation. We show the biological requirement for the fast degradation of the free IkappaBalpha protein; alteration of free IkappaBalpha degradation dampens NF-kappaB activation. In addition, we find that both free and bound IkappaBalpha are similar substrates for IKK, and the preferential phosphorylation of NF-kappaB-bound IkappaBalpha is due to stabilization of IkappaBalpha by NF-kappaB.     

Solution structure and design of dithiophosphate backbone aptamers targeting transcription factor NF-kappaB

A variety of monothio- and dithiosubstituted duplex aptamers targeting NF-kappaB have been synthesized and designed. The specificity and affinity of the dithioate aptamers of p50 and RelA(p65) NF-kappaB homodimers was determined by gel shift experiments. The NMR solution structures for several unmodified and dithioate backbone modified 14-base paired duplex aptamers have been determined by a hybrid, complete matrix (MORASS)/restrained molecular dynamics method. Structural perturbations of the dithioate substitutions support our hypothesis that the dithioate binds cations less tightly than phosphoryl groups. This increases the electrostatic repulsion across the B-form narrow minor groove and enlarges the minor groove, similar to that found in A-form duplexes. Structural analysis of modeled aptamer complexes with NF-kappaB homo- and heterodimers suggests that the dithioate backbone substitution can increase the aptamer's relative affinity to basic groups in proteins such as NF-kappaB by helping to "strip" the cations from the aptamer backbone.     

亚硒酸钠对弥漫性大B细胞淋巴瘤SU-DHL-4细胞的干预作用及其机制*

目的: 证实亚硒酸钠对弥漫性大B细胞淋巴瘤(DLBCL)的作用,并分析其作用的分子机制。方法: 分别用0、2、10、50和250 ng/ml亚硒酸钠作于96孔培养板培养24 h 的DLBCL细胞SU-DHL-4,用CCK-8法检测DLBCL细胞的增殖活性,hoechst染色和流式细胞术分析SU-DHL-4细胞的核形变化、凋亡和坏死率,RT-qPCR和Western blot法检测该细胞基因转录、表达和活化情况。结果: 亚硒酸钠可显著抑制SU-DHL-4细胞增殖活性并诱导其凋亡,抑制基因RIP2、Bcl-xL、NIK及NF-κB的表达,同时可以使SU-DHL-4细胞内RIP2、Bcl-xL、NIK、P52蛋白表达水平明显下调,并促进Caspase-3的磷酸化。结论: 亚硒酸钠可通过影响NF-κB经典和非经典通路,从而抑制SU-DHL-4细胞的增殖,并诱导其发生凋亡。