Interaction between PKR and PACT mediated by LPS-inducible NF-κB in human gingival cells

The double‐stranded RNA‐dependent protein kinase (PKR) is a serine/threonine kinase expressed constitutively in mammalian cells. PKR is activated upon virus infection by double‐stranded RNA (dsRNA), and plays a critical role in host antiviral defense mechanisms. PKR is also known to regulate various biological responses, including cell differentiation and apoptosis. However, whether PKR is involved in the progress of periodontitis is not clear. The present study explained the phosphorylation of PKR by LPS in the human gingival cell line, Sa3. Expression of genes encoding LPS receptors was detected in Sa3 cells and treatment of cells with 1 µg/mL LPS for 6 h caused PKR phosphorylation. LPS elevated the expression of the protein activator of PKR (PACT) mRNA and protein, followed by the enhanced association between PACT and PKR within 3 h. In addition, LPS treatment induced the translocation of NF‐κB to the nucleus after 30 min, and inhibition of NF‐κB decreased the PACT–PKR interaction induced by LPS. The level of pro‐inflammatory cytokine mRNA, including interleukin‐6 (IL‐6) and tumor necrosis factor alpha (TNFα), appeared within 45 min and reached at the maximal levels by 90 min after the addition of LPS. This induction of pro‐inflammatory cytokines was not affected by RNAi‐mediated silencing of PKR and a pharmacological inhibitor of PKR, whereas the inhibition of NF‐κB decreased it. These results indicated that LPS induces PKR phosphorylation and the PACT–PKR association in Sa3 cells. Our results also suggest that NF‐κB is involved in the PACT–PKR interaction and the production of pro‐inflammatory cytokines in periodontitis. J. Cell. Biochem. 113: 165–173, 2012. © 2011 Wiley Periodicals, Inc.     

Coordinate Regulation of TPL-2 and NF-κB Signaling in Macrophages by NF-κB1 p105

The role of IκB kinase (IKK)-induced proteolysis of NF-κB1 p105 in innate immune signaling was investigated using macrophages from Nfkb1(SSAA/SSAA) mice, in which the IKK target serines on p105 are mutated to alanines. We found that the IKK/p105 signaling pathway was essential for TPL-2 kinase activation of extracellular signal-regulated kinase (ERK) mitogen-activate protein (MAP) kinase and modulated the activation of NF-κB. The Nfkb1(SSAA) mutation prevented the agonist-induced release of TPL-2 from its inhibitor p105, which blocked activation of ERK by lipopolysaccharide (LPS), tumor necrosis factor (TNF), CpG, tripalmitoyl-Cys-Ser-Lys (Pam(3)CSK), poly(I · C), flagellin, and R848. The Nfkb1(SSAA) mutation also prevented LPS-induced processing of p105 to p50 and reduced p50 levels, in addition to decreasing the nuclear translocation of RelA and cRel. Reduced p50 in Nfkb1(SSAA/SSAA) macrophages significantly decreased LPS induction of the IκBζ-regulated Il6 and Csf2 genes. LPS upregulation of Il12a and Il12b mRNAs was also impaired although specific blockade of TPL-2 signaling increased expression of these genes at late time points. Activation of TPL-2/ERK signaling by IKK-induced p105 proteolysis, therefore, induced a negative feedback loop to downregulate NF-κB-dependent expression of the proinflammatory cytokine interleukin-12 (IL-12). Unexpectedly, TPL-2 promoted soluble TNF production independently of IKK-induced p105 phosphorylation and its ability to activate ERK, which has important implications for the development of anti-inflammatory drugs targeting TPL-2.     

Modeling oscillatory control in NF-κB, p53 and Wnt signaling

Oscillations are commonly observed in cellular behavior and span a wide range of timescales, from seconds in calcium signaling to 24 hours in circadian rhythms. In between lie oscillations with time periods of 1-5 hours seen in NF-κB, p53 and Wnt signaling, which play key roles in the immune system, cell growth/death and embryo development, respectively. In the first part of this article, we provide a brief overview of simple deterministic models of oscillations. In particular, we explain the mechanism of saturated degradation that has been used to model oscillations in the NF-κB, p53 and Wnt systems. The second part deals with the potential physiological role of oscillations. We use the simple models described earlier to explore whether oscillatory signals can encode more information than steady-state signals. We then discuss a few simple genetic circuits that could decode information stored in the average, amplitude or frequency of oscillations. The presence of frequency-detector circuit downstream of NF-κB or p53 would be a strong clue that oscillations are important for the physiological response of these signaling systems.     

Control of p53 and NF-κB signaling by WIP1 and MIF: role in cellular senescence and organismal aging

The stress-activated signaling pathways, p53 and NF-κB, have a major role in the regulation of cellular senescence and organismal aging. These ancient signaling networks display functional antagonism via negative autoregulatory circuits. WIP1 (wildtype p53-induced phosphatase 1) and MIF (macrophage migration inhibitory factor) are signaling molecules which link together the p53 and NF-κB pathways via positive and negative feedback loops. It seems that the efficiency of the p53 signaling pathway declines during aging whereas that of NF-κB is clearly enhanced. Moreover, p53 is an important trigger of cellular senescence while NF-κB signaling seems to be involved in the induction of the senescence-associated secretory phenotype (SASP). MIF is a pro-inflammatory cytokine which inhibits the function of p53 signaling whereas it is linked to NF-κB signaling via a positive feedback loop. MIF knockout mice are healthier and live longer than their wild-type counterparts. An increased level of MIF can support inflammatory responses via enhancing NF-κB signaling and repressing the function of p53. p53 is an inducer of the expression of WIP1 which can subsequently inhibit NF-κB signaling. Several observations indicate that the activity of WIP1 decreases during the aging process, this being probably attributable to the decline in p53 function. Decreased WIP1 activity potentiates the activity of p38MAPK and NF-κB signaling leading to premature cellular senescence as well as low-level chronic inflammation. We will review the findings linking WIP1 and MIF to specific signaling responses of p53 and NF-κB and discuss their role in the regulation of cellular senescence and organismal aging.     

Analysis of p53 and NF-κB signaling in modulating the cardiomyocyte fate during hypertrophy

Cardiac hypertrophy leading to eventual heart failure is the most common cause of mortality throughout the world. The triggering mechanisms for cardiac hypertrophy are not clear but both apoptosis and cell proliferation have been reported in sections of failing hearts. In this study, we utilized both angiotensin II (AngII) treatment of cardiomyocytes and aortic ligation in rats (Rattus norvegicus, Wistar strain) for induction of hypertrophy to understand the cellular factors responsible for activation of apoptotic or anti-apoptotic pathway. Hypertrophy markers (ANF, β-MHC), apoptotic proteins (Bax, Bad, Fas, p53, caspase-3, PARP), and anti-apoptotic or cell proliferation marker proteins (Bcl2, NF-κB, Ki-67) were induced significantly during hypertrophy, both in vitro as well as in vivo. Co-localization of both active caspase-3 and Ki-67 was observed in hypertrophied myocytes. p53 and NF-κBp65 binding to co-activator p300 was also increased in AngII treated myocytes. Inhibition of p53 resulted in downregulation of apoptosis, NF-κB activation, and NF-κB-p300 binding; however, NF-κB inhibition did not inhibit apoptosis or p53-p300 binding. Blocking of either p53 or NF-κB by specific inhibitors resulted in decrease in cell proliferation and hypertrophy markers, suggesting that p53 initially binds to p300 and then this complex recruits NF-κB. Thus, these results indicate the crucial role of p53 in regulating both apoptotic and cell proliferation during hypertrophy.     

Interaction between NF-κB signaling and Notch signaling in gliogenesis of mouse mesencephalic neural crest cells

In the present study, we elucidated that nuclear factor-κB (NF-κB) participates in the gliogenic specification of mouse mesencephalic neural crest cells. Whereas transfection of the NF-κB expression vector stimulated gliogenesis, treatment with the dominant negative NF-κB expression vector or NF-κB small interfering RNA suppressed the promotion of gliogenic specification by FGF treatment or Notch activation. This suppression was recovered by the treatment with the Deltex-1 expression vector or mammalian hairy and enhancer of split homologs expression vectors. Furthermore, transfection of the inhibitor of κB (IκB) expression vector inhibited gliogenesis. In addition, treatment with the NF-κB expression vector promoted the expression of Deltex-1. These data suggest that NF-κB signaling is implicated in the gliogenesis through the interaction with Notch signaling. Moreover, cells that contain Sox10 expressed NF-κB and Deltex-1 in the presumptive trigeminal ganglia of embryonic day 9.0-9.5 mouse embryos. This observation supports our notion that the interaction between NF-κB signaling and Notch signaling plays an important role in the gliogenic specification of mouse mesencephalic neural crest cells.     

Blockage of NF-κB Induces Serine 15 Phosphorylation of Mutant p53 by JNK Kinase in Prostate Cancer Cells

The p53 tumor suppressor gene plays an important role during induction of apoptosis in cancer. In contrast, NF-κB prevents apoptosis in response to chemotherapeutic agents and is a critical regulator of cell survival. Despite the riches of information on the regulation of wild-type p53 function by phosphorylation, nothing is known about the modulation of mutant p53 activity by phosphorylation. Here we report that inhibition of NF-κB in DU145 prostate cancer cells results in p53 mutant phosphorylation at serine 15 (Ser15), leading to an increase of p53 stability, DNA binding and gain of function. Serine 15-phosphorylation is due to GADD45a-dependent induction of JNK kinase, which can be blocked by SP600125, a JNK kinase inhibitor. Furthermore, inhibition of GADD45a by small interfering RNA blocks JNK activation and abrogates Ser15 phosphorylation. Together, these results highlight the importance of Ser15 phosphorylation in regulating the oncogenic function of mutant p53 and apoptosis induction in the context of the NF-κB/IκB signaling pathway.     

Overexpression of BLCAP induces S phase arrest and apoptosis independent of p53 and NF-κB in human tongue carcinoma

Bladder cancer-associated protein gene (BLCAP) is a novel candidate tumor suppressor gene identified from the human bladder carcinoma. Our previous studies have shown that BLCAP overexpression could inhibit cell growth by inducing apoptosis in HeLa cells [Zuo Z, Zhao M, Liu J, Gao G, Wu X: Tumor Biol 27: 221–226, 2006]. Such evidence suggests the alterations in BLCAP may play an important role in tumorigenesis. To further study the biological function of the BLCAP gene, we constructed a recombinant retroviral vector encoding BLCAP cDNA. Overexpressed BLCAP, via stable infection of exogenous BLCAP, resulted in growth inhibition of the human tongue cancer cell line Tca8113 in vitro, accompanied by S phase cell cycle arrest and apoptosis. The growth inhibition was correlated with up-regulation of p21^WAF1/CIP1 expression and down-regulation of Bcl-XL and Bcl-2 expressions. However, p53 expression and NF-κB activity remained unchanged post infection. Furthermore, no changes in p53 phosphorylation at Ser46 and nuclear localization, which are critical to p53 function, were observed in BLCAP-overexpressed cells. Taken together, BLCAP may play a role not only in regulating cell proliferation but also in coordinating apoptosis and cell cycle via a novel way independent of p53 and NF-κB. Jun Yao and Li Duan contributed equally to this work.     

Suppression of NF-κB and NF-κB-Regulated Gene Expression by Apigenin through IκBα and IKK Pathway in TRAMP Mice

Aberrant Nuclear Factor-κappaB (NF-κB) activation due to rapid IκBα turnover and high basal IκBα kinase (IKK) activity has been frequently observed in prostate cancer. Apigenin, a naturally occurring plant flavone, exhibits anti-proliferative, anti-inflammatory and anti-carcinogenic activities by inhibiting NF-κB pathway, through a mechanism not fully understood. We found that apigenin feeding in microgram doses (bioavailable in humans) inhibited prostate tumorigenesis in TRAMP mice by interfering with NF-κB signaling. Apigenin feeding to TRAMP mice (20 and 50 μg/mouse/day, 6 days/week for 20 weeks) exhibited significant decrease in tumor volumes of the prostate and completely abolished metastasis, which correlated with inhibition of NF-κB activation and binding to the DNA. Apigenin intake blocked phosphorylation and degradation of IκBα by inhibiting IKK activation, which in turn led to suppression of NF-κB activation. The expression of NF-κB-regulated gene products involved in proliferation (cyclin D1, and COX-2), anti-apoptosis (Bcl-2 and Bcl-xL), and angiogenesis (vascular endothelial growth factor) were also downregulated after apigenin feeding. These events correlated with the induction of apoptosis in tumor cells, as evident by increased cleaved caspase-3 labeling index in the dorsolateral prostate. Our results provide convincing evidence that apigenin inhibits IKK activation and restores the expression of IκBα, preventing it’s phosphorylation in a fashion similar to that elicited by IKK and proteasomal inhibitors through suppression of NF-κB signaling pathway.