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.     

Modulation of reactive oxygen species in skeletal muscle by myostatin is mediated through NF-κB

Abnormal levels of reactive oxygen species (ROS) and inflammatory cytokines have been observed in the skeletal muscle during muscle wasting including sarcopenia. However, the mechanisms that signal ROS production and prolonged maintenance of ROS levels during muscle wasting are not fully understood. Here, we show that myostatin (Mstn) is a pro-oxidant and signals the generation of ROS in muscle cells. Myostatin, a transforming growth factor-β (TGF-β) family member, has been shown to play an important role in skeletal muscle wasting by increasing protein degradation. Our results here show that Mstn induces oxidative stress by producing ROS in skeletal muscle cells through tumor necrosis factor-α (TNF-α) signaling via NF-κB and NADPH oxidase. Aged Mstn null (Mstn(-/-) ) muscles, which display reduced sarcopenia, also show an increased basal antioxidant enzyme (AOE) levels and lower NF-κB levels indicating efficient scavenging of excess ROS. Additionally, our results indicate that both TNF-α and hydrogen peroxide (H(2) O(2) ) are potent inducers of Mstn and require NF-κB signaling for Mstn induction. These results demonstrate that Mstn and TNF-α are components of a feed forward loop in which Mstn triggers the generation of second messenger ROS, mediated by TNF-α and NADPH oxidase, and the elevated TNF-α in turn stimulates Mstn expression. Higher levels of Mstn in turn induce muscle wasting by activating proteasomal-mediated catabolism of intracellular proteins. Thus, we propose that inhibition of ROS induced by Mstn could lead to reduced muscle wasting during sarcopenia.     

Thymic stromal lymphopoietin is expressed and produced by caspase-1/NF-κB pathway in mast cells

Thymic stromal lymphopoietin (TSLP) plays a pivotal role in allergic diseases such as atopic dermatitis, asthma, and chronic obstructive pulmonary disease. Although there are many reports regarding function and regulatory mechanism of TSLP in dendritic cells and/or T cells, the regulatory mechanism of TSLP in mast cells has not been fully elucidated. Here, we describe how TSLP is expressed and produced by inflammatory stimulus in mast cells. TSLP mRNA was expressed by phorbol myristate acetate (PMA) plus A23187 stimulation in HMC-1 cells and reached its peak 5h after PMA plus A23187 stimulation. The expression of TSLP mRNA was inhibited by nuclear factor (NF)-κB inhibitor. In addition, NF-κB luciferase activity was inhibited by caspase-1 inhibitor, indicating that caspase-1 is an upstream of NF-κB in mast cells. Furthermore, caspase-1 inhibitor decreased the expression of TSLP mRNA induced by PMA plus A23187. Finally, TSLP production was inhibited by both caspase-1 inhibitor and NF-κB inhibitor. These results provide proof of principle that TSLP can be expressed and produced through caspase-1 and NF-κB in mast cells and open new perspectives to pharmacologically manipulate the expression and production of TSLP by molecules acting on the caspase-1 and NF-κB pathway.     

Palmitate-induced PTP1B expression is mediated by ceramide-JNK and nuclear factor κB (NF-κB) activation

Palmitate induces PTP1B expression in skeletal muscle cells. The purpose of this study was to investigate the mechanisms responsible for palmitate-induced PTP1B expression in mouse skeletal muscle cells. Three truncated fragments of PTP1B promoter were cloned into PGL3-basic vector and the promoter activity of PTP1B was assessed in C2C12 cells exposed to palmitate either in the presence or in the absence of several inhibitors to study the biochemical pathways involved. EMSA was performed to examine binding of NF-κB to NF-κB consensus sequence and PTP1B oligonucelotides in the cells treated with palmitate. Lentiviral PTP1B-shRNA was used to knockdown PTP1B in myotubes. The phosphorylation and protein levels of IRS-1 and Akt were detected by western blot. 0.5mM palmitate induced PTP1B promoter activity in fragment -1715/+59 by 50% (p<0.01). Palmitate increased NF-κB binding to both NF-κB consensus sequence and one NF-κB sequence (-920 to -935) in PTP1B promoter. Incubation of C2C12 cells with different concentrations of C2-ceramide enhanced PTP1B promoter activity dose-dependently. Inhibitors of de novo ceramide synthesis prevented palmitate-induced PTP1B promoter activity in myotubes. In addition, inhibitor of JNK pathway prevented ceramide-induced PTP1B promoter activity in myotubes. Knockdown of PTP1B also prevented ceramide-reduced IRS-1 and Akt phosphorylations in the myotubes. Exposure of the cells to PMA and calphostin C, an inhibitor of PKC, did not affect the activity of PTP1B promoter. Our data provide the evidence that the mechanism by which palmitate increased the expression of PTP1B seems to be through a mechanism involving the activation of ceramide-JNK and NF-κB pathways.     

IκB/NF-κB mediated cisplatin resistance in HeLa cells after low-dose γ-irradiation is associated with altered SODD expression

Fractionated -irradiation (15 × 2 Gy in 3 weeks) induces a cellular resistance in HeLa cells against cisplatin exposure but not against irradiation. The mechanisms underlying this cellular resistance are associated with major changes in the TNFR1-dependent transduction pathway. The resistant HeLa/B cells exhibit increased levels of NF-B with temporally independent regulation of the subunits NF-B50 and NF-B65. Blocking IB degradation by the proteasome inhibitor PSI, which abolishes the release of the active NF-B protein, induces cell death much more effectively in the parental than in the resistant HeLa/B cells. The translocation of NF-B does not seem to be affected in a similar manner since masking of the translocation sequence by NF-B SN50 enhances cisplatin toxicity to the same degree in both cell lines and overcomes drug resistance. Changes in upstream signaling are suggested by increased sensitivity of the parental HeLa cells to cisplatin in the presence of neutralizing anti-TNFR1. In HeLa/B cells, reduced expression of the 50 kDa silencer of death domain, SODD, is accompanied by constitutive overexpression of a 40–42 kDa SODD-like protein. A possible involvement of SODD in cisplatin resistance is discussed, which may shift the balance between life and death in the TNF receptor pathway to increased NF-B activation.     

Thrombin-induced NF-κB activation and IL-8/CXCL8 release is mediated by c-Src-dependent Shc,Raf-1, and ERK pathways in lung epithelial cells

In addition to its functions in thrombosis and hemostasis, thrombin also plays an important role in lung inflammation. Our previous report showed that thrombin activates the protein kinase C (PKC)α/c-Src and Gβγ/Rac1/PI3K/Akt signaling pathways to induce IκB kinase α/β (IKKα/β) activation, NF-κB transactivation, and IL-8/CXCL8 expressions in human lung epithelial cells (ECs). In this study, we further investigated the mechanism of c-Src-dependent Shc, Raf-1, and extracellular signal-regulated kinase (ERK) signaling pathways involved in thrombin-induced NF-κB activation and IL-8/CXCL8 release. Thrombin-induced increases in IL-8/CXCL8 release and κB-luciferase activity were inhibited by the Shc small interfering RNA (siRNA), p66Shc siRNA, GW 5074 (a Raf-1 inhibitor), and PD98059 (a mitogen-activated protein kinase (MAPK) kinase (MEK) inhibitor). Treatment of A549 cells with thrombin increased p66Shc and p46/p52Shc phosphorylation at Tyr239/240 and Tyr317, which was inhibited by cell transfection with the dominant negative mutant of c-Src (c-Src DN). Thrombin caused time-dependent phosphorylation of Raf-1 and ERK, which was attenuated by the c-Src DN. Thrombin-induced IKKα/β phosphorylation was inhibited by GW 5074 and PD98059. Treatment of cells with thrombin induced Gβγ, c-Src, and p66Shc complex formation in a time-dependent manner. Taken together, these results show for the first time that thrombin activates Shc, Raf-1, and ERK through Gβγ, c-Src, and Shc complex formation to induce IKKα/β phosphorylation, NF-κB activation, and IL-8/CXCL8 release in human lung ECs.     

The regulation of the UCH-L1 gene by transcription factor NF-κB in podocytes

In kidney, the ubiquitin carboxy-terminal hydrolase 1 (UCH-L1) is involved in podocyte injury and proteinuria but details of the mechanism underlying its regulation are not known. Activation of NF-κB is thought to be the predominant risk factor for kidney disease; therefore, it is postulated that UCH-L1 may be one of the NF-κB target genes. In this study, we investigated the involvement of NF-κB activation in the regulation of UCH-L1 expression and the function of murine podocytes. Stimulation of podocytes with the cytokines TNF-α and IL-1β up-regulated UCH-L1 expression rapidly at the mRNA and protein levels and the NF-κB-specific inhibitor pyrrolidine dithiocarbamate resulted in down-regulation. NF-κB up-regulates UCH-L1 via binding the ? 300 bp and ? 109 bp sites of its promoter, which was confirmed by the electrophoretic mobility shift assay of DNA–nuclear protein binding. In the renal biopsy from lupus nephritis patients, the expressions of NF-κB and UCH-L1 increased in immunohistochestry staining and were positively correlated. Activation of NF-κB up-regulates UCH-L1 expression following changing of other podocytes molecules, such as nephrin and snail. These results suggest that activation of the NF-κB signaling pathway could be the major pathogenesis to up-regulate UCH-L1 in podocyte injury, followed by the turnover of other molecules, which might result in morphological changes and dysfunction of podocytes. This work help us to understand the effect of NF-κB on specific target molecules of podocytes, and suggest that targeting the NF-κB–UCH-L1 interaction could be a novel therapeutic strategy for the treatment of podocyte lesions and proteinuria.