NF-κB Mediates Tumor Necrosis Factor α-Induced Expression of Optineurin, a Negative Regulator of NF-κB

Optineurin is a ubiquitously expressed multifunctional cytoplasmic protein encoded by OPTN gene. The expression of optineurin is induced by various cytokines. Here we have investigated the molecular mechanisms which regulate optineurin gene expression and the relationship between optineurin and nuclear factor κB (NF-κB). We cloned and characterized human optineurin promoter. Optineurin promoter was activated upon treatment of HeLa and A549 cells with tumor necrosis factor α (TNFα). Mutation of a putative NF-κB-binding site present in the core promoter resulted in loss of basal as well as TNFα-induced activity. Overexpression of p65 subunit of NF-κB activated this promoter through NF-κB site. Oligonucleotides corresponding to this putative NF-κB-binding site showed binding to NF-κB. TNFα-induced optineurin promoter activity was inhibited by expression of inhibitor of NF-κB (IκBα) super-repressor. Blocking of NF-κB activation resulted in inhibition of TNFα-induced optineurin gene expression. Overexpressed optineurin partly inhibited TNFα-induced NF-κB activation in Hela cells. Downregulation of optineurin by shRNA resulted in an increase in TNFα-induced as well as basal NF-κB activity. These results show that optineurin promoter activity and gene expression are regulated by NF-κB pathway in response to TNFα. In addition these results suggest that there is a negative feedback loop in which TNFα-induced NF-κB activity mediates expression of optineurin, which itself functions as a negative regulator of NF-κB

Sp1-like sequences mediate human caspase-3 promoter activation by p73 and cisplatin

Caspase-3 is a cysteine protease that plays a central role in the execution of apoptosis induced by a wide variety of stimuli. However, little is known about the mechanisms involved in the regulation of caspase-3 gene transcription. This study was carried out to characterize the human caspase-3 promoter and to understand the mechanisms involved in the induction of caspase-3 gene expression in response to the anticancer drug cisplatin and p<SUP>73</SUP>. Caspase-3 gene expression was induced by treatment of cells with cisplatin, which also induced p<SUP>73</SUP> protein in HeLa and K562 cells. The human caspase-3 promoter was cloned and characterized. p<SUP>73</SUP>β strongly activated the caspase-3 promoter, whereas p<SUP>73</SUP>α showed less activation. Cisplatin treatment increased caspase-3 promoter activity. Basal and cisplatin-induced promoter activity was inhibited by the p<SUP>73</SUP> inhibitor p<SUP>73</SUP>DD. Deletion analysis defined a minimal promoter of 120 base pairs, which showed good basal and p<SUP>73</SUP>β -induced activity. The examination of the minimal promoter sequence showed several putative Sp1 sites, but no p<SUP>53</SUP>/p<SUP>73</SUP> site. The caspase-3 promoter was activated by Sp1 in Sp1-deficient Drosophila SL-2 cells. Sp1-induced promoter activity was further enhanced by p<SUP>73</SUP>β in SL-2 cells. Mutation of Sp1 sites in the minimal promoter resulted in a loss of basal and p<SUP>73</SUP>-induced promoter activity. These results show that caspase-3 gene transcription is induced by cisplatin, which is mediated partly by p<SUP>73</SUP>. We have identified p<SUP>73</SUP> and Sp1 as activators of the caspase-3 promoter. Sp1-like sequences in the minimal promoter not only sustain basal promoter activity, but also mediate p<SUP>73</SUP>-induced activation of the promoter

Knockdown of optineurin by shRNA enhances TNFα-induced NF-κB activity.

<p>(A) HeLa cells were infected with Ad-shOptn1, Ad-shOptn2 or control viruses and after 72 hours cell lysates were subjected to western blotting using antibodies for optineurin, IκBα and p65 NF-κB. Control virus (AdC sh) expresses shRNA of unrelated sequence of same length. (B) HeLa cells were infected with Ad-shOptn1, Ad-shOptn2 or control viruses. After 48 h of infection these cells were transfected with NF-κB reporter plasmid (25 ng) along with β-galactosidase expression plasmid. After 22 hours of transfection the cells were treated with TNFα (10 ng/ml) for 4 hours. Cell lysates were then made for reporter assays. The data represent luciferase activities relative to untreated control taken as 1.0 (n = 3). (C) HeLa cells were infected with indicated adenoviruses and after 72 hours treated with TNFα for 6 min or left untreated. Cell lysates were subjected to western blot analysis using antibodies for IκB, optineurin and tubulin (loading control). AdC, control virus not expressing any shRNA.</p

NF-κB mediates optineurin gene expression.

<p>(A) NF-κB p65 activates optineurin promoter through NF-κB site. Optineurin minimal promoter constructs pGL-DP or pGL-mDP (100 ng) were transfected without or with NF-κB p65 expression plasmid (100 ng) in A549 cells. After 24 hours of transfection cell lysates were prepared for reporter assays. Luciferase activities relative to control pGL-DP (taken as 1.0) are shown (n = 3). (B) IκBα inhibits TNFα-induced NF-κB activity. pGL-DP was transfected without or with IκBα super repressor expression plasmid (100 ng). TNFα was added 6 hours after transfection. Luciferase activities relative to control are shown (n = 3). (C) Blocking of NF-κB activation inhibits TNFα-induced optineurin gene expression. A549 cells were preincubated with 25 µM MG132 or solvent DMSO (0.1%) for 30 minutes prior to treatment with TNFα. After 6 hours of treatment with TNFα, RNA was isolated and the level of optineurin mRNA was determined by real time RT-PCR analysis. GAPDH was used as a control. (D) A549 cells were treated with MG132 as in panel C and after 6 hours of treatment with TNFα cell lysates were subjected to Western blotting. (E) A549 cells were treated with 100 µg/ml of SN-50 peptide for 30 minutes prior to treatment with TNFα for 6 hours. The picture shows RT-PCR analysis for optineurin gene expression.</p

Optineurin and E50K mutant inhibit TNFα-induced NF-κB activity.

<p>(A) NF-κB-Luc reporter plasmid (25 ng) was transfected without or with optineurin expression plasmid (100 ng) in HeLa cells. After 22 hours of transfection cells were treated with TNFα for 4 hours. Luciferase activities relative to untreated control are shown (n = 3). (B) Western blot showing expression of optineurin and its mutants using HA tag antibody. Cdk2 was used as control. (C) Optineurin and E50K mutant inhibit TNFα-induced nuclear translocation of NF-κB p65. HeLa cells grown on coverslips were transfected with optineurin expression plasmid. After 24 hours of transfection cells were treated with TNFα for 30 min. The cells were then fixed and stained for optineurin (HA tag, FITC green) and p65 (Cy3, red) and visualized using a fluorescence microscope. (D) HeLa cells were infected with adenoviruses for expressing optineurin (Optn-AdV), its E50K mutant (E50K-AdV) or control virus (AdC). After 36 hours of infection, the cells were treated with TNFα for 6 min or 12 min or left untreated. Cell lysates were then prepared for western blotting with antibodies for IκBα, HA tag and Cdk2.</p

Induction of optineurin gene expression and promoter activation by TNFα.

<p>(A), A549 cells were treated with TNFα (10 ng/ml) for indicated time (3–24 hours). Total RNA was then isolated and the level of optineurin mRNA was determined by real time RT-PCR. GAPDH was used as a control. (B) Effect of TNFα on optineurin protein level. A549 cells were treated with TNFα (10 ng/ml) for indicated time. The cell lysates were then prepared for immunoblotting which was performed using antibodies against optineurin, IRF-1 and tubulin (loading control). The numbers at the top indicate relative amount of protein. Activation of optineurin promoter by TNFα in A549 cells (C) or HeLa cells (D). Cells grown in 24 well plates were transfected with 100 ng of optineurin promoter-reporter plasmid (full length construct pGL-FP or deletion construct pGL-DP) along with pCMV.SPORT β-gal plasmid. After 6 hours of transfection TNFα was added (10 ng/ml). After another 18 hours cell lysates were prepared for reporter assays. Luciferase activities relative to untreated control (taken as 1.0) are shown (n = 3) after normalizing with β-galactosidase enzyme activities.</p

A New Ken-Ken Puzzle Pattern Based Reconfiguration Technique for Maximum Power Extraction in Partial Shaded Solar PV Array

Solar Photovoltaic array may often be subjected to partial shading, which may lead to uneven row current and creates local maximum power point on the power-voltage characteristics. One of the effective approaches to dilute the concentration of partial shading is the array reconfiguration technique. This study proposes a ken-ken puzzle-based reconfiguration technique for $4\times 4$ total-cross-tied configuration to rearrange the position of modules within the array and to improve the maximum power under partial shading conditions. Further, the performance of the ken-ken puzzle arrangement is compared with the total-cross-tied configuration and existing reconfiguration techniques namely odd-even, Latin Square, and Sudoku reported in the literature. The performance of all these configurations is evaluated in terms of fill factor, mismatch loss, power loss, execution ratio, and performance enhancement ratio. The proposed ken-ken puzzle-based reconfiguration technique mitigates the occurrence of local maximum power point and eliminates the need for a complex algorithm to track the global maximum power point. The simulation result shows that the KK puzzle-based reconfiguration technique has obtained an improved PE of 10.85 &#x0025; compared to TCT configuration, followed by LS, Sudoku, and OE. Also, the experimental result shows the effectiveness of the ken-ken in diluting the effects of partial shading when the rows of the photovoltaic array are shaded. The ken-ken puzzle-based reconfiguration technique reduces the complexity, maintenance and increases reliability, scalability of the PV array