Overexpression of TDP-43 on p65 activation.

<p><b>A</b>. p65 (Green) is translocated into the cells’ nuclei (Blue) after 30 minutes of 10ng/ml TNF-α treatment (n = 3 repeats). <b>B</b>. MCF-7 cells were transfected with various doses (indicated on the right) of wild type TDP-43 tagged with mCherry expressing plasmids (middle panel, red) and were treated with 10ng/ml TNF-α for 30 minutes. In the top right corner, insets of higher magnification show the localization of p65 and nuclear TDP-43 staining (n≥3 independent experiments). <b>C</b>. The quantification of B. <b>D.</b> MCF-7 Cells were transfected with 4μg plasmid encoding TDP-43 tagged with mCherry or mCherry empty plasmid (control) and treated with 10ng/ml TNF-α for 30 minutes. Nuclear proteins were isolated and detected by western immunoblotting. Histone H3 was used as a nuclear marker. The relative density was the average of 3 individual experiments. <b>E</b>. MCF-7, Neuro 2a and BV2 cells were transfected with Wild type TDP-43 tagged with mCherry expressing plasmids (middle panel, red). MCF-7 and Neuro 2a cells were treated with 10ng/ml TNF-α for 30 minutes; BV2 cells were treated with 10μg/ml LPS for 30 minutes. Arrows indicate the blocked p65 nuclear translocation by Wt TDP-43 and stars indicate the normal p65 nuclear translocation with Wt TDP-43 overexpression. n≥3 independent experiments. <b>F</b>. The plasmid encoding TDP-43 tagged with mCherry or mCherry empty plasmid (control) was cotransfected along with NF-κB-luc (containing the wild type NF-κB-binding site). Cells were treated with 10ng/ml TNF-α for 30 minutes. Luciferase activity was measured after 24 hours. The plotted error bars represent mean±SEM from 3 independent experiments; *p < 0.05 compared to control, one-way ANOVA. ** p < 0.05 compared to the TNF-α treated control samples, Student's t test.</p

TDP-43 Inhibits NF-κB Activity by Blocking p65 Nuclear Translocation

<div><p>TDP-43 (TAR DNA binding protein 43) is a heterogeneous nuclear ribonucleoprotein (hnRNP) that has been found to play an important role in neurodegenerative diseases. TDP-43’s involvement in nuclear factor-kappaB pathways has been reported in both neurons and microglial cells. The NF-κB pathway targets hundreds of genes, many of which are involved in inflammation, immunity and cancer. p50/p65 (p50/RelA) heterodimers, as the major Rel complex in the NF-κB family, are induced by diverse external physiological stimuli and modulate transcriptional activity in almost all cell types. Both p65 and TDP-43 translocation occur through the classic nuclear transportation system. In this study, we report that TDP-43 overexpression prevents TNF-α induced p65 nuclear translocation in a dose dependent manner, and that this further inhibits p65 transactivation activity. The inhibition by TDP-43 does not occur through preventing IκB degradation but probably by competing for the nuclear transporter-importin α3 (KPNA4). This competition is dependent on the presence of the nuclear localization signal (NLS) in TDP-43. Silencing TDP-43 using a specific siRNA also increased p65 nuclear localization upon TNF-α stimulation, suggesting that endogenous TDP-43 may be a default suppressor of the NF-κB pathway. Our results indicate that TDP-43 may play an important role in regulating the levels of NF-κB activity by controlling the nuclear translocation of p65.</p></div

The competition of importin α3 (KPNA4) occurs in both the cytoplasm and nucleus.

<p>The nuclear and cytoplasmic proteins of MCF-7 cells were separated after overexpression of wild type TDP-43 and TNF-α stimulation. The top panel shows the protein levels of total TDP-43 in each fraction. N: nucleus. C: cytoplasm. The bottom shows that TDP-43 interacted with importin α3 (KPNA4). p < 0.05, Student's t test.</p

Overexpressing TDP-43 inhibits NF-κB activity by competing for the nuclear transporter.

<p><b>A</b>. Cells were transfected with wild type TDP-43 expressing plasmids (tagged with mCherry) or mCherry empty plasmid (control) and were then treated with 10ng/ml TNF-α for 30 minutes. Cell lysates were subjected to coimmunoprecipitation and western blot for the presence of p65 and TDP-43. 10% cell lysate were reserved as input. p65, TDP-43 and importin α3 (KPNA4) were used to detect input. n = 3 repeats. <b>B</b>. The relative density of p65 as shown in A (IP), *p<0.05. <b>C</b>. The relative density of wild type TDP-43 as shown in A (IP), *p<0.05.</p

The blockade of NF-κB nuclear translocation by TDP-43 can be prevented by overexpression of p65.

<p>MCF-7 cells were transfected with wild type TDP-43 expressing plasmids (top row) alone or cotransfected with TDP-43 and p65 expressing plasmids (bottom row). Then MCF-7 cells were treated with TNF-α for 30 minutes. The p65 was found co-localized with TDP-43 in the nuclei (bottom row). n = 3 independent experiments.</p

The NLS mutated TDP-43 does not affect p65 nuclear translocation.

<p>MCF-7 cells were transfected with TDP-43 ΔNLS expressing plasmids (middle panel, Red). After 30 minutes of TNF-α treatment, labeled p65 (Left panel, Green) was found in the nuclei. n = 3 independent experiments.</p

Overexpression of TDP-43 accelerates IκB degradation.

<p>Cells were transfected with wild type TDP-43 expressing plasmids (tagged with mCherry) or mCherry empty plasmid (control) and were treated with 10ng/ml TNF-α for the indicated periods. Total proteins were extracted for Western blot analysis using antibodies as indicated. n = 3 independent cultures; <b>A</b>. 30 min after TNF-α treatment, the degradation of IκB occurred in both control group and TDP-43 overexpression group. <b>B</b>. Overexpression of TDP-43 facilitates the degradation of IκB within 5 minutes after TNF-α treatment.</p

Schematic hypothetical modes for TDP-43 and p65 competition.

<p>The presence of TDP-43 in the cytosol may compete with p65 for importin α3 (cytoplasmic mode) and therefore to inhibit the nuclear localization of p65. An alternative mode (nuclear mode) shows that nuclear TDP-43 may occupy importin α3 to prevent its exit and therefore to reduce the availability of free importin α3 in the cytosol for p65 nuclear translocation.</p