The NF-κB pathway is not stimulated by glutamate or pifithrin- α

4. Results

4.3. The NF-κB pathway is not stimulated by glutamate or pifithrin- α

It is commonly accepted, that p53 induced cell death relies to a great extent on the enhanced expression of various pro-apoptotic factors under transcriptional control of p53, like BAX, Bid, PUMA and Noxa (29). In addition, a complementary mechanism has been identified in tumor cells and neuronal cells that involves concomitant repression of NF-κB transcriptional activity by p53 through competitive binding of transcriptional cofactors such as p300 and CREB binding protein (CBP) (36; 50).

Consequently, survival signaling by NF-κB is increasingly impaired in the event of cell death, mirrored by decreased levels of anti-apoptotic factors, such as e.g. IAPs and MnSOD (33; 37). Following this concept of reciprocal transcriptional inhibition between p53 and NF-κB, the present study sought to elucidate how neuroprotection induced by inhibition of p53 affected NF-κB activity.

In contrast to previous work, the present investigation relied on HT-22 cells to investigate this question. The HT-22 cells were treated with glutamate and PFT (10µM) over different time intervals. Protein levels of IkB-α and pIkB-α were determined to estimate induction NF-κB activity along the canonical pathway.

Activation of this pathway is reflected by initially increased levels of phosphorylated IkB-α, followed by IkB-α degradation and subsequent release of the NF-κB transcription factors from the inhibitory IkB-complex.

As shown in figure 11 whole protein extracts were taken from HT-22 cells at the indicated time points and analyzed by Western blot for IkB-α and pIkB-α levels.

Interestingly, neither treatment caused a change in protein levels of IkB-α and pIkB-α, indicating that the canonical NF-κB pathway was not initiated in the present model system.

Figure 11: Glutamate treatment and PFT do not stimulate the canonical NF-κB pathway in HT-22 cells. (A, B) HT-22 cells were grown in 6 well plates (21,000 cells/cm²) treated with PFT (10µM) and glutamate over the indicated time course. Protein extracts were taken and analyzed by Western blot, probing for pIκB-α, IκB-α and Aktin as a loading control. All experiments were repeated three times.

Ctrl. PFT 6h PFT

12h

I!B-"

P-I!B-"

Aktin PFT15h PFT

18h GLUT 6h GLUT

12h GLUT 15h

P-I!B I!B

Aktin Ctrl. 2h 4h 6h 8h TNF-"

Glutamate

A B

Besides the most prominent canonical activation of NF-κB alternative pathways have been described, which lead to transcriptional activation of NF-κB without affecting pIκB-α and IκB-α levels. To cover these mechanisms as well, a broad approach was applied studying the individual role of all NF-κB subunits in response to PFT and glutamate.

As mentioned previously, the transcriptional response by NF-κB can be mediated by five isoform subunits, which dimerise upon activation, translocate from the cytoplasm to the nucleus and bind to NF-κB response elements for induction of transcription.

Consequently, the cytosolic/nuclear distribution of any given subunits is a first approach for estimating the transcriptional activity.

In order to investigate the diverse role of the different NF-κB subunits in HT-22 cells, all transcription factors were stained, applying immunocytochemistry and analyzed by confocal microscopy. Counterstaining of the nuclei with DAPI allowed evaluating nuclear translocation.

HT-22 cells were treated with glutamate and PFT in ibidi-slides and fixed 12 h later using PFA. In HT-22 cells, all NF-κB subunits (p50, p52, p65, cRel and RelB) were found in the cytosol but not in the nucleus under control conditions (Figure 12).

Interestingly, no marked translocation of either subunit was detected in the cells when stimulated with glutamate or PFT, showing the same distribution pattern as control cultures.

PFT mediated protection against glutamate toxicity was reflected well by unharmed cell bodies and nuclei as opposed to shrunken cells and pyknotic nuclei in the glutamate-challenged cells. The distribution of NF-κB subunits, however, was unchanged in cells protected by PFT compared to controls. These findings clearly indicated that the p53 inhibitor PFT could not enhance transcriptional activation of NF-κB in HT-22 cells and the associated protective effect was unlikely mediated by NF-κB transcriptional activity.

4 Results 52

Figure 12: NF-κB subunits do not show any sign of nuclear translocation after treatment with glutamate or PFT in HT-22 cells (ICC analysis). HT-22 cells were treated with PFT (10µM) and glutamate. NF-κB subunits were stained by immunocytochemistry (red) and nuclei were counterstained with DAPI (blue). The cytosolic/nuclear distribution pattern of HT-22 cells was not markedly altered by PFT or glutamate.

Control