The NF-κB, a transcriptional factor, regulates the production of numerous proteins including matrix metalloproteinases,
21,22 so we determined if SNC-121 regulated the expression levels and translocation of NF-κB to the nucleus. TNF-α treatment increased the expression of NF-κB to 179% ± 21% (
P = 0.033) and 139% ± 6% (
P = 0.006) at 6 and 24 hours, respectively (
Figs. 4A,
4B). Pretreatment with SNC-121 reduced the expression of NF-κB to 114% ± 10% (
P = 0.032) and 104% ± 3% (
P = 0.001) at 6 and 24 hours, respectively (
Figs. 4A,
4B). In parallel experiments, we also have observed an intense cytosolic staining of NF-κB in the resting ONH astrocytes. TNF-α treatment clearly translocated NF-κB to the nuclei, as determined by intense nuclear staining. In contrast, nuclear staining of NF-κB was reduced and dispersed to cytoplasm by SNC-121 treatment (
Fig. 5). NF-κB resides mainly in the cytosol in its inactive state, where it is bound with IκBα.
23,24 During activation, IκBα is phosphorylated and released from the complex and undergoes proteasome-dependent degradation. Subsequently, the freed NF-κB is translocated to the nucleus to induce the expression of target genes.
23 To test if NF-κB activation is mediated via IκBα-dependent pathway, we measured the effects of SNC-121 on TNF-α–induced IκBα phosphorylation. As shown in
Figure 6, TNF-α treatment increased the phosphorylation of IκBα to 344% ± 62% (
Fig. 6A) and 268% ± 38% (
Fig. 6B) over basal levels at 6 and 24 hours, respectively. TNF-α–induced IκBα phosphorylation was significantly reduced in the presence of SNC-121 (
Figs. 6A,
6B).