Tumor necrosis factor-α induces transcriptional activation of nuclear factor-κB in insulin-producing β-cells.

We previously showed that tumor necrosis factor-α (TNF-α) induces the dysregulation of intracellular calcium [Ca(2+)](i) in β-cells by decreasing the levels of the cytoplasmic Ca(2+) binding protein calbindin-D(28k). The purpose of the present study was to test the hypothesis that TNF-α-induced dysregulation of [Ca(2+)](i) in insulin-producing β-cells causes proteolytic degradation of IκBα and consequently leads to the transcriptional activation of nuclear factor-κB (NF-κB). To test this hypothesis, rat insulinoma (RINr 1046-38) cells, which are an insulin-secreting transformed β-cell line that constitutively expresses calbindin-D(28k), were treated with increasing concentrations of TNF-α. Using the FunctionELISA procedure to measure degradation of the IκBα subunit as Phospho-IκBα, it was found that, while in the control RIN cell lysate there was no Phospho-IκBα present, in the RIN cells exposed to 2, 5, 10, 20 and 30 ng/ml TNF-α, 17.176±2.85, 17.292±4.35, 53.77±5.63, 30.58±4.89 and 12±3.27 ng/ml Phospho-IκBα/mg of total cell protein was observed, respectively (n=3, P<0.05). Upon treatment of RIN cells with 2, 5, 10, 20 and 30 ng/ml TNF-α, the relative increases in the NF-κB transcriptional activities based on the DNA binding activity of NF-κB determined using an ELISA-based kit were 6.86±0.76-, 8.42±1.27-,7.8±2.32-, 10.28±1.96- and 6.3±1.57-fold, respectively (n=3, P<0.05). The nuclear translocation of NF-κB measured by immunofluorescence showed that, while the ratio of fluorescence in nuclei to that in the cytoplasm of untreated RIN cells was 0.2078±0.0778 (n=11), in RIN cells treated with 10 ng/ ml TNF-α, the ratio was 0.6267±0.1186 (n=11), indicating a statistically significant increase (P<0.05) in the nuclear translocation of NF-κB. These observations suggest that, in insulin-producing β-cells, the TNF-α-induced degradation of IκBα leads to nuclear translocation and the transcriptional activation of NF-κB.