Activation of Nuclear Factor-κB by Depolarization and Ca²⁺ Influx in MIN6 Insulinoma Cells

The cis-reporter plasmid 5X NF-κB-LUC contains the luciferase reporter gene driven by a basic promoter element (TATA box) joined to five tandem repeats of consensus NF-κB binding sites linked to a luciferase reporter (Stratagene). The plasmid containing the catalytic unit of PKA (pFC-PKA), MAPK kinase (MEK)-1, and MEK kinase (MEKK) was purchased from Stratagene. The pRL-TK control vector contains the herpes simplex virus thymidine kinase promoter upstream of Renilla luciferase (Promega).

MIN6 cells were transfected by lipofectamine and Plus reagent (Gibco) using the suggested amounts of DNA according to the manufacturer’s protocol. Briefly, 1 × 10⁵ cells were plated in 12-well plates 3 days before transfection. Cells at ∼60% confluence were transfected by mixing the indicated amount of DNA described in the figure legends and a lipid mixture containing a 1:2 ratio of lipofectamine and Plus reagent in 1 ml of OPTI-MEM media (Gibco). After 3 h of incubation, 0.5 ml of Dulbecco’s modified Eagle’s medium (DMEM) containing 5 mmol/l glucose and 2% serum was added to the cells. After 12 h, the medium-DNA complexes were replaced by preincubation media containing DMEM with 5 mmol/l glucose and 2% fetal bovine serum (FBS), and the cells were left for 24 h. At the end of the 24-h period, the specific stimulating agent was added to the media, and the cells were harvested 6 h later. For the overexpression experiments with pFC-PKA, MEK, and MEKK, MIN6 cells were transfected as above, followed by incubation in DMEM containing 5 mmol/l glucose and 2% FBS for 30 h until harvesting. Total DNA was maintained at a constant level in all of the transfection experiments by using the empty vector of the respective cDNA to be overexpressed. To correct for differences in transfection efficiencies, 2 ng of pRL-TK Renilla luciferase plasmid was simultaneously transfected. All results are normalized for transfection efficiency and expressed as the ratio of firefly to Renilla luciferase.

Cell lysis was performed using 200 μl of passive lysis buffer (Promega). Firefly and Renilla luciferase were measured by the dual-luciferase reporter assay system (Promega) using 20 μl of cell lysate. Luciferase activity was measured in a Monolight 3010 luminometer.

MIN6 cells were preincubated in Krebs-Ringer bicarbonate HEPES (KRBH) buffer and 2% albumin for 1 h, followed by stimulation with the indicated agents. Cells were lysed with buffer containing 1 × PBS, 0.1% SDS, 0.01 mol/l dithiothreitol, and one-half of a tablet of “complete” protease inhibitor cocktail (Boehringer Mannheim). After boiling, proteins were separated by electrophoresis through 10% polyacrylamide and 0.1% SDS gels and transferred to polyvinylidine fluoride membranes. Membranes were incubated overnight at 4°C in blocking buffer containing 5% milk. Subsequently, the membranes were hybridized at 4°C overnight in blocking buffer containing IκBα antibody, with the dilutions recommended by the manufacturer (New England Biolabs). After three washes at room temperature, the membranes were incubated in secondary horseradish peroxidase antibody for 1 h. After washing for 1 h, immunodetection was performed with an enhanced chemiluminescence Western blotting detection system (Amersham) following the manufacturer’s protocol.

Transcriptional activation of NF-κB has been defined by using several methods that include the use of NF-κB-dependent reporter assays (12–15). To establish a model system to study NF-κB-dependent transcription in pancreatic β-cells, MIN6 cells were transfected with a construct containing five tandem repeats of a consensus NF-κB binding site linked to a luciferase reporter (plasmid 5X NF-κB-LUC). Depolarization induced by 45 mmol/l KCl resulted in activation of this construct (3.8-fold, P < 0.01) (Fig. 1A), suggesting that this treatment induces NF-κB-dependent transcription. Tumor necrosis factor-α (TNF-α), a cytokine known to activate NF-κB-dependent transcription in β-cells, also induced this reporter system (3.4-fold, P < 0.01) (Fig. 1A) (16–18). As shown in Fig. 2B, KCl-induced depolarization activated NF-κB-dependent transcription in a dose-dependent manner. The results of these experiments suggest that both depolarization and cytokines could induce NF-κB-dependent transcriptional activation in MIN6 insulinoma cells.

In other systems it has been observed that NF-κB is usually stored in the cytosol in its inactive form, bound to the inhibitory unit IκBα, which prevents DNA binding and nuclear translocation. NF-κB activating agents initiate phosphorylation of IκBα, inducing polyubiquination at multiple sites and tagging the subunit for degradation by a 26S proteosome complex. To further substantiate the results obtained in the promoter reporter experiments, we examined the effects of KCl and glucose in the degradation of the IκBα subunit. Extracts from MIN6 cells were obtained at different time points after stimulation with glucose or KCl. As shown in Fig. 2A, glucose treatment induced IκBα degradation after 30 min of treatment. Similar results were obtained with KCl (Fig. 2B). Treatment with TNF-α, a known activator of IκBα degradation, resulted in rapid degradation of IκBα (Fig. 2C). The results of these experiments indicated that both KCl and glucose, like TNF-α, resulted in IκBα degradation in MIN6 cells. These findings provided further evidence for the induction of NF-κB signaling by these agents.

To evaluate whether the effects of KCl on the NF-κB-dependent reporter required extracellular Ca²⁺ influx, the L-type calcium-channel inhibitor verapamil was used. As shown in Fig. 3A, depolarization-induced NF-κB-dependent transcriptional activation by KCl (3.6-fold, P = 0.01) was inhibited 80% (P < 0.01) by the addition of verapamil at 10 μmol/l. No significant NF-κB-dependent transcriptional activation was observed when verapamil was used alone. These results suggest that Ca²⁺ influx from the extracellular compartment is a major component in depolarization activation of NF-κB-dependent transcription.

Calcium regulation of gene transcription in various cell types involves multiple signaling pathways (4). These include the Ser/Thr kinases PKA, MAPK, and the CaMKs. We and others have demonstrated an increase in ERK1 and -2 MAP kinase activity by glucose- and KCl-induced depolarization (3,19–21). In other systems, NF-κB activation is modulated through the MEK/ERK pathway (22). To determine whether this pathway is important for depolarization induction of NF-κB-dependent transcription in MIN6 cells, we used the specific MEK inhibitor PD98059. Depolarization by KCl resulted in NF-κB-dependent transcription that was inhibited by PD98059 (62%, P < 0.01) (Fig. 3B), suggesting that this pathway is an important mediator in this response. Activation of NF-κB-dependent transcription by TNF-α treatment was not inhibited by PD98059 (Fig. 3B). A culture of MIN6 in the presence of PD98059 alone had no effect on transcriptional activity of NF-κB (data not shown).

Activation of the ERK pathway results from phosphorylation and activation of MEK1. Further evidence supporting the role of the MAP kinase in NF-κB-dependent transcription was assessed by cotransfection experiments with a plasmid encoding MEK1 kinase. As shown in Fig. 3C, overexpression of MEK1 and MEKK resulted in a striking increase in NF-κB-dependent transcriptional activity (98-fold and 300-fold, respectively). The results of these experiments suggest that activation of the MEK/ERK pathway in insulinoma cells is an important mechanism in the activation of NF-κB-dependent transcription.

Early gene transcription induced by glucose in β-cells is mediated by multiple signaling pathways that include the activation of PKA (20,23). The effects of glucose in PKA induction could be dependent (or not) on Ca²⁺ influx, but because Ca²⁺ was required for depolarization induction of NF-κB-dependent transcription, we tested the hypothesis that PKA could be a factor in mediating depolarization induction of NF-κB-dependent transcription. Evidence that this mechanism may be operating came from our observation that activation of PKA by forskolin induced NF-κB-dependent transcription (data not shown). As shown in Fig. 3B, a cotransfection plasmid encoding the catalytic unit of PKA resulted in a robust increase in NF-κB-dependent transcription (21-fold, P < 0.001).

Previous studies have shown that NF-κB is an important mediator of cytokine signaling in β-cells. The importance of the current studies is the demonstration that KCl-induced depolarization can also result in induction of NF-κB-dependent transcription. This activation requires Ca²⁺ influx through the L-type Ca²⁺ channels. These studies also show that the MEK/ERK pathway is an important component of transcriptional activation of NF-κB by depolarization and Ca²⁺ influx. Interestingly, TNF-α treatment, unlike depolarization, activated this pathway in a MEK/ERK-independent fashion, suggesting that NF-κB-dependent transcription in islet β-cells can be induced by multiple pathways.

Depolarization activation of NF-κB-dependent transcription by 45 mmol/l KCl could be the result of an osmotic effect. Previous experiments using mannitol or NaCl to control for osmolality did not activate the MAP/ERK pathway, which has been shown to be involved in the activation of NF-κB by depolarization in the current studies (20). Although both glucose and KCl treatment were shown to result in IκBα degradation, a process associated with NF-κB activation, the direct effects of glucose on NF-κB-dependent transcription were not assessed. Thus, the glucose effect on IκBα degradation could involve alternative pathways other than depolarization and Ca²⁺ influx.

Increase of Ca²⁺ influx is an important inducer of NF-κB signaling in neuronal cells (24–28). The mechanism by which Ca²⁺ influx regulates NF-κB in non-β-cells is unclear, and there are data to suggest that activation of IκB kinase by Ca²⁺ signaling could be mediated in a calcineurin- and calpain-dependent manner (14,29–31). Experiments intended to assess the role of calcineurin and calpain in Ca²⁺ induction of NF-κB-dependent transcription need to be performed in β-cells.

The mechanisms by which extracellular and intracellular stimuli trigger NF-κB activation are not fully understood, but several common features have been described. Potent activators, such as TNF-α and interleukin-1, induce NF-κB transcription by rapid (within minutes) degradation of IκBα. Although degradation of IκB is sufficient to cause nuclear translocation, subsequent events can affect NF-κB’s ability to activate transcription. Activation of NF-κB directly by phosphorylation of p65 independent of cytosolic degradation of IκBα has been demonstrated, suggesting that phosphorylation of NF-κB proteins may modulate their transcriptional activity (32–39). Although the results of the present study indicated that IκB degradation is associated with NF-κB-dependent transcription in pancreatic islet β-cells, the results of these studies do not exclude a role of IκB-independent pathways in Ca²⁺ induction of NF-κB-dependent transcriptional activation.

Proteins of the NF-κB family have been implicated in multiple processes, such as ontogeny of the immune system, immune responses to pathogens, and oncogenesis (40). In other tissues, activation of NF-κB signaling can lead to expression of genes that are also induced in response to immune mediators, stress, inflammation, and inhibitors of apoptosis (37,40–42). However, at the present time, the consequences of depolarization activation of NF-κB transcription in pancreatic β-cells have yet to be uncovered. These questions could be addressed in animal models with gain and loss of function of NF-κB signaling.

The authors would like to thank Gary Skolnick for preparation of the manuscript. This work was supported in part by National Institutes of Health Grant DK16746 (to M.A.P.) and the Diabetes Research and Training Center for technical support.