Activation of Nuclear Factor-B by Depolarization and Ca2؉ Influx in MIN6 Insulinoma Cells Ernesto Bernal-Mizrachi, Wu Wen, Michael Shornick, and M. Alan Permutt
The purpose of the current study was to determine whether nuclear factor-B (NF-B) activation is a com- -ponent of the depolarization/Ca2؉-dependent signaling slet -cell mass is a critical factor for the develop in -cells. MIN6 cells were transfected with a plasmid ment of type 1 and type 2 diabetes. -Cell mass is containing five tandem repeats of NF-B binding sites determined by the combined rates of neogenesis, linked to a luciferase reporter. The results of these replication, and apoptosis (1). In addition to growth experiments showed that KCl induced depolarization- I factors, the concentration of glucose in plasma has been activated NF- B–dependent transcription (3.8-fold at shown to be an important determinant of pancreatic -cell 45 mmol/l, P < 0.01) in a concentration-dependent mass by neogenesis and replication, and thus by function manner. Tumor necrosis factor-␣ (TNF-␣), a known (2). Glucose induces pleiotropic effects in islet -cells that inducer of NF- B signaling, activated this construct by 2ϩ 3.4-fold (P < 0.01). The response of NF-B to depolar- are depolarization and Ca dependent, and these effects -2؉ are potentially mediated by activation of multiple intracel ization was inhibited by the Ca -channel blocker vera- ϩ pamil and by the mitogen-activated protein kinase lular signaling pathways. Depolarization-induced Ca2 in- kinase (MEK) inhibitor PD98059 (70 and 62%, respec- flux has been implicated in the activation of CaM kinase tively). TNF-␣, glucose, and KCl treatment resulted in (CaMK)-II, CaMK-IV, protein kinase A (PKA) and C (PKC), inhibitory B␣ degradation by Western blot analysis. extracellular-regulated kinase (ERK), mitogen-activated TNF-␣ treatment and depolarization activation of protein kinase (MAPK), and phosphatidylinositol (PI)-3 ␣ NF- B differed significantly in that TNF- activation kinase in islet -cells (3–6). The associations between was not blocked by PD98059. Transfection with PKA, these activated pathways, their downstream targets, and MEK, and MEK kinase induced NF-B–dependent tran- the regulation of growth and survival has not been defined. scription by 20-, 90-, and 300-fold, respectively, suggest- 2ϩ ing that these pathways contribute to the activation in The relationships between Ca -activated pathways, the depolarization response. These findings demon- their downstream targets, and the pleiotropic effects of strate that depolarization/Ca2؉ influx, as well as TNF-␣ depolarization have not been defined (7). In hippocampal treatment, can activate NF-B–dependent transcription pyramidal neurons, activation of glutamate receptors and in pancreatic -cells, but by different signaling path- membrane depolarization lead to activation of nuclear ؉ ways. The current studies show that Ca2 signals in factor-B (NF-B), suggesting that depolarization and pancreatic -cells can activate transcription factors Ca2ϩinflux could regulate NF-B–dependent transcription involved in the regulation of cell cycle and apoptosis. (8). NF-B was originally identified in B lymphocytes, These findings now add NF-B to the list of depolariza-  where it stimulates transcription of the immunoglobulin tion-induced transcription factors in pancreatic -cells. Diabetes 51 (Suppl. 3):S484–S488, 2002 light chain (8). In its inactive form, NF- B consists of a three-subunit complex consisting of two (prototypical) subunits of 50 kDa (p50) and 65 kDa (p65; RelA) and an inhibitory B(IB) subunit (IB␣ or IB). However, depending on cell type, developmental stage, and environ- mental factors, cells may express other NF-B DNA- binding subunits (e.g., p52, c-Rel, and RelB) and IBs (e.g., Bcl-3 and IB⑀). The NF-B complex is located in the cytosol and is activated when IB is induced to dissociate from the complex. NF-B is activated by various extracel- lular signals, including cytokines, neurotrophic factors, From the Washington University School of Medicine, Division of Endocrinol- and neurotransmitters (8–11). Although in neuronal cells ogy, Diabetes and Metabolism, St. Louis, Missouri. Address correspondence and reprint requests to Ernesto Bernal-Mizrachi, this pathway has been associated with survival, the con- 2ϩ 660 S. Euclid Ave., Campus Box 8127, St. Louis, MO 63110. E-mail: sequences of the depolarization/Ca activation of this [email protected]. transcription factor are currently unknown. Received for publication 19 March 2002 and accepted in revised form 1 June 2002. The present study was intended to demonstrate whether CaMK, CaM kinase; DMEM, Dulbecco’s modified Eagle’s medium; ERK, the transcription factor NF-B is a downstream target of extracellular-regulated kinase; FBS, fetal bovine serum; IB, inhibitory B; 2ϩ KRBH, Krebs-Ringer bicarbonate HEPES; MAPK, mitogen-activated protein depolarization/Ca signaling in insulinoma cells. These kinase; MEK, MAPK kinase; MEKK, MEK kinase; NF-B, nuclear factor-B; PI, experiments are the first step in elucidating the signal phosphatidylinositol; PKA, protein kinase A; PKC, protein kinase C; TNF-␣, transduction pathways involved in the regulation of NF-B tumor necrosis factor-␣. 2ϩ  The symposium and the publication of this article have been made possible by Ca in pancreatic -cells. The results suggest that by an unrestricted educational grant from Servier, Paris. activation of NF-B–dependent transcription can be a
S484 DIABETES, VOL. 51, SUPPLEMENT 3, DECEMBER 2002 E. BERNAL-MIZRACHI AND ASSOCIATES
potential mechanism involved in the regulation of signal- ing by depolarization and Ca2ϩ influx.
RESEARCH DESIGN AND METHODS Plasmids. The cis-reporter plasmid 5X NF-B-LUC contains the luciferase reporter gene driven by a basic promoter element (TATA box) joined to five tandem repeats of consensus NF-B 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). Transient transfections. MIN6 cells were transfected by lipofectamine and Plus reagent (Gibco) using the suggested amounts of DNA according to the manufacturer’s protocol. Briefly, 1 ϫ 105 cells were plated in 12-well plates 3 days before transfection. Cells at ϳ60% 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). After3hofincubation, 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 incuba- tion 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 fireflytoRenilla luciferase. Luciferase assay. Cell lysis was performed using 200 l of passive lysis buffer (Promega). Firefly and Renilla luciferase were measured by the dual-luciferase reporter assay system (Promega) using 20 l of cell lysate. Luciferase activity was measured in a Monolight 3010 luminometer. Immunoblotting. 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 ϫ PBS, 0.1% FIG. 1. A: KCl and TNF- induce NF- B–dependent transcription in MIN6 insulinoma cell lines. NF-B–dependent transcription was as- SDS, 0.01 mol/l dithiothreitol, and one-half of a tablet of “complete” protease sessed by transfecting a construct containing five tandem repeats of a inhibitor cocktail (Boehringer Mannheim). After boiling, proteins were sepa- consensus NF-B binding site linked to a luciferase reporter (5X rated by electrophoresis through 10% polyacrylamide and 0.1% SDS gels and NF-B-LUC). MIN6 cells were transiently transfected and incubated as transferred to polyvinylidine fluoride membranes. Membranes were incubated described in RESEARCH DESIGN AND METHODS. After a 24-h preincubation, overnight at 4°C in blocking buffer containing 5% milk. Subsequently, the cells were continued in preincubation media containing 5 mmol/l membranes were hybridized at 4°C overnight in blocking buffer containing glucose and 2% FBS (control) or stimulated with 45 mmol/l KCl or 10 IB␣ antibody, with the dilutions recommended by the manufacturer (New ng/ml TNF-␣ for 6 h. B: Dose-dependent activation by KCl of NF-B– England Biolabs). After three washes at room temperature, the membranes dependent transcription. NF- B–dependent transcription was as- sessed as described in A. MIN6 insulinoma cells were transfected with were incubated in secondary horseradish peroxidase antibody for 1 h. After 20 ng of 5X NF-B-LUC as described in above. After culturing in regular washing for 1 h, immunodetection was performed with an enhanced chemi- media containing 5 mmol/l glucose and 2% FBS for 24 h, the cells were luminescence Western blotting detection system (Amersham) following the continued in the same media (control) or stimulated for 6 h with the manufacturer’s protocol. indicated concentrations of KCl. To control for transfection efficiency, 2 ng of the pRL-TK luciferase construct was used. The data are expressed as the fold induction over the luciferase activity at 5 mmol/l RESULTS glucose. The results are presented as the means ؎ SE of triplicates KCl and tumor necrosis factor-␣ induce NF-B–de- from one experiment that was repeated three times. P values were determined by Student’s t test for nonpaired data. *P < 0.05; **P < pendent transcription in MIN6 insulinoma cell lines. 0.01. Transcriptional activation of NF-B has been defined by using several methods that include the use of NF-B– these experiments suggest that both depolarization and dependent reporter assays (12–15). To establish a model cytokines could induce NF-B–dependent transcriptional system to study NF-B–dependent transcription in pancre- activation in MIN6 insulinoma cells. atic -cells, MIN6 cells were transfected with a construct Time course for IB␣ degradation by glucose, KCl, containing five tandem repeats of a consensus NF-B and TNF-␣ in MIN6 cells. In other systems it has been binding site linked to a luciferase reporter (plasmid 5X observed that NF-B is usually stored in the cytosol in its NF-B-LUC). Depolarization induced by 45 mmol/l KCl inactive form, bound to the inhibitory unit IB␣, which resulted in activation of this construct (3.8-fold, P Ͻ 0.01) prevents DNA binding and nuclear translocation. NF-B (Fig. 1A), suggesting that this treatment induces NF-B– activating agents initiate phosphorylation of IB␣, induc- dependent transcription. Tumor necrosis factor-␣ (TNF- ing polyubiquination at multiple sites and tagging the ␣), a cytokine known to activate NF-B–dependent subunit for degradation by a 26S proteosome complex. To transcription in -cells, also induced this reporter system further substantiate the results obtained in the promoter (3.4-fold, P Ͻ 0.01) (Fig. 1A) (16–18). As shown in Fig. 2B, reporter experiments, we examined the effects of KCl and KCl-induced depolarization activated NF-B–dependent glucose in the degradation of the IB␣ subunit. Extracts transcription in a dose-dependent manner. The results of from MIN6 cells were obtained at different time points
DIABETES, VOL. 51, SUPPLEMENT 3, DECEMBER 2002 S485 NF-B ACTIVATION BY DEPOLARIZATION
FIG. 2. Time course for IB␣ degradation by glucose, KCl, and TNF-␣ in MIN6 cells. MIN6 cells were preincubated in KRBH buffer with 2% albumin for 1 h followed by incubation with 25 mmol/l glucose (A), 45 mmol/l KCl (B), or 10 ng/ml TNF-␣ (C) for the indicated times. Protein was subjected to Western blotting with an anti-IB␣ antibody, as described in RESEARCH DESIGN AND METHODS. The results are representa- tive of two different experiments performed in duplicate. after stimulation with glucose or KCl. As shown in Fig. 2A, glucose treatment induced IB␣ degradation after 30 min of treatment. Similar results were obtained with KCl (Fig. 2B). Treatment with TNF-␣, a known activator of IB␣ degradation, resulted in rapid degradation of IB␣ (Fig. 2C). The results of these experiments indicated that both KCl and glucose, like TNF-␣, resulted in IB␣ degradation in MIN6 cells. These findings provided further evidence for the induction of NF-B signaling by these agents. KCl induction of NF-B–dependent transcription is a -Ca2؉-dependent process. To evaluate whether the ef fects of KCl on the NF-B–dependent reporter required extracellular Ca2ϩ influx, the L-type calcium-channel in- hibitor verapamil was used. As shown in Fig. 3A, depolar- ization-induced NF-B–dependent transcriptional activa- tion by KCl (3.6-fold, P ϭ 0.01) was inhibited 80% (P Ͻ 0.01) by the addition of verapamil at 10 mol/l. No significant NF-B–dependent transcriptional activation was observed when verapamil was used alone. These results suggest that Ca2ϩ influx from the extracellular compartment is a major component in depolarization -activation of NF-B–dependent transcription. FIG. 3. A. KCl induction of NF-B–dependent transcription is a Ca2؉ dependent process. MIN6 cells were transfected with the same concen- NF- B–dependent transcription by depolarization tration of plasmids and preincubated as described under Fig. 1. Before but not by TNF-␣ is inhibited by MEK inhibition. stimulation with 45 mmol/l KCl, MIN6 cells were exposed to 10 mol/l Calcium regulation of gene transcription in various cell verapamil for 30 min where indicated. B: NF-B–dependent transcrip- tion by depolarization but not by TNF-␣ is inhibited by MEK inhibition. types involves multiple signaling pathways (4). These MIN6 insulinoma cells were transfected with 10 ng of 5X NF-B-LUC, include the Ser/Thr kinases PKA, MAPK, and the CaMKs. as described above. After culturing in regular media containing 5 mmol/l glucose and 2% FBS for 24 h, the cells were continued in the We and others have demonstrated an increase in ERK1 same media or stimulated for 6 h with KCl (45 mmol/l) and TNF-␣ (10 and -2 MAP kinase activity by glucose- and KCl-induced ng/ml) in the presence or absence of the MEK (PD98059 50 mol/l) depolarization (3,19–21). In other systems, NF-B activa- inhibitor. The MEK inhibitor was added to the media 1 h before stimulation with KCl or TNF-␣. C: Activation of the MEK and PKA tion is modulated through the MEK/ERK pathway (22). To pathway induces NF-B–dependent transcription. MIN6 insulinoma determine whether this pathway is important for depolar- cells were transfected with 10 ng of 5X NF-B-LUC, as described. ization induction of NF-B–dependent transcription in Cotransfection with a constitutively active form of PKA (50 ng), MEK (50 ng), and MEKK (50 ng) was performed. To control for transfection MIN6 cells, we used the specific MEK inhibitor PD98059. efficiency, 2 ng of pRL-TK luciferase construct was used. The data are Depolarization by KCl resulted in NF-B–dependent tran- expressed as the fold induction over the luciferase activity at 5 mmol/l Ͻ glucose. The results are presented as the means ؎ SE of triplicates scription that was inhibited by PD98059 (62%, P 0.01) from one experiment that was repeated three times. P values were (Fig. 3B), suggesting that this pathway is an important determined by Student’s t test for nonpaired data. **P < 0.01; ***P < mediator in this response. Activation of NF-B–dependent 0.001.
S486 DIABETES, VOL. 51, SUPPLEMENT 3, DECEMBER 2002 E. BERNAL-MIZRACHI AND ASSOCIATES transcription by TNF-␣ treatment was not inhibited by cineurin- and calpain-dependent manner (14,29–31). Ex- PD98059 (Fig. 3B). A culture of MIN6 in the presence of periments intended to assess the role of calcineurin and PD98059 alone had no effect on transcriptional activity of calpain in Ca2ϩ induction of NF-B–dependent transcrip- NF-B (data not shown). tion need to be performed in -cells. Activation of the ERK pathway results from phosphor- The mechanisms by which extracellular and intracellu- ylation and activation of MEK1. Further evidence support- lar stimuli trigger NF-B activation are not fully under- ing the role of the MAP kinase in NF-B–dependent stood, but several common features have been described. transcription was assessed by cotransfection experiments Potent activators, such as TNF-␣ and interleukin-1, induce with a plasmid encoding MEK1 kinase. As shown in Fig. NF-B transcription by rapid (within minutes) degradation 3C, overexpression of MEK1 and MEKK resulted in a of IB␣. Although degradation of IBissufficient to cause striking increase in NF-B–dependent transcriptional ac- nuclear translocation, subsequent events can affect NF- tivity (98-fold and 300-fold, respectively). The results of B’s ability to activate transcription. Activation of NF-B these experiments suggest that activation of the MEK/ERK directly by phosphorylation of p65 independent of cytoso- pathway in insulinoma cells is an important mechanism in lic degradation of IB␣ has been demonstrated, suggesting the activation of NF-B–dependent transcription. that phosphorylation of NF-B proteins may modulate Early gene transcription induced by glucose in -cells is their transcriptional activity (32–39). Although the results mediated by multiple signaling pathways that include the of the present study indicated that IB degradation is activation of PKA (20,23). The effects of glucose in PKA associated with NF-B–dependent transcription in pancre- induction could be dependent (or not) on Ca2ϩ influx, but atic islet -cells, the results of these studies do not exclude because Ca2ϩ was required for depolarization induction of a role of IB–independent pathways in Ca2ϩ induction of NF-B–dependent transcription, we tested the hypothesis NF-B–dependent transcriptional activation. that PKA could be a factor in mediating depolarization Proteins of the NF-B family have been implicated in induction of NF-B–dependent transcription. Evidence multiple processes, such as ontogeny of the immune that this mechanism may be operating came from our system, immune responses to pathogens, and oncogenesis observation that activation of PKA by forskolin induced (40). In other tissues, activation of NF-B signaling can NF-B–dependent transcription (data not shown). As lead to expression of genes that are also induced in shown in Fig. 3B, a cotransfection plasmid encoding the response to immune mediators, stress, inflammation, and catalytic unit of PKA resulted in a robust increase in inhibitors of apoptosis (37,40–42). However, at the NF-B–dependent transcription (21-fold, P Ͻ 0.001). present time, the consequences of depolarization activa- tion of NF-B transcription in pancreatic -cells have yet to be uncovered. These questions could be addressed in DISCUSSION animal models with gain and loss of function of NF-B Previous studies have shown that NF-B is an important signaling. mediator of cytokine signaling in -cells. The importance of the current studies is the demonstration that KCl- ACKNOWLEDGMENTS induced depolarization can also result in induction of The authors would like to thank Gary Skolnick for prepa- NF-B–dependent transcription. This activation requires 2ϩ 2ϩ ration of the manuscript. This work was supported in part Ca influx through the L-type Ca channels. These by National Institutes of Health Grant DK16746 (to M.A.P.) studies also show that the MEK/ERK pathway is an and the Diabetes Research and Training Center for tech- important component of transcriptional activation of nical support. NF-B by depolarization and Ca2ϩ influx. Interestingly, ␣ TNF- treatment, unlike depolarization, activated this REFERENCES pathway in a MEK/ERK-independent fashion, suggesting 1. Bernard C, Berthault MF, Saulnier C, Ktorza A: Neogenesis vs. apoptosis as that NF-B–dependent transcription in islet -cells can be main components of pancreatic beta cell mass changes in glucose-infused induced by multiple pathways. normal and mildly diabetic adult rats. FASEB J 13:1195–1205, 1999 Depolarization activation of NF-B–dependent tran- 2. 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