MAPK Kinase Kinase-1 Is Essential for Cytokine-Induced c-Jun NH2-Terminal Kinase and Nuclear Factor-κB Activation in Human Pancreatic Islet Cells
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Figures
- FIG. 1.
Cytokine-induced JNK phosphorylation and NF-κB activation is potentiated by MEKK-1 overexpression in βTC-6 cells. A: βTC-6 cells were transfected with GFP, GFP + wild-type MEKK-1, or GFP + kinase inactive mutant (mut) MEKK-1. The GFP-expressing cells were enriched by fluorescence-activated cell sorting. Two days after the transfection and cell sorting procedure, the cells were lysed, and proteins were separated by SDS gel electrophoresis and analyzed by immunoblotting with MEKK-1 and ERK antibodies. A representative blot from one out of three experiments is shown. B: βTC-6 cells transiently overexpressing GFP, GFP + wild-type MEKK-1, or GFP + mut MEKK-1 were either left untreated or treated with a mixture of cytokines (50 units/ml IL-1β, 1,000 units/ml IFN-γ, and 1,000 units/ml TNF-α) 2 days after the transfection and cell sorting procedure. After 30 min of cytokine exposure, the cells were lysed and proteins were separated by SDS gel electrophoresis and analyzed by immunoblotting with phospho-specific antibodies against JNK, ERK, or p38. Results from immunoblots were quantified by densitometry. Values of phospho-protein bands were related to those of non–phospho-specific protein bands. Data are presented as means ± SE for three individual experiments. *P < 0.05 using one-way ANOVA followed by Student's t test. C: βTC-6 cells transiently overexpressing GFP, GFP + wild-type MEKK-1, or GFP + mut MEKK-1 were either left untreated or treated with a cytokine mixture (50 units/ml IL-1β, 1,000 units/ml IFN-γ, and 1,000 units/ml TNF-α). After 30 min of cytokine exposure, the cells were lysed, and proteins were separated by SDS gel electrophoresis and analyzed by immunoblotting with p65 and IκB antibodies (top panels). Results from immunoblots as the one shown in the top panel were quantified by densitometry (bottom panel). Values of IκB bands were related to those of p65 bands. Data are presented as means ± SE for three individual experiments. *P < 0.05 using one-way ANOVA followed by Student's t test.
- FIG. 2.
Transient overexpression of MEKK-1 augments cytokine-induced cell death in βTC-6 cells. βTC-6 cells transiently overexpressing GFP, GFP + wild-type MEKK-1, or GFP + mut MEKK-1 were either left untreated or treated with a mixture of cytokines (50 units/ml IL-1β, 1,000 units/ml IFN-γ, and 1,000 units/ml TNF-α) for 48 h corresponding to days 3–4 after the transfection and cell sorting procedure. The cells were then analyzed for cell death by fluorescence microscopy. Results from three separate observations are presented as means ± SE. *P < 0.05 and **P < 0.01, respectively, using one-way ANOVA and Student's t test.
- FIG. 3.
Cytokine treatment induces MEKK-1 T1383 phosphorylation in βTC-6 cells. βTC-6 cells were either left untreated or treated with a mixture of cytokines (50 units/ml IL-1β, 1,000 units/ml IFN-γ, and 1,000 units/ml TNF-α) for 0.5, 1, or 3 h. After cytokine treatment, the cells were lysed and immunoprecipitated (IP) using MEKK-1 antibody or rabbit IgG as control. The immunoprecipitated proteins were separated by SDS gel electrophoresis and analyzed by immunoblotting using T1383 phospho–MEKK-1 and MEKK-1 antibodies (A). Results from immunoblots, as the one shown in the top panel, were quantified by densitometry (B). MEKK-1 phosphorylation was determined by relating P-MEKK-1 bands to those of total MEKK-1. Data are presented as means ± SE for four individual experiments. *P < 0.05 vs. control using Student's t test.
- FIG. 4.
Effects of MEKK-1 d-siRNA on MEKK-1 levels and JNK, p38, ERK, or MKK4 activation in dispersed human islet cells. A: Dispersed human islet cells were treated with GL-3 or MEKK-1 d-siRNA. Two days after the d-siRNA treatment, the cells were lysed, and proteins were separated by SDS gel electrophoresis and analyzed by immunoblotting with MEKK-1 antibodies. A representative blot from one out of two experiments is shown. B: Dispersed human islet cells were transfected with GL3 d-siRNA or MEKK-1 d-siRNA and either left untreated or treated with a mixture of cytokines (50 units/ml IL-1β, 1,000 units/ml IFN-γ, and 1,000 units/ml TNF-α). After the 30-min cytokine exposure, the cells were lysed, and proteins were separated by SDS gel electrophoresis and analyzed by immunoblotting with phospho-specific JNK, ERK, and p38 antibodies. The results were quantified by densitometric scanning of three experiments and expressed as means ± SE. C: Dispersed human islet cells were treated with GL-3 or MEKK-1 d-siRNA. Two days after the d-siRNA treatment, the cells were exposed to 50 units/ml IL-1β. After 30 min of IL-1β treatment, the cells were lysed, and proteins were separated by SDS gel electrophoresis and analyzed by immunoblotting. MKK4 activation was determined by calculating the ratios between P-MKK4 intensities and ERK intensities. Values are means ± SE for three observations.
- FIG. 5.
MEKK-1 is required for IκB degradation in human islet cells. Dispersed human islet cells were transfected with GL3 d-siRNA or MEKK-1 d-siRNA. Two days after the d-siRNA treatment, the cells were incubated with 50 units/ml IL-1β for 30 min. The cells were then lysed, and proteins were separated by SDS gel electrophoresis and analyzed by immunoblotting with p65 and IκB antibodies (top panel). Results from immunoblots as the one shown in the top panel were quantified by densitometry. Intensities of IκB and p65 bands were used for calculation of IκB over p65 ratios. Data are presented as means ± SE for three individual experiments. *P < 0.05 using one-way ANOVA followed by Student's t test.
- FIG. 6.
MEKK-1 is required for IL-1β–induced nuclear translocation of NF-κB. βTC-6 cells (A) and human islet cells (B) were treated with GL3 or MEKK-1 d-siRNA. Two days after the d-siRNA treatment, the cells were exposed to 50 units/ml IL-1β for 30 min. Nuclear extracts were prepared and NF-κB activity was determined by EMSA. The last lane contains nuclear extracts from IL-1β–exposed cells and the p65 antibody to induce a supershift. The bottom panels show densitometric scanning results. Results are means ± SE for three separate experiments. *P < 0.05 vs. GL3 + IL-1β using Student's t test.
- FIG. 7.
MEKK-1 knockdown attenuates IL-1β–induced iNOS induction in βTC-6 cells. βTC-6 cells were treated with GL3 or MEKK-1 d-siRNA. Forty-eight hours after d-siRNA treatment, the cells were either left untreated or treated with 50 units/ml IL-1β overnight. The cells were then lysed, and proteins were separated by SDS gel electrophoresis and analyzed by immunoblotting with iNOS and ERK antibodies. The percentage of iNOS induction was calculated by relating iNOS bands to those of ERK-1. Data are presented as means ± SE for three individual experiments. *P < 0.05 using Student's t test.
- FIG. 8.
MEKK-1 downregulation protects against cytokine-induced βTC-6 and mouse islet cell death. βTC-6 cells (A) and dispersed mouse islet cells (B) that had been transfected with GL3 or MEKK-1 d-siRNA were either left untreated or treated with a mixture of cytokines (50 units/ml IL-1β, 1,000 units/ml IFN-γ, and 1,000 units/ml TNF-α) for 48 h and analyzed for cell death by vital staining with bisbenzimide and propidium iodide followed by fluorescence microscopy. Results from three separate observations are shown in A and are given as means ± SE. *P < 0.05 using Student's paired t test. B: Results from eight separate observations are shown as means ± SE. *P < 0.05 using repeated measurement two-way ANOVA followed by Student's t test.
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