Radicicol Inhibits iNOS Expression in Cytokine-Stimulated Pancreatic Beta Cells

Abstract

Here, we show that radicicol, a fungal antibiotic, resulted in marked inhibition of inducible nitric oxide synthase (iNOS) transcription by the pancreatic beta cell line MIN6N8a in response to cytokine mixture (CM: TNF-α, IFN-γ, and IL-1β). Treatment of MIN6N8a cells with radicicol inhibited CM-stimulated activation of NF-κB/Rel, which plays a critical role in iNOS transcription, in a dose-related manner. Nitrite production in the presence of PD98059, a specific inhibitor of the extracellular signal-regulated protein kinase-1 and 2 (ERK1/2) pathway, was dramatically diminished, suggesting that the ERK1/2 pathway is involved in CM-induced iNOS expression. In contrast, SB203580, a specific inhibitor of p38, had no effect on nitrite generation. Collectively, this series of experiments indicates that radicicol inhibits iNOS gene expression by blocking ERK1/2 signaling. Due to the critical role that NO release plays in mediating destruction of pancreatic beta cells, the inhibitory effects of radicicol on iNOS expression suggest that radicicol may represent a useful anti-diabetic activity.

Keywords: ERK1/2; NO; iNOS; β cells.

Fig. 1

Inhibition of nitrite production by radicicol in cytokine-mixture (CM)-stimulated pancreatic beta cells. Pancreatic beta cell line, MIN6N8a, was treated with the indicated concentrations of radicicol in the presence of cytokine mixture (CM: TNF-α, 500 U/ml; IFN-γ, 100 U/ml; IL-1β, 10 U/ml) for 48 h. (A) Supernatants were subsequently isolated and analyzed for nitrite. (B) Cells were analyzed for the viablility by 0.4% trypan blue staining. Each column shows the mean±S.D. of triplicate determinations. ^*Response that is significantly different from the control group as determined by Dunnett's two-tailed t test at p<0.05.

Fig. 2

Inhibition of iNOS gene expression by radicicol in CM-stimulated MIN6N8a cells. (A) MIN6N8a cells were treated with radicicol (100 ng/ml) in the presence of CM for 24 h on a cover slide in 12-well plates. Cells were subjected to immunofluorescence staining using an antibody specific for murine iNOS. Immunoreactivity of iNOS was localized along the margins of the cytoplasm in the control group. MIN6N8a cells were treated with the indicated concentrations of radicicol in the presence of CM for 24 h (B) or 8 h (C). (B) Expression of iNOS was analyzed by Western immunoblotting using antibody specific for murine iNOS. (C) Total RNA was isolated and analyzed for mRNA expression levels of iNOS and β-actin.

Fig. 3

Inhibition of NF-κB/Rel activation by radicicol in CM-stimulated MIN6N8a cells. (A, B) Inhibition of NF-κB/Rel transcriptional activation by radicicol. MIN6N8a cells were transfected with p(NF-κB/Rel)₃-CAT (A) or p(Oct)₃-CAT (B) using the DEAE dextran method. Twenty-four hours after transfection, cells were treated with the indicated concentrations of radicicol in the presence of CM for 18 h. Cell extracts were then prepared and analyzed for the expression of CAT using a CAT ELISA kit. (C, D) Inhibition of NF-κB/Rel DNA binding by radicicol. Cells were incubated with radicicol in the presence of CM for 2 h. Nuclear extracts (5 mg/ml) were then isolated and analyzed for the activities of NF-κB/Rel (C) and Oct (D). Each column shows the mean±S.D. of triplicate determinations. ^*Response that is significantly different from the control group as determined by Dunnett's two-tailed t test at p<0.05.

Fig. 4

Inhibition of p65 nuclear translocation by radicicol in CM-stimulated MIN6N8a cells. (A) Cells (5×10⁵ cells/ml) were incubated with radicicol (100 ng/ml) in the presence of CM for 2 h on a cover slide in 12-well plates. Cells were subjected to immunofluorescence staining using an antibody specific for murine p65. One of two representative experiments is shown. (B) Cells were treated with SN50 (10 µM) for 48 h in the presence of CM. The supernatants were subsequently isolated and analyzed for nitrite. Each column shows the mean±S.D. of triplicate determinations. ^*Response that is significantly different from the control group as determined by Dunnett's two-tailed t test at p<0.05.

Fig. 5

Inhibition of p44/42 phosphorylation by Radicicol in CM-stimulated MIN6N8a cells. (A) MIN6N8a cells were treated with PD98059 (50 µM) or SB203580 (30 µM) for 48 h in the presence of CM. The supernatants were subsequently isolated and analyzed for nitrite. Each column shows the mean±S.D. of triplicate determinations. ^*Response that is significantly different from the control group as determined by Dunnett's two-tailed t test at p<0.05. (B) Cells were treated with radicicol for the indicated time in the presence of CM. (C) Cells were treated with radicicol for 20 min in the presence of CM. The phosphorylation of p44/p42 was analyzed by Western blot assay.