Korean J Physiol Pharmacol.  2013 Aug;17(4):315-320. 10.4196/kjpp.2013.17.4.315.

Radicicol Inhibits iNOS Expression in Cytokine-Stimulated Pancreatic Beta Cells

• Youn CK
• Park SJ
• Li MH
• Lee MY
• Lee KY
• Cha MJ
• Kim OH
• You HJ
• Chang IY
• Yoon SP
• Jeon YJ

• KMID: 2285466
• DOI: http://doi.org/10.4196/kjpp.2013.17.4.315

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-alpha, IFN-gamma, and IL-1beta). Treatment of MIN6N8a cells with radicicol inhibited CM-stimulated activation of NF-kappaB/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.

Keyword

beta cells; ERK1/2; iNOS; NO

MeSH Terms

Flavonoids
Gene Expression
Imidazoles
Insulin-Secreting Cells
Macrolides
Negotiating
Nitric Oxide Synthase Type II
Pyridines
Tumor Necrosis Factor-alpha
Flavonoids
Imidazoles
Macrolides
Nitric Oxide Synthase Type II
Pyridines
Tumor Necrosis Factor-alpha

Figure

• 

  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.

Reference

1. Cetkovic-Cvrlje M, Eizirik DL. TNF-alpha and IFN-gamma potentiate the deleterious effects of IL-1 beta on mouse pancreatic islets mainly via generation of nitric oxide. Cytokine. 1994; 6:399–406. PMID: 7948748.

2. Darville MI, Eizirik DL. Regulation by cytokines of the inducible nitric oxide synthase promoter in insulin-producing cells. Diabetologia. 1998; 41:1101–1108. PMID: 9754830.

3. Eizirik DL, Sandler S, Welsh N, Cetkovic-Cvrlje M, Nieman A, Geller DA, Pipeleers DG, Bendtzen K, Hellerström C. Cytokines suppress human islet function irrespective of their effects on nitric oxide generation. J Clin Invest. 1994; 93:1968–1974. PMID: 7514190.

4. Yamada K, Otabe S, Inada C, Takane N, Nonaka K. Nitric oxide and nitric oxide synthase mRNA induction in mouse islet cells by interferon-gamma plus tumor necrosis factor-alpha. Biochem Biophys Res Commun. 1993; 197:22–27. PMID: 7504484.

5. Takamura T, Kato I, Kimura N, Nakazawa T, Yonekura H, Takasawa S, Okamoto H. Transgenic mice overexpressing type 2 nitric-oxide synthase in pancreatic beta cells develop insulin-dependent diabetes without insulitis. J Biol Chem. 1998; 273:2493–2496. PMID: 9446547.

6. Flodström M, Tyrberg B, Eizirik DL, Sandler S. Reduced sensitivity of inducible nitric oxide synthase-deficient mice to multiple low-dose streptozotocin-induced diabetes. Diabetes. 1999; 48:706–713. PMID: 10102685.

7. Fukuhara Y, Turner RJ. Cation dependence of renal outer cortical brush border membrane L-glutamate transport. Am J Physiol. 1985; 248:F869–F875. PMID: 2860810.

8. Oikawa T, Ito H, Ashino H, Toi M, Tominaga T, Morita I, Murota S. Radicicol, a microbial cell differentiation modulator, inhibits in vivo angiogenesis. Eur J Pharmacol. 1993; 241:221–227. PMID: 7694864.

9. Kwon HJ, Yoshida M, Abe K, Horinouchi S, Beppu T. Radicicol, an agent inducing the reversal of transformed phenotypes of src-transformed fibroblasts. Biosci Biotechnol Biochem. 1992; 56:538–539. PMID: 1368339.

10. Kwon HJ, Yoshida M, Fukui Y, Horinouchi S, Beppu T. Potent and specific inhibition of p60v-src protein kinase both in vivo and in vitro by radicicol. Cancer Res. 1992; 52:6926–6930. PMID: 1458481.

11. Soga S, Kozawa T, Narumi H, Akinaga S, Irie K, Matsumoto K, Sharma SV, Nakano H, Mizukami T, Hara M. Radicicol leads to selective depletion of Raf kinase and disrupts K-Ras-activated aberrant signaling pathway. J Biol Chem. 1998; 273:822–828. PMID: 9422737.

12. Chanmugam P, Feng L, Liou S, Jang BC, Boudreau M, Yu G, Lee JH, Kwon HJ, Beppu T, Yoshida M, Xia Y, Wilson CB, Hwand D. Radicicol, a protein tyrosine kinase inhibitor, suppresses the expression of mitogen-inducible cyclooxygenase in macrophages stimulated with lipopolysaccharide and in experimental glomerulonephritis. J Biol Chem. 1995; 270:5418–5426. PMID: 7890656.

13. Jeon YJ, Kim YK, Lee M, Park SM, Han SB, Kim HM. Radicicol suppresses expression of inducible nitric-oxide synthase by blocking p38 kinase and nuclear factor-kappaB/Rel in lipopolysaccharide-stimulated macrophages. J Pharmacol Exp Ther. 2000; 294:548–554. PMID: 10900231.

14. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR. Analysis of nitrate, nitrite, and [15N] nitrate in biological fluids. Anal Biochem. 1982; 126:131–138. PMID: 7181105.

15. Huong PT, Lee MY, Lee KY, Chang IY, Lee SK, Yoon SP, Lee DC, Jeon YJ. Synergistic induction of iNOS by IFN-γ and glycoprotein isolated from dioscorea batatas. Korean J Physiol Pharmacol. 2012; 16:431–436. PMID: 23269906.

16. Jeon YJ, Yang KH, Pulaski JT, Kaminski NE. Attenuation of inducible nitric oxide synthase gene expression by delta 9-tetrahydrocannabinol is mediated through the inhibition of nuclear factor-kappa B/Rel activation. Mol Pharmacol. 1996; 50:334–341. PMID: 8700141.

17. Li MH, Kothandan G, Cho SJ, Huong PT, Nan YH, Lee KY, Shin SY, Yea SS, Jeon YJ. Magnolol inhibits LPS-induced NF-κB/Rel activation by blocking p38 kinase in murine macrophages. Korean J Physiol Pharmacol. 2010; 14:353–358. PMID: 21311674.

18. Xie QW, Whisnant R, Nathan C. Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide. J Exp Med. 1993; 177:1779–1784. PMID: 7684434.

19. Jeon YJ, Han SH, Lee YW, Yea SS, Yang KH. Inhibition of NF-kappa B/Rel nuclear translocation by dexamethasone: mechanism for the inhibition of iNOS gene expression. Biochem Mol Biol Int. 1998; 45:435–441. PMID: 9679644.

20. Dunnett M. A multiple comparison procedure for comparing several treatments with a control. J Am Statistics Assoc. 1955; 50:1096–1121.

21. Xie QW, Kashiwabara Y, Nathan C. Role of transcription factor NF-kappa B/Rel in induction of nitric oxide synthase. J Biol Chem. 1994; 269:4705–4708. PMID: 7508926.

22. Lin YZ, Yao SY, Veach RA, Torgerson TR, Hawiger J. Inhibition of nuclear translocation of transcription factor NF-kappa B by a synthetic peptide containing a cell membrane-permeable motif and nuclear localization sequence. J Biol Chem. 1995; 270:14255–14258. PMID: 7782278.

23. Heimberg H, Heremans Y, Jobin C, Leemans R, Cardozo AK, Darville M, Eizirik DL. Inhibition of cytokine-induced NF-kappaB activation by adenovirus-mediated expression of a NF-kappaB super-repressor prevents beta-cell apoptosis. Diabetes. 2001; 50:2219–2224. PMID: 11574401.

24. Baker MS, Chen X, Cao XC, Kaufman DB. Expression of a dominant negative inhibitor of NF-kappaB protects MIN6 beta-cells from cytokine-induced apoptosis. J Surg Res. 2001; 97:117–122. PMID: 11341786.

25. Eldor R, Yeffet A, Baum K, Doviner V, Amar D, Ben-Neriah Y, Christofori G, Peled A, Carel JC, Boitard C, Klein T, Serup P, Eizirik DL, Melloul D. Conditional and specific NF-kappaB blockade protects pancreatic beta cells from diabetogenic agents. Proc Natl Acad Sci USA. 2006; 103:5072–5077. PMID: 16551748.

26. Schmitz ML, Baeuerle PA. The p65 subunit is responsible for the strong transcription activating potential of NF-kappa B. EMBO J. 1991; 10:3805–3817. PMID: 1935902.

27. Roe SM, Prodromou C, O'Brien R, Ladbury JE, Piper PW, Pearl LH. Structural basis for inhibition of the Hsp90 molecular chaperone by the antitumor antibiotics radicicol and geldanamycin. J Med Chem. 1999; 42:260–266. PMID: 9925731.

28. Larsen L, Strling J, Darville M, Eizirik DL, Bonny C, Billestrup N, Mandrup-Poulsen T. Extracellular signal-regulated kinase is essential for interleukin-1-induced and nuclear factor kappaB-mediated gene expression in insulin-producing INS-1E cells. Diabetologia. 2005; 48:2582–2590. PMID: 16283237.