Use of RNA Interference to Investigate Cytokine Signal Transduction in Pancreatic Beta Cells
Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by immune infiltration of the pancreatic islets resulting in an inflammatory reaction named insulitis and subsequent beta cell apoptosis. During the course of insulitis beta cell death is probably caused by direct contact with activated macrophages and T-cells, and/or exposure to soluble mediators secreted by these cells, including cytokines, nitric oxide, and free oxygen radicals. In vitro exposure of beta cells to the cytokines interleukin(IL)-1β + interferon(IFN)-γ or to tumor necrosis factor(TNF)-α + IFN-γ induces beta cell dysfunction and ultimately apoptosis. The transcription factors NF-κB and STAT1 are key regulators of cytokine-induced beta cell death. However, little is known about the gene networks regulated by these (or other) transcription factors that trigger beta cell apoptosis. The recent development of RNA interference (RNAi) technology offers a unique opportunity to decipher the cytokine-activated molecular pathways responsible for beta cell death. Use of RNAi has been hampered by technical difficulties in transfecting primary beta cells, but in recent years we have succeeded in developing reliable and reproducible protocols for RNAi in beta cells. This chapter details the methods and settings used to achieve efficient and nontoxic transfection of small interfering RNA in immortal and primary beta cells.
Key wordsSmall interfering RNA siRNA Pancreatic beta cells Apoptosis Gene knockdown Inducible nitric oxide synthase Interleukin-1β Interferon-γ Tumor necrosis factor-α
This work has been supported by grants from the Fonds National de la Recherche Scientifique (FNRS – FRSM) Belgium, the Communauté Française de Belgique – Actions de Recherche Concertées (ARC), the European Union (STREP Savebeta, contract no. 036903; in the Framework Programme 6 of the European Community) and the Belgium Program on Interuniversity Poles of Attraction initiated by the Belgium State (IUAP P6/40). F.M. is the recipient of a Post-Doctoral Fellowship from FNRS, Belgium. The authors have no duality of interest associated with this manuscript. We thank M.A. Neef, G. Vandenbroeck, M. Urbain, J. Schoonheydt, R. Leeman, A. M. Musuaya, and S. Mertens from the Laboratory of Experimental Medicine, ULB, for excellent technical support, Dr. Fernanda Ortis (Laboratory of Experimental Medicine) for helpful comments and Dr. Piero Marchetti (Department of Endocrinology and Metabolism, Metabolic Unit, University of Pisa, Pisa, Italy) for providing the human islets used for siRNA testing.
- 8.Cunha, D.A., Hekerman, P., Ladriere, L., Bazarra-Castro, A., Ortis, F., Wakeham, M.C. Moore, F., Rasschaert, J., Cardozo, A.K., Bellomo, E., Overbergh, L., Mathieu, C., Lupi, R., Hai, T., Herchuelz, A., Marchetti, P., Rutter, G.A., Eizirik, D.L., Cnop, M. (2008) Initiation and execution of lipotoxic ER stress in pancreatic beta-cells. J. Cell Sci. 121: 2308–2318.PubMedCrossRefGoogle Scholar
- 11.Pipeleers, D.G., in’t Veld, P.A., Van de Winkel, M., Maes, E., Schuit, F.C., Gepts, W. (1985) A new in vitro model for the study of pancreatic A and B cells. Endocrinology 117: 806–816.Google Scholar
- 12.Rasschaert, J., Ladriere, L., Urbain, M., Dogusan, Z., Katabua, B., Sato, S., Akira, S, Gysemans, C., Mathieu, C., Eizirik, D.L. (2005) Toll-like receptor 3 and STAT-1 contribute to double-stranded RNA + interferon-gamma-induced apoptosis in primary pancreatic beta-cells. J. Biol. Chem. 280: 33984–33991.PubMedCrossRefGoogle Scholar
- 14.Lupi, R., Dotta, F., Marselli, L., Del Guerra, S., Masini, M., Santangelo, C. Patané, G., Boggi, U., Piro, S., Anello, M., Bergamini, E., Mosca, F., Di Mario, U., Del Prato, S., Marchetti, P. (2002) Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets: evidence that beta-cell death is caspase mediated, partially dependent on ceramide pathway, and Bcl-2 regulated. Diabetes 51: 1437–1442.PubMedCrossRefGoogle Scholar
- 20.Dande, P., Prakash, T.P., Sioufi, N., Gaus, H., Jarres, R., Berdeja, A., Swayze, E.E., Griffey, R.H., Bhat, B. (2006) Improving RNA interference in mammalian cells by 4′-thio-modified small interfering RNA (siRNA): effect on siRNA activity and nuclease stability when used in combination with 2′-O-alkyl modifications. J. Med. Chem. 49: 1624–1634.PubMedCrossRefGoogle Scholar
- 23.Editorial Comment (2003) Whither RNAi? Nat. Cell Biol. 5: 489–490.Google Scholar
- 24.Moore, F., Colli, M.L., Cnop, M., Esteve, M.I., Cardozo, A.K., Cunha, D.A., Buglian, M., Marchetti, P., Eizirik, D.L. (2009) PTPN2, a candidate gene for type 1 diabetes, modulates interferon-gamma-induced pancreatic beta-cell apoptosis. Diabetes 58: 1283–1291.Google Scholar
- 27.Moore, F., Naamane, N., Colli, M.L., Bouckenooghe, T., Ortis, F., Gurzov, E.N., Igoillo-Esteve, M., Mathieu, C., Bontempi, G., Thykjaer, T., Ørntoft, T.F., Eizirik, D.L. (2011) STAT1 is a master regulator of pancreatic beta cell apoptosis and islet inflammation. J. Biol. Chem. 286: 929–941.Google Scholar
- 28.Callewaert, H.I., Gysemans, C.A., Ladriere, L., D’Hertog, W., Hagenbrock, J., Overbergh, L. Eizirik, D.L., Mathieu C. (2007) Deletion of STAT-1 pancreatic islets protects against streptozotocin-induced diabetes and early graft failure but not against late rejection. Diabetes 56: 2169–2173.PubMedCrossRefGoogle Scholar
- 29.Gysemans, C.A., Ladriere, L., Callewaert, H., Rasschaert, J., Flamez, D., Levy, D.E., Matthys, P., Eizirik, D.L., Mathieu, C. (2005) Disruption of the gamma-interferon signaling pathway at the level of signal transducer and activator of transcription-1 prevents immune destruction of beta-cells. Diabetes 54: 2396–2403.PubMedCrossRefGoogle Scholar
- 32.Berns, K., Hijmans, E.M., Mullenders, J., Brummelkamp, T.R., Velds, A., Heimerikx, M. Kerkhoven, R.M., Madiredjo, M., Nijkamp, W., Weigelt, B., Agami, R., Ge, W., Cavet, G., Linsley, P.S., Beijersbergen, R.L., Bernards, R. (2004) A large-scale RNAi screen in human cells identifies new components of the p53 pathway. Nature 428: 431–437.PubMedCrossRefGoogle Scholar