title: In vitro phenotypic, genomic and proteomic characterization of a cytokine-resistant murine β-TC3 cell line.
abstract: Type 1 diabetes mellitus (T1DM) is caused by the selective destruction of insulin-producing β-cells. This process is mediated by cells of the immune system through release of nitric oxide, free radicals and pro-inflammatory cytokines, which induce a complex network of intracellular signalling cascades, eventually affecting the expression of genes involved in β-cell survival.The aim of our study was to investigate possible mechanisms of resistance to cytokine-induced β-cell death. To this purpose, we created a cytokine-resistant β-cell line (β-TC3R) by chronically treating the β-TC3 murine insulinoma cell line with IL-1β + IFN-γ. β-TC3R cells exhibited higher proliferation rate and resistance to cytokine-mediated cell death in comparison to the parental line. Interestingly, they maintained expression of β-cell specific markers, such as PDX1, NKX6.1, GLUT2 and insulin. The analysis of the secretory function showed that β-TC3R cells have impaired glucose-induced c-peptide release, which however was only moderately reduced after incubation with KCl and tolbutamide. Gene expression analysis showed that β-TC3R cells were characterized by downregulation of IL-1β and IFN-γ receptors and upregulation of SOCS3, the classical negative regulator of cytokines signaling. Comparative proteomic analysis showed specific upregulation of 35 proteins, mainly involved in cell death, stress response and folding. Among them, SUMO4, a negative feedback regulator in NF-kB and JAK/STAT signaling pathways, resulted hyper-expressed. Silencing of SUMO4 was able to restore sensitivity to cytokine-induced cell death in β-TC3R cells, suggesting it may play a key role in acquired cytokine resistance by blocking JAK/STAT and NF-kB lethal signaling.In conclusion, our study represents the first extensive proteomic characterization of a murine cytokine-resistant β-cell line, which might represent a useful tool for studying the mechanisms involved in resistance to cytokine-mediated β-cell death. This knowledge may be of potential benefit for patients with T1DM. In particular, SUMO4 could be used as a therapeutical target.
authors.affiliation: Section of Endocrinology, Dipartimento Biomedico di Medicina Interna e Specialistica (DIBIMIS), University of Palermo, Palermo, Italy.
authors.forename: Antonina
authors.initials: A
authors.lastname: Coppola
authors.forename: Laura
authors.initials: L
authors.lastname: Tomasello
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Pizzolanti
authors.forename: Ida
authors.initials: I
authors.lastname: Pucci-Minafra
authors.forename: Nadia
authors.initials: N
authors.lastname: Albanese
authors.forename: Gianluca
authors.initials: G
authors.lastname: Di Cara
authors.forename: Patrizia
authors.initials: P
authors.lastname: Cancemi
authors.forename: Maria
authors.initials: M
authors.lastname: Pitrone
authors.forename: Alessandra
authors.initials: A
authors.lastname: Bommarito
authors.forename: Elvira
authors.initials: E
authors.lastname: Carissimi
authors.forename: Giovanni
authors.initials: G
authors.lastname: Zito
authors.forename: Angela
authors.initials: A
authors.lastname: Criscimanna
authors.forename: Aldo
authors.initials: A
authors.lastname: Galluzzo
authors.forename: Carla
authors.initials: C
authors.lastname: Giordano
mesh_terms: D000818:Animals; D017209:Apoptosis; D018929:Cell Culture Techniques; D002453:Cell Cycle; D016923:Cell Death; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival; D016207:Cytokines / Q000378:metabolism*; D015536:Down-Regulation; D020868:Gene Silencing; D023281:Genomics / Q000379:methods; D007150:Immunohistochemistry / Q000379:methods; D050417:Insulin-Secreting Cells / Q000166:cytology*; D007340:Insulinoma / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D010641:Phenotype; D040901:Proteomics / Q000379:methods
chemical_list: D016207:Cytokines; D016328:NF-kappa B