Cytokines interleukin-1beta and tumor necrosis factor-alpha regulate different transcriptional and alternative splicing networks in primary beta-cells.
OBJECTIVE
Cytokines contribute to pancreatic beta-cell death in type 1 diabetes. This effect is mediated by complex gene networks that remain to be characterized. We presently utilized array analysis to define the global expression pattern of genes, including spliced variants, modified by the cytokines interleukin (IL)-1beta + interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha + IFN-gamma in primary rat beta-cells.


METHODS
Fluorescence-activated cell sorter-purified rat beta-cells were exposed to IL-1beta + IFN-gamma or TNF-alpha + IFN-gamma for 6 or 24 h, and global gene expression was analyzed by microarray. Key results were confirmed by RT-PCR, and small-interfering RNAs were used to investigate the mechanistic role of novel and relevant transcription factors identified by pathway analysis. RESULTS Nearly 16,000 transcripts were detected as present in beta-cells, with temporal differences in the number of genes modulated by IL-1beta + IFNgamma or TNF-alpha + IFN-gamma. These cytokine combinations induced differential expression of inflammatory response genes, which is related to differential induction of IFN regulatory factor-7. Both treatments decreased the expression of genes involved in the maintenance of beta-cell phenotype and growth/regeneration. Cytokines induced hypoxia-inducible factor-alpha, which in this context has a proapoptotic role. Cytokines also modified the expression of >20 genes involved in RNA splicing, and exon array analysis showed cytokine-induced changes in alternative splicing of >50% of the cytokine-modified genes.


CONCLUSIONS
The present study doubles the number of known genes expressed in primary beta-cells, modified or not by cytokines, and indicates the biological role for several novel cytokine-modified pathways in beta-cells. It also shows that cytokines modify alternative splicing in beta-cells, opening a new avenue of research for the field.
Ortis
Fernanda
F
Laboratory of Experimental Medicine, Université Libre de Bruxelles, Brussels, Belgium.
Naamane
Najib
N
Flamez
Daisy
D
Ladrière
Laurence
L
Moore
Fabrice
F
Cunha
Daniel A
DA
Colli
Maikel L
ML
Thykjaer
Thomas
T
Thorsen
Kasper
K
Orntoft
Torben F
TF
Eizirik
Decio L
DL
D017398:Alternative Splicing / Q000187:drug effects*; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002952:Citric Acid Cycle / Q000187:drug effects / Q000235:genetics; D002956:Citrulline / Q000378:metabolism; D016207:Cytokines / Q000494:pharmacology; D005091:Exons / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D018398:Homeodomain Proteins / Q000235:genetics; D054795:Incretins / Q000235:genetics; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D007371:Interferon-gamma / Q000494:pharmacology; D053583:Interleukin-1beta / Q000494:pharmacology*; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators / Q000235:genetics; D014158:Transcription, Genetic / Q000187:drug effects*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D016207:Cytokines; D018398:Homeodomain Proteins; D054795:Incretins; D053583:Interleukin-1beta; D034741:RNA, Small Interfering; D015534:Trans-Activators; D014409:Tumor Necrosis Factor-alpha; C084010:pancreatic and duodenal homeobox 1 protein; D002956:Citrulline; D007371:Interferon-gamma