Extracellular signal-regulated kinase is essential for interleukin-1-induced and nuclear factor kappaB-mediated gene expression in insulin-producing INS-1E cells.
OBJECTIVE
The beta cell destruction and insulin deficiency that characterises type 1 diabetes mellitus is partially mediated by cytokines, such as IL-1beta, and by nitric oxide (NO)-dependent and -independent effector mechanisms. IL-1beta activates mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), p38 and c-Jun NH2-terminal kinase (JNK), and the nuclear factor kappa B (NFkappaB) pathway. Both pathways are required for expression of the gene encoding inducible nitric oxide synthase (iNOS) and for IL-1beta-mediated beta cell death. The molecular mechanisms by which these two pathways regulate beta cell Nos2 expression are currently unknown. Therefore, the aim of this study was to clarify the putative crosstalk between MAPK and NFkappaB activation in beta cells.


METHODS
The MAPKs ERK, p38 and JNK were inhibited by SB203580, PD98059 or Tat-JNK binding domain or by cells overexpressing the JNK binding domain. The effects of MAPK inhibition on IL-1beta-induced iNOS production and kappa B inhibitor protein (IkappaB) degradation were examined by western blotting. NFkappaB DNA binding was investigated by electrophoretic mobility shift assay, while NFkappaB-induced gene transcription was evaluated by gene reporter assays.


RESULTS
Inhibition of the MAPKs did not affect IkappaB degradation or NFkappaB DNA binding. However, inhibition of ERK reduced NFkappaB-mediated Nos2 expression; serine 276 phosphorylation of the p65 unit of the NFkappaB complex seemed critical, as evaluated by amino acid mutation analysis.


CONCLUSIONS
ERK activity is required for NFkappaB-mediated transcription of Nos2 in insulin-producing INS-1E cells, indicating that ERK regulates Nos2 expression by increasing the transactivating capacity of NFkappaB. This may involve phosphorylation of Ser276 on p65 by an as yet unidentified kinase.
Larsen
L
L
Steno Diabetes Center, 2 Niels Steensens Vej, 2820, Gentofte, Denmark.
Størling
J
J
Darville
M
M
Eizirik
D L
DL
Bonny
C
C
Billestrup
N
N
Mandrup-Poulsen
T
T
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D015153:Blotting, Western; D045744:Cell Line, Tumor; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D005419:Flavonoids / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D007375:Interleukin-1 / Q000494:pharmacology / Q000502:physiology*; D048670:MAP Kinase Kinase 4 / Q000037:antagonists & inhibitors / Q000502:physiology; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D016328:NF-kappa B / Q000502:physiology*; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis / Q000235:genetics*; D010766:Phosphorylation; D011725:Pyridines / Q000494:pharmacology; D051381:Rats; D012694:Serine / Q000032:analysis; D050863:Synaptotagmin I / Q000737:chemistry / Q000502:physiology; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000502:physiology
D005419:Flavonoids; D007093:Imidazoles; D007328:Insulin; D007375:Interleukin-1; D016328:NF-kappa B; D011725:Pyridines; D050863:Synaptotagmin I; D012694:Serine; D052247:Nitric Oxide Synthase Type II; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one