Interleukin-1 stimulates the expression of type I and type II interleukin-1 receptors in the rat insulinoma cell line Rinm5F; sequencing a rat type II interleukin-1 receptor cDNA.
The insulin secreting rat Rinm5F cells are often used to study the cytotoxic actions of interleukin-1 (IL-1) on pancreatic beta-cells. We demonstrate here that Rinm5F insulinoma cells express both type I and type II interleukin-1 receptor (IL-1R) mRNAs and gene products. IL-1R agonists, recombinant murine IL-1 alpha (rmIL-1 alpha, 10 ng/ml) and recombinant rat IL-1 beta (rrIL-1 beta, 100 pg/ml or 10 ng/ml) induce the upregulation of mRNA expression for both types of IL-1 receptors (IL-1Rs). This effect of rrIL-1 beta is antagonised by preincubation with recombinant human interleukin 1 receptor antagonist protein (rhIL-1ra, 5 micrograms/ml). Furthermore, this rrIL-1 beta induced upregulation of IL-1R mRNAs is blocked by actinomycin D (7.5 micrograms/ml), whereas cycloheximide (20 micrograms/ml) has no effect. The phorbol ester PMA (20 nM) upregulates the expression of mRNAs both IL-1 receptors, whereas glucose (50 mM) upregulates the expression of the type I IL-1R mRNA only. Pretreatment of cells with pertussis toxin (100 ng/ml) partially blocks the rrIL-1 beta induced expression of mRNA for the type I and, to a lesser extent, the type II IL-1R. Incubation of the cells with rrIL-1 beta also induces a time-dependent expression of c-fos, interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF-alpha) mRNAs. Binding studies with 125I-recombinant human IL-1 beta (125I-rhIL-1 beta) indicate that IL-1R gene products, with the ligand binding characteristics of the type I IL-1R, are constitutively present on Rinm5F cells. Treatment with rrIL-1 beta (6h) increases the number of 125I-rhIL-1 beta binding sites on Rinm5F cells. We have also demonstrated that the number of type II IL-1R binding sites increases after induction with rrIL-1 beta (6h), by indirect immunofluorescence using a monoclonal antibody (ALVA 42) raised against the human type II IL-1R. Furthermore, we have sequenced the type II IL-1R cDNA in the rat insulinoma Rinm5F cells. The comparison of the amino acid sequence of the rat type II IL-1R with that of the mouse and human type II IL-1Rs shows 90% and 62% amino acid identity, respectively. The most important difference between the human and murine type II IL-1Rs, and this rat type II IL-1R cDNA, is an open reading frame coding for a six amino acid longer, strongly charged (QIKEMK), cytosolic domain.(ABSTRACT TRUNCATED AT 400 WORDS)
Bristulf
J
J
Department of Neurochemistry and Neurotoxicology, Arrhenius Laboratories for Natural Sciences, Stockholm University, Sweden.
Gatti
S
S
Malinowsky
D
D
Bjork
L
L
Sundgren
A K
AK
Bartfai
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D007340:Insulinoma / Q000378:metabolism*; D007375:Interleukin-1 / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000032:analysis; D012194:RNA-Directed DNA Polymerase; D051381:Rats; D017472:Receptors, Interleukin-1 / Q000096:biosynthesis / Q000187:drug effects* / Q000235:genetics; D016415:Sequence Alignment; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D007375:Interleukin-1; D012333:RNA, Messenger; D017472:Receptors, Interleukin-1; D012194:RNA-Directed DNA Polymerase