title: Interleukin-1beta mediates endotoxin- and tumor necrosis factor alpha-induced RGS16 protein expression in cultured cardiac myocytes.
abstract: Endotoxin (LPS)-induced cardiac failure is associated with an up-regulation of RGS16 protein expression and repression of phospholipase C activity in vivo. Since the release of pro-inflammatory cytokines plays an important role in mediating LPS-induced myocardial dysfunction, we examined the effect of recombinant cytokines on the expression of RGS16 protein in neonatal cardiac myocytes. Myocytes in culture were treated with 50 ng/ml recombinant tumor necrosis factor alpha (TNFalpha), 2 ng/ml interleukin 1beta (IL-1beta), interleukin 6 (IL-6), interferon gamma (IFNgamma) or diluent (NaCl) for 24 h. Before stimulation with LPS (4 micro g/ml for 24 h) cells were treated with 200 ng/ml interleukin 1-receptor antagonist (IL-1ra), 500 ng/ml soluble TNF receptor (sTNFr), or NaCl for 1 h. Isolated membrane proteins were used for Western blot analysis. Cell-associated and secreted IL-1beta and TNFalpha protein content were determined in myocyte protein homogenates and cell culture supernatants by ELISA immunoblotting 3, 6, 24, 48 and 72 h after treatment with LPS. IL-1beta (1.75-fold) and TNFalpha (1.62-fold) but not IL-6 and IFNgamma induced RGS16 protein expression. LPS stimulated intracellular IL-1beta expression within 6 h (847.1+/-172.9 pg/3x10(6) cells) followed by an increase in extracellular secretion up to 70.8+/-8.1 pg/3x10(6) cells after 48 h. In contrast, intracellular protein concentrations of TNFalpha were almost not detectable (0.03+/-0.01 pg/3x10(6) cells), but extracellular secretion was induced by LPS with a maximum at 6 h (653.9+/-36.3 pg/3x10(6) cells). The LPS-induced increase in RGS16 (1.6-fold) was blunted by IL-1ra but not by TNFalpha scavenging. Interestingly, both, the IL-1beta- and TNFalpha-effect could be blocked by IL-1ra, indicating that also the TNFalpha-induced RGS16 expression is mediated by IL-1. We therefore conclude that LPS induces RGS16 protein expression by activation of the cytokine IL-1beta in cardiac myocytes. Our data substantiate the role of IL-1beta as an important mediator in LPS-induced cardiac failure.
authors.affiliation: Zentrum für Innere Medizin, III. Medizinische Klinik, Kardiologie, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, 20246, Hamburg, Germany. patten@uke.uni-hamburg.de
authors.forename: Monica
authors.initials: M
authors.lastname: Patten
authors.forename: Sabine
authors.initials: S
authors.lastname: Stübe
authors.forename: Bryan
authors.initials: B
authors.lastname: Thoma
authors.forename: Thomas
authors.initials: T
authors.lastname: Wieland
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002478:Cells, Cultured; D007371:Interferon-gamma / Q000378:metabolism / Q000494:pharmacology; D007375:Interleukin-1 / Q000378:metabolism* / Q000494:pharmacology; D015850:Interleukin-6 / Q000378:metabolism / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D020710:RGS Proteins / Q000096:biosynthesis* / Q000235:genetics; D051381:Rats; D017472:Receptors, Interleukin-1 / Q000037:antagonists & inhibitors; D011994:Recombinant Proteins / Q000494:pharmacology; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D007375:Interleukin-1; D015850:Interleukin-6; D008070:Lipopolysaccharides; D011506:Proteins; D020710:RGS Proteins; C107413:RGS16 protein; D017472:Receptors, Interleukin-1; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma