title: p38β-regulated induction of the heat shock response by carbon monoxide releasing molecule CORM-2 mediates cytoprotection in lung cells in vitro.
abstract: The carbon monoxide releasing molecule tricarbonyldichlororuthenium (CORM-2) displays protective actions like carbon monoxide. The molecular mechanism underlying this effect remains controversial. We hypothesized that CORM-2 mediates cytoprotection via induction of heat shock proteins through activation of p38 mitogen-activated kinase. Embryonic bovine lung cells were incubated with CORM-2. Apoptosis was induced by staurosporine and analyzed by flow cytometry following annexin-V staining, caspase-3 activity assay, and by Western Blot for caspase-3 cleavage. Heat shock response was assessed by DNA-binding activity of heat shock factor 1 and by reporter gene activity. Cells were transfected with siRNA targeting p38 isoforms. Data were analyzed with ANOVA and post-hoc Holm-Sidak test. CORM-2 inhibited staurosporine-induced apoptosis (% annexin-V positive cells: staurosporine = 60 ± 4% vs. CORM-2 10 μM = 48 ± 4%, CORM-2 25 μM=42 ± 5%, CORM-2 50 μM = 40 ± 4% and CORM-2 100 μM = 38 ± 2%, mean ± S.D., P<0.001; caspase-3 activity: staurosporine=92 ± 15 RFUs vs. CORM-2 50 μM=60 ± 14 RFUs, mean ± S.D. P<0.001). CORM-2 induced phosphorylation of p38 MAPK, but not of JNK and ERK1/2. CORM-2 induced DNA-binding of heat shock factor 1 and elicited a 4-fold induction of gene activity (P<0.05). Incubation with the Hsp inhibitors KNK437 attenuated and 17-AAG abolished the anti-apoptotic effect of CORM-2 (P<0.001). p38 inhibition and silencing of p38β attenuated the anti-apoptotic effect of CORM-2 (P<0.05), most likely by abolishing CORM-2-induced HSF-1 binding activity. These findings suggest that CORM-2-mediated cytoprotection is caused by induction of the heat shock response and by p38 activation. Furthermore, the p38β isoform activation may represent an upstream mechanism of heat shock response induction.
authors.affiliation: Department of Anesthesiology and Critical Care Medicine, University Medical Center, Freiburg, Germany. nils.schallner@uniklinik-freiburg.de
authors.forename: Nils
authors.initials: N
authors.lastname: Schallner
authors.forename: Sven
authors.initials: S
authors.lastname: Schwemmers
authors.forename: Christian I
authors.initials: CI
authors.lastname: Schwer
authors.forename: Christian
authors.initials: C
authors.lastname: Froehlich
authors.forename: Patrick
authors.initials: P
authors.lastname: Stoll
authors.forename: Matjaz
authors.initials: M
authors.lastname: Humar
authors.forename: Heike L
authors.initials: HL
authors.lastname: Pahl
authors.forename: Alexander
authors.initials: A
authors.lastname: Hoetzel
authors.forename: Torsten
authors.initials: T
authors.lastname: Loop
authors.forename: Ulrich
authors.initials: U
authors.lastname: Goebel
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002245:Carbon Dioxide / Q000737:chemistry* / Q000494:pharmacology*; D002417:Cattle; D019610:Cytoprotection / Q000187:drug effects*; D004789:Enzyme Activation / Q000187:drug effects; D020868:Gene Silencing; D018869:Heat-Shock Response / Q000187:drug effects*; D008168:Lung / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D048290:Mitogen-Activated Protein Kinase 11 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D009942:Organometallic Compounds / Q000737:chemistry*; D019311:Staurosporine / Q000494:pharmacology
chemical_list: D009942:Organometallic Compounds; C447082:tricarbonyldichlororuthenium (II) dimer; D002245:Carbon Dioxide; D048290:Mitogen-Activated Protein Kinase 11; D019311:Staurosporine