title: Celastrol regulates innate immunity response via NF-κB and Hsp70 in human retinal pigment epithelial cells.
abstract: Elevated nuclear factor kappa B (NF-κB) activity and interleukin-6 (IL-6) secretion participates in the pathology of several age and inflammatory-related diseases, including age-related macular degeneration (AMD), in which retinal pigment epithelial cells are the key target. Recent findings reveal that heat shock protein 70 (Hsp70) may affect regulation of NF-κB. In the current study, effects of Hsp70 expression on NF-κB RelA/p65 activity were evaluated in human retinal pigment epithelial cells (ARPE-19) by using celastrol, a novel anti-inflammatory compound. Anti-inflammatory properties of celastrol were determined by measuring expression levels of IL-6 and endogenous NF-κB levels during lipopolysaccharide (LPS) exposure by using enzyme-linked immunosorbent assays (ELISA). Cell viability was measured by MTT and LDH assay, and Hsp70 expression levels were analyzed by Western blotting. ARPE-19 cells were transfected with hsp70 small interfering RNA (siRNA) in order to attenuate Hsp70 expression and activity of NF-κB RelA/p65 was measured using NF-κB consensus bound ELISA. Simultaneous exposures to LPS and celastrol reduced IL-6 expression levels as well as activity of phosphorylated NF-κB at serine 536 (Ser536) in ARPE-19 cells when compared to LPS exposure alone. In addition, inhibition of NF-κB RelA/p65 activity by celastrol was attenuated when Hsp70 response was silenced by siRNA. Favorable anti-inflammatory concentrations of celastrol showed no signs of cytotoxic response. Our findings reveal that celastrol is a novel plant compound which suppresses innate immunity response in human retinal pigment epithelial cells via NF-κB and Hsp70 regulation, and that Hsp70 is a critical regulator of NF-κB.
authors.affiliation: Department of Ophthalmology, Institute of Clinical Medicine, University of Eastern Finland, P.O. Box 1627, FIN-70211 Kuopio, Finland.
authors.forename: Tuomas
authors.initials: T
authors.lastname: Paimela
authors.forename: Juha M T
authors.initials: JM
authors.lastname: Hyttinen
authors.forename: Johanna
authors.initials: J
authors.lastname: Viiri
authors.forename: Tuomas
authors.initials: T
authors.lastname: Ryhänen
authors.forename: Reijo O
authors.initials: RO
authors.lastname: Karjalainen
authors.forename: Antero
authors.initials: A
authors.lastname: Salminen
authors.forename: Kai
authors.initials: K
authors.lastname: Kaarniranta
mesh_terms: D000893:Anti-Inflammatory Agents / Q000276:immunology*; D002460:Cell Line; D018840:HSP70 Heat-Shock Proteins / Q000276:immunology*; D006801:Humans; D007113:Immunity, Innate / Q000187:drug effects*; D016328:NF-kappa B / Q000276:immunology*; D053978:Pentacyclic Triterpenes; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000187:drug effects / Q000276:immunology; D029757:Tripterygium / Q000737:chemistry; D014315:Triterpenes / Q000276:immunology*
chemical_list: D000893:Anti-Inflammatory Agents; D018840:HSP70 Heat-Shock Proteins; D016328:NF-kappa B; D053978:Pentacyclic Triterpenes; D014315:Triterpenes; C050414:celastrol