title: siRNA-mediated silencing of phosphodiesterase 4B expression affects the production of cytokines in endotoxin-stimulated primary cultured microglia.
abstract: Phosphodiesterase 4 (PDE4) has four subtypes: PDE4A, PDE4B, PDE4C and PDE4D. The expression of PDE4 subtypes in microglial cells and the specific contribution of each subtype to inflammation remain unclear. In this study, the expression of PDE4 subtypes in primary microglial cells was assayed. Primary microglial cells were then transfected with specific small interfering RNA (siRNA) against each PDE4 subtype. PDE4 subtype A-D knockdown was confirmed by quantitative polymerase chain reaction. Secreted cytokines in the supernatant and intracellular cyclic adenosine monophosphate (cAMP) levels of transfected cells were measured. The effect of PDE4B siRNA on the activation of extracellular regulated protein kinase (ERK) induced by lipopolysaccharide (LPS) in microglia was further tested by western blotting. Results showed that the primary microglial cells expressed all four types of PDE4s at the protein level. Transfection with the four siRNAs inhibited PDE4 subtype A-D mRNA expression, respectively. In primary microglial cells, treatment with PDE4B siRNA significantly inhibited the expression of tumor necrosis factor-α and interleukin (IL)-1β, and enhanced the expression of cAMP, while siRNAs to other subtypes had no significant effects. However, none of the four siRNAs had any significant effect on the expression of IL-10. Furthermore, in the PDE4B group, the level of phosphorylated ERK was reduced. Among the four PDE4 subtypes, PDE4B plays an important role in regulating inflammatory responses in microglia, potentially through initially regulating the intracellular cAMP concentration.
authors.affiliation: Department of Anesthesiology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Hao
authors.initials: H
authors.lastname: Cheng
authors.affiliation: Department of Anesthesiology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Zhifang
authors.initials: Z
authors.lastname: Wu
authors.affiliation: Department of Anesthesiology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Xiaoyun
authors.initials: X
authors.lastname: He
authors.affiliation: Department of Anesthesiology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Qingzhen
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Department of Anesthesiology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Hongbin
authors.initials: H
authors.lastname: Jia
authors.affiliation: Department of Anesthesiology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Di
authors.affiliation: Department of Anesthesiology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, Jiangsu 210002, P.R. China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Ji
keywords: inflammation; microglia; phosphodiesterase 4B; small interfering RNA