title: The role of Janus kinase 2 (JAK2) activation in pneumococcal EstA protein-induced inflammatory response in RAW 264.7 macrophages.
abstract: In a previous study, we demonstrated pneumococcal EstA-induced inflammatory response through NF-κB and MAPK-dependent pathways. Herein, we tested the hypothesis that the Janus kinase 2 (JAK2) activation and associated signaling cascades may also be involved in EstA-induced inflammatory process in RAW 264.7 macrophages. Our immunoblot analysis indicated EstA-induced activation of JAK2, with the phosphorylated protein detected from 1 to 24 h post-stimulation. As type I interferon (IFN) signaling requires the JAK/STAT pathway, we investigated EstA-induced expression of INF-α4 and INF-β by semi-quantitative and quantitative RT PCR. Our results indicated both concentration- and time-dependent increases in both IFN-α4 and IFN-β mRNA expression after EstA challenge, with the highest fold-increases observed at 4 h and 6 h post-stimulation for IFN-α4 and IFN-β mRNA, respectively. Furthermore, we applied a pharmacological approach to demonstrate the effect of JAK2 inhibition on EstA-induced nitric oxide (NO) and pro-inflammatory cytokine production. The JAK2 inhibitor AG-490 reduced significantly (P < 0.05) EstA-induced NO production and the expression of iNOS mRNA in a concentration-dependent manner. Similarly, EstA-induced IL-1β and IL-6 production and their respective mRNA expression were markedly suppressed by AG-490. However, AG-490 had no inhibitory effect on both mRNA and protein levels of TNF-α. Taken together, we demonstrate that JAK2 activation and IFN I signaling are integral parts of EstA-induced inflammatory process. Further studies will elucidate the interaction of the different signaling pathways, the specific downstream targets of JAK2, the kinetics of cytokine release, and if EstA could induce the pro-inflammatory mediators to the same extent in alveolar macrophages.
authors.affiliation: Laboratory of Applied Pharmacokinetics and Pharmacodynamics, College of Veterinary Medicine, Kyungpook National University, Daegu 702-701, Republic of Korea.
authors.forename: Elias
authors.initials: E
authors.lastname: Gebru
authors.forename: Eun-Hee
authors.initials: EH
authors.lastname: Kang
authors.forename: Dereje
authors.initials: D
authors.lastname: Damte
authors.forename: Joong-Su
authors.initials: JS
authors.lastname: Lee
authors.forename: Seung-Hee
authors.initials: SH
authors.lastname: Jang
authors.forename: Myung Hee
authors.initials: MH
authors.lastname: Kim
authors.forename: Henrique
authors.initials: H
authors.lastname: Cheng
authors.forename: Seung-Chun
authors.initials: SC
authors.lastname: Park
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000633:toxicity*; D015153:Blotting, Western; D002265:Carboxylic Ester Hydrolases / Q000633:toxicity*; D002460:Cell Line; D016207:Cytokines / Q000096:biosynthesis*; D020869:Gene Expression Profiling; D053614:Janus Kinase 2 / Q000378:metabolism*; D008264:Macrophages / Q000276:immunology* / Q000382:microbiology*; D051379:Mice; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013296:Streptococcus pneumoniae / Q000472:pathogenicity*; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; D016207:Cytokines; D053614:Janus Kinase 2; D002265:Carboxylic Ester Hydrolases; C098953:EstA protein, bacteria