title: Single Cell Chemical Cytometry of Akt Activity in Rheumatoid Arthritis and Normal Fibroblast-like Synoviocytes in Response to Tumor Necrosis Factor α.
abstract: The etiology of rheumatoid arthritis (RA) is poorly understood, and 30% of patients are unresponsive to established treatments targeting tumor necrosis factor α (TNFα). Akt kinase is implicated in TNFα signaling and may act as a barometer of patient responses to biologic therapies. Fluorescent peptide sensors and chemical cytometry were employed to directly measure Akt activity as well as proteolytic activity in individual fibroblast-like synoviocytes (FLS) from RA and normal subjects. The specificity of the peptide reporter was evaluated and shown to be a valid measure of Akt activity in single cells. The effect of TNFα treatment on Akt activity was highly heterogeneous between normal and RA subjects, which was not observable in bulk analyses. In 2 RA subjects, a bimodal distribution of Akt activity was observed, primarily due to a subpopulation (21.7%: RA Subject 5; 23.8%: RA Subject 6) of cells in which >60% of the reporter was phosphorylated. These subjects also possessed statistically elevated proteolytic cleavage of the reporter relative to normal subjects, suggesting heterogeneity in Akt and protease activity that may play a role in the RA-affected joint. We expect that chemical cytometry studies pairing peptide reporters with capillary electrophoresis will provide valuable data regarding aberrant kinase activity from small samples of clinical interest.
authors.affiliation: Department of Chemistry, University of North Carolina , Chapel Hill, North Carolina 27599, United States.
authors.forename: Emilie R
authors.initials: ER
authors.lastname: Mainz
authors.affiliation: Thurston Arthritis Research Center, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
authors.forename: D Stephen
authors.initials: DS
authors.lastname: Serafin
authors.affiliation: Department of Chemistry, University of North Carolina , Chapel Hill, North Carolina 27599, United States.
authors.forename: Tuong T
authors.initials: TT
authors.lastname: Nguyen
authors.affiliation: Thurston Arthritis Research Center, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.|Department of Medicine, Division of Rheumatology, University of North Carolina at Chapel Hill School of Medicine , Chapel Hill, North Carolina 27599, United States.
authors.forename: Teresa K
authors.initials: TK
authors.lastname: Tarrant
authors.affiliation: Department of Chemistry, University of North Carolina , Chapel Hill, North Carolina 27599, United States.|Department of Medicine, Division of Rheumatology, University of North Carolina at Chapel Hill School of Medicine , Chapel Hill, North Carolina 27599, United States.
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Sims
authors.affiliation: Department of Chemistry, University of North Carolina , Chapel Hill, North Carolina 27599, United States.|Department of Biomedical Engineering, University of North Carolina , Chapel Hill, North Carolina 27599, United States and.
authors.forename: Nancy L
authors.initials: NL
authors.lastname: Allbritton
mesh_terms: D001172:Arthritis, Rheumatoid / Q000378:metabolism / Q000473:pathology*; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D019075:Electrophoresis, Capillary; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D009025:Morpholines / Q000494:pharmacology; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D059010:Single-Cell Analysis; D000070918:Synoviocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D002867:Chromones; D007328:Insulin; D009025:Morpholines; D034741:RNA, Small Interfering; D047888:Receptors, Tumor Necrosis Factor, Type I; D014409:Tumor Necrosis Factor-alpha; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D051057:Proto-Oncogene Proteins c-akt