title: Transduction of Cu, Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into human chondrocytes.
abstract: This study was performed to investigate the transduction of a full-length superoxide dismutase (SOD) protein fused to transactivator of transcription (Tat) into human chondrocytes, and to determine the regulatory function of transduced Tat-SOD in the inflammatory cytokine induced catabolic pathway. The pTat-SOD expression vector was constructed to express the basic domain of HIV-1 Tat as a fusion protein with Cu, Zn-SOD. We also purified histidine-tagged SOD without an HIV-1 Tat and Tat-GFP as control proteins. Cartilage samples were obtained from patients with osteoarthritis (OA) and chondrocytes were cultured in both a monolayer and an explant. For the transduction of fusion proteins, cells/explants were treated with a variety of concentrations of fusion proteins. The transduced protein was detected by fluorescein labeling, western blotting and SOD activity assay. Effects of transduced Tat-SOD on the regulation of IL-1 induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) mRNA expression was assessed by the Griess reaction and reverse transcriptase PCR, respectively. Tat-SOD was successfully delivered into both the monolayer and explant cultured chondrocytes, whereas the control SOD was not. The intracellular transduction of Tat-SOD into cultured chondrocytes was detected after 1 hours, and the amount of transduced protein did not change significantly after further incubation. SOD enzyme activity increased in a dose-dependent manner. NO production and iNOS mRNA expression, in response to IL-1 stimulation, was significantly down-regulated by pretreatment with Tat-SOD fusion proteins. This study shows that protein delivery employing the Tat-protein transduction domain is feasible as a therapeutic modality to regulate catabolic processes in cartilage. Construction of additional Tat-fusion proteins that can regulate cartilage metabolism favorably and application of this technology in in vivo models of arthritis are the subjects of future studies.
authors.affiliation: Department of Internal Medicine, Hallym University Sacred Heart Hospital, 896, Pyongchondong, Dongan-gu, Anyang, Kyunggi-do, 431-070, Korea. kimha@hallym.ac.kr
authors.forename: Hyun Ah
authors.initials: HA
authors.lastname: Kim
authors.forename: Dae Won
authors.initials: DW
authors.lastname: Kim
authors.forename: Jinseu
authors.initials: J
authors.lastname: Park
authors.forename: Soo Young
authors.initials: SY
authors.lastname: Choi
mesh_terms: D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D005240:Feasibility Studies; D050939:Gene Fusion; D015696:Gene Products, tat / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000494:pharmacology; D009569:Nitric Oxide / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II / Q000235:genetics; D010003:Osteoarthritis / Q000378:metabolism* / Q000473:pathology; D017434:Protein Structure, Tertiary / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000235:genetics* / Q000378:metabolism*; D014161:Transduction, Genetic / Q000379:methods; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D015696:Gene Products, tat; D007375:Interleukin-1; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; D013482:Superoxide Dismutase