Transduction of Cu, Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into human chondrocytes.
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.
Kim
Hyun Ah
HA
Department of Internal Medicine, Hallym University Sacred Heart Hospital, 896, Pyongchondong, Dongan-gu, Anyang, Kyunggi-do, 431-070, Korea. kimha@hallym.ac.kr
Kim
Dae Won
DW
Park
Jinseu
J
Choi
Soo Young
SY
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
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