PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of arthrestherBioMed Centralbiomed central web sitesearchsubmit a manuscriptregisterthis articleArthritis Research & Therapy
 
Arthritis Res Ther. 2006; 8(4): R96.
Published online Jun 22, 2006. doi:  10.1186/ar1972
PMCID: PMC1779403
Transduction of Cu, Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into human chondrocytes
Hyun Ah Kim,corresponding author1 Dae Won Kim,2 Jinseu Park,2 and Soo Young Choi2
1Department of Internal Medicine, Hallym University Sacred Heart Hospital, 896, Pyongchondong, Dongan-gu, Anyang, Kyunggi-do, 431-070, Korea
2Department of Biomedical Sciences and Research Institute for Bioscience and Biotechnology, Hallym University, Chunchon 200-702, Korea
corresponding authorCorresponding author.
Hyun Ah Kim: kimha/at/hallym.ac.kr; Dae Won Kim: dwkim/at/hallym.ac.kr; Jinseu Park: jinpark/at/hallym.ac.kr; Soo Young Choi: sychoi/at/hallym.ac.kr
Received March 6, 2006; Revisions requested April 4, 2006; Revised May 4, 2006; Accepted May 12, 2006.
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.
Articles from Arthritis Research & Therapy are provided here courtesy of
BioMed Central