Transduction of Cu, Zn-superoxide dismutase mediated by an HIV-1 Tat protein basic domain into human chondrocytes

Autor: Kim, Hyun Ah, Won Kim, Dae, Park, Jinseu, Choi, Soo Young
Jazyk: angličtina
Rok vydání: 2006
Předmět:
Zdroj: Arthritis Research & Therapy
ISSN: 1478-6362
1478-6354
Popis: 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.
Databáze: OpenAIRE
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