In this study, the transduction of the full-length SOD protein fused to HIV-1 Tat PTD into the cultured chondrocytes was examined. Non-specific results were strictly excluded by inclusion of a negative control SOD protein that carried six histidine residues, the same as Tat-SOD, without an HIV-1 Tat PTD. After extensive washing and stringent processing, the SOD signal above background was detected in none of the control SOD treated samples. In addition, Tat-GFP protein was included to demonstrate that internalization of the Tat moiety itself was not responsible for our results. Because chondrocytes are embedded in thick extracellular matrix, it is considered a challenge to deliver foreign genes or proteins into chondrocytes in cartilage. However, our findings show delivery of fusion proteins into the cartilage explant culture, demonstrating that protein delivery employing Tat-PTD is a feasible therapeutic approach for regulation of cartilage degeneration.
In our study, transduction of Tat-SOD into chondrocytes effectively inhibited the production of the proinflammatory mediator NO, which is induced by IL-1, both in monolayer and explant cultures. However, Tat-SOD transduction failed to inhibit Metalloproteinase (MMP)-1, -3 and -13 expression or prostaglandin (PG) E2
activation induced by the same cytokine (data not shown). A prior study of chondrocytes showed that treatment of chondrocytes with IL-1 induces ROS, which includes both hydrogen peroxide and superoxide, but that only superoxide mediated IL-1 induced NF-kB activation and iNOS expression [20
]. The investigators did not report whether IL-1 induced ROS or superoxide is responsible for stimulation of MMP in chondrocytes. Based on our results, it is likely that MMP production in chondrocytes in response to IL-1 is independent of superoxide.
Several strategies are currently being developed to block the proinflammatory pathway in arthritic diseases. Chemical compounds are being developed, but it is very difficult to find a compound that will specifically block a narrow target pathway. Thus, they often lead to non-specific and widespread effects that would make their clinical application difficult. Use of decoy oligonucleotides and RNA interference technology to modulate the expression of proinflammatory genes are more specific than chemical inhibitors. However, these therapies employing gene delivery still pose more problems than solutions in terms of efficiency, safety and inflammatory and immunogenic effects elicited by viral vectors. Potential advantages in regulating cellular functions using protein transduction technology are: it allows for the application, in various pathological conditions, of our accumulated knowledge on intracellular functions of particular signaling pathways; the levels of protein inside cells are directly and specifically regulated so that maximum benefit can be achieved with minimal side effects; and it is reversible and treatment can be terminated or can resume after a resting period as deemed necessary [21
]. Disadvantages include the lack of target cell specificity, potential of immunogenicity, and our incomplete knowledge of the molecular mechanisms of protein transduction. Reports of the application of PTD in potential therapeutic development are increasing in various fields and in a variety of cell systems and include inhibition of apoptosis by transduction of anti-apoptotic Bcl-XL
protein in explant cultured human chondrocytes and in human islet cells [22
], induction of chemosensitivity by transduction of cytosine deaminase in human tumor cells [24
], and inhibition of proinflammatory signaling by transduction of superreppressor IkB in Jurkat T cells [21
]. Protein transduction therapy is also showing promise in in vivo
models, such as in the reduction of inflammation in carageenan-induced pleurisy of Wistar rat and the reduction of pancreatic islet cell toxicity in streptozotocin-induced diabetic mice [25
The mechanism by which Tat mediates cell entry has been a focus of interest, and it has been shown that the entry mediated by Tat-PTD is not dependent on a specific receptor, energy generation or endocytosis [27
]. Recent data provided evidence that Tat-PTD binds to the negatively charged cell surface constituents by electrostatic interaction and then stimulates membrane ruffling to form heterogeneous vesicular structures called macropinosomes [28
]. It has also been reported that Tat also promotes gene transfer into mammalian cells, and may be a potential delivery vehicle for gene therapy as well [29
A recent report shows that extracellular SOD is decreased in both human and animal models of OA, suggesting that inadequate control of reactive oxygen species plays a role in the pathophysiology of OA [30