Inhibition of PMA-induced endothelial cell activation and adhesion by over-expression of domain negative IkappaBalpha protein.
OBJECTIVE
NF-kappaB, regulate the expression of cytokine-inducible genes involving immune and inflammatory responses, will be potential therapy approach for allograft from rejection. In this study, we use pCMV- IkappaBalphaM vector to inhibit NF-kappaB activation and investigate the effect of pCMV- IkappaBalphaM in inhibition of T cells adhesion to endothelial cells.


METHODS
The NF-kappaB activity was detected with pNF-kappaB reporter gene and electrophoretic mobility shift assay. Expression of cell surface molecules was detected by RT-PCR and flow cytometer. The cell-cell adhesion assay was performed to determine the effect of pCMV-IkappaBalphaM in inhibition of T cells adhesion to endothelial cells.


RESULTS
We could find that NF-kappaB activity is inhibited by over-expression of non-degraded IkappaBalpha protein. Expression of adhesion molecules like ICAM-1, VCAM-1, and P-selectin as well as cell-cell adhesion were inhibited significantly by transfection of the pCMV- IkappaBalphaM vector.


CONCLUSIONS
Our results indicate that the pCMV- IkappaBalphaM, which inhibit the activity of NF-kappaB through over-expression of non-degraded IkappaBalpha protein, can be used for gene therapy in diseases involving NF-kappaB activation abnormally like organ transplantation via inhibiting cell adhesion.
Wei
Jian-Feng
JF
Key Lab of Multi-organ Transplantation of Ministry, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, Zhejiang Province, China.
Sun
Ke
K
Xu
Shi-Guo
SG
Xie
Hai-Yang
HY
Zheng
Shu-Sen
SS
D002448:Cell Adhesion; D002460:Cell Line; D042783:Endothelial Cells / Q000187:drug effects / Q000276:immunology*; D004730:Endothelium, Vascular / Q000166:cytology*; D006084:Graft Rejection / Q000276:immunology; D006801:Humans; D020672:I-kappa B Proteins / Q000096:biosynthesis*; D000072000:NF-KappaB Inhibitor alpha; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; D000072000:NF-KappaB Inhibitor alpha; D013755:Tetradecanoylphorbol Acetate