Selective inhibition of monocyte chemoattractant protein-1 gene expression in human embryonal kidney cells by specific triple helix-forming oligonucleotides.
Monocyte chemoattractant protein-1 (MCP-1) is a chemokine that is expressed by a variety of tissue cells in response to inflammatory stimuli, such as IL-1beta, TNF-alpha, and IFN-gamma. A major function of MCP-1 is the recruitment and activation of monocytes and T lymphocytes. Overexpression of MCP-1 has been implicated in a number of diseases, including glomerulonephritis and rheumatoid arthritis, indicating that the modulation of MCP-1 activity and/or expression is a desired therapeutic strategy. In the present study, our aim was to test whether the MCP-1 expression could be inhibited at the transcriptional level using triple helix-forming oligonucleotides (TFOs). We designed a TFO targeted to the SP-1 binding site in the human MCP-1 gene promoter. Gel mobility shift assays demonstrated that the phosphodiester TFO formed a sequence-specific triplex with its dsDNA target with an EC50 of approximately 1.9 x 10(-7) M. The corresponding phosphorothioated oligonucleotide was also effective in this assay with an 8-fold higher EC50 value. Binding of the TFO to the target DNA prevented the binding of rSP-1 and of nuclear proteins in vitro. The TFO could also partially inhibit endogenous MCP-1 gene expression in cultured human embryonic kidney cells. Treatment of TNF-alpha-stimulated human embryonic kidney 293 cells with the TFO inhibited the secretion of MCP-1 in a dose-dependent manner (up to 45% at 5 microM oligonucleotide). The inhibition of MCP secretion was caused at the level of gene transcription, because MCP-1 mRNA levels in oligonucleotide-treated cells were also decreased by approximately 40%.
Marchand
P
P
Institute of Pharmacology, Medical School Hannover, Hannover, Germany.
Resch
K
K
Radeke
H H
HH
D001667:Binding, Competitive / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D018932:Chemokine CCL2 / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D018390:Gene Targeting; D005822:Genetic Vectors / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology* / Q000196:embryology / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000276:immunology; D016329:Sp1 Transcription Factor / Q000037:antagonists & inhibitors / Q000378:metabolism
D018932:Chemokine CCL2; D009841:Oligonucleotides; D016329:Sp1 Transcription Factor