Selective inhibition of monocyte chemoattractant protein-1 gene expressionin 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-1 beta, TNF-alpha, and IFN-gamma. A major function of MCP-1 is the recru itment and activation of monocytes and T lymphocytes, Overexpression of MCP -1 has been implicated in a number of diseases, including glomerulonephriti s and rheumatoid arthritis, indicating that the modulation of MCP-1 activit y 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 th e transcriptional level using triple helix-forming oligonucleotides (TFOs), We designed a TFO targeted to the SP-1 binding site in the human MCP-1 gen e promoter. Gel mobility shift assays demonstrated that the phosphodiester TFO formed a sequence-specific tripler with its dsDNA target with an EC50 o f similar to 1.9 x 10(-7) M, The corresponding phosphorothioated oligonucle otide was also effective in this assay with an 8-fold higher EC50 value. Bi nding of the TFO to the target DNA prevented the binding of rSP-1 and of nu clear proteins in vitro. The TFO could also partially inhibit endogenous MC P-1 gene expression in cultured human embryonic kidney cells. Treatment of TNF-alpha-stimulated human embryonic kidney 293 cells with the TFO inhibite d the secretion of MCP-1 in a dose-dependent manner (up to 45% at 5 mu M ol igonucleotide), The inhibition of MCP secretion was caused at the level of gene transcription, because MCP-1 mRNA levels in oligonucleotide-treated ce lls were also decreased by similar to 40%, The Journal of Immunology, 2000, 164: 2070-2076.