An oligonucleotide decoy for transcription factor E2F inhibits mesangial cell proliferation in vitro

Abstract

The transcription factor E2F controls expression of several genes involved in cell proliferation including c- myc, c- myb, proliferating cell nuclear antigen (PCNA), and cdk2 kinase. Having established that both PCNA and cdk2 kinase are induced in rat mesangial cells (MC) by serum stimulation, we attempted to inhibit MC proliferation in vitro by transfecting these cells with cationic liposomes containing a synthetic double-stranded oligodeoxynucleotide (ODN) with high affinity for E2F. Using a gel mobility shift assay, we detected increased specific binding of E2F in MC following serum stimulation. This binding was completely inhibited by preincubation of MC nuclear extracts with the double-stranded ODN with high affinity for E2F but not by preincubation with a missense ODN containing two point mutations. MC were also transfected with a luciferase reporter gene construct containing three E2F binding sites. Luciferase activity was enhanced by serum stimulation of MC, and this effect was specifically abolished by cotransfection of MC with E2F decoy ODN. Furthermore, RT-PCR analysis revealed that serum-induced upregulation of PCNA and cdk2 kinase gene expression was inhibited by E2F decoy ODN transfection but not by transfection of missense ODN. These changes in gene expression were paralleled by a reduction in PCNA and cdk2 kinase protein expression in E2F decoy ODN transfected cells. MC number increased following serum stimulation. This effect was blunted by transfection with E2F decoy ODN but not by transfection of missense ODN. These data suggest that the transcription factor E2F plays a crucial role in the regulation of MC proliferation and that this factor can be successfully targeted to inhibit MC cell cycle progression.

Transcription factor decoy for AP-1 reduces mesangial cell proliferation and extracellular matrix production in vitro and in vivo

“…[13][14][15][16] To date, molecular therapeutic strategies using decoy ODN in glomerulonephritis have successfully focused on the direct effect of decoy ODN for transcription factor E2F or nuclear factor (NF)-kB. [17][18][19][20] These transcription factors are related with cell cycle progression and inflammatory diseases after glomerular injury. Another transcription factor AP-1 binds to AP-1 consensus sequences present in numerous genes associated with cell-proliferative response and ECM production, such as TGF-b1, collagen, and PAI-1.…”

Role of differential and cell type-specific expression of cell cycle regulatory proteins in mediating progressive glomerular injury in human IgA nephropathy

“…27,30 E2F decoy oligonucleotides have been shown to be capable of suppressing mesangial cell proliferation in vitro and in vivo. 31,32 ECM-induced cell cycle arrest in mesangial cells was shown to be associated with transcriptional repression of E2F-responsive genes such as c-myc, cyclin A and cdc2. 33 In contrast to these reports, we did not detect a corresponding upregulation of the protein products of E2F-responsive genes in the mesangium of glomeruli in human IgAN.…”

Molecular strategy using cis-element ‘decoy’ of E2F binding site inhibits neointimal formation in porcine balloon-injured coronary artery model

“…4,5 We and others have documented the potential utility of the decoy strategy to treat various diseases. [6][7][8][9]14 One important target for the decoy strategy is the essential transcription factor, E2F, for cell cycle progression. Accordingly, we and others demonstrated the inhibition of neointimal formation after transfection of E2F decoy ODN.…”

“…Previous studies have established that E2F binds with high specificity and affinity to a double-stranded ODN containing a unique 8-bp consensus sequence. [6][7][8] Indeed, transfection of a decoy cis element to bind E2F resulted in the prevention of intimal hyperplasia in response to vascular injury in a rat carotid artery model, accompanied by a reduction of mRNA of c-myc, cdc 2 kinase and PCNA. 6 Moreover, this inhibition continued for up to 8 weeks after a single transfection in a dose-dependent manner.…”