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A DNA motif present in alpha V integrin promoter exhibits dual binding preference to distinct transcription factors
H W Sharma1, K Higgins-Sochaski, J R Perez
1Division of Oncology, Roche Research Center, Hoffmann-La Roche, Inc., Nutley, NJ 07110, USA.
Abstract:
Antisense inhibition of the RelA subunit but not the NFKB1 subunit of NK-kappa B transcription factor results in a block of cellular adhesion and inhibition of tumor cell growth in vitro and in vivo. Studies aimed at dissecting the molecular mechanism of antisense relA action led to our identification of a kappa B-like motif present in aV integrin promoter. The alpha V/kappa B motif is closely related to RelA/c-Rel-binding sequences, such as 65-2 and TF-1. However, unlike these two kappa Blike motifs, the alpha V/kappa B motif detected a nuclear Sp1 activity distinct from kappa B activity, which was subsequently confirmed to be derived from Sp1. In comparison to the conventional GC box-containing Sp1 motif, the alpha V/kappa B motif also binds in vitro to c-Rel and RelA but not to NFKB1. Antisense inhibition of RelA inhibited the alpha V/kappa B activity. Direct in vivo competition of alpha V/kappa B-binding activity by a decoy approach also resulted in inhibition of alpha V/kappa B activity in intact cells. A variant of the alpha V/kappa B motif was found to retain the dual ability to detect Sp1 and the NF-kappa B complex in the nuclear and cytoplasmic extracts. Such dual interacting ability of a DNA motif offers yet another way of gene regulation in vivo and hence can affect cellular growth. Our results identify alpha V integrin as one of the molecular targets for relA/NF-kappa B and may explain growth inhibition by antisense relA.
Insights
Antisense inhibition of RelA, a subunit of the NK-kappa B transcription factor, blocks tumor cell growth and adhesion. This occurs via a novel kappa B-like motif in the alpha V integrin promoter, revealing a new gene regulation mechanism.
Area of Science:
- Molecular Biology
- Cell Biology
- Oncology
Background:
- The transcription factor NK-kappa B regulates genes involved in cell growth, adhesion, and inflammation.
- RelA and NFKB1 are key subunits of NK-kappa B, with distinct roles in gene regulation.
- Integrins, such as alpha V integrin, are crucial for cell adhesion and migration, and their dysregulation is implicated in cancer.
Purpose of the Study:
- To investigate the molecular mechanism underlying the anti-tumor effects of antisense inhibition of RelA.
- To identify specific DNA motifs and transcription factors involved in regulating alpha V integrin expression.
- To explore the potential of targeting the RelA/NF-kappa B pathway for cancer therapy.
Main Methods:
- Antisense oligonucleotide treatment to inhibit RelA and NFKB1 subunits.
- Analysis of cellular adhesion and tumor cell growth in vitro and in vivo.
- Identification and characterization of DNA motifs in the alpha V integrin promoter using electrophoretic mobility shift assays (EMSAs) and reporter gene assays.
- In vivo competition assays using decoy oligonucleotides.
Main Results:
- Antisense inhibition of RelA, but not NFKB1, blocked cellular adhesion and tumor cell growth.
- A novel kappa B-like motif was identified in the alpha V integrin promoter, which binds Sp1 and RelA/c-Rel, but not NFKB1.
- Inhibition of RelA activity reduced the activity of this alpha V/kappa B motif.
- A variant motif demonstrated dual binding to Sp1 and NF-kappa B complexes.
Conclusions:
- Alpha V integrin is a molecular target of RelA/NF-kappa B signaling.
- The identified alpha V/kappa B motif represents a novel regulatory element with dual binding capabilities, affecting gene expression and cellular growth.
- Targeting RelA may offer a therapeutic strategy for inhibiting tumor cell growth and adhesion.