Evidence for differential functions of the p50 and p65 subunits of NF-kappa B with a cell adhesion model.

Abstract

The p5O and p65 subunits of NF-cB represent two members of a gene family that shares considerable homology to the rel oncogene. Proteins encoded by these genes form homo-and heterodimers which recognize a common DNA sequence motif. Recent data have suggested that homodimers of individual subunits of NF-KB can selectively activate gene expression in vitro. To explore this possibility in a more physiological manner, murine embryonic stem (ES) cells were treated with phosphorothio antisense oligonucleotides to either p50 or p65. Within 5 h after exposure to phosphorothio antisense p65 oligonucleotides, cells exhibited dramatic alterations in adhesion properties. Similar findings were obtained in a stable cell line that expressed a dexamethasone-inducible antisense mRNA to p65. Although antisense oligonucleotides raised against both p50 and p65 elicited a significant reduction in their respective mRNAs, only the cells treated with antisense p50 maintained a normal morphology. However, 6 days following removal of leukemia-inhibiting factor, a growth factor which suppresses embryonic stem cell differentiation, adhesion properties of cells treated with the antisense p50 oligonucleotides were markedly affected. The ability of the individual antisense oligonucleotides to elicit differential effects on cell adhesion, a property dependent upon the stage of differentiation, suggests that the p50 and p65 subunits of NF-cB regulate gene expression either as homodimers or as heterodimers with other rel family members. Furthermore, the finding that reduction in p65 expression alone had profound effects on cell adhesion properties indicates that p65 plays an important role in nonstimulated cells and cannot exist solely complexed with the cytosolic inhibitory protein IKB.The NF-KB transcription factor complex is a pleiotropic activator which participates in the induction of a wide variety of cellular and viral genes (2, 17). The active complex is composed of two subunits designated p50 and p65 (2, 7). The genes encoding p50 (8, 12) and p65 (21, 24) have been cloned, and the N termini of both proteins show considerable homology to the product of the oncogene rel.Although both subunits of NF-KB bind to a common decameric DNA motif as either homo-or heterodimers, it was recently demonstrated that the individual subunits exhibit distinct binding preferences (15). Furthermore, selected DNA motifs that bound only one of these subunits (p50 or p65) were significantly reduced in their ability to recognize NF-KB (i.e., p50/p65) and sometimes could not recognize it at all (15). These findings suggest the existence of rather distinct KB DNA motifs that have differential binding specificities for the Rel proteins, implicating a role for such motifs in selective regulation of gene expression. Consistent with this speculation, transfection studies demonstrate that the p65 subunit can function as a homodimeric transcriptional activator (3, 25). However, the physiological relevance of this observation remains unclear, as it is believed that in r...

Down-regulation of NF-κB activity and NF-κB p65 subunit expression by ras and polyoma middle T oncogenes in human colonic Caco-2 cells

“…Of interest, TNF-a-treated Caco-2-H cells also displayed signi®cantly increased cellular adhesiveness as compared to untreated cells (data not shown). These results indicate that NF-kB is involved in the control of cellular adhesion in Caco-2 cells, in accordance with what has been shown in other cell types Narayanan et al, 1993;Perez et al, 1994). Our data therefore support the notion that the decrease in the adhesive properties observed in oncogene-transfected Caco-2 cells is linked to the decreased NF-kB activity manifested by these cell lines as a result of the down-regulation of p65 expression.…”

“…In view of the role attributed to NF-kB in the control of cell adhesion Narayanan et al, 1993;Perez et al, 1994), we next examined whether the decrease in NF-kB activity observed in oncogenetransfected Caco-2 cells as a result of p65 downregulation was associated with altered adhesive proper- The amounts of the p65 transcript were quanti®ed by densitometric scanning of the autoradiographs and normalized with respect to the 28S signal ties of these cell lines. To this end, Caco-2-MT and Caco-2-T cells were compared with Caco-2-H cells for their ability to adhere to laminin and collagen IV, two of the most speci®c components of the ECM of epithelial cells (Weiser et al, 1990).…”

“…Beg et al (1995) recently reported that embryonic ®broblasts from RelA-de®cient mice exhibited no alteration of the basal level of expression of two NF-kB-regulated genes, that are IkB-a and granulocyte/macrophage colony stimulating factor. More recently, Neurath et al (1996) showed that antisense oligomer-mediated inhibition of p65 expression by more than 90% failed to modify the level of p50 in mononuclear cells. Together, these ®ndings add further support to the notion (Beg et al, 1995) that Rel A may not control the basal transcription of NF-kB-regulated genes.…”

Activation of p65 NF-κB protein by p210BCR – ABL in a myeloid cell line (P210BCR – ABL activates p65 NF-κB)

“…However, this results does not rule out the activation of other REL/ NF-kB family subunits. Indeed, the members of this family have dierent anities for various DNA binding motifs and each subunit combination may have optimal motifs (Fujita et al, 1992;Kunsch et al, 1992;Narayanan et al, 1993). In the present study, we used the representative Igk kB motif (mouse immunoglobulin k chain) which probably can not display all REL/NF-kB subunit dimers prominently.…”

NF-κB is required for H-ras oncogene induced abnormal cell proliferation and tumorigenesis

“…Earlier studies suggest that NF-kB activation may contribute to cell transformation and tumorigenicity of Ras transformed cells by regulating the expression of genes encoding cell adhesion molecules and by inhibiting transformation-associated cell death (Mayo et al, 1997;Higgins et al, 1993). It has been demonstrated that p65 antisense oligonucleotide inhibited growth and tumorigenicity of variously transformed cell lines including Ras transformed 3T3 cell, presumably by inhibiting cell adhesion processes Narayanan et al, 1993). Since a number of cell adhesion genes have been shown to contain consensus NF-kB binding sites (Ahmad et al, 1995;Ledebur and Parks, 1995;Lewis et al, 1994;Takeuchi and Baichwal, 1995) and cell adhesion molecules play important roles in tumorigenesis and metastasis, inhibition of gene expression of these molecules appears to partly contribute to anti-tumorigenic activity of this oligonucleotide.…”