Regulation of c-myc gene by nitric oxide via inactivating NF-kB complex in P19 mouse embryonal carcinoma cells

Nitric oxide (NO) may regulate gene expression by directly modifying redox state-sensitive residues of transcription factors. Here we show that the NO donor, sodium nitroprusside (SNP), rapidly represses c-myc gene transcription in a protein synthesis-independent manner in P19 embryonal carcinoma cells by inactivation of NF-kappa B. SNP treatment reduces the DNA binding ability of the constitutively active NF-kappa B heterodimer, p65/p50, and its consequent transactivation of the c-myc promoter. Repression can be blocked by the peroxynitrite scavenger, deferoxamine, but not by dithiothreitol, which triggers reduction of S-nitrosylated residues. In HEK293 cells, where tumor necrosis factor-alpha can activate NF-kappa B, SNP likewise suppresses the binding of the active NF-kappa B complex, restoring the binding of the repressive p50/p50 homodimer complex. This effect of SNP in HEK293 cells is also blocked by deferoxamine. Chromatin immunoprecipitation analysis of SNP-treated P19 cells reveals reduced association of p65, but not of p50, with the promoter region of the endogenous c-myc gene. SNP-induced p65 dissociation was associated with the recruitment of histone deacetylase 1 and 2 to the endogenous c-myc gene promoter and the subsequent deacetylation of its chromatin histone. This study is the first to demonstrate that NO modulates the transcriptional activity of the c-myc gene promoter by dissociating the active form of NF-kappa B and replacing it with a repressive NF-kappa B complex, correlated with the recruitment of gene-silencing histone deacetylases. In light of findings that NF-kappa B stimulates Myc oncoprotein expression in cancers, our findings suggest that NO should be investigated as a prospective therapeutic cancer agent.

7 oligonucleotides (URE, IRE, mt-URE, and nonspecific KOR gene promoter sequences) ( Fig. 2) were each used as probes that were labeled with [a 32 P]dCTP, or used as competitors without labeling. Whole cell lysates were analyzed on a Western blot by using anti-c-Myc antibody as described (10).
Chromatin Immunoprecipitation (ChIP) AssaysaeP19 cells were treated with SNP or SNP plus DFO for 6 h (or in a time-dependent manner), followed by cross-linking with 1% formaldehyde. ChIP assay was as described previously (10) by using 2 mg of anti-AcH4, anti-p65, anti-p50, anti-HDAC1, or anti-HDAC2 antibodies. Precipitated DNA was amplified with primers specific to the URE flanking sequences in the endogenous cmyc promoter and to its 3' untranslated region (UTR), followed by Southern blot analyses of amplified endogenous c-myc sequences. in both control cells (lanes 1-5) and cells exposed to NO (lanes 6-10). Therefore, c-myc mRNA stability is not affected by NO, whereas its gene transcription is directly repressed by NO signal.

Repression of c-myc Gene Transcription by NO through Inactivation of NF-kB
ActivityaeTo define the target of NO-mediated repression of c-myc gene transcription, the regulatory regions of c-myc gene was carefully examined. This gene utilizes two promoters located approximately 160 bp apart, with the second promoter residing in exon 1. Interestingly, two putative NF-kB binding sites, an upstream responsive element (URE) (GGGTTTCCCC) located at -1261 to -1251 bp in the 5'-flanking region of promoter 1, and an internal responsive element (IRE) (GGGAATTTTT) located at +280 to +289 relative to the promoter P2 (27, 28), were found to be relevant ( Fig. 2A). NF-kB has been shown to be a target protein of NO signal (3,42). We examined how the endogenous NF-kB activity in P19 cells might be affected by NO and augment c-myc gene expression. P19 cells were treated with SNP, followed by analyzing its nuclear extract for DNA binding ability to the kB binding sites of the c-myc promoter in EMSA. As shown  -labeled URE probe revealed a major retarded band and several minor   bands (lane 1), which could be competed out specifically by the wild type cold probe   (lane 2), but not by the mutated cold probe (mt-URE, lane 3) or nonspecific oligonucleotides (lane 4). The anti-p65 antibody was able to block the major binding species that is the heterodimeric p65/p50 NF-kB complex (lane 5), and the anti-p50 antibody was able to supershift the minor species, the p50 complex (lane 6). The control, a non-specific antibody such as the anti-Sp1, had no effect (lane 7). Therefore, the URE consensus sequences of the c-myc gene indeed can be bound by p65/p50 heterodimer and p50 homodimer of NF-kB, but the major complex in P19 cells is the transcriptionally active p65/p50 heterodimer. The second putative NF-kB binding site, IRE, was also tested in EMSA as shown in Fig. 2C, which appeared to share a very similar binding pattern as that of the URE. Furthermore, SNP decreased the intensity of the p65/p50 NF-kB band but increased the p50 complex for both NF-kB sites (lanes 1-4), revealing a decreased activator p65/p50-DNA interaction of P19 nuclear extract as a result of SNP treatment.
To determine if the transcriptional activity of endogenous NF-kB was affected by NO, transient transfection assays were conducted in P19 stem cells and fully differentiated COS-1 cells by using a specific NF-kB reporter, p(Igk) 4 -LUC (39), without introducing any other expression vectors. As shown in Fig. 3A, SNP effectively suppressed the specific reporter activity in a dose-dependent manner in P19 cells, but not in COS-1 cells, in consistence with our previous conclusion that NO suppressed KOR gene transcription only in P19 cells but not in COS-1 cells (10). This suppressive effect of NO on NF-kB reporter plasmid in P19 cells agreed with the reduced DNA binding activity of NF-kB from SNP-treated P19 cells (Fig. 2C). The resistance of the same reporter activity to SNP in COS-1 cells was due to the absence of p65/p50 species binding to the URE in COS-1 cells that contained primarily the p50 species binding to the URE site (data not shown). To further confirm that the effect of SNP was indeed mediated by p65/p50 NF-kB, the specific reporter and a pMT2T-p65 expression vector (37) were simultaneously introduced into COS-1 cells (Fig. 3A). Indeed, ectopic expression of p65 increased the specific reporter activity in COS-1 cells, which was again by guest on November 17, 2017 http://www.jbc.org/ Downloaded from suppressed by SNP in a dose-dependent manner (Fig. 3B). Thus, NO not only suppresses the DNA binding activity of p65/p50 NF-kB, but also efficiently reduces NF-kB activity in transcriptional control. The effect is primarily at the p65 subunit.

Repression of c-myc Gene Transcription by NO through PeroxynitriteaePreviously
we have suggested that S-nitrosylation was not involved in c-myc repression by SNP, a NO donor, in P19 cells because this suppressive effect could not be reversed by DTT, an agent that blocks S-nitrosylation of Cys residues by NO (10). Although it is accepted that NF-kB activity can be inhibited by NO through S-nitrosylation on a Cys residue of  4). These results rule out S-nitrosylation as the underlying mechanism, and suggest that Tyr nitration could be the actual underlying mechanism for this novel effect of NO. It is proposed that an alternative pathway of protein modification by NO, possibly through Tyr nitration of the p65 subunit of NF-kB, is able to rapidly inactivate NF-kB activity.
We are in the process of determining specific Tyr residue(s) of NF-kB that could be NO targets.
NO is known to be involved in numerous pathophysiological processes. In particular, Tyr nitration of various proteins by peroxynitrite has been detected in a number of inflammatory or degenerative diseases (54, 55), and tumors (9, 56). As such, it has generated enthusiasm for managing diseases with agents that modulate NO signaling pathways. NF-kB is an important transcription factor that coordinates the expression of a wide variety of genes (57), and is known to be an inhibitor of apoptosis, because of its ability to induce anti-apoptotic factors such as cellular inhibitors of apoptosis (cIAPs) and the members of the BCL2 family (58,59). The c-Myc protein is involved in numerous important cellular processes, including proliferation, metabolism, cell cycle control, apoptosis, differentiation, genomic stability and tumor formation (11-13, 60, 61).
The ability of a NO donor, SNP, to directly and effectively repress NF-kB activity provides a potentially important route for therapeutic intervention of numerous disease conditions. In particular, its rapid transcriptional repression of c-myc gene via inactivating