As one of the most commonly altered genes in human cancer, research on p53 has been intense for many years. It is well established that most of the biological effects of p53 are due to its function as a transcription factor. Although a variety of p53 downstream target genes have been identified, which are involved in apoptosis (e.g. BAX, DR5, BID, APAF-1, Caspase-6, PIGs, Perp, p53AIP, SCN3B and Scotin), knockout of any one individual gene has failed to protect cells completely from p53-induced death. These findings suggest that the p53 response requires a network of collaborating genes (10
). In addition, several studies have shown that some genes with antiapoptotic activity, such as HB-EGF, DcR1 and DcR2, can be transcriptionally activated by p53 (55–57
), while others, such as survivin and PTGG1, are repressed by p53 (6
). Thus, the p53 pathway is complicated, and identification of additional p53 targets is critical for understanding the pathway. Here, our results reveal that NDRG2
is a p53-inducible target gene that is transactivated by p53 and is involved in p53-mediated apoptosis.
Increased NDRG2 mRNA and protein levels occurred not only after treatment with the DNA-damaging agents or stress, which activates endogenous p53, but also following adenovirus-mediated ectopic expression of p53 in p53 null cells. Neither DNA damage in p53 mutant and p53 null cell lines nor introduction of mutant p53 could enhance NDRG2 expression; moreover, knockdown of endogenous p53 markedly attenuated Adr-induced increase of Ndrg2, indicating that the effect requires intact p53. Considering the previous finding that NDRG1, a member of NDRG family, is a direct transcriptional target gene of p53, we hypothesized that NDRG2 could also be a p53 target gene.
has a p53 responsive element in its promoter, we identified one candidate sequence for p53BSs within intron 1 of NDRG2
. The NDRG2
element contained all the conserved residues, as well as five mismatches, of the consensus p53BS. It is well documented that p53 target genes harbor p53BSs in either their promoters or intronic regions, and sequences with different mismatched bases may vary considerably in their transactivation function (52
). Interestingly, some response elements whose structures are quite different from the canonical p53 consensus site can also be recognized by p53 (52
Our experiments demonstrate that the putative p53BS within NDRG2 intron is a functional one in response to exogenous or endogenous wt p53, as shown in reporter assays. Reporter constructs carrying the endogenous NDRG2 promoter region, exon 1 and intron 1 containing p53BSs exhibited increased transcriptional activity upon p53 activation. Meanwhile, the corresponding counterpart lacking p53BSs showed no response to p53, indicating that the intronic p53BSs are essential for p53-mediated transactivation of NDRG2. Using ChIP and EMSA, we demonstrated that the p53BSs form protein–DNA complexes in Ad-p53-infected p53 null cells or Adr-treated wt p53-bearing cells, suggesting that the intronic NDRG2 p53BSs can bind to p53 after its activation and DNA damage. Collectively, we provide compelling evidence that p53 directly transactivates NDGR2 via intronic p53BSs.
As a member of the NDRG
family, the NDRG2
gene has been mapped to chromosome 14q11.2, and encodes a 41 kDa protein, which is expressed in various tissues, particularly in the brain, heart and skeletal muscle (28
). Although the precise function of Ndrg2 is still unclear, it has been shown to be intimately involved in numerous biological processes, such as cell differentiation (30
), neurodegeneration (39
), stress responses (34
), as well as carcinogenesis and cancer progression (44
). Mounting evidence has indicated that NDRG2
mRNA levels are downregulated or undetectable in a number of human cancers and cancer cell-lines, including glioblastoma, squamous cell carcinoma, pancreatic cancer, gastric cancer and colorectal carcinoma, compared to their cognate noncancerous counterparts (44
). Moreover, NDRG2
expression has been inversely correlated with tumor grade and aggressive tumor behavior (43
). In two recent reports, NDRG2
was even identified as a gene whose expression in high-grade primary astrocytomas or gastric cancers was positively correlated with survival (47
). These findings strongly implicate Ndrg2 is a tumor suppressor. However, to date, only a few studies have explored the role of Ndrg2 in tumor suppression. Ndrg2 overexpression in a human glioblastoma cell line was shown to markedly inhibit cell proliferation (43
). Another study from our laboratory demonstrated that Ndrg2 translocates from the cytosol into the nucleus upon NiCl2
treatment, where it interacts with the MSP58 oncoprotein and inhibits MSP58-induced proliferation of HeLa cells (59
). Most recently, Choi et al.
) demonstrated that NDRG2
silencing can decrease Fas-mediated cell death via a slight downregulation of Fas expression in SNU-620 gastric cancer cell lines.
Here, we showed that Ndrg2 is required for the full p53-mediated apoptotic response. Additionally, we showed that NDRG2
knockdown using siRNA markedly attenuated the p53-mediated apoptotic effect. It is possible that the attenuation was incomplete because the silencing efficiency in our current experiment was not high enough to entirely block the NDRG2
expression. Alternatively, it is likely that other apoptosis-related proteins that are downstream of p53 play a compensatory role in the case of NDRG2
knockdown by contributing to the apoptotic effect. In this regard, the function of Ndrg2 may be similar to many known p53 targets, such as BAX, BID, CD95, APAF-1 and DR5, since when each of these genes is silenced or removed from a particular model system, a partial resistance to p53-induced apoptosis is observed (17–19
). Therefore, our results together with two recent reports (23
) support the idea that p53-dependent apoptosis is a complex process, reflecting the combined effect of a number of p53-inducible targets. As for the role of Ndrg2 in the p53-initiated apoptosis pathway, it is unlikely that Ndrg2 functions as a pro-apoptotic factor because we did not observed Ndrg2-promoted apoptosis following its overexpression in p53-null Saos2 cells and p53-intact A549 cells (34
). Another possibility is that Ndrg2 may play a regulatory role either by facilitating apoptosis choice over cell-cycle arrest for p53 in response to stresses, or by regulating p53 downstream targets that are implicated directly in apoptosis, such as Bax, PUMA and NOXA. Further studies are needed to prove or disprove these hypotheses. Interestingly, our previous data showed that NDRG2
also participates in hypoxia-evoked apoptotic response in A549 cells at least partially by acting as a hypoxia-inducible HIF-1 target gene (34
), provided that hypoxia-induced p53 accumulation (60
) and subsequent induction of NDRG2
may play a role during this process. Collectively, it appears that NDRG2
can act as a stress-responsive gene to facilitate cell death in order to eliminate hazardous cells.
We have also shown that NDRG2
overexpression led to inhibition of cell proliferation, regardless of the absence or presence of p53, which is consistent with previous reports (43
). These results suggest that Ndrg2 has an inhibitory role on proliferation that might be p53 independent. Nevertheless, in case of p53-intact cells, whether the proliferation-inhibitory role of Ndrg2 influences its positive role in apoptosis and vice versa still remains to be determined.
In summary, this study demonstrated for the first time that NDRG2, a novel p53-inducible gene, is implicated in the p53-mediated apoptosis pathway in response to DNA damage and also plays a proliferation-inhibitory role that is independent of p53 status. These findings contribute insights for the function of Ndrg2, particularly its stress-responsive activity and tumor-repressive effects.