Fate decision made by cells, in terms of whether they continue to proliferate, undergo cell cycle arrest or die, is crucial during development as well as for normal tissue homeostasis / functions. When induced, the p53 transcription factor, either leads to cell cycle arrest or apoptosis and its ability to control such events depends on which of its target genes are induced under different cellular conditions (42
). While many p53 dependent target genes have been identified, it is now clear that transcriptional regulation of these target genes by p53 is highly dependent not only on the levels and localization of p53, but also on other cellular proteins that are co-expressed and physically associate with p53. For instance, whilst ASPP1/2 proteins can enhance the expression of pro-apoptotic p53 target genes such as Bax and Apaf1 (32
), co-expression of the differentiation-associated Brn-3a POU protein with p53 results in antagonism on pro-apoptotic Bax and Noxa promoters, but cooperation on p21cip1/waf1
promoter with consequent increased survival and cell cycle arrest in neuronal cells (22
In this study, we have shown that the related Brn-3b POU protein also physically interacts with p53 via the POU domain, a region with very high homology to Brn-3a. Deletion constructs that lacked the DBD and C′ terminus of p53 failed to associate with Brn-3b, suggesting that this region of the p53 protein is required for effective interaction with Brn-3b. Because of the significant similarity (95%) between the POU domains of Brn-3a and Brn-3b (45
), it is likely that Brn-3b will bind p53 via the DBD. Moreover, like Brn-3a, Brn-3b can be immunoprecipitated in complex with p53 from cellular extracts, demonstrating a physiologically relevant association of the Brn-3 proteins with p53 in intact cells. However, analysis of the functional effects of co-expressing Brn-3b with p53 showed that in contrast to Brn-3a (which prevented p53 mediated cell death but supported cell cycle arrest when co-expressed with p53 in these cells), increasing both Brn-3b and wtp53 expression, enhanced cell death in ND7 neuroblastoma cells compared with either factor alone but did not significantly alter cell cycle arrest caused by p53. At the molecular level, Brn-3b can cooperate with p53 to enhance the expression of the pro-apoptotic Bax promoter but not the p21cip1/waf1
promoter. This is in contrast to Brn-3a which antagonized p53 mediated bax expression but cooperated to enhance p21cip1/waf1
expression. Consequently, co-expression of Brn-3b with p53 resulted in increased endogenous Bax protein expression compared with p53 alone or with controls.
The stimulation of Bax expression in the presence of Brn-3b requires wt p53, since on its own, Brn-3b represses bax promoter activity but when it is co-expressed with p53, bax promoter activity is significantly enhanced. Although the experiments in ND7 cells relied on over-expression of exogenous Brn-3b and p53 and has to be interpreted with caution, we observed that induction of wt p53 in MCF7 cells which express high levels of Brn-3b resulted in higher levels of bax and accompanying apoptosis. This was also supported by findings that the p53 inhibitor, pifithrin-α-hydrobromide, prevented p53 mediated trans-activation of the Bax promoter and abolished the cooperative effects of Brn-3b when it is co-expressed with p53. Moreover the distinct effects of Brn-3a and Brn-3b upon co-expression with p53 suggest specific and significant effects that are dependent on which POU protein is co-expressed with p53.
The role for Brn-3b in regulating p53 mediated Bax expression was also demonstrated in primary cultures of DRG neurons prepared from Brn-3b KO mice. Thus, cultures prepared from WT littermates underwent apoptosis upon NGF withdrawal which is associated with increased bax mRNA levels (39
). However, this response was attenuated in Brn-3b(−/−) cultures, which showed significantly reduced death when grown under similar conditions (39), and this correlated with decreased Bax expression. This observation highlights an important mechanism by which Brn-3b may act to control cell fate during development. Brn-3b and the related Brn-3a proteins are both expressed during development in neuronal precursors and in specific neurons but evidence so far suggest that they give rise to distinct effects. The role for Brn-3a during neurogenesis in the PNS has been widely studied and this transcription factor was shown to be essential for survival and differentiation of specific neurons since Brn-3a(−/−) mutants suffer loss of somato-sensory neurons during development with resultant early lethality (46
). Although Brn-3b has been identified as being critical for survival and normal function of retinal ganglion cells (41
), there is still much to be understood about its expression and function during neurogenesis in the PNS. Our findings showing that neurons cultured from Brn-3b KO are resistant to apoptosis is very interesting particularly in view of the decreased bax mRNA expression found in these cells. It is known that p53 levels are increased during neuronal development, when it is associated with either cell cycle arrest or apoptosis (26
). Furthermore, Bax expression in DRG appears to peak between E13.5–15.5 coincident with increased death in Brn-3a KO embryos, which suffer significant neuronal apoptosis during development (36
). A number of studies have shown that Bax plays a critical role in controlling apoptosis in sensory neurons of the DRG. For instance, DRG neurons fail to undergo apoptosis in bax KO mice (38
) and in vitro
cultures of DRG neurons prepared from bax KO mice survived in the absence of the neurotrophic support, NGF which induces widespread apoptosis in WT cultures (37
). Furthermore, loss of bax prevented death in bcl-x deficient mice (54
) whereas over-expression of bax in chick embryos increases the susceptibility of sensory neurons to apoptosis (55
). Given the critical role for bax in controlling death in these neurons, we hypothesize that if p53 is induced in neuronal progenitor cells or neurons when Brn-3b is elevated there will be increased apoptosis as a consequence of elevated Bax. However, if Brn-3a is expressed at high levels, it will repress pro-apoptotic p53 target genes but cooperate to increase differentiation-associated genes to enhance cell cycle arrest and survival. Thus when p53 is induced in these neurons, the relative ratio of Brn-3a to Brn-3b will be critical in determining cell fate. The resistance of Brn-3b KO neurons to apoptosis could result either because Brn-3b is required for maximal induction of bax in these cells or in the absence of Brn-3b, the related Brn-3a factor (which may be increased in a compensatory manner) is likely to associate with p53 and repress Bax expression, even at baseline, with consequent enhanced survival.
Although these hypotheses will need to be tested further, the opposite expression and antagonistic effects of Brn-3b and related Brn-3a has previously been observed in neuroblastoma cells. Thus, Brn-3b but not Brn-3a is expressed in proliferating neuroblastoma cells. Upon induction of differentiation by growth of cells in serum free medium, Brn-3b levels are significantly decreased whilst there is a concomitant increase in Brn-3a expression (4
). Moreover, changing the relative levels of Brn-3a and Brn-3b can alter the growth and behavior of neuroblastoma cells since forced over-expression of Brn-3b prevents differentiation of ND7 cells, whereas increasing Brn-3a induces cell cycle arrest and neurite outgrowth (associated with differentiation) even under growth conditions that should maintain active proliferation (11
Because of the high homology in the conserved DNA binding POU domain, Brn-3a and Brn-3b recognize similar DNA sequences on promoters of target genes. However, there is very limited similarity outside this region in the full length protein. These differences contribute to distinct transcriptional effects by these two POU proteins that are dependent on many factors, including the target gene and cell types. For instance, whereas Brn-3a can stimulate the expression of neuronal target genes such as neurofilament, α-internexin, SNAP 25 and synaptophysin, Brn-3b either represses these genes and antagonizes the effects of Brn-3a or has no effect on the promoter activity (15
). Thus, although these two transcription factors appear to bind to p53 via their POU domains and are thus likely to make similar contacts on the DBD of p53, it is unlikely that their expression would overlap, at least in neuroblastoma cells, where Brn-3b is expressed in actively proliferating cells and will be decreased when Brn-3a is induced upon differentiation. However, it is possible that in other cell types or during development, there may be co-expression of Brn-3a and Brn-3b and thus competition for binding to p53 with consequent changes in cell fate. Under such circumstances, the relative ratio of Brn-3a to Brn-3b factors, which depends on the cellular context, will determine outcome on cellular fate, given the distinct and opposite effects of Brn-3a and Brn-3b on specific p53 mediated target gene expression.
Brn-3b but not Brn-3a protein is elevated in both breast cancers and in neuroblastoma tumours (4
). When elevated in these tumours, Brn-3b transcription factor can alter the growth and behavior of cells by modulating expression of target genes that help to confer growth advantages to the cells, e.g. increasing CDK4 (associated cell cycle progression) and HSP27 (involved in migration and drug resistance) but represses the tumour suppressor protein, BRCA1 (7
). However, its ability to alter p53 mediated effects becomes quite important in these cell types. Although on its own, Brn-3b represses Bax, when co-expressed with wt p53 there is cooperative activation to increase bax expression. This observation is very interesting as it provides a distinct mechanism by which Brn-3b can act in cancer cells in response to different conditions. For instance, many neuroblastomas express wt p53 that remains inactive (e.g. by cytoplasmic sequestration) so does not prevent the initiation or progression of tumourigenesis (56
). Such tumours are often responsive to chemotherapy that relies on induction and activation of wtp53 to give rise to apoptosis. Our results lead us to hypothesize that increasing Brn-3b in cancer cells promotes growth changes whereas induction of wtp53 (e.g. by irradiation or chemotherapy) will induce cell cycle arrest and inhibit growth. When such opposing signals arise in the cells, they triggers a crisis and cells will undergo apoptosis instead of proliferation. Such an effect was previously observed when the co-expression of the oncogenic Myc protein with p53 in quiescent cells lead to apoptosis rather than proliferation or cell cycle arrest (58
). Thus, the ability of Brn-3b to enhance p53 mediated bax transcription may help to explain the molecular mechanism by which cells that express wt p53 and Brn-3b, respond to such conflicting signals.
Although p53 can transactivate the bax promoter in the absence of Brn-3b, it is clear that high levels of both Brn-3b and p53 can significantly enhance the expression of this pro-apoptotic protein in the cells used in this study. The ability of Brn-3b proteins to associate with and enhance p53 mediated apoptosis in contrast to the related Brn-3a protein, which prevents apoptosis by p53 and promotes cell cycle arrest, may prove to be important in understanding how the molecular responses of p53 can be modified in different cell types depending on particular proteins that are co-expressed with it.