Cardiomyocyte death following ischaemic/hypoxic injury causes irreversible damage to cardiac function and contributes to chronic diseases such as heart failure. Understanding the mechanisms associated with myocyte loss under these conditions can help to identify strategies to minimise/abrogate such detrimental effects. The p53 protein can induce apoptosis or cell cycle arrest, but effects on cell fate depend on interactions with other regulators such as POU4F2/Brn-3b (Brn-3b), which co-operates with p53 to increase the expression of pro-apoptotic genes. In contrast, the related POU4F1/Brn-3a (Brn-3a) blocks p53-mediated apoptosis but co-operates with p53 to enhance cell cycle arrest. In this study, we showed that permanent coronary artery ligation in mouse hearts, which induced apoptotic markers, activated caspase-3 and -8 and necroptosis markers; RIP-1 and -3 also increased Brn-3b and Brn-3a expression. However, Brn-3a was only detected in uninjured myocardium but not at the site of injury, whereas Brn-3b showed generalised increase, including within the infarct zone. Conversely, p53 was detected in the infarct zone and in some cells adjacent to the site of injury but not in uninjured myocardium. Co-localisation studies showed Brn-3a co-expression with p53 in cardiomyocytes adjacent to the infarct zone, whereas Brn-3b was co-localised with p53 in the infarct zone only. Increased Brn-3b and p53 correlated with elevated expression of pro-apoptotic target genes, Bax, Noxa and PUMA, whereas cleaved caspase-3 confirmed the presence of apoptotic cells within this region of the injured heart. Similarly, simulated ischaemia/reoxygenation (sI/R) injury in neonatal rat ventricular cardiomyocytes (NRVM) and heart derived H9c2 myoblasts increased Brn-3b, p53 as well as apoptotic genes, and this was associated with enhanced apoptosis. Furthermore, targeted reduction of Brn-3b using shRNA caused reduction in pro-apoptotic Bax and Noxa proteins, even though p53 expression remained intact, suggesting that Brn-3b is important for controlling the fate of the myocardium in the injured heart.

IntroductionIn cancer cells, elevated transcription factor-related Brn-3a regulator isolated from brain cDNA (Brn-3b) transcription factor enhances proliferation in vitro and increases tumour growth in vivo whilst conferring drug resistance and migratory potential, whereas reducing Brn-3b slows growth both in vitro and in vivo. Brn-3b regulates distinct groups of key target genes that control cell growth and behaviour. Brn-3b is elevated in >65% of breast cancer biopsies, but mechanisms controlling its expression in these cells are not known.MethodsBioinformatics analysis was used to identify the regulatory promoter region and map transcription start site as well as transcription factor binding sites. Polymerase chain reaction (PCR) cloning was used to generate promoter constructs for reporter assays. Chromatin immunoprecipitation and site-directed mutagenesis were used to confirm the transcription start site and autoregulation. MCF-7 and Cos-7 breast cancer cells were used. Cells grown in culture were transfected with Brn-3b promoter and treated with growth factors or estradiol to test for effects on promoter activity. Quantitative reverse transcriptase PCR assays and immunoblotting were used to confirm changes in gene and protein expression.ResultsWe cloned the Brn-3b promoter, mapped the transcription start site and showed stimulation by estradiol and growth factors, nerve growth factor and epidermal growth factor, which are implicated in breast cancer initiation and/or progression. The effects of growth factors are mediated through the mitogen-activated protein kinase pathway, whereas hormone effects act via oestrogen receptor α (ERα). Brn-3b also autoregulates its expression and cooperates with ERα to further enhance levels.ConclusionsKey regulators of growth in cancer cells, for example, oestrogens and growth factors, can stimulate Brn-3b expression, and autoregulation also contributes to increasing Brn-3b in breast cancers. Since increasing Brn-3b profoundly enhances growth in these cells, understanding how Brn-3b is increased in breast cancers will help to identify strategies for reducing its expression and thus its effects on target genes, thereby reversing its effects in breast cancer cells.

POU4F2/Brn-3b transcription factor (referred to as Brn-3b) is elevated in >60% of breast cancers and profoundly alters growth and behaviour of cancer cells by regulating distinct subsets of target genes. Previous studies showed that Brn-3b was required to maximally transactivate small heat shock protein, HSPB1/Hsp-27 (referred to as , and consequently, Brn-3b expression correlated well with Hsp27 levels in human breast biopsies. In these studies, we showed that Brn-3b is increased in MCF7 breast cancer cells that survive following treatment with chemotherapeutic drug doxorubicin (Dox) with concomitant increases in Hsp-27 expression. Targeting of Brn3b using short interfering RNA reduced Hsp-27 in Doxtreated cells, suggesting that Brn-3b regulates Hsp-27 expression under these conditions. Wound healing assays showed increased Brn-3b in Dox-treated migratory cells that also express Hsp-27. Interestingly, Hsp-27 phosphorylation and cellular localisation are also significantly altered at different times following Dox treatment. Thus, phosphoHsp-27 (p-Hsp27) protein displayed widespread distribution after 24 hrs of Dox treatment but was restricted to the nucleus after 5 days. However, in drug-resistant cells (grown in Dox for >1 month), p-Hsp-27 was excluded from nuclei and most of the cytoplasm and appeared to be associated with the cell membrane. Studies to determine how this protein promotes survival and migration in breast cancer cells showed that the protective effects were conferred by unphosphorylated Hsp-27 protein.