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Transcription factor activity is often controlled through the dynamic use of post-translational modifications. Two such modifications are acetylation and sumoylation, which both occur on lysine residues, providing the opportunity for cross-talk at the molecular level. Here, we focussed on the ETS-domain transcription factor PEA3 and studied the potential interplay between these two modifications. We demonstrate that PEA3 is acetylated in a p300-dependent manner. ERK MAPK pathway signalling potentiates acetylation of PEA3, and enhances its trans-activation capacity. However, the major acetylation and sumoylation events take place on the same sites in PEA3 making simultaneous modification impossible. Indeed, manipulation of either the sumoylation or acetylation pathways causes reciprocal changes in PEA3 acetylation and sumoylation respectively. However, despite the mutually exclusive nature of these modifications, both contribute to the trans-activation capacity of PEA3, implying that a dynamic series of modification events occurs during the activation process.

Background: Many members of the ETS-domain transcription factor family are important drivers of tumourigenesis. In this context, their activation by Ras-ERK pathway signaling is particularly relevant to the tumourigenic properties of many ETS-domain transcription factors. The PEA3 subfamily of ETS-domain transcription factors have been implicated in tumour metastasis in several different cancers., Results: Here, we have studied the expression of the PEA3 subfamily members PEA3/ETV4 and ER81/ETV1 in oesophageal adenocarcinomas and determined their role in oesophageal adenocarcinoma cell function. PEA3 plays an important role in controlling both the proliferation and invasive properties of OE33 oesophageal adenocarcinoma cells. A key target gene is MMP-1. The ERK MAP kinase pathway activates PEA3 subfamily members and also plays a role in these PEA3 controlled events, establishing the ERK-PEA3-MMP-1 axis as important in OE33 cells. PEA3 subfamily members are upregulated in human adenocarcinomas and expression correlates with MMP-1 expression and late stage metastatic disease. Enhanced ERK signaling is also more prevalent in late stage oesophageal adenocarcinomas., Conclusions: This study shows that the ERK-PEA3-MMP-1 axis is upregulated in oesophageal adenocarcinoma cells and is a potentially important driver of the metastatic progression of oesophageal adenocarcinomas.

Many transcription factors are controlled through SUMO modification, and in the majority of cases this modification results in enhancements in their repressive properties. In some instances, SUMO modification and its associated repressive activities can be reversed by the action of intracellular signaling pathways, leading to enhanced transcriptional capacities of transcription factors. Here we have investigated sumoylation of the ETS domain transcription factor PEA3 and its interplay with the extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase signaling pathway. PEA3 is modified by SUMO in vitro and in vivo on multiple sites in its N-terminal region. Activation of the ERK MAP kinase pathway promotes sumoylation of PEA3. Importantly, sumoylation of PEA3 is required for maximal activation of target gene promoters, including MMP-1 and COX-2. Molecularly, sumoylation is selectively required for synergistic activation of target gene expression with the coactivator CBP. Moreover, sumoylation of PEA3 is required for ubiquitination of PEA3 and promotes its degradation, suggesting that SUMO-mediated recycling of PEA3 plays a role in PEA3-mediated promoter activation. Thus, in contrast to the majority of other transcription factors studied, sumoylation of PEA3 plays a positive role in PEA3-mediated transcriptional activation and the ERK MAP kinase pathway cooperates with rather than antagonizes this process.

PEA3 is a member of a subfamily of ETS domain transcription factors which is regulated by a number of signaling cascades, including the mitogen-activated protein (MAP) kinase pathways. PEA3 activates gene expression and is thought to play an important role in promoting tumor metastasis and also in neuronal development. Here, we have identified the LIM domain protein LPP as a novel coregulatory binding partner for PEA3. LPP has intrinsic transactivation capacity, forms a complex with PEA3, and is found associated with PEA3-regulated promoters. By manipulating LPP levels, we show that it acts to upregulate the transactivation capacity of PEA3. LPP can also functionally interact in a similar manner with the related family member ER81. Thus, we have uncovered a novel nuclear function for the LIM domain protein LPP as a transcriptional coactivator. As LPP continually shuttles between the cell periphery and the nucleus, it represents a potential novel link between cell surface events and changes in gene expression.

Hypoxia inducible factor (HIF) is a hypoxia-associated transcription factor that has a protective role against hypoxia-induced damage. Prolyl hydroxylase-2 (PHD2) is a dioxygenase enzyme that specifically hydroxylates HIF targeting it for degradation, therefore, inhibition of the PHD2 enzyme activity acts to upregulate HIF function. This study was to identify novel PHD2 inhibitors. An established fluorescence-based PHD2 activity assay was used for inhibitors screening. Western blot and quantitative real-time PCR was used to detect the protein and mRNA levels respectively. Further animal experiment was carried out. Caffeic acid was screened and identified as a novel PHD2 inhibitor. Caffeic acid treated PC12 and SH-SY5Y neuronal cell lines stabilized endogenous HIF-1α protein levels and consequently increased mRNA levels of its downstream regulated genes VEGF and EPO. Caffeic acid treatment reduced hypoxia-induced cell apoptosis and promoted HIF/BNIP3-mediated mitophagy. Moreover, animal studies indicated that caffeic acid increased the level of HIF-1α protein and mRNA levels of VEGF and EPO in the brain of mice exposed to hypoxia. Conventional brain injury markers including malondialdehyde, lactic acid and lactate dehydrogenase in the caffeic acid treated mice were shown to be reduced to the levels of the control group. This study suggests that caffeic acid inhibits PHD2 enzyme activity which then activates the hypoxia-associated transcription factor HIF leading to a neuroprotective effect against hypoxia. Caffeic acid inhibits PHD2 enzyme activity which consequently stabilizes and activates the hypoxia-associated transcription factor HIF-1α. This leads to the protection of hypoxia-induced cell apoptosis and brain injury in mice. [Display omitted] • Natural product library screening identified caffeic acid as a novel PHD2 inhibitor. • Caffeic acid treated neuronal cells stabilized endogenous HIF-1α protein level. • Caffeic acid reduced hypoxia-induced cell apoptosis and promoted mitophagy. • Caffeic acid reduced hypoxia-induced brain injury in mice. [ABSTRACT FROM AUTHOR]