FOXO forwards : novel targets and feedback regulation

Abstract

Protein kinase B (PKB)/Akt and Forkhead box-O (FOXO) transcription factors play important roles in cell cycle regulation, cell growth and apoptosis and (thereby) influence organismal health and aging. While increased FOXO activity results in prolonged life-span in organisms of varying complexity, precise PKB activity is required for normal growth and homeostasis in higher eukaryotes. More specifically, elevated PKB activity is often observed in and important for cancer cells whereas decreased PKB activity results in diabetes. PKB activity is increased in response to growth factor signalling and, among other things, stimulates cell division and cell growth while it inhibits apoptosis and FOXO transcription factors. Interestingly, in the absence of PKB inhibition, FOXO family members oppose a number of PKB downstream effects directly or indirectly through target gene regulation. Thus, simply increasing FOXO activity by dampening PKB activity has undesirable side-effects, at least in e.g. mice. Furthermore, elevated FOXO activity also results in activation of PKB, creating a negative feedback loop that presumably limits the duration of FOXO activation. Firstly, we elaborated on the regulation of PKB by FOXO transcription factors and uncovered that they their activities are interdependent. We observe that elevated expression of FOXO3 results in elevated activity of PKB, which is mediated by a number of (context specific) FOXO3 target genes. Conversely, depletion of the predominant FOXO isoforms results in decreased PKB activity, coinciding with decreased expression of FOXO target genes involved in PKB regulation. Thus these data suggest that, unlike previously thought, FOXO transcription factors are continuously active and engaged in maintaining PKB activity. Secondly, we have attempted to clarify how otherwise functionally redundant FOXO transcription factors can mediate their general effect on e.g. life-span by comparing expression changes induced by FOXO3 and FOXO4 in a number of cell lines of human and mouse origin. A small subset of genes is consistently regulated in these cell lines and we have extensively studied previously unidentified FOXO target carboxyl-terminal domain small phosphatase 2 (CTDSP2). CTDSP2 expression results in cell cycle arrest, which partially depends on increased expression of the cyclin dependent kinase inhibitor p21Cip1/WAF1. Our current data indicate that p21Cip1/WAF1 is increased due to aberrant activation of Ras, which indicates CTDSP2 mediates previously unidentified cross-talk between the PKB-FOXO pathway and the Ras-mitogen activated protein kinase (MAPK) pathway. Lastly, we have explored micro RNA regulation by FOXO3 and more extensively studied the effects of FOXO3 regulated miR-26a, of which the precursor is embedded in the transcript of CTDSP2. Interestingly, ectopic expression of miR-26a also results in elevated expression of p21Cip1/WAF1, although this is the result of decreased expression of phosphatase and tensin homolog (PTEN) and concomitant increased PKB activity. Moreover, CTDSP2 expression also results in elevated PKB activity, possibly due to activation of Ras, which indicates that the FOXO regulated CTDSP2/miR-26a cluster may also be part of the PKB-mediated negative feedback loop invoked by FOXO. Concluding, we have made progress in understanding the dynamic behaviour of FOXO transcription factors and extended our knowledge of FOXO target genes and their functions.