Novel cAMP targets in cell proliferation

Abstract

cAMP is a second messenger that plays a role in a wide variety of biological processes, one of which is the regulation of cell proliferation. Adenylate cyclases generate cAMP in the cell upon activation, followed by binding to and activation of its direct targets, PKA and Epac. PKA is a protein kinase that can regulate the activity of key signalling pathways like the MAPK and PI3K pathway. Inhibition of these pathways plays a role in the inhibition of proliferation of several cell types. This is often achieved by regulation of expression levels of important cell cycle regulators, like cyclin D1 and the CDK inhibitor p27Kip1. The importance of tight regulation of these cell cycle proteins is that aberrant expression can result in deregulated cell cycle and cancer. The second important cAMP target is Epac, which is a guanine nucleotide exchange factor for the small GTPase Rap1. Activation of Epac by cAMP leads to activation of Rap1, which has a well established role in cell adhesion. In this thesis it has been investigated whether certain proteins or genes are regulated by cAMP and whether this contributes to the regulation of cell proliferation. Chapter 2 addresses the question whether the Rap1GEF PDZ-GEF can be controlled by cAMP. It has been reported that this exchange factor can directly bind cAMP, resulting in activation of the small GTPase Ras. We show that PDZ-GEFs are specific for the Rap GTPases and that PDZ-GEFs are not activated by cAMP, do not bind directly cAMP, nor can activate Ras. In addition we searched for ligands that can activate PDZ-GEF, but so far this has not been successful. In chapter 3 the regulation of FoxO transcription factors by cAMP and their role in the anti-proliferative effect of cAMP is described. FoxOs can inhibit proliferation of various cell lines via regulation of cyclin D and p27Kip1. We show that cAMP can regulate the activity of these transcription factors in MEFs via inhibition of the PI3K/PKB pathway and that this is part of the mechanism by which cAMP upregulates p27Kip1 levels. Moreover, we show that activation of the FoxO transcription factors is an important aspect of the anti-proliferative effect of cAMP in MEFs. To identify novel genes involved in the inhibition of proliferation by cAMP, cAMP-induced expression profiles were determined. This is described in chapter 4. Several novel cAMP-regulated genes were identified, discriminated for their regulation by the MAPK pathway or not. HDGF was identified as one of these novel cAMP-regulated genes, and we show that HDGF is a candidate that may be involved in cAMP-dependent inhibition of proliferation. Addendum 1 describes that proliferation of NIH3T3-A14 cells is inhibited by cAMP via a mechanism that does not involve the PKB/FoxO pathway. In line with this, p27Kip1 protein levels are not increased upon cAMP treatment. Interestingly, we observed that in these cells cAMP regulates instead the subcellular localization of p27Kip1. In addendum 2 we describe the effect of cAMP-activated Rap1 on gene expression. The purpose was to identify novel functions of Rap1, apart from the induction of cell adhesion. We identified some genes which expression was changed upon activation of Rap1, indicating that Rap1 can be involved in signalling pathways that control transcription or mRNA stability. In addendum 3 we determine insulin-induced expression profiles as a control for cAMP-regulated genes in NIH3T3-A14 cells. In chapter 5 the achieved results are discussed. A main point is that regulation of p27Kip1 localization or regulation of novel cAMP-regulated genes, which were identified by microarray analysis, may be part of a general mechanism of cAMP-induced inhibition of cell proliferation.