Mutations in Bone Morphogenetic Protein (BMP) Receptor (BMPR)1A and SMAD4 are detected in 50% of juvenile polyposis syndrome (JPS) patients, who develop stroma-rich hamartomatous polyps. The... Show moreMutations in Bone Morphogenetic Protein (BMP) Receptor (BMPR)1A and SMAD4 are detected in 50% of juvenile polyposis syndrome (JPS) patients, who develop stroma-rich hamartomatous polyps. The established role of stromal cells in regulating BMP activity in the intestine implies a role for stromal cells in polyp development. We used conditional Cre-LoxP mice to investigate how specific loss of BMPR1A in endothelial cells, fibroblasts, or myofibroblasts/smooth muscle cells affects intestinal homeostasis. Selective loss of BMPR1A in fibroblasts causes severe histological changes in the intestines with a significant increase in stromal cell content and epithelial cell hyperproliferation, leading to numerous serrated polyps. This phenotype suggests that crucial changes occur in the fibroblast secretome that influences polyp development. Analyses of publicly available RNA expression databases identified CXCL12 as a potential candidate. RNAscope in situ hybridization showed an evident increase of Cxcl12-expressing fibroblasts. In vitro, stimulation of fibroblasts with BMPs resulted in downregulation of CXCL12, while inhibition of the BMP pathway resulted in gradual upregulation of CXCL12 over time. Moreover, neutralization of CXCL12 in vivo in the fibroblast-specific BMPR1A KO mice resulted in a significant decrease in polyp formation. Finally, in CRC patient specimens, mRNA-expression data showed that patients with high GREMLIN1 and CXCL12 expression had a significantly poorer overall survival. Significantly higher GREMLIN1, NOGGIN, and CXCL12 expression were detected in the Consensus Molecular Subtype 4 (CMS4) colorectal cancers, which are thought to arise from serrated polyps. Taken together, these data imply that fibroblast-specific BMP signaling-CXCL12 interaction could have a role in the etiology of serrated polyp formation. Show less
In this thesis the role and regulation of the negative p53 regulator Hdmx (Mdmx/Mdm4) are the main topics. Whereas the role of its close homolog Hdm2 is elaborately studied, the function of Hdmx in... Show moreIn this thesis the role and regulation of the negative p53 regulator Hdmx (Mdmx/Mdm4) are the main topics. Whereas the role of its close homolog Hdm2 is elaborately studied, the function of Hdmx in tumourigenesis less understood. Post-translational modification of Hdmx is abundant and may play an important role in the regulation of it function. However, relatively little is known about the nature of these modifications. The aim of this thesis was to provide more insight in the putative oncogenic role of Hdmx and to elucidate some of the mechanisms by which Hdmx function is regulated. Chapter 1 introduces the p53 pathway and it describes the main aspects of the regulation of p53 by Hdm2 and Hdmx. In Chapter 2 the present knowledge about the regulation of Hdmx in relation to the p53 response is presented. Chapter 3 describes the effects of c-Abl mediated phosphorylation on tyrosine residues 55 and 99 of Hdmx, which both modulate the interaction with p53. In chapter 4 the oncogenic functions of Hdmx have been investigated, using an in vitro transformation model of human primary fibroblasts and embryonic retinoblasts. The function of Hdmx overexpression and alternative splicing in osteosarcoma is described in Chapter 5. A model for a role of Hdmx in early tumours and loss of Hdmx in later stage tumours is proposed, with a key role for the change in ratio between the expression of the alternative Hdmx splice variant, Hdmx-S and full length Hdmx. Chapter 6 shows that p53 and its regulators Hdm2 and Hdmx are SUMOylated and ubiquitinated and how the SUMO-specific ubiquitin E3 ligase RNF4 might be involved in these processes. In chapter 7 the function of the FAU gene, encoding the ubiquitin-like protein FUBI and the ribosomal S30 protein, has been studied in osteosarcoma cells. Chapter 8 is a general discussion and provides a summary of the thesis. Show less
