Studies on the regulation of apolipoprotein E gene expression in macrophages

It was decided to investigate the regulation of apoE by the LXR subfamily of nuclear receptors. LXRs have atheroprotective properties due to their ability to upregulate the expression of genes involved in the reverse cholesterol transport process. ApoE is a direct target of these ligand-mediated transcription factors and a clearer understanding of the LXR-mediated transcription of apoE would aid the development of potential therapeutic agents for atherosclerosis. Therefore, the involvement of signal transduction pathways in the LXR-mediated regulation of apoE in macrophages was investigated. Through the use of commercially available inhibitors, we firstly identified a novel role for the JNK/SAPK MAPK, P13K and CK2 pathways in the LXR-mediated induction of apoE mRNA protein and secretion in human THP-1 macrophages. This inhibition of apoE induction was also shown to occur in human primary monocyte-derived macrophages and further investigations demonstrated the potential role of these cell signalling pathways in the LXR-mediated regulation of ABCA1, ABCG1 and LXRα. It was also found, for the first time, that treatment of THP-1 macrophages with the oxysterol LXR ligand, 22(R)-hydroxycholesterol, induced JNK phosphorylation and kinase activity, and the subsequent phosphorylation of the c-jun transcription factor. Treatment of THP-1 macrophages with the LXR ligand also resulted in the phosphorylation and activation of Akt, a downstream component of PI3K signalling. In addition CK2 activity was found to be increased in THP-1 macrophages treated with 22(R)-hydroxycholesterol. In conclusion, the studies presented in this thesis demonstrated, for the first time, an important role for the JNK/SAPK MAPK, P13K and CK2 pathways in the activation of macrophage apoE gene expression by the LXR subfamily of nuclear receptors. A potential role for these cell signalling pathways was also implicated in the LXR-mediated regulation of ABCA1, ABCG1 and LXRα

Epigenetic silencing of serine protease HTRA1 drives polyploidy

Background: Increased numbers and improperly positioned centrosomes, aneuploidy or polyploidy, and chromosomal instability are frequently observed characteristics of cancer cells. While some aspects of these events and the checkpoint mechanisms are well studied, not all players have yet been identified. As the role of proteases other than the proteasome in tumorigenesis is an insufficiently addressed question, we investigated the epigenetic control of the widely conserved protease HTRA1 and the phenotypes of deregulation. Methods: Mouse embryonal fibroblasts and HCT116 and SW480 cells were used to study the mechanism of epigenetic silencing of HTRA1. In addition, using cell biological and genetic methods, the phenotypes of downregulation of HTRA1 expression were investigated. Results: HTRA1 is epigenetically silenced in HCT116 colon carcinoma cells via the epigenetic adaptor protein MBD2. On the cellular level, HTRA1 depletion causes multiple phenotypes including acceleration of cell growth, centrosome amplification and polyploidy in SW480 colon adenocarcinoma cells as well as in primary mouse embryonic fibroblasts (MEFs). Conclusions: Downregulation of HTRA1 causes a number of phenotypes that are hallmarks of cancer cells suggesting that the methylation state of the HtrA1 promoter may be used as a biomarker for tumour cells or cells at risk of transformation

Identification of cellular and genetic drivers of breast cancer heterogeneity in genetically engineered mouse tumour models

The heterogeneous nature of mammary tumours may arise from different initiating genetic lesions occurring in distinct cells of origin. Here, we generated mice in which Brca2, Pten and p53 were depleted in either basal mammary epithelial cells or luminal oestrogen receptor (ER) negative cells. Basal cell-origin tumors displayed similar histological phenotypes regardless of the depleted gene. In contrast, luminal ER negative cells gave rise to diverse phenotypes, depending on the initiating lesions, including both ER negative and, strikingly, ER positive Invasive Ductal Carcinomas. Molecular profiling demonstrated that luminal ER negative cell-origin tumours resembled a range of the molecular subtypes of human breast cancer, including basal-like, luminal B and ‘normal-like’. Furthermore, a subset of these tumours resembled the ‘claudin-low’ tumour subtype. These findings demonstrate that not only do mammary tumour phenotypes depend on the interactions between cell-of-origin and driver genetic aberrations, but also that multiple mammary tumour subtypes, including both ER positive and negative disease, can originate from a single epithelial cell type. This is a fundamental advance in our understanding of tumour etiology

Studies on the regulation of apolipoprotein E gene expression in macrophages

It was decided to investigate the regulation of apoE by the LXR subfamily of nuclear receptors. LXRs have atheroprotective properties due to their ability to upregulate the expression of genes involved in the reverse cholesterol transport process. ApoE is a direct target of these ligand-mediated transcription factors and a clearer understanding of the LXR-mediated transcription of apoE would aid the development of potential therapeutic agents for atherosclerosis. Therefore, the involvement of signal transduction pathways in the LXR-mediated regulation of apoE in macrophages was investigated. Through the use of commercially available inhibitors, we firstly identified a novel role for the JNK/SAPK MAPK, P13K and CK2 pathways in the LXR-mediated induction of apoE mRNA protein and secretion in human THP-1 macrophages. This inhibition of apoE induction was also shown to occur in human primary monocyte-derived macrophages and further investigations demonstrated the potential role of these cell signalling pathways in the LXR-mediated regulation of ABCA1, ABCG1 and LXR?. It was also found, for the first time, that treatment of THP-1 macrophages with the oxysterol LXR ligand, 22(R)-hydroxycholesterol, induced JNK phosphorylation and kinase activity, and the subsequent phosphorylation of the c-jun transcription factor. Treatment of THP-1 macrophages with the LXR ligand also resulted in the phosphorylation and activation of Akt, a downstream component of PI3K signalling. In addition CK2 activity was found to be increased in THP-1 macrophages treated with 22(R)-hydroxycholesterol. In conclusion, the studies presented in this thesis demonstrated, for the first time, an important role for the JNK/SAPK MAPK, P13K and CK2 pathways in the activation of macrophage apoE gene expression by the LXR subfamily of nuclear receptors. A potential role for these cell signalling pathways was also implicated in the LXR-mediated regulation of ABCA1, ABCG1 and LXR?.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Controlling the stem cell compartment and regeneration in vivo: the role of pluripotency pathways

Since the realization that embryonic stem cells are maintained in a pluripotent state through the interplay of a number of key signal transduction pathways, it is becoming increasingly clear that stemness and pluripotency are defined by the complex molecular convergence of these pathways. Perhaps this has most clearly been demonstrated by the capacity to induce pluripotency in differentiated cell types, so termed iPS cells. We are therefore building an understanding of how cells may be maintained in a pluripotent state, and how we may manipulate cells to drive them between committed and pluripotent compartments. However, it is less clear how cells normally pass in and out of the stem cell compartment under normal and diseased physiological states in vivo, and indeed, how important these pathways are in these settings. It is also clear that there is a potential “dark side” to manipulating the stem cell compartment, as deregulation of somatic stem cells is being increasingly implicated in carcinogenesis and the generation of “cancer stem cells.” This review explores these relationships, with a particular focus on the role played by key molecular regulators of stemness in tissue repair, and the possibility that a better understanding of this control may open the door to novel repair strategies in vivo. The successful development of such strategies has the potential to replace or augment intervention-based strategies (cell replacement therapies), although it is clear they must be developed with a full understanding of how such approaches might also influence tumorigenesis

The key role of apolipoprotein E in atherosclerosis

Apolipoprotein E is a multifunctional protein that is synthesized by the liver and several peripheral tissues and cell types, including macrophages. The protein is involved in the efficient hepatic uptake of lipoprotein particles, stimulation of cholesterol efflux from macrophage foam cells in the atherosclerotic lesion, and the regulation of immune and inflammatory responses. Apolipoprotein E deficiency in mice leads to the development of atherosclerosis and re-expression of the protein reduces the extent of the disease. This review presents evidence for the potent anti-atherogenic action of apolipoprotein E and describes our current understanding of its multiple functions and regulation by factors implicated in the pathogenesis of cardiovascular disease

Chk1 deficiency in the mouse small intestine results in p53-independent crypt death and subsequent intestinal compensation

Chk1 is a serine/threonine protein kinase which is activated by a wide range of DNA damaging agents in order to slow the cell cycle during S phase and G2/M. Abrogation of these cell cycle checkpoints using Chk1 inhibitors results in hypersensitivity to DNA damaging agents in vitro and may provide a potential therapeutic tool to sensitize tumour cells in vivo. We have generated a Cre-Lox based mouse model, where Chkl can be inducibly deleted from somatic epithelial cells in the adult mouse small intestine and liver. Loss of Chk1 in the liver is tolerated with no apparent phenotype. In contrast, loss of Chk1 within the small intestine results in immediate DNA damage, and high levels of p53-independent apoptosis leading to crypt death. However, the intestine is able to compensate for this death by undergoing complete repopulation with Chk1 proficient cells. This data therefore shows that Chk1 deficiency is cell lethal, but the intestine can tolerate such lethality at the organ level

Reproductive history determines ErbB2 locus amplification, WNT signalling and tumour phenotype in a murine breast cancer model

Understanding the mechanisms underlying tumour heterogeneity is key to development of treatments that can target specific tumour subtypes. We have previously targeted CRE recombinase-dependent conditional deletion of the tumour suppressor genes Brca1, Brca2, p53 and/or Pten to basal or luminal ER- cells of the mouse mammary epithelium. We demonstrated that both the cell-of-origin and the tumour-initiating genetic lesions co-operate to influence mammary tumour phenotype. Here, we use a CRE-activated HER2 orthologue to specifically target HER2/ERBB2 oncogenic activity to basal or luminal ER- mammary epithelial cells and perform a detailed analysis of the tumours which develop. We find that in contrast to our previous studies, basal epithelial cells are less sensitive to transformation by the activated NeuKI allele, with mammary epithelial tumour formation largely confined to luminal ER- cells. Histologically, most tumours that developed were classified as either adenocarcinomas of no special type or metaplastic adenosquamous tumours. The former were typically characterised by amplification of the NeuNT/ErbB2 locus; in contrast, tumours displaying squamous metaplasia were enriched in animals that had been through at least one pregnancy and typically had lower levels of NeuNT/ErbB2 locus amplification but had activated canonical WNT signalling. Squamous changes in these tumours were associated with activation of the Epidermal Differentiation Cluster. Thus, in this model of HER2 breast cancer, cell-of-origin, reproductive history, NeuNT/ErbB2 locus amplification, and the activation of specific branches of the WNT signalling pathway all interact to drive inter-tumour heterogeneity

A novel role for c-Jun N-terminal kinase and phosphoinositide 3-kinase in the liver X receptor-mediated induction of macrophage gene expression

Liver X receptors (LXRs) are ligand-dependent transcription factors that are activated by metabolites of cholesterol, oxysterols, and a number of synthetic agonists. LXRs play potent anti-atherogenic roles in part by stimulating the efflux of cholesterol from macrophage foam cells. The LXR-induced expression of ATP-binding cassette transporter (ABC)-A1 and Apolipoprotein E (ApoE) in macrophages is essential for the stimulation of cholesterol efflux and the prevention of atherosclerotic development. Unfortunately, the signaling pathways underlying such regulation are poorly understood and were therefore investigated in human macrophages. The expression of ApoE and ABCA1 induced by synthetic or natural LXR ligands [TO901317, GW3965, and 22-(R)-hydroxycholesterol (22-(R)-HC), respectively] was attenuated by inhibitors of c-Jun N-terminal kinase (JNK) (curcumin and SP600125) and phosphoinositide 3-kinase (PI3K) (LY294002). Similar results were obtained with ABCG1 and LXR-α, two other LXR target genes. LXR agonists activated several components of the JNK pathway (SEK1, JNK and c-Jun) along with AKT, a downstream target for PI3K. In addition, dominant negative mutants of JNK and PI3K pathways inhibited the LXR-agonists-induced activity of the ABCA1 and LXR-α gene promoters in transfected cells. LXR agonists also induced the binding of activator protein-1 (AP-1), a key transcription factor family regulated by JNK, to recognition sequences present in the regulatory regions of the ApoE and ABCA1 genes. These studies reveal a novel role for JNK and PI3K/AKT signaling in the LXR-regulated expression in macrophages of several key genes implicated in atherosclerosis