Endogenous Csf1 and exogenous HIV-Tat: effectors of intestinal disease

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Genetics, School of Molecular Sciences, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

The human intestine is a complex system of cells and tissue arrangement, forming a structure that serves both as a point of nutrient absorption and as a barrier against invading pathogens. These roles, in turn, are controlled by a range of endogenous and exogenous factors. Extensive research has been done over many years to identify regulatory factors or pathways that control intestinal homeostasis. Yet, the picture is far from complete as evidenced a continuing flow of new regulatory factors and or pathways identified by investigators worldwide. This thesis aims to contribute to this knowledge base by focusing upon two currently under-explored factors in term of intestinal biology; an endogenous haemopoeitic growth factor, Macrophage Colony Stimulating Factor (Csf1) and the HIV transactivator protein, Tat, with the hypothesis that these two factors can influence intestinal epithelial homeostasis, and by implication, that these factors, when dys-regulated, may be involved in intestinal diseases. Data generated from in vivo studies using mouse models and in vitro experiments using cell lines indicated that the absence of functioning Csf1 and or its cellular receptor, Csf1r, affected all major cell types in the intestinal epithelium; all the intestinal tracts examined showed moderate to severe defects in the absence of Csf1 Csf1r signalling. The reintroduction of the cell-surface isoform of Csf1 was found to be able to rescue the observed defects in the Csf1 mutant mice. In vivo molecular analysis indicated a requirement of Csf1 receptor signalling for cellular proliferation and cell linage differentiation while in vitro examination of epithelial cells from mutant mice and from immortalised cell lines confirmed the requirement of Csf1 for cellular proliferation. Further, heterozygous loss of Csf1 was found to be protective against induced (ulcerative) colitis, this finding is concordant with many other studies implicating Csf1 in intestinal inflammation. Similar to the broad ranging effects of Csf1, data generated from in vitro analysis using two CRC cell lines, LIM1215 and LIM2537 indicated that the potential gastro-intestinal (GI) oncogene of the Human Immunodeficiency Virus (HIV), Tat, can modify normal epithelial biology by inhibiting epithelial cyto-differentiation, apoptosis and accelerates tumour progression in an in vivo mouse model. Furthermore, Tat significantly increases in vitro cellular proliferation and migration in the absence of growth supplements. These are indicative characteristics of tumourigenesis. Together, the data generated in this thesis highlighted a requirement of Csf1 in intestinal homeostasis, and that this homeostasis can be disrupted when Csf1 is dys-regulated, or, in the case of HIV-Tat, exhibit tumourigenic properties in the intestinal epithelium.