A Study of the Interactions Between Macrophage Inflammatory Protein 1 Alpha (MIP-1alpha) and Transforming Growth Factor Beta (TGF-beta) in the Control of Haemopoietic Stem Cell Proliferation

[摘要] TGF-beta1 and MIP-1alpha have recently been identified as potent inhibitors of haemopoietic stem cell proliferation. From previous studies, these molecules appear to have similar functions in the control of stem cell proliferation. This study was designed to investigate the relationship, if any, of these two negative regulators in an attempt to elucidate possible distinctive roles for each within the haemopoietic system. The results presented in this thesis demonstrate that both MIP-1alpha and TGF-beta are capable of potently inhibiting the same stem cell population (colony forming unit [CFU]-A/CFU-S). It is further shown that all three mammalian isoforms of TGF-beta are capable of potently inhibiting MIP-la gene expression in bone marrow-derived macrophages, a likely source of MIP-la in the bone marrow. These molecules differ slightly in their potency in this regard, and more diverse members of the TGF-beta superfamily such as activin and BMP-2 do not exhibit this effect within the same spectrum of concentrations as TGF-beta1, -beta2 or -beta3. This inhibition is not specific to MIP-1alpha in that expression of MIP-1beta, a related molecule that does not exhibit potent stem cell inhibitory properties, is inhibited in a similar manner. However, expression of RANTES, which is a more diverse member of the MIP-1alpha superfamily, appears to be unaffected by TGF-beta. The inhibition of MIP-1alpha gene expression is also seen as a reduction in MIP-1alpha protein production by the murine macrophage cell line RAW 264.7. These in vitro results suggest that in the presence of active TGF-beta in vivo, and in the absence of upregulators of MIP-1alpha expression, very little MIP-1alpha will be produced. To address how the target cells of MIP-1alpha, the stem cells, would respond to TGF-beta, and the consequently low levels of MIP-1alpha produced, the effect of TGF-beta on MIP-1alpha receptor levels on FDCP-MIX cells, a murine stem cell line was analysed. TGF-beta (100pM) reversibly downregulates MIP-1alpha receptor levels on these cells to a maximum of around 50-70% after 24hrs. This level of downregulation does not change upon increasing the concentration of TGF-beta or the length of time of exposure of the cells to TGF-beta. Scatchard analysis shows that TGF-beta downregulates MIP-1alpha receptor number numbers with no change in affinity of the remaining receptors for ligand. Further, this reduction in MIP-la receptor numbers by TGF-beta is reflected in reduced ability of FDCP-MIX cells to mobilise calcium in response to MIP-1alpha. These results suggest that TGF-beta may be capable of interfering with the role of MIP-1alpha as a stem cell inhibitor. Indeed, they suggest that in the presence of active TGF-beta in vivo, and in the absence of upregulators of MIP-1alpha expression, MIP-1alpha may only be a weak contributor to the overall physiological inhibition of stem cells. To fully understand the interactions between these two molecules, and to address the possibility that MIP-1alpha functions either wholly or in part through induction of TGF-beta, the effect of MIP-1alpha on TGF-beta1 gene expression and protein production was investigated. The results demonstrate that MIP-1alpha acts as an inducer of both TGF-beta gene expression and protein production in bone marrow macrophages. This suggests that MIP-1alpha may act through upregulation of TGF-beta in these and possibly other secondary cell types. However, inactivation of endogenous TGF-beta in the in vitro CFU-A assay does not reduce the ability of MIP-1alpha to inhibit CFU-A colony formation. This suggests that although MIP-1alpha can induce TGF-beta in bone marrow macrophages, it does not function as a stem cell inhibitor through this action. Indeed, the results presented in this thesis suggest that while MIP-1alpha and TGF-beta appear to have overlapping roles with respect to inhibition of stem cell proliferation, they both act via independent mechanisms. In summary, the results from my PhD suggest that in cells expressing both MIP-1alpha and TGF-beta, TGF-beta will be dominantly expressed and may therefore be the major contributor to physiological inhibition of stem cells.

[发布机构] University:University of Glasgow

[关键词] Medicine, Cellular biology, Molecular biology