Hematopoietic stem cells (HSCs) closely associate with the bone marrow (BM) extracellular matrix (ECM) as part of the stem cell """"""""niche"""""""". Interaction with the ECM can regulate the proliferation/differentiation of HSCs and the engraftment ability is critical for normal repopulating activity following transplant. Also, during development, primitive HSCs must migrate appropriately from the sites of the aorta-gonad-mesonephros (AGM) and yolk sac regions, to mature and expand as definitive HSCs in the fetal liver, and finally move to the BM in the newborn. Growth factors secreted from stromal cells can regulate the interaction of HSCs with the ECM during development and in the adult. Therefore, it is important to understand the molecular regulators of cytokine function if HSCs are to be manipulated for clinical use. Members of the matrix metalloproteinase (MMP) family are transcriptionally upregulated following growth factor stimulation. MMPs are zinc dependent proteases that cleave a wide range of ECM components. Specifically, MMP-9 is upregulated in macrophages, lymphocytes, and CD34 + cells following cytokine stimulation. MMP-9 release from neutrophils is also highly associated with HSC mobilization. We propose to test whether natural inhibitors of the MMPs called tissue inhibitors of matrix metalloproteinases (TIMPs) maintain the proteolytic balance in the BM niche by acting as negative regulators of MMP activity. In addition to the MMP inhibiting activity, TIMP-1 has MMP-independent erythroid growth promoting activity and can stimulate development of germinal center B lymphocytes. We have generated retroviral vectors expressing human TIMP-1 and found a novel differentiation inhibiting activity on murine myeloid M1 cells in vitro that was mediated through an autocrine mechanism.
We aim to determine: 1) The structure/function relationship for human TIMP-1 activity in M1 cells in vitro 2) Whether constitutive expression of human TIMP-1 in murine BM HSCs alters long-term repopulating activity and homing/engraftment properties 3) Whether endogenous Timp-1 expression is required in murine HSCs or stromal cells for normal hematopoiesis. We propose that TIMP-1 has MMP-dependent and MMP-independent functions that regulate hematopoiesis by limiting the recruitment and mobilization of stem cells in response to hematopoietic stress. Stem cell-based therapies hold tremendous promise and these studies could lead to novel approaches for increasing engraftment of expanded or embryonic-derived HSCs, for limiting infiltration of tissues with myeloid inflammatory cells, or for inhibiting tumor growth and angiogenesis, through direct effects on HSC survival and proliferation.
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