Role of TIMPs in Hematopoietic Stem Cell Biology

描述(申请人提供)：造血干细胞(HSCs)与骨髓(BM)细胞外基质(ECM)密切相关，是干细胞“利基”的一部分。与ECM的相互作用可以调节HSCs的增殖/分化，植入能力是移植后正常再填充活动的关键。此外，在发育过程中，原始的HSC必须从主动脉-性腺-中肾(AGM)和卵黄囊区适当迁移，成熟并在胎肝中扩展为最终的HSC，最后迁移到新生儿的BM。基质细胞分泌的生长因子可以调节HSCs与细胞外基质的相互作用，在发育过程中和成体。因此，如果要将造血干细胞用于临床，了解细胞因子功能的分子调控是很重要的。基质金属蛋白酶(MMPs)家族成员在生长因子刺激下转录上调。MMPs是一种锌依赖的蛋白水解酶，能裂解多种ECM成分。具体地说，在细胞因子刺激后，巨噬细胞、淋巴细胞和CD34细胞中的基质金属蛋白酶-9表达上调。中性粒细胞释放基质金属蛋白酶-9也与肝星状细胞动员高度相关。我们建议测试被称为基质金属蛋白酶组织抑制因子(TIMPs)的MMPs的天然抑制物是否通过作为MMPs活性的负调节来维持BM利基中的蛋白分解平衡。TIMP-1除了具有抑制基质金属蛋白酶的活性外，还具有基质金属蛋白酶非依赖性的促红细胞生长活性，并能刺激生发中心B淋巴细胞的发育。我们已经构建了表达人TIMP-1的逆转录病毒载体，并在体外发现了一种新的对小鼠髓系M1细胞的分化抑制活性，这种活性是通过自分泌机制介导的。我们的目标是确定：1)人TIMP-1在体外M1细胞中活性的结构/功能关系；2)人TIMP-1在小鼠骨髓HSCs中的结构性表达是否改变了小鼠HSCs的长期再填充活性和归巢/植入特性；3)正常造血是否需要内源性TIMP-1的表达。我们认为TIMP-1具有基质金属蛋白酶依赖和基质金属蛋白酶非依赖性的功能，通过限制干细胞在造血应激中的募集和动员来调节造血。基于干细胞的治疗具有巨大的前景，这些研究可能带来新的方法，通过直接影响HSC的存活和增殖，增加扩增的或胚胎来源的HSC的植入，限制髓系炎症细胞对组织的渗透，或抑制肿瘤生长和血管生成。

DESCRIPTION (provided by applicant): 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.