Role of TIMPs in Hematopoietic Stem Cell Biology

描述（由申请人提供）：造血干细胞（HSC）与骨髓（BM）细胞外基质（ECM）密切相关，作为干细胞“生态位”的一部分。 与ECM的相互作用可以调节HSC的增殖/分化，并且植入能力对于移植后的正常重建活动至关重要。此外，在发育过程中，原始HSC必须适当地从性腺-中肾（AGM）和卵黄囊区域的位点迁移，以在胎儿肝脏中成熟和扩增为最终的HSC，并最终移动到新生儿的BM。基质细胞分泌的生长因子可以调节造血干细胞在发育过程中和成年后与细胞外基质的相互作用。因此，重要的是要了解细胞因子功能的分子调节因子，如果HSC被操纵用于临床使用。基质金属蛋白酶（MMP）家族的成员在生长因子刺激后转录上调。MMP是锌依赖性蛋白酶，其切割广泛的ECM组分。具体而言，MMP-9在细胞因子刺激后在巨噬细胞、淋巴细胞和CD 34+细胞中上调。MMP-9从中性粒细胞的释放也与HSC动员高度相关。我们建议测试是否天然抑制剂的基质金属蛋白酶的基质金属蛋白酶（TIMPs）的组织抑制剂保持蛋白水解平衡的BM利基作为负调节MMP活性。除了MMP抑制活性外，TIMP-1还具有MMP非依赖性红细胞生长促进活性，并可刺激生发中心B淋巴细胞的发育。我们已经产生了表达人TIMP-1的逆转录病毒载体，并在体外发现了一种新的对鼠骨髓M1细胞的分化抑制活性，该活性是通过自分泌机制介导的。我们的目标是确定：1）体外M1细胞中人TIMP-1活性的结构/功能关系2）人TIMP-1在鼠BM HSC中的组成型表达是否改变长期再增殖活性和归巢/移植特性3）内源性Timp-1表达是否在鼠HSC或基质细胞中对于正常造血作用是必需的。我们认为TIMP-1具有MMP依赖性和MMP非依赖性的功能，通过限制造血应激时干细胞的募集和动员来调节造血。基于干细胞的疗法具有巨大的前景，这些研究可能会导致新的方法，用于增加扩增或胚胎来源的HSC的植入，用于限制骨髓炎性细胞对组织的浸润，或用于抑制肿瘤生长和血管生成，通过对HSC存活和增殖的直接影响。

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.