Control of bone homeostasis by MT1-MMP signaling

 描述（由申请人提供）：我们建议研究膜型1基质金属蛋白酶（MT1-MMP、MMP-14）控制骨形成的新机制。 MT1-MMP 是一种细胞外基质降解跨膜蛋白酶，具有 20 个氨基酸的胞质尾，在出生后骨形成中发挥关键作用。在人类中，MT1-MMP 突变会导致多中心骨质溶解和关节炎疾病（温彻斯特综合征）。 MT1-MMP 缺乏会导致小鼠侏儒症、骨质减少和严重关节炎。骨髓来源的间充质祖细胞（MSC）中的 MT1-MMP 缺失完全重现了这种表型，表明 MT1-MMP 控制 MSC 中的成骨分化。有人提出MT1-MMP-/-小鼠的表型是由于MT1-MMP缺乏蛋白水解活性造成的。然而，越来越多的证据表明 MT1-MMP 具有多种不依赖于蛋白水解的信号传导功能。 MT1-MMP 胞质尾部独特的酪氨酸残基 (Y573) 对于细胞内信号传导的控制至关重要。我们已经证明，Y573 控制 Ras-ERK1/2 通路的激活，并且 Y573 被天冬氨酸 (D)（一种带负电荷的氨基酸，如磷酸酪氨酸）取代可阻断该功能，而不影响 MT1-MMP 蛋白水解活性。因此，我们产生了 MT1-MMP 细胞质尾部带有 Y573D 取代的突变小鼠。我们对该小鼠表型的广泛研究使我们意外地发现，MT1-MMP Y573D 小鼠具有与 MT1-MMP-/- 小鼠相反的表型。 MT1-MMP Y573D 纯合子和半合子小鼠均表现出骨量增加、MSC 成骨细胞分化增加以及骨髓前体细胞破骨细胞分化减少。重要的是，MT1-MMP Y573D 表达显着上调 MSC 中的 Wnt 信号传导。这是 MT1-MMP 的一种新颖且意想不到的功能，可用于针对改变骨稳态的各种病症产生创新疗法。因此，我们建议通过制定以下具体目标来研究它： 目标 1) 生成并表征骨中条件性表达 MT1-MMP Y573D 的小鼠的表型。我们已经生成了 MT1-MMP Y573D 小鼠，其中 MT1-MMP 基因座包含 floxed neo 盒。 neo 盒半合子小鼠表型正常。我们将这些小鼠与在 MSC 或定型成骨细胞祖细胞或破骨细胞的骨髓前体中表达 Cre 重组酶的小鼠进行杂交。删除neo盒将产生杂合的MT1-MMP Y573D小鼠，其具有我们描述的表型。然后我们将分析它们的骨表型、成骨细胞和破骨细胞分化以及 Wnt 信号传导。目标 2) 表征 MT1-MMP 信号在骨发育和重塑中的作用。我们假设MT1-MMP Y573D小鼠的表型可以通过骨髓移植转移到wt小鼠身上。因此，我们将来自 wt 或 MT1-MMP Y573D 小鼠的骨髓移植到年轻或成年 wt 和 MT1-MMP Y573D 小鼠中，并在移植后 8 周分析它们的长骨。

 DESCRIPTION (provided by applicant): We propose to investigate a novel mechanism by which membrane-type 1 matrix metalloproteinase (MT1- MMP, MMP-14) controls bone formation. MT1-MMP, an extracellular matrix-degrading, transmembrane proteinase with a 20-amino acid cytoplasmic tail, plays key roles in postnatal bone formation. In humans mutation of MT1-MMP causes the multicentric osteolysis and arthritis disease, Winchester syndrome. In the mouse MT1-MMP deficiency causes dwarfism, osteopenia and severe arthritis. MT1-MMP deletion in bone marrow-derived mesenchymal progenitor cells (MSC) fully recapitulates this phenotype, showing that MT1- MMP controls osteogenic differentiation in MSC. It has been proposed that the phenotype of MT1-MMP-/- mice results from the lack of the proteolytic activity of MT1-MMP. However, mounting evidence shows a variety of proteolysis-independent signaling functions of MT1-MMP. The unique tyrosine residue (Y573) in the MT1-MMP cytoplasmic tail is fundamental for the control of intracellular signaling. We have shown that Y573 controls activation of the Ras-ERK1/2 pathway, and that Y573 substitution with aspartic acid (D), a negatively charged amino acid like phosphotyrosine, blocks this function without affecting MT1-MMP proteolytic activity. We have therefore generated a mutant mouse with the Y573D substitution in the cytoplasmic tail of MT1-MMP. Our extensive studies of the phenotype of this mouse have led us to the unexpected and surprising finding that, MT1-MMP Y573D mice have a phenotype opposite to that of MT1-MMP-/- mice. Both homo- and hemizygous MT1-MMP Y573D mice show increased bone mass, increased osteoblast differentiation from MSC and decreased osteoclast differentiation from myeloid precursors. Importantly, MT1-MMP Y573D expression dramatically upregulates Wnt signaling in MSC. This is a novel and unexpected function of MT1-MMP that can be exploited for the generation of innovative treatments for a variety of conditions that alter bone homeostasis. Therefore, we propose to study it by developing the following Specific Aims: Aim 1) To generate and characterize the phenotype of mice with conditional expression of MT1-MMP Y573D in bone. We have already generated a MT1-MMP Y573D mouse in which the MT1-MMP locus contains a floxed neo cassette. Mice hemizygous for the neo cassette are phenotypically normal. We will cross these mice with mice that express Cre recombinase in either MSC or committed osteoblast progenitors, or in myeloid precursors of osteoclasts. Deletion of the neo cassette will result in heterozygous MT1-MMP Y573D mice, which have the phenotype we described. We will then analyze their bone phenotype, osteoblast and osteoclast differentiation and Wnt signaling. Aim 2) To characterize the role of MT1-MMP signaling in bone development and remodeling. We hypothesize that the phenotype of MT1-MMP Y573D mice can be transferred to wt mice by bone marrow transplantation. We will therefore transplant bone marrow from wt or MT1-MMP Y573D mice into young or adult, wt and MT1-MMP Y573D mice, and analyze their long bones 8 weeks post transplant.