Control of bone homeostasis by MT1-MMP signaling

 描述(申请人提供)：我们建议研究一种新的机制，通过膜-类型1基质金属蛋白酶(MT1-MMPs，MMP14)控制骨形成。MT1-基质金属蛋白酶是一种降解细胞外基质的跨膜酶，具有20个氨基酸的胞浆尾巴，在出生后骨形成中起着关键作用。在人类中，MT1-MMP的突变会导致多中心骨溶解和关节炎疾病，温彻斯特综合征。在小鼠中，MT1-MMP缺乏会导致侏儒症、骨量减少和严重的关节炎。在骨髓间充质祖细胞(MSC)中MT1-基质金属蛋白酶的缺失完全概括了这种表型，表明MT1-基质金属蛋白酶控制着骨髓间充质干细胞的成骨分化。有研究认为，MT1-MMP1-/-小鼠的表型是由于缺乏MT1-MMPs的蛋白分解活性所致。然而，越来越多的证据表明，MT1-MMPs具有多种不依赖于蛋白降解的信号功能。MT1-MMP胞浆尾中独特的酪氨酸残基(Y573)是控制细胞内信号转导的基础。我们已经证明，Y573控制Ras-ERK1/2途径的激活，并且用天冬氨酸(D)取代Y573可以阻断这一功能，而不影响MT1-MMP蛋白水解酶的活性。因此，我们在MT1-MMP胞浆尾部产生了一个Y573D替换的突变小鼠。我们对这只小鼠的表型进行了广泛的研究，得出了一个意想不到的令人惊讶的发现，MT1-MMPY573D小鼠的表型与MT1-MMP1-/-小鼠相反。纯合子和半合子MT1-MMPY573D小鼠均表现出骨量增加、MSC向成骨细胞分化增加、髓系前体细胞向破骨细胞分化减少。重要的是，MT1-MMPY573D的表达显著上调了MSC中的Wnt信号。这是MT1-MMP一种新的和意想不到的功能，可以被用来产生各种改变骨骼稳态的创新治疗方法。目的1)建立并鉴定MT1-MMPY573D骨组织条件性表达小鼠的表型。我们已经产生了一只MT1-MMPY573D小鼠，其中MT1-MMP座包含一个有花环的NEO盒。小鼠半合子的neo盒是正常的表型。我们将把这些小鼠与在MSC或成骨细胞前体细胞或破骨细胞的髓系前体细胞中表达Cre重组酶的小鼠进行杂交。Neo盒的缺失将导致杂合子MT1-MMPY573D小鼠，它们具有我们所描述的表型。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。目的2)研究MT1-MMPs信号转导通路在骨发育和骨重建中的作用。我们假设MT1-MMPY573D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Wt或MT1-MMPY573D小鼠的骨髓移植到幼年或成年Wt和MT1-MMPY573D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.