Control of bone homeostasis by MT1-MMP signaling

 描述（由申请人提供）：我们拟研究膜1型基质金属蛋白酶（MT 1- MMP，MMP-14）控制骨形成的新机制。MT 1-MMP是一种降解细胞外基质的跨膜蛋白酶，具有20个氨基酸的胞质尾，在出生后骨形成中起关键作用。在人类中，MT 1-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。在小鼠中，MT 1-MMP缺乏导致侏儒症、骨质减少和严重关节炎。骨髓间充质祖细胞（MSC）中MT 1-MMP缺失完全重现了这种表型，表明MT 1- MMP控制MSC的成骨分化。已经提出MT 1-MMP-/-小鼠的表型是由于缺乏MT 1-MMP的蛋白水解活性。然而，越来越多的证据表明MT 1-MMP具有多种不依赖于蛋白水解的信号传导功能。MT 1-MMP胞质尾区中独特的酪氨酸残基（Y 573）是控制胞内信号传导的基础。我们已经证明Y 573控制Ras-ERK 1/2通路的激活，并且用天冬氨酸（D）（一种带负电荷的氨基酸，如磷酸酪氨酸）取代Y 573阻断该功能而不影响MT 1-MMP蛋白水解活性。因此，我们已经产生了突变小鼠与Y 573 D取代在胞质尾的MT 1-MMP。我们对这种小鼠的表型进行了广泛的研究，我们发现了意想不到且令人惊讶的发现，即MT 1-MMP Y 573 D小鼠具有与MT 1-MMP-/-小鼠相反的表型。同型和半合子MT 1-MMP Y 573 D小鼠均显示骨量增加，MSC的成骨细胞分化增加，髓样前体的破骨细胞分化减少。重要的是，MT 1-MMP Y 573 D表达显著上调MSC中的Wnt信号传导。这是MT 1-MMP的一种新的和意想不到的功能，可用于产生改变骨稳态的各种病症的创新治疗。因此，我们提出通过开发以下具体目的来研究它：目的1）产生并表征骨中条件性表达MT 1-MMP Y 573 D的小鼠的表型。我们已经产生了MT 1-MMP Y 573 D小鼠，其中MT 1-MMP基因座含有floxed neo盒。对于neo盒半合子的小鼠是表型正常的。我们将这些小鼠与在MSC或定向成骨细胞祖细胞或破骨细胞的髓样前体中表达Cre重组酶的小鼠杂交。neo盒的缺失将导致杂合MT 1-MMP Y 573 D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。目的2）研究MT 1-MMP信号通路在骨发育和骨重建中的作用。我们假设MT 1-MMP Y 573 D小鼠的表型可以通过骨髓移植转移到野生型小鼠。因此，我们将来自wt或MT 1-MMP Y 573 D小鼠的骨髓移植到年轻或成年的wt和MT 1-MMP Y 573 D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.