Matrix Metalloproteinases: Remodeling of the Extracellular Matrix

在这个项目中，我们继续努力确定骨骼，特别是骨骼干细胞/祖细胞群体是如何在积极重塑的细胞外基质的范围内维持和分化的。为此，我们扩展了我们的工作，以了解不同的骨骼干细胞和破骨细胞的后代，通过不同的分化阶段特异性基因表达来识别如何依赖于mt-MMP依赖的蛋白分解来进行物理基质重塑以及调节细胞信号分子来维持骨骼的动态平衡。 通过在成骨成熟不同阶段的细胞中缺失MT1-MMP的小鼠，确定MT1-MMP的表达对于从早期(OSX-CRE)骨祖细胞表面移除Rank1是必不可少的(Purcell和Kaplan，共同第一作者，等人，提交给PLoS One)。我们进一步鉴定了MT1-MMP从完全成熟的骨钙素表达细胞(OCN-CRE小鼠)中缺失的小鼠的表型。这些小鼠出生时明显正常，但发展成骨量减少的表型并不像OSX-CRE小鼠那样是由于破骨细胞生成增加所致。小鼠在6周时开始自发骨折，骨痂和骨髓腔中完全充满了黄色的富含脂肪细胞的骨髓，而不是正常的骨折骨痂和红色骨髓。对这些小鼠和对照小鼠骨髓切除的骨的表达分析表明，跨膜蛋白Delta样同源基因Dlk1显著上调。在体外，来自全球MT1-MMP缺陷小鼠的BMSCs显示出成脂分化的增加，类似于从我们的合作者，FDA的Steven Bauer和Heba Degheidy博士那里获得的Dlk1缺陷的BMSCs细胞。在缺乏MT1-MMP的情况下，Dlk1在成骨细胞中高度丰富，其中大部分是膜结合的。我们通过共表达证明了Dlk1被MT1-MMPs处理为可溶形式，并且这种形式的Dlk1有效地抑制了典型的Notch配体之一Delta-like 4(DLL4)对Notch信号的诱导。我们的结果表明，骨髓中的成脂扩张与MT1-基质金属蛋白酶将Dlk1的膜形式有效切割成可溶性成脂抑制因子的丧失是一致的。综上所述，这些观察结果强调了MT1-MMP介导的Dlk1蛋白分解作为SSCs/BMSCs向骨髓脂肪细胞默认分化的守门人的重要作用。同样重要的是，这些发现强调了成熟骨细胞(OCN+)在调节SSC/BMSC命运和通过Dlk1介导的抑制成脂分化维持多能性中所起的重要作用。这个项目现在正在与我们以前的K99/R00研究员Jason A.Horton博士和我们的FDA同事合作进行。

In this project, we have continued our efforts to establish how the skeleton and in particular the skeletal stem/progenitor population is maintained and differentiates within the confines of an actively remodeling extracellular matrix. To this end, we have extended our work to understand how the various progeny of the skeletal stem cells and osteoclastic cells, identified by distinct differentiation stage-specific gene expression rely on Mt-Mmp-dependent proteolysis for both physical matrix remodeling as well as modulation of cell-signaling molecules to maintain skeletal homeostasis. Using mice in which Mt1-Mmp was deleted in cells at different stages of osteogenic maturation, it was determined that Mt1-Mmp expression is essential for removal of Rank1 from the surface of early (OSX-Cre) osteoprogenitor cells (Purcell and Kaplan, co-first authors, et al, submitted to PLoS One). We have further characterized the phenotype of mice in which Mt1-Mmp was deleted from fully mature Osteocalcin-expressing cells (Ocn-Cre mice). These mice are born overtly normal, but develop an osteopenic phenotype that is not due to increased osteoclastogenesis as seen in the Osx-Cre mice. The mice begin to spontaneously fracture at 6 wks, and instead of a normal fracture callus and red marrow, the callus and marrow cavity are completely filled with yellow, adipocyte-rich marrow. Expression analyses of marrow-ablated bone from these mice and control littermates demonstrated a dramatic upregulation of the transmembrane protein delta-like homolog, Dlk1. In vitro, BMSCs derived from the global Mt1-Mmp-deficient mice displayed increased adipogenic differentiation, similar to what is made by Dlk1-deficient BMSCs cells obtained from our collaborators, Drs Steven Bauer and Heba Degheidy from the FDA. In the absence of Mt1-Mmp, Dlk1 was highly enriched in osteogenic cells, the majority of which was membrane-bound. We demonstrated by co-expression that Dlk1 is processed into a soluble form by Mt1-Mmp, and further, that this form of Dlk1 efficiently inhibited the induction of Notch signaling by Delta-like 4 (Dll4), one of the canonical notch ligands. Our results demonstrate that adipogenic expansion in the bone marrow is coincident with a loss of efficient cleavage of the membrane form of Dlk1 into a soluble adipogenic suppressor by Mt1-Mmp. Taken together, these observations highlight the essential role of Mt1-Mmp-mediated proteolysis of Dlk1 as a gatekeeper of default differentiation of SSCs/BMSCs into marrow adipocytes. Equally important, these findings underscore the essential role undertaken by mature bone cells (Ocn+) in the regulation of SSC/BMSC fate and maintenance of multipotency through Dlk1-mediated suppression of adipogenic differentiation. This project is now being performed in colllaboration with our former K99/R00 fellow, Dr. Jason A. Horton, and our FDA colleagues.