NOTCH SIGNALING IN REGULATION OF OSTEOGENIC FATE OF MESENCHYMAL STEM CELLS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 最近的证据表明，Notch 1信号传导，干细胞分化的关键调节因子，可能在预防瓣膜钙化中发挥作用。 然而，潜在的机制仍然不清楚。此外，Notch受体家族的其他成员在这一过程中可能发挥的作用尚未研究，尽管它们与Notch 1共享相同的配体和下游途径。我们的目的是研究其他Notch受体和配体是否在调节骨生成和促进心血管钙化中发挥作用。该项目的第二个目的是确定已知诱导动脉粥样硬化病变和钙化的炎症过程与通过翻译后修饰调节Notch信号传导之间的潜在联系。最后，第三个目的是证明Notch家族成员在激活导致血管钙化的特定下游途径中的选择性。 在一项初步研究中，我们研究了Notch受体在调节间充质干细胞（MSC）成骨分化中的潜在作用，作为血管钙化的模型。 Notch 2（N2）和Notch 3（N3）的表达模式提示Notch 2和Notch 3可能参与了成骨分化过程。 通过将N2或N3的活性胞内结构域或空载体（V）转染到小鼠MSC中，进一步研究了N2和N3的作用。 分别于培养2、3、4 d后通过碱性磷酸酶（ALP）活性和von Kossa染色检测羟基磷灰石（HA）晶体形成来检测成骨分化。 与对照MSC相比，N3转染的MSC在成骨或对照培养基中在所有时间点均显示出增强的ALP活性和HA形成，这与N3表达模式所表明的促进成骨的作用一致。 N2转染的MSC在2天时也显示出增强的ALP活性和HA晶体形成。进一步的结果表明，N2在成骨分化的后期阶段具有抑制作用。Notch 2和3还显示出在激活标准下游Notch靶标如HES转录因子方面的选择性。 负责N2和N3的选择性的机制的初步调查表明，N2和N3可能竞争Jag 1配体结合。先前已经证明Notch配体结合受糖基化调节，糖基化抑制Jag配体结合并促进Delta配体结合。与此机制一致，我们的研究结果表明，N3糖基化的减少和N2糖基化的增加可以解释我们在Jag 1结合中看到的变化，其在N3免疫复合物中逐渐增加，在N2免疫复合物中减少。初步结果表明，在成骨过程中，N3糖基化减少，N2糖基化增加。Notch糖基化反过来可以通过氧化还原机制调节，其修饰二硫键并改变Notch胞外结构域中EGF重复序列的结构。我们的主要目的是通过使用质谱法对N2和N3免疫反应性蛋白条带及其翻译后修饰（糖基化、亚硝基化）进行额外鉴定来确认初步结果。如果可能，还将鉴定翻译后修饰的位点，以提供关于Notch信号传导机制的额外信息。 此外，没有关于Notch信号传导过程中Jag配体加工的信息。因此，另一个目的是通过质谱分析Jag 1以鉴定通过糖基化和蛋白水解加工的潜在修饰。我们正在使用1D凝胶电泳和凝胶内消化来鉴定蛋白质并寻找翻译后修饰。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Recent evidence indicates that Notch1 signaling, a key regulator of stem cell differentiation, may play a role in preventing valvular calcification. However, the underlying mechanisms remain obscure. In addition, the roles that other members of Notch receptor family may play in this process have not been investigated, although they share the same ligands and downstream pathways with Notch1. Our aim is to investigate whether other Notch receptors and ligands may play a role in regulating osteogenesis and contribute to cardiovascular calcification. The second aim of this project is to identify a potential connection between inflammatory processes known to induce atherosclerotic lesions and calcification and the regulation of Notch signaling through post-translational modifications. Lastly, a third aim is to demonstrate the selectivity of Notch family members in activating specific downstream pathways leading to vascular calcification. In a preliminary study we investigated the potential role of Notch receptors in regulating the osteogenic differentiation of mesenchymal stem cells (MSC), as a model of vascular calcification. The expression patterns suggested that Notch2 (N2) and Notch3 (N3) might be involved in the osteogenic differentiation process. The roles of N2 and N3 were further investigated by transfecting the active intracellular domains of N2 or N3, or an empty vector (V) into mouse MSC. Osteogenic differentiation was measured after 2, 3 and 4 days via alkaline phosphatase (ALP) activity and hydroxyapatite (HA) crystal formation detected by von Kossa staining. N3-transfected MSC displayed enhanced ALP activity and HA formation compared to control MSC in either osteogenic or control media at all time-points, consistent with a role in promoting osteogenesis, as suggested by N3 expression pattern. N2-transfected MSC also showed enhanced ALP activity and HA crystal formation at 2 days. Further results, suggested an inhibitory role for N2 at later stages of osteogenic differentiation. Notch 2 and 3 also showed selectivity in activating standard downstream Notch targets such as HES transcription factors. Preliminary investigation of the mechanism responsible for N2 and N3 selectivity suggests that N2 and N3 may compete for Jag1 ligand binding. It has been previously demonstrated that Notch ligand binding is regulated by glycosylation, which inhibits Jag ligand binding and promotes Delta ligand binding. In agreement with this mechanism, our results suggest that a decrease in N3 glycosylation and an increase in N2 glycosylation could account for the shift we see in Jag1 binding, which gradually increases in N3 immunocomplexes and decreases in N2 immunocomplexes. Preliminary results, indicate that during osteogenesis there is a decrease in N3 glycosylation and an increase in N2 glycosylation. Notch glycosylation can in turn be regulated by redox mechanisms, which modify disulfide bridges and change the structure of the EGF repeats in the Notch extracellular domain. Our main objective is to confirm the preliminary results by additional identification of N2 and N3 immunoreactive protein bands and their post-translational modifications (glycosylation, nitrosylation) using mass-spectrometry. The sites of post-translational modifications will be also identified if possible, to provide additional information on the mechanism of Notch signaling. In addition, no information exists on the processing of Jag ligands during Notch signaling. Therefore another objective is to analyze Jag1 by mass spectrometry to identify potential modifications by glycosylation and proteolytic processing. We are using 1D gel electrophoresis and in-gel digestion to identify the proteins and search for post-translational modifications.