Abstract：
Cysteine proteases orchestrate bone remodeling, and are inhibited by cystatins. In reinforcing our hypothesis that exogenous and naturally obtained inhibitors of cysteine proteases (cystatins) act on bone remodeling, we decided to challenge osteoblasts with sugarcane-derived cystatin (CaneCPI-5) for up to 7 days. To this end, we investigated molecular issues related to the decisive, preliminary stages of osteoblast biology, such as adhesion, migration, proliferation, and differentiation. Our data showed that CaneCPI-5 negatively modulates both cofilin phosphorylation at Ser03, and the increase in cytoskeleton remodeling during the adhesion mechanism, possibly as a prerequisite to controlling cell proliferation and migration. This is mainly because CaneCPI-5 also caused the overexpression of the CDK2 gene, and greater migration of osteoblasts. Extracellular matrix remodeling was also evaluated in this study by investigating matrix metalloproteinase (MMP) activities. Our data showed that CaneCPI-5 overstimulates both MMP-2 and MMP-9 activities, and suggested that this cellular event could be related to osteoblast differentiation. Additionally, differentiation mechanisms were better evaluated by investigating Osterix and alkaline phosphatase (ALP) genes, and bone morphogenetic protein (BMP) signaling members. Altogether, our data showed that CaneCPI-5 can trigger biological mechanisms related to osteoblast differentiation, and broaden the perspectives for better exploring biotechnological approaches for bone disorders.
摘要：
半胱氨酸蛋白酶协调骨重建，并被胱抑素抑制。在加强我们的假设，即外源性和天然获得的半胱氨酸蛋白酶抑制剂（cystatins）作用于骨重建，我们决定用甘蔗源性胱抑素(CaneCPI-5)对成骨细胞进行7天的攻击.为此,我们调查了与决定性有关的分子问题，成骨细胞生物学的初步阶段，如附着力，迁移,扩散,和差异化。我们的数据显示，CaneCPI-5对Ser03上的cofilin磷酸化以及粘附机制中细胞骨架重塑的增加都有负向调节，可能作为控制细胞增殖和迁移的先决条件。这主要是因为CaneCPI-5也引起了CDK2基因的过表达,和更大的成骨细胞迁移。在这项研究中，还通过研究基质金属蛋白酶（MMP）活性来评估细胞外基质的重塑。我们的数据显示CaneCPI-5过度刺激MMP-2和MMP-9活性，并提示该细胞事件可能与成骨细胞分化有关。此外,通过研究Osterix和碱性磷酸酶（ALP）基因更好地评估了分化机制，和骨形态发生蛋白（BMP）信号成员。总之,我们的数据显示CaneCPI-5可以触发与成骨细胞分化相关的生物学机制，并拓宽了更好地探索骨骼疾病的生物技术方法的视野。
