Annexins and Osteoarthritis

描述（由申请人提供）：由于膜联蛋白能够调节参与疾病病理的重要信号通路的活动，因此成为治疗各种疾病（包括癌症、心血管疾病和糖尿病）的有吸引力的治疗靶点。Annexin (Anx) A5和AnxA6在骨关节炎（OA）软骨中高表达；然而，它们在OA病理中的作用尚不清楚。在OA中起作用的两个主要信号通路是NF-κB和Wnt/ß-catenin (Wnt)，这鼓励我们研究AnxA5和AnxA6与这些信号通路之间的潜在相互作用。NF-κB信号通路是骨性关节炎中主要的分解代谢信号通路之一，它在关节软骨细胞中被多种细胞因子（包括白细胞介素(IL)-1）激活，而Wnt信号通路最近被证明在人关节软骨细胞中具有抗分解代谢作用，而在小鼠软骨细胞中具有分解代谢作用。我们的初步研究结果表明，AnxA5和AnxA6刺激NF-κB信号传导，同时抑制Wnt信号传导。此外，我们的研究结果表明，AnxA5通过与半胱氨酸蛋白酶calpain的抑制剂calpastatin直接相互作用来调节NF-κB和Wnt信号，从而刺激calpain的活性。Calpain刺激NF-κB信号传导，抑制Wnt信号传导。另一方面，AnxA6通过直接结合NF-κB复合物的p65单元刺激NF-κB信号传导，同时通过干扰Wnt信号复合物的膜结合抑制Wnt信号传导，这是Wnt信号传导激活所必需的。基于这些发现，我们假设在OA病理过程中，AnxA5和AnxA6通过不同的机制调节NF-κB和Wnt信号，刺激软骨破坏。为了验证我们的假设，我们提出了两个目标。在第一个目标中，我们将确定AnxA5/calpastatin和AnxA6/p65相互作用的性质，以及这些相互作用如何影响NF-κB信号传导活性。此外，我们将确定AnxA5/calpastatin相互作用如何影响Wnt信号传导，以及Ca2+依赖的AnxA6质膜关联如何干扰Wnt信号传导复合物的膜关联，最终影响Wnt信号传导活性。此外，我们将确定AnxA5和AnxA6单敲除和双敲除小鼠在衰老、膝关节IL-1注射或手术诱导的OA过程中，AnxA5和AnxA6如何单独或共同影响NF-κB和Wnt信号。在Aim 2中，我们将确定膜联蛋白介导的这些信号通路对人关节软骨细胞功能和表型的影响。最后，我们将确定膜联蛋白介导的Wnt信号抑制如何影响单敲除和双敲除AnxA5和AnxA6关节软骨细胞以及过表达AnxA5和AnxA6的软骨细胞中OPG的表达，并最终影响OA病理中破骨细胞的发生和软骨下骨的改变。我们期望本研究的成功完成将为OA病理过程中刺激软骨破坏的新机制和OA治疗提供新的治疗靶点。

DESCRIPTION (provided by applicant): Annexins became attractive therapeutic targets for the treatment of various diseases, including cancer, cardiovascular diseases and diabetes, because of their ability to modulate the activities of important signaling pathways involved in disease pathology. Annexin (Anx) A5 and AnxA6 are highly expressed in osteoarthritic (OA) cartilage; however, nothing is known about their role in OA pathology. Two major signaling pathways with roles in OA are NF-κB and the Wnt/ß-catenin (Wnt), encouraging us to examine the potential interaction between AnxA5 and AnxA6 and these signaling pathways. NF-κB signaling pathway, which is being activated in articular chondrocytes by various cytokines, including interleukin (IL)-1, is one of the major catabolic signaling pathways in OA, whereas the Wnt signaling pathway has been recently shown to act anti-catabolically in human articular chondrocytes while acting catabolically in mouse chondrocytes. Our preliminary findings show that AnxA5 and AnxA6 stimulate NF-κB signaling while inhibiting Wnt signaling. In addition, our findings suggest that AnxA5 modulates NF-κB and Wnt signaling via direct interaction with calpastatin, an inhibitor of the cysteine protease calpain, thereby stimulating calpain activity. Calpain stimulates NF-κB signaling, while inhibiting Wnt signaling. AnxA6, on the other hand, stimulates NF-κB signaling via direct binding to the p65 unit of the NF-κB complex, while it inhibits Wnt signaling via interfering with membrane association of the Wnt signaling complex, which is required for Wnt signaling activation. Based on these findings, we hypothesize that AnxA5 and AnxA6 act via different mechanisms, to modulate NF-κB and Wnt signaling to stimulate cartilage destruction during OA pathology. To test our hypothesis, we are proposing two aims. In the first aim we will determine the nature of the AnxA5/calpastatin and AnxA6/p65 interactions, and how these interactions affect NF-κB signaling activity. In addition, we will determine how the AnxA5/calpastatin interaction affect Wnt signaling and how Ca2+-dependent plasma membrane association of AnxA6 interferes with the membrane association of the Wnt signaling complex and ultimately Wnt signaling activity. Furthermore, we will determine how AnxA5 and AnxA6 individually and together affect NF-κB and Wnt signaling during aging, IL-1 injection in the knee joint or surgically induced OA in AnxA5 and AnxA6 single and double knockout mice. In Aim 2, we will determine the effect of annexin-mediated modulation of these signaling pathways on the function and phenotype of human articular chondrocytes. Finally, we will determine how annexin-mediated inhibition of Wnt signaling affects OPG expression in AnxA5 and AnxA6 single and double knockout articular chondrocytes and AnxA5 and AnxA6 overexpressing chondrocytes and ultimately osteoclastogenesis and subchondral bone changes in OA pathology. We expect that the successful completion of this proposal will provide novel mechanisms stimulating cartilage destruction during OA pathology and novel therapeutic targets for the treatment of OA.