Minashima T, Campbell K A, Hadley S R, Zhang Y, Kirsch T
Musculoskeletal Research Center, Department of Orthopaedic Surgery, New York University School of Medicine, New York, USA.
Osteoarthritis Cartilage. 2014 Jun;22(6):852-61. doi: 10.1016/j.joca.2014.04.008. Epub 2014 Apr 18.
To determine the role of progressive ankylosis protein (ANK)/Myb-binding protein 1a (MYBBP1a) and sphingosine kinase 1 (SPHK1) interactions in catabolic events of articular chondrocytes.
ANK/MYBBP1a and SPHK1 interactions were identified using yeast two-hybrid screening and co-immunoprecipitation. To determine the role of these interactions in catabolic events of articular chondrocytes, ank/ank and wild type (WT) mouse chondrocytes transfected with full-length or mutant ank expression vectors (EVs) or femoral heads were treated with interleukin-1beta (IL-1β) in the absence or presence of SPHK inhibitor. Catabolic marker mRNA levels were analyzed by real time PCR; proteoglycan loss using safranin O staining and MMP-13 immunostaining were determined in femoral head explants; NF-κB activity was determined by transfecting chondrocytes with an NF-κB-specific luciferase reporter and analyzing nuclear translocation of p65 by immunoblotting; MYBBP1a nuclear or cytoplasmic amounts were determined by immunohistochemistry and immunoblotting.
The ANK N-terminal region interacted with SPHK1, whereas a cytoplasmic C-terminal loop interacted with MYBBP1a. Lack of ANK/MYBBP1a and SPHK1 interactions in ank/ank chondrocytes resulted in increased MYBBP1a nuclear amounts and decreased SPHK1 activity, and consequently decreased NF-κB activity, catabolic marker mRNA levels, proteoglycan loss, and MMP-13 immunostaining in IL-1β-treated articular chondrocytes or femoral heads. Transfection with full-length ank EV reduced nuclear MYBBP1a amounts and fully restored SPHK and NF-κB activities in IL-1β-treated ank/ank chondrocytes, whereas transfection with P5L or F376del mutant ank reduced nuclear MYBBP1a or increased SPHK activity, respectively, and consequently either transfection only partially restored NF-κB activity.
ANK/MYBBP1a and SPHK1 interactions stimulate catabolic events in IL-1β-mediated cartilage degradation.
确定进行性关节强硬蛋白（ANK）/Myb结合蛋白1a（MYBBP1a）与鞘氨醇激酶1（SPHK1）的相互作用在关节软骨细胞分解代谢事件中的作用。
采用酵母双杂交筛选和免疫共沉淀法鉴定ANK/MYBBP1a与SPHK1的相互作用。为确定这些相互作用在关节软骨细胞分解代谢事件中的作用，在有无SPHK抑制剂的情况下，用白细胞介素-1β（IL-1β）处理转染了全长或突变型ank表达载体（EVs）的ank/ank和野生型（WT）小鼠软骨细胞或股骨头。通过实时PCR分析分解代谢标志物mRNA水平；用番红O染色和MMP-13免疫染色测定股骨头外植体中的蛋白聚糖损失；通过用NF-κB特异性荧光素酶报告基因转染软骨细胞并通过免疫印迹分析p65的核转位来测定NF-κB活性；通过免疫组织化学和免疫印迹测定MYBBP1a的核或细胞质含量。
ANK的N端区域与SPHK1相互作用，而细胞质C端环与MYBBP1a相互作用。ank/ank软骨细胞中ANK/MYBBP1a和SPHK1相互作用的缺乏导致MYBBP1a核含量增加和SPHK1活性降低，进而导致IL-1β处理的关节软骨细胞或股骨头中NF-κB活性、分解代谢标志物mRNA水平、蛋白聚糖损失和MMP-13免疫染色降低。用全长ank EV转染可降低IL-1β处理的ank/ank软骨细胞中核MYBBP1a含量并完全恢复SPHK和NF-κB活性，而用P5L或F376del突变型ank转染分别降低核MYBBP1a或增加SPHK活性，因此两种转染仅部分恢复NF-κB活性。
ANK/MYBBP1a和SPHK1的相互作用刺激IL-1β介导的软骨降解中的分解代谢事件。
