The role of ANK interactions with MYSBP1a and SPHK1 in catabolic events of articular chondrocytes

Objective: To determine the role of progressive ankylosis protein (ANK)/Myb-binding protein 1 a (MYBBP1a) and sphingosine kinase 1 (SPHK1) interactions in catabolic events of articular chondrocytes. Method: ANK/MYBBP1 a 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-1 beta) in the absence or presence of SPHK inhibitor. Catabolic marker mRNA levels were analyzed by real time PCR; proteoglycan loss using safranin 0 staining and MMP-13 immunostaining were determined in femoral head explants; NF-kappa B activity was determined by transfecting chondrocytes with an NF-kappa B-specific luciferase reporter and analyzing nuclear translocation of p65 by immunoblotting; MYBBP1 a nuclear or cytoplasmic amounts were determined by immunohistochemistry and immunoblotting. Results: The ANK N-terminal region interacted with SPHK1, whereas a cytoplasmic C-terminal loop interacted with MYBBP1a. Lack of ANK/MYBBP1 a and SPHK1 interactions in ank/ank chondrocytes resulted in increased MYBBP1 a nuclear amounts and decreased SPHK1 activity, and consequently decreased NF-kappa B activity, catabolic marker mRNA levels, proteoglycan loss, and MMP-13 immunostaining in IL-1 beta-treated articular chondrocytes or femoral heads. Transfection with full-length ank EV reduced nuclear MYBBP1a amounts and fully restored SPHK and NF-kappa B activities in IL-1 beta-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-kappa B activity. Conclusion: ANK/MYBBP1 a and SPHK1 interactions stimulate catabolic events in IL-1 beta-mediated cartilage degradation. (C) 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.