The role of ank in catabolic events of articular chondrocytes

Abstract

s / Osteoarthritis and Cartilage 22 (2014) S57–S489 S133 210 THE ROLE OF ANK IN CATABOLIC EVENTS OF ARTICULAR CHONDROCYTES T. Kirsch y, T. Minashima y, K. Campbell y, S. Hadley z, Y. Zhang x. yNYU Sch. of Med., New York, NY; zBrigham Women’s Hosp., Harvard Med. Sch., Boston, MA, USA; xCtr. for Joint Surgery, Southwest Hosp., Third Military Med. Univ., Chong Qing, China Purpose: The progressive ankylosis protein (ANK) is a transmembrane protein that transports intracellular pyrophosphate (PPi) to the extracellular milieu. Lack of functional ANK in ank/ank mice or ank knockout mice leads to pathological mineralization of articular cartilage, ligaments, menisci and tendons resulting in arthritic changes because of the reduced amounts of extracellular PPi, a main mineralization inhibitor. We and others, however, have shown that ANK is expressed at low levels in healthy articular cartilage, and ANK expression levels drastically increase during osteoarthritis (OA) initiation and progression. These findings raise the possibility that ANK plays more complex and unknown functions in articular cartilage and OA pathology than only preventing basic calcium crystal formation. Therefore, the purpose of this study was to determine the role of ANK in catabolic events of articular chondrocytes. Methods: The interactions of ANK with Myb-binding protein 1a (MYBBP1a) and sphingosine kinase 1 (SPHK1)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 or femoral heads were treated with interleukin-1beta (IL-1b) in the absence or presence of the SPHK inhibitor, SKI-II. 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-kB activity was determined by transfecting chondrocytes with a NF-kBspecific luciferase reporter and analyzing nuclear translocation of p65 by immunoblotting; MYBBP1a nuclear or cytoplasmic amounts were determined by immunohistochemistry and immunoblotting. Results: Yeast two-hybrid screening and co-immunoprecipitation experiments revealed that the N-terminal region of ANK interacts with SPHK1, whereas the C-terminal region interacts withMYBBP1a. MYBBP1a isa cytoplasmicprotein that canshuttle into thenucleuswhere it represses NF-kB transcriptional activity. Consequently, the loss of ANK/MYBBP1a interactions resulted in increased MYBBP1a nuclear amounts and decreasedNF-kB activity in IL-1b-treated ank/ank chondrocytes compared to IL-1b-treated WT cells. The loss of ANK/SPHK1 interaction resulted in decreased SPHK1 activity in IL-1b-treated ank/ank chondrocytes compared to IL-1b-treated WT cells. Activation of NF-kB-dependent transcriptional activity by IL-1b involves translocation of NF-kB family members, in particular p65 (RelA) and p50, from the cytoplasm to the nucleus. The increase of the nuclear p65 amount and the decrease of the cytoplasmic p65 amount in IL-1b-treated ank/ank chondrocytes were markedly reduced compared to IL-1b-treatedWTcells. The SPHK inhibitor, SKI-II reduced the amount of nuclear p65 and increased the amount of cytoplasmicp65 in IL-1b-treatedWTchondrocytes butnot in IL-1b-treated ank/ank chondrocytes. Transfection with ank expression vector reduced nuclearMYBBP1a amounts and fully restored SPHK andNF-kB activities in IL-1b-treated ank/ank chondrocytes, whereas transfection with P5L mutant ank that doesnot interactwith SPHK1or F376del ank thatdoes not interact with MYBBP1a increased SPHK activity or reduced nuclear MYBBP1a, respectively, and consequently either transfection with P5L or F376del ank only partially restored NF-kB activity. Finally, lack of ANK/ MYBBP1a and SPHK1 interactions in ank/ank chondrocytes resulted in decreased catabolic marker mRNA levels, proteoglycan loss, and MMP-13 immunostaining in IL-1b-treated articular chondrocytes or femoral heads. Conclusion: This study shows that ANK interacts via the N-terminal and C-terminal cytoplasmic regions with SPHK1 and MYBBP1a, respectively. The ANK/MYBBP1a interaction results in the inhibition of the nuclear translocation of MYBBP1a. Nuclear MYBBP1a acts as a corepressor of NF-kB transcriptional activity. The ANK/SPHK1 interaction is a major regulator of SPHK1 activity. Both interactions stimulate NF-kB activity and ultimately catabolic events in IL-1b-treated articular chondrocytes. Since IL-1b and NF-kB play major roles in OA pathogenesis, our findings suggest that increased ANK expression in OA cartilage may be a major contributor to cartilage destruction. Future experiments are needed to fully understand the mechanisms of the interactions between ANK, MYBBP1a and SPHK1 in OA pathology and develop strategies to interfere with these interactions as a potential novel therapy for the treatment of OA. 211 OSX-CRE DIRECTED ABLATION OF SITE-1 PROTEASE IN MICE RESULTS IN KYPHOSIS AND SCOLIOSIS D. Patra, E. DeLassus, L.J. Sandell.Washington Univ. Sch. of Med., St. Louis,