title: The role of ANK interactions with MYBBP1a and SPHK1 in catabolic events of articular chondrocytes.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  ANK/MYBBP1a and SPHK1 interactions stimulate catabolic events in IL-1β-mediated cartilage degradation.
authors.affiliation: Musculoskeletal Research Center, Department of Orthopaedic Surgery, New York University School of Medicine, New York, USA. Electronic address: takeshi.minashima@nyumc.org.
authors.forename: T
authors.initials: T
authors.lastname: Minashima
authors.affiliation: Musculoskeletal Research Center, Department of Orthopaedic Surgery, New York University School of Medicine, New York, USA. Electronic address: kirk.campbell@nyumc.org.
authors.forename: K A
authors.initials: KA
authors.lastname: Campbell
authors.affiliation: Musculoskeletal Research Center, Department of Orthopaedic Surgery, New York University School of Medicine, New York, USA. Electronic address: scotthadleymd@gmail.com.
authors.forename: S R
authors.initials: SR
authors.lastname: Hadley
authors.affiliation: Musculoskeletal Research Center, Department of Orthopaedic Surgery, New York University School of Medicine, New York, USA. Electronic address: zhang.ying@live.cn.
authors.forename: Y
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Musculoskeletal Research Center, Department of Orthopaedic Surgery, New York University School of Medicine, New York, USA. Electronic address: thorsten.kirsch@nyumc.org.
authors.forename: T
authors.initials: T
authors.lastname: Kirsch
mesh_terms: D000818:Animals; D002358:Cartilage, Articular / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D005260:Female; D005270:Femur Head / Q000187:drug effects; D047468:Immunoprecipitation; D053583:Interleukin-1beta / Q000494:pharmacology*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D023421:Models, Animal; D028061:Phosphate Transport Proteins / Q000235:genetics / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012380:Role; D012680:Sensitivity and Specificity; D000099157:Sphingosine Kinase
chemical_list: D053583:Interleukin-1beta; D028061:Phosphate Transport Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D000099157:Sphingosine Kinase; C410982:ank protein, mouse
keywords: ANK (progressive ankylosis protein); Articular chondrocytes; Myb-binding protein 1a (MYBBP1a); NF-kappaB; Sphingosine kinase (SPHK)