The role of ANK interactions with MYBBP1a and SPHK1 in catabolic events of articular chondrocytes

T. Minashima, K. A. Campbell, S. R. Hadley, Y. Zhang, T. Kirsch

Research output: Contribution to journalArticlepeer-review


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. Method: 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. Conclusion: ANK/MYBBP1a and SPHK1 interactions stimulate catabolic events in IL-1β-mediated cartilage degradation.

Original languageEnglish (US)
Pages (from-to)852-861
Number of pages10
JournalOsteoarthritis and Cartilage
Issue number6
StatePublished - Jun 2014


  • ANK (progressive ankylosis protein)
  • Articular chondrocytes
  • Myb-binding protein 1a (MYBBP1a)
  • NF-kappaB
  • Sphingosine kinase (SPHK)

ASJC Scopus subject areas

  • Rheumatology
  • Biomedical Engineering
  • Orthopedics and Sports Medicine


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