Abstract
Objective
To determine 1) whether a protein interaction network exists between granulin‐epithelin precursor (GEP), ADAMTS‐7/ADAMTS‐12, and cartilage oligomeric matrix protein (COMP); 2) whether GEP interferes with the interactions between ADAMTS‐7/ADAMTS‐12 metalloproteinases and COMP substrate, including the cleavage of COMP; 3) whether GEP affects tumor necrosis factor α (TNFα)–mediated induction of ADAMTS‐7/ADAMTS‐12 expression and COMP degradation; and 4) whether GEP levels are altered during the progression of arthritis.
Methods
Yeast two‐hybrid, in vitro glutathione S‐transferase pull‐down, and coimmunoprecipitation assays were used to 1) examine the interactions between GEP, ADAMTS‐7/ADAMTS‐12, and COMP, and 2) map the binding sites required for the interactions between GEP and ADAMTS‐7/ADAMTS‐12. Immunofluorescence cell staining was performed to visualize the subcellular localization of GEP and ADAMTS‐7/ADAMTS‐12. An in vitro digestion assay was employed to determine whether GEP inhibits ADAMTS‐7/ADAMTS‐12–mediated digestion of COMP. The role of GEP in inhibiting TNFα‐induced ADAMTS‐7/ADAMTS‐12 expression and COMP degradation in cartilage explants was also analyzed.
Results
GEP bound directly to ADAMTS‐7 and ADAMTS‐12 in vitro and in chondrocytes, and the 4 C‐terminal thrombospondin motifs of ADAMTS‐7/ADAMTS‐12 and each granulin unit of GEP mediated their interactions. Additionally, GEP colocalized with ADAMTS‐7 and ADAMTS‐12 on the cell surface of chondrocytes. More importantly, GEP inhibited COMP degradation by ADAMTS‐7/ADAMTS‐12 in a dose‐dependent manner through 1) competitive inhibition through direct protein–protein interactions with ADAMTS‐7/ADAMTS‐12 and COMP, and 2) inhibition of TNFα‐induced ADAMTS‐7/ADAMTS‐12 expression. Furthermore, GEP levels were significantly elevated in patients with either osteoarthritis or rheumatoid arthritis.
Conclusion
Our observations demonstrate a novel protein–protein interaction network between GEP, ADAMTS‐7/ADAMTS‐12, and COMP. Furthermore, GEP is a novel specific inhibitor of ADAMTS‐7/ADAMTS‐12–mediated COMP degradation and may play a significant role in preventing the destruction of joint cartilage in arthritis.