Granulin-epithelin precursor binds directly to ADAMTS-7 and ADAMTS-12 and inhibits their degradation of cartilage oligomeric matrix protein.
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 alpha (TNFalpha)-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 TNFalpha-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 TNFalpha-induced ADAMTS-7/ADAMTS-12 expression. Furthermore, GEP levels were significantly elevated in patients with either osteoarthritis or rheumatoid arthritis.


CONCLUSIONS
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.
Guo
Fengjin
F
New York University School of Medicine, New York, New York 10003, USA.
Lai
Yongjie
Y
Tian
Qingyun
Q
Lin
Edward A
EA
Kong
Li
L
Liu
Chuanju
C
D051722:ADAM Proteins / Q000378:metabolism*; D000071096:ADAMTS Proteins; D000071118:ADAMTS7 Protein; D001172:Arthritis, Rheumatoid / Q000378:metabolism / Q000473:pathology; D064236:Cartilage Oligomeric Matrix Protein; D002358:Cartilage, Articular / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D007719:Knee Joint; D064235:Matrilin Proteins; D010003:Osteoarthritis / Q000378:metabolism / Q000473:pathology; D000077153:Progranulins; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011498:Protein Precursors / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D064236:Cartilage Oligomeric Matrix Protein; D004271:DNA, Fungal; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D036341:Intercellular Signaling Peptides and Proteins; D064235:Matrilin Proteins; D000077153:Progranulins; D011498:Protein Precursors; C577553:TSP5 protein, human; D051722:ADAM Proteins; D000071096:ADAMTS Proteins; C496252:ADAMTS12 protein, human; D000071118:ADAMTS7 Protein; C121534:ADAMTS7 protein, human