Membrane-type 1 matrix metalloproteinase controls osteo- and chondrogenesis by a proteolysis-independent mechanism mediated by its cytoplasmic tail

Abstract

s / Osteoarthritis and Cartilage 23 (2015) A26eA81 A64 deregulated genes included Has1, ADAMTS4, Tnf, IL6, IL18, Inhba, Cd68, Ngf, Ccr2, Wnt 16, Tnfaip6 and Il1r. Conclusions: Deletion of JNK2 retarded the development of experimental murine OA, showing the importance of the kinase for cartilage homeostasis in vivo and implicating an intracellular signalling pathway in OA for the first time. 68 MEMBRANE-TYPE 1 MATRIX METALLOPROTEINASE CONTROLS OSTEOAND CHONDROGENESIS BY A PROTEOLYSIS-INDEPENDENT MECHANISM MEDIATED BY ITS CYTOPLASMIC TAIL Q. Yang y, M. Attur y, T. Kirsch z, Y.J. Lee z, S. Yakar x, Z. Liu x, S.B. Abramson y, P. Mignatti yk. yNYU Sch. of Med. Dept. of Med., New York, NY, USA; zNYU Sch. of Med. Dept. of Orthopedic Surgery, New York, NY, USA; xNYU Coll. of Dentistry Dept. of Basic Sci. and Craniofacial Biology, New York, NY, USA; kNYU Sch. of Med. Dept. of Cell Biology, New York, NY, USA Purpose: We aimed to understand the mechanism by which membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14) controls bone and cartilage homeostasis. MT1-MMP, a cell-membrane-bound proteinase with an extracellular catalytic site and a 20-amino acid cytoplasmic tail, degrades the extracellular matrix and plays a key role in postnatal bone formation. MT1-MMP is overexpressed in osteoarthritis, and mutation of MT1-MMP in humans causes the multicentric osteolysis and arthritis disease, Winchester syndrome. The genetic deficiency of MT1-MMP (MT1-MMP-/-) in the mouse causes dwarfism, osteopenia and severe arthritis. Conditional deletion of MT1-MMP in bone marrow-derived mesenchymal progenitor cells (BM-MSC) fully recapitulates this phenotype, showing that MT1-MMP controls osteogenic differentiation inMSC. It has been proposed that the phenotype of MT1-MMP-/mice results from the lack of MT1-MMP proteolytic activity. However, mounting evidence shows a variety of proteolysisindependent functions of MT1-MMP. The unique tyrosine (Y573) in the MT1-MMP cytoplasmic tail is fundamental for the control of intracellular signaling. We have shown that Y573 mediates Ras-ERK1/2 signaling, and that Y573 mutation (Y573D) blocks this functionwithout affecting MT1-MMP proteolytic activity. Methods: We generated a mouse with the Y573D mutation in MT1MMP (MT1-MMP Y573D) and characterized its skeletal phenotype by histological and microCT analyses. We isolated BM-MSC and induced them to differentiate into osteoblasts, chondrocytes and adipocytes, using qRT-PCR to analyze gene expression. Mouse C3H10T1/2MSCwere transfected with MT1-MMP cDNA and analyzed for Wnt signaling and Runx2 activation by luciferase reporter assays. Results: Analysis of the long bones of both homozygote and heterozygote MT1-MMP Y573D mice showed increased trabecular bone relative to wt littermates (BV/TV %; wt: 10 4±8; homo: 23.2±2.0; Tb.Th (mm) wt: 20.5±3.8; homo: 40.1±7.0; Tb.N./mm wt: 10.5±4.2; homo: 19.5 4.2; Tb.Sp (mm): 325.2±52.1; 190.6 49.1; p 0.01), a phenotypeopposite to that of MT1-MMP-/mice. MT1-MMP Y573D mice also showed marked thinning of articular cartilage, with disorganized tissue architecture, clustering and cloning of chondrocytes, as well as pronounced decrease in bone marrow-associated and total body fat (fat weight/body weight wt: 0.270±0.0105; homo: 0.0135±0.006; p1⁄40.0029). These findings indicated a defect in MSC differentiation. Therefore, we isolated BM-MSC from wt and homozygous mutant littermates, and induced them to differentiate into osteoblast and chondrocytes, and myeloid precursors were induced to differentiate into osteoclasts. The Y573D mutation dramatically increased MSC expression of osteoblast markers (ALP, COL1, BSP, OCN, RUNX2) and strongly downregulated chondrocyte (ADA4, ADA5, SOX9, MMP13) and osteoclast markers (TRACP, CALCR, CTSK, DC-STAMP, MMP9, iNOS). These findings indicated thatWnt signaling is upregulated inMT1MMP Y573D-expressing MSC. The canonical Wnt pathway is indeed unique, as it exerts opposite effects on osteoblast and chondrocyte differentiation fromMSC. Therefore, we analyzedWnt signaling and Runx2 activation. We transiently transfected C3H10T1/2 MSC cells in osteoblast medium with the cDNAs for wt MT1-MMP and MT1-MMP Y573D. As controls the cellswere transfectedwith the empty vector (pcDNA) orwith MT1-MMP E240A, a mutant devoid of proteolytic activity. MT1-MMP Y573D dramatically upregulated both Wnt signaling (2.8-4.6-fold; p1⁄40.007) and Runx2 activity (8.7-46.5-fold; 1⁄40.009) relative to wt MT1MMP and MT1-MMP E240A. Conclusions: MT1-MMP controls Wnt signaling by a mechanism independent of extracellular proteolysis and mediated by its cytoplasmic tail. MT1-MMP is a bifunctional protein, with an extracellular proteolytic activity that promotes bone formation through ECM remodeling and a cytoplasmic tail that controls osteogenesis by interacting with a key pro-osteogenic signaling pathway. 69 A DUAL ROLE FOR NOTCH SIGNALING IN JOINT CARTILAGE MAINTENANCE AND OSTEOARTHRITIS Z. Liu y, J. Chen z, A.J. Mirando z, C. Wang y, M.J. Zuscik y, R.J. O'Keefe x, M.J. Hilton z. yUniv. of Rochester Med. Ctr., Rochester, NY, USA; zDuke Univ. Sch. of Med., Durham, NC, USA; xWashington Univ. Sch. of Med.,