Yuho Kadono

In autoimmune arthritis, traditionally classified as a T helper (Th) type 1 disease, the activation of T cells results in bone destruction mediated by osteoclasts, but how T cells enhance osteoclastogenesis despite the anti-osteoclastogenic effect of interferon (IFN)-gamma remains to be elucidated. Here, we examine the effect of various Th cell subsets on osteoclastogenesis and identify Th17, a specialized inflammatory subset, as an osteoclastogenic Th cell subset that links T cell activation and bone resorption. The interleukin (IL)-23-IL-17 axis, rather than the IL-12-IFN-gamma axis, is critical not only for the onset phase, but also for the bone destruction phase of autoimmune arthritis. Thus, Th17 is a powerful therapeutic target for the bone destruction associated with T cell activation.

Studies of bone and the immune system have converged in recent years under the banner of osteoimmunology. The immune system is spawned in the bone marrow reservoir, and investigators now recognize that important niches also exist there for memory lymphocytes. At the same time, various factors produced during immune responses are capable of profoundly affecting regulation of bone. Mechanisms have evolved to prevent excessive interference by the immune system with bone homeostasis, yet pathologic bone loss is a common sequela associated with autoimmunity and cancer. There are also developmental links, or parallels, between bone and the immune system. Cells that regulate bone turnover share a common precursor with inflammatory immune cells and may restrict themselves anatomically, in part by utilizing a signaling network analogous to lymphocyte costimulation. Efforts are currently under way to further characterize how these two organ systems overlap and to develop therapeutic strategies that benefit from this understanding.

Osteoclasts are derived from myeloid lineage cells, and their differentiation is supported by various osteotropic factors, including the tumor necrosis factor (TNF) family member TNF-related activation-induced cytokine (TRANCE). Genetic deletion of TRANCE or its receptor, receptor activator of nuclear factor kappaB (RANK), results in severely osteopetrotic mice with no osteoclasts in their bones. TNF receptor-associated factor (TRAF) 6 is a key signaling adaptor for RANK, and its deficiency leads to similar osteopetrosis. Hence, the current paradigm holds that TRANCE-RANK interaction and subsequent signaling via TRAF6 are essential for the generation of functional osteoclasts. Surprisingly, we show that hematopoietic precursors from TRANCE-, RANK-, or TRAF6-null mice can become osteoclasts in vitro when they are stimulated with TNF-alpha in the presence of cofactors such as TGF-beta. We provide direct evidence against the current paradigm that the TRANCE-RANK-TRAF6 pathway is essential for osteoclast differentiation and suggest the potential existence of alternative routes for osteoclast differentiation.

Receptor activator of NF-κB ligand (RANKL) is a transmembrane glycoprotein that has an essential role in the development of osteoclasts. The extracellular portion of RANKL is cleaved proteolytically to produce soluble RANKL, but definite RANKL sheddase(s) and the physiologic function of RANKL shedding have not yet been determined. In the present study, we found that matrix metalloproteinase (MMP) 14 and a disintegrin and metalloproteinase (ADAM) 10 have strong RANKL shedding activity. In Western blot analysis, soluble RANKL was detected as two different molecular weight products, and RNA interference of MMP14 and ADAM10 resulted in a reduction of both the lower and higher molecular weight products. Suppression of MMP14 in primary osteoblasts increased membrane-bound RANKL and promoted osteoclastogenesis in cocultures with macrophages. Soluble RANKL produced by osteoblasts from MMP14-deficient mice was markedly reduced, and their osteoclastogenic activity was promoted, consistent with the findings of increased osteoclastogenesis in vivo. RANKL shedding is an important process that down-regulates local osteoclastogenesis. Receptor activator of NF-κB ligand (RANKL) is a transmembrane glycoprotein that has an essential role in the development of osteoclasts. The extracellular portion of RANKL is cleaved proteolytically to produce soluble RANKL, but definite RANKL sheddase(s) and the physiologic function of RANKL shedding have not yet been determined. In the present study, we found that matrix metalloproteinase (MMP) 14 and a disintegrin and metalloproteinase (ADAM) 10 have strong RANKL shedding activity. In Western blot analysis, soluble RANKL was detected as two different molecular weight products, and RNA interference of MMP14 and ADAM10 resulted in a reduction of both the lower and higher molecular weight products. Suppression of MMP14 in primary osteoblasts increased membrane-bound RANKL and promoted osteoclastogenesis in cocultures with macrophages. Soluble RANKL produced by osteoblasts from MMP14-deficient mice was markedly reduced, and their osteoclastogenic activity was promoted, consistent with the findings of increased osteoclastogenesis in vivo. RANKL shedding is an important process that down-regulates local osteoclastogenesis. Receptor activator of NF-κB ligand (RANKL), 2The abbreviations used are: RANKL, receptor activator of NF-κB ligand; MMP, matrix metalloproteinase; ADAM, a disintegrin and metalloproteinase; OPG, osteoprotegerin; SEAP, secreted placental alkaline phosphatase; MT-MMP, membrane-type matrix metalloproteinase; siRNA, small interference RNA; PGE2, prostaglandin E2; IL-1, interleukin-1; M-CSF, macrophage colony-stimulating factor; ELISA, enzyme-linked immunosorbent assay; GFP, green fluorescent protein; TNF, tumor necrosis factor. also known as TNF-related activation-induced cytokine, osteoprotegerin ligand, and osteoclast differentiation factor, is a type II transmembrane glycoprotein of the TNF ligand family. RANKL is expressed on the plasma membrane of osteoblasts, bone marrow stromal cells, and T-lymphocytes (1Anderson D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. DuBose R.F. Cosman D. Galibert L. Nature. 1997; 390: 175-179Crossref PubMed Scopus (1946) Google Scholar, 5Kong Y.Y. Feige U. Sarosi I. Bolon B. Tafuri A. Morony S. Capparelli C. Li J. Elliott R. McCabe S. Wong T. Campagnuolo G. Moran E. Bogoch E.R. Van G. Nguyen L.T. Ohashi P.S. Lacey D.L. Fish E. Boyle W.J. Penninger J.M. Nature. 1999; 402: 304-309Crossref PubMed Scopus (1596) Google Scholar) and exerts its activity through binding to its TNF family receptor RANK, which is expressed on monocyte-macrophage lineage osteoclast precursors (3Yasuda H. Shima N. Nakagawa N. Yamaguchi K. Kinosaki M. Mochizuki S. Tomoyasu A. Yano K. Goto M. Murakami A. Tsuda E. Morinaga T. Higashio K. Udagawa N. Takahashi N. Suda T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3597-3602Crossref PubMed Scopus (3571) Google Scholar, 4Lacey D.L. Timms E. Tan H.L. Kelley M.J. Dunstan C.R. Burgess T. Elliott R. Colombero A. Elliott G. Scully S. Hsu H. Sullivan J. Hawkins N. Davy E. Capparelli C. Eli A. Qian Y.X. Kaufman S. Sarosi I. Shalhoub V. 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Dunstan C.R. Lacey D.L. Mak T.W. Boyle W.J. Penninger J.M. Nature. 1999; 397: 315-323Crossref PubMed Scopus (2865) Google Scholar, 11Dougall W.C. Glaccum M. Charrier K. Rohrbach K. Brasel K. De Smedt T. Daro E. Smith J. Tometsko M.E. Maliszewski C.R. Armstrong A. Shen V. Bain S. Cosman D. Anderson D. Morrissey P.J. Peschon J.J. Schuh J. Genes Dev. 1999; 13: 2412-2424Crossref PubMed Scopus (1205) Google Scholar). Conversely, a deficiency of osteoprotegerin (OPG), a natural inhibitor of RANKL, causes severe osteoporosis due to enhanced osteoclastogenesis (12Bucay N. Sarosi I. Dunstan C.R. Morony S. Tarpley J. Capparelli C. Scully S. Tan H.L. Xu W. Lacey D.L. Boyle W.J. Simonet W.S. Genes Dev. 1998; 12: 1260-1268Crossref PubMed Scopus (2136) Google Scholar). These findings suggest a crucial role for the RANKL/RANK/OPG axis in osteoclast development. A number of transmembrane proteins undergo proteolysis and are released from the plasma membrane, a process called ectodomain shedding. The biologic and pathologic significance of ectodomain shedding varies between substrate proteins. For example, cytokines such as TNF-α and epidermal growth factor are released from local environments and exert their activity in paracrine and endocrine signaling by ectodomain shedding (13Kriegler M. Perez C. DeFay K. Albert I. Lu S.D. Cell. 1988; 53: 45-53Abstract Full Text PDF PubMed Scopus (931) Google Scholar, 15Harris R.C. Chung E. Coffey R.J. Exp. Cell Res. 2003; 284: 2-13Crossref PubMed Scopus (615) Google Scholar). In some cases, ectodomain shedding is necessary even for the local effects of growth factors such as epidermal growth factor (16Prenzel N. Zwick E. Daub H. Leserer M. Abraham R. Wallasch C. Ullrich A. Nature. 1999; 402: 884-888Crossref PubMed Scopus (1499) Google Scholar). Although membrane-bound RANKL is converted to a soluble form through ectodomain shedding (17Lum L. Wong B.R. Josien R. Becherer J.D. Erdjument-Bromage H. Schlondorff J. Tempst P. Choi Y. Blobel C.P. J. Biol. Chem. 1999; 274: 13613-13618Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 18Nakashima T. Kobayashi Y. Yamasaki S. Kawakami A. Eguchi K. Sasaki H. Sakai H. Biochem. Biophys. Res. Commun. 2000; 275: 768-775Crossref PubMed Scopus (428) Google Scholar) similar to other TNF family members, such as TNF-α (13Kriegler M. Perez C. DeFay K. Albert I. Lu S.D. Cell. 1988; 53: 45-53Abstract Full Text PDF PubMed Scopus (931) Google Scholar, 14Black R.A. Rauch C.T. Kozlosky C.J. Peschon J.J. Slack J.L. Wolfson M.F. Castner B.J. Stocking K.L. Reddy P. Srinivasan S. Nelson N. Boiani N. Schooley K.A. Gerhart M. Davis R. Fitzner J.N. Johnson R.S. Paxton R.J. March C.J. Cerretti D.P. Nature. 1997; 385: 729-733Crossref PubMed Scopus (2715) Google Scholar) and Fas ligand (19Schneider P. Holler N. Bodmer J.L. Hahne M. Frei K. Fontana A. Tschopp J. J. Exp. Med. 1998; 187: 1205-1213Crossref PubMed Scopus (708) Google Scholar), the RANKL sheddases involved in physiologic and pathologic bone resorption, and the role of RANKL shedding is unknown. We report that RANKL shedding is regulated by matrix metalloproteinase (MMP) 14 and a disintegrin and metalloproteinase (ADAM) 10 in bone marrow stromal cells and osteoblasts. MMP14 is mainly involved in RANKL shedding in osteoblasts, and its deficiency up-regulates osteoclastogenesis by reducing RANKL shedding in the local bone milieu. Reagents— DNA polymerase, KOD plus, was purchased from TOYOBO (Osaka, Japan). Antibodies for the His-tag were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), antibodies for the V5 tag were from Invitrogen, antibodies for actin were from Sigma-Aldrich, and the antibody for RANKL was from Active Motif (Carlsbad, CA). MMP inhibitors, FR255031 and FR217840, were generous gifts from Astellas Pharma Inc. (Tokyo, Japan). Constructs—The procedures for the construction of the tRANKL-SEAP expression vector, pcDNA3.1-RANKL-V5HisB, pcDNA3.1-mMMP14-V5HisA, pcDNA3.1-mMMP13-V5HisA, and expression vectors for human MT1, MT2, MT3, MT4, MT5, and MT6-MMP were described previously (20Hikita A. Kadono Y. Chikuda H. Fukuda A. Wakeyama H. Yasuda H. Nakamura K. Oda H. Miyazaki T. Tanaka S. J. Biol. Chem. 2005; 280: 41700-41706Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 26Miyamori H. Takino T. Kobayashi Y. Tokai H. Itoh Y. Seiki M. Sato H. J. Biol. Chem. 2001; 276: 28204-28211Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). An expression vector for mouse RANKL, pSG5-RANKL, was constructed by inserting mouse RANKL cDNA into pSG5 (Stratagene, La Jolla, CA). Expression vectors for mouse MMP1, MMP2, MMP3, MMP7, MMP9, MMP11, MMP19, MMP23, MMP28, ADAM9, ADAM10, ADAM17, and ADAM19 were constructed by inserting the cDNA generated by reverse transcription-PCR of mRNA of mouse osteoblasts into pcDNA3.1-V5HisA (Invitrogen). The cDNA for ADAM10-V5His was further subcloned from pcDNA3.1- or ADAM10-V5HisA and inserted into the pSG5 vector. Small interference RNA (siRNA) plasmids for GFP, mouse ADAM10 and MMP14 were constructed using piGENE mU6 vector (iGENE Therapeutics Inc., Ibaraki, Japan) according to the manufacturer's protocol. Target sites were, for GFP, 5′-GCTACGTCCAGGAGCGCACCA-3′; for ADAM10, 5′-GGGTCTGTCATTGATGGAAGA-3′ and 5′-GCTGTGATTGCTCAGATATCC-3′; and for MMP14, 5′-GGACTGAGATCAAGGCCAATG-3′ and 5′-GGATGGACACAGAGAACTTCG-3′. The retrovirus vector for mouse MMP14 was constructed by inserting the cDNA fragment for mouse MMP14 into the BamHI and EcoRI restriction sites of pMX-puro (kindly by of The of vectors for and were constructed as cDNA for siRNA, the mouse were subcloned from piGENE by using and into II by and inserted to the EcoRI restriction of Cell human were in and the mouse bone marrow stromal from and were with and both of were with and were in a osteoblasts were from the of from Japan) and mice and mice as previously described T. E. Nakamura I. Takahashi N. 1997; PubMed Scopus Google Scholar). of RANKL alkaline of the and the Western blot were described previously (20Hikita A. Kadono Y. Chikuda H. Fukuda A. Wakeyama H. Yasuda H. Nakamura K. Oda H. Miyazaki T. Tanaka S. J. Biol. Chem. 2005; 280: 41700-41706Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, A. Miyazaki T. Kadono Y. H. T. H. T. K. Oda H. Nakamura K. Tanaka S. J. Bone Miner. Res. PubMed Scopus Google Scholar). soluble RANKL released into the of cells, of the was with of protein and of protein for by The were in and to for of the retrovirus was described previously T. J. 1998; PubMed Google Scholar). osteoblasts were with the retrovirus with for for the was using for and using the according to the manufacturer's protocol. The of used were and for mouse MMP14, and for mouse and for mouse and for mouse and and for mouse Western of were with or prostaglandin and 10 for and 10 of were Cell were using according to the manufacturer's protocol. and were using the as for of of of were to of the was and soluble RANKL was using an were to to membrane, and with The was and the N-terminal was by Japan). osteoblasts were in a of cells were with retrovirus vectors as of some were with and for The of RANKL in the and in of was by using a development of and Bone marrow were from of mice by the bone marrow with and in with and 10 Japan) for and cells were For the primary osteoblasts were in a of cells were by a as described were on the primary osteoblasts a of and with and for were with (3Yasuda H. Shima N. Nakagawa N. Yamaguchi K. Kinosaki M. Mochizuki S. Tomoyasu A. Yano K. Goto M. Murakami A. Tsuda E. Morinaga T. Higashio K. Udagawa N. Takahashi N. Suda T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3597-3602Crossref PubMed Scopus (3571) Google Scholar), and the number was For primary osteoblasts were on the CA), and bone marrow were on the of by Soluble RANKL by cells were with of pcDNA3.1-RANKL-V5HisB, and using and were Bone marrow were with a of the and with 10 for and of of MMP14-deficient MMP14-deficient mice were generated and to the as previously described Y. H. Seiki M. J. Cell Sci. PubMed Scopus Google Scholar). and were from MMP14-deficient mice and and cells were using a The cells were in with and for in and were by For the osteoclast were in a of and with 10 and RANKL for For the survival and the were with primary osteoblasts in and on with matrix Japan). osteoclasts were were released from the by with in and by For the survival cells were on for and osteoclasts were by the osteoblasts by and in osteoclasts were further in for For the cells were on the with and in and for the cells were by were by with and using CA). of to MMP14-deficient and were in and in were and with and or were to the of the the number of osteoclasts on the bone and of the bone using of were using for the alkaline and a for the and A of was to of and as RANKL previously a RANKL shedding activity using an expression vector a protein of secreted placental alkaline with the form of RANKL, which the transmembrane and of RANKL (20Hikita A. Kadono Y. Chikuda H. Fukuda A. Wakeyama H. Yasuda H. Nakamura K. Oda H. Miyazaki T. Tanaka S. J. Biol. Chem. 2005; 280: 41700-41706Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Expression vectors of or were to cells with tRANKL-SEAP and the alkaline activity of the was In increased alkaline activity in the an in RANKL shedding and tRANKL-SEAP shedding and ADAM10, MMP14 matrix MT2, MT3, and activity In MMP1, MMP2, MMP3, MMP9, MMP11, MMP23, and not tRANKL-SEAP shedding activity. MMP14 and ADAM10 RANKL in the of to RANKL, were in the mouse bone marrow stromal with Soluble RANKL was from the using an protein and to Western blot RANKL with V5 and was in cells were two different molecular and in the The N-terminal of the two are in These two were by metalloproteinase cells were with which but not TNF-α T. S. Y. K. J. M. T. Y. Y. H. Y. A. S. J. 2005; PubMed Scopus Google Scholar), the lower the other with FR217840, which not but also TNF-α T. S. Y. K. J. M. T. Y. Y. S. J. 2005; PubMed Scopus Google Scholar), both of the These suggest that the is produced by family and the lower by ADAM9, ADAM10, and ADAM19 cleaved RANKL, but ADAM10 produced soluble RANKL with the molecular weight as the in cells not MMP14, MT2, MT3, and generated soluble RANKL with the molecular weight as the lower and MT6-MMP not the soluble RANKL detected in the not that the MMP14 mRNA was higher that of or in cells and primary osteoblasts that MMP14 is mainly involved in the of the lower The mRNA of ADAM10 was to that of MMP14 in two cells not the role of ADAM10 and MMP14 in the shedding of RANKL in cells, we constructed expression vectors of for ADAM10 and MMP14 and We constructed two different and and of expression of the The of soluble RANKL the RANKL was to cells with the lower with and the was both and were The in the of FR217840, but not FR255031 These that ADAM10 and MMP14 are two RANKL sheddases in MMP14 RANKL in the RANKL sheddases in primary osteoblasts. primary osteoblasts were with PGE2, and IL-1, two in the with the molecular and as in cells with RANKL tag and not but the lower was The expression of MMP14 was also higher that of or in osteoblasts, that MMP14 is the RANKL in primary osteoblasts We constructed retrovirus vectors of that MMP14 expression in primary osteoblasts osteoblasts were with the lower was in the and membrane-bound RANKL was increased in the The of soluble RANKL in the and membrane-bound RANKL in the was also by A in soluble RANKL and an in membrane-bound RANKL was by MMP14 consistent with the of Western blot Conversely, of MMP14 in primary osteoblasts increased the lower of soluble RANKL in the and membrane-bound RANKL in the and further the role of MMP14 in RANKL shedding in osteoblasts, we primary osteoblasts from MMP14 primary osteoblasts of MMP14-deficient mice were with PGE2, and IL-1, the of soluble RANKL was in the The of soluble RANKL was markedly in the and membrane-bound RANKL was increased RANKL the role of MMP14 in the osteoclastogenic activity of osteoblasts, we osteoblasts with retrovirus with bone marrow macrophages. The osteoblasts, in which RANKL shedding activity was and membrane-bound RANKL was osteoclastogenesis cells Conversely, of MMP14 in primary osteoblasts osteoclastogenesis These suggest that membrane-bound RANKL osteoclastogenesis soluble RANKL, and the ectodomain shedding of RANKL by MMP14 osteoclastogenesis. We the osteoclastogenic activity of the soluble RANKL generated by The of cells with RANKL and MMP14 expression vectors were and bone marrow were in with 10 were in from the vector or RANKL expression osteoclasts not even in the of In the from the cells with both RANKL and MMP14 expression vectors osteoclast that the soluble RANKL generated by MMP14 osteoclastogenic activity. primary osteoblasts MMP14 by retrovirus vectors and bone marrow cells were using which the of but and were with PGE2, and IL-1, osteoclasts were These suggest that the of soluble RANKL generated in primary osteoblasts is not to osteoclastogenesis in We the osteoclastogenic activity of MMP14-deficient osteoblasts. bone marrow were with osteoblasts from MMP14-deficient osteoclasts were in cocultures with osteoblasts the other bone marrow from MMP14-deficient mice into osteoclasts with the as cells and their activity and survival were with in of MMP14-deficient mice osteoporosis and increased osteoclasts on the bone which with a report K. P. J. S. M. M. I. J.M. H. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) These that the increased osteoclast number in MMP14-deficient mice is due to the in membrane-bound RANKL in and increased osteoclastogenesis in MMP14-deficient of the of MMP14 mouse and the of of MMP14 mouse and or the and with and or and the number of osteoclasts bone is that protein ectodomain shedding a number of membrane TNF family members, but the of shedding on the substrate For example, TNF-α is cleaved by some such as TNF-α and released into the to strong effects (13Kriegler M. Perez C. DeFay K. Albert I. Lu S.D. Cell. 1988; 53: 45-53Abstract Full Text PDF PubMed Scopus (931) Google Scholar, 14Black R.A. Rauch C.T. Kozlosky C.J. Peschon J.J. Slack J.L. Wolfson M.F. Castner B.J. Stocking K.L. Reddy P. Srinivasan S. Nelson N. Boiani N. Schooley K.A. Gerhart M. Davis R. Fitzner J.N. Johnson R.S. Paxton R.J. March C.J. Cerretti D.P. Nature. 1997; 385: 729-733Crossref PubMed Scopus (2715) Google Scholar). In Fas ligand, which is a strong has effects in its soluble form (19Schneider P. Holler N. Bodmer J.L. Hahne M. Frei K. Fontana A. Tschopp J. J. Exp. Med. 1998; 187: 1205-1213Crossref PubMed Scopus (708) Google Scholar). RANKL is a of the TNF family of and that RANKL is essential in for the differentiation and of osteoclasts. RANKL is proteolytically to the soluble but the involved in RANKL shedding and its physiologic and pathologic are not Western blot that are two different cleaved in the of both bone marrow stromal cells and primary osteoblasts, and the lower was the in primary osteoblasts The N-terminal of the with the previously in cells, and that of the lower with the MMP The lower was in the of an MMP inhibitor and both were by with FR217840, which both and also that and have RANKL shedding activity (17Lum L. Wong B.R. Josien R. Becherer J.D. Erdjument-Bromage H. Schlondorff J. Tempst P. Choi Y. Blobel C.P. J. Biol. Chem. 1999; 274: 13613-13618Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, A. Kadono Y. Chikuda H. Fukuda A. Wakeyama H. Yasuda H. Nakamura K. Oda H. Miyazaki T. Tanaka S. J. Biol. Chem. 2005; 280: 41700-41706Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, A. N. J.L. B. T. M. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar, V. Becherer J.D. Y. Erdjument-Bromage H. Tempst P. Blobel C.P. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). we the RANKL shedding activity of and using the previously RANKL shedding (20Hikita A. Kadono Y. Chikuda H. Fukuda A. Wakeyama H. Yasuda H. Nakamura K. Oda H. Miyazaki T. Tanaka S. J. Biol. Chem. 2005; 280: 41700-41706Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). MMP7, MMP19, ADAM9, ADAM10, ADAM17, MMP14, MT2, MT3, MT4, MT5, and MT6-MMP tRANKL-SEAP shedding activity. ADAM9, ADAM10, MMP14, MT2, MT3, and cleaved Although and ADAM19 RANKL shedding the molecular of the cleaved were different from generated in cells and primary osteoblasts. MMP14 was expressed in and primary osteoblasts. was to RANKL in or and has an important role in A. N. J.L. B. T. M. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). Although tRANKL-SEAP shedding not RANKL in Blobel and (17Lum L. Wong B.R. Josien R. Becherer J.D. Erdjument-Bromage H. Schlondorff J. Tempst P. Choi Y. Blobel C.P. J. Biol. Chem. 1999; 274: 13613-13618Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar) that a fragment of RANKL but not RANKL J. L. Blobel C.P. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), and we that not Although deficiency leads to M. Y. C. C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), not tRANKL-SEAP or These in with the of to that ADAM10 and MMP14 were RANKL sheddases in cells and osteoblasts, is that other are involved in RANKL shedding in some pathologic in which are The between RANKL and RANKL is In the of primary osteoblasts, the lower molecular weight was the higher molecular weight that MMP14 is the RANKL in primary osteoblasts. was further by the from MMP14 osteoblasts and MMP14-deficient osteoblasts, in which the lower molecular weight was and the of soluble RANKL was MMP14 expression in primary osteoblasts by or its deficiency in MMP14 mouse osteoblasts RANKL shedding and increased membrane-bound RANKL, which to increased osteoclastogenic activity in the Although soluble RANKL produced by MMP14 osteoclastogenesis from bone marrow the of primary osteoblasts with and not osteoclastogenesis N. T. Udagawa N. Sasaki T. Yamaguchi A. J.M. Suda T. 1988; PubMed Scopus Google Scholar), even MMP14 was was due to an of soluble RANKL, the of the soluble RANKL in the was not and the of soluble RANKL necessary to osteoclastogenesis in was is that the expression of RANKL is soluble RANKL has effects on bone that D.M. Maraskovsky E. Billingsley W.L. Dougall W.C. Tometsko M.E. Roux E.R. Teepe M.C. DuBose R.F. Cosman D. Galibert L. Nature. 1997; 390: 175-179Crossref PubMed Scopus (1946) Google Scholar, A. N. J.L. B. T. M. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar) not RANKL but also MMP14 in human osteoblasts, which in the of bone by membrane-bound RANKL in the cells H. Res. PubMed Scopus Google Scholar). In MMP14-deficient was Although due to the bone as previously K. P. J. S. M. M. I. J.M. H. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), the osteoclast number was higher in due to the increased membrane-bound RANKL in the osteoblasts. In the of soluble RANKL in MMP14 mice was even were with not These are not due to in osteoclast were in osteoclast differentiation from MMP14-deficient or function of osteoclasts. We were to the role of ADAM10 in in RANKL shedding or due to in and development D. B. L. K. A. W. L. T. A. K. P. PubMed Google Scholar). using of ADAM10 are we MMP14 as a RANKL in primary osteoblasts. RANKL shedding local osteoclastogenesis by reducing membrane-bound RANKL in the We R. Yamaguchi of The of FR255031 and were by Astellas Pharma Inc. (Tokyo, Japan).