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Converting Enzyme or Endocytosed in a Clathrin-Dependent Manner1
,
* Department of Anti-Aging Orthopedic Research,
Department of Orthopedic Surgery, and
Department of Musculoskeletal Reconstruction and Regeneration Surgery, Keio University, School of Medicine, Tokyo, Japan; and
Arthritis and Tissue Degeneration Program, Hospital for Special Surgery, and Departments of Medicine and of Physiology and Biophysics, Weill Medical College of Cornell University, New York, NY 10021
CSF-1 is a hemopoietic growth factor, which plays an essential role in macrophage and osteoclast development. Alternative splice variants of CSF-1 are synthesized as soluble or membrane-anchored molecules, although membrane CSF-1 (mCSF-1) can be cleaved from the cell membrane to become soluble CSF-1. The activities involved in this proteolytic processing, also referred to as ectodomain shedding, remain poorly characterized. In the present study, we examined the properties of the mCSF-1 sheddase in cell-based assays. Shedding of mCSF-1 was up-regulated by phorbol ester treatment and was inhibited by the metalloprotease inhibitors GM6001 and tissue inhibitor of metalloproteases 3. Moreover, the stimulated shedding of mCSF-1 was abrogated in fibroblasts lacking the TNF-
converting enzyme (TACE, also known as a disintegrin and metalloprotease 17) and was rescued by expression of wild-type TACE in these cells, strongly suggesting that the stimulated shedding is TACE dependent. Additionally, we observed that mCSF-1 is predominantly localized to intracellular membrane compartments and is efficiently internalized in a clathrin-dependent manner. These results indicate that the local availability of mCSF-1 is actively regulated by ectodomain shedding and endocytosis. This mechanism may have important implications for the development and survival of monocyte lineage cells.
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1 This work was supported by Nagao Memorial Fund, The Nakatomi Foundation, and Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (19591765) to K.H.
2 Address correspondence and reprint requests to Dr. Keisuke Horiuchi, Department of Orthopedic Surgery, Keio University, School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail address: horiuchi{at}z3.keio.jp
3 Abbreviations used in this paper: mCSF-1, membrane CSF-1; ADAM, a disintegrin and metalloprotease; ADAMTS, ADAM domain with thrombospondin type I motif; AP, alkaline phosphatase; EGF, epidermal growth factor; ER, endoplasmic reticulum; HA, hemagglutinin; HB, heparin binding; MEF, mouse embryonic fibroblast; MMP, matrix metalloprotease; MT-MMP, membrane-type MMP; PFA, paraformaldehyde; TACE, TNF- converting enzyme; TIMP, tissue inhibitor of metalloproteases; WGA, wheat germ agglutinin.
This article has been cited by other articles:
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  K. Horiuchi, H. Morioka, H. Takaishi, H. Akiyama, C. P. Blobel, and Y. Toyama Ectodomain Shedding of FLT3 Ligand Is Mediated by TNF-{alpha} Converting Enzyme J. Immunol., June 15, 2009; 182(12): 7408 - 7414. [Abstract] [Full Text] [PDF]
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K. Horiuchi, T. Kimura, T. Miyamoto, K. Miyamoto, H. Akiyama, H. Takaishi, H. Morioka, T. Nakamura, Y. Okada, C. P. Blobel, et al. Conditional Inactivation of TACE by a Sox9 Promoter Leads to Osteoporosis and Increased Granulopoiesis via Dysregulation of IL-17 and G-CSF J. Immunol., February 15, 2009; 182(4): 2093 - 2101. [Abstract] [Full Text] [PDF]
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