The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 179, 6715 -6724
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Horiuchi, K.
Right arrow Articles by Toyama, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Horiuchi, K.
Right arrow Articles by Toyama, Y.

Cell Surface Colony-Stimulating Factor 1 Can Be Cleaved by TNF-{alpha} Converting Enzyme or Endocytosed in a Clathrin-Dependent Manner1

Keisuke Horiuchi2,*,{dagger}, Takeshi Miyamoto{dagger},{ddagger}, Hironari Takaishi{dagger}, Akihiro Hakozaki{dagger}, Naoto Kosaki{dagger}, Yoshiteru Miyauchi{dagger}, Mitsuru Furukawa{dagger}, Jiro Takito{dagger}, Hironori Kaneko{dagger}, Kenichiro Matsuzaki{dagger}, Hideo Morioka{dagger}, Carl P. Blobel§ and Yoshiaki Toyama{dagger}

* Department of Anti-Aging Orthopedic Research, {dagger} Department of Orthopedic Surgery, and {ddagger} 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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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-{alpha} 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Colony-stimulating factor 1 is a homodimeric protein that plays an essential role in the development, proliferation, and survival of monocyte-macrophage lineage cells (for reviews on CSF-1, see Refs. 1, 2, 3, 4). CSF-1 binds to the tyrosine kinase CSF-1 receptor or the c-fms proto-oncogene, which is highly expressed on monocyte-macrophage lineage cells. CSF-1 has also been identified as one of the key regulators in osteoclastogenesis in addition to the receptor activator of NF-{kappa}B ligand. As illustrated by the osteopetrotic mutant mouse line op/op, which lacks functional CSF-1 and has few osteoclasts, CSF-1 is crucial for osteoclastogenesis (5, 6, 7).

CSF-1 mRNA is alternatively spliced, giving rise to at least five different CSF-1 mRNA species, which in turn encode three biochemically distinct protein isoforms, as follows: a secreted glycoprotein, a secreted proteoglycan, and a cell surface glycoprotein (8, 9, 10, 11, 12). All three isoforms are biologically active and have been shown to correct the defects in op/op mice, to a different degree, when they are transgenetically introduced (13, 14, 15). Although some full-length CSF-1 has been shown to be expressed as a membrane-anchored isoform (16), the majority of membrane-bound or cell surface CSF-1 (henceforth referred to as membrane CSF-1 (mCSF-1))3 arises from alternative usage of exon 6 of the CSF-1 mRNA. It has been shown that osteoblasts and stromal cells express both the membrane-bound and the soluble CSF-1 isoforms (17, 18, 19), and that this membrane-bound isoform is capable of supporting osteoclastogenesis and CSF-1-dependent cell growth in vitro (20, 21, 22, 23). Additionally, mCSF-1 is involved in regulating body weight, which cannot be compensated by the soluble isoforms (14).

Interestingly, it has also been shown that mCSF-1 is proteolytically cleaved and released from the cell surface to become a soluble growth factor (24, 25, 26). In recent years, various membrane-bound molecules have been shown to be cleaved and released from the cell surface. The proteolytic release of extracellular domains of membrane-bound proteins, called ectodomain shedding, has emerged as an important posttranslational mechanism for regulating the availability and the function of membrane-bound proteins (27, 28, 29). The various shed molecules comprise a wide variety of functionally and structurally diverse proteins, including TNF-{alpha} and its receptors (TNFR1 and TNFR2) (30, 31, 32, 33), ligands of the epidermal growth factor (EGF) receptor, such as TGF{alpha}, heparin-binding EGF (HB-EGF), EGF, amphiregulin, betacellulin, epiregulin and epigen (34, 35, 36), Notch (37), ErbB4 (38), chemokines such as fractalkine (39) and CXCL16 (40), and CSF-1 receptor, c-fms (41), to name some examples. The contribution of ectodomain shedding to the substrate’s function may be different from one protein to another. In the case of HB-EGF and TGF{alpha}, ectodomain shedding is essential, at least during development, for these growth factors to become fully functional (31), whereas in the case of the TNFR1, ectodomain shedding has a role in regulating the sensitivity of cells to TNF-{alpha} by reducing the receptor’s availability (42).

TNF-{alpha} converting enzyme (TACE) (3), also known as a disintegrin and metalloprotease (ADAM) 17, is the first enzyme shown to function as sheddase and was originally identified as an enzyme responsible for the ectodomain shedding of the membrane-bound precursor of TNF-{alpha} (30, 33). However, subsequent studies have identified a far wider range of substrates than initially expected, including the above-mentioned molecules (TGF{alpha}, HB-EGF, amphiregulin, epiregulin, epigen, TNFRs 1 and 2, ErbB4, chemokines, L-selectin, neuregulin, amyloid precursor protein, neurotrophin receptor p75, and c-fms) (30, 31, 32, 33, 34, 35, 36, 38, 39, 40, 41, 43, 44, 45, 46). The significance of ectodomain shedding in vivo has been highlighted by the phenotype of TACE-deficient (Tace–/–) mice, which showed various developmental defects quite similar to those seen in Tgf{alpha}–/–, Hb-egf–/–, and Egfr–/– mice (31, 47).

In addition to ectodomain shedding, other mechanisms can modulate the function of membrane-bound molecules, such as regulation of cell surface availability by means of intracellular trafficking. In fact, various receptors are known to be endocytosed upon ligand binding and then targeted for degradation, or recycled to the cell surface (48, 49, 50). Additionally, recent studies have revealed that some membrane-bound ligands are also subjected to internalization, and that this also has roles in regulating their functions (51, 52).

In this study, we evaluated the biochemical properties of the ectodomain sheddase of mCSF-1, which we found to be sensitive to metalloprotease inhibitors and tissue inhibitor of metalloproteases (TIMP) 3. Stimulated shedding of mCSF-1 was almost completely abolished in fibroblasts from Tace–/– embryos, and was rescued by expression of wild-type TACE, indicating that TACE is essential for stimulated mCSF-1 shedding. Moreover, we examined the subcellular localization of mCSF-1 and found that it is predominantly localized intracellularly and is efficiently internalized from the cell surface via a clathrin-dependent pathway. These results provide new insights into the mechanisms responsible for controlling the local bioavailability of CSF-1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and reagents

TACE-deficient embryonic fibroblasts (Tace–/– mouse embryonic fibroblasts (MEFs)) derived from E13.5 embryos were immortalized, as previously described (53 , 54). ST2 and COS-7 cells were from RIKEN Cell Bank and from M. Jasin (Sloan-Kettering Institute, New York, NY), respectively. Anti-hemagglutinin (HA)-Tag mAbs and anti-Myc-Tag polyclonal Abs were purchased from MBL. Fluorescent-conjugated Abs and streptavidin used in immunostaining were purchased from Jackson ImmunoResearch Laboratories. TOTO3-, phalloidin-, and Alexa-Fluor 568-conjugated transferrin were from Molecular Probes. FITC-labeled wheat germ agglutinin (WGA) from Tritium vulgaris was from Sigma-Aldrich. Anti-calreticulin Ab was from Affinity BioReagents. Anti-mouse CSF-1 was purchased from BD Pharmingen. U0126 and SB202190 were purchased from Calbiochem. GM6001 was from Chemicon International, and human rTIMPs 1–3 were purchased from R&D Systems. All other reagents were obtained from Sigma-Aldrich, unless otherwise indicated.

Cloning of murine mCSF-1 and generation of expression vectors

A cDNA fragment including the entire coding sequence of mCSF-1 was cloned by RT-PCR using the RNA from mouse bone marrow tissue as a template, and the PCR fragment was sequenced (see Fig. 1A for the nucleotide sequence of murine and human mCSF-1). The epitope-tagged expression vectors and cytoplasmic domain deletion mutants were generated by PCR and cloned into pAPtag5 (Genhunter) and pcDNA4/Myc-His (Invitrogen Life Technologies) (see Fig. 1B for the schema of each vector).


Figure 1
View larger version (24K):
[in this window]
[in a new window]

  		FIGURE 1. A, Comparison of nucleotide sequences of murine and human mCSF-1. B, Constructs used in the current study. Schema of AP-tagged (AP-mCSF-1), HA, and Myc/His epitope doubly tagged mCSF-1 (HA-mCSF-1), and their cytoplasmic deletion mutants (AP-{Delta}CP-mCSF-1 and HA-{Delta}CP-mCSF-1, respectively). SS, Signal sequence; GFD, growth factor domain; TM, transmembrane domain; CT, cytoplasmic tail.

 
Cell culture, transfection, and shedding assay

COS-7 cells, ST2 cells, and MEFs were grown in DMEM supplemented with 5% FCS and antibiotics. The cells were transfected with alkaline phosphatase (AP)-tagged mCSF-1 (AP-mCSF-1 and AP-{Delta}CP-mCSF-1; see Fig. 1B) using lipofectamine (Invitrogen Life Technologies), as previously described (34, 55, 56). Fresh Opti-MEM (Invitrogen Life Technologies) medium with or without the indicated reagents was added 18–24 h after transfection and incubated for 1 h. The supernatants were collected and cleared by centrifugation for 15 min at 15,000 x g in a centrifuge to removed cell debris. The activity of AP in the supernatant, which is derived from AP-tagged mCSF-1 released from the cell surface by proteolysis, was measured by colorimetry, as previously described (56). In short, 100 µl of cleared cell supernatants applied in 96-well plates was mixed with the same volume of 2 mg/ml 4-nitrophenylphosphate per well. AP converts 4-nitrophenylphosphate into nitrophenol, which results in increased absorbance at 405 nm. Plates were incubated at 37°C for color development, and OD405 was measured by a microplate reader (Model 680XR; Bio-Rad). The supernatant from nontransfected cells incubated with 4-nitrophenylphosphate for the same amount of time was used as a spectrophotometric blank to normalize for background AP activity. The conditioned supernatant from cells transfected with the AP-tagged vector and incubated without addition of a stimulus such as PMA or an inhibitor of shedding for the same amount of time was used as a control to determine constitutive background shedding for each individual experiment. The average of this constitutive background shedding was set to 1, and used as a reference to calculate percent increase in shedding following stimulation, or percent decrease in shedding following inhibition. The OD405 value of a given sample was normalized by dividing by the OD405 value of the standard from the same individual experiment. To validate that the AP activity in the supernatant was derived from shedding of AP-mCSF-1, but not from cell fragments or vesicles containing AP-mCSF-1, we visualized AP-mCSF-1 from the supernatant and the cell lysate by NBT/5-bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich) on SDS-PAGE, as previously described (56), and confirmed that AP-mCSF-1 from the supernatant migrated faster than that from the cell lysate (data not shown). The faster migration of AP-mCSF-1 in the supernatant compared with the AP-mCSF-1 in the cell lysate is caused by proteolytic cleavage in the membrane-proximal ectodomain, which removes the transmembrane domain and cytoplasmic domain of AP-mCSF-1. All experiments were repeated at least three times in duplicate with similar results.

Immunostaining and confocal microscopy

Cells transiently transfected with HA-mCSF-1 or HA-{Delta}CP-mCSF-1 were incubated for 18–24 h, washed with PBS, and fixed with 4% paraformaldehyde (PFA)/PBS. Permeabilization with 0.1% Triton X-100/PBS was performed when applicable. Fixed cells were blocked with 1% BSA/PBS, incubated with primary Ab at 4°C overnight, washed three times with PBS, and incubated with secondary Ab for another hour. Cells mounted in FluoroGuard (Bio-Rad) were viewed and photographed using an Olympus FV1000 confocal microscope and Olympus Fluoview software.

mCSF-1 internalization assay

The assay was performed essentially as described previously (51). COS-7 cells or ST2 cells were transiently transfected with HA-mCSF-1 or HA-{Delta}CP-mCSF-1. Twenty hours after transfection, cells were incubated with anti-HA Ab at 4°C for 1 h. Unbound Abs were removed by washes with PBS, and the cells were further incubated at 37°C for 0–60 min. After incubation, the cells were washed several times with PBS and fixed with 4% PFA/PBS, followed by permeabilization with 0.1% Triton X-100/PBS. The anti-HA Ab/HA-mCSF-1 complex was detected with fluorophor-conjugated secondary Abs.

Flow cytometry analysis

COS-7 cells transiently expressing either HA-mCSF-1 or HA-{Delta}CP-mCSF-1 were washed twice with ice-cold PBS and then incubated with anti-HA Abs at 4°C for 30 min. After this incubation, the cells were washed with PBS, and were either immediately trypsinized or further incubated with growth medium containing GM6001 at 37°C for the indicated periods of time before trypsinization. Trypsinized cells were resuspended in ice-cold 5% FCS/PBS, incubated with fluorophore-conjugated secondary Ab, washed two times with 5% FCS/PBS, and then analyzed by flow cytometry.

Statistical analysis

Student’s t test for two samples assuming equal variances was used to calculate the p values. Values of p smaller than 0.05 were considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Cloning of murine mCSF-1

In humans, there are multiple CSF-1 mRNAs species (4.0, 3.0, 2.3, 1.9, and 1.6 kb), derived from a single gene via alternative splicing (8, 9, 11). Among these, mCSF-1 is encoded by the 1.6- and 3.0-kb mRNAs. We performed RT-PCR using RNA prepared from murine bone marrow to clone ~0.8-kb-long PCR product. Sequencing revealed that it contained a 771-bp open reading frame with 83% identity to human mCSF-1 cDNA. At the protein level, murine mCSF-1 encoded a 257-aa protein with a putative transmembrane domain, which had 77% identity with human mCSF-1, with all the cysteine residues conserved between these two species (Fig. 1A). When HA-tagged mCSF-1 was expressed in COS-7 cells, it could be detected as a 38-kDa protein by Western blot analysis, which is consistent with the predicted mass of the tagged mCSF-1 molecule (data not shown). We thus concluded that the PCR product is a murine homologue of mCSF-1 and used it for the subsequent experiments.

Ectodomain shedding of mCSF-1 is metalloprotease dependent

Previous studies showed that cleavage of human mCSF-1 can be stimulated by phorbol esters, such as PMA (25), but the nature of this enzymatic activity remained to be characterized. To facilitate the analysis of the shedding activity of mCSF-1, we took advantage of an AP reporter system for ectodomain shedding (for a detailed description of this method, see Ref. 56). Briefly, a mCSF-1 expression vector with an AP tag added to its 5' end was generated (AP-mCSF-1), and was transfected into cultured cells. Cleavage of AP-mCSF-1 resulted in the release of AP activity into the supernatant, allowing shedding of the soluble ectodomain to be assessed by spectrophotometric detection of AP activity. As described previously for untagged mCSF-1 (25), we found that the shedding of the AP-tagged version of mCSF-1 can also be stimulated with PMA (Fig. 2A). To further characterize the protease responsible for this activity, we examined whether GM6001, a broad-spectrum metalloprotease inhibitor, could inhibit this activity. As shown in Fig. 2A, addition of GM6001 effectively decreased both the constitutive and PMA-stimulated shedding of AP-mCSF-1 from transfected COS-7 cells, indicating that the shedding of mCSF-1 into the culture supernatant is metalloprotease dependent, and not caused by release of membrane vesicles or cell debris from the cells. Identical results were obtained when mCSF-1 shedding was studied in ST2 cells, which express endogenous CSF-1 and support osteoclastogenesis in vitro (57) (Fig. 2B).


Figure 2
View larger version (22K):
[in this window]
[in a new window]

  		FIGURE 2. Characterization of constitutive and PMA-stimulated shedding of mCSF-1. COS-7 cells (A) or ST2 cells (B) transiently expressing AP-tagged mCSF-1 were incubated with or without PMA (25 ng/ml) and/or GM6001 (10 µM) for 1 h at 37°C. After the incubation, supernatant was collected and subjected to a colorimetric assay for AP activity, as described in Materials and Methods. C, Effect of MAPK inhibitors on constitutive and PMA-stimulated mCSF-1 shedding. COS-7 cells transfected with AP-mCSF-1 were preincubated for 1 h at 37°C with U0126 (5 µM, a MEK1 and 2 inhibitor) and/or SB202190 (20 µM, a p38 inhibitor), followed by an additional incubation for 1 h with or without PMA in the presence or absence of the designated inhibitor(s). D and E, MAPK inhibitors inhibited PMA-stimulated shedding of mCSF-1 in a dose-dependent manner. COS-7 cells transiently expressing AP-mCSF-1 were incubated with PMA (25 ng/ml) in the presence of a different concentration of U0126 (5, 1, and 0.2 µM) and SB202190 (20, 2, and 0.2 µM), either alone (D) or in combination (E). F, Effects of TIMPs 1–3 on constitutive and PMA-stimulated shedding of mCSF-1. COS-7 cells transiently expressing AP-tagged mCSF-1 were treated with or without PMA (25 ng/ml) in the presence or absence of TIMPs 1–3 (16 nM). Each value represents the mean derived from at least three individual experiments; error bars, SD. *, p < 0.05; **, p < 0.005. ND, No significant difference.

 
MAPK inhibitors reduce PMA-stimulated mCSF-1 shedding

Various signaling pathways and stimuli have been implicated in metalloprotease-mediated ectodomain shedding. Among these, the MAPK pathway has been shown to participate in growth factor-induced and PMA-induced ectodomain shedding for several membrane-bound molecules whose shedding depends on TACE (46, 58, 59). To assess the possible involvement of the MAPK signaling pathway in the release of mCSF-1, we evaluated how a MEK-1 and -2 inhibitor, U0126, and a P38 MAPK inhibitor, SB202190, affected mCSF-1 shedding. Both U0126 and SB202190 slightly reduced the level of constitutive shedding of mCSF-1 (32 and 27%, respectively; Fig. 2C). In contrast, PMA-stimulated shedding was inhibited by 50 and 35% with each reagent, respectively, and was almost completely reduced to the level of basal constitutive shedding when these two inhibitors were used simultaneously, suggesting synergistic effects between these two inhibitors. To further validate these observations, we performed similar experiments using the inhibitors at a lower concentration, either alone (Fig. 2D) or in combination (Fig. 2E). Both U0126 and SB202190 showed statistically significant inhibitory effect at 0.2 µM. When used in combination at this concentration, PMA-stimulated shedding was inhibited by 44%, which is lower than that of these inhibitors used alone (U0126, 24%; SB202190, 30%, respectively). These observations indicate that PMA-stimulated shedding of mCSF-1 is potentially dependent on the MAPK signaling pathways.

Shedding of mCSF-1 is inhibited by TIMP3, but not by TIMP1 or 2

Because the members of the metzincin family, including matrix metalloproteases (MMPs), membrane-type (MT)-MMPs, ADAM domain with thrombospondin type I motifs (ADAMTSs), and ADAMs, show different sensitivity to TIMPs (for reviews on TIMPs, see Refs. 60 and 61), determining the TIMP-inhibitor profile can be an informative approach toward identifying a given sheddase. MMPs are sensitive to TIMPs 1–3, whereas MT1-MMP is sensitive to TIMPs 2 and 3, but not TIMP1, whereas TACE is partially inhibited by TIMP3 only. As shown in Fig. 2F, TIMP3 had a significant inhibitory effect on PMA-induced shedding, whereas TIMPs 1 and 2 had little effect, if any, indicating that the PMA-induced shedding is unlikely to be dependent on MMPs or MT1-MMP. Interestingly, the inhibitory effect of TIMP3 on constitutive shedding was only marginal, although statistically significant, compared with that of GM6001 (Fig. 2A), indicating a possible involvement of other metalloprotease(s) that is less sensitive to TIMPs 1–3 in constitutive shedding of mCSF-1.

PMA-stimulated shedding of mCSF-1 is TACE dependent

Because TACE-dependent shedding can usually be stimulated by PMA, and because TACE is sensitive to TIMP3, but not to TIMPs 1 or 2 (62, 63), we hypothesized that TACE is involved in the shedding of mCSF-1. To assess the contribution of TACE to the shedding of mCSF-1, we used immortalized embryonic fibroblasts derived from E13.5 Tace–/– embryos (Tace–/– MEFs) (54). AP-mCSF-1 was transfected into Tace–/– MEFs together with a wild-type TACE expression vector or with an empty vector. PMA-stimulated shedding was abolished in Tace–/– MEFs (Fig. 3A), and the coexpression of TACE restored PMA-induced shedding activity to a similar level as seen in wild-type control cells (Fig. 3B), suggesting that TACE indeed plays an essential role in PMA-stimulated mCSF-1 shedding.


Figure 3
View larger version (16K):
[in this window]
[in a new window]

  		FIGURE 3. Stimulated shedding of mCSF-1 is TACE dependent. AP-mCSF-1 was transfected into immortalized Tace–/– fibroblasts together with empty vector (–) or TACE expression vector (TACE) (A), or into wild-type immortalized fibroblast (B). The cells were incubated with or without PMA (25 ng/ml) at 37°C for 1 h. Each value represents the mean derived from at least four individual experiments; error bars, SD. **, p < 0.005.

 
mCSF-1 is predominantly expressed in an intracellular compartment

In an attempt to further characterize the cell-biological properties of mCSF-1, we studied its subcellular localization, about which very little has been previously reported (11). When COS-7 cells were transfected with HA-tagged mCSF-1 and immunostained without permeabilization, positive staining was observed on the cell surface (Fig. 4A, a and b). However when PFA-fixed cells were permeabilized with Triton X-100, strong staining was predominantly observed inside the cell, presumably in the Golgi apparatus and in the endoplasmic reticulum (ER) (Fig. 4A, c and d). The anti-HA Ab did not stain untransfected control cells (data not shown). To further corroborate the expression of HA-mCSF-1 on the cell surface, cells transfected with HA-mCSF-1 were trypsinized and then subjected to flow cytometry. As shown in Fig. 4B, the anti-HA Ab bound to HA-mCSF-1 on cell surface, whereas the anti-Myc Ab, which only recognizes an intracellular domain of HA-mCSF-1, did not show any detectable binding (see Fig. 1B for a diagram of the construct). A similar subcellular staining pattern was observed with ST2 cells, which endogenously express CSF-1, and with COS-7 cells transiently expressing HA-mCSF-1 after they were stained with anti-CSF-1 Abs (Fig. 4C). The subcellular localization of mCSF-1 was further examined by costaining the ER with anti-calreticulin, and the Golgi apparatus with WGA. Fig. 4D shows a significant overlap of the staining pattern between mCSF-1 and these two organelle markers. To examine whether PMA has an impact on the cellular localization of mCSF-1, cell staining was also performed with or without incubation with PMA. However, as shown in Fig. 4E (a, b, d, e, g, and h), there was no evident change in the subcellular localization of CSF-1 in the presence or absence of PMA. Additionally, in all cases, there was no major difference in the staining pattern observed with an anti-HA Ab (which recognizes the extracellular domain of HA-mCSF-1) compared with an anti-Myc Ab (which recognizes the cytoplasmic domain of HA-mCSF-1). This observation indicates that a relatively small proportion of the cell-associated mCSF-1 molecules is subjected to ectodomain shedding, and hence, most remain uncleaved (because HA-mCSF-1 is dually tagged by HA and Myc epitopes, any dissociation in the staining pattern between these two epitopes reflects the presence of cleaved mCSF-1 or the membrane-bound cleaved stalk). Alternatively, shed mCSF-1 could be rapidly released from the cell and the membrane-bound stalk could then be immediately degraded. We next examined whether inhibiting mCSF-1 shedding would lead to accumulation of uncleaved mCSF-1 on the cell surface, which should result in stronger staining on the plasma membrane. However, there was little, if any, difference in the staining pattern of mCSF-1 after incubation in the presence of GM6001 for up to 5 h (Fig. 4E, c, f, and i). Consistent with these results, flow cytometric analysis did not reveal any significant difference in the amount of HA-mCSF-1 on the cell surface of COS-7 cells treated with PMA to stimulate shedding, or with GM6001 to block shedding (Fig. 4F). The ratio of HA-mCSF-1-positive COS-7 cells relative to the untreated control (=100%) was 96.8% ± 1.0 SD for PMA-treated cells and 101.2% ± 4.0 SD for GM6001-treated cells (n = 3, p > 0.9), respectively. Up-regulation (by PMA) and inhibition (by GM6001) of the release of HA-mCSF-1 in the supernatant were confirmed by ELISA against murine CSF-1 (data not shown). These results suggest that either a limited amount of the total mCSF-1 is subjected to shedding, as mentioned above, or that uncleaved mCSF-1 is rapidly removed from the cell surface via endocytosis.


Figure 4
View larger version (44K):
[in this window]
[in a new window]

  		FIGURE 4. Evaluation of the subcellular localization of mCSF-1. A, HA-mCSF-1 was transfected into COS-7 cells and incubated overnight. PFA-fixed cells were incubated with an anti-HA mAb (HA), which recognizes the ectodomain of HA-mCSF-1 (see Fig. 1B for a diagram of this construct), with (c and d) or without (a and b) permeabilization. The nucleus and actin filaments were counterstained with TOTO3 and phalloidin, respectively (b and d). Bar, 20 µm. B, Cell surface expression of HA-mCSF-1 in COS-7 cells transfected with the expression vector was evaluated by flow cytometry, using anti-HA Ab or anti-Myc Ab, which recognizes the cytoplasmic domain of HA-mCSF-1. C, Nontransfected ST2 cells (a) and COS-7 cell transfected with HA-mCSF-1 (b) were stained with anti-CSF-1 Ab and counterstained with TOTO3. Bar, 20 µm. D, COS-7 cells transiently expressing HA-mCSF-1 were costained with anti-HA Ab (a and d) and WGA (Golgi apparatus, b) or anti-calreticulin Ab (ER, e). Merged images (c and f) show significant overlap of the staining pattern between mCSF-1 and these organelle markers. E, COS-7 cells transfected with HA-mCSF-1 were incubated with fresh medium (–; a, d, and g), with PMA (25 ng/ml) for 1 h (b, e, and h), or with GM6001 (10 µM) for 5 h (c, f, and i). Cells were PFA fixed and stained with anti-Myc (a–c), anti-HA (d–f), and TOTO3 (g–i). Bar, 20 µm. F, The same cells as in E were similarly treated with/without PMA or GM6001 and subsequently immunostained with anti-HA Ab. The bound Ab was detected with fluorescent-conjugated secondary Ab and evaluated by flow cytometry. Shown here is a representative result derived from three independent experiments.

 
mCSF-1 is efficiently endocytosed via a clathrin-dependent mechanism

To address the hypothesis that uncleaved mCSF-1 is internalized, as is the case for some other membrane-tethered molecules such as TGF{alpha} and the EGFR (49, 51), we incubated live COS-7 cells transiently expressing HA-mCSF-1 with an anti-HA Ab at 4°C and examined whether the bound Abs would be endocytosed and thereby relocalized to an intracellular vesicular compartment. As shown in Fig. 5A (a and b), the HA-mCSF-1/anti-HA-Ab complex was efficiently internalized when COS-7 cells were subsequently incubated at 37°C, but not when they were immediately fixed after incubation at 4°C. Identical results were also observed in ST2 cells (Fig. 5A, c and d), indicating that this cellular mechanism is also active in a cell line used as a model for osteoblasts and stromal cells. Because internalization of a given ligand or a receptor can be triggered by addition of an Ab, we next examined whether the internalization of mCSF-1 occurs independently of Ab binding. COS-7 cells transiently expressing HA-mCSF-1 were cell surface biotinylated and incubated at 37°C. We then immunostained the cells with anti-HA- and FITC-conjugated secondary Abs (to detect HA-mCSF-1) and rhodamine-conjugated streptavidin (to detect biotinylated molecules), and compared their subcellular localization. As shown in Fig. 5B, numerous costained spots were found in the merged images. This result implies that the internalization of mCSF-1 can also occur independently of Ab stimulation.


Figure 5
View larger version (43K):
[in this window]
[in a new window]

  		FIGURE 5. mCSF-1 is internalized in a clathrin-dependent manner. A, COS-7 cells (a and b) or ST2 cells (c and d) were transfected with HA-mCSF-1. Twenty hours after transfection, the cells were incubated with an anti-HA Ab at 4°C for 60 min. After removal of unbound Abs, the plates were either incubated at 37°C for another 45 min and fixed (b and d), or subsequently fixed without further incubation (a and c). The HA-mCSF-1/Ab complex was then detected with fluorescent-conjugated anti-mouse IgG Ab. Bar, 20 µm. B, COS-7 cells transfected with HA-mCSF-1 were cell surface biotinylated at 4°C and incubated for 45 min at 37°C. The cells were then fixed and HA-mCSF-1 was visualized with FITC-conjugated Ab (a), and biotin was detected with rhodamine-conjugated streptavidin (b). The merged images (c and d (boxed area in c)) show a number of costained spots. Bar, 20 µm. C, COS-7 cells transiently expressing HA-mCSF-1 were labeled with anti-HA Ab and incubated with fluorescent-conjugated transferrin for 1 h at 37°C. The cells were fixed, permeabilized, and immunostained with fluorescent-conjugated anti-mouse IgG Ab. Both HA-mCSF-1 (a) and transferrin were efficiently endocytosed. The merged image (c) shows significant colocalization of transferrin and internalized HA-mCSF-1 (arrowheads). Bar, 20 µm. D, COS-7 cells transiently expressing HA-mCSF-1 were labeled with anti-HA Ab and incubated with or without the clathrin inhibitors monodansylcadaverine (300 µM) and sucrose (0.45 M) for 1 h at 37°C. Note that very few intracellular spots are present in the cells treated with the inhibitors (b), compared with the untreated control (a, arrowheads). Bar, 20 µm.

 
The internalization of various proteins and lipids from the cell surface occurs through clathrin-dependent and clathrin-independent, lipid-raft endocytic pathways (64, 65, 66). Clathrin-mediated endocytosis is a vesicular transport event and involves several accessory proteins essential for the formation of clathrin-coated pits. To examine the potential involvement of this pathway in the internalization of mCSF-1, we compared the distribution of internalized HA-mCSF-1/Ab complexes with that of the transferrin receptor, a well-established marker for the clathrin-mediated endocytic pathway. We used fluorescent-conjugated human transferrin to track the endocytosed transferrin receptor and double stained the cells with HA-mCSF-1/Abs. As shown in Fig. 5C, there was a significant colocalization between internalized mCSF-1 and transferrin, indicating that mCSF-1 internalization occurred in a clathrin-dependent manner. Consistently, pretreatment and incubation with a combination of clathrin inhibitors monodansylcadaverine and sucrose (67) effectively inhibited the internalization of mCSF-1 in this assay (Fig. 5D).

Deletion of the cytoplasmic domain leads to faster internalization/degradation of mCSF-1

Because the cytoplasmic domain of membrane proteins is essential for proper endocytic sorting, we next asked whether the deletion of the cytoplasmic domain of mCSF-1 has an impact on the subcellular localization or turnover of mCSF-1. We transfected mCSF-1 lacking its cytoplasmic domain (HA-{Delta}CP-mCSF-1) and HA-mCSF-1 into COS-7 cells and performed an internalization assay with a 0- to 60-min time course (Fig. 6A). Both HA-{Delta}CP-mCSF-1 and HA-mCSF-1 had a comparable cell surface staining pattern at the outset of this experiment, yet HA-{Delta}CP-mCSF-1 was internalized more rapidly than HA-mCSF-1. Internalization of HA-{Delta}CP-mCSF-1 was seen after 5 min, whereas most of the Ab-bound HA-mCSF-1 still remained on the cell surface, with only very little positive staining of intracellular membranes (Fig. 6A, b, c, h, and i). The internalization of HA-{Delta}CP-mCSF-1 appeared to be increased throughout this experiment, and there were many cells with only a few positive spots left at 60 min, indicating increased degradation of HA-{Delta}CP-mCSF-1 in addition to increased endocytosis. In contrast, in cells transfected with HA-mCSF-1, strong positive staining was seen in endosomes and on the cell surface during the same time period. Because the rapid disappearance of HA-{Delta}CP-mCSF-1 from the cell surface could also be caused by accelerated shedding, we performed a similar assay in the presence of GM6001. However, as shown in Fig. 6B, addition of the metalloprotease inhibitor GM6001 had no detectable effect on the removal of HA-{Delta}CP-mCSF-1. Furthermore, deletion of the cytoplasmic domain of mCSF-1 did not significantly affect its shedding efficiency compared with wild-type mCSF-1 (Fig. 6C). To quantitatively validate the more rapid disappearance of HA-{Delta}CP-mCSF-1 compared with HA-mCSF-1, we repeated the experiments described above using a flow cytometric analysis as a readout for the surface expression levels of both membrane proteins. COS-7 cells transiently expressing either HA-mCSF-1 or HA-{Delta}CP-mCSF-1 were labeled with anti-HA Ab at 4°C and were subsequently incubated at 37°C for 0–6 h in the presence of GM6001. Following the incubation, the cells were trypsinized and resuspended in 5% FCS/PBS. As shown in Fig. 6, D and E, flow cytometric analysis demonstrated that HA-{Delta}CP-mCSF-1 disappears more rapidly from the cell surface than HA-mCSF-1 (79.5% ± 3.3 SD and 42.0% ± 13.7 SD at 6 h, respectively), which corroborates the results obtained with confocal microscopy (see above). Taken together, these observations indicate that the cytoplasmic domain of mCFS-1 has a role in endocytic sorting and in the regulation of the cell surface expression levels, but does not affect the susceptibility of mCSF-1 to ectodomain shedding.


Figure 6
View larger version (38K):
[in this window]
[in a new window]

  		FIGURE 6. Removal of the cytoplasmic domain from mCSF-1 leads to rapid internalization and degradation. A, COS-7 cells were transfected with HA-mCSF-1 (a–f) or the cytoplasmic-domain deletion mutant HA-{Delta}CP-mCSF-1 (g–l). After incubation with anti-HA Abs at 4°C, the cells were incubated at 37°C for the indicated time period. Note that intracellular spots are already present after 5 min of incubation in HA-{Delta}CP-mCSF-1-transfected cells (h and i (boxed area in h), but not in wild-type mCSF-1-transfected cells (b and c (boxed area in b)). B, HA-mCSF-1 (a) or HA-{Delta}CP-mCSF-1 (b) expressed in COS-7 cells were immunolabeled with anti-HA Ab and incubated with GM6001 (10 µM) for 1 h. C, COS-7 cells transiently expressing either AP-tagged wild-type mCSF-1 (WT) or AP-tagged cytoplasmic deletion mutant (AP-{Delta}CP-mCSF-1, {Delta}CP) were incubated with or without PMA (25 ng/ml) and/or GM6001 (10 µM) for 1 h. The shedding of the AP-tagged ectodomain was evaluated, as described in Materials and Methods. D and E, COS-7 cells transiently expressing HA-mCSF-1 or HA-{Delta}CP-mCSF-1 were labeled with anti-HA-Ab at 4°C for 30 min and incubated in the presence of GM6001 (10 µM) for 0–6 h at 37°C. The expression levels of HA-mCSF-1 or HA-{Delta}CP-mCSF-1 remaining on the cell surface were evaluated by flow cytometry. Note that the number of cells expressing high levels of HA-{Delta}CP-mCSF-1 decreases more rapidly than the number of cells expressing HA-mCSF-1. Shown here is a representative result (D) and the mean (E) derived from four independent experiments; error bars, SD. **, p < 0.005.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
CSF-1 plays indispensable roles in development and survival of monocytes and osteoclasts, as illustrated by the deficiencies in the number of monocyte lineage cells and severe osteopetrosis in op/op mice, which lack CSF-1 (5, 6, 7). Although CSF-1 derives from a single gene, there are multiple mRNAs resulting from alternative splicing, which in turn give rise to three biochemically distinct protein isoforms (8, 9, 10, 11, 12). It has been shown that mCSF-1 is biologically active in in vitro models (21, 23) and partially restores CSF-1 function when transgenically expressed in CSF-1-deficient mice (13, 15). Previously, the membrane-anchored form of CSF-1 was shown to be released from cells (25, 26); yet little is known about the proteolytic activity responsible for this ectodomain shedding and its posttranslational regulation. The current study is the first to characterize and identify the CSF-1 sheddase and to show that mCSF-1 that is not shed is effectively internalized from the cell surface via a clathrin-dependent pathway.

As was shown previously (25), the phorbol ester PMA strongly stimulates mCSF-1 shedding. To further characterize this proteolytic activity, we explored the inhibitory effects of several reagents. Both constitutive and PMA-stimulated shedding of mCSF-1 was sensitive to the metalloprotease inhibitor GM6001 in COS-7 cells and ST2 cells, demonstrating that it depends on a metalloprotease(s). Because GM6001 can inhibit the proteolytic activity of several different enzymes, including MMPs, MT-MMPs, ADAMTSs, and ADAMs, we next used TIMPs 1–3 to narrow down the candidate proteases. TIMPs are the major cellular inhibitors of metalloproteases and have been shown to exhibit distinct efficacies against different metalloprotease subfamily members (61). PMA-stimulated shedding of mCSF-1 was reduced to the levels of constitutive shedding by TIMP3, but not by TIMP1 or 2. This result excludes MMPs, which are sensitive to all three TIMPs, and MT1-MMP, which is sensitive to TIMPs 2 and 3, leaving ADAMs and ADAMTSs as candidate sheddases for mCSF-1.

Because the proteolytic activity of TACE can be highly stimulated by phorbol ester and is sensitive to TIMP3 (34, 46, 55, 62, 63), we hypothesized that TACE is, at least in part, responsible for mCSF-1 shedding. To explore this possibility, we used immortalized fibroblasts derived from Tace–/– embryos. Cells lacking TACE showed little PMA-stimulated increase in mCSF-1 shedding. The PMA-stimulated shedding of mCSF-1 from these cells could be rescued by cotransfection of wild-type TACE, demonstrating that TACE is required for PMA-regulated shedding of mCSF-1. Intriguingly, although constitutive shedding of mCSF-1 was inhibited by GM6001, it was less sensitive to TIMP3. Consistent with this observation, GM6001 showed an inhibitory effect on constitutive shedding even in the absence of TACE (data not shown), and the expression of wild-type TACE in Tace–/– MEFs only led to a minor up-regulation in the constitutive shedding of mCSF-1. The properties of the mCSF-1 sheddases thus closely resemble those of the sheddases for the p75 neurotrophin receptor, for which the PMA-stimulated sheddase is also TACE, whereas the constitutive sheddase(s) is (are) distinct from TACE (46).

For several TACE substrates, such as TGF{alpha}, L-selectin, TNF-{alpha}, and the neurotrophin receptor p75, it has been shown that regulated and constitutive shedding activities depend on MAPK signaling (46, 59). The current study shows that PMA-stimulated mCSF-1 shedding is also potentially dependent on signaling via both MEK and P38 MAPK pathways. This is supported by the observation that the PMA-stimulated component of CSF-1 shedding was strongly reduced when these two pathways are simultaneously blocked. In contrast, the inhibitors of these two MAPKs showed only limited effects on constitutive shedding compared with that of GM6001. This observation further supports the hypothesis that there are distinct sheddases responsible for constitutive and stimulated shedding of mCSF-1.

Processing of membrane proteins by TACE usually occurs in the juxtamembrane region of the substrates. In the case of EGFR-ligand family, the cleavage site is located 7–14 aa residues from the transmembrane domain (68). Similarly, the cleavage site of human mCSF-1 is located at ~8 aa residues from transmembrane domain (69). To our knowledge, the cleavage site of murine mCSF-1 had not been identified yet; however, because the amino acid residues around the putative cleavage site are identical (EGSS; see Fig. 1A) in mouse and human mCSF-1, it is likely that both have the same cleavage site. In contrast, five residues in the juxtamembrane domain of human mCSF-1 (PQLQE), which are essential for human mCSF-1 cleavage (69), are not conserved in mouse (PQIPE). We found that the deletion of these five residues from mouse mCSF-1 had little impact on the efficiency of its ectodomain shedding (data not shown).

Although the membrane-bound isoform of CSF-1 was identified several years ago, very little is known about the subcellular localization of mCSF-1 (11). Our data show that mCSF-1 is detectable on the cell surface; however, the predominant localization of mCSF-1 appears to be in an intracellular compartment. One possible explanation for the relatively low amount of mCSF-1 on the cell surface might be that it is efficiently shed from the cell surface. In this case, blocking mCSF-1 should lead to an accumulation of uncleaved mCSF-1 on the cell surface. However, treatment with GM6001, which blocks mCSF-1 shedding, or with PMA, which enhances its shedding, had little effects on the subcellular expression pattern of mCSF-1. To further explore the fate of uncleaved mCSF-1, we labeled HA-tagged mCSF-1 with anti-HA Abs and tracked its subcellular localization. This demonstrated that uncleaved mCSF-1 is efficiently endocytosed in a clathrin-dependent manner. Taken together, these observations suggest a model in which the availability of soluble mCSF-1 derived from the membrane-anchored form can be controlled by ectodomain shedding, whereas the levels of mCSF-1 on the cell surface appear to be regulated by internalization via clathrin-coated pits. In addition, shedding could also affect the amounts of mCSF-1 on the cell surface, even though this was difficult to detect in the experiments presented in this study, presumably because any mCSF-1 molecules that escaped ectodomain shedding would be rapidly endocytosed.

Previous studies have identified several motifs, which encode endocytic signals and have essential roles in targeting the receptors/ligands to their subcellular destination. These include tyrosine-based (NPXY and YXX{phi}; {phi}, a bulky hydrophobic side chain) (70) and dileucine ([DE]XXXL[LI] and DXXLL) (71, 72) signals. The cytoplasmic domain of mCSF-1 is relatively short (37 aa; see Fig. 1A) and lacks any of these well-characterized motifs. Further studies will therefore be necessary to identify the motif(s) required for intracellular trafficking and endocytosis of mCSF-1.

The exact role of ectodomain shedding of mCSF-1 in vivo remains to be determined. Uncleaved mCSF-1 is at least as potent as the soluble isoforms or has synergistic effects in its ability to support osteoclastogenesis in vitro (21, 73, 74, 75). However, the bioactivity of CSF-1 is retained even after it is shed to become soluble (25). Thus, the shedding mechanism does not seem to be simply involved in switching the function of mCSF-1 on or off, as is the case for TGF{alpha} and HB-EGF, in which ectodomain shedding is essential for their functional activation during development (31, 47). One possible purpose of mCSF-1 shedding might be to down-regulate c-fms signaling elicited by cell-cell contact, which is thought to generate an even more potent signal than that induced by the interaction between soluble CSF-1 and c-fms (21, 76). However, because blocking shedding did not lead to a detectable increase in mCSF-1 levels on the cell surface, one would have to postulate that even small increases in mCSF-1 amounts on the cell surface, which might be difficult to detect with the assays used in this study, would affect its ability to participate in juxtacrine signaling. It is also possible that shedding regulates specific, nonredundant functions of the membrane-bound isoform, such as the maintenance of a normal growth rate (14). Finally, shedding of mCSF-1 could contribute to paracrine signaling, and thereby have similar functions as the soluble splice variants. This would provide an additional mechanism to generate soluble mCSF-1, which would be subjected to regulation by metalloproteases instead of alternative splicing. Further studies, including the generation of knock-in mice carrying uncleavable mCSF-1, will be required to learn more about the role of ectodomain shedding in regulating the function of mCSF-1.

In summary, we present an evaluation of the ectodomain shedding and endocytosis of mCSF-1. Pharmacological inhibitors and a mutant cell line were used to identify TACE as the major sheddase for stimulated shedding for mCSF-1. Moreover, our data show for the first time that uncleaved mCSF-1 is internalized via a clathrin-dependent mechanism, indicating that the bioactivity and/or availability of cell surface mCSF-1 are regulated by two different mechanisms, as follows: namely, ectodomain shedding and endocytosis. These observations are likely to contribute to a better understanding of the local regulation of CSF-1 and of its role in osteoclastogenesis and the survival and development of monocyte lineage cells.


   Acknowledgments
 
We thank Dr. Steven Swendeman for critically reading the manuscript, and Takafumi Yamaguchi, Fumiyo Fukui, and Shizue Tomita for their excellent technical assistance.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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. Back

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 Back

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-{alpha} converting enzyme; TIMP, tissue inhibitor of metalloproteases; WGA, wheat germ agglutinin. Back

Received for publication April 10, 2007. Accepted for publication August 31, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Pixley, F. J., E. R. Stanley. 2004. CSF-1 regulation of the wandering macrophage: complexity in action. Trends Cell Biol. 14: 628-638. [Medline]
  2. Ross, F. P., S. L. Teitelbaum. 2005. {alpha}vβ3 and macrophage CSF: partners in osteoclast biology. Immunol. Rev. 208: 88-105. [Medline]
  3. Chitu, V., E. R. Stanley. 2006. CSF-1 in immunity and inflammation. Curr. Opin. Immunol. 18: 39-48. [Medline]
  4. Fixe, P., V. Praloran. 1998. M-CSF: hemopoietic growth factor or inflammatory cytokine?. Cytokine 10: 32-37. [Medline]
  5. Yoshida, H., S. Hayashi, T. Kunisada, M. Ogawa, S. Nishikawa, H. Okamura, T. Sudo, L. D. Shultz, S. Nishikawa. 1990. The murine mutation osteopetrosis is in the coding region of the macrophage CSF gene. Nature 345: 442-444. [Medline]
  6. Wiktor-Jedrzejczak, W. W., A. Ahmed, C. Szczylik, R. R. Skelly. 1982. Hematological characterization of congenital osteopetrosis in op/op mouse: possible mechanism for abnormal macrophage differentiation. J. Exp. Med. 156: 1516-1527. [Abstract/Free Full Text]
  7. Wiktor-Jedrzejczak, W., A. Bartocci, A. W. Ferrante, Jr, A. Ahmed-Ansari, K. W. Sell, J. W. Pollard, E. R. Stanley. 1990. Total absence of CSF 1 in the macrophage-deficient osteopetrotic (op/op) mouse. Proc. Natl. Acad. Sci. USA 87: 4828-4832. [Abstract/Free Full Text]
  8. Kawasaki, E. S., M. B. Ladner, A. M. Wang, J. Van Arsdell, M. K. Warren, M. Y. Coyne, V. L. Schweickart, M. T. Lee, K. J. Wilson, A. Boosman, et al 1985. Molecular cloning of a complementary DNA encoding human macrophage-specific CSF (CSF-1). Science 230: 291-296. [Abstract/Free Full Text]
  9. Ladner, M. B., G. A. Martin, J. A. Noble, D. M. Nikoloff, R. Tal, E. S. Kawasaki, T. J. White. 1987. Human CSF-1: gene structure and alternative splicing of mRNA precursors. EMBO J. 6: 2693-2698. [Medline]
  10. Wong, G. G., P. A. Temple, A. C. Leary, J. S. Witek-Giannotti, Y. C. Yang, A. B. Ciarletta, M. Chung, P. Murtha, R. Kriz, R. J. Kaufman, et al 1987. Human CSF-1: molecular cloning and expression of 4-kb cDNA encoding the human urinary protein. Science 235: 1504-1508. [Abstract/Free Full Text]
  11. Cerretti, D. P., J. Wignall, D. Anderson, R. J. Tushinski, B. M. Gallis, M. Stya, S. Gillis, D. L. Urdal, D. Cosman. 1988. Human macrophage-CSF: alternative RNA and protein processing from a single gene. Mol. Immunol. 25: 761-770. [Medline]
  12. Pampfer, S., S. Tabibzadeh, F. C. Chuan, J. W. Pollard. 1991. Expression of CSF-1 (CSF-1) messenger RNA in human endometrial glands during the menstrual cycle: molecular cloning of a novel transcript that predicts a cell surface form of CSF-1. Mol. Endocrinol. 5: 1931-1938. [Abstract/Free Full Text]
  13. Yao, G. Q., J. J. Wu, B. H. Sun, N. Troiano, M. A. Mitnick, K. Insogna. 2003. The cell surface form of CSF-1 is biologically active in bone in vivo. Endocrinology 144: 3677-3682. [Abstract/Free Full Text]
  14. Nandi, S., M. P. Akhter, M. F. Seifert, X. M. Dai, E. R. Stanley. 2006. Developmental and functional significance of the CSF-1 proteoglycan chondroitin sulfate chain. Blood 107: 786-795. [Abstract/Free Full Text]
  15. Dai, X. M., X. H. Zong, V. Sylvestre, E. R. Stanley. 2004. Incomplete restoration of CSF 1 (CSF-1) function in CSF-1-deficient Csf1op/Csf1op mice by transgenic expression of cell surface CSF-1. Blood 103: 1114-1123. [Abstract/Free Full Text]
  16. Ryan, G. R., X. M. Dai, M. G. Dominguez, W. Tong, F. Chuan, O. Chisholm, R. G. Russell, J. W. Pollard, E. R. Stanley. 2001. Rescue of the CSF 1 (CSF-1)-nullizygous mouse (Csf1(op)/Csf1(op)) phenotype with a CSF-1 transgene and identification of sites of local CSF-1 synthesis. Blood 98: 74-84. [Abstract/Free Full Text]
  17. Weir, E. C., M. C. Horowitz, R. Baron, M. Centrella, B. M. Kacinski, K. L. Insogna. 1993. Macrophage CSF release and receptor expression in bone cells. J. Bone Miner. Res. 8: 1507-1518. [Medline]
  18. Elford, P. R., R. Felix, M. Cecchini, U. Trechsel, H. Fleisch. 1987. Murine osteoblastlike cells and the osteogenic cell MC3T3-E1 release a macrophage colony-stimulating activity in culture. Calcif. Tissue Int. 41: 151-156. [Medline]
  19. Kaplan, D. L., C. M. Eielson, M. C. Horowitz, K. L. Insogna, E. C. Weir. 1996. TNF-{alpha} induces transcription of the CSF-1 gene in murine osteoblasts. J. Cell. Physiol. 168: 199-208. [Medline]
  20. Rubin, J., X. Fan, D. Thornton, R. Bryant, D. Biskobing. 1996. Regulation of murine osteoblast macrophage CSF production by 1,25(OH)2D3. Calcif. Tissue Int. 59: 291-296. [Medline]
  21. Yao, G. Q., B. Sun, E. E. Hammond, E. N. Spencer, M. C. Horowitz, K. L. Insogna, E. C. Weir. 1998. The cell-surface form of CSF-1 is regulated by osteotropic agents and supports formation of multinucleated osteoclast-like cells. J. Biol. Chem. 273: 4119-4128. [Abstract/Free Full Text]
  22. Felix, R., J. Halasy-Nagy, A. Wetterwald, M. G. Cecchini, H. Fleisch, W. Hofstetter. 1996. Synthesis of membrane- and matrix-bound CSF-1 by cultured osteoblasts. J. Cell. Physiol. 166: 311-322. [Medline]
  23. Stein, J., G. V. Borzillo, C. W. Rettenmier. 1990. Direct stimulation of cells expressing receptors for macrophage CSF (CSF-1) by a plasma membrane-bound precursor of human CSF-1. Blood 76: 1308-1314. [Abstract/Free Full Text]
  24. Tuck, D. P., D. P. Cerretti, A. Hand, A. Guha, S. Sorba, N. Dainiak. 1994. Human macrophage CSF is expressed at and shed from the cell surface. Blood 84: 2182-2188. [Abstract/Free Full Text]
  25. Stein, J., C. W. Rettenmier. 1991. Proteolytic processing of a plasma membrane-bound precursor to human macrophage CSF (CSF-1) is accelerated by phorbol ester. Oncogene 6: 601-605. [Medline]
  26. Rettenmier, C. W., M. F. Roussel, R. A. Ashmun, P. Ralph, K. Price, C. J. Sherr. 1987. Synthesis of membrane-bound CSF 1 (CSF-1) and down-modulation of CSF-1 receptors in NIH 3T3 cells transformed by cotransfection of the human CSF-1 and c-fms (CSF-1 receptor) genes. Mol. Cell. Biol. 7: 2378-2387. [Abstract/Free Full Text]
  27. Schlondorff, J., C. P. Blobel. 1999. Metalloprotease-disintegrins: modular proteins capable of promoting cell-cell interactions and triggering signals by protein-ectodomain shedding. J. Cell Sci. 112: 3603-3617. [Abstract]
  28. Blobel, C. P.. 2000. Remarkable roles of proteolysis on and beyond the cell surface. Curr. Opin. Cell Biol. 12: 606-612. [Medline]
  29. Blobel, C. P.. 2005. ADAMs: key components in EGFR signaling and development. Nat. Rev. Mol. Cell Biol. 6: 32-43. [Medline]
  30. Black, R. A., C. T. Rauch, C. J. Kozlosky, J. J. Peschon, J. L. Slack, M. F. Wolfson, B. J. Castner, K. L. Stocking, P. Reddy, S. Srinivasan, et al 1997. A metalloproteinase disintegrin that releases tumor-necrosis factor-{alpha} from cells. Nature 385: 729-733. [Medline]
  31. Peschon, J. J., J. L. Slack, P. Reddy, K. L. Stocking, S. W. Sunnarborg, D. C. Lee, W. E. Russell, B. J. Castner, R. S. Johnson, J. N. Fitzner, et al 1998. An essential role for ectodomain shedding in mammalian development. Science 282: 1281-1284. [Abstract/Free Full Text]
  32. Solomon, K. A., N. Pesti, G. Wu, R. C. Newton. 1999. Cutting edge: a dominant negative form of TNF-{alpha} converting enzyme inhibits proTNF and TNFRII secretion. J. Immunol. 163: 4105-4108. [Abstract/Free Full Text]
  33. Moss, M. L., S. L. Jin, M. E. Milla, D. M. Bickett, W. Burkhart, H. L. Carter, W. J. Chen, W. C. Clay, J. R. Didsbury, D. Hassler, et al 1997. Cloning of a disintegrin metalloproteinase that processes precursor tumor-necrosis factor-{alpha}. Nature 385: 733-736. [Medline]
  34. Sahin, U., G. Weskamp, K. Kelly, H. M. Zhou, S. Higashiyama, J. Peschon, D. Hartmann, P. Saftig, C. P. Blobel. 2004. Distinct roles for ADAM10 and ADAM17 in ectodomain shedding of six EGFR ligands. J. Cell Biol. 164: 769-779. [Abstract/Free Full Text]
  35. Sunnarborg, S. W., C. L. Hinkle, M. Stevenson, W. E. Russell, C. S. Raska, J. J. Peschon, B. J. Castner, M. J. Gerhart, R. J. Paxton, R. A. Black, D. C. Lee. 2002. TNF-{alpha} converting enzyme (TACE) regulates epidermal growth factor receptor ligand availability. J. Biol. Chem. 277: 12838-12845. [Abstract/Free Full Text]
  36. Sahin, U., C. P. Blobel. 2007. Ectodomain shedding of the EGF-receptor ligand epigen is mediated by ADAM17. FEBS Lett. 581: 41-44. [Medline]
  37. Hartmann, D., B. de Strooper, L. Serneels, K. Craessaerts, A. Herreman, W. Annaert, L. Umans, T. Lubke, A. Lena Illert, K. von Figura, P. Saftig. 2002. The disintegrin/metalloprotease ADAM 10 is essential for Notch signaling but not for {alpha}-secretase activity in fibroblasts. Hum. Mol. Genet. 11: 2615-2624. [Abstract/Free Full Text]
  38. Rio, C., J. D. Buxbaum, J. J. Peschon, G. Corfas. 2000. TNF-{alpha}-converting enzyme is required for cleavage of erbB4/HER4. J. Biol. Chem. 275: 10379-10387. [Abstract/Free Full Text]
  39. Garton, K. J., P. J. Gough, C. P. Blobel, G. Murphy, D. R. Greaves, P. J. Dempsey, E. W. Raines. 2001. TNF-{alpha}-converting enzyme (ADAM17) mediates the cleavage and shedding of fractalkine (CX3CL1). J. Biol. Chem. 276: 37993-38001. [Abstract/Free Full Text]
  40. Gough, P. J., K. J. Garton, P. T. Wille, M. Rychlewski, P. J. Dempsey, E. W. Raines. 2004. A disintegrin and metalloproteinase 10-mediated cleavage and shedding regulates the cell surface expression of CXCL 16. J. Immunol. 172: 3678-3685. [Abstract/Free Full Text]
  41. Rovida, E., A. Paccagnini, M. Del Rosso, J. Peschon, P. Dello Sbarba. 2001. TNF-{alpha}-converting enzyme cleaves the macrophage CSF receptor in macrophages undergoing activation. J. Immunol. 166: 1583-1589. [Abstract/Free Full Text]
  42. McDermott, M. F., I. Aksentijevich, J. Galon, E. M. McDermott, B. W. Ogunkolade, M. Centola, E. Mansfield, M. Gadina, L. Karenko, T. Pettersson, et al 1999. Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define a family of dominantly inherited autoinflammatory syndromes. Cell 97: 133-144. [Medline]
  43. Buxbaum, J. D., K. N. Liu, Y. Luo, J. L. Slack, K. L. Stocking, J. J. Peschon, R. S. Johnson, B. J. Castner, D. P. Cerretti, R. A. Black. 1998. Evidence that TNF {alpha} converting enzyme is involved in regulated {alpha}-secretase cleavage of the Alzheimer amyloid protein precursor. J. Biol. Chem. 273: 27765-27767. [Abstract/Free Full Text]
  44. Horiuchi, K., H. M. Zhou, K. Kelly, K. Manova, C. P. Blobel. 2005. Evaluation of the contributions of ADAMs 9, 12, 15, 17, and 19 to heart development and ectodomain shedding of neuregulins β1 and β2. Dev. Biol. 283: 459-471. [Medline]
  45. Condon, T. P., S. Flournoy, G. J. Sawyer, B. F. Baker, T. K. Kishimoto, C. F. Bennett. 2001. ADAM17 but not ADAM10 mediates TNF-{alpha} and L-selectin shedding from leukocyte membranes. Antisense Nucleic Acid Drug Dev. 11: 107-116. [Medline]
  46. Weskamp, G., J. Schlondorff, L. Lum, J. D. Becherer, T. W. Kim, P. Saftig, D. Hartmann, G. Murphy, C. P. Blobel. 2004. Evidence for a critical role of the TNF {alpha} convertase (TACE) in ectodomain shedding of the p75 neurotrophin receptor (p75NTR). J. Biol. Chem. 279: 4241-4249. [Abstract/Free Full Text]
  47. Jackson, L. F., T. H. Qiu, S. W. Sunnarborg, A. Chang, C. Zhang, C. Patterson, D. C. Lee. 2003. Defective valvulogenesis in HB-EGF and TACE-null mice is associated with aberrant BMP signaling. EMBO J. 22: 2704-2716. [Medline]
  48. Tsao, P., T. Cao, M. von Zastrow. 2001. Role of endocytosis in mediating down-regulation of G-protein-coupled receptors. Trends Pharmacol. Sci. 22: 91-96. [Medline]
  49. Sorkin, A., M. Von Zastrow. 2002. Signal transduction and endocytosis: close encounters of many kinds. Nat. Rev. Mol. Cell Biol. 3: 600-614. [Medline]
  50. Seto, E. S., H. J. Bellen, T. E. Lloyd. 2002. When cell biology meets development: endocytic regulation of signaling pathways. Genes Dev. 16: 1314-1336. [Free Full Text]
  51. Martinez-Arca, S., J. J. Bech-Serra, M. Hurtado-Kuttner, A. Borroto, J. Arribas. 2005. Recycling of cell surface pro-TGF-({alpha}) regulates epidermal growth factor receptor activation. J. Biol. Chem. 280: 36970-36977. [Abstract/Free Full Text]
  52. Tat, S. K., M. Padrines, S. Theoleyre, S. Couillaud-Battaglia, D. Heymann, F. Redini, Y. Fortun. 2006. OPG/membranous-RANKL complex is internalized via the clathrin pathway before a lysosomal and a proteasomal degradation. Bone 39: 706-715. [Medline]
  53. Weskamp, G., H. Cai, T. A. Brodie, S. Higashyama, K. Manova, T. Ludwig, C. P. Blobel. 2002. Mice lacking the metalloprotease-disintegrin MDC9 (ADAM9) have no evident major abnormalities during development or adult life. Mol. Cell. Biol. 22: 1537-1544. [Abstract/Free Full Text]
  54. Horiuchi, K., T. Kimura, T. Miyamoto, H. Takaishi, Y. Okada, Y. Toyama, C. P. Blobel. 2007. TNF{alpha}-converting enzyme (TACE/ADAM17) inactivation in mouse myeloid cells prevents lethality from endotoxin shock. J. Immunol. 179: 2686-2689. [Abstract/Free Full Text]
  55. Zheng, Y., P. Saftig, D. Hartmann, C. Blobel. 2004. Evaluation of the contribution of different ADAMs to tumor necrosis factor {alpha} (TNF{alpha}) shedding and of the function of the TNF{alpha} ectodomain in ensuring selective stimulated shedding by the TNF{alpha} convertase (TACE/ADAM17). J. Biol. Chem. 279: 42898-42906. [Abstract/Free Full Text]
  56. Sahin, U., G. Weskamp, Y. Zheng, V. Chesneau, K. Horiuchi, C. P. Blobel. 2006. A sensitive method to monitor ectodomain shedding of ligands of the epidermal growth factor receptor. Methods Mol. Biol. 327: 99-113. [Medline]
  57. Udagawa, N., N. Takahashi, T. Akatsu, T. Sasaki, A. Yamaguchi, H. Kodama, T. J. Martin, T. Suda. 1989. The bone marrow-derived stromal cell lines MC3T3-G2/PA6 and ST2 support osteoclast-like cell differentiation in cocultures with mouse spleen cells. Endocrinology 125: 1805-1813. [Abstract/Free Full Text]
  58. Montero, J. C., L. Yuste, E. Diaz-Rodriguez, A. Esparis-Ogando, A. Pandiella. 2002. MAPK-dependent and -independent routes control shedding of transmembrane growth factors through multiple secretases. Biochem. J. 363: 211-221. [Medline]
  59. Fan, H., R. Derynck. 1999. Ectodomain shedding of TGF-{alpha} and other transmembrane proteins is induced by receptor tyrosine kinase activation and MAP kinase signaling cascades. EMBO J. 18: 6962-6972. [Medline]
  60. Lambert, E., E. Dasse, B. Haye, E. Petitfrere. 2004. TIMPs as multifacial proteins. Crit. Rev. Oncol. Hematol. 49: 187-198. [Medline]
  61. Baker, A. H., D. R. Edwards, G. Murphy. 2002. Metalloproteinase inhibitors: biological actions and therapeutic opportunities. J. Cell Sci. 115: 3719-3727. [Abstract/Free Full Text]
  62. Amour, A., P. M. Slocombe, A. Webster, M. Butler, C. G. Knight, B. J. Smith, P. E. Stephens, C. Shelley, M. Hutton, V. Knauper, et al 1998. TNF-{alpha} converting enzyme (TACE) is inhibited by TIMP-3. FEBS Lett. 435: 39-44. [Medline]
  63. Horiuchi, K., S. Le Gall, M. Schulte, T. Yamaguchi, K. Reiss, G. Murphy, Y. Toyama, D. Hartmann, P. Saftig, C. P. Blobel. 2007. Substrate selectivity of epidermal growth factor-receptor ligand sheddases and their regulation by phorbol esters and calcium influx. Mol. Biol. Cell 18: 176-188. [Abstract/Free Full Text]
  64. Le Roy, C., J. L. Wrana. 2005. Clathrin- and non-clathrin-mediated endocytic regulation of cell signaling. Nat. Rev. Mol. Cell Biol. 6: 112-126. [Medline]
  65. Takei, K., V. Haucke. 2001. Clathrin-mediated endocytosis: membrane factors pull the trigger. Trends Cell Biol. 11: 385-391. [Medline]
  66. Johannes, L., C. Lamaze. 2002. Clathrin-dependent or not: is it still the question?. Traffic 3: 443-451. [Medline]
  67. Rapacciuolo, A., S. Suvarna, L. Barki-Harrington, L. M. Luttrell, M. Cong, R. J. Lefkowitz, H. A. Rockman. 2003. Protein kinase A and G protein-coupled receptor kinase phosphorylation mediates β-1 adrenergic receptor endocytosis through different pathways. J. Biol. Chem. 278: 35403-35411. [Abstract/Free Full Text]
  68. Harris, R. C., E. Chung, R. J. Coffey. 2003. EGF receptor ligands. Exp. Cell Res. 284: 2-13. [Medline]
  69. Deng, P., C. W. Rettenmier, P. K. Pattengale. 1996. Structural requirements for the ectodomain cleavage of human cell surface macrophage CSF. J. Biol. Chem. 271: 16338-16343. [Abstract/Free Full Text]
  70. Bonifacino, J. S., L. M. Traub. 2003. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu. Rev. Biochem. 72: 395-447. [Medline]
  71. Letourneur, F., R. D. Klausner. 1992. A novel di-leucine motif and a tyrosine-based motif independently mediate lysosomal targeting and endocytosis of CD3 chains. Cell 69: 1143-1157. [Medline]
  72. Sandoval, I. V., O. Bakke. 1994. Targeting of membrane proteins to endosomes and lysosomes. Trends Cell Biol. 4: 292-297. [Medline]
  73. Yao, G. Q., B. H. Sun, E. C. Weir, K. L. Insogna. 2002. A role for cell-surface CSF-1 in osteoblast-mediated osteoclastogenesis. Calcif. Tissue Int. 70: 339-346. [Medline]
  74. Fan, X., D. Fan, H. Gewant, C. L. Royce, M. S. Nanes, J. Rubin. 2001. Increasing membrane-bound MCSF does not enhance OPGL-driven osteoclastogenesis from marrow cells. Am. J. Physiol. 280: E103-E111.
  75. Takahashi, N., N. Udagawa, T. Akatsu, H. Tanaka, Y. Isogai, T. Suda. 1991. Deficiency of osteoclasts in osteopetrotic mice is due to a defect in the local microenvironment provided by osteoblastic cells. Endocrinology 128: 1792-1796. [Abstract/Free Full Text]
  76. Itoh, K., N. Udagawa, K. Matsuzaki, M. Takami, H. Amano, T. Shinki, Y. Ueno, N. Takahashi, T. Suda. 2000. Importance of membrane- or matrix-associated forms of M-CSF and RANKL/ODF in osteoclastogenesis supported by SaOS-4/3 cells expressing recombinant PTH/PTHrP receptors. J. Bone Miner. Res. 15: 1766-1775. [Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
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]

Home page
 J. Immunol.Home page
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]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Horiuchi, K.
Right arrow Articles by Toyama, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Horiuchi, K.
Right arrow Articles by Toyama, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS